WorldWideScience

Sample records for n-terminal cytoplasmic fragment

  1. Secondary structure, stability and tetramerisation of recombinant Kv1.1 potassium channel cytoplasmic N-terminal fragment.

    NARCIS (Netherlands)

    Abbott, G.W.; Bloemendal, M.; van Stokkum, I.H.M.; Mercer, E.A.J.; Miller, R.T.; Sewing, S.; Wolters, M.J.J.; Pongs, O.; Srai, S.K.S.

    1997-01-01

    The recombinant N-terminal fragment (amino acids 14-162) of a tetrameric voltage-gated potassium channel (K(V)1.1) has been studied using spectroscopic techniques. Evidence is presented that it forms a tetramer in aqueous solution, whereas when solubilised in 1% Triton X-100 it remains monomeric.

  2. Antral content, secretion and peripheral metabolism of N-terminal progastrin fragments

    DEFF Research Database (Denmark)

    Goetze, Jens Peter; Hansen, Carsten Palnaes; Rehfeld, Jens F

    2006-01-01

    OBJECTIVES: In addition to the acid-stimulatory gastrins, progastrin also release N-terminal fragments. In order to examine the cellular content, secretion and peripheral metabolism of these fragments, we developed an immunoassay specific for the N-terminal sequence of human progastrin. RESULTS......-terminal progastrin fragments. The basal concentration of N-terminal fragments in normal human plasma was almost 30-fold higher than that of the amidated, acid-stimulatory gastrins (286 pmol/l versus 9.8 pmol/l, n=26, P...-35 in circulation was 30 min, and a pig model revealed the kidneys and the vasculature to the head as the primary sites of degradation. CONCLUSION: The cellular and circulatory concentration profiles of N-terminal progastrin fragments differ markedly from those of the acid-stimulatory gastrins. The high basal...

  3. Expanded Polyglutamine-containing N-terminal Huntingtin Fragments Are Entirely Degraded by Mammalian Proteasomes

    NARCIS (Netherlands)

    Juenemann, Katrin; Schipper-Krom, Sabine; Wiemhoefer, Anne; Kloss, Alexander; Sanz Sanz, Alicia; Reits, Eric A. J.

    2013-01-01

    Huntington disease is a neurodegenerative disorder caused by an expanded polyglutamine (polyQ) repeat within the protein huntingtin (Htt). N-terminal fragments of the mutant Htt (mHtt) proteins containing the polyQ repeat are aggregation-prone and form intracellular inclusion bodies. Improving the

  4. Regulation of presynaptic Ca2+, synaptic plasticity and contextual fear conditioning by a N-terminal β-amyloid fragment.

    Science.gov (United States)

    Lawrence, James L M; Tong, Mei; Alfulaij, Naghum; Sherrin, Tessi; Contarino, Mark; White, Michael M; Bellinger, Frederick P; Todorovic, Cedomir; Nichols, Robert A

    2014-10-22

    Soluble β-amyloid has been shown to regulate presynaptic Ca(2+) and synaptic plasticity. In particular, picomolar β-amyloid was found to have an agonist-like action on presynaptic nicotinic receptors and to augment long-term potentiation (LTP) in a manner dependent upon nicotinic receptors. Here, we report that a functional N-terminal domain exists within β-amyloid for its agonist-like activity. This sequence corresponds to a N-terminal fragment generated by the combined action of α- and β-secretases, and resident carboxypeptidase. The N-terminal β-amyloid fragment is present in the brains and CSF of healthy adults as well as in Alzheimer's patients. Unlike full-length β-amyloid, the N-terminal β-amyloid fragment is monomeric and nontoxic. In Ca(2+) imaging studies using a model reconstituted rodent neuroblastoma cell line and isolated mouse nerve terminals, the N-terminal β-amyloid fragment proved to be highly potent and more effective than full-length β-amyloid in its agonist-like action on nicotinic receptors. In addition, the N-terminal β-amyloid fragment augmented theta burst-induced post-tetanic potentiation and LTP in mouse hippocampal slices. The N-terminal fragment also rescued LTP inhibited by elevated levels of full-length β-amyloid. Contextual fear conditioning was also strongly augmented following bilateral injection of N-terminal β-amyloid fragment into the dorsal hippocampi of intact mice. The fragment-induced augmentation of fear conditioning was attenuated by coadministration of nicotinic antagonist. The activity of the N-terminal β-amyloid fragment appears to reside largely in a sequence surrounding a putative metal binding site, YEVHHQ. These findings suggest that the N-terminal β-amyloid fragment may serve as a potent and effective endogenous neuromodulator. Copyright © 2014 the authors 0270-6474/14/3414210-09$15.00/0.

  5. Structure of the N-terminal fragment of topoisomerase V reveals a new family of topoisomerases

    Energy Technology Data Exchange (ETDEWEB)

    Taneja, Bhupesh; Patel, Asmita; Slesarev, Alexei; Mondragon, Alfonso (NWU); (FSI)

    2010-09-02

    Topoisomerases are involved in controlling and maintaining the topology of DNA and are present in all kingdoms of life. Unlike all other types of topoisomerases, similar type IB enzymes have only been identified in bacteria and eukarya. The only putative type IB topoisomerase in archaea is represented by Methanopyrus kandleri topoisomerase V. Despite several common functional characteristics, topoisomerase V shows no sequence similarity to other members of the same type. The structure of the 61 kDa N-terminal fragment of topoisomerase V reveals no structural similarity to other topoisomerases. Furthermore, the structure of the active site region is different, suggesting no conservation in the cleavage and religation mechanism. Additionally, the active site is buried, indicating the need of a conformational change for activity. The presence of a topoisomerase in archaea with a unique structure suggests the evolution of a separate mechanism to alter DNA.

  6. Prokaryotic Expression and Purification of Human TLE1 N-terminal Q Domain Fragment and Production of its Polyclonal Antibody

    Directory of Open Access Journals (Sweden)

    Su WANG

    2010-11-01

    Full Text Available Background and objective TLE1 is an important protein in regulating Wnt, Notch and EGFR signaling pathways. The TLE1 N-terminal Q domain regulates the pathways by mediating its oligomerization and interaction with LEF1. The aim of this study is to construct the human TLE1 N-terminal Q domain fragment in prokaryotic expression system, express and purify protein TLE1 N-terminal Q domain and prepare its polyclonal antibody. Methods The sequence of TLE1 N-terminal Q domain obtained by PCR from human lung adenocarcinoma cDNA, was cloned into the prokaryotic expression vector pGEX-4T-1 containing Glutathione S-transferase (GST. Vector pGEX-4T1-TLE1-Q was transformed into E.coli BL21 condon plus. The GST-TLE1-Q(1-136 fusion protein was induced by IPTG, digested by Thrombin, purified with glutathione-sepharose beads and FPLC, identified by SDS-PAGE. Then rabbits were immunized with the purified protein TLE1-Q(1-136 for obtaining the antiserum. The titers and specificity of antibodies were measured by ELISA and Western blot. Results The PCR identification and the sequencing of recombinant plasmid demonstrated that vector pGEX-4T1-TLE1-Q was successfully constructed. The SDS-PAGE shows target protein (14 000 Da is the interest protein TLE1-Q(1-136. The TLE1 N-terminal Q domain fragment TLE1-Q(1-136 and its polyclonal antibody have been acquired, with an antibody titer of 1:20 000. Conclusion Expression vector pGEX-4T1-TLE1-Q is correctly constructed. The TLE1 N-terminal Q domain fragment TLE1-Q(1-136 and its polyclonal antibody have been acquired. These work established the foundation for further biological study between TLE1 and lung cancers.

  7. The N-terminal cytoplasmic region of NCBE displays features of an intrinsic disordered structure and represents a novel target for specific drug screening

    Science.gov (United States)

    Bjerregaard-Andersen, Kaare; Perdreau-Dahl, Harmonie; Guldsten, Hanne; Praetorius, Jeppe; Jensen, Jan K.; Morth, Jens P.

    2013-01-01

    The sodium dependent bicarbonate transporter NCBE/NBCn2 is predominantly expressed in the central nervous system (CNS). The highest protein concentrations are found in the choroid plexus. The primary function of this integral plasma membrane transport protein is to regulate intracellular neuronal pH and also probably to maintain the pH homeostasis across the blood-cerebrospinal fluid barrier. NCBE is predicted to contain at least 10 transmembrane helices. The N- and C- termini are both cytoplasmic, with a large N-terminal domain (Nt-NCBE) and a relatively small C-terminal domain (Ct-NCBE). The Nt-NCBE is likely to be involved in bicarbonate recognition and transport and contains key areas of regulation involving pH sensing and protein-protein interactions. Intrinsic disordered protein regions (IDPRs) are defined as protein regions having no rigid three-dimensional structure under physiological conditions. They are believed to be involved in signaling networks in which specific, low affinity, protein-protein interactions play an important role. We predict that NCBE and other SoLute Carrier 4 (SLC4) family members have a high level of intrinsic disorder in their cytoplasmic regions. To provide biophysical evidence for the IDPRs predicted in Nt-NCBE, we produced pure (>99%), recombinant Nt-NCBE using E. coli as the expression host. The protein was used to perform differential scanning fluorescence spectroscopy (DSF), in order to search for small molecules that would induce secondary or tertiary structure in the IDPRs. We expect this to assist the development of selective pharmaceutical compounds against individual SLC4 family members. We have also determined a low resolution (4 Å) X-ray crystal structure of the N-terminal core domain. The N-terminal cytoplasmic domain (cdb3) of anion exchanger 1 (AE1) shares a similar fold with the N-terminal core domain of NCBE. Crystallization conditions for the full-length N-terminal domain have been sought, but only the core

  8. A role for galanin N-terminal fragment (1-15) in anxiety- and depression-related behaviors in rats.

    Science.gov (United States)

    Millón, Carmelo; Flores-Burgess, Antonio; Narváez, Manuel; Borroto-Escuela, Dasiel O; Santín, Luis; Parrado, Concepción; Narváez, José Angel; Fuxe, Kjell; Díaz-Cabiale, Zaida

    2014-10-31

    Galanin (GAL) plays a role in mood regulation. In this study we analyzed the action of the active N-terminal fragment [GAL(1-15)] in anxiety- and depression-related behavioral tests in rats. The effect of GAL(1-15) was analyzed in the forced swimming test, tail suspension test, open field test, and light/dark test. The proximity of GAL1 and GAL2 receptors was examined with the proximity ligation assay (PLA). We tested the GAL receptors involved in GAL(1-15) effects with the GAL2 receptor antagonist M871 and with an in vivo model of siRNA GAL2 receptor knockdown or siRNA GAL1 receptor knockdown rats. The effects of GAL(1-15) were also studied in the cell line RN33B. GAL(1-15) induced strong depression-like and anxiogenic-like effects in all the tests. These effects were stronger than the ones induced by GAL. The involvement of the GAL2 receptor was demonstrated with M871 and with the siRNA GAL2 receptor knockdown rats. The PLA indicated the possible existence of GAL1 and GAL2 heteroreceptor complexes in the dorsal hippocampus and especially in the dorsal raphe nucleus. In the siRNA GAL1 receptor knockdown rats the behavioral actions of GAL(1-15) disappeared, and in the siRNA GAL2 receptor knockdown rats the reductions of the behavioral actions of GAL(1-15) was linked to a disappearance of PLA. In the cell line RN33B, GAL(1-15) decreased 5-HT immunoreactivity more strongly than GAL. Our results indicate that GAL(1-15) exerts strong depression-related and anxiogenic-like effects and may give the basis for the development of drugs targeting GAL1 and GAL2 heteroreceptor complexes in the raphe-limbic system for the treatment of depression and anxiety. © The Author 2015. Published by Oxford University Press on behalf of CINP.

  9. A Role for Galanin N-Terminal Fragment (1–15) in Anxiety- and Depression-Related Behaviors in Rats

    Science.gov (United States)

    Millón, Carmelo; Flores-Burgess, Antonio; Narváez, Manuel; Borroto-Escuela, Dasiel O.; Santín, Luis; Parrado, Concepción; Narváez, José Angel; Fuxe, Kjell

    2015-01-01

    Background: Galanin (GAL) plays a role in mood regulation. In this study we analyzed the action of the active N-terminal fragment [GAL(1–15)] in anxiety- and depression-related behavioral tests in rats. Methods: The effect of GAL(1–15) was analyzed in the forced swimming test, tail suspension test, open field test, and light/dark test. The proximity of GAL1 and GAL2 receptors was examined with the proximity ligation assay (PLA). We tested the GAL receptors involved in GAL(1–15) effects with the GAL2 receptor antagonist M871 and with an in vivo model of siRNA GAL2 receptor knockdown or siRNA GAL1 receptor knockdown rats. The effects of GAL(1–15) were also studied in the cell line RN33B. Results: GAL(1–15) induced strong depression-like and anxiogenic-like effects in all the tests. These effects were stronger than the ones induced by GAL. The involvement of the GAL2 receptor was demonstrated with M871 and with the siRNA GAL2 receptor knockdown rats. The PLA indicated the possible existence of GAL1 and GAL2 heteroreceptor complexes in the dorsal hippocampus and especially in the dorsal raphe nucleus. In the siRNA GAL1 receptor knockdown rats the behavioral actions of GAL(1–15) disappeared, and in the siRNA GAL2 receptor knockdown rats the reductions of the behavioral actions of GAL(1–15) was linked to a disappearance of PLA. In the cell line RN33B, GAL(1–15) decreased 5-HT immunoreactivity more strongly than GAL. Conclusions: Our results indicate that GAL(1–15) exerts strong depression-related and anxiogenic-like effects and may give the basis for the development of drugs targeting GAL1 and GAL2 heteroreceptor complexes in the raphe-limbic system for the treatment of depression and anxiety. PMID:25522404

  10. Analysis of proteolytic processes and enzymatic activities in the generation of huntingtin n-terminal fragments in an HEK293 cell model.

    Directory of Open Access Journals (Sweden)

    Andrew T N Tebbenkamp

    Full Text Available N-terminal fragments of mutant huntingtin (htt that terminate between residues 90-115, termed cleavage product A or 1 (cp-A/1, form intracellular and intranuclear inclusion bodies in the brains of patients with Huntington's disease (HD. These fragments appear to be proteolytic products of the full-length protein. Here, we use an HEK293 cell culture model to investigate huntingtin proteolytic processing; previous studies of these cells have demonstrated cleavage of htt to cp-A/1 like htt fragments.Recombinant N-terminal htt fragments, terminating at residue 171 (also referred to as cp-B/2 like, were efficiently cleaved to produce cp-A/1 whereas fragments representing endogenous caspase, calpain, and metalloproteinase cleavage products, terminating between residues 400-600, were inefficiently cleaved. Using cysteine-labeling techniques and antibody binding mapping, we localized the C-terminus of the cp-A/1 fragments produced by HEK293 cells to sequences minimally limited by cysteine 105 and an antibody epitope composed of residues 115-124. A combination of genetic and pharmacologic approaches to inhibit potential proteases, including γ-secretase and calpain, proved ineffective in preventing production of cp-A/1.Our findings indicate that HEK293 cells express a protease that is capable of efficiently cleaving cp-B/2 like fragments of htt with normal or expanded glutamine repeats. For reasons that remain unclear, this protease cleaves longer htt fragments, with normal or expanded glutamine expansions, much less efficiently. The protease in HEK293 cells that is capable of generating a cp-A/1 like htt fragment may be a novel protease with a high preference for a cp-B/2-like htt fragment as substrate.

  11. Purification and antimicrobial activity studies of the N-terminal fragment of ubiquitin from human amniotic fluid.

    Science.gov (United States)

    Kim, Jin-Young; Lee, Sun Young; Park, Seong-Cheol; Shin, Song Yub; Choi, Sang Joon; Park, Yoonkyung; Hahm, Kyung-Soo

    2007-09-01

    A 4.3-kDa antimicrobial peptide was isolated from human amniotic fluid by dialysis, ultrafiltration, and C18 reversed-phase high performance liquid chromatography. This peptide, which we named Amniotic Fluid Peptide-1 (AFP-1), possessed antimicrobial activity but lacked hemolytic activity. In addition, AFP-1 potently inhibited the growth of a variety of bacteria (Escherichia coli, Salmonella typhimurium, Listeria monocytogenes and Staphylococcus aureus), filamentous fungi (Botrytis cinerea, Aspergillus fumigatus, Neurospora crassa and Fusarium oxysporum) and yeast cells (Candida albicans and Cryptococcus neoformans). Automated Edman degradation showed that the N-terminal sequence of AFP-1 was NH(2)-Met-Gln-Ile-Phe-Val-Lys-Thr-Leu-Thr-Gly-Lys-Thr-Ile-Thr-Leu-Glu-Val-Glu-. The partial sequence had 100% homology to the N-terminal sequence of ubiquitin. Matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry revealed that the molecular mass of AFP-1 was 4280.2 Da. Our data show an antimicrobial activity of ubiquitin N-terminal derived peptide that makes it suitable for use as an antimicrobial agent.

  12. Structure of a two-domain N-terminal fragment of ribosomal protein L10 from Methanococcus jannaschii reveals a specific piece of the archaeal ribosomal stalk.

    Science.gov (United States)

    Kravchenko, Olesya; Mitroshin, Ivan; Nikonov, Stanislav; Piendl, Wolfgang; Garber, Maria

    2010-06-04

    Ribosomal stalk is involved in the formation of the so-called "GTPase-associated site" and plays a key role in the interaction of ribosome with translation factors and in the control of translation accuracy. The stalk is formed by two or three copies of the L7/L12 dimer bound to the C-terminal tail of protein L10. The N-terminal domain of L10 binds to a segment of domain II of 23S rRNA near the binding site for ribosomal protein L11. The structure of bacterial L10 in complex with three L7/L12 N-terminal dimers has been determined in the isolated state, and the structure of the first third of archaeal L10 bound to domain II of 23S rRNA has been solved within the Haloarcula marismortui 50S ribosomal subunit. A close structural similarity between the RNA-binding domain of archaeal L10 and the RNA-binding domain of bacterial L10 has been demonstrated. In this work, a long RNA-binding N-terminal fragment of L10 from Methanococcus jannaschii has been isolated and crystallized. The crystal structure of this fragment (which encompasses two-thirds of the protein) has been solved at 1.6 A resolution. The model presented shows the structure of the RNA-binding domain and the structure of the adjacent domain that exist in archaeal L10 and eukaryotic P0 proteins only. Furthermore, our model incorporated into the structure of the H. marismortui 50S ribosomal subunit allows clarification of the structure of the archaeal ribosomal stalk base. Copyright (c) 2010 Elsevier Ltd. All rights reserved.

  13. Structure and function of the N-terminal 40 kDa fragment of human PMS2: a monomeric GHL ATPase

    OpenAIRE

    Guarné, Alba; Junop, Murray S.; Yang, Wei

    2001-01-01

    Human MutLα, a heterodimer of hMLH1 and hPMS2, is essential for DNA mismatch repair. Inactivation of the hmlh1 or hpms2 genes by mutation or epigenesis causes genomic instability and a predisposition to hereditary non-polyposis cancer. We report here the X-ray crystal structures of the conserved N-terminal 40 kDa fragment of hPMS2, NhPMS2, and its complexes with ATPγS and ADP at 1.95, 2.7 and 2.7 Å resolution, respectively. The NhPMS2 structures closely resemble the ATPase fragment of Escheri...

  14. Supramaximal elevation in B-type natriuretic peptide and its N-terminal fragment levels in anephric patients with heart failure: a case series

    Directory of Open Access Journals (Sweden)

    Ting John YC

    2012-10-01

    Full Text Available Abstract Introduction Little is known about the responses of natriuretic peptides to developing congestive heart failure in ‘anephric’ end-stage kidney disease. Case presentation We present three consecutive cases of surgically-induced anephric patients in a critical care environment: a 28-year-old Caucasian woman (with congestive heart failure, a 42-year-old Caucasian woman (without congestive heart failure, and a 23-year-old Caucasian woman (without congestive heart failure. Our limited study data indicate that cut-off values advocated for B-type natriuretic peptide and its N-terminal fragment to ‘rule out’ congestive heart failure in two of our end-stage kidney disease patients (without congestive heart failure are largely appropriate for anephric patients. However, our index (first patient developed congestive heart failure accompanied by the phenomenon of massive and persistent elevation of these natriuretic levels. Conclusion Our findings suggest that patients from the anephric subclass suffering from congestive heart failure will develop supramaximal elevation of B-type natriuretic peptide and its N-terminal fragment, implying the need for dramatically higher cut-off values with respective magnitudes of the order of 50-fold (B-type natriuretic peptide ~5780pmol/L; 20,000ng/L to 100-fold (N-terminal fragment ~11,800pmol/L; 100,000ng/L higher than current values used to ‘rule in’ congestive heart failure. Further research will be required to delineate those cut-off values. The role of our devised ‘Blood Volume – B-type natriuretic peptide feedback control system’ on ‘anatomical’ and ‘functional’ anephric patients led to significant mathematically-enriched arguments supporting our proposal that this model provides plausible explanations for the study findings, and the model lends support to the important hypothesis that these two groups of anephric patients inflicted with congestive heart failure should effectively

  15. Structure and function of the N-terminal 40 kDa fragment of human PMS2: a monomeric GHL ATPase.

    Science.gov (United States)

    Guarné, A; Junop, M S; Yang, W

    2001-10-01

    Human MutLalpha, a heterodimer of hMLH1 and hPMS2, is essential for DNA mismatch repair. Inactivation of the hmlh1 or hpms2 genes by mutation or epigenesis causes genomic instability and a predisposition to hereditary non-polyposis cancer. We report here the X-ray crystal structures of the conserved N-terminal 40 kDa fragment of hPMS2, NhPMS2, and its complexes with ATPgammaS and ADP at 1.95, 2.7 and 2.7 A resolution, respectively. The NhPMS2 structures closely resemble the ATPase fragment of Escherichia coli MutL, which coordinates protein-protein interactions in mismatch repair by undergoing structural transformation upon binding of ATP. Unlike the E.coli MutL, whose ATPase activity requires protein dimerization, the monomeric form of NhPMS2 is active both in ATP hydrolysis and DNA binding. NhPMS2 is the first example of a GHL ATPase active as a monomer, suggesting that its activity may be modulated by hMLH1 in MutLalpha, and vice versa. The potential heterodimer interface revealed by crystallography provides a mutagenesis target for functional studies of MutLalpha.

  16. Crystal structure of the human CD4 N-terminal two-domain fragment complexed to a class II MHC molecule.

    Energy Technology Data Exchange (ETDEWEB)

    Wang, J.-H.; Meijers, R.; Xiong, Y.; Liu, J.-H.; Sakihama, T.; Zhang, R.-G.; Joachimiak, A.; Reinherz, E. L.; Biosciences Division; Dana-Farber Cancer Inst.; Harvard Medical School

    2001-09-11

    The structural basis of the interaction between the CD4 coreceptor and a class II major histocompatibility complex (MHC) is described. The crystal structure of a complex containing the human CD4 N-terminal two-domain fragment and the murine I-A{sup k }class II MHC molecule with associated peptide (pMHCII) shows that only the 'top corner' of the CD4 molecule directly contacts pMHCII. The CD4 Phe-43 side chain extends into a hydrophobic concavity formed by MHC residues from both {alpha}2 and {beta}2 domains. A ternary model of the CD4-pMHCII-T-cell receptor (TCR) reveals that the complex appears V-shaped with the membrane-proximal pMHCII at the apex. This configuration excludes a direct TCR-CD4 interaction and suggests how TCR and CD4 signaling is coordinated around the antigenic pMHCII complex. Human CD4 binds to HIV gp120 in a manner strikingly similar to the way in which CD4 interacts with pMHCII. Additional contacts between gp120 and CD4 give the CD4-gp120 complex a greater affinity. Thus, ligation of the viral envelope glycoprotein to CD4 occludes the pMHCII-binding site on CD4, contributing to immunodeficiency.

  17. Crystallization of the two-domain N-terminal fragment of the archaeal ribosomal protein L10(P0) in complex with a specific fragment of 23S rRNA

    Energy Technology Data Exchange (ETDEWEB)

    Kravchenko, O. V.; Mitroshin, I. V.; Gabdulkhakov, A. G.; Nikonov, S. V.; Garber, M. B., E-mail: garber@vega.protres.ru [Institute of Protein Research RAS (Russian Federation)

    2011-07-15

    Lateral L12-stalk (P1-stalk in Archaea, P1/P2-stalk in eukaryotes) is an obligatory morphological element of large ribosomal subunits in all organisms studied. This stalk is composed of the complex of ribosomal proteins L10(P0) and L12(P1) and interacts with 23S rRNA through the protein L10(P0). L12(P1)-stalk is involved in the formation of GTPase center of the ribosome and plays an important role in the ribosome interaction with translation factors. High mobility of this stalk puts obstacles in determination of its structure within the intact ribosome. Crystals of a two-domain N-terminal fragment of ribosomal protein L10(P0) from the archaeon Methanococcus jannaschii in complex with a specific fragment of rRNA from the same organism have been obtained. The crystals diffract X-rays at 3.2 Angstrom-Sign resolution.

  18. Crystallization of the two-domain N-terminal fragment of the archaeal ribosomal protein L10(P0) in complex with a specific fragment of 23S rRNA

    Science.gov (United States)

    Kravchenko, O. V.; Mitroshin, I. V.; Gabdulkhakov, A. G.; Nikonov, S. V.; Garber, M. B.

    2011-07-01

    Lateral L12-stalk (P1-stalk in Archaea, P1/P2-stalk in eukaryotes) is an obligatory morphological element of large ribosomal subunits in all organisms studied. This stalk is composed of the complex of ribosomal proteins L10(P0) and L12(P1) and interacts with 23S rRNA through the protein L10(P0). L12(P1)-stalk is involved in the formation of GTPase center of the ribosome and plays an important role in the ribosome interaction with translation factors. High mobility of this stalk puts obstacles in determination of its structure within the intact ribosome. Crystals of a two-domain N-terminal fragment of ribosomal protein L10(P0) from the archaeon Methanococcus jannaschii in complex with a specific fragment of rRNA from the same organism have been obtained. The crystals diffract X-rays at 3.2 Å resolution.

  19. Myocardial infarction-induced N-terminal fragment of cardiac myosin-binding protein C (cMyBP-C) impairs myofilament function in human myocardium.

    Science.gov (United States)

    Witayavanitkul, Namthip; Ait Mou, Younss; Kuster, Diederik W D; Khairallah, Ramzi J; Sarkey, Jason; Govindan, Suresh; Chen, Xin; Ge, Ying; Rajan, Sudarsan; Wieczorek, David F; Irving, Thomas; Westfall, Margaret V; de Tombe, Pieter P; Sadayappan, Sakthivel

    2014-03-28

    Myocardial infarction (MI) is associated with depressed cardiac contractile function and progression to heart failure. Cardiac myosin-binding protein C, a cardiac-specific myofilament protein, is proteolyzed post-MI in humans, which results in an N-terminal fragment, C0-C1f. The presence of C0-C1f in cultured cardiomyocytes results in decreased Ca(2+) transients and cell shortening, abnormalities sufficient for the induction of heart failure in a mouse model. However, the underlying mechanisms remain unclear. Here, we investigate the association between C0-C1f and altered contractility in human cardiac myofilaments in vitro. To accomplish this, we generated recombinant human C0-C1f (hC0C1f) and incorporated it into permeabilized human left ventricular myocardium. Mechanical properties were studied at short (2 μm) and long (2.3 μm) sarcomere length (SL). Our data demonstrate that the presence of hC0C1f in the sarcomere had the greatest effect at short, but not long, SL, decreasing maximal force and myofilament Ca(2+) sensitivity. Moreover, hC0C1f led to increased cooperative activation, cross-bridge cycling kinetics, and tension cost, with greater effects at short SL. We further established that the effects of hC0C1f occur through direct interaction with actin and α-tropomyosin. Our data demonstrate that the presence of hC0C1f in the sarcomere is sufficient to induce depressed myofilament function and Ca(2+) sensitivity in otherwise healthy human donor myocardium. Decreased cardiac function post-MI may result, in part, from the ability of hC0C1f to bind actin and α-tropomyosin, suggesting that cleaved C0-C1f could act as a poison polypeptide and disrupt the interaction of native cardiac myosin-binding protein C with the thin filament.

  20. N-terminal nesprin-2 variants regulate β-catenin signalling

    Energy Technology Data Exchange (ETDEWEB)

    Zhang, Qiuping; Minaisah, Rose-Marie; Ferraro, Elisa; Li, Chen; Porter, Lauren J.; Zhou, Can; Gao, Fang; Zhang, Junyi; Rajgor, Dipen; Autore, Flavia; Shanahan, Catherine M.; Warren, Derek T., E-mail: derek.warren@kcl.ac.uk

    2016-07-15

    The spatial compartmentalisation of biochemical signalling pathways is essential for cell function. Nesprins are a multi-isomeric family of proteins that have emerged as signalling scaffolds, herein, we investigate the localisation and function of novel nesprin-2 N-terminal variants. We show that these nesprin-2 variants display cell specific distribution and reside in both the cytoplasm and nucleus. Immunofluorescence microscopy revealed that nesprin-2 N-terminal variants colocalised with β-catenin at cell-cell junctions in U2OS cells. Calcium switch assays demonstrated that nesprin-2 and β-catenin are lost from cell-cell junctions in low calcium conditions whereas emerin localisation at the NE remained unaltered, furthermore, an N-terminal fragment of nesprin-2 was sufficient for cell-cell junction localisation and interacted with β-catenin. Disruption of these N-terminal nesprin-2 variants, using siRNA depletion resulted in loss of β-catenin from cell-cell junctions, nuclear accumulation of active β-catenin and augmented β-catenin transcriptional activity. Importantly, we show that U2OS cells lack nesprin-2 giant, suggesting that the N-terminal nesprin-2 variants regulate β-catenin signalling independently of the NE. Together, these data identify N-terminal nesprin-2 variants as novel regulators of β-catenin signalling that tether β-catenin to cell-cell contacts to inhibit β-catenin transcriptional activity. - Highlights: • N-terminal nesprin-2 variants display cell specific expression patterns. • N-terminal spectrin repeats of nesprin-2 interact with β-catenin. • N-terminal nesprin-2 variants scaffold β-catenin at cell-cell junctions.. • Nesprin-2 variants play multiple roles in β-catenin signalling.

  1. N-terminal nesprin-2 variants regulate β-catenin signalling

    International Nuclear Information System (INIS)

    Zhang, Qiuping; Minaisah, Rose-Marie; Ferraro, Elisa; Li, Chen; Porter, Lauren J.; Zhou, Can; Gao, Fang; Zhang, Junyi; Rajgor, Dipen; Autore, Flavia; Shanahan, Catherine M.; Warren, Derek T.

    2016-01-01

    The spatial compartmentalisation of biochemical signalling pathways is essential for cell function. Nesprins are a multi-isomeric family of proteins that have emerged as signalling scaffolds, herein, we investigate the localisation and function of novel nesprin-2 N-terminal variants. We show that these nesprin-2 variants display cell specific distribution and reside in both the cytoplasm and nucleus. Immunofluorescence microscopy revealed that nesprin-2 N-terminal variants colocalised with β-catenin at cell-cell junctions in U2OS cells. Calcium switch assays demonstrated that nesprin-2 and β-catenin are lost from cell-cell junctions in low calcium conditions whereas emerin localisation at the NE remained unaltered, furthermore, an N-terminal fragment of nesprin-2 was sufficient for cell-cell junction localisation and interacted with β-catenin. Disruption of these N-terminal nesprin-2 variants, using siRNA depletion resulted in loss of β-catenin from cell-cell junctions, nuclear accumulation of active β-catenin and augmented β-catenin transcriptional activity. Importantly, we show that U2OS cells lack nesprin-2 giant, suggesting that the N-terminal nesprin-2 variants regulate β-catenin signalling independently of the NE. Together, these data identify N-terminal nesprin-2 variants as novel regulators of β-catenin signalling that tether β-catenin to cell-cell contacts to inhibit β-catenin transcriptional activity. - Highlights: • N-terminal nesprin-2 variants display cell specific expression patterns. • N-terminal spectrin repeats of nesprin-2 interact with β-catenin. • N-terminal nesprin-2 variants scaffold β-catenin at cell-cell junctions.. • Nesprin-2 variants play multiple roles in β-catenin signalling.

  2. Dynorphin 1-17 and Its N-Terminal Biotransformation Fragments Modulate Lipopolysaccharide-Stimulated Nuclear Factor-kappa B Nuclear Translocation, Interleukin-1beta and Tumor Necrosis Factor-alpha in Differentiated THP-1 Cells.

    Directory of Open Access Journals (Sweden)

    Siti Sarah Fazalul Rahiman

    Full Text Available Dynorphin 1-17, (DYN 1-17 opioid peptide produces antinociception following binding to the kappa-opioid peptide (KOP receptor. Upon synthesis and release in inflamed tissues by immune cells, DYN 1-17 undergoes rapid biotransformation and yields a unique set of opioid and non-opioid fragments. Some of these major fragments possess a role in immunomodulation, suggesting that opioid-targeted therapeutics may be effective in diminishing the severity of inflammatory disorders. This study aimed to examine the immunomodulatory effects of DYN 1-17 and major N-terminal fragments found in the inflammatory environment on nuclear factor-kappaB/p65 (NF-κB/p65 nuclear translocation and the release of interleukin-1beta (IL-1β and tumor necrosis factor-alpha (TNF-α from lipopolysaccharide (LPS-stimulated, differentiated THP-1 cells. The results demonstrate that NF-κB/p65 nuclear translocation was significantly attenuated following treatment with DYN 1-17 and a specific range of fragments, with the greatest reduction observed with DYN 1-7 at a low concentration (10 nM. Antagonism with a selective KOP receptor antagonist, ML-190, significantly reversed the inhibitory effects of DYN 1-17, DYN 1-6, DYN 1-7 and DYN 1-9, but not other DYN 1-17 N-terminal fragments (DYN 1-10 and 1-11 on NF-κB/p65 nuclear translocation. DYN 1-17 and selected fragments demonstrated differential modulation on the release of IL-1β and TNF-α with significant inhibition observed with DYN 1-7 at low concentrations (1 nM and 10 pM. These effects were blocked by ML-190, suggesting a KOP receptor-mediated pathway. The results demonstrate that DYN 1-17 and certain N-terminal fragments, produced in an inflamed environment, play an anti-inflammatory role by inhibiting NF-κB/p65 translocation and the subsequent cytokine release through KOP receptor-dependent and independent pathways.

  3. Spatial structure of oligopeptide PAP(248-261), the N-terminal fragment of the HIV enhancer prostatic acid phosphatase peptide PAP(248-286), in aqueous and SDS micelle solutions

    Science.gov (United States)

    Blokhin, Dmitriy S.; Filippov, Andrei V.; Antzutkin, Oleg N.; Karataeva, Farida Kh.; Klochkov, Vladimir V.

    2014-07-01

    Prostatic acid phosphatase (PAP) is an enzyme that facilitates infection of cells by HIV. Its peptide fragment PAP(248-286) forms amyloid fibrils known as SEVI, which enhance attachment of the virus by viral adhesion to the host cell prior to receptor-specific binding via reducing the electrostatic repulsion between the membranes of the virus and the target cell. The secondary structure of PAP(248-286) in aqueous and SDS solutions can be divided into an N-terminal disordered region, an α-helical central part and an α/310-helical C-terminal region (Nanga et al., 2009). In this work, we used NMR spectroscopy to study the spatial structure of the isolated N-terminal fragment of PAP(248-286), PAP(248-261) (GIHKQKEKSRLQGG), in aqueous and SDS micelle solutions. Formation of a PAP(248-261)-SDS complex was confirmed by chemical shift alterations in the 1H NMR spectra of the peptide, as well as by the signs and values of Nuclear Overhauser Effect (NOE). In addition, the PAP(248-261) peptide does not form any specified secondary structure in either aqueous or SDS solutions.

  4. Plasma Levels of Monocyte Chemoattractant Protein-1, n-Terminal Fragment of Brain Natriuretic Peptide and Calcidiol Are Independently Associated with the Complexity of Coronary Artery Disease.

    Directory of Open Access Journals (Sweden)

    Roberto Martín-Reyes

    Full Text Available We investigated the relationship of the Syntax Score (SS and coronary artery calcification (CAC, with plasma levels of biomarkers related to cardiovascular damage and mineral metabolism, as there is sparse information in this field.We studied 270 patients with coronary disease that had an acute coronary syndrome (ACS six months before. Calcidiol, fibroblast growth factor-23, parathormone, phosphate and monocyte chemoattractant protein-1 [MCP-1], high-sensitivity C-reactive protein, galectin-3, and N-terminal pro-brain natriuretic peptide [NT-proBNP] levels, among other biomarkers, were determined. CAC was assessed by coronary angiogram as low-grade (0-1 and high-grade (2-3 calcification, measured with a semiquantitative scale ranging from 0 (none to 3 (severe. For the SS study patients were divided in SS<14 and SS≥14. Multivariate linear and logistic regression analyses were performed.MCP-1 predicted independently the SS (RC = 1.73 [95%CI = 0.08-3.39]; p = 0.040, along with NT-proBNP (RC = 0.17 [95%CI = 0.05-0.28]; p = 0.004, male sex (RC = 4.15 [95%CI = 1.47-6.83]; p = 0.003, age (RC = 0.13 [95%CI = 0.02-0.24]; p = 0.020, hypertension (RC = 3.64, [95%CI = 0.77-6.50]; p = 0.013, hyperlipidemia (RC = 2.78, [95%CI = 0.28-5.29]; p = 0.030, and statins (RC = 6.12 [95%CI = 1.28-10.96]; p = 0.013. Low calcidiol predicted high-grade calcification independently (OR = 0.57 [95% CI = 0.36-0.90]; p = 0.013 along with ST-elevation myocardial infarction (OR = 0.38 [95%CI = 0.19-0.78]; p = 0.006, diabetes (OR = 2.35 [95%CI = 1.11-4.98]; p = 0.028 and age (OR = 1.37 [95%CI = 1.18-1.59]; p<0.001. During follow-up (1.79 [0.94-2.86] years, 27 patients developed ACS, stroke, or transient ischemic attack. A combined score using SS and CAC predicted independently the development of the outcome.MCP-1 and NT-proBNP are independent predictors of SS, while low calcidiol plasma levels are associated with CAC. More studies are needed to confirm these data.

  5. Protection efficacy of the Brucella abortus ghost vaccine candidate lysed by the N-terminal 24-amino acid fragment (GI24) of the 36-amino acid peptide PMAP-36 (porcine myeloid antimicrobial peptide 36) in murine models.

    Science.gov (United States)

    Kwon, Ae Jeong; Moon, Ja Young; Kim, Won Kyong; Kim, Suk; Hur, Jin

    2016-11-01

    Brucella abortus cells were lysed by the N-terminal 24-amino acid fragment (GI24) of the 36-amino acid peptide PMAP-36 (porcine myeloid antimicrobial peptide 36). Next, the protection efficacy of the lysed fragment as a vaccine candidate was evaluated. Group A mice were immunized with sterile PBS, group B mice were intraperitoneally (ip) immunized with 3 × 10 8 colony-forming units (CFUs) of B. abortus strain RB51, group C mice were immunized ip with 3 × 10 8 cells of the B. abortus vaccine candidate, and group D mice were orally immunized with 3 × 10 9 cells of the B. abortus vaccine candidate. Brucella lipopolysaccharide (LPS)-specific serum IgG titers were considerably higher in groups C and D than in group A. The levels of interleukin (IL)-4, IL-10, tumor necrosis factor alpha (TNF-α) and interferon gamma (IFN-γ) were significantly higher in groups B-D than in group A. After an ip challenge with B. abortus 544, only group C mice showed a significant level of protection as compared to group A. Overall, these results show that ip immunization with a vaccine candidate lysed by GI24 can effectively protect mice from systemic infection with virulent B. abortus.

  6. Evaluation of the ability of N-terminal fragment of lethal factor of Bacillus anthracis for delivery of Mycobacterium T cell antigen ESAT-6 into cytosol of antigen presenting cells to elicit effective cytotoxic T lymphocyte response

    International Nuclear Information System (INIS)

    Chandra, Subhash; Kaur, Manpreet; Midha, Shuchi; Bhatnagar, Rakesh; Banerjee-Bhatnagar, Nirupama

    2006-01-01

    We report the ability of N-terminal fragment of lethal factor of Bacillus anthracis to deliver genetically fused ESAT-6 (early secretory antigen target), a potent T cell antigen of Mycobacterium tuberculosis, into cytosol to elicit Cytotoxic T lymphocyte (CTL) response. In vitro Th1 cytokines data and CTL assay proved that efficient delivery of LFn.ESAT-6 occurs in cytosol, in the presence of protective antigen (PA), and leads to generation of effective CTL response. Since CTL response is essential for protection against intracellular pathogens and, it is well known that only single T cell epitope or single antigenic protein is not sufficient to elicit protective CTL response due to variation or polymorphism in MHC-I alleles among the individuals, we suggest that as a fusion protein LFn can be used to deliver multiepitopes of T cells or multiproteins which can generate effective CTLs against intracellular pathogens like M. tuberculosis. It can be used to enhance the protective efficacy of BCG vaccine

  7. An Intrabody Drug (rAAV6-INT41 Reduces the Binding of N-Terminal Huntingtin Fragment(s to DNA to Basal Levels in PC12 Cells and Delays Cognitive Loss in the R6/2 Animal Model

    Directory of Open Access Journals (Sweden)

    I. Alexandra Amaro

    2016-01-01

    Full Text Available Huntington’s disease (HD is a fatal progressive disease linked to expansion of glutamine repeats in the huntingtin protein and characterized by the progressive loss of cognitive and motor function. We show that expression of a mutant human huntingtin exon-1-GFP fusion construct results in nonspecific gene dysregulation that is significantly reduced by 50% due to coexpression of INT41, an intrabody specific for the proline-rich region of the huntingtin protein. Using stable PC12 cell lines expressing either inducible human mutant huntingtin (mHtt, Q73 or normal huntingtin (nHtt, Q23, we investigated the effect of rAAV6-INT41, an adeno-associated virus vector with the INT41 coding sequence, on the subcellular distribution of Htt. Compartmental fractionation 8 days after induction of Htt showed a 6-fold increased association of a dominate N-terminal mHtt fragment with DNA compared to N-terminal nHtt. Transduction with rAAV6-INT41 reduced DNA binding of N-terminal mHtt 6.5-fold in the nucleus and reduced nuclear translocation of the detected fragments. Subsequently, when rAAV6-INT41 is delivered to the striatum in the R6/2 mouse model, treated female mice exhibited executive function statistically indistinguishable from wild type, accompanied by reductions in Htt aggregates in the striatum, suggesting that rAAV6-INT41 is promising as a gene therapy for Huntington’s disease.

  8. Functional expression of single-chain variable fragment antibody against c-Met in the cytoplasm of Escherichia coli.

    Science.gov (United States)

    Heo, Mi-Ae; Kim, Su-Hyun; Kim, So-Yeon; Kim, Yu-Jin; Chung, Junho; Oh, Min-Kyu; Lee, Sun-Gu

    2006-05-01

    c-Met, a high affinity receptor for hepatocyte growth factor/scatter factor, shown to be overexpressed in a variety of malignant cells, is a potential biomarker as well as a therapeutic target. Thus, single-chain antibody fragment (scFv) specific for c-Met is expected to be efficiently employed in the clinical treatment or imaging of many cancer cells. Here, we constructed the expression system for anti-c-Met scFv fused with T7 tag at its N-terminus using pET vector and investigated the expression conditions to achieve a functional and soluble expression of the scFv in the cytoplasm of recombinant Escherichia coli. The redox potential of E. coli cytoplasm was the most critical factor for the functional expression of anti-c-Met scFv. The employment of a host with oxidizing cytoplasm, E. coli trxB/gor double mutant, improved the productivity of functional anti-c-Met scFv by approximately 10-fold compared to the production of anti-c-Met scFv in the reducing cytoplasm of wild type E. coli. Productivity of functional anti-c-Met scFv could be further enhanced by co-expressing molecular chaperones such as GroELS, trigger factor, and DsbC with the scFv. Coexpression of DsbC increased the yield of functional anti-c-Met scFv about 2.5-fold in the cytoplasm of E. coli trxB/gor mutant compared to the production of scFv without DsbC coexpression. Lowering the IPTG concentration from 1 to 0.05 mM led to the slight enhancement, approximately 1.6-fold, of productivity of functional scFv. Although the use of low temperature for anti-c-Met scFv expression increased the ratio of soluble scFv fraction to insoluble fraction, productivity of soluble scFv decreased owing to the significant reduction of expression rate. The addition of 0.5 M sucrose in the medium inhibited the formation of intracellular insoluble anti-c-Met scFv. To purify the anti-c-Met scFv simply, we fused hexahistidine at the C-terminus of scFv and purified the scFv showing 98% of purity through the interaction

  9. A Chlamydia trachomatis OmcB C-Terminal Fragment Is Released into the Host Cell Cytoplasm and Is Immunogenic in Humans ▿

    OpenAIRE

    Qi, Manli; Gong, Siqi; Lei, Lei; Liu, Quanzhong; Zhong, Guangming

    2011-01-01

    The Chlamydia trachomatis outer membrane complex protein B (OmcB) is an antigen with diagnostic and vaccine relevance. To further characterize OmcB, we generated antibodies against OmcB C-terminal (OmcBc) and N-terminal (OmcBn) fragments. Surprisingly, the anti-OmcBc antibody detected dominant signals in the host cell cytosol, while the anti-OmcBn antibody exclusively labeled intrainclusion signals in C. trachomatis-infected cells permeabilized with saponin. Western blot analyses revealed tha...

  10. Cytoplasmic location of α1A voltage-gated calcium channel C-terminal fragment (Cav2.1-CTF aggregate is sufficient to cause cell death.

    Directory of Open Access Journals (Sweden)

    Makoto Takahashi

    Full Text Available The human α1A voltage-dependent calcium channel (Cav2.1 is a pore-forming essential subunit embedded in the plasma membrane. Its cytoplasmic carboxyl(C-tail contains a small poly-glutamine (Q tract, whose length is normally 4∼19 Q, but when expanded up to 20∼33Q, the tract causes an autosomal-dominant neurodegenerative disorder, spinocerebellar ataxia type 6 (SCA6. A recent study has shown that a 75-kDa C-terminal fragment (CTF containing the polyQ tract remains soluble in normal brains, but becomes insoluble mainly in the cytoplasm with additional localization to the nuclei of human SCA6 Purkinje cells. However, the mechanism by which the CTF aggregation leads to neurodegeneration is completely elusive, particularly whether the CTF exerts more toxicity in the nucleus or in the cytoplasm. We tagged recombinant (rCTF with either nuclear-localization or nuclear-export signal, created doxycyclin-inducible rat pheochromocytoma (PC12 cell lines, and found that the CTF is more toxic in the cytoplasm than in the nucleus, the observations being more obvious with Q28 (disease range than with Q13 (normal-length. Surprisingly, the CTF aggregates co-localized both with cAMP response element-binding protein (CREB and phosphorylated-CREB (p-CREB in the cytoplasm, and Western blot analysis showed that the quantity of CREB and p-CREB were both decreased in the nucleus when the rCTF formed aggregates in the cytoplasm. In human brains, polyQ aggregates also co-localized with CREB in the cytoplasm of SCA6 Purkinje cells, but not in other conditions. Collectively, the cytoplasmic Cav2.1-CTF aggregates are sufficient to cause cell death, and one of the pathogenic mechanisms may be abnormal CREB trafficking in the cytoplasm and reduced CREB and p-CREB levels in the nuclei.

  11. Molecular determinants of interactions between the N-terminal domain and the transmembrane core that modulate hERG K+ channel gating.

    Directory of Open Access Journals (Sweden)

    Jorge Fernández-Trillo

    Full Text Available A conserved eag domain in the cytoplasmic amino terminus of the human ether-a-go-go-related gene (hERG potassium channel is critical for its slow deactivation gating. Introduction of gene fragments encoding the eag domain are able to restore normal deactivation properties of channels from which most of the amino terminus has been deleted, and also those lacking exclusively the eag domain or carrying a single point mutation in the initial residues of the N-terminus. Deactivation slowing in the presence of the recombinant domain is not observed with channels carrying a specific Y542C point mutation in the S4-S5 linker. On the other hand, mutations in some initial positions of the recombinant fragment also impair its ability to restore normal deactivation. Fluorescence resonance energy transfer (FRET analysis of fluorophore-tagged proteins under total internal reflection fluorescence (TIRF conditions revealed a substantial level of FRET between the introduced N-terminal eag fragments and the eag domain-deleted channels expressed at the membrane, but not between the recombinant eag domain and full-length channels with an intact amino terminus. The FRET signals were also minimized when the recombinant eag fragments carried single point mutations in the initial portion of their amino end, and when Y542C mutated channels were used. These data suggest that the restoration of normal deactivation gating by the N-terminal recombinant eag fragment is an intrinsic effect of this domain directed by the interaction of its N-terminal segment with the gating machinery, likely at the level of the S4-S5 linker.

  12. Conformational changes of the N-terminal part of Mason-Pfizer monkey virus p12 protein during multimerization

    International Nuclear Information System (INIS)

    Knejzlik, Zdenek; Ulbrich, Pavel; Strohalm, Martin; Lastuvkova, Hana; Kodicek, Milan; Sakalian, Michael; Ruml, Tomas

    2009-01-01

    The Mason-Pfizer monkey virus is a prototype Betaretrovirus with the defining characteristic that it assembles spherical immature particles from Gag-related polyprotein precursors within the cytoplasm of the infected cell. It was shown previously that the N-terminal part of the Gag p12 domain (wt-Np12) is required for efficient assembly. However, the precise role for p12 in mediating Gag-Gag interaction is still poorly understood. In this study we employed detailed circular dichroism spectroscopy, electron microscopy and ultracentrifugation analyses of recombinant wt-Np12 prepared by in vitro transcription and translation. The wt-Np12 domain fragment forms fibrillar structures in a concentration-dependent manner. Assembly into fibers is linked to a conformational transition from unfolded or another non-periodical state to α-helix during multimerization.

  13. Macrophage conditioned medium induced cellular network formation in MCF-7 cells through enhanced tunneling nanotube formation and tunneling nanotube mediated release of viable cytoplasmic fragments

    Energy Technology Data Exchange (ETDEWEB)

    Patheja, Pooja, E-mail: pooja.patheja8@gmail.com [Laser Biomedical Applications Section, Raja Ramanna Centre for Advanced Technology, Indore 452013, Madhya Pradesh (India); Homi Bhabha National Institute, Training School Complex, Anushaktinagar, Mumbai 400094, Maharashtra (India); Sahu, Khageswar [Laser Biomedical Applications Section, Raja Ramanna Centre for Advanced Technology, Indore 452013, Madhya Pradesh (India)

    2017-06-15

    Infiltrating macrophages in tumor microenvironment, through their secreted cytokines and growth factors, regulate several processes of cancer progression such as cancer cell survival, proliferation, invasion, metastasis and angiogenesis. Recently, intercellular cytoplasmic bridges between cancer cells referred as tunneling nanotubes (TNTs) have been recognized as novel mode of intercellular communication between cancer cells. In this study, we investigated the effect of inflammatory mediators present in conditioned medium derived from macrophages on the formation of TNTs in breast adenocarcinoma cells MCF-7. Results show that treatment with macrophage conditioned medium (MφCM) not only enhanced TNT formation between cells but also stimulated the release of independently migrating viable cytoplasmic fragments, referred to as microplasts, from MCF-7 cells. Time lapse microscopy revealed that microplasts were released from parent cancer cells in extracellular space through formation of TNT-like structures. Mitochondria, vesicles and cytoplasm could be transferred from parent cell body to microplasts through connecting TNTs. The microplasts could also be resorbed into the parent cell body by retraction of the connecting TNTs. Microplast formation inhibited in presence cell migration inhibitor, cytochalasin-B. Notably by utilizing migratory machinery within microplasts, distantly located MCF-7 cells formed several TNT based intercellular connections, leading to formation of physically connected network of cells. Together, these results demonstrate novel role of TNTs in microplast formation, novel modes of TNT formation mediated by microplasts and stimulatory effect of MφCM on cellular network formation in MCF-7 cells mediated through enhanced TNT and microplast formation.

  14. Effects of thymosin β4 and its N-terminal fragment Ac-SDKP on TGF-β-treated human lung fibroblasts and in the mouse model of bleomycin-induced lung fibrosis.

    Science.gov (United States)

    Conte, Enrico; Iemmolo, Maria; Fruciano, Mary; Fagone, Evelina; Gili, Elisa; Genovese, Tiziana; Esposito, Emanuela; Cuzzocrea, Salvatore; Vancheri, Carlo

    2015-01-01

    Thymosin β4 (Tβ4) and its amino-terminal fragment comprising N-acetyl-seryl-aspartyl-lysyl-proline (Ac-SDKP) have been reported to act as anti-inflammatory and anti-fibrotic agents in vitro and in vivo. In recent papers, we have shown that Tβ4 exerts a widely protective role in mice treated with bleomycin, and in particular, we have demonstrated its inhibitory effects on both inflammation and early fibrosis. In this study, the putative anti-proliferative and anti-fibrogenic effects of Tβ4 and Ac-SDKP were evaluated in vitro. In addition, the effects of Tβ4 up to 21 days were evaluated in the bleomycin mouse model of lung fibrosis. We utilized both control and TGF-β-stimulated primary human lung fibroblasts isolated from both idiopathic pulmonary fibrosis (IPF) and control tissues. The in vivo effects of Tβ4 were assessed in CD1 mice treated with bleomycin. In the in vitro experiments, we observed significant anti-proliferative effects of Ac-SDKP in IPF fibroblasts. In those cells, Ac-SDKP significantly inhibited TGF-β-induced α-SMA and collagen expression, hallmarks of fibroblast differentiation into myofibroblasts triggered by TGF-β. In vivo, despite its previously described protective role in mice treated with bleomycin at 7 days, Tβ4 failed to prevent fibrosis induced by the drug at 14 and 21 days. We conclude that, compared to Tβ4, Ac-SDKP may have greater potential as an anti-fibrotic agent in the lung. Further in vivo experiments are warranted.

  15. Cytoplasmic bacteriophage display system

    Science.gov (United States)

    Studier, F. William; Rosenberg, Alan H.

    1998-06-16

    Disclosed are display vectors comprising DNA encoding a portion of a structural protein from a cytoplasmic bacteriophage, joined covalently to a protein or peptide of interest. Exemplified are display vectors wherein the structural protein is the T7 bacteriophage capsid protein. More specifically, in the exemplified display vectors the C-terminal amino acid residue of the portion of the capsid protein is joined to the N-terminal residue of the protein or peptide of interest. The portion of the T7 capsid protein exemplified comprises an N-terminal portion corresponding to form 10B of the T7 capsid protein. The display vectors are useful for high copy number display or lower copy number display (with larger fusion). Compositions of the type described herein are useful in connection with methods for producing a virus displaying a protein or peptide of interest.

  16. Towards the N-terminal acetylome

    DEFF Research Database (Denmark)

    Zhang, Xumin; Højrup, Peter

    2013-01-01

    Protein N-terminal acetylation (N(α)-acetylation) is observed widely from prokaryotes to eukaryotes. It gains increased importance in biological field, due to its multiple roles in many aspects of the protein life, such as assembly, stability, activity, and location. Today, mass spectrometry (MS...

  17. Carbamylation of N-terminal proline.

    Science.gov (United States)

    Olajuyigbe, Folasade M; Demitri, Nicola; Ajele, Joshua O; Maurizio, Elisa; Randaccio, Lucio; Geremia, Silvano

    2010-09-09

    Protein carbamylation is of great concern both in vivo and in vitro. Here, we report the first structural characterization of a protein carbamylated at the N-terminal proline. The unexpected carbamylation of the α-amino group of the least reactive codified amino acid has been detected in high-resolution electron density maps of a new crystal form of the HIV-1 protease/saquinavir complex. The carbamyl group is found coplanar to the proline ring with a trans conformation. The reaction of N-terminal with cyanate ion derived from the chaotropic agent urea was confirmed by mass spectra analysis on protease single crystals. Implications of carbamylation process in vitro and in vivo are discussed.

  18. Modulation of mutant huntingtin N-terminal cleavage and its effect on aggregation and cell death

    NARCIS (Netherlands)

    Juenemann, Katrin; Weisse, Christina; Reichmann, Denise; Kaether, Christoph; Calkhoven, Cornelis F.; Schilling, Gabriele

    2011-01-01

    Huntington's disease (HD) is a neurodegenerative disorder caused by a polyglutamine expansion near the N-terminus of huntingtin. A neuropathological hallmark of Huntington's disease is the presence of intracellular aggregates composed of mutant huntingtin N-terminal fragments in human postmortem

  19. The Association of Five-Year Changes in the Levels of N-Terminal Fragment of the Prohormone Brain-Type Natriuretic Peptide (NT-proBNP) with Subsequent Heart Failure and Death in Patients with Stable Coronary Artery Disease: The Heart and Soul Study.

    Science.gov (United States)

    Mishra, Rakesh K; Judson, Gregory; Christenson, Robert H; DeFilippi, Christopher; Wu, Alan H B; Whooley, Mary A

    The N-terminal fragment of the prohormone brain-type natriuretic peptide (NT-proBNP) is a powerful predictor of adverse outcomes in patients with coronary artery disease (CAD). However, little is known regarding the prognostic significance of longitudinal changes in NT-proBNP levels. We evaluated the ability of 5-year changes in NT-proBNP levels to predict subsequent heart failure (HF) hospitalization or cardiovascular (CV) death in 635 participants with stable CAD enrolled in the Heart and Soul Study. The median (IQR) 5-year change in NT-proBNP was 50 pg/mL (-5 to +222). During an average of 4.0 ± 1.4 years follow-up (i.e., 9 years from the baseline measurement), there were 67 events. Participants with 5-year changes in the highest quartile (≥ 223 pg/mL increase in NT-proBNP) had an almost 4-fold greater risk of subsequent HF or CV death than those in the lowest quartile of ≤-5 pg/mL (HR 3.8; 95% CI 2.0-7.3; p < 0.001). This association remained strong after adjustment for demographic variables, comorbidities, left ventricular mass index, systolic and diastolic function, and baseline and follow-up NT-proBNP levels (HR 3.9; 95% CI 1.1-13.4; p = 0.01). Changes in NT-proBNP levels at 5 years predict subsequent HF or CV death in patients with stable CAD, independent of other prognostic markers, including baseline and follow-up NT-proBNP levels. A stable NT-proBNP level predicts a low risk of subsequent events. © 2017 S. Karger AG, Basel.

  20. Diversity of the marine picocyanobacteria Prochlorococcus and Synechococcus assessed by terminal restriction fragment length polymorphisms of 16S-23S rRNA internal transcribed spacer sequences Diversidad de las picocianobacterias marinas Prochlorococcus y Synechococcus por medio de polimorfismos de longitud de fragmentos de restricción terminal en secuencias del espaciador transcrito interno del ARNr 16S - 23S

    Directory of Open Access Journals (Sweden)

    PARIS LAVIN

    2008-12-01

    distribution of these organisms.Con el fin de evaluar la utilización de secuencias del espaciador interno transcrito (ITS en estudios de genética de población de cianobacterias marinas, se amplificó y clonó la secuencia del gen ARNr 16S junto a la region espadadora 16S-23S ARNr de seis cepas de Prochlorococcus y Synechococcus. Se analizaron los amplicones del ITS por electroforesis en gel de gradiente de desnaturalización (DGGE y por polimorfismos de longitud de fragmentos de restricción terminal (T-RFLP. Al aplicar los métodos estándares de estas técnicas, se obtuvo más de una banda o fragmento de restricción terminal (T-RF por cepa o secuencia clonada. Informes en la literatura han sugerido que estas anomalías podrían ser atribuidas a la formación de estructuras secundarias. Por consiguiente, la estructura secundaria de las secuencias de ITS de las cepas de Prochlorococcus y Synechococcus fue modelada a las diferentes temperaturas que se utilizaron durante la reacción en cadena de polimerasa (PCR. Dicho modelamiento predijo la existencia de bucles que podrían persistir incluso durante la temperatura de extensión. Es probable que estos bucles generen productos de PCR con fragmentos incompletos y hebras simples. En este trabajo se modificó el procedimiento del método de T-RFLP añadiendo el partidor marcado en los últimos dos ciclos. Esto resultó, para la mayoría de los casos, la obtención de un solo fragmento de restricción por ribotipo. La aplicación de esta técnica a una muestra del medio ambiente obtenida frente al norte de Chile, demostró que es posible identificar la presencia, y determinar la abundancia relativa, de varios linajes filogenéticos de los géneros Prochlorococcus y Synechococcus que habitan la zona eufótica. El análisis filogenético de las secuencias de ITS obtenidos por clonación y secuenciación de ADN a partir de la misma muestra confirmó la presencia de los diferentes genotipos. Con la modificación propuesta, el m

  1. N-terminal Pro-B-type natriuretic peptide: a measure of significant patent cuctus arteriosus

    LENUS (Irish Health Repository)

    OFarombi-Oghuvbu, IO

    2008-01-24

    Background: B type natriuretic peptide (BNP) is a marker for ventricular dysfunction secreted as a pre-prohormone, Pro-B-type natriuretic peptide (ProBNP), and cleaved into BNP and a biologically inactive fragment, N-terminal pro-B-type natriuretic peptide (NT-proBNP). Little is known about the clinical usefulness of NT-proBNP in preterm infants.\\r\

  2. The influence of the N-terminal region of antimicrobial peptide pleurocidin on fungal apoptosis.

    Science.gov (United States)

    Choi, Hyemin; Lee, Dong Gun

    2013-10-28

    In our previous study, the 25-mer antimicrobial peptide pleurocidin (Ple) had been thought to induce apoptosis in Candida albicans. This study demonstrated that reactive oxygen species (ROS) production was a major cause of Ple-induced apoptosis. Four truncated analogs were synthesized to understand the functional roles in the N- and C-terminal regions of Ple on the apoptosis. Ple, Ple (4-25), Ple (1-22), and Ple (1-19) produced ROS, including hydroxyl radicals, on the order of [Ple > Ple (1-22) > Ple (4-25) > Ple (1-19)], whereas Ple (7-25) did not induce any ROS production. The results suggested that the N-terminal deletion affected the ROS-inducing activities much more than that of the C-terminal deletion, and net hydrophobicity [Ple > Ple (1-22) > Ple (4-25) > Ple (1-19) > Ple (7-25)] was related to ROS generation rather than other primary factors like net charge. Hence, we focused on the N-terminal-truncated peptides, Ple (4-25) and Ple (7-25), and examined other apoptotic features, including mitochondrial membrane depolarization, caspase activation, phosphatidylserine externalization, and DNA and nuclear fragmentation. The results also confirmed the disappearance of apoptotic activity of Ple (7-25) by the truncation of the N-terminal region (1-6) and the specific activity patterns between Ple and analogs. In conclusion, the N-terminal region of Ple played an important role in apoptosis.

  3. Isotope-coded N-terminal sulfonation of peptides allows quantitative proteomic analysis with increased de novo peptide sequencing capability.

    Science.gov (United States)

    Lee, Yong Ho; Han, Hoon; Chang, Seok-Bok; Lee, Sang-Won

    2004-01-01

    Recently various methods for the N-terminal sulfonation of peptides have been developed for the mass spectrometric analyses of proteomic samples to facilitate de novo sequencing of the peptides produced. This paper describes the isotope-coded N-terminal sulfonation (ICenS) of peptides; this procedure allows both de novo peptide sequencing and quantitative proteomics to be studied simultaneously. As N-terminal sulfonation reagents, 13C-labeled 4-sulfophenyl[13C6]isothiocyanate (13C-SPITC) and unlabeled 4-sulfophenyl isothiocyanate (12C-SPITC) were synthesized. The experimental and reference peptide mixtures were derivatized independently using 13C-SPITC and 12C-SPITC and then combined to generate an isotopically labeled peptide mixture in which each isotopic pair differs in mass by 6 Da. Capillary reverse-phase liquid chromatography/tandem mass spectrometry experiments on the resulting peptide mixtures revealed several immediate advantages of ICenS in addition to the de novo sequencing capability of N-terminal sulfonation, namely, differentiation between N-terminal sulfonated peptides and unmodified peptides in mass spectra, differentiation between N- and C-terminal fragments in tandem mass spectra of multiply protonated peptides by comparing fragmentations of the isotopic pairs, and relative peptide quantification between proteome samples. We demonstrate that the combination of N-terminal sulfonation and isotope coding in the mass spectrometric analysis of proteomic samples is a viable method that overcomes many problems associated with current N-terminal sulfonation methods. Copyright 2004 John Wiley & Sons, Ltd.

  4. N-terminally truncated POM121C inhibits HIV-1 replication.

    Directory of Open Access Journals (Sweden)

    Hideki Saito

    Full Text Available Recent studies have identified host cell factors that regulate early stages of HIV-1 infection including viral cDNA synthesis and orientation of the HIV-1 capsid (CA core toward the nuclear envelope, but it remains unclear how viral DNA is imported through the nuclear pore and guided to the host chromosomal DNA. Here, we demonstrate that N-terminally truncated POM121C, a component of the nuclear pore complex, blocks HIV-1 infection. This truncated protein is predominantly localized in the cytoplasm, does not bind to CA, does not affect viral cDNA synthesis, reduces the formation of 2-LTR and diminished the amount of integrated proviral DNA. Studies with an HIV-1-murine leukemia virus (MLV chimeric virus carrying the MLV-derived Gag revealed that Gag is a determinant of this inhibition. Intriguingly, mutational studies have revealed that the blockade by N-terminally-truncated POM121C is closely linked to its binding to importin-β/karyopherin subunit beta 1 (KPNB1. These results indicate that N-terminally-truncated POM121C inhibits HIV-1 infection after completion of reverse transcription and before integration, and suggest an important role for KPNB1 in HIV-1 replication.

  5. N-terminally truncated POM121C inhibits HIV-1 replication

    Science.gov (United States)

    Saito, Hideki; Masuda, Takao; Noda, Takeshi; Yamaoka, Shoji

    2017-01-01

    Recent studies have identified host cell factors that regulate early stages of HIV-1 infection including viral cDNA synthesis and orientation of the HIV-1 capsid (CA) core toward the nuclear envelope, but it remains unclear how viral DNA is imported through the nuclear pore and guided to the host chromosomal DNA. Here, we demonstrate that N-terminally truncated POM121C, a component of the nuclear pore complex, blocks HIV-1 infection. This truncated protein is predominantly localized in the cytoplasm, does not bind to CA, does not affect viral cDNA synthesis, reduces the formation of 2-LTR and diminished the amount of integrated proviral DNA. Studies with an HIV-1-murine leukemia virus (MLV) chimeric virus carrying the MLV-derived Gag revealed that Gag is a determinant of this inhibition. Intriguingly, mutational studies have revealed that the blockade by N-terminally-truncated POM121C is closely linked to its binding to importin-β/karyopherin subunit beta 1 (KPNB1). These results indicate that N-terminally-truncated POM121C inhibits HIV-1 infection after completion of reverse transcription and before integration, and suggest an important role for KPNB1 in HIV-1 replication. PMID:28873410

  6. The N-Terminal of Aquareovirus NS80 Is Required for Interacting with Viral Proteins and Viral Replication.

    Directory of Open Access Journals (Sweden)

    Jie Zhang

    Full Text Available Reovirus replication and assembly occurs within viral inclusion bodies that formed in specific intracellular compartments of cytoplasm in infected cells. Previous study indicated that aquareovirus NS80 is able to form inclusion bodies, and also can retain viral proteins within its inclusions. To better understand how NS80 performed in viral replication and assembly, the functional regions of NS80 associated with other viral proteins in aquareovirus replication were investigated in this study. Deletion mutational analysis and rotavirus NSP5-based protein association platform were used to detect association regions. Immunofluorescence images indicated that different N-terminal regions of NS80 could associate with viral proteins VP1, VP4, VP6 and NS38. Further co-immunoprecipitation analysis confirmed the interaction between VP1, VP4, VP6 or NS38 with different regions covering the N-terminal amino acid (aa, 1-471 of NS80, respectively. Moreover, removal of NS80 N-terminal sequences required for interaction with proteins VP1, VP4, VP6 or NS38 not only prevented the capacity of NS80 to support viral replication in NS80 shRNA-based replication complementation assays, but also inhibited the expression of aquareovirus proteins, suggesting that N-terminal regions of NS80 are necessary for viral replication. These results provided a foundational basis for further understanding the role of NS80 in viral replication and assembly during aquareovirus infection.

  7. The N-terminal strand modulates immunoglobulin light chain fibrillogenesis

    Energy Technology Data Exchange (ETDEWEB)

    Pozo-Yauner, Luis del, E-mail: ldelpozo@inmegen.gob.mx [Instituto Nacional de Medicina Genómica, Periférico Sur No. 4809, Col. Arenal Tepepan, Delegación Tlalpan, México, D.F. C.P. 14610 (Mexico); Wall, Jonathan S. [Departments of Radiology and Medicine, The University of Tennessee Medical Center, 1924 Alcoa Highway, Knoxville, TN (United States); González Andrade, Martín [Instituto Nacional de Medicina Genómica, Periférico Sur No. 4809, Col. Arenal Tepepan, Delegación Tlalpan, México, D.F. C.P. 14610 (Mexico); Sánchez-López, Rosana [Instituto de Biotecnología, Universidad Nacional Autónoma de México, Av. Universidad 2001, Col. Chamilpa Cuernavaca, Morelos C.P. 62210 (Mexico); Rodríguez-Ambriz, Sandra L. [Centro de Desarrollo de Productos Bióticos, Instituto Politécnico Nacional, Calle CEPROBI No. 8, Col. San Isidro, Yautepec, Morelos C.P. 62731 (Mexico); Pérez Carreón, Julio I. [Instituto Nacional de Medicina Genómica, Periférico Sur No. 4809, Col. Arenal Tepepan, Delegación Tlalpan, México, D.F. C.P. 14610 (Mexico); and others

    2014-01-10

    Highlights: •We evaluated the impact of mutations in the N-terminal strand of 6aJL2 protein. •Mutations destabilized the protein in a position-dependent manner. •Destabilizing mutations accelerated the fibrillogenesis by shortening the lag time. •The effect on the kinetic of fibril elongation by seeding was of different nature. •The N-terminal strand is buried in the fibrillar state of 6aJL2 protein. -- Abstract: It has been suggested that the N-terminal strand of the light chain variable domain (V{sub L}) protects the molecule from aggregation by hindering spurious intermolecular contacts. We evaluated the impact of mutations in the N-terminal strand on the thermodynamic stability and kinetic of fibrillogenesis of the V{sub L} protein 6aJL2. Mutations in this strand destabilized the protein in a position-dependent manner, accelerating the fibrillogenesis by shortening the lag time; an effect that correlated with the extent of destabilization. In contrast, the effect on the kinetics of fibril elongation, as assessed in seeding experiments was of different nature, as it was not directly dependant on the degree of destabilization. This finding suggests different factors drive the nucleation-dependent and elongation phases of light chain fibrillogenesis. Finally, taking advantage of the dependence of the Trp fluorescence upon environment, four single Trp substitutions were made in the N-terminal strand, and changes in solvent exposure during aggregation were evaluated by acrylamide-quenching. The results suggest that the N-terminal strand is buried in the fibrillar state of 6aJL2 protein. This finding suggest a possible explanation for the modulating effect exerted by the mutations in this strand on the aggregation behavior of 6aJL2 protein.

  8. Asymmetric configurations and N-terminal rearrangements in connexin26 gap junction channels.

    Science.gov (United States)

    Oshima, Atsunori; Tani, Kazutoshi; Toloue, Masoud M; Hiroaki, Yoko; Smock, Amy; Inukai, Sayaka; Cone, Angela; Nicholson, Bruce J; Sosinsky, Gina E; Fujiyoshi, Yoshinori

    2011-01-21

    Gap junction channels are unique in that they possess multiple mechanisms for channel closure, several of which involve the N terminus as a key component in gating, and possibly assembly. Here, we present electron crystallographic structures of a mutant human connexin26 (Cx26M34A) and an N-terminal deletion of this mutant (Cx26M34Adel2-7) at 6-Å and 10-Å resolutions, respectively. The three-dimensional map of Cx26M34A was improved by data from 60° tilt images and revealed a breakdown of the hexagonal symmetry in a connexin hemichannel, particularly in the cytoplasmic domain regions at the ends of the transmembrane helices. The Cx26M34A structure contained an asymmetric density in the channel vestibule ("plug") that was decreased in the Cx26M34Adel2-7 structure, indicating that the N terminus significantly contributes to form this plug feature. Functional analysis of the Cx26M34A channels revealed that these channels are predominantly closed, with the residual electrical conductance showing normal voltage gating. N-terminal deletion mutants with and without the M34A mutation showed no electrical activity in paired Xenopus oocytes and significantly decreased dye permeability in HeLa cells. Comparing this closed structure with the recently published X-ray structure of wild-type Cx26, which is proposed to be in an open state, revealed a radial outward shift in the transmembrane helices in the closed state, presumably to accommodate the N-terminal plug occluding the pore. Because both Cx26del2-7 and Cx26M34Adel2-7 channels are closed, the N terminus appears to have a prominent role in stabilizing the open configuration. Copyright © 2010 Elsevier Ltd. All rights reserved.

  9. Distinct Molecular Regulation of Glycogen Synthase Kinase-3α Isozyme Controlled by Its N-terminal Region

    Science.gov (United States)

    Azoulay-Alfaguter, Inbar; Yaffe, Yakey; Licht-Murava, Avital; Urbanska, Malgorzata; Jaworski, Jacek; Pietrokovski, Shmuel; Hirschberg, Koret; Eldar-Finkelman, Hagit

    2011-01-01

    Glycogen synthase kinase-3 (GSK-3) is expressed as two isozymes α and β. They share high similarity in their catalytic domains but differ in their N- and C-terminal regions, with GSK-3α having an extended glycine-rich N terminus. Here, we undertook live cell imaging combined with molecular and bioinformatic studies to understand the distinct functions of the GSK-3 isozymes focusing on GSK-3α N-terminal region. We found that unlike GSK-3β, which shuttles between the nucleus and cytoplasm, GSK-3α was excluded from the nucleus. Deletion of the N-terminal region of GSK-3α resulted in nuclear localization, and treatment with leptomycin B resulted in GSK-3α accumulation in the nucleus. GSK-3α rapidly accumulated in the nucleus in response to calcium or serum deprivation, and accumulation was strongly inhibited by the calpain inhibitor calpeptin. This nuclear accumulation was not mediated by cleavage of the N-terminal region or phosphorylation of GSK-3α. Rather, we show that calcium-induced GSK-3α nuclear accumulation was governed by GSK-3α binding with as yet unknown calpain-sensitive protein or proteins; this binding was mediated by the N-terminal region. Bioinformatic and experimental analyses indicated that nuclear exclusion of GSK-3α was likely an exclusive characteristic of mammalian GSK-3α. Finally, we show that nuclear localization of GSK-3α reduced the nuclear pool of β-catenin and its target cyclin D1. Taken together, these data suggest that the N-terminal region of GSK-3α is responsible for its nuclear exclusion and that binding with a calcium/calpain-sensitive product enables GSK-3α nuclear retention. We further uncovered a novel link between calcium and nuclear GSK-3α-mediated inhibition of the canonical Wnt/β-catenin pathway. PMID:21266584

  10. Miro's N-Terminal GTPase Domain Is Required for Transport of Mitochondria into Axons and Dendrites

    Science.gov (United States)

    Babic, Milos; Russo, Gary J.; Wellington, Andrea J.; Sangston, Ryan M.; Gonzalez, Migdalia

    2015-01-01

    Mitochondria are dynamically transported in and out of neuronal processes to maintain neuronal excitability and synaptic function. In higher eukaryotes, the mitochondrial GTPase Miro binds Milton/TRAK adaptor proteins linking microtubule motors to mitochondria. Here we show that Drosophila Miro (dMiro), which has previously been shown to be required for kinesin-driven axonal transport, is also critically required for the dynein-driven distribution of mitochondria into dendrites. In addition, we used the loss-of-function mutations dMiroT25N and dMiroT460N to determine the significance of dMiro's N-terminal and C-terminal GTPase domains, respectively. Expression of dMiroT25N in the absence of endogenous dMiro caused premature lethality and arrested development at a pupal stage. dMiroT25N accumulated mitochondria in the soma of larval motor and sensory neurons, and prevented their kinesin-dependent and dynein-dependent distribution into axons and dendrites, respectively. dMiroT25N mutant mitochondria also were severely fragmented and exhibited reduced kinesin and dynein motility in axons. In contrast, dMiroT460N did not impair viability, mitochondrial size, or the distribution of mitochondria. However, dMiroT460N reduced dynein motility during retrograde mitochondrial transport in axons. Finally, we show that substitutions analogous to the constitutively active Ras-G12V mutation in dMiro's N-terminal and C-terminal GTPase domains cause neomorphic phenotypic effects that are likely unrelated to the normal function of each GTPase domain. Overall, our analysis indicates that dMiro's N-terminal GTPase domain is critically required for viability, mitochondrial size, and the distribution of mitochondria out of the neuronal soma regardless of the employed motor, likely by promoting the transition from a stationary to a motile state. PMID:25855186

  11. 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

  12. Powdery mildew fungal effector candidates share N-terminal Y/F/WxC-motif

    Directory of Open Access Journals (Sweden)

    Emmersen Jeppe

    2010-05-01

    Full Text Available Abstract Background Powdery mildew and rust fungi are widespread, serious pathogens that depend on developing haustoria in the living plant cells. Haustoria are separated from the host cytoplasm by a plant cell-derived extrahaustorial membrane. They secrete effector proteins, some of which are subsequently transferred across this membrane to the plant cell to suppress defense. Results In a cDNA library from barley epidermis containing powdery mildew haustoria, two-thirds of the sequenced ESTs were fungal and represented ~3,000 genes. Many of the most highly expressed genes encoded small proteins with N-terminal signal peptides. While these proteins are novel and poorly related, they do share a three-amino acid motif, which we named "Y/F/WxC", in the N-terminal of the mature proteins. The first amino acid of this motif is aromatic: tyrosine, phenylalanine or tryptophan, and the last is always cysteine. In total, we identified 107 such proteins, for which the ESTs represent 19% of the fungal clones in our library, suggesting fundamental roles in haustoria function. While overall sequence similarity between the powdery mildew Y/F/WxC-proteins is low, they do have a highly similar exon-intron structure, suggesting they have a common origin. Interestingly, searches of public fungal genome and EST databases revealed that haustoria-producing rust fungi also encode large numbers of novel, short proteins with signal peptides and the Y/F/WxC-motif. No significant numbers of such proteins were identified from genome and EST sequences from either fungi which do not produce haustoria or from haustoria-producing Oomycetes. Conclusion In total, we identified 107, 178 and 57 such Y/F/WxC-proteins from the barley powdery mildew, the wheat stem rust and the wheat leaf rust fungi, respectively. All together, our findings suggest the Y/F/WxC-proteins to be a new class of effectors from haustoria-producing pathogenic fungi.

  13. Interaction of N-terminal peptide analogues of the Na+,K+-ATPase with membranes.

    Science.gov (United States)

    Nguyen, Khoa; Garcia, Alvaro; Sani, Marc-Antoine; Diaz, Dil; Dubey, Vikas; Clayton, Daniel; Poggetto, Giovanni Dal; Cornelius, Flemming; Payne, Richard J; Separovic, Frances; Khandelia, Himanshu; Clarke, Ronald J

    2018-03-06

    The Na + ,K + -ATPase, which is present in the plasma membrane of all animal cells, plays a crucial role in maintaining the Na + and K + electrochemical potential gradients across the membrane. Recent studies have suggested that the N-terminus of the protein's catalytic α-subunit is involved in an electrostatic interaction with the surrounding membrane, which controls the protein's conformational equilibrium. However, because the N-terminus could not yet be resolved in any X-ray crystal structures, little information about this interaction is so far available. In measurements utilising poly-l-lysine as a model of the protein's lysine-rich N-terminus and using lipid vesicles of defined composition, here we have identified the most likely origin of the interaction as one between positively charged lysine residues of the N-terminus and negatively charged headgroups of phospholipids (notably phosphatidylserine) in the surrounding membrane. Furthermore, to isolate which segments of the N-terminus could be involved in membrane binding, we chemically synthesized N-terminal fragments of various lengths. Based on a combination of results from RH421 UV/visible absorbance measurements and solid-state 31 P and 2 H NMR using these N-terminal fragments as well as MD simulations it appears that the membrane interaction arises from lysine residues prior to the conserved LKKE motif of the N-terminus. The MD simulations indicate that the strength of the interaction varies significantly between different enzyme conformations. Copyright © 2018. Published by Elsevier B.V.

  14. N-terminal N-myristoylation of proteins: prediction of substrate proteins from amino acid sequence.

    Science.gov (United States)

    Maurer-Stroh, Sebastian; Eisenhaber, Birgit; Eisenhaber, Frank

    2002-04-05

    Myristoylation by the myristoyl-CoA:protein N-myristoyltransferase (NMT) is an important lipid anchor modification of eukaryotic and viral proteins. Automated prediction of N-terminal N-myristoylation from the substrate protein sequence alone is necessary for large-scale sequence annotation projects but it requires a low rate of false positive hits in addition to a sufficient sensitivity. Our previous analysis of substrate protein sequence variability, NMT sequences and 3D structures has revealed motif properties in addition to the known PROSITE motif that are utilized in a new predictor described here. The composite prediction function (with separate ad hoc parameterization (a) for queries from non-fungal eukaryotes and their viruses and (b) for sequences from fungal species) consists of terms evaluating amino acid type preferences at sequences positions close to the N terminus as well as terms penalizing deviations from the physical property pattern of amino acid side-chains encoded in multi-residue correlation within the motif sequence. The algorithm has been validated with a self-consistency and two jack-knife tests for the learning set as well as with kinetic data for model substrates. The sensitivity in recognizing documented NMT substrates is above 95 % for both taxon-specific versions. The corresponding rate of false positive prediction (for sequences with an N-terminal glycine residue) is close to 0.5 %; thus, the technique is applicable for large-scale automated sequence database annotation. The predictor is available as public WWW-server with the URL http://mendel.imp.univie.ac.at/myristate/. Additionally, we propose a version of the predictor that identifies a number of proteolytic protein processing sites at internal glycine residues and that evaluates possible N-terminal myristoylation of the protein fragments.A scan of public protein databases revealed new potential NMT targets for which the myristoyl modification may be of critical importance for

  15. Crystal Structure of the N-terminal Domain of the Group B Streptococcus Alpha C Protein

    Energy Technology Data Exchange (ETDEWEB)

    Auperin,T.; Bolduc, G.; Baron, M.; Heroux, A.; Filman, D.; Madoff, L.; Hogle, J.

    2005-01-01

    Group B Streptococcus (GBS) is the leading cause of bacterial pneumonia, sepsis, and meningitis among neonates and an important cause of morbidity among pregnant women and immunocompromised adults. Invasive diseases due to GBS are attributed to the ability of the pathogen to translocate across human epithelial surfaces. The alpha C protein (ACP) has been identified as an invasin that plays a role in internalization and translocation of GBS across epithelial cells. The soluble N-terminal domain of ACP (NtACP) blocks the internalization of GBS. We determined the 1.86-{angstrom} resolution crystal structure of NtACP comprising residues Ser{sup 52} through Leu{sup 225} of the full-length ACP. NtACP has two domains, an N-terminal {beta}-sandwich and a C-terminal three-helix bundle. Structural and topological alignments reveal that the {beta}-sandwich shares structural elements with the type III fibronectin fold (FnIII), but includes structural elaborations that make it unique. We have identified a potential integrin-binding motif consisting of Lys-Thr-Asp{sup 146}, Arg{sup 110}, and Asp{sup 118}. A similar arrangement of charged residues has been described in other invasins. ACP shows a heparin binding activity that requires NtACP. We propose a possible heparin-binding site, including one surface of the three-helix bundle, and nearby portions of the sandwich and repeat domains. We have validated this prediction using assays of the heparin binding and cell-adhesion properties of engineered fragments of ACP. This is the first crystal structure of a member of the highly conserved Gram-positive surface alpha-like protein family, and it will enable the internalization mechanism of GBS to be dissected at the atomic level.

  16. Dynamics and molecular determinants of cytoplasmic lipid droplet clustering and dispersion.

    Directory of Open Access Journals (Sweden)

    David J Orlicky

    Full Text Available Perilipin-1 (Plin1, a prominent cytoplasmic lipid droplet (CLD binding phosphoprotein and key physiological regulator of triglyceride storage and lipolysis in adipocytes, is thought to regulate the fragmentation and dispersion of CLD that occurs in response to β-adrenergic activation of adenylate cyclase. Here we investigate the dynamics and molecular determinants of these processes using cell lines stably expressing recombinant forms of Plin1 and/or other members of the perilipin family. Plin1 and a C-terminal CLD-binding fragment of Plin1 (Plin1CT induced formation of single dense CLD clusters near the microtubule organizing center, whereas neither an N-terminal CLD-binding fragment of Plin1, nor Plin2 or Plin3 induced clustering. Clustered CLD coated by Plin1, or Plin1CT, dispersed in response to isoproterenol, or other agents that activate adenylate cyclase, in a process inhibited by the protein kinase A inhibitor, H89, and blocked by microtubule disruption. Isoproterenol-stimulated phosphorylation of CLD-associated Plin1 on serine 492 preceded their dispersion, and live cell imaging showed that cluster dispersion involved initial fragmentation of tight clusters into multiple smaller clusters, which then fragmented into well-dispersed individual CLD. siRNA knockdown of the cortical actin binding protein, moesin, induced disaggregation of tight clusters into multiple smaller clusters, and inhibited the reaggregation of dispersed CLD into tight clusters. Together these data suggest that the clustering and dispersion processes involve a complex orchestration of phosphorylation-dependent, microtubule-dependent and independent, and microfilament dependent steps.

  17. N-Terminal Tau Fragments as Biomarkers for Alzheimer’s Disease and Neurotrauma

    Science.gov (United States)

    2014-10-01

    with inducible expression of E2- and E2+ tau isoforms and the culturing of P19s in media suitable for exosome collection (i.e. exosome- free FBS...Sabatini DD, De Robertis EM (2010) Wnt signaling requires sequestration of glycogen synthase kinase 3 inside multivesicular endosomes. Cell 143, 1136-1148...Strautman AF, Cork RJ and Robinson KR. The distribution of free calcium in transected spinal axons and its modulation by applied electrical fields

  18. N-Terminal Tau Fragments as Biomarkers for Alzheimer’s Disease and Neurotrauma

    Science.gov (United States)

    2017-12-01

    established AD) and ischemia control (VaD/MID) multisample CSF EMV30-200 proteomes. Proteins involved in macromolecular turnover mechanisms (proteostasis... minority in the DG. When present, SPs in the DG molecular layers colocalized with some, but not all molecular layer FTLs in both CTE3/4 and AD samples...A significant minority of the proteins in our fingerprint have been identified previously as potential candidate biomarkers for early AD. • ELISA

  19. Distinct molecular regulation of glycogen synthase kinase-3alpha isozyme controlled by its N-terminal region: functional role in calcium/calpain signaling.

    Science.gov (United States)

    Azoulay-Alfaguter, Inbar; Yaffe, Yakey; Licht-Murava, Avital; Urbanska, Malgorzata; Jaworski, Jacek; Pietrokovski, Shmuel; Hirschberg, Koret; Eldar-Finkelman, Hagit

    2011-04-15

    Glycogen synthase kinase-3 (GSK-3) is expressed as two isozymes α and β. They share high similarity in their catalytic domains but differ in their N- and C-terminal regions, with GSK-3α having an extended glycine-rich N terminus. Here, we undertook live cell imaging combined with molecular and bioinformatic studies to understand the distinct functions of the GSK-3 isozymes focusing on GSK-3α N-terminal region. We found that unlike GSK-3β, which shuttles between the nucleus and cytoplasm, GSK-3α was excluded from the nucleus. Deletion of the N-terminal region of GSK-3α resulted in nuclear localization, and treatment with leptomycin B resulted in GSK-3α accumulation in the nucleus. GSK-3α rapidly accumulated in the nucleus in response to calcium or serum deprivation, and accumulation was strongly inhibited by the calpain inhibitor calpeptin. This nuclear accumulation was not mediated by cleavage of the N-terminal region or phosphorylation of GSK-3α. Rather, we show that calcium-induced GSK-3α nuclear accumulation was governed by GSK-3α binding with as yet unknown calpain-sensitive protein or proteins; this binding was mediated by the N-terminal region. Bioinformatic and experimental analyses indicated that nuclear exclusion of GSK-3α was likely an exclusive characteristic of mammalian GSK-3α. Finally, we show that nuclear localization of GSK-3α reduced the nuclear pool of β-catenin and its target cyclin D1. Taken together, these data suggest that the N-terminal region of GSK-3α is responsible for its nuclear exclusion and that binding with a calcium/calpain-sensitive product enables GSK-3α nuclear retention. We further uncovered a novel link between calcium and nuclear GSK-3α-mediated inhibition of the canonical Wnt/β-catenin pathway.

  20. N-Terminal Plasmodium vivax Merozoite Surface Protein-1, a Potential Subunit for Malaria Vivax Vaccine

    Directory of Open Access Journals (Sweden)

    Fernanda G. Versiani

    2013-01-01

    Full Text Available The human malaria is widely distributed in the Middle East, Asia, the western Pacific, and Central and South America. Plasmodium vivax started to have the attention of many researchers since it is causing diseases to millions of people and several reports of severe malaria cases have been noticed in the last few years. The lack of in vitro cultures for P. vivax represents a major delay in developing a functional malaria vaccine. One of the major candidates to antimalarial vaccine is the merozoite surface protein-1 (MSP1, which is expressed abundantly on the merozoite surface and capable of activating the host protective immunity. Studies have shown that MSP-1 possesses highly immunogenic fragments, capable of generating immune response and protection in natural infection in endemic regions. This paper shows humoral immune response to different proteins of PvMSP1 and the statement of N-terminal to be added to the list of potential candidates for malaria vivax vaccine.

  1. The outermost N-terminal region of tapasin facilitates folding of major histocompatibility complex class I

    DEFF Research Database (Denmark)

    Røder, Gustav Andreas; Geironson, Linda; Darabi, Anna

    2009-01-01

    ). Using a biochemical peptide-MHC-I-binding assay, recombinant Tpn(1-87) was found to specifically facilitate peptide-dependent folding of HLA-A*0201. Furthermore, we used Tpn(1-87) to generate a monoclonal antibody, alphaTpn(1-87)/80, specific for natural human Tpn and capable of cellular staining of ER......Tapasin (Tpn) is an ER chaperone that is uniquely dedicated to MHC-I biosynthesis. It binds MHC-I molecules, integrates them into peptide-loading complexes, and exerts quality control of the bound peptides; only when an "optimal peptide" is bound will the MHC-I be released and exported to the cell...... surface for presentation to T cells. The exact mechanisms of Tpn quality control and the criteria for being an optimal peptide are still unknown. Here, we have generated a recombinant fragment of human Tpn, Tpn(1-87) (representing the 87 N-terminal and ER-luminal amino acids of the mature Tpn protein...

  2. N-terminal pro brain natriuretic peptide as a cardiac biomarker in Japanese hemodialysis patients.

    Science.gov (United States)

    Shimizu, Minako; Doi, Shigehiro; Nakashima, Ayumu; Naito, Takayuki; Masaki, Takao

    2018-03-01

    This study examined the clinical significance of N-terminal pro brain natriuretic peptide level as a cardiac marker in Japanese hemodialysis patients. This was a multicenter cross-sectional study involving 1428 Japanese hemodialysis patients. Ultrasonic cardiography data at post-hemodialysis were obtained from 395 patients. We examined whether serum N-terminal pro brain natriuretic peptide levels were associated with cardiac parameters and assessed cut-off values and investigated factors associated with a reduced ratio of N-terminal pro brain natriuretic peptide levels pre- and post-hemodialysis. Multivariate logistic regression analysis showed that pre- and post-hemodialysis N-terminal pro brain natriuretic peptide levels were associated with left ventricular hypertrophy on electrocardiogram (odds ratio: 3.10; p N-terminal pro brain natriuretic peptide levels were also significantly associated with ejection fraction on urine chorionic gonadotrophin (ultrasonic cardiography; odds ratio: 35.83; p N-terminal pro brain natriuretic peptide reduction ratio during a hemodialysis session correlated with Kt/V, membrane area, membrane type, modality, body weight gain ratio, treatment time, and ultrafiltration rate with multiple linear regression ( R: 0.53; p N-terminal pro brain natriuretic peptide are associated with the presence of left ventricular hypertrophy in this population. The post-hemodialysis N-terminal pro brain natriuretic peptide level is a useful marker for systolic dysfunction.

  3. Molecular basis for the anchoring of proto-oncoprotein Nup98 to the cytoplasmic face of the nuclear pore complex.

    Science.gov (United States)

    Stuwe, Tobias; von Borzyskowski, Lennart Schada; Davenport, Andrew M; Hoelz, André

    2012-06-22

    The cytoplasmic filament nucleoporins of the nuclear pore complex (NPC) are critically involved in nuclear export and remodeling of mRNA ribonucleoprotein particles and are associated with various human malignancies. Here, we report the crystal structure of the Nup98 C-terminal autoproteolytic domain, frequently missing from leukemogenic forms of the protein, in complex with the N-terminal domain of Nup82 and the C-terminal tail fragment of Nup159. The Nup82 β propeller serves as a noncooperative binding platform for both binding partners. Interaction of Nup98 with Nup82 occurs through a reciprocal exchange of loop structures. Strikingly, the same Nup98 groove promiscuously interacts with Nup82 and Nup96 in a mutually excusive fashion. Simultaneous disruption of both Nup82 interactions in yeast causes severe defects in mRNA export, while the severing of a single interaction is tolerated. Thus, the cytoplasmic filament network of the NPC is robust, consistent with its essential function in nucleocytoplasmic transport. Published by Elsevier Ltd.

  4. Characterization of the part of N-terminal PIP2 binding site of the TRPM1 channel.

    Science.gov (United States)

    Jirku, Michaela; Bumba, Ladislav; Bednarova, Lucie; Kubala, Martin; Sulc, Miroslav; Franek, Miloslav; Vyklicky, Ladislav; Vondrasek, Jiri; Teisinger, Jan; Bousova, Kristyna

    2015-12-01

    Transient receptor potential melastatin-1 (TRPM1) is a calcium channel that is essential for the depolarization of photo-responsive retinal bipolar cells, but most of the physiological functions and cellular roles of this channel are still poorly understood. Most transient receptor potential (TRP) channels are typically regulated by intracellular proteins and other signaling molecules. Phosphatidylinositol-4,5 bisphosphate (PIP2), a minor phospholipid component of cell membranes, has previously been shown to directly bind TRP channels and to play a unique role in modulating receptor function. To characterize the binding of PIP2 as a potential regulator of TRPM1, we utilized biophysical methods and molecular modeling to study the interactions of PIP2 with an N-terminal fragment of TRPM1 (residues A451-N566). The basic N-terminal residue K464 of TRPM1 suggests that it is part of putative pleckstrin homology (PH) domain and is involved in the interactions with PIP2. This is the first report detailing the binding of PIP2 at the N-terminus of the TRPM1 receptor. Copyright © 2015 Elsevier B.V. All rights reserved.

  5. Depletion of the human N-terminal acetyltransferase hNaa30 disrupts Golgi integrity and ARFRP1 localization.

    Science.gov (United States)

    Starheim, Kristian K; Kalvik, Thomas V; Bjørkøy, Geir; Arnesen, Thomas

    2017-04-30

    The organization of the Golgi apparatus (GA) is tightly regulated. Golgi stack scattering is observed in cellular processes such as apoptosis and mitosis, and has also been associated with disruption of cellular lipid metabolism and neurodegenerative diseases. Our studies show that depletion of the human N-α-acetyltransferase 30 (hNaa30) induces fragmentation of the Golgi stack in HeLa and CAL-62 cell lines. The GA associated GTPase ADP ribosylation factor related protein 1 (ARFRP1) was previously shown to require N-terminal acetylation for membrane association and based on its N-terminal sequence, it is likely to be a substrate of hNaa30. ARFRP1 is involved in endosome-to- trans -Golgi network (TGN) traffic. We observed that ARFRP1 shifted from a predominantly cis -Golgi and TGN localization to localizing both Golgi and non-Golgi vesicular structures in hNaa30-depleted cells. However, we did not observe loss of membrane association of ARFRP1. We conclude that hNaa30 depletion induces Golgi scattering and induces aberrant ARFRP1 Golgi localization. © 2017 The Author(s).

  6. The Herpes Simplex Virus Protein pUL31 Escorts Nucleocapsids to Sites of Nuclear Egress, a Process Coordinated by Its N-Terminal Domain.

    Directory of Open Access Journals (Sweden)

    Christina Funk

    2015-06-01

    Full Text Available Progeny capsids of herpesviruses leave the nucleus by budding through the nuclear envelope. Two viral proteins, the membrane protein pUL34 and the nucleo-phosphoprotein pUL31 form the nuclear egress complex that is required for capsid egress out of the nucleus. All pUL31 orthologs are composed of a diverse N-terminal domain with 1 to 3 basic patches and a conserved C-terminal domain. To decipher the functions of the N-terminal domain, we have generated several Herpes simplex virus mutants and show here that the N-terminal domain of pUL31 is essential with basic patches being critical for viral propagation. pUL31 and pUL34 entered the nucleus independently of each other via separate routes and the N-terminal domain of pUL31 was required to prevent their premature interaction in the cytoplasm. Unexpectedly, a classical bipartite nuclear localization signal embedded in this domain was not required for nuclear import of pUL31. In the nucleus, pUL31 associated with the nuclear envelope and newly formed capsids. Viral mutants lacking the N-terminal domain or with its basic patches neutralized still associated with nucleocapsids but were unable to translocate them to the nuclear envelope. Replacing the authentic basic patches with a novel artificial one resulted in HSV1(17+Lox-UL31-hbpmp1mp2, that was viable but delayed in nuclear egress and compromised in viral production. Thus, while the C-terminal domain of pUL31 is sufficient for the interaction with nucleocapsids, the N-terminal domain was essential for capsid translocation to sites of nuclear egress and a coordinated interaction with pUL34. Our data indicate an orchestrated sequence of events with pUL31 binding to nucleocapsids and escorting them to the inner nuclear envelope. We propose a common mechanism for herpesviral nuclear egress: pUL31 is required for intranuclear translocation of nucleocapsids and subsequent interaction with pUL34 thereby coupling capsid maturation with primary

  7. Peptides derived from human galectin-3 N-terminal tail interact with its carbohydrate recognition domain in a phosphorylation-dependent manner

    Energy Technology Data Exchange (ETDEWEB)

    Berbís, M. Álvaro [Chemical and Physical Biology Department, Centro de Investigaciones Biológicas, CSIC, 28040 Madrid (Spain); André, Sabine [Institute of Physiological Chemistry, Faculty of Veterinary Medicine, Ludwig-Maximilians University, 80539 Munich (Germany); Cañada, F. Javier [Chemical and Physical Biology Department, Centro de Investigaciones Biológicas, CSIC, 28040 Madrid (Spain); Pipkorn, Rüdiger [Central Peptide Synthesis Unit, German Cancer Research Center, 69120 Heidelberg (Germany); Ippel, Hans [Department of Biochemistry, CARIM, University of Maastricht, Maastricht (Netherlands); Department of Biochemistry, Molecular Biology and Biophysics, University of Minnesota, Minneapolis, MN 55455 (United States); Mayo, Kevin H. [Department of Biochemistry, Molecular Biology and Biophysics, University of Minnesota, Minneapolis, MN 55455 (United States); Kübler, Dieter [Biomolecular Interactions, German Cancer Research Center, 69120 Heidelberg (Germany); Gabius, Hans-Joachim [Institute of Physiological Chemistry, Faculty of Veterinary Medicine, Ludwig-Maximilians University, 80539 Munich (Germany); Jiménez-Barbero, Jesús, E-mail: jjbarbero@cib.csic.es [Chemical and Physical Biology Department, Centro de Investigaciones Biológicas, CSIC, 28040 Madrid (Spain)

    2014-01-03

    Highlights: •Galectin-3 is composed of a carbohydrate recognition domain and an N-terminal tail. •Synthetic peptides derived from the tail are shown to interact with the CRD. •This interaction is modulated by Ser- and Tyr-phosphorylation of the peptides. -- Abstract: Galectin-3 (Gal-3) is a multi-functional effector protein that functions in the cytoplasm and the nucleus, as well as extracellularly following non-classical secretion. Structurally, Gal-3 is unique among galectins with its carbohydrate recognition domain (CRD) attached to a rather long N-terminal tail composed mostly of collagen-like repeats (nine in the human protein) and terminating in a short non-collagenous terminal peptide sequence unique in this lectin family and not yet fully explored. Although several Ser and Tyr sites within the N-terminal tail can be phosphorylated, the physiological significance of this post-translational modification remains unclear. Here, we used a series of synthetic (phospho)peptides derived from the tail to assess phosphorylation-mediated interactions with {sup 15}N-labeled Gal-3 CRD. HSQC-derived chemical shift perturbations revealed selective interactions at the backface of the CRD that were attenuated by phosphorylation of Tyr 107 and Tyr 118, while phosphorylation of Ser 6 and Ser 12 was essential. Controls with sequence scrambling underscored inherent specificity. Our studies shed light on how phosphorylation of the N-terminal tail may impact on Gal-3 function and prompt further studies using phosphorylated full-length protein.

  8. NMR structural characterization of the N-terminal domain of the adenylyl cyclase-associated protein (CAP) from Dictyostelium discoideum

    Energy Technology Data Exchange (ETDEWEB)

    Mavoungou, Chrystelle [Max Planck Institute for Biochemistry (Germany); Israel, Lars [Ludwig Maximilians-University, Adolf Butenandt Institute, Cell Biology (Germany); Rehm, Till; Ksiazek, Dorota; Krajewski, Marcin; Popowicz, Grzegorz [Max Planck Institute for Biochemistry (Germany); Noegel, Angelika A. [University of Cologne, Institute for Biochemistry (Germany); Schleicher, Michael [Ludwig Maximilians-University, Adolf Butenandt Institute, Cell Biology (Germany); Holak, Tad A. [Max Planck Institute for Biochemistry (Germany)

    2004-05-15

    Cyclase-associated proteins (CAPs) are highly conserved, ubiquitous actin binding proteins that are involved in microfilament reorganization. The N-termini of CAPs play a role in Ras signaling and bind adenylyl cyclase; the C-termini bind to G-actin. We report here the NMR characterization of the amino-terminal domain of CAP from Dictyostelium discoideum (CAP(1-226)). NMR data, including the steady state {sup 1}H-{sup 15}N heteronuclear NOE experiments, indicate that the first 50 N-terminal residues are unstructured and that this highly flexible serine-rich fragment is followed by a stable, folded core starting at Ser 51. The NMR structure of the folded core is an {alpha}-helix bundle composed of six antiparallel helices, in a stark contrast to the recently determined CAP C-terminal domain structure, which is solely built by {beta}-strands.

  9. N-terminal pro-B-type natriuretic peptide and long-term mortality in stable coronary heart disease

    DEFF Research Database (Denmark)

    Kragelund, Charlotte; Grønning, Bjørn; Køber, Lars

    2005-01-01

    BACKGROUND: The level of the inactive N-terminal fragment of pro-brain (B-type) natriuretic peptide (BNP) is a strong predictor of mortality among patients with acute coronary syndromes and may be a strong prognostic marker in patients with chronic coronary heart disease as well. We assessed...... quartile was 2.4 (95 percent confidence interval, 1.5 to 4.0; Prisk factors, including the patient's age; sex; family history with respect to ischemic heart disease; the presence or absence of a history......-term mortality in patients with stable coronary disease and provides prognostic information above and beyond that provided by conventional cardiovascular risk factors and the degree of left ventricular systolic dysfunction....

  10. Crystal Structure of the N-Terminal Half of the Traffic Controller UL37 from Herpes Simplex Virus 1.

    Science.gov (United States)

    Koenigsberg, Andrea L; Heldwein, Ekaterina E

    2017-10-15

    Inner tegument protein UL37 is conserved among all three subfamilies of herpesviruses. Studies of UL37 homologs from two alphaherpesviruses, herpes simplex virus 1 (HSV-1) and pseudorabies virus (PRV), have suggested that UL37 plays an essential albeit poorly defined role in intracellular capsid trafficking. At the same time, HSV and PRV homologs cannot be swapped, which suggests that in addition to a conserved function, UL37 homologs also have divergent virus-specific functions. Accurate dissection of UL37 functions requires detailed maps in the form of atomic-resolution structures. Previously, we reported the crystal structure of the N-terminal half of UL37 (UL37N) from PRV. Here, we report the crystal structure of HSV-1 UL37N. Comparison of the two structures reveals that UL37 homologs differ in their overall shapes, distributions of surface charges, and locations of projecting loops. In contrast, the previously identified R2 surface region is structurally conserved. We propose that within the N-terminal half of UL37, functional conservation is centered within the R2 surface region, whereas divergent structural elements pinpoint regions mediating virus-specific functions and may engage different binding partners. Together, the two structures can now serve as templates for a structure-guided exploration of both conserved and virus-specific functions of UL37. IMPORTANCE The ability to move efficiently within host cell cytoplasm is essential for replication in all viruses. It is especially important in the neuroinvasive alphaherpesviruses, such as human herpes simplex virus 1 (HSV-1), HSV-2, and veterinarian pseudorabies virus (PRV), that infect the peripheral nervous system and have to travel long distances along axons. Capsid movement in these viruses is controlled by capsid-associated tegument proteins, yet their specific roles have not yet been defined. Systematic exploration of the roles of tegument proteins in capsid trafficking requires detailed navigational

  11. Crystal Structure of the N-Terminal Half of the Traffic Controller UL37 from Herpes Simplex Virus 1

    Energy Technology Data Exchange (ETDEWEB)

    Koenigsberg, Andrea L.; Heldwein, Ekaterina E.; Sandri-Goldin, Rozanne M.

    2017-08-02

    Inner tegument protein UL37 is conserved among all three subfamilies of herpesviruses. Studies of UL37 homologs from two alphaherpesviruses, herpes simplex virus 1 (HSV-1) and pseudorabies virus (PRV), have suggested that UL37 plays an essential albeit poorly defined role in intracellular capsid trafficking. At the same time, HSV and PRV homologs cannot be swapped, which suggests that in addition to a conserved function, UL37 homologs also have divergent virus-specific functions. Accurate dissection of UL37 functions requires detailed maps in the form of atomic-resolution structures. Previously, we reported the crystal structure of the N-terminal half of UL37 (UL37N) from PRV. Here, we report the crystal structure of HSV-1 UL37N. Comparison of the two structures reveals that UL37 homologs differ in their overall shapes, distributions of surface charges, and locations of projecting loops. In contrast, the previously identified R2 surface region is structurally conserved. We propose that within the N-terminal half of UL37, functional conservation is centered within the R2 surface region, whereas divergent structural elements pinpoint regions mediating virus-specific functions and may engage different binding partners. Together, the two structures can now serve as templates for a structure-guided exploration of both conserved and virus-specific functions of UL37.

    IMPORTANCEThe ability to move efficiently within host cell cytoplasm is essential for replication in all viruses. It is especially important in the neuroinvasive alphaherpesviruses, such as human herpes simplex virus 1 (HSV-1), HSV-2, and veterinarian pseudorabies virus (PRV), that infect the peripheral nervous system and have to travel long distances along axons. Capsid movement in these viruses is controlled by capsid-associated tegument proteins, yet their specific roles have not yet been defined. Systematic exploration of the roles of tegument proteins in capsid trafficking requires

  12. BNP and N-terminal proBNP are both extracted in the normal kidney

    DEFF Research Database (Denmark)

    Goetze, J P; Jensen, G; Møller, S

    2006-01-01

    BACKGROUND: Increased plasma concentrations of cardiac-derived B-type natriuretic peptide (BNP) and N-terminal pro-B-type natriuretic peptide (proBNP) are both associated with left ventricular dysfunction. Information on the regional elimination of the peptides is, however, still scarce. We...... with catheterization of the femoral artery and femoral and renal veins. Blood sampling from the catheters allowed determination of the arteriovenous extraction ratio of N-terminal proBNP and BNP. RESULTS: Neither the peripheral N-terminal proBNP (13, 11, 19 pmol L(-1), NS) nor the BNP plasma concentrations (4, 12, 9...... compared with BNP (0.00 vs. 0.125, P = 0.007). CONCLUSIONS: A comparable renal elimination of N-terminal proBNP and BNP is contrasted by a selective extraction of BNP in the lower extremity. Our results suggest a different elimination mechanism in the renal and peripheral circulation, which partly may...

  13. BNP and N-terminal proBNP are both extracted in the normal kidney

    DEFF Research Database (Denmark)

    Gøtze, Jens Peter; Jensen, Gorm Boje; Møller, Søren

    2006-01-01

    Background Increased plasma concentrations of cardiac-derived B-type natriuretic peptide (BNP) and N-terminal pro-B-type natriuretic peptide (proBNP) are both associated with left ventricular dysfunction. Information on the regional elimination of the peptides is, however, still scarce. We...... with catheterization of the femoral artery and femoral and renal veins. Blood sampling from the catheters allowed determination of the arteriovenous extraction ratio of N-terminal proBNP and BNP. Results Neither the peripheral N-terminal proBNP (13, 11, 19 pmol L(-1), NS) nor the BNP plasma concentrations (4, 12, 9...... compared with BNP (0.00 vs. 0.125, P = 0.007). Conclusions A comparable renal elimination of N-terminal proBNP and BNP is contrasted by a selective extraction of BNP in the lower extremity. Our results suggest a different elimination mechanism in the renal and peripheral circulation, which partly may...

  14. N-terminal Slit2 inhibits HIV-1 replication by regulating the actin cytoskeleton

    Directory of Open Access Journals (Sweden)

    Anand Appakkudal R

    2013-01-01

    Full Text Available Abstract Background Slit2 is a ~ 200 kDa secreted glycoprotein that has been recently shown to regulate immune functions. However, not much is known about its role in HIV (human immunodeficiency virus-1 pathogenesis. Results In the present study, we have shown that the N-terminal fragment of Slit2 (Slit2N (~120 kDa inhibits replication of both CXCR4 and CCR5-tropic HIV-1 viruses in T-cell lines and peripheral blood T-cells. Furthermore, we demonstrated inhibition of HIV-1 infection in resting CD4+ T-cells. In addition, we showed that Slit2N blocks cell-to-cell transmission of HIV-1. We have shown that Slit2N inhibits HIV-1 infection by blocking viral entry into T-cells. We also ruled out Slit2N-mediated inhibition of various other steps in the life cycle including binding, integration and viral transcription. Elucidation of the molecular mechanism revealed that Slit2N mediates its functional effects by binding to Robo1 receptor. Furthermore, we found that Slit2N inhibited Gp120-induced Robo1-actin association suggesting that Slit2N may inhibit cytoskeletal rearrangements facilitating HIV-1 entry. Studies into the mechanism of inhibition of HIV-1 revealed that Slit2N abrogated HIV-1 envelope-induced actin cytoskeletal dynamics in both T-cell lines and primary T-cells. We further showed that Slit2N specifically attenuated the HIV-1 envelope-induced signaling pathway consisting of Rac1, LIMK and cofilin that regulates actin polymerization. Conclusions Taken together, our results show that Slit2N inhibits HIV-1 replication through novel mechanisms involving modulation of cytoskeletal dynamics. Our study, thus, provides insights into the role of Slit2N in HIV-1 infection and underscores its potential in limiting viral replication in T-cells.

  15. The N-terminal domain of human hemokinin-1 influences functional selectivity property for tachykinin receptor neurokinin-1.

    Science.gov (United States)

    Mou, Lingyun; Xing, Yanhong; Kong, Ziqing; Zhou, Ying; Chen, Zongyao; Wang, Rui

    2011-03-01

    Human hemokinin-1 (hHK-1) is a substance P-like tachykinin peptide preferentially expressed in non-neuronal tissues. It is involved in multiple physiological functions such as inflammation, hematopoietic cells development and vasodilatation via the interaction with tachykinin receptor neurokinin-1 (NK1). To further understand the intracellular signal transduction mechanism under such functional multiplicity, current study was focused on the differential activation of Gs and Gq pathways by hHK-1 and its C-terminal fragments, which is termed as functional selectivity. We demonstrated these hHK-1 and related peptide fragments can independently activate Gs and Gq pathways, showing a relative bias toward Gq over Gs pathway. The T1, K3 and Q6 of hHK-1 might play roles in the activation of adenylate cyclase mediated by Gs, while having negligible effect on Gq mediated intracellular calcium release. The stepwise truncation of N-terminal amino acid of hHK-1 caused gradual decrease in ERK1/2 phosphorylation level and NF-κB activity. However, it had little influence on the induction of NK1 receptor desensitization and internalization. Taken together these data support that hHK-1 and its C-terminal fragments are human NK1 receptor agonists with different functional selectivity properties and that such functional selectivity leads to differential activation of downstream signaling and receptor trafficking. Copyright © 2010 Elsevier Inc. All rights reserved.

  16. Host factors that interact with the pestivirus N-terminal protease, Npro, are components of the ribonucleoprotein complex.

    Science.gov (United States)

    Jefferson, Matthew; Donaszi-Ivanov, Andras; Pollen, Sean; Dalmay, Tamas; Saalbach, Gerhard; Powell, Penny P

    2014-09-01

    The viral N-terminal protease N(pro) of pestiviruses counteracts cellular antiviral defenses through inhibition of IRF3. Here we used mass spectrometry to identify a new role for N(pro) through its interaction with over 55 associated proteins, mainly ribosomal proteins and ribonucleoproteins, including RNA helicase A (DHX9), Y-box binding protein (YBX1), DDX3, DDX5, eIF3, IGF2BP1, multiple myeloma tumor protein 2, interleukin enhancer binding factor 3 (IEBP3), guanine nucleotide binding protein 3, and polyadenylate-binding protein 1 (PABP-1). These are components of the translation machinery, ribonucleoprotein particles (RNPs), and stress granules. Significantly, we found that stress granule formation was inhibited in MDBK cells infected with a noncytopathic bovine viral diarrhea virus (BVDV) strain, Kyle. However, ribonucleoproteins binding to N(pro) did not inhibit these proteins from aggregating into stress granules. N(pro) interacted with YBX1 though its TRASH domain, since the mutant C112R protein with an inactive TRASH domain no longer redistributed to stress granules. Interestingly, RNA helicase A and La autoantigen relocated from a nuclear location to form cytoplasmic granules with N(pro). To address a proviral role for N(pro) in RNP granules, we investigated whether N(pro) affected RNA interference (RNAi), since interacting proteins are involved in RISC function during RNA silencing. Using glyceraldehyde-3-phosphate dehydrogenase (GAPDH) silencing with small interfering RNAs (siRNAs) followed by Northern blotting of GAPDH, expression of N(pro) had no effect on RNAi silencing activity, contrasting with other viral suppressors of interferon. We propose that N(pro) is involved with virus RNA translation in the cytoplasm for virus particle production, and when translation is inhibited following stress, it redistributes to the replication complex. Although the pestivirus N-terminal protease, N(pro), has been shown to have an important role in degrading IRF3 to

  17. Structural and functional analysis of an essential nucleoporin heterotrimer on the cytoplasmic face of the nuclear pore complex

    Energy Technology Data Exchange (ETDEWEB)

    Yoshida, Kimihisa; Seo, Hyuk-Soo; Debler, Erik W.; Blobel, Günter; Hoelz, André (Rockefeller)

    2012-07-25

    So far, only a few of the interactions between the {approx}30 nucleoporins comprising the modular structure of the nuclear pore complex have been defined at atomic resolution. Here we report the crystal structure, at 2.6 {angstrom} resolution, of a heterotrimeric complex, composed of fragments of three cytoplasmically oriented nucleoporins of yeast: Nup82, Nup116, and Nup159. Our data show that the Nup82 fragment, representing more than the N-terminal half of the molecule, folds into an extensively decorated, seven-bladed {beta}-propeller that forms the centerpiece of this heterotrimeric complex and anchors both a C-terminal fragment of Nup116 and the C-terminal tail of Nup159. Binding between Nup116 and Nup82 is mutually reinforced via two loops, one emanating from the Nup82 {beta}-propeller and the other one from the {beta}-sandwich fold of Nup116, each contacting binding pockets in their counterparts. The Nup82-Nup159 interaction occurs through an amphipathic {alpha}-helix of Nup159, which is cradled in a large hydrophobic groove that is generated from several large surface decorations of the Nup82 {beta}-propeller. Although Nup159 and Nup116 fragments bind to the Nup82 {beta}-propeller in close vicinity, there are no direct contacts between them, consistent with the noncooperative binding that was detected biochemically. Extensive mutagenesis delineated hot-spot residues for these interactions. We also showed that the Nup82 {beta}-propeller binds to other yeast Nup116 family members, Nup145N, Nup100 and to the mammalian homolog, Nup98. Notably, each of the three nucleoporins contains additional nuclear pore complex binding sites, distinct from those that were defined here in the heterotrimeric Nup82 {center_dot} Nup159 {center_dot} Nup116 complex.

  18. N-terminal Proteomics Assisted Profiling of the Unexplored Translation Initiation Landscape in Arabidopsis thaliana.

    Science.gov (United States)

    Willems, Patrick; Ndah, Elvis; Jonckheere, Veronique; Stael, Simon; Sticker, Adriaan; Martens, Lennart; Van Breusegem, Frank; Gevaert, Kris; Van Damme, Petra

    2017-06-01

    Proteogenomics is an emerging research field yet lacking a uniform method of analysis. Proteogenomic studies in which N-terminal proteomics and ribosome profiling are combined, suggest that a high number of protein start sites are currently missing in genome annotations. We constructed a proteogenomic pipeline specific for the analysis of N-terminal proteomics data, with the aim of discovering novel translational start sites outside annotated protein coding regions. In summary, unidentified MS/MS spectra were matched to a specific N-terminal peptide library encompassing protein N termini encoded in the Arabidopsis thaliana genome. After a stringent false discovery rate filtering, 117 protein N termini compliant with N-terminal methionine excision specificity and indicative of translation initiation were found. These include N-terminal protein extensions and translation from transposable elements and pseudogenes. Gene prediction provided supporting protein-coding models for approximately half of the protein N termini. Besides the prediction of functional domains (partially) contained within the newly predicted ORFs, further supporting evidence of translation was found in the recently released Araport11 genome re-annotation of Arabidopsis and computational translations of sequences stored in public repositories. Most interestingly, complementary evidence by ribosome profiling was found for 23 protein N termini. Finally, by analyzing protein N-terminal peptides, an in silico analysis demonstrates the applicability of our N-terminal proteogenomics strategy in revealing protein-coding potential in species with well- and poorly-annotated genomes. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

  19. Identification and functional characterization of N-terminally acetylated proteins in Drosophila melanogaster.

    Directory of Open Access Journals (Sweden)

    Sandra Goetze

    2009-11-01

    Full Text Available Protein modifications play a major role for most biological processes in living organisms. Amino-terminal acetylation of proteins is a common modification found throughout the tree of life: the N-terminus of a nascent polypeptide chain becomes co-translationally acetylated, often after the removal of the initiating methionine residue. While the enzymes and protein complexes involved in these processes have been extensively studied, only little is known about the biological function of such N-terminal modification events. To identify common principles of N-terminal acetylation, we analyzed the amino-terminal peptides from proteins extracted from Drosophila Kc167 cells. We detected more than 1,200 mature protein N-termini and could show that N-terminal acetylation occurs in insects with a similar frequency as in humans. As the sole true determinant for N-terminal acetylation we could extract the (XPX rule that indicates the prevention of acetylation under all circumstances. We could show that this rule can be used to genetically engineer a protein to study the biological relevance of the presence or absence of an acetyl group, thereby generating a generic assay to probe the functional importance of N-terminal acetylation. We applied the assay by expressing mutated proteins as transgenes in cell lines and in flies. Here, we present a straightforward strategy to systematically study the functional relevance of N-terminal acetylations in cells and whole organisms. Since the (XPX rule seems to be of general validity in lower as well as higher eukaryotes, we propose that it can be used to study the function of N-terminal acetylation in all species.

  20. The N-terminal 45-kDa Domain of Dna2 Endonuclease/Helicase Targets the Enzyme to Secondary Structure DNA*

    Science.gov (United States)

    Lee, Chul-Hwan; Lee, Miju; Kang, Hyo-Jin; Kim, Do-Hyung; Kang, Young-Hoon; Bae, Sung-Ho; Seo, Yeon-Soo

    2013-01-01

    The removal of initiating primers from the 5′-ends of each Okazaki fragment, required for the generation of contiguous daughter strands, can be catalyzed by the combined action of DNA polymerase δ and Fen1. When the flaps generated by displacement of DNA synthesis activity of polymerase δ become long enough to bind replication protein A or form hairpin structures, the helicase/endonuclease enzyme, Dna2, becomes critical because of its ability to remove replication protein A-coated or secondary structure flaps. In this study, we show that the N-terminal 45-kDa domain of Dna2 binds hairpin structures, allowing the enzyme to target secondary structure flap DNA. We found that this activity was essential for the efficient removal of hairpin flaps by the endonuclease activity of Dna2 with the aid of its helicase activity. Thus, the efficient removal of hairpin structure flaps requires the coordinated action of all three functional domains of Dna2. We also found that deletion of the N-terminal 45-kDa domain of Dna2 led to a partial loss of the intra-S-phase checkpoint function and an increased rate of homologous recombination in yeast. We discuss the potential roles of the N-terminal domain of Dna2 in the maintenance of genomic stability. PMID:23344960

  1. N-terminal modifications of cellular proteins: The enzymes involved, their substrate specificities and biological effects

    Science.gov (United States)

    Varland, Sylvia; Osberg, Camilla; Arnesen, Thomas

    2015-01-01

    The vast majority of eukaryotic proteins are N-terminally modified by one or more processing enzymes. Enzymes acting on the very first amino acid of a polypeptide include different peptidases, transferases, and ligases. Methionine aminopeptidases excise the initiator methionine leaving the nascent polypeptide with a newly exposed amino acid that may be further modified. N-terminal acetyl-, methyl-, myristoyl-, and palmitoyltransferases may attach an acetyl, methyl, myristoyl, or palmitoyl group, respectively, to the α-amino group of the target protein N-terminus. With the action of ubiquitin ligases, one or several ubiquitin molecules are transferred, and hence, constitute the N-terminal modification. Modifications at protein N-termini represent an important contribution to proteomic diversity and complexity, and are essential for protein regulation and cellular signaling. Consequently, dysregulation of the N-terminal modifying enzymes is implicated in human diseases. We here review the different protein N-terminal modifications occurring co- or post-translationally with emphasis on the responsible enzymes and their substrate specificities. PMID:25914051

  2. Blocking an N-terminal acetylation–dependent protein interaction inhibits an E3 ligase

    Energy Technology Data Exchange (ETDEWEB)

    Scott, Daniel C.; Hammill, Jared T.; Min, Jaeki; Rhee, David Y.; Connelly, Michele; Sviderskiy, Vladislav O.; Bhasin, Deepak; Chen, Yizhe; Ong, Su-Sien; Chai, Sergio C.; Goktug, Asli N.; Huang, Guochang; Monda, Julie K.; Low, Jonathan; Kim, Ho Shin; Paulo, Joao A.; Cannon, Joe R.; Shelat, Anang A.; Chen, Taosheng; Kelsall, Ian R.; Alpi, Arno F.; Pagala, Vishwajeeth; Wang, Xusheng; Peng, Junmin; Singh , Bhuvanesh; Harper, J. Wade; Schulman, Brenda A.; Guy, R. Kip (MSKCC); (Dundee); (SJCH); (Harvard-Med); (MXPL)

    2017-06-05

    N-terminal acetylation is an abundant modification influencing protein functions. Because ~80% of mammalian cytosolic proteins are N-terminally acetylated, this modification is potentially an untapped target for chemical control of their functions. Structural studies have revealed that, like lysine acetylation, N-terminal acetylation converts a positively charged amine into a hydrophobic handle that mediates protein interactions; hence, this modification may be a druggable target. We report the development of chemical probes targeting the N-terminal acetylation–dependent interaction between an E2 conjugating enzyme (UBE2M or UBC12) and DCN1 (DCUN1D1), a subunit of a multiprotein E3 ligase for the ubiquitin-like protein NEDD8. The inhibitors are highly selective with respect to other protein acetyl-amide–binding sites, inhibit NEDD8 ligation in vitro and in cells, and suppress anchorage-independent growth of a cell line with DCN1 amplification. Overall, our data demonstrate that N-terminal acetyl-dependent protein interactions are druggable targets and provide insights into targeting multiprotein E2–E3 ligases.

  3. Palladium Mediated Rapid Deprotection of N-Terminal Cysteine under Native Chemical Ligation Conditions for the Efficient Preparation of Synthetically Challenging Proteins.

    Science.gov (United States)

    Jbara, Muhammad; Maity, Suman Kumar; Seenaiah, Mallikanti; Brik, Ashraf

    2016-04-20

    Facilitating the process of chemical protein synthesis is an important goal in order to enable the efficient preparation of large and novel protein analogues. Native chemical ligation, which is widely used in the synthesis and semisynthesis of proteins, has been going through several developments to expedite the synthetic process and to obtain the target protein in high yield. A key aspect of this approach is the utilization of protecting groups for the N-terminal Cys in the middle fragments, which bear simultaneously the two reactive groups, i.e., N-terminal Cys and C-terminal thioester. Despite important progress in this area, as has been demonstrated in the use of thiazolidine protecting group in the synthesis of over 100 proteins, finding optimal protecting group(s) remains a challenge. For example, the thiazolidine removal step is very slow (>8 h), and in some cases the applied conditions lead to undesired side reactions. Here we show that water-soluble palladium(II) complexes are excellent reagents for the effective unmasking of thiazolidine, enabling its complete removal within 15 min under native chemical ligation conditions. Moreover, palladium is also able to rapidly remove propargyloxycarbonyl-protecting group from the N-terminal Cys in a similar efficiency. The utility of the new removal conditions for both protecting groups is exemplified in the rapid and efficient synthesis of Lys34-ubiquitinated H2B and for the first time neddlyated peptides derived from cullin1. The current approach expands the use of palladium in protein chemistry and should significantly facilitate the chemical and semisynthesis of synthetically challenging proteins from multiple fragments.

  4. N-terminal pro-C-type natriuretic peptide in serum associated with bone destruction in patients with multiple myeloma

    DEFF Research Database (Denmark)

    Mylin, Anne K; Goetze, Jens P; Heickendorff, Lene

    2015-01-01

    AIM: To examine whether N-terminal proCNP concentrations in serum is associated with bone destruction in patients with multiple myeloma. MATERIALS & METHODS: N-terminal proCNP and biochemical bone markers were measured in 153 patients. Radiographic bone disease and skeletal-related events were...... evaluated at specific time-points. RESULTS: N-terminal proCNP concentrations increased with age. High N-terminal proCNP concentrations were associated with high-risk disease and renal impairment. Renal function explained 22% of the variation. N-terminal proCNP concentrations correlated with serum bone ALP...... and serum PINP, but lacked association with bone resorption markers, radiographic bone disease and skeletal-related events. CONCLUSION: Serum N-terminal proCNP are associated with bone formation activity in patients with multiple myeloma, but should be interpreted with caution in patients with renal...

  5. An N-terminal glycine to cysteine mutation in the collagen COL1A1 gene produces moderately severe osteogenesis imperfecta

    Energy Technology Data Exchange (ETDEWEB)

    Wilcox, W.; Scott, L.; Cohn, D. [Cedars-Sinai Medical Center, Los Angeles, CA (United States)

    1994-09-01

    Osteogenesis imperfecta (OI) is usually due to mutations in the type I procollagen genes COL1A1 and COL1A2. Point mutations close to the N-terminus are generally milder than those near the C-terminus of the molecule (the gradient hypothesis of collagen mutations). We describe a patient with moderately severe OI due to a mutation in the N-terminal portion of the triple helical domain of the {alpha}1(I) chain. Electrophoretic analysis of collagen isolated from fibroblast cultures suggested the abnormal presence of a cysteine in the N-terminal portion of the {alpha}1(I) chain. Five overlapping DNA fragments amplified from fibroblast RNA were screened for mutations using single strand conformational polymorphism (SSCP) and heteroduplex analyses. Direct DNA sequence analysis of the single positive fragment demonstrated a G to T transversion, corresponding to a glycine to cysteine substitution at position 226 of the triple helical domain of the {alpha}1(I) chain. The mutation was confirmed by restriction enzyme analysis of amplified genomic DNA. The mutation was not present in fibroblasts from either phenotypically normal parent. Combining this mutation with other reported mutations, glycine to cysteine substitutions at positions 205, 211, 223, and 226 produce a moderately severe phenotype whereas flanking mutations at positions 175 and 382 produce a mild phenotype. This data supports a regional rather than a gradient model of the relationship between the nature and location of type I collagen mutations and OI phenotype.

  6. The membranotropic activity of N-terminal peptides from the pore ...

    Indian Academy of Sciences (India)

    The membranotropic activity of N-terminal peptides from the pore-forming proteins sticholysin I and II is modulated by hydrophobic and electrostatic interactions ... Center for Protein Studies, Biology Faculty, University of Havana, Havana, Cuba; Department of Applied Physics, Institute of Physics, University of São Paulo, São ...

  7. BNP and N-terminal proBNP are both extracted in the normal kidney

    DEFF Research Database (Denmark)

    Gøtze, Jens Peter; Jensen, Gorm Boje; Møller, Søren

    2006-01-01

    therefore examined the renal and peripheral extraction of N-terminal proBNP and BNP. Materials and methods The study comprised 18 patients with essential arterial hypertension, 51 with cirrhosis, and 18 control patients without kidney or liver disease. All patients underwent a haemodynamic investigation...

  8. Urinary N-Terminal Prohormone Brain Natriuretic Peptide Excretion in Patients With Chronic Heart Failure

    NARCIS (Netherlands)

    Linssen, Gerard C. M.; Damman, Kevin; Hillege, Hans L.; Navis, Gerjan; van Veldhuisen, Dirk J.; Voors, Adriaan A.

    2009-01-01

    Background-Urinary excretion is currently regarded as the main mechanism of elimination of N-terminal prohormone brain natriuretic peptide (NT-proBNP). The clinical implications and the value of measurement of urinary NT-proBNP in patients with heart failure are largely unknown. Methods and

  9. The membranotropic activity of N-terminal peptides from the pore ...

    Indian Academy of Sciences (India)

    ... activity of N-terminal peptides from the pore-forming proteins sticholysin I and II is modulated by hydrophobic and electrostatic interactions as well as lipid composition. Uris Ros Lohans Pedrera Daylín Díaz Juan C De Karam Tatiane P Sudbrack Pedro A Valiente Diana Martínez Eduardo M Cilli Fabiola Pazos Rosangela ...

  10. Diagnostic Usefulness of N-terminal Pro-brain Natriuretic Peptide ...

    African Journals Online (AJOL)

    BACKGROUND: N-terminal pro-brain natriuretic peptide (NTproBNP) is useful in the diagnosis and management of adult patients with heart failure. OBJECTIVE: The objective of the study was to determine the usefulness of NT-proBNP in diagnosing congestive heart failure (CHF) in children and its correlation with left ...

  11. Molecular dynamics simulations of N-terminal peptides from a nucleotide binding protein

    NARCIS (Netherlands)

    van der Spoel, D.; Vogel, H.J.; Berendsen, H.J.C.

    Molecular dynamics (MD) simulations of N-terminal peptides from lactate dehydrogenase (LDH) with increasing length and individual secondary structure elements were used to study their stability in relation to folding, Ten simulations of 1-2 ns of different peptides in water starting from the

  12. Membrane anchoring of the AgrD N-terminal amphipathic region is required for its processing to produce a quorum-sensing pheromone in Staphylococcus aureus.

    Science.gov (United States)

    Zhang, Linsheng; Lin, Jianqun; Ji, Guangyong

    2004-05-07

    Quorum-sensing pheromones are signal molecules that are secreted from Gram-positive bacteria and utilized by these bacteria to communicate among individual cells to regulate their activities as a group through a cell density-sensing mechanism. Typically, these pheromones are processed from precursor polypeptides. The mechanisms of trafficking, processing, and modification of the precursor to generate a mature pheromone are unclear. In Staphylococcus aureus, AgrD is the propeptide for an autoinducing peptide (AIP) pheromone that triggers the Agr cell density-sensing system upon reaching a threshold and subsequently regulates expression of virulence factor genes. The transmembrane protein AgrB, encoded in the agr locus, is necessary for the processing of AgrD to produce mature AIP; however, it is not clear how AgrD interacts with AgrB and how this interaction results in the generation of mature AIP. In this study, we found that the AgrD propeptide was integrated into the cytoplasmic membrane by a conserved alpha-helical amphipathic motif in its N-terminal region. We demonstrated that membrane targeting of AgrD by this motif was required for the stabilization of AgrD and the production of mature AIP, although this region was not specifically involved in the interaction with AgrB. An artificial amphipathic peptide replacing the N-terminal amphipathic motif of AgrD directed the protein to the cytoplasmic membrane and enabled the production of AIP. Analysis of Bacillus ComX precursor protein sequences suggested that the amphipathic membrane-targeting motif might also exist in pheromone precursors of other Gram-positive bacteria.

  13. Clinical correlation between N-terminal pro-b-type natriuretic peptide and angiographic coronary atherosclerosis

    Directory of Open Access Journals (Sweden)

    Demóstenes G.L. Ribeiro

    2014-06-01

    Full Text Available OBJECTIVES:This study aimed to investigate the clinical correlation between angiographic coronary atherosclerosis and N-terminal pro-B-type natriuretic peptide along with other known correlated factors.METHODS:In total, 153 patients with a diagnostic hypothesis of stable angina, unstable angina or acute myocardial infarction were classified as group A (patients with angiographically normal coronary arteries or group B (patients with angiographic coronary atherosclerosis. The two groups were analyzed with respect to the following factors: gender, age, body mass index, abdominal circumference, smoking, diabetes mellitus, arterial hypertension, early family history of atherosclerosis, statin use, the presence of metabolic syndrome, clinical presentation and biochemical factors, including cholesterol, creatinine and fibrinogen plasma concentrations, monocyte counts and N-terminal pro-B-type natriuretic peptide.RESULTS:Univariate analyses comparing the two groups revealed that group B patients more frequently had diabetes, used statins and had systolic dysfunction, N-terminal pro-B-type natriuretic peptide levels ≥250 pg/mL, fibrinogen levels >500 mg/dL and ≥501 monocytes/mm3 compared with group A patients (p<0.05. Nevertheless, multivariate logistic regression analysis demonstrated that the independent predictors of angiographic coronary atherosclerosis were an N-terminal pro-B-type natriuretic peptide level ≥250 pg/mL, diabetes mellitus and increased monocyte numbers and fibrinogen plasma concentration, regardless of the creatinine level or the presence of systolic dysfunction.CONCLUSIONS:An N-terminal pro-B-type natriuretic peptide plasma concentration of ≥250 pg/mL is an independent predictor of angiographic coronary atherosclerosis.

  14. Site directed spin labeling studies of Escherichia coli dihydroorotate dehydrogenase N-terminal extension

    Energy Technology Data Exchange (ETDEWEB)

    Couto, Sheila G. [Instituto de Fisica de Sao Carlos, Universidade de Sao Paulo, Av. Trabalhador Sao-carlense 400, C.P. 369, 13560-970, Sao Carlos, SP (Brazil); Grupo de Biofisica e Fisica Aplicada a Medicina, Instituto de Fisica, Universidade Federal de Goias, Campus Samambaia, C.P. 131, 74001-970, Goiania, GO (Brazil); Cristina Nonato, M. [Laboratorio de Cristalografia de Proteinas, Faculdade de Ciencias Farmaceuticas de Ribeirao Preto, Universidade de Sao Paulo, Av. do Cafe S/N, 14040-903, Ribeirao Preto, SP (Brazil); Costa-Filho, Antonio J., E-mail: ajcosta@ffclrp.usp.br [Instituto de Fisica de Sao Carlos, Universidade de Sao Paulo, Av. Trabalhador Sao-carlense 400, C.P. 369, 13560-970, Sao Carlos, SP (Brazil); Departamento de Fisica, Faculdade de Filosofia, Ciencias e Letras de Ribeirao Preto, Av. Bandeirantes 3900, 14040-901, Ribeirao Preto, SP (Brazil)

    2011-10-28

    Highlights: Black-Right-Pointing-Pointer EcDHODH is a membrane-associated enzyme and a promising target for drug design. Black-Right-Pointing-Pointer Enzyme's N-terminal extension is responsible for membrane association. Black-Right-Pointing-Pointer N-terminal works as a molecular lid regulating access to the protein interior. -- Abstract: Dihydroorotate dehydrogenases (DHODHs) are enzymes that catalyze the fourth step of the de novo synthesis of pyrimidine nucleotides. In this reaction, DHODH converts dihydroorotate to orotate, using a flavine mononucleotide as a cofactor. Since the synthesis of nucleotides has different pathways in mammals as compared to parasites, DHODH has gained much attention as a promising target for drug design. Escherichia coli DHODH (EcDHODH) is a family 2 DHODH that interacts with cell membranes in order to promote catalysis. The membrane association is supposedly made via an extension found in the enzyme's N-terminal. In the present work, we used site directed spin labeling (SDSL) to specifically place a magnetic probe at positions 2, 5, 19, and 21 within the N-terminal and thus monitor, by using Electron Spin Resonance (ESR), dynamics and structural changes in this region in the presence of a membrane model system. Overall, our ESR spectra show that the N-terminal indeed binds to membranes and that it experiences a somewhat high flexibility that could be related to the role of this region as a molecular lid controlling the entrance of the enzyme's active site and thus allowing the enzyme to give access to quinones that are dispersed in the membrane and that are necessary for the catalysis.

  15. Inhibition of N-terminal lysines acetylation and transcription factor assembly by epirubicin induced deranged cell homeostasis.

    Directory of Open Access Journals (Sweden)

    Shahper N Khan

    Full Text Available Epirubicin (EPI, an anthracycline antitumour antibiotic, is a known intercalating and DNA damaging agent. Here, we study the molecular interaction of EPI with histones and other cellular targets. EPI binding with histone core protein was predicted with spectroscopic and computational techniques. The molecular distance r, between donor (histone H3 and acceptor (EPI was estimated using Förster's theory of non-radiation energy transfer and the detailed binding phenomenon is expounded. Interestingly, the concentration dependent reduction in the acetylated states of histone H3 K9/K14 was observed suggesting more repressed chromatin state on EPI treatment. Its binding site near N-terminal lysines is further characterized by thermodynamic determinants and molecular docking studies. Specific DNA binding and inhibition of transcription factor (Tf-DNA complex formation implicates EPI induced transcriptional inhibition. EPI also showed significant cell cycle arrest in drug treated cells. Chromatin fragmentation and loss of membrane integrity in EPI treated cells is suggestive of their commitment to cell death. This study provides an analysis of nucleosome dynamics during EPI treatment and provides a novel insight into its action.

  16. N-terminal pro-brain natriuretic peptide and abnormal brain aging: The AGES-Reykjavik Study.

    Science.gov (United States)

    Sabayan, Behnam; van Buchem, Mark A; de Craen, Anton J M; Sigurdsson, Sigurdur; Zhang, Qian; Harris, Tamara B; Gudnason, Vilmundur; Arai, Andrew E; Launer, Lenore J

    2015-09-01

    To investigate the independent association of serum N-terminal fragment of the prohormone natriuretic peptide (NT-proBNP) with structural and functional features of abnormal brain aging in older individuals. In this cross-sectional study based on the Age, Gene/Environment Susceptibility (AGES)-Reykjavik Study, we included 4,029 older community-dwelling individuals (born 1907 to 1935) with a measured serum level of NT-proBNP. Outcomes included parenchymal brain volumes estimated from brain MRI, cognitive function measured by tests of memory, processing speed, and executive functioning, and presence of depressive symptoms measured using the Geriatric Depression Scale. In a substudy, cardiac output of 857 participants was assessed using cardiac MRI. In multivariate analyses, adjusted for sociodemographic and cardiovascular factors, higher levels of NT-proBNP were independently associated with lower total (p brain volumes. Likewise, in multivariate analyses, higher levels of NT-proBNP were associated with worse scores in memory (p = 0.005), processing speed (p = 0.001), executive functioning (p brain parenchymal volumes, impaired executive function and processing speed, and higher depressive symptoms were independent of the level of cardiac output. Higher serum levels of NT-proBNP, independent of cardiovascular risk factors and a measure of cardiac function, are linked with alterations in brain structure and function. Roles of natriuretic peptides in the process of brain aging need to be further elucidated. © 2015 American Academy of Neurology.

  17. Molecular Interaction between the Chaperone Hsc70 and the N-terminal Flank of Huntingtin Exon 1 Modulates Aggregation*

    Science.gov (United States)

    Monsellier, Elodie; Redeker, Virginie; Ruiz-Arlandis, Gemma; Bousset, Luc; Melki, Ronald

    2015-01-01

    The aggregation of polyglutamine (polyQ)-containing proteins is at the origin of nine neurodegenerative diseases. Molecular chaperones prevent the aggregation of polyQ-containing proteins. The exact mechanism by which they interact with polyQ-containing, aggregation-prone proteins and interfere with their assembly is unknown. Here we dissect the mechanism of interaction between a huntingtin exon 1 fragment of increasing polyQ lengths (HttEx1Qn), the aggregation of which is tightly associated with Huntington's disease, and molecular chaperone Hsc70. We show that Hsc70, together with its Hsp40 co-chaperones, inhibits HttEx1Qn aggregation and modifies the structural, seeding, and infectious properties of the resulting fibrils in a polyQ-independent manner. We demonstrate that Hsc70 binds the 17-residue-long N-terminal flank of HttEx1Qn, and we map Hsc70-HttEx1Qn surface interfaces at the residue level. Finally, we show that this interaction competes with homotypic interactions between the N termini of different HttEx1Qn molecules that trigger the aggregation process. Our results lay the foundations of future therapeutic strategies targeting huntingtin aggregation in Huntington disease. PMID:25505179

  18. Cyclization of the N-Terminal X-Asn-Gly Motif during Sample Preparation for Bottom-Up Proteomics

    DEFF Research Database (Denmark)

    Zhang, Xumin; Højrup, Peter

    2010-01-01

    We, herein, report a novel -17 Da peptide modification corresponding to an N-terminal cyclization of peptides possessing the N-terminal motif of X-Asn-Gly. The cyclization occurs spontaneously during sample preparation for bottom-up proteomics studies. Distinct from the two well-known N...

  19. Plant cytoplasm preserved by lightning.

    Science.gov (United States)

    Wang, X

    2004-10-01

    Usually only an organism with hard parts may be preserved in the fossil record. Cytoplasm, which is a physiologically active part of a plant, is rarely seen in the fossil record. Two Cretaceous plant fossils older than 100 million years with exceptional preservation of cytoplasm are reported here. Some cytoplasm is well preserved with subcellular details while other cytoplasm is highly hydrolyzed in the cortex of the same fossil even though both of preservations may be less than 2 microm away. The unique preservation pattern, sharp contrast of preservation in adjacent cells and the exceptional preservation of cytoplasm in the cortex suggest that lightning should play an important role in the preservation of cytoplasm and that cytoplasmic membranes may be more stable than the cell contents. Interpreting the preservation needs knowledge scattering in several formerly unrelated fields of science, including geophysics, botany, biophysics, cytology and microwave fixation technology. This new interpretation of fossilization will shed new light on preservation of cytoplasm and promote cytoplasm fossils from a position of rarity to a position of common research objects available for biological research. The importance of the identification of cytoplasm in fossil lies not in itself but in how much it influences the future research in paleobotany.

  20. Molecular characterization of the 30-AA N-terminal mineral interaction domain of the biomineralization protein AP7.

    Science.gov (United States)

    Kim, Il Won; Morse, Daniel E; Evans, John Spencer

    2004-12-21

    The AP7 protein is one of several mollusk shell proteins which are responsible for aragonite polymorph formation and stabilization within the nacre layer of the Pacific red abalone, H. rufescens. Previously, we demonstrated that the 30-AA N-terminal domain of AP7, denoted as AP7-1, exists as an unfolded sequence and possesses the capability of inhibiting calcium carbonate crystal growth in vitro via growth step frustration or interruption. However, very little is known with regard to the interactive capabilities of this sequence with Ca(II) and with calcium carbonates. Using multidisciplinary techniques, we determine that the AP7-1 polypeptide interacts with Ca(II) ions at the -DD- sequence clusters, yet retains its unfolded, conformationally labile structure in the presence of Ca(II) ions. Further, NMR experiments reveal that the extended structured sequence blocks, -GNGM-, -SVRTQG-, and -ISYL, exhibit motional, chemical exchange, and/or backbone geometry perturbations in response to Ca(II) interactions with AP7-1. Solid-state NMR magic angle spinning studies verify that during the course of in vitro calcium carbonate crystal growth, AP7-1 becomes bound to calcite fragments and cannot be entirely displaced from the mineral fragments using competitive Ca(II) washing. Finally, using a scrambled sequence version of the AP7-1 polypeptide, we observe that sequence scrambling does not adversely affect the crystal growth inhibitory activity of AP7-1, suggesting that the amino acid composition of AP7-1 may be more critical to growth step inhibition than the linear ordering of amino acids.

  1. The S-Layer Proteins of Two Bacillus stearothermophilus Wild-Type Strains Are Bound via Their N-Terminal Region to a Secondary Cell Wall Polymer of Identical Chemical Composition

    Science.gov (United States)

    Egelseer, Eva Maria; Leitner, Karl; Jarosch, Marina; Hotzy, Christoph; Zayni, Sonja; Sleytr, Uwe B.; Sára, Margit

    1998-01-01

    Two Bacillus stearothermophilus wild-type strains were investigated regarding a common recognition and binding mechanism between the S-layer protein and the underlying cell envelope layer. The S-layer protein from B. stearothermophilus PV72/p6 has a molecular weight of 130,000 and assembles into a hexagonally ordered lattice. The S-layer from B. stearothermophilus ATCC 12980 shows oblique lattice symmetry and is composed of subunits with a molecular weight of 122,000. Immunoblotting, peptide mapping, N-terminal sequencing of the whole S-layer protein from B. stearothermophilus ATCC 12980 and of proteolytic cleavage fragments, and comparison with the S-layer protein from B. stearothermophilus PV72/p6 revealed that the two S-layer proteins have identical N-terminal regions but no other extended structurally homologous domains. In contrast to the heterogeneity observed for the S-layer proteins, the secondary cell wall polymer isolated from peptidoglycan-containing sacculi of the different strains showed identical chemical compositions and comparable molecular weights. The S-layer proteins could bind and recrystallize into the appropriate lattice type on native peptidoglycan-containing sacculi from both organisms but not on those extracted with hydrofluoric acid, leading to peptidoglycan of the A1γ chemotype. Affinity studies showed that only proteolytic cleavage fragments possessing the complete N terminus of the mature S-layer proteins recognized native peptidoglycan-containing sacculi as binding sites or could associate with the isolated secondary cell wall polymer, while proteolytic cleavage fragments missing the N-terminal region remained unbound. From the results obtained in this study, it can be concluded that S-layer proteins from B. stearothermophilus wild-type strains possess an identical N-terminal region which is responsible for anchoring the S-layer subunits to a secondary cell wall polymer of identical chemical composition. PMID:9515918

  2. Characterization of immune cell function in N-terminal fragment minipig model of Huntington´s disease

    Czech Academy of Sciences Publication Activity Database

    Valeková, Ivona; Butalová, N.; Vidinská, Daniela; Juhás, Štefan; Kovářová, Hana; Motlík, Jan

    2015-01-01

    Roč. 78, Suppl 2 (2015), s. 24-25 ISSN 1210-7859. [Conference on Animal Models for neurodegenerative Diseases /3./. 08.11.2015-10.11.2015, Liblice] R&D Projects: GA MŠk ED2.1.00/03.0124; GA MŠk(CZ) 7F14308 Institutional support: RVO:67985904 Keywords : neuroinflammation * innate immune system * microglia Subject RIV: FH - Neurology

  3. The role of N-terminal phosphorylation on Huntingtin oligomerization, aggregation and toxicity

    OpenAIRE

    Santos, Joana Margarida Marques Branco dos, 1987-

    2012-01-01

    Tese de mestrado, Neurociências, Faculdade de Medicina, Universidade de Lisboa, 2012 Huntington’s disease (HD) is a hereditary disorder caused by a mutation in the exon-1 of the IT-15 gene, which encodes for a protein called huntingtin. The N-terminal region of mutant huntingtin is prone to aggregate and is toxic for specific types of neurons in the striatum and cortex, leading to the involuntary movements and psychiatric disturbances that characterize HD. Growing evidence indicates that t...

  4. N-terminal PDZ-like domain of chromatin organizer SATB1 ...

    Indian Academy of Sciences (India)

    as a global repressor via recruitment of CtBP1:HDAC1-containing co-repressors to its binding targets. The. N-terminal PSD95/Dlg-A/ZO-1 (PDZ)-like domain of SATB1 mediates interactions with several chromatin proteins. In the present study, we set out to address whether the PDZ-domain-mediated interactions of SATB1 ...

  5. Jun N-terminal protein kinase enhance middle ear mucosal proliferation during bacterial otitis media

    OpenAIRE

    Furukawa, Masayuki; Ebmayer, Jörg; Pak , Kwang; Austin, Darrell A.; Melhus , Åsa; Webster, Nicholas J. G.; Ryan, Allen F.

    2007-01-01

    Mucosal hyperplasia is a characteristic component of otitis media. The present study investigated the participation of signaling via the Jun N-terminal protein kinase (JNK) mitogen-activated protein kinase in middle ear mucosal hyperplasia in animal models of bacterial otitis media. Otitis media was induced by the inoculation of nontypeable Haemophilus influenzae into the middle ear cavity. Western blotting revealed that phosphorylation of JNK isoforms in the middle ear mucosa preceded but pa...

  6. Receptor binding and adenylate cyclase activities of glucagon analogues modified in the N-terminal region

    International Nuclear Information System (INIS)

    McKee, R.L.; Pelton, J.T.; Trivedi, D.; Johnson, D.G.; Coy, D.H.; Sueiras-Diaz, J.; Hruby, V.J.

    1986-01-01

    In this study, we determined the ability of four N-terminally modified derivatives of glucagon, [3-Me-His1,Arg12]-, [Phe1,Arg12]-, [D-Ala4,Arg12]-, and [D-Phe4]glucagon, to compete with 125I-glucagon for binding sites specific for glucagon in hepatic plasma membranes and to activate the hepatic adenylate cyclase system, the second step involved in producing many of the physiological effects of glucagon. Relative to the native hormone, [3-Me-His1,Arg12]glucagon binds approximately twofold greater to hepatic plasma membranes but is fivefold less potent in the adenylate cyclase assay. [Phe1,Arg12]glucagon binds threefold weaker and is also approximately fivefold less potent in adenylate cyclase activity. In addition, both analogues are partial agonists with respect to adenylate cyclase. These results support the critical role of the N-terminal histidine residue in eliciting maximal transduction of the hormonal message. [D-Ala4,Arg12]glucagon and [D-Phe4]glucagon, analogues designed to examine the possible importance of a beta-bend conformation in the N-terminal region of glucagon for binding and biological activities, have binding potencies relative to glucagon of 31% and 69%, respectively. [D-Ala4,Arg12]glucagon is a partial agonist in the adenylate cyclase assay system having a fourfold reduction in potency, while the [D-Phe4] derivative is a full agonist essentially equipotent with the native hormone. These results do not necessarily support the role of an N-terminal beta-bend in glucagon receptor recognition. With respect to in vivo glycogenolysis activities, all of the analogues have previously been reported to be full agonists

  7. Accessibility of the Shine-Dalgarno sequence dictates N-terminal codon bias in E. coli

    OpenAIRE

    Shakhnovich, Eugene; Zhang, Wenli; Yan, Jin; Adkar, Bharat; Jacobs, William; Bhattacharyya, Sanchari; Adkar, Bharat

    2018-01-01

    Despite considerable efforts, no physical mechanism has been shown to explain N-terminal codon bias in prokaryotic genomes. Using a systematic study of synonymous substitutions in two endogenous E. coli genes, we show that interactions between the coding region and the upstream Shine-Dalgarno (SD) sequence modulate the efficiency of translation initiation, affecting both intracellular mRNA and protein levels due to the inherent coupling of transcription and translation in E. coli. We further ...

  8. Structure of the human histone chaperone FACT Spt16 N-terminal domain

    International Nuclear Information System (INIS)

    Marcianò, G.; Huang, D. T.

    2016-01-01

    The Spt16–SSRP1 heterodimer is a histone chaperone that plays an important role in regulating chromatin assembly. Here, a crystal structure of the N-terminal domain of human Spt16 is presented and it is shown that this domain may contribute to histone binding. The histone chaperone FACT plays an important role in facilitating nucleosome assembly and disassembly during transcription. FACT is a heterodimeric complex consisting of Spt16 and SSRP1. The N-terminal domain of Spt16 resembles an inactive aminopeptidase. How this domain contributes to the histone chaperone activity of FACT remains elusive. Here, the crystal structure of the N-terminal domain (NTD) of human Spt16 is reported at a resolution of 1.84 Å. The structure adopts an aminopeptidase-like fold similar to those of the Saccharomyces cerevisiae and Schizosaccharomyces pombe Spt16 NTDs. Isothermal titration calorimetry analyses show that human Spt16 NTD binds histones H3/H4 with low-micromolar affinity, suggesting that Spt16 NTD may contribute to histone binding in the FACT complex. Surface-residue conservation and electrostatic analysis reveal a conserved acidic patch that may be involved in histone binding

  9. Structure of the human histone chaperone FACT Spt16 N-terminal domain

    Energy Technology Data Exchange (ETDEWEB)

    Marcianò, G.; Huang, D. T., E-mail: d.huang@beatson.gla.ac.uk [Cancer Research UK Beatson Institute, Garscube Estate, Switchback Road, Glasgow G61 1BD, Scotland (United Kingdom)

    2016-01-22

    The Spt16–SSRP1 heterodimer is a histone chaperone that plays an important role in regulating chromatin assembly. Here, a crystal structure of the N-terminal domain of human Spt16 is presented and it is shown that this domain may contribute to histone binding. The histone chaperone FACT plays an important role in facilitating nucleosome assembly and disassembly during transcription. FACT is a heterodimeric complex consisting of Spt16 and SSRP1. The N-terminal domain of Spt16 resembles an inactive aminopeptidase. How this domain contributes to the histone chaperone activity of FACT remains elusive. Here, the crystal structure of the N-terminal domain (NTD) of human Spt16 is reported at a resolution of 1.84 Å. The structure adopts an aminopeptidase-like fold similar to those of the Saccharomyces cerevisiae and Schizosaccharomyces pombe Spt16 NTDs. Isothermal titration calorimetry analyses show that human Spt16 NTD binds histones H3/H4 with low-micromolar affinity, suggesting that Spt16 NTD may contribute to histone binding in the FACT complex. Surface-residue conservation and electrostatic analysis reveal a conserved acidic patch that may be involved in histone binding.

  10. Copper(II) Binding Sites in N-Terminally Acetylated α-Synuclein: A Theoretical Rationalization.

    Science.gov (United States)

    Ramis, Rafael; Ortega-Castro, Joaquín; Vilanova, Bartolomé; Adrover, Miquel; Frau, Juan

    2017-08-03

    The interactions between N-terminally acetylated α-synuclein and Cu(II) at several binding sites have been studied with DFT calculations, specifically with the M06 hybrid functional and the ωB97X-D DFT-D functional. In previous experimental studies, Cu(II) was shown to bind several α-synuclein residues, including Met1-Asp2 and His50, forming square planar coordination complexes. Also, it was determined that a low-affinity binding site exists in the C-terminal domain, centered on Asp121. However, in the N-terminally acetylated protein, present in vivo, the Met1 site is blocked. In this work, we simplify the representation of the protein by modeling each experimentally found binding site as a complex between an N-terminally acetylated α-synuclein dipeptide (or several independent residues) and a Cu(II) cation, and compare the results with a number of additional, structurally analogous sites not experimentally found. This way of representing the binding sites, although extremely simple, allows us to reproduce experimental results and to provide a theoretical rationale to explain the preference of Cu(II) for certain sites, as well as explicit geometrical structures for the complexes formed. These results are important to understand the interactions between α-synuclein and Cu(II), one of the factors inducing structural changes in the protein and leading to aggregated forms of it which may play a role in neurodegeneration.

  11. Analysis of the intracellular localization of p73 N-terminal protein isoforms TAp73 and ∆Np73 in medulloblastoma cell lines.

    Science.gov (United States)

    Nekulová, Marta; Zitterbart, Karel; Sterba, Jaroslav; Veselská, Renata

    2010-10-01

    The protein homologous to the tumor suppressor p53, p73, has essential roles in development and tumorigenesis. This protein exists in a wide range of isoforms with different, even antagonistic, functions. However, there are virtually no detailed morphological studies analyzing the endogenous expression of p73 isoforms at the cellular level in cancer cells. In this study, we investigated the expression and subcellular distribution of two N-terminal isoforms, TAp73 and ΔNp73, in medulloblastoma cells using immunofluorescence microscopy. Both proteins were observed in all cell lines examined, but differences were noted in their intracellular localization between the reference Daoy cell line and four newly established medulloblastoma cell lines (MBL-03, MBL-06, MBL-07 and MBL-10). In the new cell lines, TAp73 and ΔNp73 were located predominantly in cell nuclei. However, there was heterogeneity in TAp73 distribution in the cells of all MBL cell lines, with the protein located in the nucleus and also in a limited non-random area in the cytoplasm. In a small percentage of cells, we detected cytoplasmic localization of TAp73 only, i.e., nuclear exclusion was observed. Our results provide a basis for future studies on the causes and function of distinct intracellular localization of p73 protein isoforms with respect to different protein-protein interactions in medulloblastoma cells.

  12. Characterization of cDNA for human tripeptidyl peptidase II: The N-terminal part of the enzyme is similar to subtilisin

    International Nuclear Information System (INIS)

    Tomkinson, B.; Jonsson, A-K

    1991-01-01

    Tripeptidyl peptidase II is a high molecular weight serine exopeptidase, which has been purified from rat liver and human erythrocytes. Four clones, representing 4453 bp, or 90% of the mRNA of the human enzyme, have been isolated from two different cDNA libraries. One clone, designated A2, was obtained after screening a human B-lymphocyte cDNA library with a degenerated oligonucleotide mixture. The B-lymphocyte cDNA library, obtained from human fibroblasts, were rescreened with a 147 bp fragment from the 5' part of the A2 clone, whereby three different overlapping cDNA clones could be isolated. The deduced amino acid sequence, 1196 amino acid residues, corresponding to the longest open rading frame of the assembled nucleotide sequence, was compared to sequences of current databases. This revealed a 56% similarity between the bacterial enzyme subtilisin and the N-terminal part of tripeptidyl peptidase II. The enzyme was found to be represented by two different mRNAs of 4.2 and 5.0 kilobases, respectively, which probably result from the utilziation of two different polyadenylation sites. Futhermore, cDNA corresponding to both the N-terminal and C-terminal part of tripeptidyl peptidase II hybridized with genomic DNA from mouse, horse, calf, and hen, even under fairly high stringency conditions, indicating that tripeptidyl peptidase II is highly conserved

  13. N-Terminal Prodomain of Pfs230 Synthesized Using a Cell-Free System Is Sufficient To Induce Complement-Dependent Malaria Transmission-Blocking Activity▿

    Science.gov (United States)

    Tachibana, Mayumi; Wu, Yimin; Iriko, Hideyuki; Muratova, Olga; MacDonald, Nicholas J.; Sattabongkot, Jetsumon; Takeo, Satoru; Otsuki, Hitoshi; Torii, Motomi; Tsuboi, Takafumi

    2011-01-01

    The aim of a malaria transmission-blocking vaccine is to block the development of malaria parasites in the mosquito and thus prevent subsequent infection of the human host. Previous studies have demonstrated that the gametocyte/gamete surface protein Pfs230 can induce transmission-blocking immunity and have evaluated Escherichia coli-produced Pfs230 as a transmission-blocking vaccine candidate. In this study, we used the wheat germ cell-free expression system to produce N-terminal fragments of Pfs230 and evaluated the transmission-blocking activity of antisera raised against the recombinant Pfs230 protein. The rabbit antisera reacted to the surface of cultured gametocytes and gametes of the Plasmodium falciparum NF54 line, recognized the 360-kDa form of parasite-produced Pfs230 by Western blot assay, and reduced the infectivity of NF54 parasites to Anopheles stefensi mosquitoes in the presence of complement in a standard membrane feeding assay. Thus, our data demonstrate that the N-terminal pro domain of Pfs230 is sufficient to induce complement-dependent transmission-blocking activity against P. falciparum. PMID:21715579

  14. N-terminal prodomain of Pfs230 synthesized using a cell-free system is sufficient to induce complement-dependent malaria transmission-blocking activity.

    Science.gov (United States)

    Tachibana, Mayumi; Wu, Yimin; Iriko, Hideyuki; Muratova, Olga; MacDonald, Nicholas J; Sattabongkot, Jetsumon; Takeo, Satoru; Otsuki, Hitoshi; Torii, Motomi; Tsuboi, Takafumi

    2011-08-01

    The aim of a malaria transmission-blocking vaccine is to block the development of malaria parasites in the mosquito and thus prevent subsequent infection of the human host. Previous studies have demonstrated that the gametocyte/gamete surface protein Pfs230 can induce transmission-blocking immunity and have evaluated Escherichia coli-produced Pfs230 as a transmission-blocking vaccine candidate. In this study, we used the wheat germ cell-free expression system to produce N-terminal fragments of Pfs230 and evaluated the transmission-blocking activity of antisera raised against the recombinant Pfs230 protein. The rabbit antisera reacted to the surface of cultured gametocytes and gametes of the Plasmodium falciparum NF54 line, recognized the 360-kDa form of parasite-produced Pfs230 by Western blot assay, and reduced the infectivity of NF54 parasites to Anopheles stefensi mosquitoes in the presence of complement in a standard membrane feeding assay. Thus, our data demonstrate that the N-terminal pro domain of Pfs230 is sufficient to induce complement-dependent transmission-blocking activity against P. falciparum.

  15. Highly informative proteome analysis by combining improved N-terminal sulfonation for de novo peptide sequencing and online capillary reverse-phase liquid chromatography/tandem mass spectrometry.

    Science.gov (United States)

    Lee, Yong Ho; Kim, Min-Sik; Choie, Woo-Suk; Min, Hye-Ki; Lee, Sang-Won

    2004-06-01

    Recently, various chemical modifications of peptides have been incorporated into mass spectrometric analyses of proteome samples, predominantly in conjunction with matrix-assisted laser desorption/ionization mass spectrometry (MALDI MS), to facilitate de novo sequencing of peptides. In this work, we investigate systematically the utility of N-terminal sulfonation of tryptic peptides by 4-sulfophenyl isothiocyanate (SPITC) for proteome analysis by capillary reverse-phase liquid chromatography/tandem mass spectrometry (cRPLC/MS/MS). The experimental conditions for the sulfonation were carefully adjusted so that SPITC reacts selectively with the N-terminal amino groups, even in the presence of the epsilon-amino groups of lysine residues. Mass spectrometric analyses of the modified peptides by cRPLC/MS/MS indicated that SPITC derivatization proceeded toward near completion under the experimental conditions employed here. The SPITC-derivatized peptides underwent facile fragmentation, predominantly resulting in y-series ions in the MS/MS spectra. Combining SPITC derivatization and cRPLC/MS/MS analyses facilitated the acquisition of sequence information for lysine-terminated tryptic peptides as well as arginine-terminated peptides without the need for additional peptide pretreatment, such as guanidination of lysine amino group. This process alleviated the biased detection of arginine-terminated peptides that is often observed in MALDI MS experiments. We will discuss the utility of the technique as a viable method for proteome analyses and present examples of its application in analyzing samples having different levels of complexity.

  16. Differential isotope dansylation labeling combined with liquid chromatography mass spectrometry for quantification of intact and N-terminal truncated proteins

    International Nuclear Information System (INIS)

    Tang, Yanan; Li, Liang

    2013-01-01

    Graphical abstract: -- Highlights: •LC–MS was developed for quantifying protein mixtures containing both intact and N-terminal truncated proteins. • 12 C 2 -Dansylation of the N-terminal amino acid of proteins was done first, followed by microwave-assisted acid hydrolysis. •The released 12 C 2 -dansyl labeled N-terminal amino acid was quantified using 13 C 2 -dansyl labeled amino acid standards. •The method provided accurate and precise results for quantifying intact and N-terminal truncated proteins within 8 h. -- Abstract: The N-terminal amino acids of proteins are important structure units for maintaining the biological function, localization, and interaction networks of proteins. Under different biological conditions, one or several N-terminal amino acids could be cleaved from an intact protein due to processes, such as proteolysis, resulting in the change of protein properties. Thus, the ability to quantify the N-terminal truncated forms of proteins is of great importance, particularly in the area of development and production of protein-based drugs where the relative quantity of the intact protein and its truncated form needs to be monitored. In this work, we describe a rapid method for absolute quantification of protein mixtures containing intact and N-terminal truncated proteins. This method is based on dansylation labeling of the N-terminal amino acids of proteins, followed by microwave-assisted acid hydrolysis of the proteins into amino acids. It is shown that dansyl labeled amino acids are stable in acidic conditions and can be quantified by liquid chromatography mass spectrometry (LC–MS) with the use of isotope analog standards

  17. Nematode development after removal of egg cytoplasm: absence of localized unbound determinants.

    Science.gov (United States)

    Laufer, J S; von Ehrenstein, G

    1981-01-23

    Embryos of Caenorhabditis elegans develop into fertile adults after cell fragments, containing presumptive cytoplasm of somatic and germ line precursors, are extruded from uncleaved eggs or early blastomeres through laser-induced holes in the eggshells. This suggests that the determinate development of this worm is not dependent on the prelocalization of determinants in specific regions of the egg cytoplasm.

  18. Functional analysis of the N-terminal region of endolysin Lyb5 encoded by Lactobacillus fermentum bacteriophage φPYB5.

    Science.gov (United States)

    Guo, Tingting; Zhang, Chenchen; Liu, Wei; Wang, Shaohua; Kong, Jian

    2015-06-16

    Lactobacillus fermentum temperate bacteriophage φPYB5 uses endolysin Lyb5 and holin Hyb5 to burst the host cell. Previous results showed that expression of Lyb5 in Escherichia coli caused host cell lysis slowly, leading us to suppose that Lyb5 could pass the cytoplasmic membrane partly. In this work, the function of a putative signal peptide (SPLyb5) at the N-terminal of Lyb5 was investigated. In E. coli, the cell adopted a spherical shape during induction of Lyb5 protein, while morphological changes were not observed during expression of the SPLyb5 truncation, indicating that the SPLyb5 motif may serve as a functional signal peptide. However, SPLyb5 was not proteolytically cleaved at the predicted site during the translocation of Lyb5, and the expressed Lyb5 protein appeared in the cytoplasm, cytoplasmic membrane and periplasm fractions with the same molecular mass. Similar results were obtained using Lactococcus lactis as a host to express Lyb5. These results indicated that SPLyb5 could direct Lyb5 to the periplasm in a membrane-tethered form, and then release it as a soluble active enzyme into the periplasm. In addition, SPLyb5 could also drive the fused NucleaseB protein to the extracytoplasm environment in E. coli as well as in L. lactis. We proposed that in Gram-negative and Gram-positive hosts SPLyb5 acted as a signal-anchor-release domain, which was firstly identified here by experimental evidences in lactic acid bacteria phages. The application of signal-anchor-release domain for endolysin export in bacteriophages infecting Gram-positive and Gram-negative hosts was discussed. Copyright © 2015 Elsevier B.V. All rights reserved.

  19. The multifaceted nature of amyloid precursor protein and its proteolytic fragments: friends and foes.

    Science.gov (United States)

    Nhan, Hoang S; Chiang, Karen; Koo, Edward H

    2015-01-01

    The amyloid precursor protein (APP) has occupied a central position in Alzheimer's disease (AD) pathophysiology, in large part due to the seminal role of amyloid-β peptide (Aβ), a proteolytic fragment derived from APP. Although the contribution of Aβ to AD pathogenesis is accepted by many in the research community, recent studies have unveiled a more complicated picture of APP's involvement in neurodegeneration in that other APP-derived fragments have been shown to exert pathological influences on neuronal function. However, not all APP-derived peptides are neurotoxic, and some even harbor neuroprotective effects. In this review, we will explore this complex picture by first discussing the pleiotropic effects of the major APP-derived peptides cleaved by multiple proteases, including soluble APP peptides (sAPPα, sAPPβ), various C- and N-terminal fragments, p3, and APP intracellular domain fragments. In addition, we will highlight two interesting sequences within APP that likely contribute to this duality in APP function. First, it has been found that caspase-mediated cleavage of APP in the cytosolic region may release a cytotoxic peptide, C31, which plays a role in synapse loss and neuronal death. Second, recent studies have implicated the -YENPTY- motif in the cytoplasmic region as a domain that modulates several APP activities through phosphorylation and dephosphorylation of the first tyrosine residue. Thus, this review summarizes the current understanding of various APP proteolytic products and the interplay among them to gain deeper insights into the possible mechanisms underlying neurodegeneration and AD pathophysiology.

  20. Aldehyde stress-mediated novel modification of proteins: epimerization of the N-terminal amino acid.

    Science.gov (United States)

    Kajita, Ryo; Goto, Takaaki; Lee, Seon Hwa; Oe, Tomoyuki

    2013-12-16

    Various kinds of aldehyde-mediated chemical modifications of proteins have been identified as being exclusively covalent. We report a unique noncovalent modification: the aldehyde-mediated epimerization of the N-terminal amino acid. Epimerization of amino acids is thought to cause conformational changes that alter their biological activity. However, few mechanistic studies have been performed, because epimerization of an amino acid is a miniscule change in a whole protein. Furthermore, it does not produce a mass shift, making mass spectrometric analysis difficult. Here, we have demonstrated epimerization mediated by endogenous aldehydes. A model peptide, with an N-terminal l- or d-FMRFamide, was incubated with an endogenous or synthetic aldehyde [acetaldehyde, methylglyoxal, pyridoxal 5'-phosphate (PLP), 4-oxo-2(E)-nonenal, 4-hydroxy-2(E)-nonenal, d-glucose (Glc), 4- or 2-pyridinecarboxaldehyde] under physiological conditions. Each reaction mixture was analyzed by liquid chromatography with ultraviolet detection and/or electrospray ionization mass spectrometry. Considerable epimerization occurred after incubation with some endogenous aldehydes (PLP, 40.6% after 1 day; Glc with copper ions, 6.5% after 7 days). Moreover, the epimerization also occurred in whole proteins (human serum albumin and PLP, 26.3% after 1 day). Tandem mass spectrometric studies, including deuterium labeling and sodium borohydride reduction, suggested that the epimerization results from initial Schiff base formation followed by tautomerization to ketimine that causes the chirality to be lost. This suggests that the epimerization of the N-terminal amino acid can also occur in vivo as a post-translational modification under a high level of aldehyde stress.

  1. KV4.3 N-terminal deletion mutant Δ2–39

    Science.gov (United States)

    Hovind, Laura J; Skerritt, Matthew R

    2011-01-01

    Gating transitions in the KV4.3 N-terminal deletion mutant Δ2–39 were characterized in the absence and presence of KChIP2b. We particularly focused on gating characteristics of macroscopic (open state) versus closed state inactivation (CSI) and recovery. In the absence of KChIP2b Δ2–39 did not significantly alter the steady-state activation “a4” relationship or general CSI characteristics, but it did slow the kinetics of deactivation, macroscopic inactivation and macroscopic recovery. Recovery kinetics (for both WT KV4.3 and Δ2–39) were complicated and displayed sigmoidicity, a process which was enhanced by Δ2–39. Deletion of the proximal N-terminal domain therefore appeared to specifically slow mechanisms involved in regulating gating transitions occurring after the channel open state(s) had been reached. In the presence of KChIP2b Δ2–39 recovery kinetics (from both macroscopic and CSI) were accelerated, with an apparent reduction in initial sigmoidicity. Hyperpolarizing shifts in both “a4” and isochronal inactivation “i” were also produced. KChIP2b-mediated remodeling of KV4.3 gating transitions was therefore not obligatorily dependent upon an intact N-terminus. To account for these effects we propose that KChIP2 regulatory domains exist in KV4.3 α subunit regions outside of the proximal N-terminal. In addition to regulating macroscopic inactivation, we also propose that the KV4.3 N-terminus may act as a novel regulator of deactivation-recovery coupling. PMID:21057209

  2. Peptidase family U34 belongs to the superfamily of N-terminal nucleophile hydrolases

    Science.gov (United States)

    Pei, Jimin; Grishin, Nick V.

    2003-01-01

    Peptidase family U34 consists of enzymes with unclear catalytic mechanism, for instance, dipeptidase A from Lactobacillus helveticus. Using extensive sequence similarity searches, we infer that U34 family members are homologous to penicillin V acylases (PVA) and thus potentially adopt the N-terminal nucleophile (Ntn) hydrolase fold. Comparative sequence and structural analysis reveals a cysteine as the catalytic nucleophile as well as other conserved residues important for catalysis. The PVA/U34 family is variable in sequence and exhibits great diversity in substrate specificity, to include enzymes such as choloyglycine hydrolases, acid ceramidases, isopenicillin N acyltransferases, and a subgroup of eukaryotic proteins with unclear function. PMID:12717035

  3. Crystal Structure of the Full-Length Feline Immunodeficiency Virus Capsid Protein Shows an N-Terminal β-Hairpin in the Absence of N-Terminal Proline

    Directory of Open Access Journals (Sweden)

    Christelle Folio

    2017-11-01

    Full Text Available Feline immunodeficiency virus (FIV is a member of the Retroviridae family. It is the causative agent of an acquired immunodeficiency syndrome (AIDS in cats and wild felines. Its capsid protein (CA drives the assembly of the viral particle, which is a critical step in the viral replication cycle. Here, the first atomic structure of full-length FIV CA to 1.67 Å resolution is determined. The crystallized protein exhibits an original tetrameric assembly, composed of dimers which are stabilized by an intermolecular disulfide bridge induced by the crystallogenesis conditions. The FIV CA displays a standard α-helical CA topology with two domains, separated by a linker shorter than other retroviral CAs. The β-hairpin motif at its amino terminal end, which interacts with nucleotides in HIV-1, is unusually long in FIV CA. Interestingly, this functional β-motif is formed in this construct in the absence of the conserved N-terminal proline. The FIV CA exhibits a cis Arg–Pro bond in the CypA-binding loop, which is absent in known structures of lentiviral CAs. This structure represents the first tri-dimensional structure of a functional, full-length FIV CA.

  4. Crystal Structure of the Full-Length Feline Immunodeficiency Virus Capsid Protein Shows an N-Terminal β-Hairpin in the Absence of N-Terminal Proline.

    Science.gov (United States)

    Folio, Christelle; Sierra, Natalia; Dujardin, Marie; Alvarez, Guzman; Guillon, Christophe

    2017-11-09

    Feline immunodeficiency virus (FIV) is a member of the Retroviridae family. It is the causative agent of an acquired immunodeficiency syndrome (AIDS) in cats and wild felines. Its capsid protein (CA) drives the assembly of the viral particle, which is a critical step in the viral replication cycle. Here, the first atomic structure of full-length FIV CA to 1.67 Å resolution is determined. The crystallized protein exhibits an original tetrameric assembly, composed of dimers which are stabilized by an intermolecular disulfide bridge induced by the crystallogenesis conditions. The FIV CA displays a standard α-helical CA topology with two domains, separated by a linker shorter than other retroviral CAs. The β-hairpin motif at its amino terminal end, which interacts with nucleotides in HIV-1, is unusually long in FIV CA. Interestingly, this functional β-motif is formed in this construct in the absence of the conserved N-terminal proline. The FIV CA exhibits a cis Arg-Pro bond in the CypA-binding loop, which is absent in known structures of lentiviral CAs. This structure represents the first tri-dimensional structure of a functional, full-length FIV CA.

  5. Structure-activity relationships of the antimicrobial peptide arasin 1 - and mode of action studies of the N-terminal, proline-rich region.

    Directory of Open Access Journals (Sweden)

    Victoria S Paulsen

    Full Text Available Arasin 1 is a 37 amino acid long proline-rich antimicrobial peptide isolated from the spider crab, Hyas araneus. In this work the active region of arasin 1 was identified through structure-activity studies using different peptide fragments derived from the arasin 1 sequence. The pharmacophore was found to be located in the proline/arginine-rich NH(2 terminus of the peptide and the fragment arasin 1(1-23 was almost equally active to the full length peptide. Arasin 1 and its active fragment arasin 1(1-23 were shown to be non-toxic to human red blood cells and arasin 1(1-23 was able to bind chitin, a component of fungal cell walls and the crustacean shell. The mode of action of the fully active N-terminal arasin 1(1-23 was explored through killing kinetic and membrane permeabilization studies. At the minimal inhibitory concentration (MIC, arasin 1(1-23 was not bactericidal and had no membrane disruptive effect. In contrast, at concentrations of 5×MIC and above it was bactericidal and interfered with membrane integrity. We conclude that arasin 1(1-23 has a different mode of action than lytic peptides, like cecropin P1. Thus, we suggest a dual mode of action for arasin 1(1-23 involving membrane disruption at peptide concentrations above MIC, and an alternative mechanism of action, possibly involving intracellular targets, at MIC.

  6. Cytoplasmic chromatin triggers inflammation in senescence and cancer.

    Science.gov (United States)

    Dou, Zhixun; Ghosh, Kanad; Vizioli, Maria Grazia; Zhu, Jiajun; Sen, Payel; Wangensteen, Kirk J; Simithy, Johayra; Lan, Yemin; Lin, Yanping; Zhou, Zhuo; Capell, Brian C; Xu, Caiyue; Xu, Mingang; Kieckhaefer, Julia E; Jiang, Tianying; Shoshkes-Carmel, Michal; Tanim, K M Ahasan Al; Barber, Glen N; Seykora, John T; Millar, Sarah E; Kaestner, Klaus H; Garcia, Benjamin A; Adams, Peter D; Berger, Shelley L

    2017-10-19

    Chromatin is traditionally viewed as a nuclear entity that regulates gene expression and silencing. However, we recently discovered the presence of cytoplasmic chromatin fragments that pinch off from intact nuclei of primary cells during senescence, a form of terminal cell-cycle arrest associated with pro-inflammatory responses. The functional significance of chromatin in the cytoplasm is unclear. Here we show that cytoplasmic chromatin activates the innate immunity cytosolic DNA-sensing cGAS-STING (cyclic GMP-AMP synthase linked to stimulator of interferon genes) pathway, leading both to short-term inflammation to restrain activated oncogenes and to chronic inflammation that associates with tissue destruction and cancer. The cytoplasmic chromatin-cGAS-STING pathway promotes the senescence-associated secretory phenotype in primary human cells and in mice. Mice deficient in STING show impaired immuno-surveillance of oncogenic RAS and reduced tissue inflammation upon ionizing radiation. Furthermore, this pathway is activated in cancer cells, and correlates with pro-inflammatory gene expression in human cancers. Overall, our findings indicate that genomic DNA serves as a reservoir to initiate a pro-inflammatory pathway in the cytoplasm in senescence and cancer. Targeting the cytoplasmic chromatin-mediated pathway may hold promise in treating inflammation-related disorders.

  7. Cellular Subcompartments through Cytoplasmic Streaming.

    Science.gov (United States)

    Pieuchot, Laurent; Lai, Julian; Loh, Rachel Ann; Leong, Fong Yew; Chiam, Keng-Hwee; Stajich, Jason; Jedd, Gregory

    2015-08-24

    Cytoplasmic streaming occurs in diverse cell types, where it generally serves a transport function. Here, we examine streaming in multicellular fungal hyphae and identify an additional function wherein regimented streaming forms distinct cytoplasmic subcompartments. In the hypha, cytoplasm flows directionally from cell to cell through septal pores. Using live-cell imaging and computer simulations, we identify a flow pattern that produces vortices (eddies) on the upstream side of the septum. Nuclei can be immobilized in these microfluidic eddies, where they form multinucleate aggregates and accumulate foci of the HDA-2 histone deacetylase-associated factor, SPA-19. Pores experiencing flow degenerate in the absence of SPA-19, suggesting that eddy-trapped nuclei function to reinforce the septum. Together, our data show that eddies comprise a subcellular niche favoring nuclear differentiation and that subcompartments can be self-organized as a consequence of regimented cytoplasmic streaming. Copyright © 2015 Elsevier Inc. All rights reserved.

  8. The AAA+ ATPase TRIP13 remodels HORMA domains through N-terminal engagement and unfolding

    Energy Technology Data Exchange (ETDEWEB)

    Ye, Qiaozhen; Kim, Dong Hyun; Dereli, Ihsan; Rosenberg, Scott C.; Hagemann, Goetz; Herzog, Franz; Tóth, Attila; Cleveland, Don W.; Corbett, Kevin D.

    2017-06-28

    Proteins of the conserved HORMA domain family, including the spindle assembly checkpoint protein MAD2 and the meiotic HORMADs, assemble into signaling complexes by binding short peptides termed “closure motifs”. The AAA+ ATPase TRIP13 regulates both MAD2 and meiotic HORMADs by disassembling these HORMA domain–closure motif complexes, but its mechanisms of substrate recognition and remodeling are unknown. Here, we combine X-ray crystallography and crosslinking mass spectrometry to outline how TRIP13 recognizes MAD2 with the help of the adapter protein p31comet. We show that p31comet binding to the TRIP13 N-terminal domain positions the disordered MAD2 N-terminus for engagement by the TRIP13 “pore loops”, which then unfold MAD2 in the presence of ATP. N-terminal truncation of MAD2 renders it refractory to TRIP13 action in vitro, and in cells causes spindle assembly checkpoint defects consistent with loss of TRIP13 function. Similar truncation of HORMAD1 in mouse spermatocytes compromises its TRIP13-mediated removal from meiotic chromosomes, highlighting a conserved mechanism for recognition and disassembly of HORMA domain–closure motif complexes by TRIP13.

  9. Novel Insights into Structure-Activity Relationships of N-Terminally Modified PACE4 Inhibitors.

    Science.gov (United States)

    Kwiatkowska, Anna; Couture, Frédéric; Levesque, Christine; Ly, Kévin; Beauchemin, Sophie; Desjardins, Roxane; Neugebauer, Witold; Dory, Yves L; Day, Robert

    2016-02-04

    PACE4 plays important roles in prostate cancer cell proliferation. The inhibition of this enzyme has been shown to slow prostate cancer progression and is emerging as a promising therapeutic strategy. In previous work, we developed a highly potent and selective PACE4 inhibitor, the multi-Leu (ML) peptide, an octapeptide with the sequence Ac-LLLLRVKR-NH2 . Here, with the objective of developing a useful compound for in vivo administration, we investigate the effect of N-terminal modifications. The inhibitory activity, toxicity, stability, and cell penetration properties of the resulting analogues were studied and compared to the unmodified inhibitor. Our results show that the incorporation of a polyethylene glycol (PEG) moiety leads to a loss of antiproliferative activity, whereas the attachment of a lipid chain preserves or improves it. However, the lipidated peptides are significantly more toxic when compared with their unmodified counterparts. Therefore, the best results were achieved not by the N-terminal extension but by the protection of both ends with the d-Leu residue and 4-amidinobenzylamide, which yielded the most stable inhibitor, with an excellent activity and toxicity profile. © 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  10. The unique N-terminal zinc finger of synaptotagmin-like protein 4 reveals FYVE structure.

    Science.gov (United States)

    Miyamoto, Kazuhide; Nakatani, Arisa; Saito, Kazuki

    2017-12-01

    Synaptotagmin-like protein 4 (Slp4), expressed in human platelets, is associated with dense granule release. Slp4 is comprised of the N-terminal zinc finger, Slp homology domain, and C2 domains. We synthesized a compact construct (the Slp4N peptide) corresponding to the Slp4 N-terminal zinc finger. Herein, we have determined the solution structure of the Slp4N peptide by nuclear magnetic resonance (NMR). Furthermore, experimental, chemical modification of Cys residues revealed that the Slp4N peptide binds two zinc atoms to mediate proper folding. NMR data showed that eight Cys residues coordinate zinc atoms in a cross-brace fashion. The Simple Modular Architecture Research Tool database predicted the structure of Slp4N as a RING finger. However, the actual structure of the Slp4N peptide adopts a unique C 4 C 4 -type FYVE fold and is distinct from a RING fold. To create an artificial RING finger (ARF) with specific ubiquitin-conjugating enzyme (E2)-binding capability, cross-brace structures with eight zinc-ligating residues are needed as the scaffold. The cross-brace structure of the Slp4N peptide could be utilized as the scaffold for the design of ARFs. © 2017 The Protein Society.

  11. In Silico Identification and Characterization of N-Terminal Acetyltransferase Genes of Poplar (Populus trichocarpa

    Directory of Open Access Journals (Sweden)

    Hang-Yong Zhu

    2014-01-01

    Full Text Available N-terminal acetyltransferase (Nats complex is responsible for protein N-terminal acetylation (Nα-acetylation, which is one of the most common covalent modifications of eukaryotic proteins. Although genome-wide investigation and characterization of Nat catalytic subunits (CS and auxiliary subunits (AS have been conducted in yeast and humans they remain unexplored in plants. Here we report on the identification of eleven genes encoding eleven putative Nat CS polypeptides, and five genes encoding five putative Nat AS polypeptides in Populus. We document that the expansion of Nat CS genes occurs as duplicated blocks distributed across 10 of the 19 poplar chromosomes, likely only as a result of segmental duplication events. Based on phylogenetic analysis, poplar Nat CS were assigned to six subgroups, which corresponded well to the Nat CS types (CS of Nat A–F, being consistent with previous reports in humans and yeast. In silico analysis of microarray data showed that in the process of normal development of the poplar, their Nat CS and AS genes are commonly expressed at one relatively low level but share distinct tissue-specific expression patterns. This exhaustive survey of Nat genes in poplar provides important information to assist future studies on their functional role in poplar.

  12. Specificity of N-terminal methionyl peptidase: analysis by site-directed mutagenesis

    International Nuclear Information System (INIS)

    Kasper, T.J.; Boissel, J.P.; Bunn, H.F.

    1987-01-01

    The start site of eukaryotic translation is normally an AUG codon. The corresponding N-terminal methionine is most often removed when the nascent chain reaches about 30 residues. Data from a survey of 1764 eukaryotic protein sequences suggest that the residue adjacent to the initiator Met determines Met cleavage. In order to investigate the mechanism of this reaction, the authors have prepared oligonucleotide-directed mutants of human β-globin from gapped heteroduplexes of a T3/T7 plasmid containing a globin cDNA clone. To date, the authors have produced mutants encoding for 15 of 19 possible amino acid replacements at position 1 in the β-globin chain. These mutants have been confirmed by dideoxy sequencing, transcribed in vitro, and translated in a rabbit reticulocyte lysate in the presence of 35 S-methionine. Labeled translation products were then isolated by cation exchange HPLC, and tryptic peptides were analyzed by RP-HPLC. Thus far, this structural analysis has shown that for β-1 Val, Ala, and Ser, the initiator Met is cleaved, whereas for β-1 Lys, Met, Glu, Trp, Asn, Tyr, and Glu, initiator Met is retained. For β-1 Leu initiator Met is cleaved with a frequency of about 50%. These results are consistent with the data obtained from the previous survey. The expression of site-directed mutants in a cell-free system can also be used to investigate other N-terminal processing events, such as acetylation and myristylation

  13. The N-terminal domain of apolipoprotein B-100: structural characterization by homology modeling

    Directory of Open Access Journals (Sweden)

    Khachfe Hassan M

    2007-07-01

    Full Text Available Abstract Background Apolipoprotein B-100 (apo B-100 stands as one of the largest proteins in humans. Its large size of 4536 amino acids hampers the production of X-ray diffraction quality crystals and hinders in-solution NMR analysis, and thus necessitates a domain-based approach for the structural characterization of the multi-domain full-length apo B. Results The structure of apo B-17 (the N-terminal 17% of apolipoprotein B-100 was predicted by homology modeling based on the structure of the N-terminal domain of lipovitellin (LV, a protein that shares not only sequence similarity with B17, but also a functional aspect of lipid binding and transport. The model structure was first induced to accommodate the six disulfide bonds found in that region, and then optimized using simulated annealing. Conclusion The content of secondary structural elements in this model structure correlates well with the reported data from other biophysical probes. The overall topology of the model conforms with the structural outline corresponding to the apo B-17 domain as seen in the EM representation of the complete LDL structure.

  14. Molecular cloning and biologically active production of IpaD N-terminal region.

    Science.gov (United States)

    Hesaraki, Mahdi; Saadati, Mojtaba; Honari, Hossein; Olad, Gholamreza; Heiat, Mohammad; Malaei, Fatemeh; Ranjbar, Reza

    2013-07-01

    Shigella is known as pathogenic intestinal bacteria in high dispersion and pathogenic bacteria due to invasive plasmid antigen (Ipa). So far, a number of Ipa proteins have been studied to introduce a new candidate vaccine. Here, for the first time, we examined whether the N-terminal region of IpaD(72-162) could be a proper candidate for Shigella vaccine. Initially, the DNA sequence coding N-terminal region was isolated by PCR from Shigella dysenteriae type I and cloned into pET-28a expression vector. Then, the heterologous protein was expressed, optimized and purified by affinity Ni-NTA column. Western blot analysis using, His-tag and IpaD(72-162) polyclonal antibodies, confirmed the purity and specificity of the recombinant protein, respectively. Subsequently, the high immunogenicity of the antigen was shown by ELISA. The results of the sereny test in Guinea pigs showed that IpaD(72-162) provides a protective system against Shigella flexneri 5a and S. dysenteriae type I. Copyright © 2013. Published by Elsevier Ltd.

  15. Isolation and N-terminal sequencing of a novel cadmium-binding protein from Boletus edulis

    Science.gov (United States)

    Collin-Hansen, C.; Andersen, R. A.; Steinnes, E.

    2003-05-01

    A Cd-binding protein was isolated from the popular edible mushroom Boletus edulis, which is a hyperaccumulator of both Cd and Hg. Wild-growing samples of B. edulis were collected from soils rich in Cd. Cd radiotracer was added to the crude protein preparation obtained from ethanol precipitation of heat-treated cytosol. Proteins were then further separated in two consecutive steps; gel filtration and anion exchange chromatography. In both steps the Cd radiotracer profile showed only one distinct peak, which corresponded well with the profiles of endogenous Cd obtained by atomic absorption spectrophotometry (AAS). Concentrations of the essential elements Cu and Zn were low in the protein fractions high in Cd. N-terminal sequencing performed on the Cd-binding protein fractions revealed a protein with a novel amino acid sequence, which contained aromatic amino acids as well as proline. Both the N-terminal sequencing and spectrofluorimetric analysis with EDTA and ABD-F (4-aminosulfonyl-7-fluoro-2, 1, 3-benzoxadiazole) failed to detect cysteine in the Cd-binding fractions. These findings conclude that the novel protein does not belong to the metallothionein family. The results suggest a role for the protein in Cd transport and storage, and they are of importance in view of toxicology and food chemistry, but also for environmental protection.

  16. UV laser-induced histone-DNA crosslinking proceeds via the N-terminal tails

    International Nuclear Information System (INIS)

    Stefanovski, V.; Dimitrov, S.; Angelov, D.; Keskinova, E.; Pashev, I.

    1990-01-01

    The covalent crosslinking of histones to DNA by UV laser irradiation is accomplished solely via the N-terminal part of the molecule. Irradiated isolated calfthymus nuclei are treated with clostripain. The crosslinked protein-DNA complexes are isolated and the presence of each core histone analyzed by dot-immunoassay using antibodies, specific to the central globular domain of the respective histone. The reaction is negative for all core histones i.e. the globular domain is absent. It means that this domain has not been crosslinked to DNA and, once cleaved by clostripain, it has been stripped from DNA during the centrigugation in CsCl. This peculiar property of the crosslinked procedure makes it particularly useful in addressing some yet unanswered questions concerning histone-DNA interactions, such as the interaction of the N-terminal tails with linker DNA, the effect of the transient postsynthetic histone acetylation on its interaction with DNA, etc. These questions are now under study. 1 fig., 6 refs

  17. Human TRPA1 is intrinsically cold- and chemosensitive with and without its N-terminal ankyrin repeat domain.

    Science.gov (United States)

    Moparthi, Lavanya; Survery, Sabeen; Kreir, Mohamed; Simonsen, Charlotte; Kjellbom, Per; Högestätt, Edward D; Johanson, Urban; Zygmunt, Peter M

    2014-11-25

    We have purified and reconstituted human transient receptor potential (TRP) subtype A1 (hTRPA1) into lipid bilayers and recorded single-channel currents to understand its inherent thermo- and chemosensory properties as well as the role of the ankyrin repeat domain (ARD) of the N terminus in channel behavior. We report that hTRPA1 with and without its N-terminal ARD (Δ1-688 hTRPA1) is intrinsically cold-sensitive, and thus, cold-sensing properties of hTRPA1 reside outside the N-terminal ARD. We show activation of hTRPA1 by the thiol oxidant 2-((biotinoyl)amino)ethyl methanethiosulfonate (MTSEA-biotin) and that electrophilic compounds activate hTRPA1 in the presence and absence of the N-terminal ARD. The nonelectrophilic compounds menthol and the cannabinoid Δ(9)-tetrahydrocannabiorcol (C16) directly activate hTRPA1 at different sites independent of the N-terminal ARD. The TRPA1 antagonist HC030031 inhibited cold and chemical activation of hTRPA1 and Δ1-688 hTRPA1, supporting a direct interaction with hTRPA1 outside the N-terminal ARD. These findings show that hTRPA1 is an intrinsically cold- and chemosensitive ion channel. Thus, second messengers, including Ca(2+), or accessory proteins are not needed for hTRPA1 responses to cold or chemical activators. We suggest that conformational changes outside the N-terminal ARD by cold, electrophiles, and nonelectrophiles are important in hTRPA1 channel gating and that targeting chemical interaction sites outside the N-terminal ARD provides possibilities to fine tune TRPA1-based drug therapies (e.g., for treatment of pain associated with cold hypersensitivity and cardiovascular disease).

  18. Mitochondrial DNA variation within P-type cytoplasmic male sterility of Plantago lanceolata L

    NARCIS (Netherlands)

    Groenendijk, C.F.M.; Sandbrink, J.M.; Van Brederode, J.; Van Damme, J.M.M.

    1997-01-01

    MtDNA restriction fragment polymorphisms were found between cytoplasmic male-sterility types P and R of Plantago lanceolata with the homologous probe pPl311 and maize mtDNA fragments derived from the regions of atp1, cox1 and cox2. No mtDNA differences were observed between male-sterile and restored

  19. Latency of transcription factor Stp1 depends on a modular regulatory motif that functions as cytoplasmic retention determinant and nuclear degron

    Science.gov (United States)

    Omnus, Deike J.; Ljungdahl, Per O.

    2014-01-01

    The Ssy1-Ptr3-Ssy5 (SPS)–sensing pathway enables yeast to respond to extracellular amino acids. Stp1, the effector transcription factor, is synthesized as a latent cytoplasmic precursor with an N-terminal regulatory domain that restricts its nuclear accumulation. The negative regulatory mechanisms impinging on the N-terminal domain are poorly understood. However, Stp1 latency depends on three inner nuclear membrane proteins, Asi1, Asi2, and Asi3. We report that the N-terminal domain of Stp1 contains a small motif, designated RI, that fully accounts for latency. RI is modular, mediates interactions with the plasma membrane, and can retain histone Htb2 in the cytoplasm. A novel class of STP1 mutations affecting RI were isolated that are less efficiently retained in the cytoplasm but remain under tight negative control by the Asi proteins. Intriguingly, these mutant proteins exhibit enhanced stability in strains lacking ASI1. Our results indicate that RI mediates latency by two distinct activities: it functions as a cytoplasmic retention determinant and an Asi-dependent degron. These findings provide novel insights into the SPS-sensing pathway and demonstrate for the first time that the inner nuclear membrane Asi proteins function in a degradation pathway in the nucleus. PMID:25253722

  20. Structure of N-Terminal Sequence Asp-Ala-Glu-Phe-Arg-His-Asp-Ser of Aβ-Peptide with Phospholipase A2 from Venom of Andaman Cobra Sub-Species Naja naja sagittifera at 2.0 Å Resolution

    Directory of Open Access Journals (Sweden)

    Zeenat Mirza

    2014-03-01

    Full Text Available Alzheimer’s disease (AD is one of the most significant social and health burdens of the present century. Plaques formed by extracellular deposits of amyloid β (Aβ are the prime player of AD’s neuropathology. Studies have implicated the varied role of phospholipase A2 (PLA2 in brain where it contributes to neuronal growth and inflammatory response. Overall contour and chemical nature of the substrate-binding channel in the low molecular weight PLA2s are similar. This study involves the reductionist fragment-based approach to understand the structure adopted by N-terminal fragment of Alzheimer’s Aβ peptide in its complex with PLA2. In the current communication, we report the structure determined by X-ray crystallography of N-terminal sequence Asp-Ala-Glu-Phe-Arg-His-Asp-Ser (DAEFRHDS of Aβ-peptide with a Group I PLA2 purified from venom of Andaman Cobra sub-species Naja naja sagittifera at 2.0 Å resolution (Protein Data Bank (PDB Code: 3JQ5. This is probably the first attempt to structurally establish interaction between amyloid-β peptide fragment and hydrophobic substrate binding site of PLA2 involving H bond and van der Waals interactions. We speculate that higher affinity between Aβ and PLA2 has the therapeutic potential of decreasing the Aβ–Aβ interaction, thereby reducing the amyloid aggregation and plaque formation in AD.

  1. A 19-kDa C-terminal tryptic fragment of the α chain of Na/K-ATPase is essential for occlusion and transport of cations

    International Nuclear Information System (INIS)

    Karlish, S.J.D.; Goldshleger, R.; Stein, W.D.

    1990-01-01

    Tryptic digestion of pig renal Na/K-ATPase in the presence of Rb and absence of Ca ions removes about half of the protein but leaves a stable 19-kDa membrane-embedded fragment derived from the α chain, a largely intact β chain, and essentially normal Rb- and Na-occlusion capacity. Subsequent digestion with trypsin in the presence of Ca or absence of Rb ions leads to rapid loss of the 19-kDa fragment and a parallel loss of Rb occlusion, demonstrating that the fragment is essential for occlusion. The N-terminal sequence of the 19-kDa fragment is Asn-Pro-Lys-Thr-Asp-Lys-Leu-Val-Asn-Glu-Arg-Leu-Ile-Ser-Met-Ala, beginning at residue 830 and extending toward the C terminus. Membranes containing the 19-kDa fragment have the following functional properties. (i) ATP-dependent functions are absent. (ii) The apparent affinity for occluding Rb is unchanged, the affinity for Na is lower than in the control enzyme, and activation is now strongly sigmoidal rather than hyperbolic. (iii) Membranes containing the 19-kDa fragment can be reconstituted into phospholipid vesicles and sustain slow Rb-Rb exchange. Thus the transport pathway is retained. The authors conclude that cation occlusion sites and the transport pathway within transmembrane segments are quite separate from the ATP binding sites, located on the cytoplasmic domain of the α chain. Interactions between cation and ATP sites, the heart of active transport, must be indirect - mediated, presumably, by conformational changes of the protein

  2. A 19-kDa C-terminal tryptic fragment of the. alpha. chain of Na/K-ATPase is essential for occlusion and transport of cations

    Energy Technology Data Exchange (ETDEWEB)

    Karlish, S.J.D.; Goldshleger, R. (Weizmann Institute of Science, Rehovot (Israel)); Stein, W.D. (Hebrew Univ. Jerusalem (Israel))

    1990-06-01

    Tryptic digestion of pig renal Na/K-ATPase in the presence of Rb and absence of Ca ions removes about half of the protein but leaves a stable 19-kDa membrane-embedded fragment derived from the {alpha} chain, a largely intact {beta} chain, and essentially normal Rb- and Na-occlusion capacity. Subsequent digestion with trypsin in the presence of Ca or absence of Rb ions leads to rapid loss of the 19-kDa fragment and a parallel loss of Rb occlusion, demonstrating that the fragment is essential for occlusion. The N-terminal sequence of the 19-kDa fragment is Asn-Pro-Lys-Thr-Asp-Lys-Leu-Val-Asn-Glu-Arg-Leu-Ile-Ser-Met-Ala, beginning at residue 830 and extending toward the C terminus. Membranes containing the 19-kDa fragment have the following functional properties. (i) ATP-dependent functions are absent. (ii) The apparent affinity for occluding Rb is unchanged, the affinity for Na is lower than in the control enzyme, and activation is now strongly sigmoidal rather than hyperbolic. (iii) Membranes containing the 19-kDa fragment can be reconstituted into phospholipid vesicles and sustain slow Rb-Rb exchange. Thus the transport pathway is retained. The authors conclude that cation occlusion sites and the transport pathway within transmembrane segments are quite separate from the ATP binding sites, located on the cytoplasmic domain of the {alpha} chain. Interactions between cation and ATP sites, the heart of active transport, must be indirect - mediated, presumably, by conformational changes of the protein.

  3. Annexin A2 is SUMOylated on its N-terminal domain: regulation by insulin.

    Science.gov (United States)

    Caron, Danielle; Boutchueng-Djidjou, Martial; Tanguay, Robert M; Faure, Robert L

    2015-04-13

    Insulin receptor (IR) endocytosis requires a remodelling of the actin cytoskeleton. We show here that ANXA2 is SUMOylated at the K10 located in a non-consensus SUMOylation motif in the N-terminal domain. The Y24F mutation decreased the SUMOylation signal, whereas insulin stimulation increased ANXA2 SUMOylation. A survey of protein SUMOylation in hepatic Golgi/endosome (G/E) fractions after insulin injections revealed the presence of a SUMOylation pattern and confirmed the SUMOylation of ANXA2. The construction of an IR/ANXA2/SUMO network (IRASGEN) in the G/E context reveals the presence of interacting nodes whereby SUMO1 connects ANXA2 to actin and microtubule-mediated changes in membrane topology. Heritable variants associated with type 2 diabetes represent 41% of the IRASGEN thus pointing out the physio-pathological importance of this subnetwork. Copyright © 2015 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.

  4. Dynamic enzyme docking to the ribosome coordinates N-terminal processing with polypeptide folding.

    Science.gov (United States)

    Sandikci, Arzu; Gloge, Felix; Martinez, Michael; Mayer, Matthias P; Wade, Rebecca; Bukau, Bernd; Kramer, Günter

    2013-07-01

    Newly synthesized polypeptides undergo various cotranslational maturation steps, including N-terminal enzymatic processing, chaperone-assisted folding and membrane targeting, but the spatial and temporal coordination of these steps is unclear. We show that Escherichia coli methionine aminopeptidase (MAP) associates with ribosomes through a charged loop that is crucial for nascent-chain processing and cell viability. MAP competes with peptide deformylase (PDF), the first enzyme to act on nascent chains, for binding sites at the ribosomal tunnel exit. PDF has extremely fast association and dissociation kinetics, which allows it to frequently sample ribosomes and ensure the processing of nascent chains after their emergence. Premature recruitment of the chaperone trigger factor, or polypeptide folding, negatively affect processing efficiency. Thus, the fast ribosome association kinetics of PDF and MAP are crucial for the temporal separation of nascent-chain processing from later maturation events, including chaperone recruitment and folding.

  5. The Use of N-Terminal-Pro-BNP in Preterm Infants

    Directory of Open Access Journals (Sweden)

    Afif EL-Khuffash

    2009-01-01

    Full Text Available The use of natriuretic peptides in the neonatal population is emerging. B-type Natriuretic Peptide (BNP and N-terminal-Pro-BNP (NTpBNP are used in the adult population to assess myocardial function and volume loading. Their role in prognosis following cardiac surgery has also been identified. In preterm infants NTpBNP is becoming increasingly recognised as a potential screening tool for patent ductus arteriosus (PDA, and a marker for myocardial performance. In addition, NTpBNP may provide prognostic information in preterm infants and term infants with congenital diaphragmatic hernia (CDH. In this paper, the role of NTpBNP in the preterm population will be discussed.

  6. N-terminal region of human ameloblastin synthetic peptide promotes bone formation.

    Science.gov (United States)

    Kitagawa, Masae; Ando, Toshinori; Subarnbhesaj, Ajiravudh; Uchida, Takashi; Miyauchi, Mutsumi; Takata, Takashi

    2017-01-01

    The aim of this study was to examine the effect of 16 amino acids of the N-terminal region of human ameloblastin (16N-AMBN) synthetic peptide, on the proliferation and differentiation of MC3T3-E1 cells and bone regeneration. While 16N-AMBN did not affect the proliferation, it induced mRNA expression of type I collagen, alkaline phosphatase (ALP), bone sialoprotein, and osteocalcin. 16N-AMBN also stimulated ALP activity and promoted mineralized nodule formation. On the other hand, these activities were inhibited by anti-16N-AMBN antibody. Treatment of rat calvarial bone defects with 16N-AMBN resulted in almost complete healing compared to that of the control treatments. These findings suggest that 16N-AMBN may be applicable for regeneration therapy of bone defects.

  7. The vasorelaxant effect of adrenomedullin, proadrenomedullin N-terminal 20 peptide and amylin in human skin

    DEFF Research Database (Denmark)

    Hasbak, Philip; Eskesen, Karen; Lind, Peter Henrik

    2006-01-01

    ) and substance P and to examine the mRNA expression of calcitonin receptor-like receptor (CL-R) and receptor-activity modifying proteins, RAMP1, RAMP 2 and RAMP3 in human subcutaneous arteries. Changes in skin blood flow of the forearm were measured using a Laser Doppler Imager after intradermal injection......In this study we aimed to assess in vivo, the vasodilator effects of adrenomedullin, proadrenomedullin N-terminal 20 peptide (PAMP) and amylin in human skin vasculature and compare the responses to the effects mediated by the endogenous neuropeptides calcitonin gene-related peptide (CGRP...... of the peptides. The mRNA expression was assessed by real-time reverse transcriptase-polymerase chain reaction (real-time PCR). CGRP, adrenomedullin and amylin induced concentration-dependent, long-lasting increases in skin blood flow. The response to PAMP was shorter in duration appearing similar...

  8. Site-directed fluorescence labeling reveals a revised N-terminal membrane topology and functional periplasmic residues in the Escherichia coli cell division protein FtsK.

    Science.gov (United States)

    Berezuk, Alison M; Goodyear, Mara; Khursigara, Cezar M

    2014-08-22

    In Escherichia coli, FtsK is a large integral membrane protein that coordinates chromosome segregation and cell division. The N-terminal domain of FtsK (FtsKN) is essential for division, and the C terminus (FtsKC) is a well characterized DNA translocase. Although the function of FtsKN is unknown, it is suggested that FtsK acts as a checkpoint to ensure DNA is properly segregated before septation. This may occur through modulation of protein interactions between FtsKN and other division proteins in both the periplasm and cytoplasm; thus, a clear understanding of how FtsKN is positioned in the membrane is required to characterize these interactions. The membrane topology of FtsKN was initially determined using site-directed reporter fusions; however, questions regarding this topology persist. Here, we report a revised membrane topology generated by site-directed fluorescence labeling. The revised topology confirms the presence of four transmembrane segments and reveals a newly identified periplasmic loop between the third and fourth transmembrane domains. Within this loop, four residues were identified that, when mutated, resulted in the appearance of cellular voids. High resolution transmission electron microscopy of these voids showed asymmetric division of the cytoplasm in the absence of outer membrane invagination or visible cell wall ingrowth. This uncoupling reveals a novel role for FtsK in linking cell envelope septation events and yields further evidence for FtsK as a critical checkpoint of cell division. The revised topology of FtsKN also provides an important platform for future studies on essential interactions required for this process. © 2014 by The American Society for Biochemistry and Molecular Biology, Inc.

  9. Crystallization of Galectin-8 Linker Reveals Intricate Relationship between the N-terminal Tail and the Linker

    Directory of Open Access Journals (Sweden)

    Yunlong Si

    2016-12-01

    Full Text Available Galectin-8 (Gal-8 plays a significant role in normal immunological function as well as in cancer. This lectin contains two carbohydrate recognition domains (CRD connected by a peptide linker. The N-terminal CRD determines ligand binding specificity, whereas the linker has been proposed to regulate overall Gal-8 function, including multimerization and biological activity. Here, we crystallized the Gal-8 N-terminal CRD with the peptide linker using a crystallization condition that contains Ni2+. The Ni2+ ion was found to be complexed between two CRDs via crystal packing contacts. The coordination between Ni2+ and Asp25 plays an indirect role in determining the structure of β-strand F0 and in influencing the linker conformation which could not be defined due to its dynamic nature. The linker was also shortened in situ and crystallized under a different condition, leading to a higher resolution structure refined to 1.08 Å. This crystal structure allowed definition of a short portion of the linker interacting with the Gal-8 N-terminal tail via ionic interactions and hydrogen bonds. Observation of two Gal-8 N-terminal CRD structures implies that the N-terminal tail and the linker may influence each other’s conformation. In addition, under specific crystallization conditions, glycerol could replace lactose and was observed at the carbohydrate binding site. However, glycerol did not show inhibition activity in hemagglutination assay.

  10. Antimicrobial activity of human prion protein is mediated by its N-terminal region.

    Directory of Open Access Journals (Sweden)

    Mukesh Pasupuleti

    Full Text Available BACKGROUND: Cellular prion-related protein (PrP(c is a cell-surface protein that is ubiquitously expressed in the human body. The multifunctionality of PrP(c, and presence of an exposed cationic and heparin-binding N-terminus, a feature characterizing many antimicrobial peptides, made us hypothesize that PrP(c could exert antimicrobial activity. METHODOLOGY AND PRINCIPAL FINDINGS: Intact recombinant PrP exerted antibacterial and antifungal effects at normal and low pH. Studies employing recombinant PrP and N- and C-terminally truncated variants, as well as overlapping peptide 20mers, demonstrated that the antimicrobial activity is mediated by the unstructured N-terminal part of the protein. Synthetic peptides of the N-terminus of PrP killed the Gram-negative bacteria Escherichia coli and Pseudomonas aeruginosa, and the Gram-positive Bacillus subtilis and Staphylococcus aureus, as well as the fungus Candida parapsilosis. Fluorescence studies of peptide-treated bacteria, paired with analysis of peptide effects on liposomes, showed that the peptides exerted membrane-breaking effects similar to those seen after treatment with the "classical" human antimicrobial peptide LL-37. In contrast to LL-37, however, no marked helix induction was detected for the PrP-derived peptides in presence of negatively charged (bacteria-mimicking liposomes. PrP furthermore showed an inducible expression during wounding of human skin ex vivo and in vivo, as well as stimulation of keratinocytes with TGF-alpha in vitro. CONCLUSIONS: The demonstration of an antimicrobial activity of PrP, localisation of its activity to the N-terminal and heparin-binding region, combined with results showing an increased expression of PrP during wounding, indicate that PrPs could have a previously undisclosed role in host defense.

  11. Unusual chemical properties of N-terminal histidine residues of glucagon and vasoactive intestinal peptide

    International Nuclear Information System (INIS)

    Hefford, M.A.; Evans, R.M.; Oda, G.; Kaplan, H.

    1985-01-01

    An N-terminal histidine residue of a protein or peptide has two functional groups, viz., an alpha-amino group and an imidazole group. A new procedure, based on the competitive labeling approach described by Duggleby and Kaplan has been developed by which the chemical reactivity of each functional group in such a residue can be determined as a function of pH. Only very small amounts of material are required, which makes it possible to determine the chemical properties in dilute solution or in proteins and polypeptides that can be obtained in only minute quantities. With this approach, the reactivity of the alpha-amino group of histidylglycine toward 1-fluoro-2,4-dinitrobenzene gave an apparent pK /sub a/ value of 7.64 +/- 0.07 at 37 degrees C, in good agreement with a value of 7.69 +/- 0.02 obtained by acid-base titration. However, the reactivity of the imidazole function gave an apparent pK /sub a/ value of 7.16 +/- 0.07 as compared to the pK /sub a/ value of 5.85 +/- 0.01 obtained by acid-base titration. Similarly, in glucagon and vasoactive intestinal peptide (VIP), apparent pKa values of 7.60 +/- 0.04 and 7.88 +/- 0.18, respectively, were obtained for the alpha-amino of their N-terminal histidine, and pKa values of 7.43 +/- 0.09 and 7.59 +/- 0.18 were obtained for the imidazole function

  12. NMR and structural data for Connexin 32 and Connexin 26 N-terminal peptides

    Directory of Open Access Journals (Sweden)

    Yuksel Batir

    2016-12-01

    Full Text Available In this article we present 1H and 13C chemical shift assignments, secondary structural propensity data and normalized temperature coefficient data for N-terminal peptides of Connexin 26 (Cx26, Cx26G12R and Cx32G12R mutants seen in syndromic deafness and Charcot Marie Tooth Disease respectively, published in “Structural Studies of N-Terminal Mutants of Connexin 26 and Connexin 32 Using 1H NMR Spectroscopy” (Y. Batir, T.A. Bargiello, T.L. Dowd, 2016 [1]. The mutation G12R affects the structure of both Cx26 and Cx32 peptides differently. We present data from secondary structure propensity chemical shift analysis which calculates a secondary structure propensity (SSP score for both disordered or folded peptides and proteins using the difference between the 13C secondary chemical shifts of the Cα and Cβ protons. This data supplements the calculated NMR structures from NOESY data [1]. We present and compare the SSP data for the Cx26 vs Cx26G12R peptides and the Cx32 and Cx32G12R peptides. In addition, we present plots of temperature coefficients obtained for Cx26, Cx26G12R and Cx32G12R peptides collected previously [1] and normalized to their random coil temperature coefficients, “Random coil 1H chemical shifts obtained as a function of temperature and trifluoroethanol concentration for the peptide series GGXGG” (G. Merutka, H.J. Dyson, P.E. Wright, 1995 [2]. Reductions in these normalized temperature coefficients are directly observable for residues in different segments of the peptide and this data informs on solvent accessibility of the NH protons and NH protons which may be more constrained due to the formation of H bonds.

  13. Correlation between spina bifida manifesta in fetal rats and c-Jun N-terminal kinase signaling★

    Science.gov (United States)

    Ma, Yinghuan; Bao, Yongxin; Li, Chenghao; Jiao, Fubin; Xin, Hongjie; Yuan, Zhengwei

    2012-01-01

    Fetal rat models with neural tube defects were established by injection with retinoic acid at 10 days after conception. The immunofluorescence assay and western blot analysis showed that the number of caspase-3 positive cells in myeloid tissues for spina bifida manifesta was increased. There was also increased phosphorylation of c-Jun N-terminal kinase, a member of the mitogen activated protein kinase family. The c-Jun N-terminal kinase phosphorylation level was positively correlated with caspase-3 expression in myeloid tissues for spina bifida manifesta. Experimental findings indicate that abnormal apoptosis is involved in retinoic acid-induced dominant spina bifida formation in fetal rats, and may be associated with the c-Jun N-terminal kinase signal transduction pathway. PMID:25337099

  14. Intrinsic structural differences in the N-terminal segment of pulmonary surfactant protein SP-C from different species

    DEFF Research Database (Denmark)

    Plasencia, I; Rivas, L; Casals, C

    2001-01-01

    Predictive studies suggest that the known sequences of the N-terminal segment of surfactant protein SP-C from animal species have an intrinsic tendency to form beta-turns, but there are important differences on the probable location of these motifs in different SP-C species. Our hypothesis...... is that intrinsic structural determinants of the sequence of the N-terminal region of SP-C could define conformation, acylation and perhaps surface properties of the mature protein. To test this hypothesis we have synthesized peptides corresponding to the 13-residue N-terminal sequence of porcine and canine SP......-C, and studied their structural behaviour in solution and in phospholipid bilayers and monolayers. In these peptides, leucine at position 1 of both sequences has been replaced by tryptophan in order to allow their study by fluorescence spectroscopy. Far-u.v. circular dichroism spectra of the peptides in aqueous...

  15. Variations in the cytoplasmic region account for the heterogeneity of the chicken MHC class I (B-F) molecules

    DEFF Research Database (Denmark)

    Møller, L B; Kaufman, J; Verland, S

    1991-01-01

    . Unlike the parent proteins, the Mr 36,000 fragment derived from isolated variants yielded identical, simple patterns in two-dimensional gel electrophoresis and identical finger prints in peptide mapping. This, together with N-terminal amino acid sequencing, as well as comparison of hydrophobicity...... comprised by several isoelectric focusing variants. This heterogeneity could not be reduced by enzymatic deglycosylation. By contrast, proteolytic removal of a small (Mr 1000-4000) fragment from the alpha chain resulted in the generation of a Mr 36,000 fragment, common to all the molecular mass variants...

  16. Redox-linked Gating of Nucleotide Binding by the N-terminal Domain of Adenosine 5′-Phosphosulfate Kinase*

    Science.gov (United States)

    Ravilious, Geoffrey E.; Westfall, Corey S.; Jez, Joseph M.

    2013-01-01

    Adenosine 5′-phosphosulfate kinase (APSK) catalyzes the phosphorylation of adenosine 5′-phosphosulfate (APS) to 3′-phosphoadenosine-5′-phosphosulfate (PAPS). Crystallographic studies of APSK from Arabidopsis thaliana revealed the presence of a regulatory intersubunit disulfide bond (Cys86–Cys119). The reduced enzyme displayed improved catalytic efficiency and decreased effectiveness of substrate inhibition by APS compared with the oxidized form. Here we examine the effect of disulfide formation and the role of the N-terminal domain on nucleotide binding using isothermal titration calorimetry (ITC) and steady-state kinetics. Formation of the disulfide bond in A. thaliana APSK (AtAPSK) inverts the binding affinities at the ATP/ADP and APS/PAPS sites from those observed in the reduced enzyme, consistent with initial binding of APS as inhibitory, and suggests a role for the N-terminal domain in guiding nucleotide binding order. To test this, an N-terminal truncation variant (AtAPSKΔ96) was generated. The resulting protein was completely insensitive to substrate inhibition by APS. ITC analysis of AtAPSKΔ96 showed decreased affinity for APS binding, although the N-terminal domain does not directly interact with this ligand. Moreover, AtAPSKΔ96 displayed reduced affinity for ADP, which corresponds to a loss of substrate inhibition by formation of an E·ADP·APS dead end complex. Examination of the AtAPSK crystal structure suggested Arg93 as important for positioning of the N-terminal domain. ITC and kinetic analysis of the R93A mutant also showed a complete loss of substrate inhibition and altered nucleotide binding affinities, which mimics the effect of the N-terminal deletion. These results show how thiol-linked changes in AtAPSK alter the energetics of binding equilibria to control its activity. PMID:23322773

  17. Intrinsic structural differences in the N-terminal segment of pulmonary surfactant protein SP-C from different species

    DEFF Research Database (Denmark)

    Plasencia, I; Rivas, L; Casals, C

    2001-01-01

    is that intrinsic structural determinants of the sequence of the N-terminal region of SP-C could define conformation, acylation and perhaps surface properties of the mature protein. To test this hypothesis we have synthesized peptides corresponding to the 13-residue N-terminal sequence of porcine and canine SP...... the packing of dipalmitoylphosphatidylcholine (DPPC) and dipalmitoylphosphatidylglycerol (DPPG) monolayers, the effects being always higher in anionic than in zwitterionic lipids, and also substantially higher in films containing canine peptide in comparison to porcine peptide. Acylation of cysteines at the N...

  18. Altering the N-terminal arms of the polymerase manager protein UmuD modulates protein interactions.

    Directory of Open Access Journals (Sweden)

    David A Murison

    Full Text Available Escherichia coli cells that are exposed to DNA damaging agents invoke the SOS response that involves expression of the umuD gene products, along with more than 50 other genes. Full-length UmuD is expressed as a 139-amino-acid protein, which eventually cleaves its N-terminal 24 amino acids to form UmuD'. The N-terminal arms of UmuD are dynamic and contain recognition sites for multiple partner proteins. Cleavage of UmuD to UmuD' dramatically affects the function of the protein and activates UmuC for translesion synthesis (TLS by forming DNA Polymerase V. To probe the roles of the N-terminal arms in the cellular functions of the umuD gene products, we constructed additional N-terminal truncated versions of UmuD: UmuD 8 (UmuD Δ1-7 and UmuD 18 (UmuD Δ1-17. We found that the loss of just the N-terminal seven (7 amino acids of UmuD results in changes in conformation of the N-terminal arms, as determined by electron paramagnetic resonance spectroscopy with site-directed spin labeling. UmuD 8 is cleaved as efficiently as full-length UmuD in vitro and in vivo, but expression of a plasmid-borne non-cleavable variant of UmuD 8 causes hypersensitivity to UV irradiation, which we determined is the result of a copy-number effect. UmuD 18 does not cleave to form UmuD', but confers resistance to UV radiation. Moreover, removal of the N-terminal seven residues of UmuD maintained its interactions with the alpha polymerase subunit of DNA polymerase III as well as its ability to disrupt interactions between alpha and the beta processivity clamp, whereas deletion of the N-terminal 17 residues resulted in decreases in binding to alpha and in the ability to disrupt the alpha-beta interaction. We find that UmuD 8 mimics full-length UmuD in many respects, whereas UmuD 18 lacks a number of functions characteristic of UmuD.

  19. Nuclear fragmentation

    International Nuclear Information System (INIS)

    Chung, K.C.

    1989-01-01

    An introduction to nuclear fragmentation, with emphasis in percolation ideas, is presented. The main theoretical models are discussed and as an application, the uniform expansion approximation is presented and the statistical multifragmentation model is used to calculate the fragment energy spectra. (L.C.)

  20. N-terminal amino acid sequence of Bacillus licheniformis alpha-amylase: comparison with Bacillus amyloliquefaciens and Bacillus subtilis Enzymes.

    OpenAIRE

    Kuhn, H; Fietzek, P P; Lampen, J O

    1982-01-01

    The thermostable, liquefying alpha-amylase from Bacillus licheniformis was immunologically cross-reactive with the thermolabile, liquefying alpha-amylase from Bacillus amyloliquefaciens. Their N-terminal amino acid sequences showed extensive homology with each other, but not with the saccharifying alpha-amylases of Bacillus subtilis.

  1. X-ray vs. NMR structure of N-terminal domain of delta-subunit of RNA polymerase

    Czech Academy of Sciences Publication Activity Database

    Demo, G.; Papoušková, V.; Komárek, J.; Kadeřávek, P.; Otrusinová, O.; Srb, P.; Rabatinová, Alžběta; Krásný, Libor; Žídek, L.; Sklenář, V.; Wimmerová, M.

    2014-01-01

    Roč. 187, č. 2 (2014), s. 174-186 ISSN 1047-8477 R&D Projects: GA ČR GA13-16842S Institutional support: RVO:61388971 Keywords : Protein crystallography * Nuclear magnetic resonance * N-terminal domain Subject RIV: EE - Microbiology, Virology Impact factor: 3.231, year: 2014

  2. 1H, 13C, and 15N resonance assignments of the N-terminal domain of human TIG3.

    Science.gov (United States)

    Wang, Lei; Yu, Wenyu; Ren, Xiaobai; Lin, Jian; Jin, Changwen; Xia, Bin

    2012-10-01

    Human TIG3 protein is a member of H-REV107 protein family which belongs to the type II tumor suppressor family. TIG3 can induce apoptosis in cancer cells, and it also possesses Ca(2+)-independent phospholipase A(1/2) activity. The NMR assignments of the N-terminal domain of TIG3 are essential for its solution structure determination.

  3. Cutting edge: HLA-B27 acquires many N-terminal dibasic peptides: coupling cytosolic peptide stability to antigen presentation

    NARCIS (Netherlands)

    Herberts, Carla A.; Neijssen, Joost J.; de Haan, Jolanda; Janssen, Lennert; Drijfhout, Jan Wouter; Reits, Eric A.; Neefjes, Jacques J.

    2006-01-01

    Ag presentation by MHC class I is a highly inefficient process because cytosolic peptidases destroy most peptides after proteasomal generation. Various mechanisms shape the MHC class I peptidome. We define a new one: intracellular peptide stability. Peptides with two N-terminal basic amino acids are

  4. N-terminal propeptide of type III procollagen as a biomarker of anabolic response to recombinant human GH and testosterone

    Science.gov (United States)

    Context: Biomarkers that predict musculoskeletal response to anabolic therapies should expedite drug development. During collagen synthesis in soft lean tissue, N-terminal propeptide of type III procollagen (P3NP) is released into circulation. We investigated P3NP as a biomarker of lean body mass (L...

  5. FUNCTIONAL-ANALYSIS OF THE N-TERMINAL PREPEPTIDES OF WATERMELON MITOCHONDRIAL AND GLYOXYSOMAL MALATE-DEHYDROGENASES

    NARCIS (Netherlands)

    LEHNERER, M; KEIZERGUNNIK, [No Value; VEENHUIS, M; GIETL, C

    1994-01-01

    Mitochondrial and glyoxysomal malate dehydrogenase (mMDH; gMDH; L-malate : NAD(+) oxidoreductase; EC 1.1.1.37) of watermelon (Citrullus vulgaris) cotyledons are synthesized with N-terminal cleavable presequences which are shown to specify sorting of the two proteins. The two presequences differ in

  6. Mutational analysis of the N-terminal topogenic signal of watermelon glyoxysomal malate dehydrogenase using the heterologous host Hansenula polymorpha

    NARCIS (Netherlands)

    Gietl, Christine; Faber, Klaas Nico; Klei, Ida J. van der; Veenhuis, Marten

    1994-01-01

    We have studied the significance of the N-terminal presequence of watermelon (Citrullus vulgaris) glyoxysomal malate dehydrogenase [gMDH; (S)-malate:NAD+ oxidoreductase; EC 1.1.1.37] in microbody targeting. The yeast Hansenula polymorpha was used as heterologous host for the in vivo expression of

  7. Functional and structural characterization of a synthetic peptide representing the N-terminal domain of prokaryotic pyruvate dehydrogenase

    NARCIS (Netherlands)

    Hengeveld, A.F.; Mierlo, van C.P.M.; Hooven, van den H.W.; Visser, A.J.W.G.; Kok, de A.

    2002-01-01

    A synthetic peptide (Nterm-E1p) is used to characterize the structure and function of the N-terminal region (amino acid residues 4-45) of the pyruvate dehydrogenase component (E1p) from the pyruvate dehydrogenase multienzyme complex (PDHC) from Azotobacter vinelandii. Activity and binding studies

  8. Glutamate dehydrogenase isoforms with N-terminal (His)6- or FLAG-tag retain their kinetic properties and cellular localization

    DEFF Research Database (Denmark)

    Pajęcka, Kamilla; Nielsen, Camilla Wendel; Hauge, Anne

    2014-01-01

    containing N-terminal (His)6 tags were successfully expressed in Sf9 cells and the recombinant proteins were isolated to ≥95 % purity in a two-step procedure involving ammonium sulfate precipitation and Ni(2+)-based immobilized metal ion affinity chromatography. To explore whether the presence of the FLAG...

  9. N-terminal amino acid sequence of Bacillus licheniformis alpha-amylase: comparison with Bacillus amyloliquefaciens and Bacillus subtilis Enzymes.

    Science.gov (United States)

    Kuhn, H; Fietzek, P P; Lampen, J O

    1982-01-01

    The thermostable, liquefying alpha-amylase from Bacillus licheniformis was immunologically cross-reactive with the thermolabile, liquefying alpha-amylase from Bacillus amyloliquefaciens. Their N-terminal amino acid sequences showed extensive homology with each other, but not with the saccharifying alpha-amylases of Bacillus subtilis. PMID:6172418

  10. Elevated plasma N-terminal ProBNP levels in unmedicated patients with major depressive disorder.

    Science.gov (United States)

    Politi, Pierluigi; Minoretti, Piercarlo; Piaggi, Noemi; Brondino, Natascia; Emanuele, Enzo

    2007-05-07

    There is considerable evidence that cardiovascular diseases are more prevalent in patients with major depressive disorder (MDD). Secretion of N-terminal pro-B-type natriuretic peptide (NT-proBNP) increases in several cardiac illnesses, making this neurohormone a reliable diagnostic and prognostic biomarker of cardiovascular risk. We measured plasma NT-proBNP levels in the following three groups of subjects free of overt cardiovascular disease: unmedicated patients with MDD (n=40), unmedicated patients with schizophrenia (n=44), and normal control subjects (n=42). The severity of depressive symptoms was rated using the Hamilton Depression Rating Scale (HAMD). Plasma NT-proBNP levels were assayed by ELISA. Plasma NT-proBNP levels were significantly higher in the MDD group (median: 217.1 pmol/L; interquartile range: 179.4-277.1 pmol/L) than in patients with schizophrenia (175.7 pmol/L [139.0-218.9]; P<0.05) or in the control group (158.9 pmol/L [98.3-212.1]; P<0.001). Among patients with MDD, there was a significant positive correlation (Spearman's rank correlation=0.422, P=0.008) between plasma NT-proBNP and HAMD scores. Altogether, our results indicate that elevated NT-proBNP levels may play a role in linking MDD with increased cardiovascular risk.

  11. Solution structure of Atg8 reveals conformational polymorphism of the N-terminal domain

    Energy Technology Data Exchange (ETDEWEB)

    Schwarten, Melanie, E-mail: m.schwarten@fz-juelich.de [Institut fuer Strukturbiologie und Biophysik, ISB-3, Forschungszentrum Juelich, 52425 Juelich (Germany); Institut fuer Physikalische Biologie und BMFZ, Heinrich-Heine-Universitaet Duesseldorf, 40225 Duesseldorf (Germany); Stoldt, Matthias, E-mail: m.stoldt@fz-juelich.de [Institut fuer Strukturbiologie und Biophysik, ISB-3, Forschungszentrum Juelich, 52425 Juelich (Germany); Institut fuer Physikalische Biologie und BMFZ, Heinrich-Heine-Universitaet Duesseldorf, 40225 Duesseldorf (Germany); Mohrlueder, Jeannine, E-mail: j.mohrlueder@fz-juelich.de [Institut fuer Strukturbiologie und Biophysik, ISB-3, Forschungszentrum Juelich, 52425 Juelich (Germany); Willbold, Dieter, E-mail: dieter.willbold@uni-duesseldorf.de [Institut fuer Strukturbiologie und Biophysik, ISB-3, Forschungszentrum Juelich, 52425 Juelich (Germany); Institut fuer Physikalische Biologie und BMFZ, Heinrich-Heine-Universitaet Duesseldorf, 40225 Duesseldorf (Germany)

    2010-05-07

    During autophagy a crescent shaped like membrane is formed, which engulfs the material that is to be degraded. This membrane grows further until its edges fuse to form the double membrane covered autophagosome. Atg8 is a protein, which is required for this initial step of autophagy. Therefore, a multistage conjugation process of newly synthesized Atg8 to phosphatidylethanolamine is of critical importance. Here we present the high resolution structure of unprocessed Atg8 determined by nuclear magnetic resonance spectroscopy. Its C-terminal subdomain shows a well-defined ubiquitin-like fold with slightly elevated mobility in the pico- to nanosecond timescale as determined by heteronuclear NOE data. In comparison to unprocessed Atg8, cleaved Atg8{sup G116} shows a decreased mobility behaviour. The N-terminal domain adopts different conformations within the micro- to millisecond timescale. The possible biological relevance of the differences in dynamic behaviours between both subdomains as well as between the cleaved and uncleaved forms is discussed.

  12. N-terminal truncated UCH-L1 prevents Parkinson's disease associated damage.

    Directory of Open Access Journals (Sweden)

    Hee-Jung Kim

    Full Text Available Ubiquitin C-terminal hydrolase-L1 (UCH-L1 has been proposed as one of the Parkinson's disease (PD related genes, but the possible molecular connection between UCH-L1 and PD is not well understood. In this study, we discovered an N-terminal 11 amino acid truncated variant UCH-L1 that we called NT-UCH-L1, in mouse brain tissue as well as in NCI-H157 lung cancer and SH-SY5Y neuroblastoma cell lines. In vivo experiments and hydrogen-deuterium exchange (HDX with tandem mass spectrometry (MS studies showed that NT-UCH-L1 is readily aggregated and degraded, and has more flexible structure than UCH-L1. Post-translational modifications including monoubiquitination and disulfide crosslinking regulate the stability and cellular localization of NT-UCH-L1, as confirmed by mutational and proteomic studies. Stable expression of NT-UCH-L1 decreases cellular ROS levels and protects cells from H2O2, rotenone and CCCP-induced cell death. NT-UCH-L1-expressing transgenic mice are less susceptible to degeneration of nigrostriatal dopaminergic neurons seen in the MPTP mouse model of PD, in comparison to control animals. These results suggest that NT-UCH-L1 may have the potential to prevent neural damage in diseases like PD.

  13. N-Terminal-Based Targeted, Inducible Protein Degradation in Escherichia coli.

    Directory of Open Access Journals (Sweden)

    Karthik Sekar

    Full Text Available Dynamically altering protein concentration is a central activity in synthetic biology. While many tools are available to modulate protein concentration by altering protein synthesis rate, methods for decreasing protein concentration by inactivation or degradation rate are just being realized. Altering protein synthesis rates can quickly increase the concentration of a protein but not decrease, as residual protein will remain for a while. Inducible, targeted protein degradation is an attractive option and some tools have been introduced for higher organisms and bacteria. Current bacterial tools rely on C-terminal fusions, so we have developed an N-terminal fusion (Ntag strategy to increase the possible proteins that can be targeted. We demonstrate Ntag dependent degradation of mCherry and beta-galactosidase and reconfigure the Ntag system to perform dynamic, exogenously inducible degradation of a targeted protein and complement protein depletion by traditional synthesis repression. Model driven analysis that focused on rates, rather than concentrations, was critical to understanding and engineering the system. We expect this tool and our model to enable inducible protein degradation use particularly in metabolic engineering, biological study of essential proteins, and protein circuits.

  14. NMR structure of the N-terminal domain of the replication initiator protein DnaA

    Energy Technology Data Exchange (ETDEWEB)

    Wemmer, David E.; Lowery, Thomas J.; Pelton, Jeffrey G.; Chandonia, John-Marc; Kim, Rosalind; Yokota, Hisao; Wemmer, David E.

    2007-08-07

    DnaA is an essential component in the initiation of bacterial chromosomal replication. DnaA binds to a series of 9 base pair repeats leading to oligomerization, recruitment of the DnaBC helicase, and the assembly of the replication fork machinery. The structure of the N-terminal domain (residues 1-100) of DnaA from Mycoplasma genitalium was determined by NMR spectroscopy. The backbone r.m.s.d. for the first 86 residues was 0.6 +/- 0.2 Angstrom based on 742 NOE, 50 hydrogen bond, 46 backbone angle, and 88 residual dipolar coupling restraints. Ultracentrifugation studies revealed that the domain is monomeric in solution. Features on the protein surface include a hydrophobic cleft flanked by several negative residues on one side, and positive residues on the other. A negatively charged ridge is present on the opposite face of the protein. These surfaces may be important sites of interaction with other proteins involved in the replication process. Together, the structure and NMR assignments should facilitate the design of new experiments to probe the protein-protein interactions essential for the initiation of DNA replication.

  15. Tor forms a dimer through an N-terminal helical solenoid with a complex topology

    Science.gov (United States)

    Baretić, Domagoj; Berndt, Alex; Ohashi, Yohei; Johnson, Christopher M.; Williams, Roger L.

    2016-04-01

    The target of rapamycin (Tor) is a Ser/Thr protein kinase that regulates a range of anabolic and catabolic processes. Tor is present in two complexes, TORC1 and TORC2, in which the Tor-Lst8 heterodimer forms a common sub-complex. We have determined the cryo-electron microscopy (EM) structure of Tor bound to Lst8. Two Tor-Lst8 heterodimers assemble further into a dyad-symmetry dimer mediated by Tor-Tor interactions. The first 1,300 residues of Tor form a HEAT repeat-containing α-solenoid with four distinct segments: a highly curved 800-residue N-terminal 'spiral', followed by a 400-residue low-curvature 'bridge' and an extended `railing' running along the bridge leading to the 'cap' that links to FAT region. This complex topology was verified by domain insertions and offers a new interpretation of the mTORC1 structure. The spiral of one TOR interacts with the bridge of another, which together form a joint platform for the Regulatory Associated Protein of TOR (RAPTOR) regulatory subunit.

  16. PrP N-terminal domain triggers PrP(Sc)-like aggregation of Dpl.

    Science.gov (United States)

    Erlich, Paul; Cesbron, Jean-Yves; Lemaire-Vieille, Catherine; Curt, Aurélie; Andrieu, Jean-Pierre; Schoehn, Guy; Jamin, Marc; Gagnon, Jean

    2008-01-18

    Transmissible spongiform encephalopathies are fatal neurodegenerative disorders thought to be transmitted by self-perpetuating conformational conversion of a neuronal membrane glycoprotein (PrP(C), for "cellular prion protein") into an abnormal state (PrP(Sc), for "scrapie prion protein"). Doppel (Dpl) is a protein that shares significant biochemical and structural homology with PrP(C). In contrast to its homologue PrP(C), Dpl is unable to participate in prion disease progression or to achieve an abnormal PrP(Sc)-like state. We have constructed a chimeric mouse protein, composed of the N-terminal domain of PrP(C) (residues 23-125) and the C-terminal part of Dpl (residues 58-157). This chimeric protein displays PrP-like biochemical and structural features; when incubated in presence of NaCl, the alpha-helical monomer forms soluble beta-sheet-rich oligomers which acquire partial resistance to pepsin proteolysis in vitro, as do PrP oligomers. Moreover, the presence of aggregates akin to protofibrils is observed in soluble oligomeric species by electron microscopy.

  17. Structure of the N-terminal region of Haemophilus Influenzae HI0017: Implications for function

    Energy Technology Data Exchange (ETDEWEB)

    Yu Liping; Mack, Jamey; Hajduk, Phil; Fesik, Stephen W. [Abbott Laboratories, Pharmaceutical Discovery Division, D46Y, AP10/LL (United States)

    2001-06-15

    Haemophilus influenzae is a gram-negative pathogen that causes infections ranging from asymptomatic colonization of the human upper respiratory tract to serious invasive diseases such as meningitis. Although the genome of Haemophilus influenzae has been completely sequenced, the structure and function of many of these proteins are unknown. HI0017 is one of these uncharacterized proteins. Here we describe the three-dimensional solution structure of the N-terminal portion of HI0017 as determined by NMR spectroscopy. The structure consists of a five-stranded antiparallel {beta}-sheet and two short {alpha}-helices. It is similar to the C-terminal domain of Diphtheria toxin repressor (DtxR). The C-terminal portion of HI0017 has an amino acid sequence that closely resembles pyruvate formate-lyase - an enzyme that converts pyruvate and CoA into acetyl-CoA and formate by a radical mechanism. Based on structural and sequence comparisons, we propose that the C-terminus of HI0017 functions as an enzyme with a glycyl radical mechanism, while the N-terminus participates in protein/protein interactions involving an activase (iron-sulfur protein) and/or the substrate.

  18. Structure of the N-terminal region of Haemophilus Influenzae HI0017: Implications for function

    International Nuclear Information System (INIS)

    Yu Liping; Mack, Jamey; Hajduk, Phil; Fesik, Stephen W.

    2001-01-01

    Haemophilus influenzae is a gram-negative pathogen that causes infections ranging from asymptomatic colonization of the human upper respiratory tract to serious invasive diseases such as meningitis. Although the genome of Haemophilus influenzae has been completely sequenced, the structure and function of many of these proteins are unknown. HI0017 is one of these uncharacterized proteins. Here we describe the three-dimensional solution structure of the N-terminal portion of HI0017 as determined by NMR spectroscopy. The structure consists of a five-stranded antiparallel β-sheet and two short α-helices. It is similar to the C-terminal domain of Diphtheria toxin repressor (DtxR). The C-terminal portion of HI0017 has an amino acid sequence that closely resembles pyruvate formate-lyase - an enzyme that converts pyruvate and CoA into acetyl-CoA and formate by a radical mechanism. Based on structural and sequence comparisons, we propose that the C-terminus of HI0017 functions as an enzyme with a glycyl radical mechanism, while the N-terminus participates in protein/protein interactions involving an activase (iron-sulfur protein) and/or the substrate

  19. Identification of evolutionarily conserved non-AUG-initiated N-terminal extensions in human coding sequences.

    LENUS (Irish Health Repository)

    Ivanov, Ivaylo P

    2011-05-01

    In eukaryotes, it is generally assumed that translation initiation occurs at the AUG codon closest to the messenger RNA 5\\' cap. However, in certain cases, initiation can occur at codons differing from AUG by a single nucleotide, especially the codons CUG, UUG, GUG, ACG, AUA and AUU. While non-AUG initiation has been experimentally verified for a handful of human genes, the full extent to which this phenomenon is utilized--both for increased coding capacity and potentially also for novel regulatory mechanisms--remains unclear. To address this issue, and hence to improve the quality of existing coding sequence annotations, we developed a methodology based on phylogenetic analysis of predicted 5\\' untranslated regions from orthologous genes. We use evolutionary signatures of protein-coding sequences as an indicator of translation initiation upstream of annotated coding sequences. Our search identified novel conserved potential non-AUG-initiated N-terminal extensions in 42 human genes including VANGL2, FGFR1, KCNN4, TRPV6, HDGF, CITED2, EIF4G3 and NTF3, and also affirmed the conservation of known non-AUG-initiated extensions in 17 other genes. In several instances, we have been able to obtain independent experimental evidence of the expression of non-AUG-initiated products from the previously published literature and ribosome profiling data.

  20. Mcm10 self-association is mediated by an N-terminal coiled-coil domain.

    Directory of Open Access Journals (Sweden)

    Wenyue Du

    Full Text Available Minichromosome maintenance protein 10 (Mcm10 is an essential eukaryotic DNA-binding replication factor thought to serve as a scaffold to coordinate enzymatic activities within the replisome. Mcm10 appears to function as an oligomer rather than in its monomeric form (or rather than as a monomer. However, various orthologs have been found to contain 1, 2, 3, 4, or 6 subunits and thus, this issue has remained controversial. Here, we show that self-association of Xenopus laevis Mcm10 is mediated by a conserved coiled-coil (CC motif within the N-terminal domain (NTD. Crystallographic analysis of the CC at 2.4 Å resolution revealed a three-helix bundle, consistent with the formation of both dimeric and trimeric Mcm10 CCs in solution. Mutation of the side chains at the subunit interface disrupted in vitro dimerization of both the CC and the NTD as monitored by analytical ultracentrifugation. In addition, the same mutations also impeded self-interaction of the full-length protein in vivo, as measured by yeast-two hybrid assays. We conclude that Mcm10 likely forms dimers or trimers to promote its diverse functions during DNA replication.

  1. Autocatalytic activity and substrate specificity of the pestivirus N-terminal protease Npro

    International Nuclear Information System (INIS)

    Gottipati, Keerthi; Acholi, Sudheer; Ruggli, Nicolas; Choi, Kyung H.

    2014-01-01

    Pestivirus N pro is the first protein translated in the viral polypeptide, and cleaves itself off co-translationally generating the N-terminus of the core protein. Once released, N pro blocks the host's interferon response by inducing degradation of interferon regulatory factor-3. N pro' s intracellular autocatalytic activity and lack of trans-activity have hampered in vitro cleavage studies to establish its substrate specificity and the roles of individual residues. We constructed N pro -GFP fusion proteins that carry the authentic cleavage site and determined the autoproteolytic activities of N pro proteins containing substitutions at the predicted catalytic sites Glu22 and Cys69, at Arg100 that forms a salt bridge with Glu22, and at the cleavage site Cys168. Contrary to previous reports, we show that N pro' s catalytic activity does not involve Glu22, which may instead be involved in protein stability. Furthermore, N pro does not have specificity for Cys168 at the cleavage site even though this residue is conserved throughout the pestivirus genus. - Highlights: • N pro' s autoproteolysis is studied using N pro -GFP fusion proteins. • N-terminal 17 amino acids are dispensable without loss of protease activity. • The putative catalytic residue Glu22 is not involved in protease catalysis. • No specificity for Cys168 at the cleavage site despite evolutionary conservation. • N pro prefers small amino acids with non-branched beta carbons at the P1 position

  2. Bifunctional anti-huntingtin proteasome-directed intrabodies mediate efficient degradation of mutant huntingtin exon 1 protein fragments.

    Directory of Open Access Journals (Sweden)

    David C Butler

    Full Text Available Huntington's disease (HD is a fatal autosomal dominant neurodegenerative disorder caused by a trinucleotide (CAG(n repeat expansion in the coding sequence of the huntingtin gene, and an expanded polyglutamine (>37Q tract in the protein. This results in misfolding and accumulation of huntingtin protein (htt, formation of neuronal intranuclear and cytoplasmic inclusions, and neuronal dysfunction/degeneration. Single-chain Fv antibodies (scFvs, expressed as intrabodies that bind htt and prevent aggregation, show promise as immunotherapeutics for HD. Intrastriatal delivery of anti-N-terminal htt scFv-C4 using an adeno-associated virus vector (AAV2/1 significantly reduces the size and number of aggregates in HDR6/1 transgenic mice; however, this protective effect diminishes with age and time after injection. We therefore explored enhancing intrabody efficacy via fusions to heterologous functional domains. Proteins containing a PEST motif are often targeted for proteasomal degradation and generally have a short half life. In ST14A cells, fusion of the C-terminal PEST region of mouse ornithine decarboxylase (mODC to scFv-C4 reduces htt exon 1 protein fragments with 72 glutamine repeats (httex1-72Q by ~80-90% when compared to scFv-C4 alone. Proteasomal targeting was verified by either scrambling the mODC-PEST motif, or via proteasomal inhibition with epoxomicin. For these constructs, the proteasomal degradation of the scFv intrabody proteins themselves was reduced<25% by the addition of the mODC-PEST motif, with or without antigens. The remaining intrabody levels were amply sufficient to target N-terminal httex1-72Q protein fragment turnover. Critically, scFv-C4-PEST prevents aggregation and toxicity of httex1-72Q fragments at significantly lower doses than scFv-C4. Fusion of the mODC-PEST motif to intrabodies is a valuable general approach to specifically target toxic antigens to the proteasome for degradation.

  3. Feline Immunodeficiency Virus Vif N-Terminal Residues Selectively Counteract Feline APOBEC3s.

    Science.gov (United States)

    Gu, Qinyong; Zhang, Zeli; Cano Ortiz, Lucía; Franco, Ana Cláudia; Häussinger, Dieter; Münk, Carsten

    2016-12-01

    Feline immunodeficiency virus (FIV) Vif protein counteracts feline APOBEC3s (FcaA3s) restriction factors by inducing their proteasomal degradation. The functional domains in FIV Vif for interaction with FcaA3s are poorly understood. Here, we have identified several motifs in FIV Vif that are important for selective degradation of different FcaA3s. Cats (Felis catus) express three types of A3s: single-domain A3Z2, single-domain A3Z3, and double-domain A3Z2Z3. We proposed that FIV Vif would selectively interact with the Z2 and the Z3 A3s. Indeed, we identified two N-terminal Vif motifs (12LF13 and 18GG19) that specifically interacted with the FcaA3Z2 protein but not with A3Z3. In contrast, the exclusive degradation of FcaA3Z3 was regulated by a region of three residues (M24, L25, and I27). Only a FIV Vif carrying a combination of mutations from both interaction sites lost the capacity to degrade and counteract FcaA3Z2Z3. However, alterations in the specific A3s interaction sites did not affect the cellular localization of the FIV Vif protein and binding to feline A3s. Pulldown experiments demonstrated that the A3 binding region localized to FIV Vif residues 50 to 80, outside the specific A3 interaction domain. Finally, we found that the Vif sites specific to individual A3s are conserved in several FIV lineages of domestic cat and nondomestic cats, while being absent in the FIV Vif of pumas. Our data support a complex model of multiple Vif-A3 interactions in which the specific region for selective A3 counteraction is discrete from a general A3 binding domain. Both human immunodeficiency virus (HIV) and feline immunodeficiency virus (FIV) Vif proteins counteract their host's APOBEC3 restriction factors. However, these two Vif proteins have limited sequence homology. The molecular interaction between FIV Vif and feline APOBEC3s are not well understood. Here, we identified N-terminal FIV Vif sites that regulate the selective interaction of Vif with either feline APOBEC3Z

  4. Characterization of niphatenones that inhibit androgen receptor N-terminal domain.

    Directory of Open Access Journals (Sweden)

    Carmen A Banuelos

    Full Text Available Androgen ablation therapy causes a temporary reduction in tumor burden in patients with advanced prostate cancer. Unfortunately the malignancy will return to form lethal castration-recurrent prostate cancer (CRPC. The androgen receptor (AR remains transcriptionally active in CRPC in spite of castrate levels of androgens in the blood. AR transcriptional activity resides in its N-terminal domain (NTD. Possible mechanisms of continued AR transcriptional activity may include, at least in part, expression of constitutively active splice variants of AR that lack the C-terminal ligand-binding domain (LBD. Current therapies that target the AR LBD, would not be effective against these AR variants. Currently no drugs are clinically available that target the AR NTD which should be effective against these AR variants as well as full-length AR. Niphatenones were originally isolated and identified in active extracts from Niphates digitalis marine sponge. Here we begin to characterize the mechanism of niphatenones in blocking AR transcriptional activity. Both enantiomers had similar IC50 values of 6 µM for inhibiting the full-length AR in a functional transcriptional assay. However, (S-niphatenone had significantly better activity against the AR NTD compared to (R-niphatenone. Consistent with niphatenones binding to and inhibiting transactivation of AR NTD, niphatenones inhibited AR splice variant. Niphatenone did not affect the transcriptional activity of the related progesterone receptor, but slightly decreased glucocorticoid receptor (GR activity and covalently bound to GR activation function-1 (AF-1 region. Niphatenone blocked N/C interactions of AR without altering either AR protein levels or its intracellular localization in response to androgen. Alkylation with glutathione suggests that niphatenones are not a feasible scaffold for further drug development.

  5. Biologic variability of N-terminal pro-brain natriuretic peptide in adult healthy cats.

    Science.gov (United States)

    Harris, Autumn N; Estrada, Amara H; Gallagher, Alexander E; Winter, Brandy; Lamb, Kenneth E; Bohannon, Mary; Hanscom, Jancy; Mainville, Celine A

    2017-02-01

    Objectives The biologic variability of N-terminal pro-brain natriuretic peptide (NT-proBNP) and its impact on diagnostic utility is unknown in healthy cats and those with cardiac disease. The purpose of this study was to determine the biologic variation of NT-proBNP within-day and week-to-week in healthy adult cats. Methods Adult cats were prospectively evaluated by complete blood count (CBC), biochemistry, total thyroxine, echocardiography, electrocardiography and blood pressure, to exclude underlying systemic or cardiac disease. Adult healthy cats were enrolled and blood samples were obtained at 11 time points over a 6 week period (0, 2 h, 4 h, 6 h, 8 h, 10 h and at weeks 2, 3, 4, 5 and 6). The intra-individual (coefficient of variation [CV I ]) biologic variation along with index of individuality and reference change values (RCVs) were calculated. Univariate models were analyzed and included comparison of the six different time points for both daily and weekly samples. This was followed by a Tukey's post-hoc adjustment, with a P value of <0.05 being significant. Results The median daily and weekly CV I for the population were 13.1% (range 0-28.7%) and 21.2% (range 3.9-68.1%), respectively. The index of individuality was 0.99 and 1 for daily and weekly samples, respectively. The median daily and weekly RCVs for the population were 39.8% (range 17.0-80.5%) and 60.5% (range 20.1-187.8%), respectively. Conclusions and relevance This study demonstrates high individual variability for NT-proBNP concentrations in a population of adult healthy cats. Further research is warranted to evaluate NT-proBNP variability, particularly how serial measurements of NT-proBNP may be used in the diagnosis and management of cats with cardiac disease.

  6. Suppression of cell death by the secretory form of N-terminal ERC/mesothelin.

    Science.gov (United States)

    Wang, Tegexibaiyin; Kajino, Kazunori; Abe, Masaaki; Tan, Ke; Maruo, Masumi; Sun, Guodong; Hagiwara, Yoshiaki; Maeda, Masahiro; Hino, Okio

    2010-08-01

    ERC/mesothelin is highly expressed in malignant mesothelioma, pancreatic cancer, and ovarian cancer. It is cleaved to a 30 kDa N-terminal secretory form (N-ERC) and a 40 kDa C-terminal membranous form (C-ERC). Several functions have been reported for full-length ERC (full-ERC) and C-ERC/mesothelin, such as in cell adhesion and invasion, stimulation of cell proliferation, and the suppression of cell death. However, there have been no studies to date on the function of secretory N-ERC, despite the fact that it is abundantly secreted into the sera of mesothelioma patients. In this study, we investigated whether N-ERC could function as a secretory factor to stimulate tumor progression. Full-, N, or C-ERC was overexpressed in the human hepatocellular carcinoma cell line Huh7 that lacks endogenous expression of ERC/mesothelin. Changes in the rates of cell proliferation and cell death were determined, and the state of signal transducers was examined using various endpoints: total cell counts, trypan blue exclusion rate, BrdU incorporation rate, TUNEL assay, and the phosphorylation of ERK1/2 and Stat3. In cells overexpressing N-ERC, phosphorylation of ERK1/2 was enhanced and the rate of cell death decreased, leading to the increase of cell number. The culture medium containing the secretory N-ERC also had the activity to increase the number of cells. Our data suggested that one of the full-ERC functions reported previously was mediated by the secretory N-ERC.

  7. Investigating the DNA-binding ability of GATA-1-N-terminal zinc finger

    International Nuclear Information System (INIS)

    Wong, R.; Newton, A.; Crossley, M.; Mackay, J.

    2001-01-01

    Erythroid transcription factor GATA-1 interacts with both DNA and other proteins through its zinc finger domains (ZnFs). While it has been known for me time that the C-terminal ZnF binds DNA at GATA sites, only recently has it been observed that the N-terminal finger (NF) is capable of interacting with GATC sites. Further, a number of naturally occurring mutations in NF (V205M, G208S, R216Q, D218G) that lead to anaemia and thrombocytopenia have been identified. We are interested in characterising the NF-DNA interaction and determining the effects of mutation upon this interaction. Using nuclear magnetic resonance (NMR) spectroscopy, we have observed an interaction between recombinant NF and a 16-mer DNA duplex containing a core GATC sequence. This result forms the basis from which residues in NF involved in DNA binding can be identified, and work is being carried out to improve the quality of the NMR data with the aim of determining the solution structure of the NF-DNA complex. The DNA-binding affinity of both wild-type and mutant NFs mentioned above is also being investigated using isothermal titration calorimetry. These data suggest that the strength of the interaction between NF and the 16-mer DNA duplex is in the sub-micromolar range, and comparisons between the DNA-binding affinities of the NF mutants are being made. Together, these studies will help us to understand how GATA-1 acts as a transcriptional regulator and how mutations in NF domain of GATA-1 may lead to blood disorders

  8. Involvement of c-Jun N-Terminal Kinase in TNF-α-Driven Remodeling.

    Science.gov (United States)

    Eurlings, Irene M J; Reynaert, Niki L; van de Wetering, Cheryl; Aesif, Scott W; Mercken, Evi M; de Cabo, Rafael; van der Velden, Jos L; Janssen-Heininger, Yvonne M; Wouters, Emiel F M; Dentener, Mieke A

    2017-03-01

    Lung tissue remodeling in chronic obstructive pulmonary disease (COPD) is characterized by airway wall thickening and/or emphysema. Although the bronchial and alveolar compartments are functionally independent entities, we recently showed comparable alterations in matrix composition comprised of decreased elastin content and increased collagen and hyaluronan contents of alveolar and small airway walls. Out of several animal models tested, surfactant protein C (SPC)-TNF-α mice showed remodeling in alveolar and airway walls similar to what we observed in patients with COPD. Epithelial cells are able to undergo a phenotypic shift, gaining mesenchymal properties, a process in which c-Jun N-terminal kinase (JNK) signaling is involved. Therefore, we hypothesized that TNF-α induces JNK-dependent epithelial plasticity, which contributes to lung matrix remodeling. To this end, the ability of TNF-α to induce a phenotypic shift was assessed in A549, BEAS2B, and primary bronchial epithelial cells, and phenotypic markers were studied in SPC-TNF-α mice. Phenotypic markers of mesenchymal cells were elevated both in vitro and in vivo, as shown by the expression of vimentin, plasminogen activator inhibitor-1, collagen, and matrix metalloproteinases. Concurrently, the expression of the epithelial markers, E-cadherin and keratin 7 and 18, was attenuated. A pharmacological inhibitor of JNK attenuated this phenotypic shift in vitro, demonstrating involvement of JNK signaling in this process. Interestingly, activation of JNK signaling was also clearly present in lungs of SPC-TNF-α mice and patients with COPD. Together, these data show a role for TNF-α in the induction of a phenotypic shift in vitro, resulting in increased collagen production and the expression of elastin-degrading matrix metalloproteinases, and provide evidence for involvement of the TNF-α-JNK axis in extracellular matrix remodeling.

  9. Autocatalytic activity and substrate specificity of the pestivirus N-terminal protease N{sup pro}

    Energy Technology Data Exchange (ETDEWEB)

    Gottipati, Keerthi; Acholi, Sudheer [Department of Biochemistry and Molecular Biology, Sealy Center for Structural Biology and Molecular Biophysics, The University of Texas Medical Branch, Galveston, TX 77555-0647 (United States); Ruggli, Nicolas [Institute of Virology and Immunology, CH-3147 Mittelhäusern (Switzerland); Choi, Kyung H., E-mail: kychoi@utmb.edu [Department of Biochemistry and Molecular Biology, Sealy Center for Structural Biology and Molecular Biophysics, The University of Texas Medical Branch, Galveston, TX 77555-0647 (United States)

    2014-03-15

    Pestivirus N{sup pro} is the first protein translated in the viral polypeptide, and cleaves itself off co-translationally generating the N-terminus of the core protein. Once released, N{sup pro} blocks the host's interferon response by inducing degradation of interferon regulatory factor-3. N{sup pro'}s intracellular autocatalytic activity and lack of trans-activity have hampered in vitro cleavage studies to establish its substrate specificity and the roles of individual residues. We constructed N{sup pro}-GFP fusion proteins that carry the authentic cleavage site and determined the autoproteolytic activities of N{sup pro} proteins containing substitutions at the predicted catalytic sites Glu22 and Cys69, at Arg100 that forms a salt bridge with Glu22, and at the cleavage site Cys168. Contrary to previous reports, we show that N{sup pro'}s catalytic activity does not involve Glu22, which may instead be involved in protein stability. Furthermore, N{sup pro} does not have specificity for Cys168 at the cleavage site even though this residue is conserved throughout the pestivirus genus. - Highlights: • N{sup pro'}s autoproteolysis is studied using N{sup pro}-GFP fusion proteins. • N-terminal 17 amino acids are dispensable without loss of protease activity. • The putative catalytic residue Glu22 is not involved in protease catalysis. • No specificity for Cys168 at the cleavage site despite evolutionary conservation. • N{sup pro} prefers small amino acids with non-branched beta carbons at the P1 position.

  10. [Structure of tryptic fragments of a neurotoxin from black widow spider venom].

    Science.gov (United States)

    Volkova, T M; Galkina, T G; Kudelin, A B; Grishin, E V

    1991-04-01

    The N-terminal amino acid sequence of a neurotoxin from the venom of Latrodectus mactans tredecimguttatus (alpha-latrotoxin) was determined. Latrotoxin was subjected to the tryptic cleavage and total or partial amino acid sequences of 25 peptides were established. In total the tryptic fragments contained 252 amino acid residues. Essential structural information on cloning of the latrotoxin structural gene was obtained.

  11. Gamma-carboxylation and fragmentation of osteocalcin in human serum defined by mass spectrometry

    Science.gov (United States)

    Serum osteocalcin (Oc) concentration is a highly specific measure of bone turnover, but its circulating proteoform(s) have not been well defined. Based on immunological methods, the major forms are thought to be the intact polypeptide and a large N-terminal-mid molecule fragment for which there is n...

  12. Properties, production and applications of camelid single-domain antibody fragments

    NARCIS (Netherlands)

    Harmsen, M.M.; Haard, de H.J.

    2007-01-01

    Camelids produce functional antibodies devoid of light chains of which the single N-terminal domain is fully capable of antigen binding. These single-domain antibody fragments (VHHs or Nanobodies®) have several advantages for biotechnological applications. They are well expressed in microorganisms

  13. Accessory factors of cytoplasmic viral RNA sensors required for antiviral innate immune response

    Directory of Open Access Journals (Sweden)

    Hiroyuki eOshiumi

    2016-05-01

    Full Text Available Type I interferon (IFN induces many antiviral factors in host cells. RIG-I-like receptors (RLRs are cytoplasmic viral RNA sensors that trigger the signal to induce the innate immune response that includes type I IFN production. RIG-I and MDA5 are RLRs that form nucleoprotein filaments along viral double-stranded RNA, resulting in the activation of MAVS adaptor molecule. The MAVS protein forms a prion-like aggregation structure, leading to type I IFN production. RIG-I and MDA5 undergo post-translational modification. TRIM25 and Riplet ubiquitin ligases deliver a K63-linked polyubiquitin moiety to the RIG-I N-terminal caspase activation and recruitment domains (CARDs and C-terminal region; the polyubiquitin chain then stabilizes the two-CARD tetramer structure required for MAVS assembly. MDA5 activation is regulated by phosphorylation. RIOK3 is a protein kinase that phosphorylates the MDA5 protein in a steady state, and PP1α/γ dephosphorylate this protein, resulting in its activation. RIG-I and MDA5 require cytoplasmic RNA helicases for their efficient activation. LGP2, another RLR, is an RNA helicase involved in RLR signaling. This protein does not possess N-terminal CARDs and thus cannot trigger downstream signaling by itself. Recent studies have revealed that this protein modulates MDA5 filament formation, resulting in enhanced type I IFN production. Several other cytoplasmic RNA helicases are involved in RLR signaling. DDX3, DHX29, DHX36, and DDX60 RNA helicases have been reported to be involved in RLR-mediated type I IFN production after viral infection. However, the underlying mechanism is largely unknown. Future studies are required to reveal the role of RNA helicases in the RLR signaling pathway.

  14. [Expression, structure and antigenicity analysis of N51 derived from the N-terminal heptad repeat domain in gp41 of HIV-1 CRF07_BC strain].

    Science.gov (United States)

    Shao, Jiping; Jiang, Shibo; Liu, Shuwen

    2012-12-01

    To express N51 derived from the N-terminal heptad repeat (NHR) domain in gp41 of the HIV-1 CRF07_BC strain and analyze its molecular structure and antigenicity. Overlapping PCR was used to amplify the DNA fragment encoding N51Fd gene, which was then subcloned into the vector pFUSE-hIgG1-Fc2. The construct was confirmed by DNA sequencing. The structure and antigenicity of the recombinant protein N51FdFc-BC were analyzed using bioinformatic software, circular dichroism, and Western blotting. A recombinant expression vector pFUSE/N51Fd-BC was successfully constructed. N51FdFc-BC recombinant protein with a relative molecular mass of about 35 000 was effectively expressed in mammalian 293T cells and could be recognized by rabbit antibodies against HIV-1 gp41 N/C peptides as shown by Western blotting. Bioinformatic analysis showed that the recombinant protein N51FdFc-BC, with a relative molecular mass of 34 315.1 and a PI of 7.59, formed a secondary structure of random coil to allow its interactions as an antigen with antibodies. Circular dichroism measurement confirmed the random coil structure of N51FdFc-BC protein. The recombinant protein N51FdFc-BC has a random coil structure and can be used as an immunogen for development of HIV-1 subunit vaccine.

  15. The N-terminal domain of the Drosophila retinoblastoma protein Rbf1 interacts with ORC and associates with chromatin in an E2F independent manner.

    Directory of Open Access Journals (Sweden)

    Joseph Ahlander

    2008-07-01

    Full Text Available The retinoblastoma (Rb tumor suppressor protein can function as a DNA replication inhibitor as well as a transcription factor. Regulation of DNA replication may occur through interaction of Rb with the origin recognition complex (ORC.We characterized the interaction of Drosophila Rb, Rbf1, with ORC. Using expression of proteins in Drosophila S2 cells, we found that an N-terminal Rbf1 fragment (amino acids 1-345 is sufficient for Rbf1 association with ORC but does not bind to dE2F1. We also found that the C-terminal half of Rbf1 (amino acids 345-845 interacts with ORC. We observed that the amino-terminal domain of Rbf1 localizes to chromatin in vivo and associates with chromosomal regions implicated in replication initiation, including colocalization with Orc2 and acetylated histone H4.Our results suggest that Rbf1 can associate with ORC and chromatin through domains independent of the E2F binding site. We infer that Rbf1 may play a role in regulating replication directly through its association with ORC and/or chromatin factors other than E2F. Our data suggest an important role for retinoblastoma family proteins in cell proliferation and tumor suppression through interaction with the replication initiation machinery.

  16. N-terminal arginines modulate plasma-membrane localization of Kv7.1/KCNE1 channel complexes.

    Directory of Open Access Journals (Sweden)

    Zenawit Girmatsion

    Full Text Available BACKGROUND AND OBJECTIVE: The slow delayed rectifier current (I(Ks is important for cardiac action potential termination. The underlying channel is composed of Kv7.1 α-subunits and KCNE1 β-subunits. While most evidence suggests a role of KCNE1 transmembrane domain and C-terminus for the interaction, the N-terminal KCNE1 polymorphism 38G is associated with reduced I(Ks and atrial fibrillation (a human arrhythmia. Structure-function relationship of the KCNE1 N-terminus for I(Ks modulation is poorly understood and was subject of this study. METHODS: We studied N-terminal KCNE1 constructs disrupting structurally important positively charged amino-acids (arginines at positions 32, 33, 36 as well as KCNE1 constructs that modify position 38 including an N-terminal truncation mutation. Experimental procedures included molecular cloning, patch-clamp recording, protein biochemistry, real-time-PCR and confocal microscopy. RESULTS: All KCNE1 constructs physically interacted with Kv7.1. I(Ks resulting from co-expression of Kv7.1 with non-atrial fibrillation '38S' was greater than with any other construct. Ionic currents resulting from co-transfection of a KCNE1 mutant with arginine substitutions ('38G-3xA' were comparable to currents evoked from cells transfected with an N-terminally truncated KCNE1-construct ('Δ1-38'. Western-blots from plasma-membrane preparations and confocal images consistently showed a greater amount of Kv7.1 protein at the plasma-membrane in cells co-transfected with the non-atrial fibrillation KCNE1-38S than with any other construct. CONCLUSIONS: The results of our study indicate that N-terminal arginines in positions 32, 33, 36 of KCNE1 are important for reconstitution of I(Ks. Furthermore, our results hint towards a role of these N-terminal amino-acids in membrane representation of the delayed rectifier channel complex.

  17. Inhibition of c-Jun N-terminal kinase (JNK) suppresses porcine oocyte ageing in vitro

    Czech Academy of Sciences Publication Activity Database

    Sedmíková, M.; Petr, J.; Dörflerová, A.; Nevoral, J.; Novotná, Božena; Krejčová, T.; Chmelíková, E.; Tůmová, L.

    2013-01-01

    Roč. 58, č. 12 (2013), s. 535-545 ISSN 1212-1819 Grant - others:CIGA(CZ) 20122034; GAGA(CZ) 20122038 Institutional support: RVO:68378041 Keywords : MAPK * DNA fragmentation * apoptosis Subject RIV: EB - Genetics ; Molecular Biology Impact factor: 0.871, year: 2013

  18. Formation of large viroplasms and virulence of Cauliflower mosaic virus in turnip plants depend on the N-terminal EKI sequence of viral protein TAV.

    Directory of Open Access Journals (Sweden)

    Angèle Geldreich

    Full Text Available Cauliflower mosaic virus (CaMV TAV protein (TransActivator/Viroplasmin plays a pivotal role during the infection cycle since it activates translation reinitiation of viral polycistronic RNAs and suppresses RNA silencing. It is also the major component of cytoplasmic electron-dense inclusion bodies (EDIBs called viroplasms that are particularly evident in cells infected by the virulent CaMV Cabb B-JI isolate. These EDIBs are considered as virion factories, vehicles for CaMV intracellular movement and reservoirs for CaMV transmission by aphids. In this study, focused on different TAV mutants in vivo, we demonstrate that three physically separated domains collectively participate to the formation of large EDIBs: the N-terminal EKI motif, a sequence of the MAV domain involved in translation reinitiation and a C-terminal region encompassing the zinc finger. Surprisingly, EKI mutant TAVm3, corresponding to a substitution of the EKI motif at amino acids 11-13 by three alanines (AAA, which completely abolished the formation of large viroplasms, was not lethal for CaMV but highly reduced its virulence without affecting the rate of systemic infection. Expression of TAVm3 in a viral context led to formation of small irregularly shaped inclusion bodies, mild symptoms and low levels of viral DNA and particles accumulation, despite the production of significant amounts of mature capsid proteins. Unexpectedly, for CaMV-TAVm3 the formation of viral P2-containing electron-light inclusion body (ELIB, which is essential for CaMV aphid transmission, was also altered, thus suggesting an indirect role of the EKI tripeptide in CaMV plant-to-plant propagation. This important functional contribution of the EKI motif in CaMV biology can explain the strict conservation of this motif in the TAV sequences of all CaMV isolates.

  19. Domain swapping reveals that the N-terminal domain of the sensor kinase KdpD in Escherichia coli is important for signaling

    Directory of Open Access Journals (Sweden)

    Lippert Marie-Luise

    2009-07-01

    Full Text Available Abstract Background The KdpD/KdpE two-component system of Escherichia coli regulates expression of the kdpFABC operon encoding the high affinity K+ transport system KdpFABC. The input domain of KdpD comprises a domain that belongs to the family of universal stress proteins (Usp. It has been previously demonstrated that UspC binds to this domain, resulting in KdpD/KdpE scaffolding under salt stress. However the mechanistic significance of this domain for signaling remains unclear. Here, we employed a "domain swapping" approach to replace the KdpD-Usp domain with four homologous domains or with the six soluble Usp proteins of E. coli. Results Full response to salt stress was only achieved with a chimera that contains UspC, probably due to unaffected scaffolding of the KdpD/KdpE signaling cascade by soluble UspC. Unexpectedly, chimeras containing either UspF or UspG not only prevented kdpFABC expression under salt stress but also under K+ limiting conditions, although these hybrid proteins exhibited kinase and phosphotransferase activities in vitro. These are the first KdpD derivatives that do not respond to K+ limitation due to alterations in the N-terminal domain. Analysis of the KdpD-Usp tertiary structure revealed that this domain has a net positively charged surface, while UspF and UspG are characterized by net negative surface charges. Conclusion The Usp domain within KdpD not only functions as a binding surface for the scaffold UspC, but it is also important for KdpD signaling. We propose that KdpD sensing/signaling involves alterations of electrostatic interactions between the large N- and C-terminal cytoplasmic domains.

  20. Chameleon fragmentation

    International Nuclear Information System (INIS)

    Brax, Philippe; Upadhye, Amol

    2014-01-01

    A scalar field dark energy candidate could couple to ordinary matter and photons, enabling its detection in laboratory experiments. Here we study the quantum properties of the chameleon field, one such dark energy candidate, in an ''afterglow'' experiment designed to produce, trap, and detect chameleon particles. In particular, we investigate the possible fragmentation of a beam of chameleon particles into multiple particle states due to the highly non-linear interaction terms in the chameleon Lagrangian. Fragmentation could weaken the constraints of an afterglow experiment by reducing the energy of the regenerated photons, but this energy reduction also provides a unique signature which could be detected by a properly-designed experiment. We show that constraints from the CHASE experiment are essentially unaffected by fragmentation for φ 4 and 1/φ potentials, but are weakened for steeper potentials, and we discuss possible future afterglow experiments

  1. Chameleon fragmentation

    Energy Technology Data Exchange (ETDEWEB)

    Brax, Philippe [Institut de Physique Théorique, CEA, IPhT, CNRS, URA 2306, F-91191Gif/Yvette Cedex (France); Upadhye, Amol, E-mail: philippe.brax@cea.fr, E-mail: aupadhye@anl.gov [Institute for the Early Universe, Ewha University, International Education, Building #601, 11-1, Daehyun-Dong Seodaemun-Gu, Seoul 120-750 (Korea, Republic of)

    2014-02-01

    A scalar field dark energy candidate could couple to ordinary matter and photons, enabling its detection in laboratory experiments. Here we study the quantum properties of the chameleon field, one such dark energy candidate, in an ''afterglow'' experiment designed to produce, trap, and detect chameleon particles. In particular, we investigate the possible fragmentation of a beam of chameleon particles into multiple particle states due to the highly non-linear interaction terms in the chameleon Lagrangian. Fragmentation could weaken the constraints of an afterglow experiment by reducing the energy of the regenerated photons, but this energy reduction also provides a unique signature which could be detected by a properly-designed experiment. We show that constraints from the CHASE experiment are essentially unaffected by fragmentation for φ{sup 4} and 1/φ potentials, but are weakened for steeper potentials, and we discuss possible future afterglow experiments.

  2. Tetrahydrocannabinol-induced neurotoxicity depends on CB1 receptor-mediated c-Jun N-terminal kinase activation in cultured cortical neurons

    Science.gov (United States)

    Downer, Eric J; Fogarty, Marie P; Campbell, Veronica A

    2003-01-01

    Δ9-Tetrahydrocannabinol (THC), the main psychoactive ingredient of marijuana, induces apoptosis in cultured cortical neurons. THC exerts its apoptotic effects in cortical neurons by binding to the CB1 cannabinoid receptor. The CB1 receptor has been shown to couple to the stress-activated protein kinase, c-Jun N-terminal kinase (JNK). However, the involvement of specific JNK isoforms in the neurotoxic properties of THC remains to be established. The present study involved treatment of rat cultured cortical neurons with THC (0.005–50 μM), and combinations of THC with the CB1 receptor antagonist, AM 251 (10 μM) and pertussis toxin (PTX; 200 ng ml−1). Antisense oligonucleotides (AS) were used to deplete neurons of JNK1 and JNK2 in order to elucidate their respective roles in THC signalling. Here we report that THC induces the activation of JNK via the CB1 receptor and its associated G-protein, Gi/o. Treatment of cultured cortical neurons with THC resulted in a differential timeframe of activation of the JNK1 and JNK2 isoforms. Use of specific JNK1 and JNK2 AS identified activation of caspase-3 and DNA fragmentation as downstream consequences of JNK1 and JNK2 activation. The results from this study demonstrate that activation of the CB1 receptor induces JNK and caspase-3 activation, an increase in Bax expression and DNA fragmentation. The data demonstrate that the activation of both JNK1 and JNK2 isoforms is central to the THC-induced activation of the apoptotic pathway in cortical neurons. PMID:14522843

  3. Synergy between the N-terminal and C-terminal domains of Mycobacterium tuberculosis HupB is essential for high-affinity binding, DNA supercoiling and inhibition of RecA-promoted strand exchange.

    Science.gov (United States)

    Sharadamma, N; Khan, Krishnendu; Kumar, Sandeep; Patil, K Neelakanteshwar; Hasnain, Seyed E; Muniyappa, K

    2011-09-01

    The occurrence of DNA architectural proteins containing two functional domains derived from two different architectural proteins is an interesting emerging research theme in the field of nucleoid structure and function. Mycobacterium tuberculosis HupB, unlike Escherichia coli HU, is a two-domain protein that, in the N-terminal region, shows broad sequence homology with bacterial HU. The long C-terminal extension, on the other hand, contains seven PAKK/KAAK motifs, which are characteristic of the histone H1/H5 family of proteins. In this article, we describe several aspects of HupB function, in comparison with its truncated derivatives lacking either the C-terminus or N-terminus. We found that HupB binds a variety of DNA repair and replication intermediates with K(d) values in the nanomolar range. By contrast, the N-terminal fragment of M. tuberculosis HupB (HupB(MtbN)) showed diminished DNA-binding activity, with K(d) values in the micromolar range, and the C-terminal domain was completely devoid of DNA-binding activity. Unlike HupB(MtbN) , HupB was able to constrain DNA in negative supercoils and introduce negative superhelical turns into relaxed DNA. Similarly, HupB exerted a robust inhibitory effect on DNA strand exchange promoted by cognate and noncognate RecA proteins, whereas HupB(MtbN), even at a 50-fold molar excess, had no inhibitory effect. Considered together, these results suggest that synergy between the N-terminal and C-terminal domains of HupB is essential for its DNA-binding ability, and to modulate the topological features of DNA, which has implications for processes such as DNA compaction, gene regulation, homologous recombination, and DNA repair. © 2011 The Authors Journal compilation © 2011 FEBS.

  4. Structure of a double hexamer of the Pyrococcus furiosus minichromosome maintenance protein N-terminal domain

    Energy Technology Data Exchange (ETDEWEB)

    Meagher, Martin; Enemark, Eric J.

    2016-06-22

    The crystal structure of the N-terminal domain of thePyrococcus furiosusminichromosome maintenance (MCM) protein as a double hexamer is described. The MCM complex is a ring-shaped helicase that unwinds DNA at the replication fork of eukaryotes and archaea. Prior to replication initiation, the MCM complex assembles as an inactive double hexamer at specific sites of DNA. The presented structure is highly consistent with previous MCM double-hexamer structures and shows two MCM hexamers with a head-to-head interaction mediated by the N-terminal domain. Minor differences include a diminished head-to-head interaction and a slightly reduced inter-hexamer rotation.

  5. Interaction of the N-terminal segment of pulmonary surfactant protein SP-C with interfacial phospholipid films

    DEFF Research Database (Denmark)

    Plasencia, Inés; Keough, Kevin M W; Perez-Gil, Jesus

    2005-01-01

    Pulmonary surfactant protein SP-C is a 35-residue polypeptide composed of a hydrophobic transmembrane alpha-helix and a polycationic, palmitoylated-cysteine containing N-terminal segment. This segment is likely the only structural motif the protein projects out of the bilayer in which SP-C is ins...... related with the ability of SP-C to facilitate reinsertion of surface active lipid molecules into the lung interface during respiratory compression-expansion cycling. Udgivelsesdato: 2005-Jul-30...

  6. A novel calmodulin-regulated Ca2+-ATPase (ACA2) from Arabidopsis with an N-terminal autoinhibitory domain

    Science.gov (United States)

    Harper, J. F.; Hong, B.; Hwang, I.; Guo, H. Q.; Stoddard, R.; Huang, J. F.; Palmgren, M. G.; Sze, H.; Evans, M. L. (Principal Investigator)

    1998-01-01

    To study transporters involved in regulating intracellular Ca2+, we isolated a full-length cDNA encoding a Ca2+-ATPase from a model plant, Arabidopsis, and named it ACA2 (Arabidopsis Ca2+-ATPase, isoform 2). ACA2p is most similar to a "plasma membrane-type" Ca2+-ATPase, but is smaller (110 kDa), contains a unique N-terminal domain, and is missing a long C-terminal calmodulin-binding regulatory domain. In addition, ACA2p is localized to an endomembrane system and not the plasma membrane, as shown by aqueous-two phase fractionation of microsomal membranes. ACA2p was expressed in yeast as both a full-length protein (ACA2-1p) and an N-terminal truncation mutant (ACA2-2p; Delta residues 2-80). Only the truncation mutant restored the growth on Ca2+-depleted medium of a yeast mutant defective in both endogenous Ca2+ pumps, PMR1 and PMC1. Although basal Ca2+-ATPase activity of the full-length protein was low, it was stimulated 5-fold by calmodulin (50% activation around 30 nM). In contrast, the truncated pump was fully active and insensitive to calmodulin. A calmodulin-binding sequence was identified within the first 36 residues of the N-terminal domain, as shown by calmodulin gel overlays on fusion proteins. Thus, ACA2 encodes a novel calmodulin-regulated Ca2+-ATPase distinguished by a unique N-terminal regulatory domain and a non-plasma membrane localization.

  7. Autoantibodies to N-terminally truncated GAD improve clinical phenotyping of individuals with adult-onset diabetes: Action LADA 12.

    Science.gov (United States)

    Achenbach, Peter; Hawa, Mohammed I; Krause, Stephanie; Lampasona, Vito; Jerram, Samuel T; Williams, Alistair J K; Bonifacio, Ezio; Ziegler, Anette G; Leslie, R David

    2018-04-04

    Adult-onset type 1 diabetes, in which the 65 kDa isoform of GAD (GAD65) is a major autoantigen, has a broad clinical phenotype encompassing variable need for insulin therapy. This study aimed to evaluate whether autoantibodies against N-terminally truncated GAD65 more closely defined a type 1 diabetes phenotype associated with insulin therapy. Of 1114 participants with adult-onset diabetes from the Action LADA (latent autoimmune diabetes in adults) study with sufficient sera, we selected those designated type 1 (n = 511) or type 2 diabetes (n = 603) and retested the samples in radiobinding assays for human full-length GAD65 autoantibodies (f-GADA) and N-terminally truncated (amino acids 96-585) GAD65 autoantibodies (t-GADA). Individuals' clinical phenotypes were analysed according to antibody binding patterns. Overall, 478 individuals were f-GADA-positive, 431 were t-GADA-positive and 628 were negative in both assays. Risk of insulin treatment was augmented in t-GADA-positive individuals (OR 4.69 [95% CI 3.57, 6.17]) compared with f-GADA-positive individuals (OR 3.86 [95% CI 2.95, 5.06]), irrespective of diabetes duration. Of 55 individuals who were f-GADA-positive but t-GADA-negative, i.e. with antibody binding restricted to the N-terminus of GAD65, the phenotype was similar to type 2 diabetes with low risk of progression to insulin treatment. Compared with these individuals with N-terminal GAD65-restricted GADA, t-GADA-positive individuals were younger at diagnosis (p = 0.005), leaner (p N-terminally truncated GAD65 autoantibodies is associated with the clinical phenotype of autoimmune type 1 diabetes and predicts insulin therapy.

  8. Fine tuning of the catalytic activity of colicin E7 nuclease domain by systematic N-terminal mutations

    Czech Academy of Sciences Publication Activity Database

    Németh, E.; Körtvélyesi, T.; Thulstrup, P. W.; Christensen, H. E. M.; Kožíšek, Milan; Nagata, K.; Czene, A.; Gyurcsik, B.

    2014-01-01

    Roč. 23, č. 8 (2014), s. 1113-1122 ISSN 0961-8368 Grant - others:Seventh Framework Programme of the European Union(XE) FP7-312284; OPPC(CZ) CZ.2.16/3.1.00/24016 Institutional support: RVO:61388963 Keywords : DNA cleavage * flow linear dichroism * isothermal calorimetry * positively charged N-terminal residues * Zn2+ binding Subject RIV: CE - Biochemistry Impact factor: 2.854, year: 2014

  9. Plasmatic levels of N-terminal pro-atrial natriuretic peptide in preeclamptic patients and healthy normotensive pregnant women.

    Science.gov (United States)

    Reyna-Villasmil, Eduardo; Mejia-Montilla, Jorly; Reyna-Villasmil, Nadia; Mayner-Tresol, Gabriel; Herrera-Moya, Pedro; Fernández-Ramírez, Andreina; Rondón-Tapía, Marta

    2018-05-11

    To compare plasma N-terminal pro-atrial natriuretic peptide concentrations in preeclamptic patients and healthy normotensive pregnant women. A cases-controls study was done with 180 patients at Hospital Central Dr. Urquinaona, Maracaibo, Venezuela, that included 90 preeclamptic patients (group A; cases) and 90 healthy normotensive pregnant women selected with the same age and body mass index similar to group A (group B; controls). Blood samples were collected one hour after admission and prior to administration of any medication in group A to determine plasma N-terminal pro-atrial natriuretic peptide and other laboratory parameters. Plasma N-terminal pro-atrial natriuretic peptide concentrations in group A (mean 1.01 [0.26] pg/mL) showed a significant difference when compared with patients in group B (mean 0.55 [0.07] pg/mL; P<.001]. There was no significant correlation with systolic and diastolic blood pressure values in preeclamptic patients (P=ns). A cut-off value of 0.66ng/mL had an area under the curve of 0.93, sensitivity of 87.8%, specificity of 83.3%, a positive predictive value of 84.0% and a negative predictive value of 87.2%, with a diagnostic accuracy of 85.6%. Preeclamptic patients have significantly higher concentrations of plasma N-terminal pro-atrial natriuretic peptide compared with healthy normotensive pregnant women, with high predictive values for diagnosis. Copyright © 2017 Elsevier España, S.L.U. All rights reserved.

  10. N-Terminal Pro–B-Type Natriuretic Peptide Variability in Stable Dialysis Patients

    Science.gov (United States)

    Hayen, Andrew; Horvath, Andrea R.; Dimeski, Goce; Coburn, Amanda; Johnson, David W.; Hawley, Carmel M.; Campbell, Scott B.; Craig, Jonathan C.

    2015-01-01

    Background and objectives Monitoring N-terminal pro–B-type natriuretic peptide (NT-proBNP) may be useful for assessing cardiovascular risk in dialysis patients. However, its biologic variation is unknown, hindering the accurate interpretation of serial concentrations. The aims of this prospective cohort study were to estimate the within- and between-person coefficients of variation of NT-proBNP in stable dialysis patients, and derive the critical difference between measurements needed to exclude biologic and analytic variation. Design, setting, participants, & measurements Fifty-five prevalent hemodialysis and peritoneal dialysis patients attending two hospitals were assessed weekly for 5 weeks and then monthly for 4 months between October 2010 and April 2012. Assessments were conducted at the same time in the dialysis cycle and entailed NT-proBNP testing, clinical review, electrocardiography, and bioimpedance spectroscopy. Patients were excluded if they became unstable. Results This study analyzed 136 weekly and 113 monthly NT-proBNP measurements from 40 and 41 stable patients, respectively. Results showed that 22% had ischemic heart disease; 9% and 87% had left ventricular systolic and diastolic dysfunction, respectively. Respective between- and within-person coefficients of variation were 153% and 27% for weekly measurements, and 148% and 35% for monthly measurements. Within-person variation was unaffected by dialysis modality, hydration status, inflammation, or cardiac comorbidity. NT-proBNP concentrations measured at weekly intervals needed to increase by at least 46% or decrease by 84% to exclude change due to biologic and analytic variation alone with 90% certainty, whereas monthly measurements needed to increase by at least 119% or decrease by 54%. Conclusions The between-person variation of NT-proBNP was large and markedly greater than within-person variation, indicating that NT-proBNP testing might better be applied in the dialysis population using a

  11. N-terminal pro-B-type natriuretic peptide variability in stable dialysis patients.

    Science.gov (United States)

    Fahim, Magid A; Hayen, Andrew; Horvath, Andrea R; Dimeski, Goce; Coburn, Amanda; Johnson, David W; Hawley, Carmel M; Campbell, Scott B; Craig, Jonathan C

    2015-04-07

    Monitoring N-terminal pro-B-type natriuretic peptide (NT-proBNP) may be useful for assessing cardiovascular risk in dialysis patients. However, its biologic variation is unknown, hindering the accurate interpretation of serial concentrations. The aims of this prospective cohort study were to estimate the within- and between-person coefficients of variation of NT-proBNP in stable dialysis patients, and derive the critical difference between measurements needed to exclude biologic and analytic variation. Fifty-five prevalent hemodialysis and peritoneal dialysis patients attending two hospitals were assessed weekly for 5 weeks and then monthly for 4 months between October 2010 and April 2012. Assessments were conducted at the same time in the dialysis cycle and entailed NT-proBNP testing, clinical review, electrocardiography, and bioimpedance spectroscopy. Patients were excluded if they became unstable. This study analyzed 136 weekly and 113 monthly NT-proBNP measurements from 40 and 41 stable patients, respectively. Results showed that 22% had ischemic heart disease; 9% and 87% had left ventricular systolic and diastolic dysfunction, respectively. Respective between- and within-person coefficients of variation were 153% and 27% for weekly measurements, and 148% and 35% for monthly measurements. Within-person variation was unaffected by dialysis modality, hydration status, inflammation, or cardiac comorbidity. NT-proBNP concentrations measured at weekly intervals needed to increase by at least 46% or decrease by 84% to exclude change due to biologic and analytic variation alone with 90% certainty, whereas monthly measurements needed to increase by at least 119% or decrease by 54%. The between-person variation of NT-proBNP was large and markedly greater than within-person variation, indicating that NT-proBNP testing might better be applied in the dialysis population using a relative-change strategy. Serial NT-proBNP concentrations need to double or halve to confidently

  12. Antigenic modules in the N-terminal S1 region of the transmissible gastroenteritis virus spike protein

    Science.gov (United States)

    Reguera, Juan; Ordoño, Desiderio; Santiago, César; Enjuanes, Luis

    2011-01-01

    The N-terminal S1 region of the transmissible gastroenteritis virus (TGEV) spike (S) glycoprotein contains four antigenic sites (C, B, D and A, from the N- to the C-terminal end) and is engaged in host-cell receptor recognition. The most N-terminal portion of the S1 region, which comprises antigenic sites C and B, is needed for the enteric tropism of TGEV, whereas the major antigenic site A at the C-terminal moiety is required for both respiratory and enteric cell tropism, and is engaged in recognition of the aminopeptidase N (APN) receptor. This study determined the kinetics for binding of a soluble S1 protein to the APN protein. Moreover, the S1 region of the TGEV S protein was dissected, with the aim of identifying discrete modules displaying unique antigenic sites and receptor-binding functions. Following protease treatments and mammalian cell expression methods, four modules or domains (D1–D4) were defined at the S1 region. Papain treatment identified an N-terminal domain (D1) resistant to proteolysis, whereas receptor binding defined a soluble and functional APN receptor-binding domain (D3). This domain was recognized by neutralizing antibodies belonging to the antigenic site A and therefore could be used as an immunogen for the prevention of viral infection. The organization of the four modules in the S1 region of the TGEV S glycoprotein is discussed. PMID:21228126

  13. The first N-terminal unprotected (Gly-Aib)n peptide: H-Gly-Aib-Gly-Aib-OtBu.

    Science.gov (United States)

    Gessmann, Renate; Brückner, Hans; Petratos, Kyriacos

    2015-12-01

    Glycine (Gly) is incorporated in roughly half of all known peptaibiotic (nonribosomally biosynthesized antibiotic peptides of fungal origin) sequences and is the residue with the greatest conformational flexibility. The conformational space of Aib (α-aminoisobutyric acid) is severely restricted by the second methyl group attached to the Cα atom. Most of the crystal structures containing Aib are N-terminal protected. Deprotection of the N- or C-terminus of peptides may alter the hydrogen-bonding scheme and/or the structure and may facilitate crystallization. The structure reported here for glycyl-α-aminoisobutyrylglycyl-α-aminoisobutyric acid tert-butyl ester, C16H30N4O5, describes the first N-terminal-unprotected (Gly-Aib)n peptide. The achiral peptide could form an intramolecular hydrogen bond between the C=O group of Gly1 and the N-H group of Aib4. This hydrogen bond is found in all tetrapeptides and N-terminal-protected tripeptides containing Aib, apart from one exception. In the present work, this hydrogen bond is not observed (N...O = 5.88 Å). Instead, every molecule is hydrogen bonded to six other symmetry-related molecules with a total of eight hydrogen bonds per molecule. The backbone conformation starts in the right-handed helical region (and the left-handed helical region for the inverted molecule) and reverses the screw sense in the last two residues.

  14. N-Terminal Coiled-Coil Structure of ATPase Subunits of 26S Proteasome Is Crucial for Proteasome Function

    Science.gov (United States)

    Inobe, Tomonao; Genmei, Reiko

    2015-01-01

    The proteasome is an essential proteolytic machine in eukaryotic cells, where it removes damaged proteins and regulates many cellular activities by degrading ubiquitinated proteins. Its heterohexameric AAA+ ATPase Rpt subunits play a central role in proteasome activity by the engagement of substrate unfolding and translocation for degradation; however, its detailed mechanism remains poorly understood. In contrast to AAA+ ATPase domains, their N-terminal regions of Rpt subunits substantially differ from each other. Here, to investigate the requirements and roles of the N-terminal regions of six Rpt subunits derived from Saccharomyces cerevisiae, we performed systematic mutational analysis using conditional knockdown yeast strains for each Rpt subunit and bacterial heterologous expression system of the base subcomplex. We showed that the formation of the coiled-coil structure was the most important for the N-terminal region of Rpt subunits. The primary role of coiled-coil structure would be the maintenance of the ring structure with the defined order. However, the coiled-coil region would be also be involved in substrate recognition and an interaction between lid and base subcomplexes. PMID:26208326

  15. Intermediate Fragment

    DEFF Research Database (Denmark)

    Kruse Aagaard, Anders

    2015-01-01

    ‘Engaging Through Architecture’ in 2015 by Aarhus School of Architecture as a part of the Ventura Lambrate Milan Design Week, where it was exhibited under the name Concrete. The fundamental pool of techniques and knowledge that set the agenda for the fragment was established before the intentions...

  16. Framing Fragmentation

    DEFF Research Database (Denmark)

    Bundgaard, Charlotte

    2009-01-01

    to create architectural meaning and give character to an architecture of fragmentation. Layers are both seen as conceptual as well as material frames which define certain strong properties or meanings in the architectural work. Defining layers is a way of separating and organizing; it both defines...

  17. Rock fragmentation

    Energy Technology Data Exchange (ETDEWEB)

    Brown, W.S.; Green, S.J.; Hakala, W.W.; Hustrulid, W.A.; Maurer, W.C. (eds.)

    1976-01-01

    Experts in rock mechanics, mining, excavation, drilling, tunneling and use of underground space met to discuss the relative merits of a wide variety of rock fragmentation schemes. Information is presented on novel rock fracturing techniques; tunneling using electron beams, thermocorer, electric spark drills, water jets, and diamond drills; and rock fracturing research needs for mining and underground construction. (LCL)

  18. N-Terminal Lipid Modification Is Required for the Stable Accumulation of CyanoQ in Synechocystis sp. PCC 6803.

    Directory of Open Access Journals (Sweden)

    Andrea D Juneau

    Full Text Available The CyanoQ protein has been demonstrated to be a component of cyanobacterial Photosystem II (PS II, but there exist a number of outstanding questions concerning its physical association with the complex. CyanoQ is a lipoprotein; upon cleavage of its transit peptide by Signal Peptidase II, which targets delivery of the mature protein to the thylakoid lumenal space, the N-terminal cysteinyl residue is lipid-modified. This modification appears to tether this otherwise soluble component to the thylakoid membrane. To probe the functional significance of the lipid anchor, mutants of the CyanoQ protein have been generated in Synechocystis sp. PCC 6803 to eliminate the N-terminal cysteinyl residue, preventing lipid modification. Substitution of the N-terminal cysteinyl residue with serine (Q-C22S resulted in a decrease in the amount of detectable CyanoQ protein to 17% that of the wild-type protein. Moreover, the physical properties of the accumulated Q-C22S protein were consistent with altered processing of the CyanoQ precursor. The Q-C22S protein was shifted to a higher apparent molecular mass and partitioned in the hydrophobic phase in TX-114 phase-partitioning experiments. These results suggest that the hydrophobic N-terminal 22 amino acids were not properly cleaved by a signal peptidase. Substitution of the entire CyanoQ transit peptide with the transit peptide of the soluble lumenal protein PsbO yielded the Q-SS mutant and resulted in no detectable accumulation of the modified CyanoQ protein. Finally, the CyanoQ protein was present at normal amounts in the PS II mutant strains ΔpsbB and ΔpsbO, indicating that an association with PS II was not a prerequisite for stable CyanoQ accumulation. Together these results indicate that CyanoQ accumulation in Synechocystis sp. PCC 6803 depends on the presence of the N-terminal lipid anchor, but not on the association of CyanoQ with the PS II complex.

  19. Fragmented Authoritarianism or Integrated Fragmentation

    DEFF Research Database (Denmark)

    Brødsgaard, Kjeld Erik

    or from a position as business leader to a position in the state apparatus or in the Party and vice versa. To conceptualize the coexistence of the contradicting forces for further enterprise autonomy and continued central control that characterizes the evolving relationship between business groups...... and the Party-state, I suggest the notion of integrated fragmentation....

  20. Influence of the N-terminal domain on the aggregation properties of the prion protein

    OpenAIRE

    Frankenfield, Kristen N.; Powers, Evan T.; Kelly, Jeffery W.

    2005-01-01

    Prion diseases appear to be caused by the aggregation of the cellular prion protein (PrPC) into an infectious form denoted PrPSc. The in vitro aggregation of the prion protein has been extensively investigated, yet many of these studies utilize truncated polypeptides. Because the C-terminal portion of PrPSc is protease-resistant and retains infectivity, it is assumed that studies on this fragment are most relevant. The full-length protein can be distinguished from the truncated protein becaus...

  1. Molecular analysis of a new cytoplasmic male sterile genotype in sunflower

    NARCIS (Netherlands)

    Spassova, Mariana; Christov, Michail; Bohorova, Natasha; Petrov, Peter; Dudov, Kalin; Atanassov, Atanas; Nijkamp, H. John J.; Hille, Jaques

    1992-01-01

    Mitochondrial DNA from 1 fertile and 6 cytoplasmic male sterile (CMS) sunflower genotypes was studied. The CMS genotypes had been obtained either by specific crosses between different Helianthus species or by mutagenesis. CMS-associated restriction fragment length polymorphisms (RFLPs) were found in

  2. An N-terminal Region of Mot-2 Binds to p53 In Vitro

    Directory of Open Access Journals (Sweden)

    Sunil C. Kaul

    2001-01-01

    Full Text Available The mouse mot-2 protein was earlier shown to bind to the tumor suppressor protein, p53. The mot-2 binding site of p53 was mapped to C-terminal amino acid residues 312–352, which includes the cytoplasmic sequestration domain. In the present study, we have found that both mot-1 and mot-2 bind to p53 in vitro. By using His-tagged deletion mutant proteins, the p53-binding domain of mot-2 was mapped to its Nterminal amino acid residues 253–282, which are identical in mot-1 and mot-2 proteins. Some peptides containing the p53-binding region of mot-2 were able to compete with the full-length protein for p53 binding. The data provided rationale for in vitro binding of mot-1 and mot-2 proteins to p53 and supported the conclusion that inability of mot-1 protein to bind p53 in vivo depends on secondary structure or its binding to other cellular factors. Most interestingly, the p53-binding region of mot-2 was common to its MKT-077, a cationic dye that exhibits antitumor activity, binding region. Therefore it is most likely that MKT-077-induced nuclear translocation and restoration of wild-type p53 function in transformed cells takes place by a competitional mechanism.

  3. X-ray crystal structure of the N-terminal region of Moloney murine leukemia virus integrase and its implications for viral DNA recognition: N-Terminal Region of M-MuLV Integrase

    Energy Technology Data Exchange (ETDEWEB)

    Guan, Rongjin [Center for Advanced Biotechnology and Medicine, Rutgers, The State University of New Jersey, Piscataway New Jersey 08854; Northeast Structural Genomics Consortium, Rutgers, The State University of New Jersey, Piscataway New Jersey 08854; Department of Molecular Biology and Biochemistry, Rutgers, The State University of New Jersey, Piscataway New Jersey 08854; Aiyer, Sriram [Department of Pharmacology, Robert Wood Johnson Medical School, Rutgers, The State University of New Jersey, Piscataway New Jersey 08854; Cote, Marie L. [Department of Biochemistry, Robert Wood Johnson Medical School, UMDNJ, Piscataway New Jersey 08854; Xiao, Rong [Center for Advanced Biotechnology and Medicine, Rutgers, The State University of New Jersey, Piscataway New Jersey 08854; Northeast Structural Genomics Consortium, Rutgers, The State University of New Jersey, Piscataway New Jersey 08854; Department of Molecular Biology and Biochemistry, Rutgers, The State University of New Jersey, Piscataway New Jersey 08854; Jiang, Mei [Center for Advanced Biotechnology and Medicine, Rutgers, The State University of New Jersey, Piscataway New Jersey 08854; Northeast Structural Genomics Consortium, Rutgers, The State University of New Jersey, Piscataway New Jersey 08854; Department of Molecular Biology and Biochemistry, Rutgers, The State University of New Jersey, Piscataway New Jersey 08854; Acton, Thomas B. [Center for Advanced Biotechnology and Medicine, Rutgers, The State University of New Jersey, Piscataway New Jersey 08854; Northeast Structural Genomics Consortium, Rutgers, The State University of New Jersey, Piscataway New Jersey 08854; Department of Molecular Biology and Biochemistry, Rutgers, The State University of New Jersey, Piscataway New Jersey 08854; Roth, Monica J. [Department of Pharmacology, Robert Wood Johnson Medical School, Rutgers, The State University of New Jersey, Piscataway New Jersey 08854; Montelione, Gaetano T. [Center for Advanced Biotechnology and Medicine, Rutgers, The State University of New Jersey, Piscataway New Jersey 08854; Northeast Structural Genomics Consortium, Rutgers, The State University of New Jersey, Piscataway New Jersey 08854; Department of Molecular Biology and Biochemistry, Rutgers, The State University of New Jersey, Piscataway New Jersey 08854; Department of Biochemistry, Robert Wood Johnson Medical School, UMDNJ, Piscataway New Jersey 08854

    2017-02-03

    The retroviral integrase (IN) carries out the integration of a dsDNA copy of the viral genome into the host DNA, an essential step for viral replication. All IN proteins have three general domains, the N-terminal domain (NTD), the catalytic core domain, and the C-terminal domain. The NTD includes an HHCC zinc finger-like motif, which is conserved in all retroviral IN proteins. Two crystal structures of Moloney murine leukemia virus (M-MuLV) IN N-terminal region (NTR) constructs that both include an N-terminal extension domain (NED, residues 1–44) and an HHCC zinc-finger NTD (residues 45–105), in two crystal forms are reported. The structures of IN NTR constructs encoding residues 1–105 (NTR1–105) and 8–105 (NTR8–105) were determined at 2.7 and 2.15 Å resolution, respectively and belong to different space groups. While both crystal forms have similar protomer structures, NTR1–105 packs as a dimer and NTR8–105 packs as a tetramer in the asymmetric unit. The structure of the NED consists of three anti-parallel β-strands and an α-helix, similar to the NED of prototype foamy virus (PFV) IN. These three β-strands form an extended β-sheet with another β-strand in the HHCC Zn2+ binding domain, which is a unique structural feature for the M-MuLV IN. The HHCC Zn2+ binding domain structure is similar to that in HIV and PFV INs, with variations within the loop regions. Differences between the PFV and MLV IN NEDs localize at regions identified to interact with the PFV LTR and are compared with established biochemical and virological data for M-MuLV. Proteins 2017; 85:647–656.

  4. Variations in the cytoplasmic region account for the heterogeneity of the chicken MHC class I (B-F) molecules

    DEFF Research Database (Denmark)

    Møller, L B; Kaufman, J; Verland, S

    1991-01-01

    . Unlike the parent proteins, the Mr 36,000 fragment derived from isolated variants yielded identical, simple patterns in two-dimensional gel electrophoresis and identical finger prints in peptide mapping. This, together with N-terminal amino acid sequencing, as well as comparison of hydrophobicity...... comprised by several isoelectric focusing variants. This heterogeneity could not be reduced by enzymatic deglycosylation. By contrast, proteolytic removal of a small (Mr 1000-4000) fragment from the alpha chain resulted in the generation of a Mr 36,000 fragment, common to all the molecular mass variants...... properties of fragments obtained by gradual proteolytic digestion, indicated that the small peptide responsible for the major B-F heterogeneity was situated in the intracellular, C-terminal part. Udgivelsesdato: 1991-null...

  5. N-tail translocation of mature beta-lactamase across the Escherichia coli cytoplasmic membrane.

    Science.gov (United States)

    Mitsopoulos, C; Hashemzadeh-Bonehi, L; Broome-Smith, J K

    1997-12-08

    Mature beta-lactamase was attached to the N-terminus of human glycophorin C, an N-out membrane protein lacking a cleavable signal peptide (an N-tail membrane protein). When synthesised in Escherichia coli more than 30% of the intact mature beta-lactamase-glycophorin C molecules assembled N-out, C-in into the cytoplasmic membrane. The N-tail translocated beta-lactamase folded into an enzymatically active form, but it was more susceptible to proteolysis than the equivalent portion of beta-lactamase-glycophorin C synthesised with an N-terminal signal peptide. Its translocation was virtually abolished when the N-out domain of glycophorin C was truncated or when the basic residues C-terminally flanking the glycophorin C membrane-spanning segment were replaced with neutral ones.

  6. The presequence of Euglena LHCPII, a cytoplasmically synthesized chloroplast protein, contains a functional endoplasmic reticulum-targeting domain.

    Science.gov (United States)

    Kishore, R; Muchhal, U S; Schwartzbach, S D

    1993-01-01

    The precursor to the Euglena light-harvesting chlorophyll a/b-binding protein of photosystem II (pLHCPII) is unique; it is a polyprotein, synthesized on membrane-bound ribosomes and transported to the Golgi apparatus prior to chloroplast localization. A cDNA corresponding to the 5' end of LHCPII mRNA has been isolated and sequenced. The deduced amino acid sequence of this cDNA indicates that Euglena pLHCPII contains a 141-amino acid N-terminal extension. The N-terminal extension contains three hydrophobic domains and a potential signal peptidase cleavage site at amino acid 35. Cotranslational processing by canine microsomes removed approximately 35 amino acids from an in vitro synthesized 33-kDa pLHCPII composed of a 141-amino acid N-terminal extension and a 180-amino acid partial LHCPII unit truncated at the beginning of the third membrane-spanning hydrophobic domain. Processed pLHCPII was degraded by exogenous protease, indicating that it had not been translocated to the microsomal lumen. Extraction with 0.1 M Na2CO3, pH 11.5, did not remove the processed pLHCPII from the microsomal membrane. A stop-transfer membrane anchor sequence appears to anchor the nascent protein within the membrane, preventing translocation into the lumen. Taken together, these results provide biochemical evidence for a functional cleaved signal sequence within the N-terminal extension of a Euglena cytoplasmically synthesized chloroplast-localized protein. Images Fig. 2 Fig. 3 PMID:8265635

  7. The regulation of N-terminal Huntingtin (Htt552) accumulation by Beclin1

    Science.gov (United States)

    Wu, Jun-chao; Qi, Lin; Wang, Yan; Kegel, Kimberly B; Yoder, Jennifer; Difiglia, Marian; Qin, Zheng-hong; Lin, Fang

    2012-01-01

    Aim: Huntingtin protein (Htt) was a neuropathological hallmark in human Huntington's Disease. The study aimed to investigate whether the macroautophagy regulator, Beclin1, was involved in the degradation of Htt. Methods: PC12 cells and primary cultured brain neurons of rats were examined. pDC316 adenovirus shuttle plasmid was used to mediate the expression of wild-type Htt-18Q-552 or mutant Htt-100Q-552 in PC12 cells. The expression of the autophagy-related proteins LC3 II and Beclin1, as well as the lysosome-associated enzymes Cathepsin B and L was evaluated using Western blotting. The locations of Beclin1 and Htt were observed with immunofluorescence and confocal microscope. Results: Htt552 expression increased the expression of LC3 II, Beclin1, cathepsin B and L in autophagy/lysosomal degradation pathway. Treatment with the autophagy inhibitor 3-MA or the proteasome inhibitors lactacystin and MG-132 increased Htt552 levels in PC12 cells infected with Ad-Htt-18Q-552 or Ad-Htt-100Q-552. The proteasome inhibitor caused a higher accumulation of Htt552-18Q than Htt552-100Q, and the autophagy inhibitor resulted in a higher accumulation of Htt552-100Q than Htt552-18Q. Similar results were observed in primary cultured neurons infected with adenovirus. In Htt552-expressing cells, Beclin1 was redistributed from the nucleus to the cytoplasm. Htt siRNA prevented Beclin1 redistribution in starvation conditions. Blockade of Beclin1 nuclear export by leptomycin B or Beclin1 deficiency caused by RNA interference induced the formation of mHtt552 aggregates. Conclusion: Beclin1 regulates the accumulation of Htt via macroautophagy. PMID:22543707

  8. Phosphorylation Regulates Interaction of 210-kDa Myosin Light Chain Kinase N-terminal Domain with Actin Cytoskeleton.

    Science.gov (United States)

    Vilitkevich, E L; Khapchaev, A Y; Kudryashov, D S; Nikashin, A V; Schavocky, J P; Lukas, T J; Watterson, D M; Shirinsky, V P

    2015-10-01

    High molecular weight myosin light chain kinase (MLCK210) is a multifunctional protein involved in myosin II activation and integration of cytoskeletal components in cells. MLCK210 possesses actin-binding regions both in the central part of the molecule and in its N-terminal tail domain. In HeLa cells, mitotic protein kinase Aurora B was suggested to phosphorylate MLCK210 N-terminal tail at serine residues (Dulyaninova, N. G., and Bresnick, A. R. (2004) Exp. Cell Res., 299, 303-314), but the functional significance of the phosphorylation was not established. We report here that in vitro, the N-terminal actin-binding domain of MLCK210 is located within residues 27-157 (N27-157, avian MLCK210 sequence) and is phosphorylated by cAMP-dependent protein kinase (PKA) and Aurora B at serine residues 140/149 leading to a decrease in N27-157 binding to actin. The same residues are phosphorylated in a PKA-dependent manner in transfected HeLa cells. Further, in transfected cells, phosphomimetic mutants of N27-157 showed reduced association with the detergent-stable cytoskeleton, whereas in vitro, the single S149D mutation reduced N27-157 association with F-actin to a similar extent as that achieved by N27-157 phosphorylation. Altogether, our results indicate that phosphorylation of MLCK210 at distinct serine residues, mainly at S149, attenuates the interaction of MLCK210 N-terminus with the actin cytoskeleton and might serve to regulate MLCK210 microfilament cross-linking activity in cells.

  9. Restricted N-terminal truncation of cardiac troponin T: a novel mechanism for functional adaptation to energetic crisis.

    Science.gov (United States)

    Feng, Han-Zhong; Biesiadecki, Brandon J; Yu, Zhi-Bin; Hossain, M Moazzem; Jin, J-P

    2008-07-15

    The N-terminal variable region of cardiac troponin T (TnT) is a regulatory structure that can be selectively removed during myocardial ischaemia reperfusion by mu-calpain proteolysis. Here we investigated the pathophysiological significance of this post-translational modification that removes amino acids 1-71 of cardiac TnT. Working heart preparations were employed to study rat acute myocardial infarction and transgenic mouse hearts over-expressing the N-terminal truncated cardiac TnT (cTnT-ND). Ex vivo myocardial infarction by ligation of the left anterior descending coronary artery induced heart failure and produced cTnT-ND not only in the infarct but also in remote zones, including the right ventricular free wall, indicating a whole organ response in the absence of systemic neurohumoral mechanisms. Left ventricular pressure overload in mouse working hearts produced increased cTnT-ND in both ventricles, suggesting a role of haemodynamic stress in triggering an acute whole organ proteolytic regulation. Transgenic mouse hearts in which the endogenous intact cardiac TnT was partially replaced by cTnT-ND showed lowered contractile velocity. When afterload increased from 55 mmHg to 90 mmHg, stroke volume decreased in the wild type but not in the transgenic mouse hearts. Correspondingly, the left ventricular rapid-ejection time of the transgenic mouse hearts was significantly longer than that of wild type hearts, especially at high afterload. The restricted deletion of the N-terminal variable region of cardiac troponin T demonstrates a novel mechanism by which the thin filament regulation adapts to sustain cardiac function under stress conditions.

  10. Affects of N-terminal variation in the SeM protein of Streptococcus equi on antibody and fibrinogen binding.

    Science.gov (United States)

    Timoney, John F; DeNegri, Rafaela; Sheoran, Abhineet; Forster, Nathalie

    2010-02-10

    The clonal Streptococcus equi causes equine strangles, a highly contagious suppurative lymphadenopathy and rhinopharyngitis. An important virulence factor and vaccine component, the antiphagocytic fibrinogen binding SeM of S. equi is a surface anchored fibrillar protein. Two recent studies of N. American, Japanese and European isolates have revealed a high frequency of N-terminal amino acid variation in SeM of S. equi CF32 that suggests this region of the protein is subject to immunologic selection pressure. The aims of the present study were firstly to map regions of SeM reactive with convalescent equine IgG and IgA and stimulatory for lymph node cells and secondly to determine effects of N-terminal variation on the functionality of SeM. Variation did not significantly affect fibrinogen binding or susceptibility of S. equi to an opsonic equine serum. Linear epitopes reactive with convalescent IgG and mucosal IgA were concentrated toward the conserved center of SeM. However, IgA but not IgG from every horse reacted with at least one peptide that contained variable sequence. Lymph node cells (CD4+) from horses immunized with SeM were strongly responsive to a peptide (alphaalpha36-138) encoding the entire variable region. SeM (CF32) specific mouse Mab 04D11 which reacted strongly with this larger peptide but not with shorter peptides within that sequence reacted strongly with whole cells of S. equi CF32 but only weakly with cells of any of 14 isolates of S. equi expressing different variants of SeM. These results in combination suggest that N-terminal variation alters a conformational epitope of significance in mucosal IgA and systemic T cell responses but does not affect antibody mediated phagocytosis and killing. Copyright (c) 2009 Elsevier Ltd. All rights reserved.

  11. Functional Analysis of γ-Tubulin Complex Proteins Indicates Specific Lateral Association via Their N-terminal Domains.

    Science.gov (United States)

    Farache, Dorian; Jauneau, Alain; Chemin, Cécile; Chartrain, Marine; Rémy, Marie-Hélène; Merdes, Andreas; Haren, Laurence

    2016-10-28

    Microtubules are nucleated from multiprotein complexes containing γ-tubulin and associated γ-tubulin complex proteins (GCPs). Small complexes (γTuSCs) comprise two molecules of γ-tubulin bound to the C-terminal domains of GCP2 and GCP3. γTuSCs associate laterally into helical structures, providing a structural template for microtubule nucleation. In most eukaryotes γTuSCs associate with additional GCPs (4, 5, and 6) to form the core of the so-called γ-tubulin ring complex (γTuRC). GCPs 2-6 constitute a family of homologous proteins. Previous structural analysis and modeling of GCPs suggest that all family members can potentially integrate into the helical structure. Here we provide experimental evidence for this model. Using chimeric proteins in which the N- and C-terminal domains of different GCPs are swapped, we show that the N-terminal domains define the functional identity of GCPs, whereas the C-terminal domains are exchangeable. FLIM-FRET experiments indicate that GCP4 and GCP5 associate laterally within the complex, and their interaction is mediated by their N-terminal domains as previously shown for γTuSCs. Our results suggest that all GCPs are incorporated into the helix via lateral interactions between their N-terminal domains, whereas the C-terminal domains mediate longitudinal interactions with γ-tubulin. Moreover, we show that binding to γ-tubulin is not essential for integrating into the helical complex. © 2016 by The American Society for Biochemistry and Molecular Biology, Inc.

  12. Fragmentation based

    Directory of Open Access Journals (Sweden)

    Shashank Srivastava

    2014-01-01

    Gaining the understanding of mobile agent architecture and the security concerns, in this paper, we proposed a security protocol which addresses security with mitigated computational cost. The protocol is a combination of self decryption, co-operation and obfuscation technique. To circumvent the risk of malicious code execution in attacking environment, we have proposed fragmentation based encryption technique. Our encryption technique suits the general mobile agent size and provides hard and thorny obfuscation increasing attacker’s challenge on the same plane providing better performance with respect to computational cost as compared to existing AES encryption.

  13. Architectural fragments

    DEFF Research Database (Denmark)

    Bang, Jacob Sebastian

    2018-01-01

    the photographs as a starting point for a series of paintings. This way of creating representations of something that already exists is for me to see a way forward in the "decoding" of my own models into other depictions. The models are analyzed through a series of representations in different types of drawings....... I try to invent the ways of drawing the models - that decode and unfold them into architectural fragments- into future buildings or constructions in the landscape. [1] Luigi Moretti: Italian architect, 1907 - 1973 [2] Man Ray: American artist, 1890 - 1976. in 2015, I saw the wonderful exhibition...

  14. Ketamine inhibits tumor necrosis factor-α and interleukin-6 gene expressions in lipopolysaccharide-stimulated macrophages through suppression of toll-like receptor 4-mediated c-Jun N-terminal kinase phosphorylation and activator protein-1 activation

    International Nuclear Information System (INIS)

    Wu, G.-J.; Chen, T.-L.; Ueng, Y.-F.; Chen, R.-M.

    2008-01-01

    Our previous study showed that ketamine, an intravenous anesthetic agent, has anti-inflammatory effects. In this study, we further evaluated the effects of ketamine on the regulation of tumor necrosis factor-α (TNF-α) and interlukin-6 (IL-6) gene expressions and its possible signal-transducing mechanisms in lipopolysaccharide (LPS)-activated macrophages. Exposure of macrophages to 1, 10, and 100 μM ketamine, 100 ng/ml LPS, or a combination of ketamine and LPS for 1, 6, and 24 h was not cytotoxic to macrophages. A concentration of 1000 μM of ketamine alone or in combined treatment with LPS caused significant cell death. Administration of LPS increased cellular TNF-α and IL-6 protein levels in concentration- and time-dependent manners. Meanwhile, treatment with ketamine concentration- and time-dependently alleviated the enhanced effects. LPS induced TNF-α and IL-6 mRNA syntheses. Administration of ketamine at a therapeutic concentration (100 μM) significantly inhibited LPS-induced TNF-α and IL-6 mRNA expressions. Application of toll-like receptor 4 (TLR4) small interfering (si)RNA into macrophages decreased cellular TLR4 levels. Co-treatment of macrophages with ketamine and TLR4 siRNA decreased the LPS-induced TNF-α and IL-6 productions more than alone administration of TLR4 siRNA. LPS stimulated phosphorylation of c-Jun N-terminal kinase and translocation of c-Jun and c-Fos from the cytoplasm to nuclei. However, administration of ketamine significantly decreased LPS-induced activation of c-Jun N-terminal kinase and translocation of c-Jun and c-Fos. LPS increased the binding of nuclear extracts to activator protein-1 consensus DNA oligonucleotides. Administration of ketamine significantly ameliorated LPS-induced DNA binding activity of activator protein-1. Therefore, a clinically relevant concentration of ketamine can inhibit TNF-α and IL-6 gene expressions in LPS-activated macrophages. The suppressive mechanisms occur through suppression of TLR4-mediated

  15. Lacking deoxygenation-linked interaction between cytoplasmic domain of band 3 and HbF from fetal red blood cells

    DEFF Research Database (Denmark)

    Weber, Roy E.

    2007-01-01

    Aim: Several of the red blood cell's metabolic and membrane functions display dependence on haemoglobin oxygenation. In adult human red cells, the increased glycolytic rate at low O2 tension results from binding of deoxygenated HbA at negatively charged, N-terminal, cytoplasmic domain of the memb......Aim: Several of the red blood cell's metabolic and membrane functions display dependence on haemoglobin oxygenation. In adult human red cells, the increased glycolytic rate at low O2 tension results from binding of deoxygenated HbA at negatively charged, N-terminal, cytoplasmic domain...... of the membrane protein band 3, which liberates glycolytic enzymes from this site. This study aims to investigate the role of fetal HbF (that has lower anion-binding capacity than HbA) in fetal red cells (that are subjected to low O2 tensions), and to elucidate possible linkage (e.g. via the major red cell...... membrane organising centre, band 3) between the individual oxygenation-linked reactions encountered in red cells. Methods: The interaction between band 3 and Hb is analysed in terms of the effects, measured under different conditions, of a 10-mer peptide that corresponds to the N-terminus of human band 3...

  16. Conserved N-terminal negative charges support optimally efficient N-type inactivation of Kv1 channels.

    Directory of Open Access Journals (Sweden)

    Alison Prince

    Full Text Available N-type inactivation is produced by the binding of a potassium channel's N-terminus within the open pore, blocking conductance. Previous studies have found that introduction of negative charges into N-terminal inactivation domains disrupts inactivation; however, the Aplysia AKv1 N-type inactivation domain contains two negatively charged residues, E2 and E9. Rather than being unusual, sequence analysis shows that this N-terminal motif is highly conserved among Kv1 sequences across many phyla. Conservation analysis shows some tolerance at position 9 for other charged residues, like D9 and K9, whereas position 2 is highly conserved as E2. To examine the functional importance of these residues, site directed mutagenesis was performed and effects on inactivation were recorded by two electrode voltage clamp in Xenopus oocytes. We find that inclusion of charged residues at positions 2 and 9 prevents interactions with non-polar sites along the inactivation pathway increasing the efficiency of pore block. In addition, E2 appears to have additional specific electrostatic interactions that stabilize the inactivated state likely explaining its high level of conservation. One possible explanation for E2's unique importance, consistent with our data, is that E2 interacts electrostatically with a positive charge on the N-terminal amino group to stabilize the inactivation domain at the block site deep within the pore. Simple electrostatic modeling suggests that due to the non-polar environment in the pore in the blocked state, even a 1 Å larger separation between these charges, produced by the E2D substitution, would be sufficient to explain the 65× reduced affinity of the E2D N-terminus for the pore. Finally, our studies support a multi-step, multi-site N-type inactivation model where the N-terminus interacts deep within the pore in an extended like structure placing the most N-terminal residues 35% of the way across the electric field in the pore blocked

  17. Barley polyamine oxidase: Characterisation and analysis of the cofactor and the N-terminal amino acid sequence

    DEFF Research Database (Denmark)

    Radova, A.; Sebela, M.; Galuszka, P.

    2001-01-01

    was further purified to a final homogeneity (by the criteria of isoelectric focusing and SDS-PAGE) using techniques of low pressure chromatography followed by two FPLC steps. The purified yellow enzyme showed visible absorption maxima of a flavoprotein at 380 and 450 nm: the presence of FAD as the cofactor...... was further confirmed by measuring the fluorescence spectra, Barley PAO is an acidic protein (pI 5.4) containing 3% of neutral sugars: its molecular mass determined by SDS-PAGE was 56 kDa, whilst gel permeation chromatography revealed the higher value of 76 kDa. The N-terminal amino acid sequence of barley...

  18. Mutational analysis of Escherichia coli elongation factor Tu in search of a role for the N-terminal region

    DEFF Research Database (Denmark)

    Mansilla, Francisco; Knudsen, Charlotte Rohde; Laurberg, M

    1998-01-01

    We have mutated lysine 2 and arginine 7 in elongation factor Tu from Escherichia coli separately either to alanine or glutamic acid. The aim of the work was to reveal the possible interactions between the conserved N-terminal part of the molecule, which is rich in basic residues and aminoacyl...... this activity. Furthermore, arginine 7 seems to play a role in regulating the binding of GTP. The three-dimensional structure of the ternary complex, EF-Tu:GTP:Phe-tRNAPhe, involving Thermus aquaticus EF-Tu and yeast Phe-tRNA(Phe), shows that Arg7 is in a position which permits salt bridge formation with Asp284...

  19. N-terminal pro-brain natriuretic peptide levels associated with severe hand, foot and mouth disease

    OpenAIRE

    Deng, Hui-Ling; Zhang, Yu-Feng; Li, Ya-Ping; Zhang, Yu; Xie, Yan; Wang, Jun; Wang, Xiao-Yan; Dang, Shuang-Suo

    2016-01-01

    Background Severe hand, foot, and mouth disease (HFMD) is sometimes associated with serious complications such as acute heart failure that can cause substantial child mortality. N-terminal pro-brain natriuretic peptide (NT-proBNP) is a sensitive and specific biomarker of congestive heart failure. The aim of this study was to use plasma NT-proBNP levels to establish the severity of childhood HFMD. Methods A retrospective study was performed in 128 Chinese patients with severe HFMD and 88 patie...

  20. N-TERMINAL PRO-BRAIN NATRIURETIC PEPTIDE (NT-PROBNP) SERUM CONCENTRATIONS IN APPARENTLY HEALTHY BOSNIAN WOMEN

    OpenAIRE

    Hadžović-Džuvo, Almira; Kučukalić-Selimović, Elma; Nakaš-Ićindić, Emina; Zaćiragić, Asija; Dražeta, Zdenka

    2007-01-01

    Brain natriuretic peptide (BNP) is a cardiac hormone secreted predominantly from the ventricles. This hormone is produced as pre-prohormone BNP (pro BNP), than cleaved by corine to biologically active 32-aminoacid BNP and non-biologically active N-terminal-pro brain natriuretic peptide (NTproBNP). NTproBNP has been found to be a useful marker for the diagnosis of heart failure and left ventricular systolic dysfunction. Recent studies showed that concentration of BNP and NTproBNP predict cardi...

  1. Prognostic usefulness of anemia and N-terminal pro-brain natriuretic peptide in outpatients with systolic heart failure

    DEFF Research Database (Denmark)

    Schou, Morten; Gustafsson, Finn; Kistorp, Caroline N

    2007-01-01

    N-terminal pro-brain natriuretic peptide (NT-pro-BNP) and anemia are predictors of outcome in systolic heart failure. It is currently unclear how these 2 markers interact in particular with regard to the prognostic information carried by each risk marker. We therefore tested the hypothesis...... that anemia (World Health Organization criteria, hemoglobin levels ... prospectively at the baseline visit to our heart failure clinic (inclusion criterion left ventricular ejection fraction anemia was 27%. In a multivariate logistic regression model, anemia (p = 0...

  2. Isolation of three allergenic fractions of the major allergen from Olea europea pollen and N-terminal amino acid sequence.

    Science.gov (United States)

    Villalba, M; López-Otín, C; Martín-Orozco, E; Monsalve, R I; Palomino, P; Lahoz, C; Rodríguez, R

    1990-10-30

    A method to isolate the major allergen from olive pollen (Ole e I) in high yield is described. The allergenic fraction has been separated into 3 subfractions by reverse-phase HPLC. All these fractions were reactive to allergic sera from olive-sensitized patients, giving similar responses. No significant differences were observed between the amino acid compositions of these three proteins. The amino acid sequence of the first 27 amino acid residues from the N-terminal end is given. No homologies have been detected between Ole e I and other known allergens obtained from pollen.

  3. Curious Sex Ratios and Cytoplasmic Genes

    Indian Academy of Sciences (India)

    formation of sperm from mat,!re male germline cells most cytoplasm is lost, including al/ endosym- bionts. In contrast, when eggs are formed from mature female germ line cells, they retain a signi- ficant amount of cytoplasm, and this is likely to contain endosymbionts. As a con- sequence, individuals appearing at each new.

  4. Immunogenetic markers associated with a naturally acquired humoral immune response against an N-terminal antigen of Plasmodium vivax merozoite surface protein 1 (PvMSP-1).

    Science.gov (United States)

    Cassiano, Gustavo Capatti; Furini, Adriana A C; Capobianco, Marcela P; Storti-Melo, Luciane M; Almeida, Maria E; Barbosa, Danielle R L; Póvoa, Marinete M; Nogueira, Paulo A; Machado, Ricardo L D

    2016-06-03

    Humoral immune responses against proteins of asexual blood-stage malaria parasites have been associated with clinical immunity. However, variations in the antibody-driven responses may be associated with a genetic component of the human host. The objective of the present study was to evaluate the influence of co-stimulatory molecule gene polymorphisms of the immune system on the magnitude of the humoral immune response against a Plasmodium vivax vaccine candidate antigen. Polymorphisms in the CD28, CTLA4, ICOS, CD40, CD86 and BLYS genes of 178 subjects infected with P. vivax in an endemic area of the Brazilian Amazon were genotyped by polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP). The levels of IgM, total IgG and IgG subclasses specific for ICB2-5, i.e., the N-terminal portion of P. vivax merozoite surface protein 1 (PvMSP-1), were determined by enzyme-linked immuno assay. The associations between the polymorphisms and the antibody response were assessed by means of logistic regression models. After correcting for multiple testing, the IgG1 levels were significantly higher in individuals recessive for the single nucleotide polymorphism rs3116496 in CD28 (p = 0.00004). Furthermore, the interaction between CD28 rs35593994 and BLYS rs9514828 had an influence on the IgM levels (p = 0.0009). The results of the present study support the hypothesis that polymorphisms in the genes of co-stimulatory components of the immune system can contribute to a natural antibody-driven response against P. vivax antigens.

  5. The pestivirus N terminal protease N(pro redistributes to mitochondria and peroxisomes suggesting new sites for regulation of IRF3 by N(pro..

    Directory of Open Access Journals (Sweden)

    Matthew Jefferson

    Full Text Available The N-terminal protease of pestiviruses, N(pro is a unique viral protein, both because it is a distinct autoprotease that cleaves itself from the following polyprotein chain, and also because it binds and inactivates IRF3, a central regulator of interferon production. An important question remains the role of N(pro in the inhibition of apoptosis. In this study, apoptotic signals induced by staurosporine, interferon, double stranded RNA, sodium arsenate and hydrogen peroxide were inhibited by expression of wild type N(pro, but not by mutant protein N(pro C112R, which we show is less efficient at promoting degradation of IRF3, and led to the conclusion that N(pro inhibits the stress-induced intrinsic mitochondrial pathway through inhibition of IRF3-dependent Bax activation. Both expression of N(pro and infection with Bovine Viral Diarrhea Virus (BVDV prevented Bax redistribution and mitochondrial fragmentation. Given the role played by signaling platforms during IRF3 activation, we have studied the subcellular distribution of N(pro and we show that, in common with many other viral proteins, N(pro targets mitochondria to inhibit apoptosis in response to cell stress. N(pro itself not only relocated to mitochondria but in addition, both N(pro and IRF3 associated with peroxisomes, with over 85% of N(pro puncta co-distributing with PMP70, a marker for peroxisomes. In addition, peroxisomes containing N(pro and IRF3 associated with ubiquitin. IRF3 was degraded, whereas N(pro accumulated in response to cell stress. These results implicate mitochondria and peroxisomes as new sites for IRF3 regulation by N(pro, and highlight the role of these organelles in the anti-viral pathway.

  6. The pestivirus N terminal protease N(pro) redistributes to mitochondria and peroxisomes suggesting new sites for regulation of IRF3 by N(pro.).

    Science.gov (United States)

    Jefferson, Matthew; Whelband, Matthew; Mohorianu, Irina; Powell, Penny P

    2014-01-01

    The N-terminal protease of pestiviruses, N(pro) is a unique viral protein, both because it is a distinct autoprotease that cleaves itself from the following polyprotein chain, and also because it binds and inactivates IRF3, a central regulator of interferon production. An important question remains the role of N(pro) in the inhibition of apoptosis. In this study, apoptotic signals induced by staurosporine, interferon, double stranded RNA, sodium arsenate and hydrogen peroxide were inhibited by expression of wild type N(pro), but not by mutant protein N(pro) C112R, which we show is less efficient at promoting degradation of IRF3, and led to the conclusion that N(pro) inhibits the stress-induced intrinsic mitochondrial pathway through inhibition of IRF3-dependent Bax activation. Both expression of N(pro) and infection with Bovine Viral Diarrhea Virus (BVDV) prevented Bax redistribution and mitochondrial fragmentation. Given the role played by signaling platforms during IRF3 activation, we have studied the subcellular distribution of N(pro) and we show that, in common with many other viral proteins, N(pro) targets mitochondria to inhibit apoptosis in response to cell stress. N(pro) itself not only relocated to mitochondria but in addition, both N(pro) and IRF3 associated with peroxisomes, with over 85% of N(pro) puncta co-distributing with PMP70, a marker for peroxisomes. In addition, peroxisomes containing N(pro) and IRF3 associated with ubiquitin. IRF3 was degraded, whereas N(pro) accumulated in response to cell stress. These results implicate mitochondria and peroxisomes as new sites for IRF3 regulation by N(pro), and highlight the role of these organelles in the anti-viral pathway.

  7. Analysis of the distribution of charged residues in the N-terminal region of signal sequences: implications for protein export in prokaryotic and eukaryotic cells.

    OpenAIRE

    von Heijne, G

    1984-01-01

    A statistical analysis of the distribution of charged residues in the N-terminal region of 39 prokaryotic and 134 eukaryotic signal sequences reveals a remarkable similarity between the two samples, both in terms of net charge and in terms of the position of charged residues within the N-terminal region, and suggests that the formyl group on Metf is not removed in prokaryotic signal sequences.

  8. Cytoplasmic Streaming - Skylab Student Experiment ED-63

    Science.gov (United States)

    1973-01-01

    This chart describes the Skylab student experiment (ED-63), Cytoplasmic Streaming, proposed by Cheryl A. Peitz of Arapahoe High School, Littleton, Colorado. Experiment ED-63 was to observe the effect of zero-gravity on cytoplasmic streaming in the aquatic plant named Elodea, commonly called water weed or water thyme. The phenomenon of cytoplasmic streaming is not well understood, but it is recognized as the circulation mechanism of the internal materials or cytoplasm of a cell. Cytoplasm is a gelatinous substance that has the ability to change its viscosity and flow, carrying various cell materials with it. The activity can be stimulated by sunlight or heat. In March 1972, NASA and the National Science Teachers Association selected 25 experiment proposals for flight on Skylab. Science advisors from the Marshall Space Flight Center aided and assisted the students in developing the proposals for flight on Skylab.

  9. Linked production of pyroglutamate-modified proteins via self-cleavage of fusion tags with TEV protease and autonomous N-terminal cyclization with glutaminyl cyclase in vivo.

    Directory of Open Access Journals (Sweden)

    Yan-Ping Shih

    Full Text Available Overproduction of N-terminal pyroglutamate (pGlu-modified proteins utilizing Escherichia coli or eukaryotic cells is a challenging work owing to the fact that the recombinant proteins need to be recovered by proteolytic removal of fusion tags to expose the N-terminal glutaminyl or glutamyl residue, which is then converted into pGlu catalyzed by the enzyme glutaminyl cyclase. Herein we describe a new method for production of N-terminal pGlu-containing proteins in vivo via intracellular self-cleavage of fusion tags by tobacco etch virus (TEV protease and then immediate N-terminal cyclization of passenger target proteins by a bacterial glutaminyl cyclase. To combine with the sticky-end PCR cloning strategy, this design allows the gene of target proteins to be efficiently inserted into the expression vector using two unique cloning sites (i.e., SnaB I and Xho I, and the soluble and N-terminal pGlu-containing proteins are then produced in vivo. Our method has been successfully applied to the production of pGlu-modified enhanced green fluorescence protein and monocyte chemoattractant proteins. This design will facilitate the production of protein drugs and drug target proteins that possess an N-terminal pGlu residue required for their physiological activities.

  10. N-Terminal Domains in Two-Domain Proteins Are Biased to Be Shorter and Predicted to Fold Faster Than Their C-Terminal Counterparts

    Directory of Open Access Journals (Sweden)

    Etai Jacob

    2013-04-01

    Full Text Available Computational analysis of proteomes in all kingdoms of life reveals a strong tendency for N-terminal domains in two-domain proteins to have shorter sequences than their neighboring C-terminal domains. Given that folding rates are affected by chain length, we asked whether the tendency for N-terminal domains to be shorter than their neighboring C-terminal domains reflects selection for faster-folding N-terminal domains. Calculations of absolute contact order, another predictor of folding rate, provide additional evidence that N-terminal domains tend to fold faster than their neighboring C-terminal domains. A possible explanation for this bias, which is more pronounced in prokaryotes than in eukaryotes, is that faster folding of N-terminal domains reduces the risk for protein aggregation during folding by preventing formation of nonnative interdomain interactions. This explanation is supported by our finding that two-domain proteins with a shorter N-terminal domain are much more abundant than those with a shorter C-terminal domain.

  11. Characterization of cytoplasmic male sterility of rice with Lead Rice cytoplasm in comparison with that with Chinsurah Boro II cytoplasm.

    Science.gov (United States)

    Itabashi, Etsuko; Kazama, Tomohiko; Toriyama, Kinya

    2009-02-01

    Rice with LD-type cytoplasmic male sterility (CMS) possesses the cytoplasm of 'Lead Rice' and its fertility is recovered by a nuclear fertility restorer gene Rf1. Rf1 promotes processing of a CMS-associated mitochondrial RNA of atp6-orf79, which consists of atp6 and orf79, in BT-CMS with the cytoplasm of 'Chinsurah Boro II'. In this study, we found that LD-cytoplasm contained a sequence variant of orf79 downstream of atp6. Northern blot analysis showed that atp6-orf79 RNA of LD-cytoplasm was co-transcribed and was processed in the presence of Rf1 in the same manner as in BT-cytoplasm. Western blot analysis showed that the ORF79 peptide did not accumulate in an LD-CMS line, while ORF79 accumulated in a BT-CMS line and was diminished by Rf1. These results suggest that accumulation of ORF79 is not the cause of CMS in LD-cytoplasm and the mechanism of male-sterility induction/fertility restoration in LD-CMS is different from that in BT-CMS.

  12. The role of the N-terminal loop in the function of the colicin E7 nuclease domain

    DEFF Research Database (Denmark)

    Czene, Anikó; Németh, Eszter; Zóka, István G.

    2013-01-01

    Colicin E7 (ColE7) is a metallonuclease toxin of Escherichia coli belonging to the HNH superfamily of nucleases. It contains highly conserved amino acids in its HHX14NX8HX3H ββα-type metal ion binding C-terminal active centre. However, the proximity of the arginine at the N-terminus of the nuclease...... domain of ColE7 (NColE7, 446–576) is necessary for the hydrolytic activity. This poses a possibility of allosteric activation control in this protein. To obtain more information on this phenomenon, two protein mutants were expressed, i.e. four and 25 N-terminal amino acids were removed from NColE7....... The effect of the N-terminal truncation on the Zn2+ ion and DNA binding as well as on the activity was investigated in this study by mass spectrometry, synchrotron-radiation circular dichroism and fluorescence spectroscopy and agarose gel mobility shift assays. The dynamics of protein backbone movement...

  13. The Dahlia mosaic virus gene VI product N-terminal region is involved in self-association.

    Science.gov (United States)

    Raikhy, Gaurav; Krause, Charles; Leisner, Scott

    2011-07-01

    The genome of the floriculture pathogen Dahlia mosaic caulimovirus (DMV) encodes six open reading frames. Generally, caulimovirus gene VI products (P6s) are thought to be multifunctional proteins required for viral infection and it is likely that self-association is required for some of these functions. In this study, yeast two-hybrid and maltose binding protein (MBP) pull-down assays indicated that full-length DMV P6 specifically self-associates. Further analyses indicated that only the DMV P6 N-terminal region, consisting of 115 amino acids, interacts with full-length P6 and with itself. This distinguishes the DMV P6 from its Cauliflower mosaic virus counterpart, which contains four regions involved in self-association. Thus, our results suggest that each caulimovirus P6 may possess a unique pattern of protein-protein interactions. Bioinformatic tools identified a putative nuclear exclusion signal located between amino acid residues 10-20, suggesting another possible function for the P6 N-terminal region. Copyright © 2011 Elsevier B.V. All rights reserved.

  14. N-Terminal His-Tagged AtTPR7 Interactions with Hsp70 and Hsp90 Proteins

    Directory of Open Access Journals (Sweden)

    ANANDAYU PRADITA

    2014-12-01

    Full Text Available Post-translational protein import into organelles is an important process to maintain cellular functions. During preprotein transport in the cytosol, chaperones, such as heat shock protein 70 (Hsp70 and heat shock protein 90 (Hsp90, are functioning to prevent aggregation and to maintain the correct protein folding of preproteins. This research was conducted in order to understand the chaperone-mediated, post-translational import of preproteins into the endoplasmic reticulum of Arabidopsis thaliana. AtTPR7 (Arabidopsis thaliana Tetratrico Peptide Repeat 7 is found in the endoplasmic reticulum and contains TPR domain, which mediates protein interaction with cytosolic Hsp70 and Hsp90. In this study, recombinant AtTPR7 was expressed in E. coli BL21 (DE3-RIPL cells and purified using an N-terminal His-tag. In order to study the interactions of the protein with the chaperones, we used pulldown and Western blot assays. We could thereby show that the N-terminally His-tagged AtTPR7 protein interacted with Hsp70 and Hsp90.

  15. Identification of a Major Dimorphic Region in the Functionally Critical N-Terminal ID1 Domain of VAR2CSA.

    Directory of Open Access Journals (Sweden)

    Justin Doritchamou

    Full Text Available The VAR2CSA protein of Plasmodium falciparum is transported to and expressed on the infected erythrocyte surface where it plays a key role in placental malaria (PM. It is the current leading candidate for a vaccine to prevent PM. However, the antigenic polymorphism integral to VAR2CSA poses a challenge for vaccine development. Based on detailed analysis of polymorphisms in the sequence of its ligand-binding N-terminal region, currently the main focus for vaccine development, we assessed var2csa from parasite isolates infecting pregnant women. The results reveal for the first time the presence of a major dimorphic region in the functionally critical N-terminal ID1 domain. Parasite isolates expressing VAR2CSA with particular motifs present within this domain are associated with gravidity- and parasite density-related effects. These observations are of particular interest in guiding efforts with respect to optimization of the VAR2CSA-based vaccines currently under development.

  16. Functional characterization of heat-shock protein 90 from Oryza sativa and crystal structure of its N-terminal domain.

    Science.gov (United States)

    Raman, Swetha; Suguna, Kaza

    2015-06-01

    Heat-shock protein 90 (Hsp90) is an ATP-dependent molecular chaperone that is essential for the normal functioning of eukaryotic cells. It plays crucial roles in cell signalling, cell-cycle control and in maintaining proteome integrity and protein homeostasis. In plants, Hsp90s are required for normal plant growth and development. Hsp90s are observed to be upregulated in response to various abiotic and biotic stresses and are also involved in immune responses in plants. Although there are several studies elucidating the physiological role of Hsp90s in plants, their molecular mechanism of action is still unclear. In this study, biochemical characterization of an Hsp90 protein from rice (Oryza sativa; OsHsp90) has been performed and the crystal structure of its N-terminal domain (OsHsp90-NTD) was determined. The binding of OsHsp90 to its substrate ATP and the inhibitor 17-AAG was studied by fluorescence spectroscopy. The protein also exhibited a weak ATPase activity. The crystal structure of OsHsp90-NTD was solved in complex with the nonhydrolyzable ATP analogue AMPPCP at 3.1 Å resolution. The domain was crystallized by cross-seeding with crystals of the N-terminal domain of Hsp90 from Dictyostelium discoideum, which shares 70% sequence identity with OsHsp90-NTD. This is the second reported structure of a domain of Hsp90 from a plant source.

  17. Characterization of four new monoclonal antibodies against the distal N-terminal region of PrPc

    Directory of Open Access Journals (Sweden)

    Alessandro Didonna

    2015-03-01

    Full Text Available Prion diseases are a group of fatal neurodegenerative disorders that affect humans and animals. They are characterized by the accumulation in the central nervous system of a pathological form of the host-encoded prion protein (PrPC. The prion protein is a membrane glycoprotein that consists of two domains: a globular, structured C-terminus and an unstructured N-terminus. The N-terminal part of the protein is involved in different functions in both health and disease. In the present work we discuss the production and biochemical characterization of a panel of four monoclonal antibodies (mAbs against the distal N-terminus of PrPC using a well-established methodology based on the immunization of Prnp0/0 mice. Additionally, we show their ability to block prion (PrPSc replication at nanomolar concentrations in a cell culture model of prion infection. These mAbs represent a promising tool for prion diagnostics and for studying the physiological role of the N-terminal domain of PrPC.

  18. Neurospora tryptophan synthase: N-terminal analysis and the sequence of the pyridoxal phosphate active site peptide

    International Nuclear Information System (INIS)

    Pratt, M.L.; Hsu, P.Y.; DeMoss, J.A.

    1986-01-01

    Tryptophan synthase (TS), which catalyzes the final step of tryptophan biosynthesis, is a multifunctional protein requiring pyridoxal phosphate (B6P) for two of its three distinct enzyme activities. TS from Neurospora has a blocked N-terminal, is a homodimer of 150 KDa and binds one mole of B6P per mole of subunit. The authors shown the N-terminal residue to be acyl-serine. The B6P-active site of holoenzyme was labelled by reduction of the B6P-Schiff base with [ 3 H]-NaBH 4 , and resulted in a proportionate loss of activity in the two B6P-requiring reactions. SDS-polyacrylamide gel electrophoresis of CNBr-generated peptides showed the labelled, active site peptide to be 6 KDa. The sequence of this peptide, purified to apparent homogeneity by a combination of C-18 reversed phase and TSK gel filtration HPLC is: gly-arg-pro-gly-gln-leu-his-lys-ala-glu-arg-leu-thr-glu-tyr-ala-gly-gly-ala-gln-ile-xxx-leu-lys-arg-glu-asp-leu-asn-his-xxx-gly-xxx-his-/sub ***/-ile-asn-asn-ala-leu. Although four residues (xxx, /sub ***/) are unidentified, this peptide is minimally 78% homologous with the corresponding peptide from yeast TS, in which residue (/sub ***/) is the lysine that binds B6P

  19. The N-terminal pro region mediates retention of unprocessed type-I PME in the Golgi apparatus.

    Science.gov (United States)

    Wolf, Sebastian; Rausch, Thomas; Greiner, Steffen

    2009-05-01

    The pectin matrix of the cell wall, a complex and dynamic network, impacts on cell growth, cell shape and signaling processes. A hallmark of pectin structure is the methylesterification status of its major component, homogalacturonan (HGA), which affects the biophysical properties and enzymatic turnover of pectin. The pectin methylesterases (PMEs), responsible for de-esterification, encompass a protein family of more than 60 isoforms in the Arabidopsis genome. The pivotal role of PME in the regulation of pectin properties also requires tight control at the post-translational level. Type-I PMEs are characterized by an N-terminal pro region, which exhibits homology with pectin methylesterase inhibitors (PMEIs). Here, we demonstrate that the proteolytic removal of the N-terminal pro region depends on conserved basic tetrad motifs, occurs in the early secretory pathway, and is required for the subsequent export of the PME core domain to the cell wall. In addition, we demonstrate the involvement of AtS1P, a subtilisin-like protease, in Arabidopsis PME processing. Our results indicate that the pro region operates as an effective retention mechanism, keeping unprocessed PME in the Golgi apparatus. Consequently, pro-protein processing could constitute a post-translational mechanism regulating PME activity.

  20. Structures of the N-terminal modules imply large domain motions during catalysis by methionine synthase.

    Science.gov (United States)

    Evans, John C; Huddler, Donald P; Hilgers, Mark T; Romanchuk, Gail; Matthews, Rowena G; Ludwig, Martha L

    2004-03-16

    B(12)-dependent methionine synthase (MetH) is a large modular enzyme that utilizes the cobalamin cofactor as a methyl donor or acceptor in three separate reactions. Each methyl transfer occurs at a different substrate-binding domain and requires a different arrangement of modules. In the catalytic cycle, the cobalamin-binding domain carries methylcobalamin to the homocysteine (Hcy) domain to form methionine and returns cob(I)alamin to the folate (Fol) domain for remethylation by methyltetrahydrofolate (CH(3)-H(4)folate). Here, we describe crystal structures of a fragment of MetH from Thermotoga maritima comprising the domains that bind Hcy and CH(3)-H(4)folate. These substrate-binding domains are (beta alpha)(8) barrels packed tightly against one another with their barrel axes perpendicular. The properties of the domain interface suggest that the two barrels remain associated during catalysis. The Hcy and CH(3)-H(4)folate substrates are bound at the C termini of their respective barrels in orientations that position them for reaction with cobalamin, but the two active sites are separated by approximately 50 A. To complete the catalytic cycle, the cobalamin-binding domain must travel back and forth between these distant active sites.

  1. Hydrodynamic property of the cytoplasm is sufficient to mediate cytoplasmic streaming in the Caenorhabiditis elegans embryo

    Science.gov (United States)

    Niwayama, Ritsuya; Shinohara, Kyosuke; Kimura, Akatsuki

    2011-01-01

    Cytoplasmic streaming is a type of intracellular transport widely seen in nature. Cytoplasmic streaming in Caenorhabditis elegans at the one-cell stage is bidirectional; the flow near the cortex (“cortical flow”) is oriented toward the anterior, whereas the flow in the central region (“cytoplasmic flow”) is oriented toward the posterior. Both cortical flow and cytoplasmic flow depend on non-muscle-myosin II (NMY-2), which primarily localizes in the cortex. The manner in which NMY-2 proteins drive cytoplasmic flow in the opposite direction from remote locations has not been fully understood. In this study, we demonstrated that the hydrodynamic properties of the cytoplasm are sufficient to mediate the forces generated by the cortical myosin to drive bidirectional streaming throughout the cytoplasm. We quantified the flow velocities of cytoplasmic streaming using particle image velocimetry (PIV) and conducted a three-dimensional hydrodynamic simulation using the moving particle semiimplicit method. Our simulation quantitatively reconstructed the quantified flow velocity distribution resolved through PIV analysis. Furthermore, our PIV analyses detected microtubule-dependent flows during the pronuclear migration stage. These flows were reproduced via hydrodynamic interactions between moving pronuclei and the cytoplasm. The agreement of flow dynamics in vivo and in simulation indicates that the hydrodynamic properties of the cytoplasm are sufficient to mediate cytoplasmic streaming in C. elegans embryos. PMID:21730185

  2. Preventive and therapeutic effects of thymosin β4 N-terminal fragment Ac-SDKP in the bleomycin model of pulmonary fibrosis.

    Science.gov (United States)

    Conte, Enrico; Fagone, Evelina; Gili, Elisa; Fruciano, Mary; Iemmolo, Maria; Pistorio, Maria Provvidenza; Impellizzeri, Daniela; Cordaro, Marika; Cuzzocrea, Salvatore; Vancheri, Carlo

    2016-06-07

    In this study, the bleomycin model of pulmonary fibrosis was utilized to investigate putative anti-fibrotic activity of Ac-SDKP in vivo. Male CD-1 mice received intra-tracheal bleomycin (BLEO, 1 mg/kg) instillation in the absence or presence of Ac-SDKP (a dose of 0.6 mg/kg delivered intra-peritoneally on the day of BLEO treatment, d0, followed by bi-weekly additional doses). To evaluate therapeutic effects in a subset of mice, Ac-SDKP was administered one week after BLEO instillation (d7). Animals were sacrificed at one, two, or three weeks later. Measurement of fluid and collagen content in the lung, Broncho Alveolar Lavage Fluid (BALF) analysis, lung histology, immunohistochemistry (IHC), and molecular analysis were performed. Compared to BLEO-treated mice, animals that received also Ac-SDKP (at both d0 and d7) had significantly decreased mortality, weight loss, inflammation (edema, and leukocyte lung infiltration), lung damage (histological evidence of lung injury), and fibrosis (collagen histological staining and soluble collagen content in the lung) at up to 21 days. Moreover, IHC and quantitative RT-PCR results demonstrated a significant decrease in BLEO-induced IL-17 and TGF-β expression in lung tissue. Importantly, α-SMA expression, the hallmark of myofibroblast differentiation, was also decreased. This is the first report showing not only a preventive protective role of Ac-SDKP but also its significant therapeutic effects in the bleomycin model of pulmonary fibrosis, thus supporting further preclinical and clinical studies.

  3. The early noncoding region of human papillomavirus type 16 is regulated by cytoplasmic polyadenylation factors

    DEFF Research Database (Denmark)

    Glahder, Jacob-Andreas Harald; Kristiansen, Karen; Durand, Marjorie

    2010-01-01

    (CPEs). We show here that a fragment of the early 3'end comprising four of the five CPE-like regions when inserted downstream of a reporter gene confers regulation of the gene expression. A key protein involved in cytoplasmic polyadenylation is CPEB. We show that the human CPEB1 can repress the activity...... of the reporter construct containing the HPV-16 early sequences. This repression can be counteracted by a human cytoplasmic poly(A) polymerase, hGLD-2 fused to CPEB1. The hGLD-2/CPEB1 fusion protein facilitates furthermore poly(A) elongation of early HPV transcripts....

  4. Fragmented Authoritarianism or Integrated Fragmentation

    DEFF Research Database (Denmark)

    Brødsgaard, Kjeld Erik

    proved their influence by obstructing the creation of new ministries and regulatory commissions that would limit their powers. The heads of these business groups often outrank their counterparts in state administrative organs and bureaus that are supposed to regulate their activities. The increased role...... of these business leaders prompts the question of whether we are seeing the development of distinct interest groups that could challenge Party and state authority and create a fragmented polity. However, through the nomenklatura system the Party has an important instrument of control to wield over business groups....... Through this system the Party controls the appointment and promotion of the heads of the most important state-owned enterprises. The nomenklatura system also enables the Party to rotate leaders in big business from a position as CEO in one company to a similar position in another state-owned company...

  5. Deletion of a 197-Amino-Acid Region in the N-Terminal Domain of Spike Protein Attenuates Porcine Epidemic Diarrhea Virus in Piglets.

    Science.gov (United States)

    Hou, Yixuan; Lin, Chun-Ming; Yokoyama, Masaru; Yount, Boyd L; Marthaler, Douglas; Douglas, Arianna L; Ghimire, Shristi; Qin, Yibin; Baric, Ralph S; Saif, Linda J; Wang, Qiuhong

    2017-07-15

    We previously isolated a porcine epidemic diarrhea virus (PEDV) strain, PC177, by blind serial passaging of the intestinal contents of a diarrheic piglet in Vero cell culture. Compared with the highly virulent U.S. PEDV strain PC21A, the tissue culture-adapted PC177 (TC-PC177) contains a 197-amino-acid (aa) deletion in the N-terminal domain of the spike (S) protein. We orally inoculated neonatal, conventional suckling piglets with TC-PC177 or PC21A to compare their pathogenicities. Within 7 days postinoculation, TC-PC177 caused mild diarrhea and lower fecal viral RNA shedding, with no mortality, whereas PC21A caused severe clinical signs and 55% mortality. To investigate whether infection with TC-PC177 can induce cross-protection against challenge with a highly virulent PEDV strain, all the surviving piglets were challenged with PC21A at 3 weeks postinoculation. Compared with 100% protection in piglets initially inoculated with PC21A, 88% and 100% TC-PC177- and mock-inoculated piglets had diarrhea following challenge, respectively, indicating incomplete cross-protection. To investigate whether this 197-aa deletion was the determinant for the attenuation of TC-PC177, we generated a mutant (icPC22A-S1Δ197) bearing the 197-aa deletion from an infectious cDNA clone of the highly virulent PEDV PC22A strain (infectious clone PC22A, icPC22A). In neonatal gnotobiotic pigs, the icPC22A-S1Δ197 virus caused mild to moderate diarrhea, lower titers of viral shedding, and no mortality, whereas the icPC22A virus caused severe diarrhea and 100% mortality. Our data indicate that deletion of this 197-aa fragment in the spike protein can attenuate a highly virulent PEDV, but the virus may lose important epitopes for inducing robust protective immunity. IMPORTANCE The emerging, highly virulent PEDV strains have caused substantial economic losses worldwide. However, the virulence determinants are not established. In this study, we found that a 197-aa deletion in the N-terminal region

  6. Fractal organization of feline oocyte cytoplasm

    Directory of Open Access Journals (Sweden)

    G De Vico

    2009-06-01

    Full Text Available The present study aimed at verifying whether immature cat oocytes with morphologic irregular cytoplasm display selfsimilar features which can be analytically described by fractal analysis. Original images of oocytes collected by ovariectomy were acquired at a final magnification of 400 X with a CCD video camera connected to an optic microscope. After greyscale thresholding segmentation of cytoplasm, image profiles were submitted to fractal analysis using FANAL++, a program which provided an analytical standard procedure for determining the fractal dimension (FD. The presentation of the oocyte influenced the magnitude of the fractal dimension with the highest FD of 1.91 measured on grey-dark cytoplasm characterized by a highly connected network of lipid droplets and intracellular membranes. Fractal analysis provides an effective quantitative descriptor of the real cytoplasm morphology, which can influence the acquirement of in vitro developmental competence, without introducing any bias or shape approximation and thus contributes to an objective and reliable classification of feline oocytes.

  7. Basolateral localisation of KCNQ1 potassium channels in MDCK cells: molecular identification of an N-terminal targeting motif

    DEFF Research Database (Denmark)

    Jespersen, Thomas; Rasmussen, Hanne B; Grunnet, Morten

    2004-01-01

    of the tyrosine residue at position 51 resulted in a non-polarized steady-state distribution of the channel. The importance of tyrosine 51 in basolateral localisation was emphasized by the fact that a short peptide comprising this tyrosine was able to redirect the p75 neurotrophin receptor, an otherwise apically......KCNQ1 potassium channels are expressed in many epithelial tissues as well as in the heart. In epithelia KCNQ1 channels play an important role in salt and water transport and the channel has been reported to be located apically in some cell types and basolaterally in others. Here we show that KCNQ1...... channels are located basolaterally when expressed in polarised MDCK cells. The basolateral localisation of KCNQ1 is not affected by co-expression of any of the five KCNE beta-subunits. We characterise two independent basolateral sorting signals present in the N-terminal tail of KCNQ1. Mutation...

  8. Structure of the mouse galectin-4 N-terminal carbohydrate-recognition domain reveals the mechanism of oligosaccharide recognition

    Energy Technology Data Exchange (ETDEWEB)

    Krejciríková, Veronika; Pachl, Petr; Fábry, Milan; Malý, Petr; Rezácová, Pavlína; Brynda, Jirí (Czech Academy)

    2011-11-18

    Galectin-4, a member of the tandem-repeat subfamily of galectins, participates in cell-membrane interactions and plays an important role in cell adhesion and modulation of immunity and malignity. The oligosaccharide specificity of the mouse galectin-4 carbohydrate-recognition domains (CRDs) has been reported previously. In this work, the structure and binding properties of the N-terminal domain CRD1 were further investigated and the crystal structure of CRD1 in complex with lactose was determined at 2.1 {angstrom} resolution. The lactose-binding affinity was characterized by fluorescence measurements and two lactose-binding sites were identified: a high-affinity site with a K{sub d} value in the micromolar range (K{sub d1} = 600 {+-} 70 {mu}M) and a low-affinity site with K{sub d2} = 28 {+-} 10 mM.

  9. The Drosophila Microtubule-Associated Protein Mars Stabilizes Mitotic Spindles by Crosslinking Microtubules through Its N-Terminal Region

    Science.gov (United States)

    Zhang, Gang; Beati, Hamze; Nilsson, Jakob; Wodarz, Andreas

    2013-01-01

    Correct segregation of genetic material relies on proper assembly and maintenance of the mitotic spindle. How the highly dynamic microtubules (MTs) are maintained in stable mitotic spindles is a key question to be answered. Motor and non-motor microtubule associated proteins (MAPs) have been reported to stabilize the dynamic spindle through crosslinking adjacent MTs. Mars, a novel MAP, is essential for the early development of Drosophila embryos. Previous studies showed that Mars is required for maintaining an intact mitotic spindle but did not provide a molecular mechanism for this function. Here we show that Mars is able to stabilize the mitotic spindle in vivo. Both in vivo and in vitro data reveal that the N-terminal region of Mars functions in the stabilization of the mitotic spindle by crosslinking adjacent MTs. PMID:23593258

  10. Crystal structure of the N-terminal domain of human SIRT7 reveals a three-helical domain architecture.

    Science.gov (United States)

    Priyanka, Anu; Solanki, Vipul; Parkesh, Raman; Thakur, Krishan Gopal

    2016-10-01

    Human SIRT7 is an NAD(+) dependent deacetylase, which belongs to sirtuin family of proteins. SIRT7, like other sirtuins has conserved catalytic domain and is flanked by N- and C-terminal domains reported to play vital functional roles. Here, we report the crystal structure of the N-terminal domain of human SIRT7 (SIRT7(NTD) ) at 2.3 Å resolution as MBP-SIRT7(NTD) fusion protein. SIRT7(NTD) adopts three-helical domain architecture and comparative structural analyses suggest similarities to some DNA binding motifs and transcription regulators. We also report here the importance of N- and C-terminal domains in soluble expression of SIRT7. Proteins 2016; 84:1558-1563. © 2016 Wiley Periodicals, Inc. © 2016 Wiley Periodicals, Inc.

  11. Detection of left ventricular enlargement and impaired systolic function with plasma N-terminal pro brain natriuretic peptide concentrations

    DEFF Research Database (Denmark)

    Grønning, Bjørn Aaris; Nilsson, Jens C.; Søndergaard, Lars

    2002-01-01

    BACKGROUND: Brain- and N-terminal pro brain natriuretic peptide (NT-proBNP) have been identified as promising markers for heart failure. However, previous studies have revealed that they may hold insufficient diagnostic power for implementation into clinical practice because of a significant...... overlap in the range of plasma levels between healthy subjects and subjects with heart failure. We hypothesized that imprecision of the reference method (ie, the echocardiographic evaluation of left ventricular [LV] function) may have affected results from those earlier studies. We therefore wanted...... to investigate the diagnostic potential of NT-proBNP with magnetic resonance imaging as the reference method for the cardiac measurements. METHODS: Forty-eight patients with stable symptomatic heart failure in New York Heart Association functional classifications II to IV were examined once with blood samples...

  12. N-terminal tagging of human P2X7 receptor disturbs calcium influx and dye uptake

    DEFF Research Database (Denmark)

    Dreisig, Karin; Kristensen, Nikolaj Pagh; Dommer, Maja Wallentin

    2018-01-01

    uptake in response to BzATP stimulation in transfected cells. We found that tagging at the N-terminal of the human P2X7 receptor with the enhanced green fluorescent protein (eGFP) disturbed channel opening and pore formation despite intact surface expression. A triple hemagglutinin (3HA) fused to the N......The P2X7 receptor is a frequently studied member of the purinergic receptor family signalling via channel opening and membrane pore formation. Fluorescent imaging is an important molecular method for studying cellular receptor expression and localization. Fusion of receptors to fluorescent proteins...... might cause major functional changes and requires careful functional evaluation such as has been done for the rat P2X7 receptor. This study examines fusion constructs of the human P2X7 receptor. We assessed surface expression, channel opening with calcium influx, and pore formation using YO-PRO-1 dye...

  13. N-terminal pro-atrial natriuretic peptide response to acute exercise in depressed patients and healthy controls

    DEFF Research Database (Denmark)

    Krogh, Jesper; Ströhle, Andreas; Westrin, Asa

    2011-01-01

    BACKGROUND: The dysfunction of hypothalamic-pituitary-adrenal (HPA) axis in major depression includes hyperactivity and reduced feedback inhibition. Atrial natriuretic peptide (ANP) is able to reduce the HPA-axis response to stress and has an anxiolytic effect in rodents and humans. We hypothesized...... that patients with depression would have an attenuated N-terminal proANP (NT-proANP) response to acute exercise compared to healthy controls. Secondly, we aimed to assess the effect of antidepressants on NT-proANP response to acute exercise. METHODS: We examined 132 outpatients with mild to moderate depression...... depressed subjects and healthy controls (group×time; F(4,162.9)=10.92; p...

  14. N-terminal pro-atrial natriuretic peptide response to acute exercise in depressed patients and healthy controls

    DEFF Research Database (Denmark)

    Krogh, Jesper; Ströhle, Andreas; Westrin, Asa

    2011-01-01

    BACKGROUND: The dysfunction of hypothalamic-pituitary-adrenal (HPA) axis in major depression includes hyperactivity and reduced feedback inhibition. Atrial natriuretic peptide (ANP) is able to reduce the HPA-axis response to stress and has an anxiolytic effect in rodents and humans. We hypothesized...... that patients with depression would have an attenuated N-terminal proANP (NT-proANP) response to acute exercise compared to healthy controls. Secondly, we aimed to assess the effect of antidepressants on NT-proANP response to acute exercise. METHODS: We examined 132 outpatients with mild to moderate depression...... (ICD-10) and 44 healthy controls, group matched for age, sex, and BMI. We used an incremental bicycle ergometer test as a physical stressor. Blood samples were drawn at rest, at exhaustion, and 15, 30, and 60min post-exercise. RESULTS: The NT-proANP response to physical exercise differed between...

  15. The Drosophila microtubule-associated protein mars stabilizes mitotic spindles by crosslinking microtubules through its N-terminal region.

    Science.gov (United States)

    Zhang, Gang; Beati, Hamze; Nilsson, Jakob; Wodarz, Andreas

    2013-01-01

    Correct segregation of genetic material relies on proper assembly and maintenance of the mitotic spindle. How the highly dynamic microtubules (MTs) are maintained in stable mitotic spindles is a key question to be answered. Motor and non-motor microtubule associated proteins (MAPs) have been reported to stabilize the dynamic spindle through crosslinking adjacent MTs. Mars, a novel MAP, is essential for the early development of Drosophila embryos. Previous studies showed that Mars is required for maintaining an intact mitotic spindle but did not provide a molecular mechanism for this function. Here we show that Mars is able to stabilize the mitotic spindle in vivo. Both in vivo and in vitro data reveal that the N-terminal region of Mars functions in the stabilization of the mitotic spindle by crosslinking adjacent MTs.

  16. The Drosophila microtubule-associated protein mars stabilizes mitotic spindles by crosslinking microtubules through its N-terminal region.

    Directory of Open Access Journals (Sweden)

    Gang Zhang

    Full Text Available Correct segregation of genetic material relies on proper assembly and maintenance of the mitotic spindle. How the highly dynamic microtubules (MTs are maintained in stable mitotic spindles is a key question to be answered. Motor and non-motor microtubule associated proteins (MAPs have been reported to stabilize the dynamic spindle through crosslinking adjacent MTs. Mars, a novel MAP, is essential for the early development of Drosophila embryos. Previous studies showed that Mars is required for maintaining an intact mitotic spindle but did not provide a molecular mechanism for this function. Here we show that Mars is able to stabilize the mitotic spindle in vivo. Both in vivo and in vitro data reveal that the N-terminal region of Mars functions in the stabilization of the mitotic spindle by crosslinking adjacent MTs.

  17. A pathogenicity determinant maps to the N-terminal coat protein region of the Pepino mosaic virus genome.

    Science.gov (United States)

    Duff-Farrier, Celia R A; Bailey, Andy M; Boonham, Neil; Foster, Gary D

    2015-04-01

    Pepino mosaic virus (PepMV) poses a worldwide threat to the tomato industry. Considerable differences at the genetic level allow for the distinction of four main genotypic clusters; however, the basis of the phenotypic outcome is difficult to elucidate. This work reports the generation of wild-type PepMV infectious clones of both EU (mild) and CH2 (aggressive) genotypes, from which chimeric infectious clones were created. Phenotypic analysis in three solanaceous hosts, Nicotiana benthamiana, Datura stramonium and Solanum lycopersicum, indicated that a PepMV pathogenicity determinant mapped to the 3'-terminal region of the genome. Increased aggression was only observed in N. benthamiana, showing that this factor is host specific. The determinant was localized to amino acids 11-26 of the N-terminal coat protein (CP) region; this is the first report of this region functioning as a virulence factor in PepMV. © 2014 BSPP AND JOHN WILEY & SONS LTD.

  18. The chondroitin sulfate A-binding site of the VAR2CSA protein involves multiple N-terminal domains

    DEFF Research Database (Denmark)

    Dahlbäck, Madeleine; Jørgensen, Lars M; Nielsen, Morten A

    2011-01-01

    by a parasite expressed protein named VAR2CSA. A vaccine protecting pregnant women against placental malaria should induce antibodies inhibiting the interaction between VAR2CSA and CSA. Much effort has been put into defining the part of the 350 kDa VAR2CSA protein that is responsible for binding. It has been...... of truncated VAR2CSA proteins. The experiments indicate that the core of the CSA-binding site is situated in three domains, DBL2X-CIDR(PAM) and a flanking domain, located in the N-terminal part of VAR2CSA. Furthermore, recombinant VAR2CSA subfragments containing this region elicit antibodies with high parasite...

  19. Investigation of the N-terminal coding region of MUC7 alterations in dentistry students with and without caries

    Directory of Open Access Journals (Sweden)

    Koç Öztürk L

    2016-06-01

    Full Text Available Human low-molecular weight salivary mucin (MUC7 is a small, secreted glycoprotein coded by MUC7. In the oral cavity, they inhibit the colonization of oral bacteria, including cariogenic ones, by masking their surface adhesions, thus helping saliva to avoid dental caries. The N-terminal domain is important for low-molecular weight (MG2 mucins to contact with oral microorganisms. In this study, we aimed to identify the N-terminal coding region of the MUC7 gene between individuals with and without caries. Forty-four healthy dental students were enrolled in this study; 24 of them were classified to have caries [decayed, missing, filled-teeth (DMFT = 5.6] according to the World Health Organization (WHO criteria, and 20 of them were caries-free (DMFT = 0. Simplified oral hygiene index (OHI-S and gingival index (GI were used to determine the oral hygiene and gingival conditions. Total protein levels and salivary total protein levels and salivary buffer capacity (SBC were determined by Lowry and Ericsson methods. DNA was extracted from peripheral blood cells of all the participants and genotyping was carried out by a polymerase chain reaction (PCR-sequencing method. No statistical differences were found between two groups in the terms of salivary parameters, oral hygiene and gingival conditions. We detected one common single nucleotide polymorphism (SNP that leads to a change of asparagine to lysine at codon 80. This substitution was found in 29.0 and 40.0%, respectively, of the groups with and without caries. No other sequence variations were detected. The SNP found in this study may be a specific polymorphism affecting the Turkish population. Further studies with extended numbers are necessary in order to clarify this finding.

  20. Characterization of an extensin-modifying metalloprotease: N-terminal processing and substrate cleavage pattern of Pectobacterium carotovorum Prt1.

    Science.gov (United States)

    Feng, Tao; Nyffenegger, Christian; Højrup, Peter; Vidal-Melgosa, Silvia; Yan, Kok-Phen; Fangel, Jonatan Ulrik; Meyer, Anne S; Kirpekar, Finn; Willats, William G; Mikkelsen, Jørn D

    2014-12-01

    Compared to other plant cell wall-degrading enzymes, proteases are less well understood. In this study, the extracellular metalloprotease Prt1 from Pectobacterium carotovorum (formerly Erwinia carotovora) was expressed in Escherichia coli and characterized with respect to N-terminal processing, thermal stability, substrate targets, and cleavage patterns. Prt1 is an autoprocessing protease with an N-terminal signal pre-peptide and a pro-peptide which has to be removed in order to activate the protease. The sequential cleavage of the N-terminus was confirmed by mass spectrometry (MS) fingerprinting and N-terminus analysis. The optimal reaction conditions for the activity of Prt1 on azocasein were at pH 6.0, 50 °C. At these reaction conditions, K M was 1.81 mg/mL and k cat was 1.82 × 10(7) U M(-1). The enzyme was relatively stable at 50 °C with a half-life of 20 min. Ethylenediaminetetraacetic acid (EDTA) treatment abolished activity; Zn(2+) addition caused regain of the activity, but Zn(2+)addition decreased the thermal stability of the Prt1 enzyme presumably as a result of increased proteolytic autolysis. In addition to casein, the enzyme catalyzed degradation of collagen, potato lectin, and plant extensin. Analysis of the cleavage pattern of different substrates after treatment with Prt1 indicated that the protease had a substrate cleavage preference for proline in substrate residue position P1 followed by a hydrophobic residue in residue position P1' at the cleavage point. The activity of Prt1 against plant cell wall structural proteins suggests that this enzyme might become an important new addition to the toolbox of cell-wall-degrading enzymes for biomass processing.

  1. Cytoplasmic-anti-neutrophil cytoplasmic antibodies targeting myeloperoxidase in Wegener′s granulomatosis: A rare phenomenon

    Directory of Open Access Journals (Sweden)

    Bhavana M Venkatesh

    2014-01-01

    Full Text Available Wegener′s granulomatosis (WG patients can rarely have antineutrophil cytoplasmic antibodies (ANCAs directed against myeloperoxidase (MPO, producing a cytoplasmic pattern on indirect immunofluorescence (IIF. This has important implications in the diagnosis and pathophysiology of the disease. We present to you a report of three cases of WG, demonstrating a cytoplasmic-ANCA pattern on indirect IIF, but directed against MPO. It is necessary to diagnose a patient taking into account both the autoimmune test results and the clinical features.

  2. Secretion of Bacterial Lipoproteins: Through the Cytoplasmic Membrane, the Periplasm and Beyond

    Science.gov (United States)

    Zückert, Wolfram R.

    2014-01-01

    Bacterial lipoproteins are peripherally anchored membrane proteins that play a variety of roles in bacterial physiology and virulence in monoderm (single membrane-enveloped, e.g., grampositive) and diderm (double membrane-enveloped, e.g., gram-negative) bacteria. After export of prolipoproteins through the cytoplasmic membrane, which occurs predominantly but not exclusively via the general secretory or Sec pathway, the proteins are lipid-modified at the cytoplasmic membrane in a multistep process that involves sequential modification of a cysteine residue and cleavage of the signal peptide by the signal II peptidase Lsp. In both monoderms and diderms, signal peptide processing is preceded by acylation with a diacylglycerol through preprolipoprotein diacylglycerol transferase (Lgt). In diderms but also some monoderms, lipoproteins are further modified with a third acyl chain through lipoprotein N-acyl transferase (Lnt). Fully modified lipoproteins that are destined to be anchored in the inner leaflet of the outer membrane (OM) are selected, transported and inserted by the Lol (lipoprotein outer membrane localization) pathway machinery, which consists of the inner-membrane (IM) ABC transporterlike LolCDE complex, the periplasmic LolA chaperone and the OM LolB lipoprotein receptor. Retention of lipoproteins in the cytoplasmic membrane results from Lol avoidance signals that were originally described as the “+2 rule”. Surface localization of lipoproteins in diderms is rare in most bacteria, with the exception of several spirochetal species. Type 2 (T2SS) and type 5 (T5SS) secretion systems are involved in secretion of specific surface lipoproteins of γ-proteobacteria. In the model spirochete Borrelia burgdorferi, surface lipoprotein secretion does not follow established sorting rules, but remains dependent on N-terminal peptide sequences. Secretion through the outer membrane requires maintenance of lipoproteins in a translocation-competent unfolded conformation

  3. Structural and biophysical characterization of the cytoplasmic domains of human BAP29 and BAP31.

    Directory of Open Access Journals (Sweden)

    Esben M Quistgaard

    Full Text Available Two members of the B-cell associated 31 (BAP31 family are found in humans; BAP29 and BAP31. These are ubiquitously expressed receptors residing in the endoplasmic reticulum. BAP31 functions in sorting of membrane proteins and in caspase-8 mediated apoptosis, while BAP29 appears to mainly corroborate with BAP31 in sorting. The N-terminal half of these proteins is membrane-bound while the C-terminal half is cytoplasmic. The latter include the so called variant of death effector domain (vDED, which shares weak sequence homology with DED domains. Here we present two structures of BAP31 vDED determined from a single and a twinned crystal, grown at pH 8.0 and pH 4.2, respectively. These structures show that BAP31 vDED forms a dimeric parallel coiled coil with no structural similarity to DED domains. Solution studies support this conclusion and strongly suggest that an additional α-helical domain is present in the C-terminal cytoplasmic region, probably forming a second coiled coil. The thermal stability of BAP31 vDED is quite modest at neutral pH, suggesting that it may assemble in a dynamic fashion in vivo. Surprisingly, BAP29 vDED is partially unfolded at pH 7, while a coiled coil is formed at pH 4.2 in vitro. It is however likely that folding of the domain is triggered by other factors than low pH in vivo. We found no evidence for direct interaction of the cytoplasmic domains of BAP29 and BAP31.

  4. Cytoplasmic Streaming in the Drosophila Oocyte.

    Science.gov (United States)

    Quinlan, Margot E

    2016-10-06

    Objects are commonly moved within the cell by either passive diffusion or active directed transport. A third possibility is advection, in which objects within the cytoplasm are moved with the flow of the cytoplasm. Bulk movement of the cytoplasm, or streaming, as required for advection, is more common in large cells than in small cells. For example, streaming is observed in elongated plant cells and the oocytes of several species. In the Drosophila oocyte, two stages of streaming are observed: relatively slow streaming during mid-oogenesis and streaming that is approximately ten times faster during late oogenesis. These flows are implicated in two processes: polarity establishment and mixing. In this review, I discuss the underlying mechanism of streaming, how slow and fast streaming are differentiated, and what we know about the physiological roles of the two types of streaming.

  5. Equivalent potency and pharmacokinetics of recombinant human growth hormones with or without an N-terminal methionine.

    Science.gov (United States)

    Moore, J A; Rudman, C G; MacLachlan, N J; Fuller, G B; Burnett, B; Frane, J W

    1988-06-01

    Two forms of human GH (hGH) have been produced by recombinant DNA technology. One form has an amino acid sequence identical to that of the natural pituitary hormone (rhGH) and the other form has an additional N-terminal methionine (Met-hGH). The biological potencies of these 2 polypeptides have been compared in hypophysectomized rats in a multidose study measuring body weights and several long bone growth parameters. The pharmacokinetic profiles after iv and sc injection were determined in cynomolgus monkeys in a 4-period cross-over study. All of the measured parameters in all the studies indicated that there was no difference in the two forms of hGH. Measurements taken after 27 daily injections of rhGH or Met-hGH (30-500 micrograms/kg.day) indicated that femur length and width of the proliferative zone in the tibial epiphysis showed dose-related effects for both forms of hGH but no difference between them. The relative potency, based on body weight gain, was calculated using a parallel line bioassay. Weight gain after 8 daily injections in the 5-dose long bone growth study indicated a rhGH potency of 0.80 (95% confidence interval, 0.5-1.23) relative to Met-hGH. It was concluded that the presence of an N-terminal methionine on hGH has no effect on potency in this model. The pharmacokinetic parameters after iv administration were estimated by fitting serum concentration-time data to a 2-compartment model. Parameters after sc injection were computed by compartment-independent methods. Met-hGH and rhGH had very similar pharmacokinetic profiles after both routes of administration. Comparison of the pharmacokinetic parameters indicated that the clearance after iv administration (rhGH, 15 ml/min; Met-hGH, 13 ml/min) and the sc bioavailability (rhGH, 0.72 +/- 0.21; Met-hGH, 0.59 +/- 0.21) were not significantly different for the 2 forms of hGH. It was concluded that rhGH and Met-hGH have equivalent bioavailability and pharmacokinetics in cynomolgus monkeys.

  6. The scavenger receptor SSc5D physically interacts with bacteria through the SRCR-containing N-terminal domain

    Directory of Open Access Journals (Sweden)

    Catarina Bessa-Pereira

    2016-10-01

    Full Text Available The scavenger receptor cysteine-rich (SRCR family comprises a group of membrane-attached or secreted proteins that contain one or more modules/domains structurally similar to the membrane distal domain of type I macrophage scavenger receptor. Although no all-inclusive biological function has been ascribed to the SRCR family, some of these receptors have been shown to recognize pathogen-associated molecular patterns (PAMP of bacteria, fungi or other microbes. SSc5D is a recently described soluble SRCR receptor produced by monocytes/macrophages and T lymphocytes, consisting of an N-terminal portion which contains five SRCR modules, and a large C-terminal mucin-like domain. Towards establishing a global common role for SRCR domains, we interrogated whether the set of five SRCR domains of SSc5D displayed pattern recognition receptor (PRR properties. For that purpose, we have expressed in a mammalian expression system the N-terminal SRCR-containing moiety of SSC5D (N-SSc5D, thus excluding the mucin-like domain likely by nature to bind microorganisms, and tested the capacity of the SRCR functional groups to physically interact with bacteria. Using conventional protein-bacteria binding assays, we showed that N-SSc5D had a superior capacity to bind to E. coli strains RS218 and IHE3034 compared with that of the extracellular domains of the SRCR proteins CD5 and CD6 (sCD5 and sCD6, respectively, and similar E. coli-binding properties as Spα, a proven PRR of the SRCR family. We have further designed a more sensitive, real-time and label-free surface plasmon resonance (SPR-based assay, and examined the capacity of N-SSc5D, Spα, sCD5 and sCD6 to bind to different bacteria. We demonstrated that the N-SSc5D compares with Spα in the capacity to bind to E. coli and L. monocytogenes, and further that it can distinguish between pathogenic E. coli RS218 and IHE3034 strains and the non-pathogenic laboratory E. coli strain BL21(DE3. Our work thus advocates the

  7. Binding of pRNA to the N-terminal 14 amino acids of connector protein of bacteriophage phi29.

    Science.gov (United States)

    Xiao, Feng; Moll, Wulf-Dieter; Guo, Songchuan; Guo, Peixuan

    2005-01-01

    During assembly, bacterial virus phi29 utilizes a motor to insert genomic DNA into a preformed protein shell called the procapsid. The motor contains one twelve-subunit connector with a 3.6 nm central channel for DNA transportation, six viral-encoded RNA (packaging RNA or pRNA) and a protein, gp16, with unknown stoichiometry. Recent DNA-packaging models proposed that the 5-fold procapsid vertexes and 12-fold connector (or the hexameric pRNA ring) represented a symmetry mismatch enabling production of a force to drive a rotation motor to translocate and compress DNA. There was a discrepancy regarding the location of the foothold for the pRNA. One model [C. Chen and P. Guo (1997) J. Virol., 71, 3864-3871] suggested that the foothold for pRNA was the connector and that the pRNA-connector complex was part of the rotor. However, one other model suggested that the foothold for pRNA was the 5-fold vertex of the capsid protein and that pRNA was the stator. To elucidate the mechanism of phi29 DNA packaging, it is critical to confirm whether pRNA binds to the 5-fold vertex of the capsid protein or to the 12-fold symmetrical connector. Here, we used both purified connector and purified procapsid for binding studies with in vitro transcribed pRNA. Specific binding of pRNA to the connector in the procapsid was found by photoaffinity crosslinking. Removal of the N-terminal 14 amino acids of the gp10 protein by proteolytic cleavage resulted in undetectable binding of pRNA to either the connector or the procapsid, as investigated by agarose gel electrophoresis, SDS-PAGE, sucrose gradient sedimentation and N-terminal peptide sequencing. It is therefore concluded that pRNA bound to the 12-fold symmetrical connector to form a pRNA-connector complex and that the foothold for pRNA is the connector but not the capsid protein.

  8. The thyroxine-binding site of human apolipoprotein-A-I: Location in the N-terminal domain

    International Nuclear Information System (INIS)

    Benvenga, S.; Cahnmann, H.J.; Robbins, J.

    1991-01-01

    We tested the ability of nine monoclonal antibodies (MAb) against human apolipoprotein-A-I (apoA-I), the 28.3-kDa major apoprotein of high density lipoproteins (HDL), to inhibit its photoaffinity labeling with [125I]T4. Two forms were evaluated: isolated lipid-free apoA-I (Sigma or Calbiochem) and lipid-complexed apoA-I [HDL2, (density, 1.063-1.125 g/ml) and HDL3 (density, 1.125-1.210 g/ml)]. After labeling with 0.5 nM [125I]T4 in the presence of MAb or normal mouse IgG, the products were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and subsequent densitometric quantitation of radioactivity associated with the 28.3-kDa band. Group I MAbs, namely those having epitopes in the N-terminal portion of apoA-I, include MAb 16 (epitopes at residues 1-16), 4 and 14 (residues 1-86), and 18 (residues 98-105); group II includes MAbs 7,10, 15, and 17 (epitopes at residues 87-148); group III includes MAb 9 (residues 149-243). All group I MAbs inhibited [125I]T4 binding to isolated apoA-I with this order of potency: MAb 16 greater than MAb 14 greater than MAb 4 greater than MAb 18. In the case of lipid-associated apoA-I, the pattern of hierarchy was variable, presumably related to the known markedly polydisperse nature of HDL, but a constant feature, in contrast to the case of isolated apoA-I, was that MAb 4 was more potent than MAb 14. Group II MAbs gave less than 3% inhibition in both isolated and lipid-complexed apoA-I. Group III MAb 9 either failed to inhibit or gave 18-27% inhibition (one preparation each of HDL2 and HDL3). We conclude that the T4 site of apoA-I is in the N-terminal domain of apoA-I, closer to the epitope for MAb 16 than to that for MAb 18, and that conformational changes occurring when apoA-I is associated with lipids in the HDL particle alter the spatial relationship between some epitopes and the T4 site

  9. ANNEXIN A1 N-TERMINAL DERIVED PEPTIDE AC2-26 EXERTS CHEMOKINETIC EFFECTS ON HUMAN NEUTROPHILS

    Directory of Open Access Journals (Sweden)

    Jesmond eDalli

    2012-02-01

    Full Text Available It is postulated that peptides derived from the N-terminal region of Annexin A1, a glucocorticoid-regulated 37-kDa protein, could act as biomimetics of the parent protein. However, recent evidence, amongst which the ability to interact with distinct receptors other then that described for Annexin A1, suggest that these peptides might fulfil other functions at variance to those reported for the parent protein. Here we tested the ability of peptide Ac2-26 to induce chemotaxis of human neutrophils, showing that this peptide can elicit responses comparable to those produced by the canonical activator formyl-Met-Leu-Phe (or FMLP. However, whilst disruption of the chemical gradient abolished the FMLP response, addition of peptide Ac2-26 in the top well of the chemotaxis chamber did not affect (10 µM or augmented (at 30 µM the neutrophil locomotion to the bottom well, as elicited by 10 µM peptide Ac2-26. Intriguingly, the sole addition of peptide Ac2-26 in the top wells produced a marked migration of neutrophils. A similar behaviour was observed when human primary monocytes were used. Thus, peptide Ac2-26 is a genuine chemokinetic agent towards human blood leukocytes.Neutralization strategies indicated that engagement of either the GPCR termed FPR1 or its cognate receptor FPR2/ALX was sufficient to sustain peptide Ac2-26 induced neutrophil migration. Similarly, application of pharmacological inhibitors showed that cell locomotion to peptide Ac2-26 was mediated primarily by the ERK, but not the JNK and p38 pathways.In conclusion, we report here novel in vitro properties for peptide Ac2-26, promoting neutrophil and monocyte chemokinesis, a process that may contribute to accelerate the resolution phase of inflammation. Here we postulate that the generation Annexin A1 N-terminal peptides at the site of inflammation may expedite the egress of migrated leukocytes thus promoting the return to homeostasis.

  10. A Proline-Rich N-Terminal Region of the Dengue Virus NS3 Is Crucial for Infectious Particle Production.

    Science.gov (United States)

    Gebhard, Leopoldo G; Iglesias, Néstor G; Byk, Laura A; Filomatori, Claudia V; De Maio, Federico A; Gamarnik, Andrea V

    2016-06-01

    Dengue virus is currently the most important insect-borne viral human pathogen. Viral nonstructural protein 3 (NS3) is a key component of the viral replication machinery that performs multiple functions during viral replication and participates in antiviral evasion. Using dengue virus infectious clones and reporter systems to dissect each step of the viral life cycle, we examined the requirements of different domains of NS3 on viral particle assembly. A thorough site-directed mutagenesis study based on solvent-accessible surface areas of NS3 revealed that, in addition to being essential for RNA replication, different domains of dengue virus NS3 are critically required for production of infectious viral particles. Unexpectedly, point mutations in the protease, interdomain linker, or helicase domain were sufficient to abolish infectious particle formation without affecting translation, polyprotein processing, or RNA replication. In particular, we identified a novel proline-rich N-terminal unstructured region of NS3 that contains several amino acid residues involved in infectious particle formation. We also showed a new role for the interdomain linker of NS3 in virion assembly. In conclusion, we present a comprehensive genetic map of novel NS3 determinants for viral particle assembly. Importantly, our results provide evidence of a central role of NS3 in the coordination of both dengue virus RNA replication and particle formation. Dengue virus is an important human pathogen, and its prominence is expanding globally; however, basic aspects of its biology are still unclear, hindering the development of effective therapeutic and prophylactic treatments. Little is known about the initial steps of dengue and other flavivirus particle assembly. This process involves a complex interplay between viral and cellular components, making it an attractive antiviral target. Unpredictably, we identified spatially separated regions of the large NS3 viral protein as determinants for

  11. Modulating the activity of short arginine-tryptophan containing antibacterial peptides with N-terminal metallocenoyl groups

    Directory of Open Access Journals (Sweden)

    H. Bauke Albada

    2012-10-01

    Full Text Available A series of small synthetic arginine and tryptophan containing peptides was prepared and analyzed for their antibacterial activity. The effect of N-terminal substitution with metallocenoyl groups such as ferrocene (FcCO and ruthenocene (RcCO was investigated. Antibacterial activity in different media, growth inhibition, and killing kinetics of the most active peptides were determined. The toxicity of selected derivatives was determined against erythrocytes and three human cancer cell lines. It was shown that the replacement of an N-terminal arginine residue with a metallocenoyl moiety modulates the activity of WRWRW-peptides against Gram-positive and Gram-negative bacteria. MIC values of 2–6 µM for RcCO-W(RW2 and 1–11 µM for (RW3 were determined. Interestingly, W(RW2-peptides derivatized with ferrocene were significantly less active than those derivatized with ruthenocene which have similar structural but different electronic properties, suggesting a major influence of the latter. The high activities observed for the RcCO-W(RW2- and (RW3-peptides led to an investigation of the origin of activity of these peptides using several important activity-related parameters. Firstly, killing kinetics of the RcCO-W(RW2-peptide versus killing kinetics of the (RW3 derivative showed faster reduction of the colony forming units for the RcCO-W(RW2-peptide, although MIC values indicated higher activity for the (RW3-peptide. This was confirmed by growth inhibition studies. Secondly, hemolysis studies revealed that both peptides did not lead to significant destruction of erythrocytes, even up to 500 µg/mL for (RW3 and 250 µg/mL for RcCO-W(RW2. In addition, toxicity against three human cancer cell lines (HepG2, HT29, MCF7 showed that the (RW3-peptide had an IC50 value of ~140 µM and the RcW(RW2 one of ~90 µM, indicating a potentially interesting therapeutic window. Both the killing kinetics and growth inhibition studies presented in this work point to a

  12. Successful Pregnancy in a Couple with Severe Male Factor Infertility after Selection of Sperm with Cytoplasmic Droplets

    OpenAIRE

    Bellish, Jenna; McCulloh, David H.; Ahmad, Khaliq; McGovern, Peter G.

    2015-01-01

    We present live births resulting from two separate IVF cycles in a couple in which ICSI was performed with sperm specifically selected for presence of small cytoplasmic droplets. These cycles followed previous cycles using standard sperm selection methods in which very poor embryo development and no pregnancies ensued. The male partner was diagnosed with severe male factor infertility including elevated DNA fragmentation.

  13. The desensitization gating of the MthK K+ channel is governed by its cytoplasmic amino terminus.

    Directory of Open Access Journals (Sweden)

    Mario Meng-Chiang Kuo

    2008-10-01

    Full Text Available The RCK-containing MthK channel undergoes two inactivation processes: activation-coupled desensitization and acid-induced inactivation. The acid inactivation is mediated by the C-terminal RCK domain assembly. Here, we report that the desensitization gating is governed by a desensitization domain (DD of the cytoplasmic N-terminal 17 residues. Deletion of DD completely removes the desensitization, and the process can be fully restored by a synthetic DD peptide added in trans. Mutagenesis analyses reveal a sequence-specific determinant for desensitization within the initial hydrophobic segment of DD. Proton nuclear magnetic resonance ((1H NMR spectroscopy analyses with synthetic peptides and isolated RCK show interactions between the two terminal domains. Additionally, we show that deletion of DD does not affect the acid-induced inactivation, indicating that the two inactivation processes are mutually independent. Our results demonstrate that the short N-terminal DD of MthK functions as a complete moveable module responsible for the desensitization. Its interaction with the C-terminal RCK domain may play a role in the gating process.

  14. Human endotoxemia activates p38 MAP kinase and p42/44 MAP kinase, but not c-Jun N-terminal kinase

    NARCIS (Netherlands)

    van den Blink, B.; Branger, J.; Weijer, S.; Deventer, S. H.; van der Poll, T.; Peppelenbosch, M. P.

    2001-01-01

    All three major members of the MAPK family (i.e., p38 MAPK, p42/p44 MAPK, and c-Jun N terminal kinase (JNK)) have been shown to control cellular responses to inflammation in vitro. Therefore these kinases have been designated suitable targets for anti-inflammatory therapy. However, the extent to

  15. Structure of the starch-debranching enzyme barley limit dextrinase reveals homology of the N-terminal domain to CBM21

    DEFF Research Database (Denmark)

    Møller, Marie Sofie; Abou Hachem, Maher; Svensson, Birte

    2012-01-01

    molecule in the active site and is virtually identical to the structures of HvLD in complex with the competitive inhibitors α-cyclodextrin and β-cyclodextrin solved to 2.5 and 2.1 Å resolution, respectively. However, three loops in the N-terminal domain that are shown here to resemble carbohydrate...

  16. Comparison of N-terminal pro-atrial natriuretic peptide and atrial natriuretic peptide in human plasma as measured with commercially available radioimmunoassay kits

    NARCIS (Netherlands)

    F. Boomsma (Frans); U.M. Bhaggoe (Usha); A.J. Man in 't Veld (Arie); M.A.D.H. Schalekamp (Maarten)

    1996-01-01

    textabstractAtrial natriuretic peptide (ANP) has become an important parameter for assessing the condition of patients with cardia disease. Recently, attention has also focused on N-terminal pro-atrial natriuretic peptide (NtproANP) in this context. NtproANP circulates in plasma in higher

  17. The major peanut allergen Ara h 1 and its cleaved-off N-terminal peptide; possible implications for peanut allergen detection

    NARCIS (Netherlands)

    Wichers, H.J.; Beijer, de T.; Savelkoul, H.F.J.; Amerongen, van A.

    2004-01-01

    Ara h 1 was purified from raw peanuts (Arachis hypogaea L.) in the presence or absence of protease inhibitors. N-Terminal amino acid sequences were determined after western blotting. Both purification procedures proved to be very consistent and resulted in identical chromatographic and

  18. Troponin T, N-terminal pro natriuretic peptide and a patent ductus arteriosus scoring system predict death before discharge or neurodevelopmental outcome at 2 years in preterm infants.

    LENUS (Irish Health Repository)

    El-Khuffash, Afif F

    2011-03-01

    There is little consensus regarding the use of echocardiography in patent ductus arteriosus (PDA) treatment in preterm infants. The use of troponin T (cTnT) and N-terminal Pro-BNP (NTpBNP) in combination with echocardiography assessment may facilitate the development of a superior predictive model.

  19. The N-Terminal Flanking Region of the Invariant Chain Peptide Augments the Immunogenicity of a Cryptic “Self” Epitope from a Tumor-Associated Antigen

    NARCIS (Netherlands)

    Hess, A.D.; Thoburn, C.; Chen, W.; Miura, Y.; Wall, E. van der

    2001-01-01

    The N-terminal flanking region of the invariant chain peptide termed CLIP appears to have superagonistic properties interacting with the T cell receptor and the MHC class II molecule at or near the binding site for the bacterial superantigen Staphylococcal enterotoxin B (SEB). The present studies

  20. N-terminal-pro-brain natriuretic peptide elevations in the course of septic and non-septic shock reflect systolic left ventricular dysfunction assessed by transpulmonary thermodilution

    NARCIS (Netherlands)

    A.J. Groeneveld; R.J. Trof (R.)

    2016-01-01

    textabstractBackground: The cardiac correlates, if any, of N-terminal probrain natriuretic peptide (NT-proBNP) levels in septic and non-septic shock patients remain controversial. Methods: In the 38 septic and 22 non-septic shock patients in the transpulmonary thermodilution arm of a previous

  1. Procollagen type I N-terminal propeptide (PINP) as an indicator of type I collagen metabolism: ELISA development, reference interval, and hypovitaminosis D induced hyperparathyroidism

    DEFF Research Database (Denmark)

    Orum, O; Hansen, M; Jensen, Charlotte Harken

    1996-01-01

    A sandwich enzyme-linked immunosorbent assay (ELISA) for quantification of the N-terminal propeptide of human procollagen type I (PINP) utilizing purified alpha 1-chain specific rabbit antibodies is described. The ELISA measured the content of the alpha 1-chain of PINP independent of the molecula...

  2. Unbiased Selective Isolation of Protein N-Terminal Peptides from Complex Proteome Samples Using Phospho Tagging PTAG) and TiO2-based Depletion

    NARCIS (Netherlands)

    Mommen, G.P.M.; Waterbeemd, van de B.; Meiring, H.D.; Kersten, G.; Heck, A.J.R.; Jong, de A.P.J.M.

    2012-01-01

    A positional proteomics strategy for global N-proteome analysis is presented based on phospho tagging (PTAG) of internal peptides followed by depletion by titanium dioxide (TiO2) affinity chromatography. Therefore, N-terminal and lysine amino groups are initially completely dimethylated with

  3. Cardiovascular risk prediction by N-terminal pro brain natriuretic peptide and high sensitivity C-reactive protein is affected by age and sex

    DEFF Research Database (Denmark)

    Olsen, M.H.; Hansen, T.W.; Christensen, M.K.

    2008-01-01

    BACKGROUND: Previous studies have shown that the urine albumin/creatinine ratio (UACR), high sensitivity C-reactive protein (hsCRP) and N-terminal pro brain natriuretic peptide (Nt-proBNP) predict cardiovascular events in a general population aged 41, 51, 61 or 71 years. This study investigated...

  4. Calcium has a permissive role in interleukin-1beta-induced c-jun N-terminal kinase activation in insulin-secreting cells

    DEFF Research Database (Denmark)

    Størling, Joachim; Zaitsev, Sergei V; Kapelioukh, Iouri L

    2005-01-01

    The c-jun N-terminal kinase (JNK) signaling pathway mediates IL-1beta-induced apoptosis in insulin-secreting cells, a mechanism relevant to the destruction of pancreatic beta-cells in type 1 and 2 diabetes. However, the mechanisms that contribute to IL-1beta activation of JNK in beta-cells are la...

  5. Site-specific quantification of lysine acetylation in the N-terminal tail of histone H4 using a double-labelling, targeted UHPLC MS/MS approach

    NARCIS (Netherlands)

    D'Urzo, Annalisa; Boichenko, Alexander P.; van den Bosch, Thea; Hermans, Jos; Dekker, Frank; Andrisano, Vincenza; Bischoff, Rainer

    We developed a targeted liquid chromatography-tandem mass spectrometry (LC-MS/MS) method for the site-specific quantification of lysine acetylation in the N-terminal region of histone H4 by combining chemical derivatization at the protein and peptide levels with digestion using chymotrypsin and

  6. Locus-specific detection of HLA-DQ and -DR antigens by antibodies against synthetic N-terminal octapeptides of the beta chain

    DEFF Research Database (Denmark)

    Deufel, T; Grove, A; Kofod, Hans

    1985-01-01

    Antibodies against synthetic peptides representing the class-II antigen HLA-DR and -DQ beta chain N-terminal sequences were prepared in rabbits. The two octapeptides only share two amino acids and enzyme-linked immuno-assays showed the antisera only to bind to its own antigen. Both peptide antisera...

  7. Quantification of the N-terminal propeptide of human procollagen type I (PINP): comparison of ELISA and RIA with respect to different molecular forms

    DEFF Research Database (Denmark)

    Jensen, Charlotte Harken; Hansen, M; Brandt, J

    1998-01-01

    This paper compares the results of procollagen type I N-terminal propeptide (PINP) quantification by radioimmunoassay (RIA) and enzyme linked immunosorbent assay (ELISA). PINP in serum from a patient with uremic hyperparathyroidism was measured in RIA and ELISA to 20 micrograms l-1 and 116...

  8. The role of n terminal - probrain natriuretic peptide in the diagnosis of hemodynamic persistent asrteriosus ductus in premature neonates patient

    Science.gov (United States)

    Dasraf, D.; Djer, M. M.; Advani, N.

    2017-08-01

    Persistent ductus arteriosus is one of the most frequent congenital heart diseases found in infants, mainly in preterms. Echocardiography is the gold standard for the diagnosis of hemodynamically significant patent ductus arteriosus (hs-PDA) in preterm neonates. A few studies have suggested that the use of a simple blood assay to detect N-terminal pro-brain natriuretic peptide (NT-proBNP) may be useful in determining the diagnosis and management of hs-PDA. No such studies have been conducted in Indonesia, although the assay kit and characteristics of the patient (gestational age and chronological age) influence the accuracy of NT-proBNP levels in determining hs-PDA. The aim of this study was to determine the association between the NT-proBNP level and the prevalence of hs-PDA in an Indonesian patient population. A cross-sectional study was conducted at Dr. Cipto Mangunkusumo Hospital. PDA was determined using echocardiography in 49 preterm neonates (gestational age groups: non-PDA, non-hsPDA, and hs-PDA. The blood NT-proBNP level was then determined in the non-hsPDA and hs-PDA groups, and between-group differences were compared. Among the 49 neonates, 33 patients had PDA, and 16 of these had hs-PDA. The results revealed a significant association between the NT-proBNP level and hs-PDA (p < 0.001).

  9. Structures of minute virus of mice replication initiator protein N-terminal domain: Insights into DNA nicking and origin binding

    Energy Technology Data Exchange (ETDEWEB)

    Tewary, Sunil K.; Liang, Lingfei; Lin, Zihan; Lynn, Annie [Department of Molecular Biosciences, University of Kansas, Lawrence, KS 66045 (United States); Cotmore, Susan F. [Departments of Laboratory Medicine, Yale University Medical School, New Haven, CT 06510 (United States); Tattersall, Peter [Departments of Laboratory Medicine, Yale University Medical School, New Haven, CT 06510 (United States); Departments of Genetics, Yale University Medical School, New Haven, CT 06510 (United States); Zhao, Haiyan, E-mail: zhaohy@ku.edu [Department of Molecular Biosciences, University of Kansas, Lawrence, KS 66045 (United States); Tang, Liang, E-mail: tangl@ku.edu [Department of Molecular Biosciences, University of Kansas, Lawrence, KS 66045 (United States)

    2015-02-15

    Members of the Parvoviridae family all encode a non-structural protein 1 (NS1) that directs replication of single-stranded viral DNA, packages viral DNA into capsid, and serves as a potent transcriptional activator. Here we report the X-ray structure of the minute virus of mice (MVM) NS1 N-terminal domain at 1.45 Å resolution, showing that sites for dsDNA binding, ssDNA binding and cleavage, nuclear localization, and other functions are integrated on a canonical fold of the histidine-hydrophobic-histidine superfamily of nucleases, including elements specific for this Protoparvovirus but distinct from its Bocaparvovirus or Dependoparvovirus orthologs. High resolution structural analysis reveals a nickase active site with an architecture that allows highly versatile metal ligand binding. The structures support a unified mechanism of replication origin recognition for homotelomeric and heterotelomeric parvoviruses, mediated by a basic-residue-rich hairpin and an adjacent helix in the initiator proteins and by tandem tetranucleotide motifs in the replication origins. - Highlights: • The structure of a parvovirus replication initiator protein has been determined; • The structure sheds light on mechanisms of ssDNA binding and cleavage; • The nickase active site is preconfigured for versatile metal ligand binding; • The binding site for the double-stranded replication origin DNA is identified; • A single domain integrates multiple functions in virus replication.

  10. Loss of Nat4 and its associated histone H4 N-terminal acetylation mediates calorie restriction-induced longevity.

    Science.gov (United States)

    Molina-Serrano, Diego; Schiza, Vassia; Demosthenous, Christis; Stavrou, Emmanouil; Oppelt, Jan; Kyriakou, Dimitris; Liu, Wei; Zisser, Gertrude; Bergler, Helmut; Dang, Weiwei; Kirmizis, Antonis

    2016-12-01

    Changes in histone modifications are an attractive model through which environmental signals, such as diet, could be integrated in the cell for regulating its lifespan. However, evidence linking dietary interventions with specific alterations in histone modifications that subsequently affect lifespan remains elusive. We show here that deletion of histone N-alpha-terminal acetyltransferase Nat4 and loss of its associated H4 N-terminal acetylation (N-acH4) extend yeast replicative lifespan. Notably, nat4Δ-induced longevity is epistatic to the effects of calorie restriction (CR). Consistent with this, (i) Nat4 expression is downregulated and the levels of N-acH4 within chromatin are reduced upon CR, (ii) constitutive expression of Nat4 and maintenance of N-acH4 levels reduces the extension of lifespan mediated by CR, and (iii) transcriptome analysis indicates that nat4Δ largely mimics the effects of CR, especially in the induction of stress-response genes. We further show that nicotinamidase Pnc1, which is typically upregulated under CR, is required for nat4Δ-mediated longevity. Collectively, these findings establish histone N-acH4 as a regulator of cellular lifespan that links CR to increased stress resistance and longevity. © 2016 The Authors. Published under the terms of the CC BY 4.0 license.

  11. Cardiac involvement in myotonic dystrophy: The role of troponins and N-terminal pro B-type natriuretic peptide.

    Science.gov (United States)

    Valaperta, Rea; De Siena, Claudia; Cardani, Rosanna; Lombardia, Fortunata; Cenko, Edina; Rampoldi, Benedetta; Fossati, Barbara; Brigonzi, Elisa; Rigolini, Roberta; Gaia, Paola; Meola, Giovanni; Costa, Elena; Bugiardini, Raffaele

    2017-12-01

    Myotonic dystrophy type 1 (DM1) and type 2 (DM2) are dominant inherited muscular dystrophies with multiple systemic involvement, often producing cardiac injury. This study sought to determine the clinical significance of elevated high sensitivity cardiac troponin T and I (hs-cTnT and hs-cTnI), and N-terminal pro B-type natriuretic peptide (NT-pro-BNP) in this population. Sixty DM patients (35 men and 25 women; mean age: 45.1 years, range: 12-73 years) underwent clinical cardiac investigations and measurements of serum hs-cTnT, hs-cTnI, creatine kinase (CK), and NT-proBNP. Left ventricular (LV) ejection fraction (EF) was assessed by echocardiography. Genetic analysis revealed that 46 of the 60 patients were DM1, and 14 DM2. Blood measurements showed persistent elevation of hs-cTnT and CK in 55/60 DM patients (91.73%). In contrast, hs-cTnI values were persistently normal throughout the study. Only 2 patients showed an EF 125 pg/mL was an independent predictor of ECG abnormalities. NT-pro-BNP levels may be considered to be used clinically to identify DM patients at increased risk of developing myocardial conduction abnormalities. Copyright © 2017 Elsevier B.V. All rights reserved.

  12. Structure of N-Terminal Domain of NPC1 Reveals Distinct Subdomains for Binding and Transfer of Cholesterol

    Energy Technology Data Exchange (ETDEWEB)

    Kwon, Hyock Joo; Abi-Mosleh, Lina; Wang, Michael L.; Deisenhofer, Johann; Goldstein, Joseph L.; Brown, Michael S.; Infante, Rodney E.; (UTSMC)

    2010-09-21

    LDL delivers cholesterol to lysosomes by receptor-mediated endocytosis. Exit of cholesterol from lysosomes requires two proteins, membrane-bound Niemann-Pick C1 (NPC1) and soluble NPC2. NPC2 binds cholesterol with its isooctyl side chain buried and its 3{beta}-hydroxyl exposed. Here, we describe high-resolution structures of the N-terminal domain (NTD) of NPC1 and complexes with cholesterol and 25-hydroxycholesterol. NPC1(NTD) binds cholesterol in an orientation opposite to NPC2: 3{beta}-hydroxyl buried and isooctyl side chain exposed. Cholesterol transfer from NPC2 to NPC1(NTD) requires reorientation of a helical subdomain in NPC1(NTD), enlarging the opening for cholesterol entry. NPC1 with point mutations in this subdomain (distinct from the binding subdomain) cannot accept cholesterol from NPC2 and cannot restore cholesterol exit from lysosomes in NPC1-deficient cells. We propose a working model wherein after lysosomal hydrolysis of LDL-cholesteryl esters, cholesterol binds NPC2, which transfers it to NPC1(NTD), reversing its orientation and allowing insertion of its isooctyl side chain into the outer lysosomal membranes.

  13. Release kinetics of N-terminal pro-B-type natriuretic peptide in a clinical model of acute myocardial infarction.

    Science.gov (United States)

    Liebetrau, Christoph; Gaede, Luise; Dörr, Oliver; Troidl, Christian; Voss, Sandra; Hoffmann, Jedrzej; Paszko, Agata; Weber, Michael; Rolf, Andreas; Hamm, Christian; Nef, Holger; Möllmann, Helge

    2014-02-15

    N-terminal segment of B-type natriuretic peptide prohormone (NT-proBNP) is elevated in patients with acute myocardial infarction (AMI) thus providing both diagnostic information and prognostic information. The aim of the present study was to determine the time course of NT-proBNP release in patients undergoing transcoronary ablation of septal hypertrophy (TASH) a procedure mimicking AMI. We analyzed the release kinetics of NT-proBNP in 18 consecutive patients with hypertrophic obstructive cardiomyopathy undergoing TASH. Serum samples were collected prior to and at 15, 30, 45, 60, 75, 90, and 105 min, and 2, 4, 8, and 24h after TASH. NT-proBNP concentrations showed a continuous increase during the first 75 min with a significant percent change compared to baseline value already 15 min after TASH (105.6% [IQR 102.2-112.7]; Pmax]: 103.5-137.2%; range of absolute increase [min-max]: 23.5-304.0 ng/L). NT-proBNP concentrations decreased below the baseline value until the 8th h after initiation of myocardial infarction. NT-proBNP concentration increases immediately after induction of myocardial infarction proving early evidence of myocardial injury despite the decrease of the left ventricular wall stress due to the TASH related reduction of the left ventricular outflow gradient. Copyright © 2013 Elsevier B.V. All rights reserved.

  14. c-Jun N-terminal kinase mediates AML1-ETO protein-induced connexin-43 expression

    International Nuclear Information System (INIS)

    Gao Fenghou; Wang Qiong; Wu Yingli; Li Xi; Zhao Kewen; Chen Guoqiang

    2007-01-01

    AML1-ETO fusion protein, a product of leukemia-related chromosomal translocation t(8;21), was reported to upregulate expression of connexin-43 (Cx43), a member of gap junction-constituted connexin family. However, its mechanism(s) remains unclear. By bioinformatic analysis, here we showed that there are two putative AML1-binding consensus sequences followed by two activated protein (AP)1 sites in the 5'-flanking region upstream to Cx43 gene. AML1-ETO could directly bind to these two AML1-binding sites in electrophoretic mobility shift assay, but luciferase reporter assay revealed that the AML1 binding sites were not indispensable for Cx43 induction by AML1-ETO protein. Conversely, AP1 sites exerted an important role in this event. In agreement, AML1-ETO overexpression in leukemic U937 cells activated c-Jun N-terminal kinase (JNK), while its specific inhibitor SP600125 effectively abrogated AML1-ETO-induced Cx43 expression, indicating that JNK signaling pathway contributes to AML1-ETO induced Cx43 expression. These results would shed new insights for understanding mechanisms of AML1-ETO-associated leukemogenesis

  15. Blood N-terminal Pro-brain Natriuretic Peptide and Interleukin-17 for Distinguishing Incomplete Kawasaki Disease from Infectious Diseases.

    Science.gov (United States)

    Wu, Ling; Chen, Yuanling; Zhong, Shiling; Li, Yunyan; Dai, Xiahua; Di, Yazhen

    2015-06-01

    To explore the diagnostic value of blood N-terminal pro-brain natriuretic peptide (NT-proBNP) and interleukin-17(IL-17) for incomplete Kawasaki disease. Patients with Kawasaki disease, Incomplete Kawasaki disease and unclear infectious fever were included in this retrospective study. Their clinical features, and laboratory test results of blood NT-proBNP and IL-17 were collected and compared. 766 patients with complete clinical information were recruited, consisting of 291 cases of Kawasaki disease, 74 cases of incomplete Kawasaki disease, and 401 cases of unclear infectious diseases. When the consistency with indicator 2 and 3 in Kawasaki disease diagnosis criteria was assessed with blood IL-17 ?11.55 pg/mL and blood NT-proBNP ? 225.5 pg/dL as the criteria, the sensitivity and specificity for distinguishing incomplete Kawasaki disease and infectious diseases reached 86.5% and 94.8%, respectively. When we chose the consistency with indicator 1 and 2 in Kawasaki disease diagnosis criteria, the appearance of decrustation and/or the BCG erythema, blood IL-17 ?11.55 pg/mL and blood NT-Pro BNP ?225.5 pg/dL as the criteria, the sensitivity and specificity for distinguishing incomplete Kawasaki disease and infectious diseases was 43.2% and 100%, respectively. Blood NT-proBNP and IL-17 are useful laboratory indicators for distinguishing incomplete Kawasaki disease and infectious diseases at the early stage.

  16. Analyses of Compact Trichinella Kinomes Reveal a MOS-Like Protein Kinase with a Unique N-Terminal Domain

    Science.gov (United States)

    Stroehlein, Andreas J.; Young, Neil D.; Korhonen, Pasi K.; Chang, Bill C. H.; Sternberg, Paul W.; La Rosa, Giuseppe; Pozio, Edoardo; Gasser, Robin B.

    2016-01-01

    Parasitic worms of the genus Trichinella (phylum Nematoda; class Enoplea) represent a complex of at least twelve taxa that infect a range of different host animals, including humans, around the world. They are foodborne, intracellular nematodes, and their life cycles differ substantially from those of other nematodes. The recent characterization of the genomes and transcriptomes of all twelve recognized taxa of Trichinella now allows, for the first time, detailed studies of their molecular biology. In the present study, we defined, curated, and compared the protein kinase complements (kinomes) of Trichinella spiralis and T. pseudospiralis using an integrated bioinformatic workflow employing transcriptomic and genomic data sets. We examined how variation in the kinome might link to unique aspects of Trichinella morphology, biology, and evolution. Furthermore, we utilized in silico structural modeling to discover and characterize a novel, MOS-like kinase with an unusual, previously undescribed N-terminal domain. Taken together, the present findings provide a basis for comparative investigations of nematode kinomes, and might facilitate the identification of Enoplea-specific intervention and diagnostic targets. Importantly, the in silico modeling approach assessed here provides an exciting prospect of being able to identify and classify currently unknown (orphan) kinases, as a foundation for their subsequent structural and functional investigation. PMID:27412987

  17. Novel role of c-jun N-terminal kinase in regulating the initiation of cap-dependent translation.

    Science.gov (United States)

    Patel, Manish R; Sadiq, Ahad A; Jay-Dixon, Joe; Jirakulaporn, Tanawat; Jacobson, Blake A; Farassati, Faris; Bitterman, Peter B; Kratzke, Robert A

    2012-02-01

    Initiation of protein translation by the 5' mRNA cap is a tightly regulated step in cell growth and proliferation. Aberrant activation of cap-dependent translation is a hallmark of many cancers including non-small cell lung cancer. The canonical signaling mechanisms leading to translation initiation include activation of the Akt/mTOR pathway in response to the presence of nutrients and growth factors. We have previously observed that inhibition of c-jun N-terminal kinase (JNK) leads to inactivation of cap-dependent translation in mesothelioma cells. Since JNK is involved in the genesis of non-small cell lung cancer (NSCLC), we hypothesized that JNK could also be involved in activating cap-dependent translation in NSCLC cells and could represent an alternative pathway regulating translation. In a series of NSCLC cell lines, inhibition of JNK using SP600125 resulted in inhibition of 4E-BP1 phosphorylation and a decrease in formation of the cap-dependent translation complex, eIF4F. Furthermore, we show that JNK-mediated inhibition of translation is independent of mTOR. Our data provide evidence that JNK is involved in the regulation of translation and has potential as a therapeutic target in NSCLC.

  18. Hsp90 is cleaved by reactive oxygen species at a highly conserved N-terminal amino acid motif.

    Directory of Open Access Journals (Sweden)

    Raphaël Beck

    Full Text Available Hsp90 is an essential chaperone that is necessary for the folding, stability and activity of numerous proteins. In this study, we demonstrate that free radicals formed during oxidative stress conditions can cleave Hsp90. This cleavage occurs through a Fenton reaction which requires the presence of redox-active iron. As a result of the cleavage, we observed a disruption of the chaperoning function of Hsp90 and the degradation of its client proteins, for example, Bcr-Abl, RIP, c-Raf, NEMO and hTert. Formation of Hsp90 protein radicals on exposure to oxidative stress was confirmed by immuno-spin trapping. Using a proteomic analysis, we determined that the cleavage occurs in a conserved motif of the N-terminal nucleotide binding site, between Ile-126 and Gly-127 in Hsp90β, and between Ile-131 and Gly-132 in Hsp90α. Given the importance of Hsp90 in diverse biological functions, these findings shed new light on how oxidative stress can affect cellular homeostasis.

  19. A Clinical Study of the N-Terminal pro-Brain Natriuretic Peptide in Myocardial Injury after Neonatal Asphyxia.

    Science.gov (United States)

    Zhu, Rui; Nie, Zhenhong

    2016-04-01

    We aimed to study the changes of serum N-terminal pro-brain natriuretic to peptide (NT-proBNP) levels after asphyxia-induced myocardial injury in children and explore the relationship between serum NT-proBNP levels and neonatal asphyxia. One hundred and six cases of neonatal asphyxia were randomly selected for the study, including 46 severe cases with myocardial injury and 60 mild cases with no cardiac injury. Sixty-three healthy newborns were selected as the control group. The serum NT-proBNP level was detected using electrochemiluminescence. Creatine kinase MB (CK-MB) and serum sodium and calcium were measured simultaneously. The serum NT-proBNP level in the myocardial injury group was significantly higher than that of the noncardiac injury and control groups (p Asphyxia serum NT-proBNP and cardiac enzymes were significantly correlated. The median value of neonatal NT-proBNP was 1491 pg/mL at postnatal Day 3 (P3) and 1077 pg/mL at postnatal Day 14 (P14). The cutoff value for children with myocardial injury was 3612.5 pg/mL; the area under the receiver operating characteristic curve was 0.80 (p neonates with asphyxia and can guide its diagnosis. Copyright © 2016. Published by Elsevier B.V.

  20. The telomerase essential N-terminal domain promotes DNA synthesis by stabilizing short RNA–DNA hybrids

    Science.gov (United States)

    Akiyama, Benjamin M.; Parks, Joseph W.; Stone, Michael D.

    2015-01-01

    Telomerase is an enzyme that adds repetitive DNA sequences to the ends of chromosomes and consists of two main subunits: the telomerase reverse transcriptase (TERT) protein and an associated telomerase RNA (TER). The telomerase essential N-terminal (TEN) domain is a conserved region of TERT proposed to mediate DNA substrate interactions. Here, we have employed single molecule telomerase binding assays to investigate the function of the TEN domain. Our results reveal telomeric DNA substrates bound to telomerase exhibit a dynamic equilibrium between two states: a docked conformation and an alternative conformation. The relative stabilities of the docked and alternative states correlate with the number of basepairs that can be formed between the DNA substrate and the RNA template, with more basepairing favoring the docked state. The docked state is further buttressed by the TEN domain and mutations within the TEN domain substantially alter the DNA substrate structural equilibrium. We propose a model in which the TEN domain stabilizes short RNA–DNA duplexes in the active site of the enzyme, promoting the docked state to augment telomerase processivity. PMID:25940626

  1. Thermodynamic stability, unfolding kinetics, and aggregation of the N-terminal actin binding domains of utrophin and dystrophin†

    Science.gov (United States)

    Singh, Surinder M.; Molas, Justine F.; Kongari, Narsimulu; Bandi, Swati; Armstrong, Geoffrey S.; Winder, Steve J.; Mallela, Krishna M.G.

    2012-01-01

    Muscular dystrophy (MD) is the most common genetic lethal disorder in children. Mutations in dystrophin trigger the most common form of MD, Duchenne and its allelic variant Becker MD. Utrophin is the closest homologue and has been shown to compensate for the loss of dystrophin in human disease animal models. However, the structural and functional similarities and differences between utrophin and dystrophin are less understood. Both proteins interact with actin through their N-terminal actin-binding domain (N-ABD). In this study, we examined the thermodynamic stability and aggregation of utrophin N-ABD and compared with that of dystrophin. Our results show that utrophin N-ABD has spectroscopic properties similar to dystrophin N-ABD. However, utrophin N-ABD has decreased denaturant and thermal stability, unfolds faster, and is correspondingly more susceptible to proteolysis, which might account for its decreased in-vivo half-life compared to dystrophin. In addition, utrophin N-ABD aggregates to a lesser extent compared with dystrophin N-ABD, contrary to the general behavior of proteins in which decreased stability enhances protein aggregation. Despite these differences in stability and aggregation, both proteins exhibit deleterious effects of mutations. When utrophin N-ABD mutations analogous in position to the dystrophin disease-causing mutations were generated, they behaved similarly to dystrophin mutants in terms of decreased stability and the formation of cross-β aggregates, indicating a possible role for utrophin mutations in disease mechanisms. PMID:22275054

  2. Analysis of N-terminal pro-brain natriuretic peptide levels in patients with chronic heart failure

    International Nuclear Information System (INIS)

    Fu Xiao; Zhang Xingping; Zhou Kejian

    2011-01-01

    To investigate the changes and its clinical significance of serum N-terminal pro-brain natriuretic peptide (NT-proBNP) levels in patients with chronic heart failure(CHF), 128 patients with decompensated CHF and 20 patients without structural heart disease were selected as CHF and control group. All subjects were evaluated heart function by New York Heart Association (NYHA) class. The serum NT-proBNP levels were assayed by electrochemiluminescence double antibody sandwich immunoassay. Left ventricular ejection fraction (LVEF) was detected by color Doppler ultrasound. The results showed that the NT-proBNP levels in CHF group were significantly higher than that of in the control group (P<0.05). Further, the NT-proBNP levels showed an increased tendency accompanied by the severity of heart failure (P<0.05) and lowering of LVEF (r=-0.595, P<0.05). The serum NT-proBNP levels can reflect the state of cardiac function in patients with decompensated DHF, and useful in the diagnosis and severity assessment of CHF. (authors)

  3. Troponin T and N-terminal pro B-Type natriuretic peptide and presence of coronary artery disease

    DEFF Research Database (Denmark)

    Mouridsen, Mette R; Sajadieh, Ahmad; Carlsen, Christian M

    2015-01-01

    BACKGROUND: We tested the effects of exercise intensity, sampling intervals, degree of coronary artery stenosis, and demographic factors on circulating N-terminal pro B-Type natriuretic peptide (NT-pro-BNP) and cardiac Troponin T (cTnT) in subjects suspected of coronary artery disease (CAD......). MATERIALS AND METHODS: A total of 242 subjects referred for diagnostic evaluation of possible CAD had blood samples obtained before, 5 min after, and again 20 h after a symptom-limited exercise test. RESULTS: Totally 40 subjects had CAD with ≥ 50% stenosis, 115 subjects had no stenosis and 87 subjects...... similarly after exercise in CAD-subjects, non-CAD-subjects, and controls (median increase 8.14 ng/L) and the increase was positively associated with baseline NT-pro-BNP but not presence of CAD. Median baseline cTnT was 6.25 ng/L in CAD-subjects and 3.00 ng/L in non-CAD-subjects as well as controls, both p...

  4. Curious Sex Ratios and Cytoplasmic Genes

    Indian Academy of Sciences (India)

    Home; Journals; Resonance – Journal of Science Education; Volume 2; Issue 6. Curious Sex Ratios and Cytoplasmic Genes Microbes Can Distort the Sex Ratio of Populations. Stephen J Freeland Laurence D Hurst. General Article Volume 2 Issue 6 June 1997 pp 68-78 ...

  5. Detection of antineutrophil cytoplasmic antibodies (ANCAs)

    DEFF Research Database (Denmark)

    Damoiseaux, Jan; Csernok, Elena; Rasmussen, Niels

    2017-01-01

    of diagnosis) from 251 patients with ANCA-associated vasculitis (AAV), including granulomatosis with polyangiitis and microscopic polyangiitis, and from 924 disease controls were tested for the presence of cytoplasmic pattern/perinuclear pattern and atypical ANCA (A-ANCA) by indirect immunofluorescence (IIF...

  6. How crowded is the prokaryotic cytoplasm?

    NARCIS (Netherlands)

    Spitzer, Jan; Poolman, Bert; Ferguson, Stuart

    2013-01-01

    We consider biomacromolecular crowding within the cytoplasm of prokaryotic cells as a two-phase system of 'supercrowded' cytogel and 'dilute' cytosol; we simplify and quantify this model for a coccoid cell over a wide range of biomacromolecular crowding. The key result shows that the supercrowded

  7. The unique C- and N-terminal sequences of Metallothionein isoform 3 mediate growth inhibition and Vectorial active transport in MCF-7 cells.

    Science.gov (United States)

    Voels, Brent; Wang, Liping; Sens, Donald A; Garrett, Scott H; Zhang, Ke; Somji, Seema

    2017-05-25

    The 3rd isoform of the metallothionein (MT3) gene family has been shown to be overexpressed in most ductal breast cancers. A previous study has shown that the stable transfection of MCF-7 cells with the MT3 gene inhibits cell growth. The goal of the present study was to determine the role of the unique C-terminal and N-terminal sequences of MT3 on phenotypic properties and gene expression profiles of MCF-7 cells. MCF-7 cells were transfected with various metallothionein gene constructs which contain the insertion or the removal of the unique MT3 C- and N-terminal domains. Global gene expression analysis was performed on the MCF-7 cells containing the various constructs and the expression of the unique C- and N- terminal domains of MT3 was correlated to phenotypic properties of the cells. The results of the present study demonstrate that the C-terminal sequence of MT3, in the absence of the N-terminal sequence, induces dome formation in MCF-7 cells, which in cell cultures is the phenotypic manifestation of a cell's ability to perform vectorial active transport. Global gene expression analysis demonstrated that the increased expression of the GAGE gene family correlated with dome formation. Expression of the C-terminal domain induced GAGE gene expression, whereas the N-terminal domain inhibited GAGE gene expression and that the effect of the N-terminal domain inhibition was dominant over the C-terminal domain of MT3. Transfection with the metallothionein 1E gene increased the expression of GAGE genes. In addition, both the C- and the N-terminal sequences of the MT3 gene had growth inhibitory properties, which correlated to an increased expression of the interferon alpha-inducible protein 6. Our study shows that the C-terminal domain of MT3 confers dome formation in MCF-7 cells and the presence of this domain induces expression of the GAGE family of genes. The differential effects of MT3 and metallothionein 1E on the expression of GAGE genes suggests unique roles of

  8. Requirement of the N-terminal residues of human cytomegalovirus UL112-113 proteins for viral growth and oriLyt-dependent DNA replication.

    Science.gov (United States)

    Kim, Young-Eui; Park, Mi Young; Kang, Kyeong Jin; Han, Tae Hee; Lee, Chan Hee; Ahn, Jin-Hyun

    2015-08-01

    The UL112-113 region of the human cytomegalovirus (HCMV) genome encodes four phosphoproteins of 34, 43, 50, and 84 kDa that promote viral DNA replication. Co-transfection assays have demonstrated that self-interaction of these proteins via the shared N-termini is necessary for their intranuclear distribution as foci and for the efficient relocation of a viral DNA polymerase processivity factor (UL44) to the viral replication sites. However, the requirement of UL112-113 N-terminal residues for viral growth and DNA replication has not been fully elucidated. Here, we investigated the effect of deletion of the N-terminal regions of UL112-113 proteins on viral growth and oriLyt-dependent DNA replication. A deletion of the entire UL112 region or the region encoding the 25 N-terminal amino-acid residues from the HCMV (Towne strain) bacmid impaired viral growth in bacmid-transfected human fibroblast cells, indicating their requirement for viral growth. In co-immunoprecipitation assays using the genomic gene expressing the four UL112-113 proteins together, the 25 N-terminal amino-acid residues were found to be necessary for stable expression of UL112-113 proteins and their self-interaction. These residues were also required for efficient binding to and relocation of UL44, but not for interaction with IE2, an origin-binding transcription factor. In co-transfection/replication assays, replication of the oriLyt-containing plasmid was promoted by expression of intact UL112-113 proteins, but not by the expression of 25-amino-acid residue-deleted proteins. Our results demonstrate that the 25 N-terminal amino-acid residues of UL112-113 proteins that mediate self-interaction contribute to viral growth by promoting their binding to UL44 and the initiation of oriLyt-dependent DNA replication.

  9. Comparative studies on tree pollen allergens. X. Further purification and N-terminal amino acid sequence analyses of the major allergen of birch pollen (Betula verrucosa).

    Science.gov (United States)

    Vik, H; Elsayed, S

    1986-01-01

    The previously isolated major allergen of birch pollen (fraction BV45), Int. Archs Allergy appl. Immun. 68: 70-78 (1982), was further purified by recycling chromatography. The purified preparation was run on a high-performance liquid chromatography (HPLC) TSK-G-2000 gel filtration chromatography column and, finally, on paper high-volt electrophoresis. The protein recovered met the homogeneity criteria required for performing the N-terminal sequence analysis. The allergenic and antigenic reactivities of the HPLC-purified protein, designated BV45B, was examined. A single homogeneous precipitation line in crossed immunoelectrophoresis (CIE) was shown. Specific IgE-inhibition tests and immuno-autoradiographic prints indicated that this allergen could bind reaginic IgE specificially and with good affinity. The homogeneity of BV45B was examined by isoelectric focusing (IEF). Several minor bands of pI differences of less than 0.1 units were visible, demonstrating the existence of some molecular variants of this protein. The N-terminal sequence analysis of the molecule was performed, and the following four amino acids were tentatively shown by sequential cleavage: NH2-Ala-Gly-Ile-Val-. The demonstration of one dominant N-terminal 1-dimethyl-amino-5-naphthalene sulphonyl (DNS)-amino acid by polyamide thin-layer chromatography at each sequence step confirmed that the N-terminal residue of the protein was not blocked; the heterogeneity shown by the IEF system was merely due to the presence of several homologous polymorphic proteins with identical N-terminal amino acid, the adequacy of the purification repertoire used.

  10. Arrest of cytoplasmic streaming induces algal proliferation in green paramecia.

    Directory of Open Access Journals (Sweden)

    Toshiyuki Takahashi

    Full Text Available A green ciliate Paramecium bursaria, bearing several hundreds of endosymbiotic algae, demonstrates rotational microtubule-based cytoplasmic streaming, in which cytoplasmic granules and endosymbiotic algae flow in a constant direction. However, its physiological significance is still unknown. We investigated physiological roles of cytoplasmic streaming in P. bursaria through host cell cycle using video-microscopy. Here, we found that cytoplasmic streaming was arrested in dividing green paramecia and the endosymbiotic algae proliferated only during the arrest of cytoplasmic streaming. Interestingly, arrest of cytoplasmic streaming with pressure or a microtubule drug also induced proliferation of endosymbiotic algae independently of host cell cycle. Thus, cytoplasmic streaming may control the algal proliferation in P. bursaria. Furthermore, confocal microscopic observation revealed that a division septum was formed in the constricted area of a dividing paramecium, producing arrest of cytoplasmic streaming. This is a first report to suggest that cytoplasmic streaming controls proliferation of eukaryotic cells.

  11. The Informative Value of N-Terminal Pro-type B Natriuretic Peptide in Cardiac Surgical Patients with Hypercreatininemia

    Directory of Open Access Journals (Sweden)

    M. G. Burzhunova

    2011-01-01

    Full Text Available Objective: to study the informative value of a dramatic increase in the preoperative blood level of the inactive moiety of the precursor of N-terminal pro-type B natriuretic peptide (NT-proBNP in cardiac surgical patients with hypercreatininemia. Subjects and materials. Twenty-one patients with a preoperative NT-proBNP level of 1000 pg/ml or more, who underwent myocardial revascularization under extracorporeal circulation (ECC, were examined. The patients were divided into groups with normal (up to 120 ^mol/l (Group 1; n=11 and elevated (Group 2; n=10 creatinine concentrations. The values of circulation were processed after skin incision and at the end of surgery. The clinical features of a perioperative period were analyzed. Results. Creatininemia was 103±3.3 and 183±12.9 ^mol/l in Groups 1 and 2, respectively (p<0.05; NT-proBNP was 1397±139 and 1908±170 pg/ml (p<0.05. EuroSCORE-predicted mortality ran to 9.8±1.6 and 9.1±1.7% (p>0.05. There were no intergroup differences in intraoperative circulatory parameters. The intensity of sympatomimetic therapy after ECC was equal in the identified patient groups and there were either no differences (p>0.05 in the frequency of intra-aortic balloon counterpulsation (18.2 and 10.0%, the length of mechanical ventilation (15±1.5 and 18.7±2.3 hours and intensive care unit stay (1.8±0.5 and 2.0±0.7 days in survivors, and inpatient mortality (23.7 and 20.0% that proved to be substantially higher than the EuroSCORE-predicted one. Regression analysis showed that in the entire group of operated patients, the level of NT-proBNP turned out to be a more significant predictor of inpatient mortality (p=0.012 than EuroSCORE-predicted one (p = 0.04. The similar regularity was characteristic for patients with hypercreatininemia. In the patients with hypercholesterolemia, the EuroSCORE-predicted mortality completely lost its significance (p=0.61 in predicting actual mortality rates. In this group, NT

  12. Characterization of N-terminally mutated cardiac Na+ channels associated with long QT syndrome 3 and Brugada syndrome

    Directory of Open Access Journals (Sweden)

    Christian eGütter

    2013-06-01

    Full Text Available Mutations in SCN5A, the gene encoding the cardiac voltage-gated Na+ channel hNav1.5, can result in life-threatening arrhythmias including long QT syndrome 3 (LQT3 and Brugada syndrome (BrS. Numerous mutant hNav1.5 channels have been characterized upon heterologous expression and patch-clamp recordings during the last decade. These studies revealed functionally important regions in hNav1.5 and provided insight into gain-of-function or loss-of-function channel defects underlying LQT3 or BrS, respectively. The N-terminal region of hNav1.5, however, has not yet been investigated in detail, although several mutations were reported in the literature. In the present study we investigated three mutant channels, previously associated with LQT3 (G9V, R18W, V125L, and six mutant channels, associated with BrS (R18Q, R27H, G35S, V95I, R104Q, K126E. We applied both the two-microelectrode voltage clamp technique, using cRNA-injected Xenopus oocytes, and the whole-cell patch clamp technique using transfected HEK293 cells. Surprisingly, four out of the nine mutations did not affect channel properties. Gain-of-function, as typically observed in LQT3 mutant channels, was observed only in R18W and V125L, whereas loss-of-function, frequently found in BrS mutants, was found only in R27H, R104Q, and K126E. Our results indicate that the hNav1.5 N-terminus plays an important role for channel kinetics and stability. At the same time, we suggest that additional mechanisms, as e.g. disturbed interactions of the Na+ channel N-terminus with other proteins, contribute to severe clinical phenotypes.

  13. N-terminal pro-brain natriuretic peptide levels associated with severe hand, foot and mouth disease.

    Science.gov (United States)

    Deng, Hui-Ling; Zhang, Yu-Feng; Li, Ya-Ping; Zhang, Yu; Xie, Yan; Wang, Jun; Wang, Xiao-Yan; Dang, Shuang-Suo

    2016-10-19

    Severe hand, foot, and mouth disease (HFMD) is sometimes associated with serious complications such as acute heart failure that can cause substantial child mortality. N-terminal pro-brain natriuretic peptide (NT-proBNP) is a sensitive and specific biomarker of congestive heart failure. The aim of this study was to use plasma NT-proBNP levels to establish the severity of childhood HFMD. A retrospective study was performed in 128 Chinese patients with severe HFMD and 88 patients with mild HFMD treated between January 2014 and October 2015. Univariate and multiple logistic regression analyses were used to analyze the risk factors for severe HFMD. NT-proBNP levels were analyzed in 128 severe HFMD patients, and the predictive value of NT-proBNP was assessed by receiver operating characteristic analyses. Multivariate analysis controlling for several potential confounders showed that enterovirus 71 infection [odds ratio (OR) 19.944, 95 % confidence interval (CI) 6.492-61.271], peripheral WBC count (OR 3.428, 95 % CI 1.186-9.914), fasting glucose (OR 19.428, 95 % CI 2.236-168.784), procalcitonin (OR 9.084, 95 % CI 3.462-23.837, and NT-proBNP (>125 pg/mL) (OR 16.649, 95 % CI 4.731-58.585) were each associated with the severity of HFMD. The 45 dead severe patients had higher pre-procedural levels of NT-proBNP than the 83 cured severe patients (12776 ± 13115 versus 1435 ± 4201 pg/mL, P < 0.001). An NT-proBNP cutoff value of 982 pg/mL predicted mortality with 87 % sensitivity and 86 % specificity. Plasma NT-pro-BNP level appears to be a useful biological marker for predicting the severity and mortality of HFMD.

  14. Mutation of androgen receptor N-terminal phosphorylation site Tyr-267 leads to inhibition of nuclear translocation and DNA binding.

    Directory of Open Access Journals (Sweden)

    Mehmet Karaca

    Full Text Available Reactivation of androgen receptor (AR may drive recurrent prostate cancer in castrate patients. Ack1 tyrosine kinase is overexpressed in prostate cancer and promotes castrate resistant xenograft tumor growth and enhances androgen target gene expression and AR recruitment to enhancers. Ack1 phosphorylates AR at Tyr-267 and possibly Tyr-363, both in the N-terminal transactivation domain. In this study, the role of these phosphorylation sites was investigated by characterizing the phosphorylation site mutants in the context of full length and truncated AR lacking the ligand-binding domain. Y267F and Y363F mutants showed decreased transactivation of reporters. Expression of wild type full length and truncated AR in LNCaP cells increased cell proliferation in androgen-depleted conditions and increased colony formation. However, the Y267F mutant of full length and truncated AR was defective in stimulating cell proliferation. The Y363F mutant was less severely affected than the Y267F mutant. The full length AR Y267F mutant was defective in nuclear translocation induced by androgen or Ack1 kinase. The truncated AR was constitutively localized to the nucleus. Chromatin immunoprecipitation analysis showed that it was recruited to the target enhancers without androgen. The truncated Y267F AR mutant did not exhibit constitutive nuclear localization and androgen enhancer binding activity. These results support the concept that phosphorylation of Tyr-267, and to a lesser extent Tyr-363, is required for AR nuclear translocation and recruitment and DNA binding and provide a rationale for development of novel approaches to inhibit AR activity.

  15. Association of N-terminal domain polymorphisms of the porcine glucocorticoid receptor with carcass composition and meat quality traits.

    Science.gov (United States)

    Reyer, Henry; Ponsuksili, Siriluck; Wimmers, Klaus; Murani, Eduard

    2014-02-01

    The glucocorticoid receptor (GR) is a ubiquitously acting transcription factor that is responsible for mediating the physiological response to stress and adaptation to environmental conditions. Genetic variation of a GR gene (NR3C1) may therefore contribute to multiple phenotypic alterations and influence relevant traits of animal production. Here, we examined effects of two non-synonymous mutations of the porcine NR3C1, leading to amino acid exchanges p.Glu13Asp (c.39A>C) and p.Val19Leu (c.55G>C) in the N-terminal domain of the GR, on meat quality and carcass composition. In addition, we explored their influence on transcriptional activity of GR in vitro. A commercial crossbreed Pietrain × (German Large White × German Landrace) herd (n = 545) in which genotypes and relevant traits had been collected was used to perform the association analysis. The single nucleotide polymorphism (SNP) c.55G>C was significantly associated with conductivity and meat color scores. These effects were highly consistent considering the physiological relationship between these traits. Association analysis of SNP c.39A>C also revealed significant effects on closely connected meat quality traits. In addition, SNP c.55G>C showed association with carcass traits, mainly those related to muscle deposition. The molecular mechanism of action of both amino acid substitutions remains obscure because neither showed significant influence on transcriptional activity of GR. Our study emphasizes NR3C1 as an important candidate gene for muscle-related traits in pigs, but further work is necessary to clarify the molecular background of the identified associations. © 2013 Stichting International Foundation for Animal Genetics.

  16. Substitutions of PrP N-terminal histidine residues modulate scrapie disease pathogenesis and incubation time in transgenic mice.

    Directory of Open Access Journals (Sweden)

    Sabina Eigenbrod

    Full Text Available Prion diseases have been linked to impaired copper homeostasis and copper induced-oxidative damage to the brain. Divalent metal ions, such as Cu2+ and Zn2+, bind to cellular prion protein (PrPC at octapeptide repeat (OR and non-OR sites within the N-terminal half of the protein but information on the impact of such binding on conversion to the misfolded isoform often derives from studies using either OR and non-OR peptides or bacterially-expressed recombinant PrP. Here we created new transgenic mouse lines expressing PrP with disrupted copper binding sites within all four histidine-containing OR's (sites 1-4, H60G, H68G, H76G, H84G, "TetraH>G" allele or at site 5 (composed of residues His-95 and His-110; "H95G" allele and monitored the formation of misfolded PrP in vivo. Novel transgenic mice expressing PrP(TetraH>G at levels comparable to wild-type (wt controls were susceptible to mouse-adapted scrapie strain RML but showed significantly prolonged incubation times. In contrast, amino acid replacement at residue 95 accelerated disease progression in corresponding PrP(H95G mice. Neuropathological lesions in terminally ill transgenic mice were similar to scrapie-infected wt controls, but less severe. The pattern of PrPSc deposition, however, was not synaptic as seen in wt animals, but instead dense globular plaque-like accumulations of PrPSc in TgPrP(TetraH>G mice and diffuse PrPSc deposition in (TgPrP(H95G mice, were observed throughout all brain sections. We conclude that OR and site 5 histidine substitutions have divergent phenotypic impacts and that cis interactions between the OR region and the site 5 region modulate pathogenic outcomes by affecting the PrP globular domain.

  17. N-terminal pro-brain natriuretic peptide and renal insufficiency as predictors of mortality in pulmonary hypertension.

    Science.gov (United States)

    Leuchte, Hanno H; El Nounou, Michal; Tuerpe, Juergen Christian; Hartmann, Bertram; Baumgartner, Rainer A; Vogeser, Michael; Muehling, Olaf; Behr, Jürgen

    2007-02-01

    N-terminal pro-brain natriuretic peptide (NT-proBNP) is a byproduct of the brain natriuretic peptide (BNP) that was shown to be of prognostic value in pulmonary hypertension (PH). The role of NT-proBNP in PH has to be determined, especially under the influence of renal impairment that might lead to an accumulation of the peptide, and may be a sign of increased mortality per se. We assessed NT-proBNP, BNP, renal function, and hemodynamic parameters (during right-heart catheterization) in 118 consecutive patients with isolated PH, excluding left-heart disease. Depending on the calculated creatinine clearance, patients were classified into different groups of renal function. Correlation analysis was performed on all key parameters. Results were then compared between the levels of renal function. The prognostic value of each parameter was assessed during a mean follow-up period of 10 months. Twenty-two patients (approximately 19%) had significantly impaired renal function (creatinine clearance < 60 mL/min). Although the overall levels of NT-proBNP were correlated with hemodynamics, we observed no correlation in the group with significant renal dysfunction. Moreover, NT-proBNP was related to creatinine clearance. Finally, NT-proBNP and renal insufficiency were independent predictors of death during univariate and multivariate analysis, whereas BNP only predicted mortality in univariate analysis. The diagnostic accuracy of NT-proBNP as a parameter of the hemodynamic status is diminished by renal function. However, NT-proBNP could be superior to BNP as a survival parameter in PH because it integrates hemodynamic impairment and renal insufficiency, which serves as a sign of increased mortality per se.

  18. Association of menopause age and N-terminal pro brain natriuretic peptide: the Multi-Ethnic Study of Atherosclerosis.

    Science.gov (United States)

    Ebong, Imo A; Watson, Karol E; Goff, David C; Bluemke, David A; Srikanthan, Preethi; Horwich, Tamara; Bertoni, Alain G

    2015-05-01

    Menopause age can affect the risk of developing cardiovascular disease (CVD). The purpose of this study was to investigate the associations of early menopause (menopause occurring before age 45 y) and menopause age with N-terminal pro brain natriuretic peptide (NT-proBNP), a potential risk marker of CVD and heart failure. Our cross-sectional study included 2,275 postmenopausal women, aged 45 to 85 years and without clinical CVD (2000-2002), from the Multi-Ethnic Study of Atherosclerosis. Participants were classified as having or not having early menopause. NT-proBNP was log-transformed. Multivariable linear regression was used for analysis. Five hundred sixty-one women had early menopause. The median (25th-75th percentiles) NT-proBNP value was 79.0 (41.1-151.6) pg/mL for all participants, 83.4 (41.4-164.9) pg/mL for women with early menopause, and 78.0 (40.8-148.3) pg/mL for women without early menopause. The mean (SD) age was 65 (10.1) and 65 (8.9) years for women with and without early menopause, respectively. No significant interactions between menopause age and ethnicity were observed. In multivariable analysis, early menopause was associated with a 10.7% increase in NT-proBNP levels, whereas each 1-year increase in menopause age was associated with a 0.7% decrease in NT-proBNP levels. Early menopause is associated with greater NT-proBNP levels, whereas each 1-year increase in menopause age is associated with lower NT-proBNP levels, in postmenopausal women.

  19. Substitutions of PrP N-terminal histidine residues modulate scrapie disease pathogenesis and incubation time in transgenic mice.

    Science.gov (United States)

    Eigenbrod, Sabina; Frick, Petra; Bertsch, Uwe; Mitteregger-Kretzschmar, Gerda; Mielke, Janina; Maringer, Marko; Piening, Niklas; Hepp, Alexander; Daude, Nathalie; Windl, Otto; Levin, Johannes; Giese, Armin; Sakthivelu, Vignesh; Tatzelt, Jörg; Kretzschmar, Hans; Westaway, David

    2017-01-01

    Prion diseases have been linked to impaired copper homeostasis and copper induced-oxidative damage to the brain. Divalent metal ions, such as Cu2+ and Zn2+, bind to cellular prion protein (PrPC) at octapeptide repeat (OR) and non-OR sites within the N-terminal half of the protein but information on the impact of such binding on conversion to the misfolded isoform often derives from studies using either OR and non-OR peptides or bacterially-expressed recombinant PrP. Here we created new transgenic mouse lines expressing PrP with disrupted copper binding sites within all four histidine-containing OR's (sites 1-4, H60G, H68G, H76G, H84G, "TetraH>G" allele) or at site 5 (composed of residues His-95 and His-110; "H95G" allele) and monitored the formation of misfolded PrP in vivo. Novel transgenic mice expressing PrP(TetraH>G) at levels comparable to wild-type (wt) controls were susceptible to mouse-adapted scrapie strain RML but showed significantly prolonged incubation times. In contrast, amino acid replacement at residue 95 accelerated disease progression in corresponding PrP(H95G) mice. Neuropathological lesions in terminally ill transgenic mice were similar to scrapie-infected wt controls, but less severe. The pattern of PrPSc deposition, however, was not synaptic as seen in wt animals, but instead dense globular plaque-like accumulations of PrPSc in TgPrP(TetraH>G) mice and diffuse PrPSc deposition in (TgPrP(H95G) mice), were observed throughout all brain sections. We conclude that OR and site 5 histidine substitutions have divergent phenotypic impacts and that cis interactions between the OR region and the site 5 region modulate pathogenic outcomes by affecting the PrP globular domain.

  20. N-terminal prohormone brain natriuretic peptide (NT-proBNP as a noninvasive marker for restrictive syndromes

    Directory of Open Access Journals (Sweden)

    C. Mady

    2008-08-01

    Full Text Available Constrictive pericarditis (CP and restrictive cardiomyopathy share many similarities in both their clinical and hemodynamic characteristics and N-terminal prohormone brain natriuretic peptide (NT-proBNP is a sensitive marker of cardiac diastolic dysfunction. The objectives of the present study were to determine whether serum NT-proBNP was high in patients with endomyocardial fibrosis (EMF and CP, and to investigate how this relates to diastolic dysfunction. Thirty-three patients were divided into two groups: CP (16 patients and EMF (17 patients. The control group consisted of 30 healthy individuals. Patients were evaluated by bidimensional echocardiography, with restriction syndrome evaluated by pulsed Doppler of the mitral flow and serum NT-proBNP measured by immunoassay and detected by electrochemiluminescence. Spearman correlation coefficient was used to analyze the association between log NT-proBNP and echocardiographic parameters. Log NT-proBNP was significantly higher (P < 0.05 in CP patients (log mean: 2.67 pg/mL; 95%CI: 2.43-2.92 log pg/mL and in EMF patients (log mean: 2.91 pg/mL; 95%CI: 2.70-3.12 log pg/mL compared with the control group (log mean: 1.45; 95%CI: 1.32-1.60 log pg/mL. There were no statistical differences between EMF and CP patients (P = 0.689 in terms of NT-proBNP. The NT-proBNP log tended to correlate with peak velocity of the E wave (r = 0.439; P = 0.060, but not with A wave (r = -0.399; P = 0.112. Serum NT-proBNP concentration can be used as a marker to detect the presence of diastolic dysfunction in patients with restrictive syndrome; however, serum NT-proBNP levels cannot be used to differentiate restrictive cardiomyopathy from CP.

  1. N-terminal-pro-brain natriuretic peptide, but not brain natriuretic peptide, is increased in patients with severe obesity

    Directory of Open Access Journals (Sweden)

    F. Fernandes

    2007-02-01

    Full Text Available Elevated body mass index (BMI has been reported as a risk factor for heart failure. Prevention of heart failure through identification and management of risk factors and preclinical phases of the disease is a priority. Levels of natriuretic peptides as well as activity of their receptors have been found altered in obese persons with some conflicting results. We investigated cardiac involvement in severely obese patients by determining N-terminal-pro-brain natriuretic peptide (NT-proBNP and brain natriuretic peptide (BNP and attempting to correlate the levels of these peptides in serum and plasma, respectively, with BMI, duration of obesity, waist circumference, and echocardiographic parameters. Thirty-three patients with severe obesity (mean BMI: 46.39 kg/m², mean age: 39 years were studied. The control group contained 30 healthy age-matched individuals (BMI: <25 kg/m², mean age: 43 years. The t-test and Spearman correlation were used for statistical analysis. Log-NT-proBNP was significantly higher (P = 0.003 in obese patients (mean 1.67, 95% CI: 1.50-1.83 log pg/mL compared to controls (mean: 1.32, 95% CI: 1.17-1.47 log pg/mL. The Log-NT-proBNP concentration correlated with duration of obesity (r = 0.339, P < 0.004. No difference was detected in the Log-BNP concentration (P = 0.63 of obese patients (mean: 0.73, 95% CI: 0.46-1.00 log pg/mL compared to controls (mean: 0.66, 95% CI: 0.51-0.81 log pg/mL. NT-proBNP, but not BNP, is increased in severely obese patients and its concentration in serum is correlated with duration of obesity. NT-proBNP may be useful as an early diagnostic tool for the detection of cardiac burden due to severe obesity.

  2. Type I Collagen Synthesis Marker Procollagen I N-Terminal Peptide (PINP) in Prostate Cancer Patients Undergoing Intermittent Androgen Suppression

    Energy Technology Data Exchange (ETDEWEB)

    Hamilton, Gerhard, E-mail: gerhard.hamilton@toc.lbg.ac.at; Olszewski-Hamilton, Ulrike [Ludwig Boltzmann Cluster of Translational of Oncology, Nussdorfer Strasse 64, Vienna A-1090 (Austria); Theyer, Gerhard [Hospital Kittsee, Kittsee A-2421, Burgenland (Austria)

    2011-09-15

    Intermittent androgen suppression (IAS) therapy for prostate cancer patients attempts to maintain the hormone dependence of the tumor cells by cycles alternating between androgen suppression (AS) and treatment cessation till a certain prostate-specific antigen (PSA) threshold is reached. Side effects are expected to be reduced, compared to standard continuous androgen suppression (CAS) therapy. The present study examined the effect of IAS on bone metabolism by determinations of serum procollagen I N-terminal peptide (PINP), a biochemical marker of collagen synthesis. A total of 105 treatment cycles of 58 patients with prostate cancer stages ≥pT2 was studied assessing testosterone, PSA and PINP levels at monthly intervals. During phases of AS lasting for up to nine months PSA levels were reversibly reduced, indicating apoptotic regression of the prostatic tumors. Within the first cycle PINP increased at the end of the AS period and peaked in the treatment cessation phase. During the following two cycles a similar pattern was observed for PINP, except a break in collagen synthesis as indicated by low PINP levels in the first months off treatment. Therefore, measurements of the serum PINP concentration indicated increased bone matrix synthesis in response to >6 months of AS, which uninterruptedly continued into the first treatment cessation phase, with a break into each of the following two pauses. In summary, synthesis of bone matrix collagen increases while degradation decreases during off-treatment phases in patients undergoing IAS. Although a direct relationship between bone matrix turnover and risk of fractures is difficult to establish, IAS for treatment of biochemical progression of prostate tumors is expected to reduce osteoporosis in elderly men often at high risk for bone fractures representing a highly suitable patient population for this kind of therapy.

  3. The Tobacco Smoke Component, Acrolein, Suppresses Innate Macrophage Responses by Direct Alkylation of c-Jun N-Terminal Kinase

    Science.gov (United States)

    Hristova, Milena; Spiess, Page C.; Kasahara, David I.; Randall, Matthew J.; Deng, Bin

    2012-01-01

    The respiratory innate immune system is often compromised by tobacco smoke exposure, and previous studies have indicated that acrolein, a reactive electrophile in tobacco smoke, may contribute to the immunosuppressive effects of smoking. Exposure of mice to acrolein at concentrations similar to those in cigarette smoke (5 ppm, 4 h) significantly suppressed alveolar macrophage responses to bacterial LPS, indicated by reduced induction of nitric oxide synthase 2, TNF-α, and IL-12p40. Mechanistic studies with bone marrow–derived macrophages or MH-S macrophages demonstrated that acrolein (1–30 μM) attenuated these LPS-mediated innate responses in association with depletion of cellular glutathione, although glutathione depletion itself was not fully responsible for these immunosuppressive effects. Inhibitory actions of acrolein were most prominent after acute exposure (acrolein with critical signaling pathways. Among the key signaling pathways involved in innate macrophage responses, acrolein marginally affected LPS-mediated activation of nuclear factor (NF)-κB, and significantly suppressed phosphorylation of c-Jun N-terminal kinase (JNK) and activation of c-Jun. Using biotin hydrazide labeling, NF-κB RelA and p50, as well as JNK2, a critical mediator of innate macrophage responses, were revealed as direct targets for alkylation by acrolein. Mass spectrometry analysis of acrolein-modified recombinant JNK2 indicated adduction to Cys41 and Cys177, putative important sites involved in mitogen-activated protein kinase (MAPK) kinase (MEK) binding and JNK2 phosphorylation. Our findings indicate that direct alkylation of JNK2 by electrophiles, such as acrolein, may be a prominent and hitherto unrecognized mechanism in their immunosuppressive effects, and may be a major factor in smoking-induced effects on the immune system. PMID:21778411

  4. Ribonucleocapsid Formation of SARS-COV Through Molecular Action of the N-Terminal Domain of N Protein

    Energy Technology Data Exchange (ETDEWEB)

    Saikatendu, K.S.; Joseph, J.S.; Subramanian, V.; Neuman, B.W.; Buchmeier, M.J.; Stevens, R.C.; Kuhn, P.; /Scripps Res. Inst.

    2007-07-12

    Conserved amongst all coronaviruses are four structural proteins, the matrix (M), small envelope (E) and spike (S) that are embedded in the viral membrane and the nucleocapsid phosphoprotein (N), which exists in a ribonucleoprotein complex in their lumen. The N terminal domain of coronaviral N proteins (N-NTD) provides a scaffold for RNA binding while the C-terminal domain (N-CTD) mainly acts as oligomerization modules during assembly. The C-terminus of N protein anchors it to the viral membrane by associating with M protein. We characterized the structures of N-NTD from severe acute respiratory syndrome coronavirus (SARS-CoV) in two crystal forms, at 1.17A (monoclinic) and 1.85 A (cubic) respectively, solved by molecular replacement using the homologous avian infectious bronchitis virus (IBV) structure. Flexible loops in the solution structure of SARS-CoV N-NTD are now shown to be well ordered around the beta-sheet core. The functionally important positively charged beta-hairpin protrudes out of the core and is oriented similar to that in the IBV N-NTD and is involved in crystal packing in the monoclinic form. In the cubic form, the monomers form trimeric units that stack in a helical array. Comparison of crystal packing of SARS-CoV and IBV N-NTDs suggest a common mode of RNA recognition, but probably associate differently in vivo during the formation of the ribonucleoprotein complex. Electrostatic potential distribution on the surface of homology models of related coronaviral N-NTDs hints that they employ different modes of both RNA recognition as well as oligomeric assembly, perhaps explaining why their nucleocapsids have different morphologies.

  5. Performance of N-terminal-pro-B-type natriuretic peptide in critically ill patients: a prospective observational cohort study.

    Science.gov (United States)

    Coquet, Isaline; Darmon, Michael; Doise, Jean-Marc; Degrès, Michel; Blettery, Bernard; Schlemmer, Benoît; Gambert, Philippe; Quenot, Jean-Pierre

    2008-01-01

    The purpose of this study was to assess the accuracy of N-terminal-pro-B-type natriuretic peptide (NT-proBNP) as a diagnostic tool to recognize acute respiratory failure of cardiac origin in an unselected cohort of critically ill patients. We conducted a prospective observational study of medical ICU patients. NT-proBNP was measured at ICU admission, and diagnosis of cardiac dysfunction relied on the patient's clinical presentation and echocardiography. Of the 198 patients included in this study, 102 (51.5%) had evidence of cardiac dysfunction. Median NT-proBNP concentrations were 5,720 ng/L (1,430 to 15,698) and 854 ng/L (190 to 3,560) in patients with and without cardiac dysfunction, respectively (P < 0.0001). In addition, NT-proBNP concentrations were correlated with age (rho = 0.43, P < 0.0001) and inversely correlated with creatinine clearance (rho = -0.58, P < 0.0001). When evaluating the performance of NT-proBNP concentrations to detect cardiac dysfunction, the area under the receiver operating characteristic (ROC) curve was 0.76 (95% confidence interval (CI) 0.69 to 0.83). In addition, a stepwise logistic regression model revealed that NT-proBNP (odds ratio (OR) = 1.01 per 100 ng/L, 95% CI 1.002 to 1.02), electrocardiogram modifications (OR = 11.03, 95% CI 5.19 to 23.41), and severity assessed by organ system failure score (OR = 1.63 per point, 95% CI 1.17 to 2.41) adequately predicted cardiac dysfunction. The area under the ROC curve of this model was 0.83 (95% CI 0.77 to 0.90). NT-proBNP measured at ICU admission might represent a useful marker to exclude cardiac dysfunction in critically ill patients.

  6. C-Jun N-Terminal Kinase 2 Promotes Liver Injury via the Mitochondrial Permeability Transition after Hemorrhage and Resuscitation

    Directory of Open Access Journals (Sweden)

    Christoph Czerny

    2012-01-01

    Full Text Available Hemorrhagic shock leads to hepatic hypoperfusion and activation of mitogen-activated stress kinases (MAPK like c-Jun N-terminal kinase (JNK 1 and 2. Our aim was to determine whether mitochondrial dysfunction leading to hepatic necrosis and apoptosis after hemorrhage/resuscitation (H/R was dependent on JNK2. Under pentobarbital anesthesia, wildtype (WT and JNK2 deficient (KO mice were hemorrhaged to 30 mm Hg for 3 h and then resuscitated with shed blood plus half the volume of lactated Ringer’s solution. Serum alanine aminotransferase (ALT, necrosis, apoptosis and oxidative stress were assessed 6 h after resuscitation. Mitochondrial polarization was assessed by intravital microscopy. After H/R, ALT in WT-mice increased from 130 U/L to 4800 U/L. In KO-mice, ALT after H/R was blunted to 1800 U/l (P<0.05. Necrosis, caspase-3 activity and ROS were all substantially decreased in KO compared to WT mice after H/R. After sham operation, intravital microscopy revealed punctate mitochondrial staining by rhodamine 123 (Rh123, indicating normal mitochondrial polarization. At 4 h after H/R, Rh123 staining became dim and diffuse in 58% of hepatocytes, indicating depolarization and onset of the mitochondrial permeability transition (MPT. By contrast, KO mice displayed less depolarization after H/R (23%, P<0.05. In conclusion, JNK2 contributes to MPT-mediated liver injury after H/R.

  7. Spinal release of tumour necrosis factor activates c-Jun N-terminal kinase and mediates inflammation-induced hypersensitivity.

    Science.gov (United States)

    Bas, D B; Abdelmoaty, S; Sandor, K; Codeluppi, S; Fitzsimmons, B; Steinauer, J; Hua, X Y; Yaksh, T L; Svensson, C I

    2015-02-01

    Mounting evidence points to individual contributions of tumour necrosis factor-alpha (TNF) and the c-Jun N-terminal kinase (JNK) pathway to the induction and maintenance of various pain states. Here we explore the role of spinal TNF and JNK in carrageenan-induced hypersensitivity. As links between TNF and JNK have been demonstrated in vitro, we investigated if TNF regulates spinal JNK activity in vivo. TNF levels in lumbar cerebrospinal fluid (CSF) were measured by enzyme-linked immunosorbent assay, spinal TNF gene expression by real-time polymerase chain reaction and TNF protein expression, JNK and c-Jun phosphorylation by western blotting. The role of spinal TNF and JNK in inflammation-induced mechanical and thermal hypersensitivity was assessed by injecting the TNF inhibitor etanercept and the JNK inhibitors SP600125 and JIP-1 intrathecally (i.t.). TNF-mediated regulation of JNK activity was examined by assessing the effect of i.t. etanercept on inflammation-induced spinal JNK activity. TNF levels were increased in CSF and spinal cord following carrageenan-induced inflammation. While JNK phosphorylation followed the same temporal pattern as TNF, c-jun was only activated at later time points. Intrathecal injection of TNF and JNK inhibitors attenuated carrageenan-induced mechanical and thermal hypersensitivity. TNF stimulation induced JNK phosphorylation in cultured spinal astrocytes and blocking the spinal actions of TNF in vivo by i.t. injection of etanercept reduced inflammation-induced spinal JNK activity. Here we show that spinal JNK activity is dependent on TNF and that both TNF and the JNK signalling pathways modulate pain-like behaviour induced by peripheral inflammation. © 2014 The Authors. European Journal of Pain published by John Wiley & Sons Ltd on behalf of European Pain Federation - EFIC®.

  8. N-terminal pro-brain natriuretic peptide and high-sensitivity troponin in the evaluation of acute chest pain of uncertain etiology. A PITAGORAS substudy.

    Science.gov (United States)

    Sanchis, Juan; Bardají, Alfredo; Bosch, Xavier; Loma-Osorio, Pablo; Marín, Francisco; Sánchez, Pedro L; Calvo, Francisco; Avanzas, Pablo; Hernández, Carolina; Serrano, Silvia; Carratalá, Arturo; Barrabés, José A

    2013-07-01

    High-sensitivity troponin assays have improved the diagnosis of acute coronary syndrome in patients presenting with chest pain and normal troponin levels as measured by conventional assays. Our aim was to investigate whether N-terminal pro-brain natriuretic peptide provides additional information to troponin determination in these patients. A total of 398 patients, included in the PITAGORAS study, presenting to the emergency department with chest pain and normal troponin levels as measured by conventional assay in 2 serial samples (on arrival and 6 h to 8h later) were studied. The samples were also analyzed in a central laboratory for high-sensitivity troponin T (both samples) and for N-terminal pro-brain natriuretic peptide (second sample). The endpoints were diagnosis of acute coronary syndrome and the composite endpoint of in-hospital revascularization or a 30-day cardiac event. Acute coronary syndrome was adjudicated to 79 patients (20%) and the composite endpoint to 59 (15%). When the N-terminal pro-brain natriuretic peptide quartile increased, the diagnosis of acute coronary syndrome also increased (12%, 16%, 23% and 29%; P=.01), as did the risk of the composite endpoint (6%, 13%, 16% and 24%; P=.004). N-terminal pro-brain natriuretic peptide elevation (>125ng/L) was associated with both endpoints (relative risk= 2.0; 95% confidence interval, 1.2-3.3; P=.02; relative risk=2.4; 95% confidence interval, 1.4-4.2; P=.004). However, in the multivariable models adjusted by clinical and electrocardiographic data, a predictive value was found for high-sensitivity T troponin but not for N-terminal pro-brain natriuretic peptide. In low-risk patients with chest pain of uncertain etiology evaluated using high-sensitivity T troponin, N-terminal pro-brain natriuretic peptide does not contribute additional predictive value to diagnosis or the prediction of short-term outcomes. Copyright © 2012 Sociedad Española de Cardiología. Published by Elsevier Espana. All rights

  9. Cytoplasmic lipid bodies of human neutrophilic leukocytes

    International Nuclear Information System (INIS)

    Weller, P.F.; Ackerman, S.J.; Nicholson-Weller, A.; Dvorak, A.M.

    1989-01-01

    The morphology and function of cytoplasmic lipid bodies in human neutrophils were evaluated. By transmission electron microscopy, neutrophil lipid bodies were cytoplasmic inclusions, usually several microns in diameter, that occasionally coalesced to attain a diameter up to 7 microM. Neutrophil lipid bodies were not enveloped by membrane but were often surrounded by a more electron-dense shell at their periphery. Normal peripheral blood neutrophils contained an average of approximately one lipid body per cell. Lipid bodies appeared in greater numbers in neutrophils from inflammatory lesions. Perturbation of neutrophils during conventional methods of cell isolation and purification modestly increased lipid body numbers in neutrophils, whereas incubation of neutrophils with 1 microM oleic acid rapidly induced lipid body formation over 30 to 60 minutes. After granulocytes were incubated for 2 hours with 3H-fatty acids, including arachidonic, oleic, and palmitic acids, electron microscopic autoradiography demonstrated that lipid bodies represented the predominant intracellular sites of localization of each of the three 3H-fatty acids. There was lesser labeling noted in the perinuclear cisterna, but not in cell membranes. Virtually all of each of the three 3H-fatty acids incorporated by the neutrophils were esterified into chromatographically resolved classes of neutral lipids or phospholipids. These findings indicate that cytoplasmic lipid bodies are more prominent in neutrophils in vivo engaged in inflammatory responses and that these organelles in human neutrophils function as sites of deposition of esterified, incorporated fatty acids

  10. Rho kinase regulates fragmentation and phagocytosis of apoptotic cells

    International Nuclear Information System (INIS)

    Orlando, Kelly A.; Stone, Nicole L.; Pittman, Randall N.

    2006-01-01

    During the execution phase of apoptosis, a cell undergoes cytoplasmic and nuclear changes that prepare it for death and phagocytosis. The end-point of the execution phase is condensation into a single apoptotic body or fragmentation into multiple apoptotic bodies. Fragmentation is thought to facilitate phagocytosis; however, mechanisms regulating fragmentation are unknown. An isoform of Rho kinase, ROCK-I, drives membrane blebbing through its activation of actin-myosin contraction; this raises the possibility that ROCK-I may regulate other execution phase events, such as cellular fragmentation. Here, we show that COS-7 cells fragment into a number of small apoptotic bodies during apoptosis; treating with ROCK inhibitors (Y-27632 or H-1152) prevents fragmentation. Latrunculin B and blebbistatin, drugs that interfere with actin-myosin contraction, also inhibit fragmentation. During apoptosis, ROCK-I is cleaved and activated by caspases, while ROCK-II is not activated, but rather translocates to a cytoskeletal fraction. siRNA knock-down of ROCK-I but not ROCK-II inhibits fragmentation of dying cells, consistent with ROCK-I being required for apoptotic fragmentation. Finally, cells dying in the presence of the ROCK inhibitor Y-27632 are not efficiently phagocytized. These data show that ROCK plays an essential role in fragmentation and phagocytosis of apoptotic cells

  11. Improving the production of the denatured recombinant N-terminal domain of rhoptry-associated protein 2 from a Plasmodium falciparum target in the pathology of anemia in falciparum malaria

    Directory of Open Access Journals (Sweden)

    Luis Andre Mariuba

    2008-09-01

    Full Text Available Rhoptry-associated protein 2 (RAP2 is known to be discharged from rhoptry onto the membrane surface of infected and uninfected erythrocytes (UEs ex vivo and in vitro and this information provides new insights into the understanding of the pathology of severe anemia in falciparum malaria. In this study, a hexahistidine-tagged recombinant protein corresponding to residues 5-190 of the N-terminal of Plasmodium falciparum RAP2 (rN-RAP2 was produced using a new method of solubilization and purification. Expression was induced with D-lactose, a less expensive alternative inducer to the more common isopropyl-²-D-thio-galactopyranosidase. The recombinant protein was purified using two types of commercially-available affinity columns, iminodiacetic and nitrilotriacetic. rN-RAP2 had immunogenic potential, since it induced high titers of anti-RAP2 antibodies in mice. These antibodies recognized full-length RAP2 prepared from Triton X-100 extracts from two strains of P. falciparum. In fact, the antibody recognized a 29-kDa product of RAP2 cleavage as well as 82 and 70-kDa products of RAP1 cleavage. These results indicate that the two antigens share sequence epitopes. Our expressed protein fragment was shown to contain a functional epitope that is also present in rhoptry-derived ring surface protein 2 which attaches to the surface of both infected and UEs and erythroid precursor cells in the bone marrow of malaria patients. Serum from malaria patients who developed anemia during infection recognized rN-RAP2, suggesting that this protein fragment may be important for epidemiological studies investigating whether immune responses to RAP2 exacerbate hemolysis in falciparum malaria patients.

  12. Circulating forms of immunoreactive parathyroid hormone-related protein for identifying patients with humoral hypercalcemia of malignancy. A comparative study with C-terminal (109-141)- and N-terminal (1-86)-region-specific PTHrP radioassay

    Energy Technology Data Exchange (ETDEWEB)

    Suehiro, Mitsuko; Murakami, Minoru; Fukuchi, Minoru (Hyogo Coll. of Medicine, Nishinomiya (Japan))

    1994-11-01

    We evaluated the circulating forms of immunoreactive parathyroid hormone-related protein(PTHrP) in 115 healthy subjects and 122 patients with malignant diseases by using radioassay systems (RAS) specific for the C-terminal (109-141) fragment of PTHrP (C-RAS) and for the N-terminal(1-86) (N-RAS). PTHrP levels in healthy controls ranged from 1.5 to 38.2 (mean: 24.5) pmol/L with the C-RAS and from 0.9 to 2.5 (mean: 1.7) pmol/L with the N-RAS. The ratio of circulating N-terminal fragment (N) to C-terminal fragment (C) of PTHrP was calculated to be about 1 : 14.4 in the healthy subjects. Of the 122 patients with malignant diseases, 40 (32.8%) had circulating PTHrP levels undetectable with the N-RAS, but only 11 (9.0%) patients had levels undetectable with the C-RAS. Of the former 122 patients, 41 (33.6%) had high PTHrP as determined with the C-RAS, and 10 (8.2%) had high PTHrP as determined with the N-RAS. The former of these included only 8 (19.5%) humoral hypercalcemia malignancy(HHM) patients, while the latter included 8 (80.0%) HHM patients. The circulating N to C ratio was about 1 : 70.7 in the HHM patients. The N and C obtained with the different RASs showed a close correlation (r=0.86). The values also showed a close correlation with serum Ca; r=0.75 for C-RAS and r=0.81 for N-RAS. In addition, the correlation between the PTHrP reading obtained with the different RASs and serum Cr were: r=0.42 with C-RAS and r=0.26 with N-RAS. The circulating form of immunoreactive PTHrP fragments is therefore comprised mainly of PTHrP (109-141). In contrast, circulating concentrations of the PTHrP (1-86) fragment are very low, but detection of the PTHrP (1-86) fragment with the N-RAS is a more useful indicator of HHM with fewer false positive results and is less likely to be influenced by renal function than the detection of the PHPrP (109-141) fragment with C-RAS. (author).

  13. Circulating forms of immunoreactive parathyroid hormone-related protein for identifying patients with humoral hypercalcemia of malignancy. A comparative study with C-terminal (109-141)- and N-terminal (1-86)-region-specific PTHrP radioassay

    International Nuclear Information System (INIS)

    Suehiro, Mitsuko; Murakami, Minoru; Fukuchi, Minoru

    1994-01-01

    We evaluated the circulating forms of immunoreactive parathyroid hormone-related protein(PTHrP) in 115 healthy subjects and 122 patients with malignant diseases by using radioassay systems (RAS) specific for the C-terminal (109-141) fragment of PTHrP (C-RAS) and for the N-terminal(1-86) (N-RAS). PTHrP levels in healthy controls ranged from 1.5 to 38.2 (mean: 24.5) pmol/L with the C-RAS and from 0.9 to 2.5 (mean: 1.7) pmol/L with the N-RAS. The ratio of circulating N-terminal fragment (N) to C-terminal fragment (C) of PTHrP was calculated to be about 1 : 14.4 in the healthy subjects. Of the 122 patients with malignant diseases, 40 (32.8%) had circulating PTHrP levels undetectable with the N-RAS, but only 11 (9.0%) patients had levels undetectable with the C-RAS. Of the former 122 patients, 41 (33.6%) had high PTHrP as determined with the C-RAS, and 10 (8.2%) had high PTHrP as determined with the N-RAS. The former of these included only 8 (19.5%) humoral hypercalcemia malignancy(HHM) patients, while the latter included 8 (80.0%) HHM patients. The circulating N to C ratio was about 1 : 70.7 in the HHM patients. The N and C obtained with the different RASs showed a close correlation (r=0.86). The values also showed a close correlation with serum Ca; r=0.75 for C-RAS and r=0.81 for N-RAS. In addition, the correlation between the PTHrP reading obtained with the different RASs and serum Cr were: r=0.42 with C-RAS and r=0.26 with N-RAS. The circulating form of immunoreactive PTHrP fragments is therefore comprised mainly of PTHrP (109-141). In contrast, circulating concentrations of the PTHrP (1-86) fragment are very low, but detection of the PTHrP (1-86) fragment with the N-RAS is a more useful indicator of HHM with fewer false positive results and is less likely to be influenced by renal function than the detection of the PHPrP (109-141) fragment with C-RAS. (author)

  14. Anti-neutrophil cytoplasm autoantibodies (ANCA) in autoimmune liver diseases

    NARCIS (Netherlands)

    Roozendaal, C.; Kallenberg, Cees

    1999-01-01

    Anti-neutrophil cytoplasm antibodies (ANCA) are autoantibodies directed against cytoplasmic constituents of neutrophil granulocytes and monocytes. ANCA have been detected in serum from patients with inflammatory bowel diseases (mainly ulcerative colitis) and autoimmune mediated liver diseases

  15. Sequencing and annotation of the chloroplast DNAs and identification of polymorphisms distinguishing normal male-fertile and male-sterile cytoplasms of onion.

    Science.gov (United States)

    von Kohn, Christopher; Kiełkowska, Agnieszka; Havey, Michael J

    2013-12-01

    Male-sterile (S) cytoplasm of onion is an alien cytoplasm introgressed into onion in antiquity and is widely used for hybrid seed production. Owing to the biennial generation time of onion, classical crossing takes at least 4 years to classify cytoplasms as S or normal (N) male-fertile. Molecular markers in the organellar DNAs that distinguish N and S cytoplasms are useful to reduce the time required to classify onion cytoplasms. In this research, we completed next-generation sequencing of the chloroplast DNAs of N- and S-cytoplasmic onions; we assembled and annotated the genomes in addition to identifying polymorphisms that distinguish these cytoplasms. The sizes (153 538 and 153 355 base pairs) and GC contents (36.8%) were very similar for the chloroplast DNAs of N and S cytoplasms, respectively, as expected given their close phylogenetic relationship. The size difference was primarily due to small indels in intergenic regions and a deletion in the accD gene of N-cytoplasmic onion. The structures of the onion chloroplast DNAs were similar to those of most land plants with large and small single copy regions separated by inverted repeats. Twenty-eight single nucleotide polymorphisms, two polymorphic restriction-enzyme sites, and one indel distributed across 20 chloroplast genes in the large and small single copy regions were selected and validated using diverse onion populations previously classified as N or S cytoplasmic using restriction fragment length polymorphisms. Although cytoplasmic male sterility is likely associated with the mitochondrial DNA, maternal transmission of the mitochondrial and chloroplast DNAs allows for polymorphisms in either genome to be useful for classifying onion cytoplasms to aid the development of hybrid onion cultivars.

  16. Frequent Nuclear/Cytoplasmic Localization of β-Catenin without Exon 3 Mutations in Malignant Melanoma

    Science.gov (United States)

    Rimm, David L.; Caca, Karel; Hu, Gang; Harrison, Frank B.; Fearon, Eric R.

    1999-01-01

    β-Catenin has a critical role in E-cadherin-mediated cell-cell adhesion, and it also functions as a downstream signaling molecule in the wnt pathway. Mutations in the putative glycogen synthase kinase 3β phosphorylation sites near the β-catenin amino terminus have been found in some cancers and cancer cell lines. The mutations render β-catenin resistant to regulation by a complex containing the glycogen synthase kinase 3β, adenomatous polyposis coli, and axin proteins. As a result, β-catenin accumulates in the cytosol and nucleus and activates T-cell factor/lymphoid enhancing factor transcription factors. Previously, 6 of 27 melanoma cell lines were found to have β-catenin exon 3 mutations affecting the N-terminal phosphorylation sites (Rubinfeld B, Robbins P, Elgamil M, Albert I, Porfiri E, Polakis P: Stabilization of beta-catenin by genetic defects in melanoma cell lines. Science 1997, 275:1790–1792). To assess the role of β-catenin defects in primary melanomas, we undertook immunohistochemical and DNA sequencing studies in 65 melanoma specimens. Nuclear and/or cytoplasmic localization of β-catenin, a potential indicator of wnt pathway activation, was seen focally within roughly one third of the tumors, though a clonal somatic mutation in β-catenin was found in only one case (codon 45 Ser→Pro). Our findings demonstrate that β-catenin mutations are rare in primary melanoma, in contrast to the situation in melanoma cell lines. Nonetheless, activation of β-catenin, as indicated by its nuclear and/or cytoplasmic localization, appears to be frequent in melanoma, and in some cases, it may reflect focal and transient activation of the wnt pathway within the tumor. PMID:10027390

  17. ¹H, ¹³C and ¹⁵N resonance assignments of an N-terminal domain of CHD4.

    Science.gov (United States)

    Silva, Ana P G; Kwan, Ann H; Mackay, Joel P

    2014-04-01

    Chromatin-remodeling proteins have a pivotal role in normal cell function and development, catalyzing conformational changes in DNA that ultimately result in changes in gene expression patterns. Chromodomain helicase DNA-binding protein 4 (CHD4), the defining subunit of the nucleosome remodeling and deacetylase (NuRD) complex, is a nucleosome-remodeling protein of the SNF2/ISWI2 family, members of which contain two chromo domains and an ATP-dependent helicase module. CHD3, CHD4 and CHD5 also contain two contiguous PHD domains and have an extended N-terminal region that has not previously been characterized. We have identified a stable domain in the N-terminal region of CHD4 and report here the backbone and side chain resonance assignments for this domain at pH 7.5 and 25 °C (BMRB No. 18906).

  18. Stabilization of peptide guinea pig myelin basic protein 72-85 by N-terminal acetylation-implications for immunological studies.

    Science.gov (United States)

    de Haan, Ellen C; Wauben, Marca H M; Wagenaar-Hilbers, Josée P A; Grosfeld-Stulemeyer, Mayken C; Rijkers, Dirk T S; Moret, Ed E; Liskamp, Rob M J

    2004-02-01

    Peptide gpMBP72-85, containing amino acids 72-85 of guinea pig myelin basic protein is commonly used to induce experimental autoimmune encephalomyelitis in Lewis rats. The N-terminal glutamine in this peptide can cyclize to pyroglutamic acid, leading to loss of the first MHC anchor for binding to MHC class II. Acetylation of the peptide N-terminus prevents pyroglutamic acid formation and ensures a constant quality. An increased MHC binding affinity after N-terminal acetylation was observed. This modification also enhanced T cell proliferation of a gpMBP reactive T cell clone. The encephalitogenicity of peptide gpMBP72-85 was unaffected by acetylation. It is concluded that acetylation improves the chemical stability of gpMBP72-85, and is not detrimental but rather favorable for its biochemical and immunological, in vitro, and in vivo behavior.

  19. Radioimmunological assay of the biologically active fragment of the human parathyroid hormone

    International Nuclear Information System (INIS)

    Desplan, C.; Jullienne, A.; Raulais, D.; Rivaille, P.; Barlet, J.P.; Moukthar, M.S.; Milhaud, G.

    1977-01-01

    The authors describe a RIA of the biologically active fraction (N-terminal) of human parathyroid hormone. This homologous test uses antibodies obtained in goats against a N-terminal 1-34 fragment of hPTH synthetised according to the method of Niall and Coll. In this system, natural hPTH of different origin (extracts from parathyroid adenomas, adenomal culture medium, hyperparathyroid plasma, adsorption chromatography extract of normal human plasma) behaved in the same manner as the synthetic reference hormone 1-34 hPTHN. The RIA detected PTH in 65% of the normal subjects and distinguished the normal values from the values of hyperparathyroid patients, which makes it suitable for clinical practice. (AJ) [de

  20. Universal elements of fragmentation

    International Nuclear Information System (INIS)

    Yanovsky, V. V.; Tur, A. V.; Kuklina, O. V.

    2010-01-01

    A fragmentation theory is proposed that explains the universal asymptotic behavior of the fragment-size distribution in the large-size range, based on simple physical principles. The basic principles of the theory are the total mass conservation in a fragmentation process and a balance condition for the energy expended in increasing the surface of fragments during their breakup. A flux-based approach is used that makes it possible to supplement the basic principles and develop a minimal theory of fragmentation. Such a supplementary principle is that of decreasing fragment-volume flux with increasing energy expended in fragmentation. It is shown that the behavior of the decreasing flux is directly related to the form of a power-law fragment-size distribution. The minimal theory is used to find universal asymptotic fragment-size distributions and to develop a natural physical classification of fragmentation models. A more general, nonlinear theory of strong fragmentation is also developed. It is demonstrated that solutions to a nonlinear kinetic equation consistent with both basic principles approach a universal asymptotic size distribution. Agreement between the predicted asymptotic fragment-size distributions and experimental observations is discussed.

  1. Biochemical Characterization of Mycobacterium smegmatis RnhC (MSMEG_4305), a Bifunctional Enzyme Composed of Autonomous N-Terminal Type I RNase H and C-Terminal Acid Phosphatase Domains.

    Science.gov (United States)

    Jacewicz, Agata; Shuman, Stewart

    2015-08-01

    Mycobacterium smegmatis encodes several DNA repair polymerases that are adept at incorporating ribonucleotides, which raises questions about how ribonucleotides in DNA are sensed and removed. RNase H enzymes, of which M. smegmatis encodes four, are strong candidates for a surveillance role. Here, we interrogate the biochemical activity and nucleic acid substrate specificity of M. smegmatis RnhC, a bifunctional RNase H and acid phosphatase. We report that (i) the RnhC nuclease is stringently specific for RNA:DNA hybrid duplexes; (ii) RnhC does not selectively recognize and cleave DNA-RNA or RNA-DNA junctions in duplex nucleic acid; (iii) RnhC cannot incise an embedded monoribonucleotide or diribonucleotide in duplex DNA; (iv) RnhC can incise tracts of 4 or more ribonucleotides embedded in duplex DNA, leaving two or more residual ribonucleotides at the cleaved 3'-OH end and at least one or two ribonucleotides on the 5'-PO4 end; (v) the RNase H activity is inherent in an autonomous 140-amino-acid (aa) N-terminal domain of RnhC; and (vi) the C-terminal 211-aa domain of RnhC is an autonomous acid phosphatase. The cleavage specificity of RnhC is clearly distinct from that of Escherichia coli RNase H2, which selectively incises at an RNA-DNA junction. Thus, we classify RnhC as a type I RNase H. The properties of RnhC are consistent with a role in Okazaki fragment RNA primer removal or in surveillance of oligoribonucleotide tracts embedded in DNA but not in excision repair of single misincorporated ribonucleotides. RNase H enzymes help cleanse the genome of ribonucleotides that are present either as ribotracts (e.g., RNA primers) or as single ribonucleotides embedded in duplex DNA. Mycobacterium smegmatis encodes four RNase H proteins, including RnhC, which is characterized in this study. The nucleic acid substrate and cleavage site specificities of RnhC are consistent with a role in initiating the removal of ribotracts but not in single-ribonucleotide surveillance. Rnh

  2. The N-terminal pleckstrin, coiled-coil, and IQ domains of the exchange factor Ras-GRF act cooperatively to facilitate activation by calcium.

    Science.gov (United States)

    Buchsbaum, R; Telliez, J B; Goonesekera, S; Feig, L A

    1996-09-01

    We have recently shown that the neuronal exchange factor p140 Ras-GRF becomes activated in vivo in response to elevated calcium levels [C. L. Farnsworth, N. W. Freshney, L. B. Rosen, A. Ghosh, M. E. Greenberg, and L. A. Feig, Nature (London) 376:524-527, 1995]. Activation is mediated by calcium-induced calmodulin binding to an IQ domain near the N terminus of Ras-GRF. Here we show that the adjacent N-terminal pleckstrin homology (PH), coiled-coil, and IQ domains function cooperatively to allow Ras-GRF activation. Deletion of the N-terminal PH domain redistributes a large percentage of Ras-GRF from the particulate to the cytosolic fraction of cells and renders the protein insensitive to calcium stimulation. A similar cellular distribution and biological activity are observed when only the core catalytic domain is expressed. Although the PH domain is necessary for particulate association of Ras-GRF, it is not sufficient for targeting the core catalytic domain to this cellular location. This requires the PH domain and the adjacent coiled-coil and IQ sequences. Remarkably, this form of Ras-GRF is constitutively activated. The PH and coiled-coil domains must also perform an additional function, since targeting to the particulate fraction of cells is not sufficient to allow Ras-GRF activation by calcium. A Ras-GRF mutant containing the PH domain from Ras-GTPase-activating protein in place of its own N-terminal PH domain localizes to the particulate fraction of cells but does not respond to calcium. Similar phenotypes are seen with mutant Ras-GRFs containing point mutations in either the PH or coiled-coil domain. These findings argue that the N-terminal PH, coiled-coil, and IQ domains of Ras-GRF function together to connect Ras-GRF to multiple components in the particulate fractions of cells that are required for responsiveness of the protein to calcium signaling.

  3. N-terminal pro-B-type natriuretic peptide for the prognostic prediction of severe enterovirus 71-associated hand, foot, and mouth disease

    OpenAIRE

    Jun Qiu; Xiulan Lu; Pingping Liu; Xinping Zhang; Chao Zuo; Zhenghui Xiao

    2017-01-01

    Objective: The aim of this study was to determine whether N-terminal pro-B-type natriuretic peptide (NT-proBNP) can predict impending brainstem encephalitis, pulmonary edema, pulmonary hemorrhage, cardiopulmonary failure, and death in children with severe enterovirus 71 (EV71)-associated hand, foot, and mouth disease (HFMD). Methods: Plasma NT-proBNP levels of 282 children with severe EV71-associated HFMD were measured. Results: NT-proBNP levels were significantly higher in patients wit...

  4. Critical structural and functional roles for the N-terminal insertion sequence in surfactant protein B analogs.

    Directory of Open Access Journals (Sweden)

    Frans J Walther

    2010-01-01

    Full Text Available Surfactant protein B (SP-B; 79 residues belongs to the saposin protein superfamily, and plays functional roles in lung surfactant. The disulfide cross-linked, N- and C-terminal domains of SP-B have been theoretically predicted to fold as charged, amphipathic helices, suggesting their participation in surfactant activities. Earlier structural studies with Mini-B, a disulfide-linked construct based on the N- and C-terminal regions of SP-B (i.e., approximately residues 8-25 and 63-78, confirmed that these neighboring domains are helical; moreover, Mini-B retains critical in vitro and in vivo surfactant functions of the native protein. Here, we perform similar analyses on a Super Mini-B construct that has native SP-B residues (1-7 attached to the N-terminus of Mini-B, to test whether the N-terminal sequence is also involved in surfactant activity.FTIR spectra of Mini-B and Super Mini-B in either lipids or lipid-mimics indicated that these peptides share similar conformations, with primary alpha-helix and secondary beta-sheet and loop-turns. Gel electrophoresis demonstrated that Super Mini-B was dimeric in SDS detergent-polyacrylamide, while Mini-B was monomeric. Surface plasmon resonance (SPR, predictive aggregation algorithms, and molecular dynamics (MD and docking simulations further suggested a preliminary model for dimeric Super Mini-B, in which monomers self-associate to form a dimer peptide with a "saposin-like" fold. Similar to native SP-B, both Mini-B and Super Mini-B exhibit in vitro activity with spread films showing near-zero minimum surface tension during cycling using captive bubble surfactometry. In vivo, Super Mini-B demonstrates oxygenation and dynamic compliance that are greater than Mini-B and compare favorably to full-length SP-B.Super Mini-B shows enhanced surfactant activity, probably due to the self-assembly of monomer peptide into dimer Super Mini-B that mimics the functions and putative structure of native SP-B.

  5. N-Terminal Pro-B-Type Natriuretic Peptide in the Emergency Department: The ICON-RELOADED Study.

    Science.gov (United States)

    Januzzi, James L; Chen-Tournoux, Annabel A; Christenson, Robert H; Doros, Gheorghe; Hollander, Judd E; Levy, Phillip D; Nagurney, John T; Nowak, Richard M; Pang, Peter S; Patel, Darshita; Peacock, W Franklin; Rivers, E Joy; Walters, Elizabeth L; Gaggin, Hanna K

    2018-03-20

    Contemporary reconsideration of diagnostic N-terminal pro-B-type natriuretic peptide (NT-proBNP) cutoffs for diagnosis of heart failure (HF) is needed. This study sought to evaluate the diagnostic performance of NT-proBNP for acute HF in patients with dyspnea in the emergency department (ED) setting. Dyspneic patients presenting to 19 EDs in North America were enrolled and had blood drawn for subsequent NT-proBNP measurement. Primary endpoints were positive predictive values of age-stratified cutoffs (450, 900, and 1,800 pg/ml) for diagnosis of acute HF and negative predictive value of the rule-out cutoff to exclude acute HF. Secondary endpoints included sensitivity, specificity, and positive (+) and negative (-) likelihood ratios (LRs) for acute HF. Of 1,461 subjects, 277 (19%) were adjudicated as having acute HF. The area under the receiver-operating characteristic curve for diagnosis of acute HF was 0.91 (95% confidence interval [CI]: 0.90 to 0.93; p < 0.001). Sensitivity for age stratified cutoffs of 450, 900, and 1,800 pg/ml was 85.7%, 79.3%, and 75.9%, respectively; specificity was 93.9%, 84.0%, and 75.0%, respectively. Positive predictive values were 53.6%, 58.4%, and 62.0%, respectively. Overall LR+ across age-dependent cutoffs was 5.99 (95% CI: 5.05 to 6.93); individual LR+ for age-dependent cutoffs was 14.08, 4.95, and 3.03, respectively. The sensitivity and negative predictive value for the rule-out cutoff of 300 pg/ml were 93.9% and 98.0%, respectively; LR- was 0.09 (95% CI: 0.05 to 0.13). In acutely dyspneic patients seen in the ED setting, age-stratified NT-proBNP cutpoints may aid in the diagnosis of acute HF. An NT-proBNP <300 pg/ml strongly excludes the presence of acute HF. Copyright © 2018 American College of Cardiology Foundation. Published by Elsevier Inc. All rights reserved.

  6. N-terminal Gly(224-Gly(411 domain in Listeria adhesion protein interacts with host receptor Hsp60.

    Directory of Open Access Journals (Sweden)

    Balamurugan Jagadeesan

    Full Text Available Listeria adhesion protein (LAP is a housekeeping bifunctional enzyme consisting of N-terminal acetaldehyde dehydrogenase (ALDH and C-terminal alcohol dehydrogenase (ADH. It aids Listeria monocytogenes in crossing the epithelial barrier through a paracellular route by interacting with its host receptor, heat shock protein 60 (Hsp60. To gain insight into the binding interaction between LAP and Hsp60, LAP subdomain(s participating in the Hsp60 interaction were investigated.Using a ModBase structural model, LAP was divided into 4 putative subdomains: the ALDH region contains N1 (Met(1-Pro(223 and N2 (Gly(224-Gly(411, and the ADH region contains C1 (Gly(412-Val(648 and C2 (Pro(649-Val(866. Each subdomain was cloned and overexpressed in Escherichia coli and purified. Purified subdomains were used in ligand overlay, immunofluorescence, and bead-based epithelial cell adhesion assays to analyze each domain's affinity toward Hsp60 protein or human ileocecal epithelial HCT-8 cells.The N2 subdomain exhibited the greatest affinity for Hsp60 with a K(D of 9.50±2.6 nM. The K(D of full-length LAP (7.2±0.5 nM to Hsp60 was comparable to the N2 value. Microspheres (1 µm diameter coated with N2 subdomain showed significantly (P<0.05 higher binding to HCT-8 cells than beads coated with other subdomains and this binding was inhibited when HCT-8 cells were pretreated with anti-Hsp60 antibody to specifically block epithelial Hsp60. Furthermore, HCT-8 cells pretreated with purified N2 subdomain also reduced L. monocytogenes adhesion by about 4 log confirming its involvement in interaction with epithelial cells.These data indicate that the N2 subdomain in the LAP ALDH domain is critical in initiating interaction with mammalian cell receptor Hsp60 providing insight into the molecular mechanism of pathogenesis for the development of potential anti-listerial control strategies.

  7. N-terminal pro-B-type natriuretic peptide as a marker of blunt cardiac contusion in trauma

    Science.gov (United States)

    Dogan, Halil; Sarikaya, Sezgin; Neijmann, Sebnem Tekin; Uysal, Emin; Yucel, Neslihan; Ozucelik, Dogac Niyazi; Okuturlar, Yıldız; Solak, Suleyman; Sever, Nurten; Ayan, Cem

    2015-01-01

    Cardiac contusion is usually caused by blunt chest trauma and, although it is potentially a life-threatening condition, the diagnosis of a myocardial contusion is difficult because of non-specific symptoms and the lack of an ideal test to detect myocardial damage. Cardiac enzymes, such as creatine kinase (CK), creatine kinase MB fraction (CK-MB), cardiac troponin I (cTn-I), and cardiac troponin T (cTn-T) were used in previous studies to demonstrate the blunt cardiac contusion (BCC). Each of these diagnostic tests alone is not effective for diagnosis of BCC. The aim of this study was to investigate the serum heart-type fatty acid binding protein (h-FABP), N-terminal pro-B-type natriuretic peptide (NT-proBNP), CK, CK-MB, and cTn-I levels as a marker of BCC in blunt chest trauma in rats. The eighteen Wistar albino rats were randomly allocated to two groups; group I (control) (n=8) and group II (blunt chest trauma) (n=10). Isolated BCC was induced by the method described by Raghavendran et al. (2005). All rats were observed in their cages and blood samples were collected after five hours of trauma for the analysis of serum h-FABP, NT-pro BNP, CK, CK-MB, and cTn-I levels. The mean serum NT-pro BNP was significantly different between group I and II (10.3±2.10 ng/L versus 15.4±3.68 ng/L, respectively; P=0.0001). NT-pro BNP level >13 ng/ml had a sensitivity of 87.5%, a specificity of 70%, a positive predictive value of 70%, and a negative predictive value of 87.5% for predicting blunt chest trauma (area under curve was 0.794 and P=0.037). There was no significant difference between two groups in serum h-FABP, CK, CK-MB and c Tn-I levels. A relation between NT-Pro BNP and BCC was shown in this study. Serum NT-proBNP levels significantly increased with BCC after 5 hours of the blunt chest trauma. The use of NT-proBNP as an adjunct to other diagnostic tests, such as troponins, electrocardiography (ECG), chest x-ray and echocardiogram may be beneficial for diagnosis of BCC

  8. c-Jun N-terminal kinase is required for thermotherapy-induced apoptosis in human gastric cancer cells.

    Science.gov (United States)

    Xiao, Feng; Liu, Bin; Zhu, Qing-Xian

    2012-12-28

    To investigate the role of c-Jun N-terminal kinase (JNK) in thermotherapy-induced apoptosis in human gastric cancer SGC-7901 cells. Human gastric cancer SGC-7901 cells were cultured in vitro. Following thermotherapy at 43°C for 0, 0.5, 1, 2 or 3 h, the cells were cultured for a further 24 h with or without the JNK specific inhibitor, SP600125 for 2 h. Apoptosis was evaluated by immunohistochemistry [terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL)] and flow cytometry (Annexin vs propidium iodide). Cell proliferation was determined by 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide. The production of p-JNK, Bcl-2, Bax and caspase-3 proteins was evaluated by Western blotting. The expression of JNK at mRNA level was determined by reverse transcription polymerase chain reaction. The proliferation of gastric carcinoma SGC-7901 cells was significantly inhibited following thermotherapy, and was 32.7%, 30.6%, 43.8% and 52.9% at 0.5, 1, 2 and 3 h post-thermotherapy, respectively. Flow cytometry analysis revealed an increased population of SGC-790l cells in G0/G1 phase, but a reduced population in S phase following thermotherapy for 1 or 2 h, compared to untreated cells (P thermotherapy for 0.5, 1, 2 or 3 h, compared to the untreated group (46.5% ± 0.23%, 39.9% ± 0.53%, 56.6% ± 0.35% and 50.4% ± 0.29% vs 7.3% ± 0.10%, P thermotherapy, compared to mock-inhibitor treatment, which was in line with the decreased rate of apoptosis. The expression of Bcl-2 was consistent with thermotherapy alone. Thermotherapy induced apoptosis in gastric cancer cells by promoting p-JNK at the mRNA and protein levels, and up-regulated the expression of Bax and caspase-3 proteins. Bcl-2 may play a protective role during thermotherapy. Activation of JNK via the Bax-caspase-3 pathway may be important in thermotherapy-induced apoptosis in gastric cancer cells.

  9. Annexin A1 N-terminal derived peptide Ac2-26 stimulates fibroblast migration in high glucose conditions.

    Directory of Open Access Journals (Sweden)

    Valentina Bizzarro

    Full Text Available Deficient wound healing in diabetic patients is very frequent, but the cellular and molecular causes are poorly defined. In this study, we have evaluated whether Annexin A1 derived peptide Ac2-26 stimulates fibroblast migration in high glucose conditions. Using normal human skin fibroblasts WS1 in low glucose (LG or high glucose (HG we observed the enrichment of Annexin A1 protein at cell movement structures like lamellipodial extrusions and interestingly, a significant decrease in levels of the protein in HG conditions. The analysis of the translocation of Annexin A1 to cell membrane showed lower levels of Annexin A1 in both membrane pool and supernatants of WS1 cells treated with HG. Wound-healing assays using cell line transfected with Annexin A1 siRNAs indicated a slowing down in migration speed of cells suggesting that Annexin A1 has a role in the migration of WS1 cells. In order to analyze the role of extracellular Annexin A1 in cell migration, we have performed wound-healing assays using Ac2-26 showing that peptide was able to increase fibroblast cell migration in HG conditions. Experiments on the mobilization of intracellular calcium and analysis of p-ERK expression confirmed the activity of the FPR1 following stimulation with the peptide Ac2-26. A wound-healing assay on WS1 cells in the presence of the FPR agonist fMLP, of the FPR antagonist CsH and in the presence of Ac2-26 indicated that Annexin A1 influences fibroblast cell migration under HG conditions acting through FPR receptors whose expression was slightly increased in HG. In conclusion, these data demonstrate that (i Annexin A1 is involved in migration of WS1 cells, through interaction with FPRs; (ii N- terminal peptide of Annexin A1 Ac2-26 is able to stimulate direct migration of WS1 cells in high glucose treatment possibly due to the increased receptor expression observed in hyperglycemia conditions.

  10. N-Terminal Pro-B-Type Natriuretic Peptide and Subclinical Brain Damage in the General Population.

    Science.gov (United States)

    Zonneveld, Hazel I; Ikram, M Arfan; Hofman, Albert; Niessen, Wiro J; van der Lugt, Aad; Krestin, Gabriel P; Franco, Oscar H; Vernooij, Meike W

    2017-04-01

    Purpose To investigate the association between N-terminal pro-B-type natriuretic peptide (NT-proBNP), which is a marker of heart disease, and markers of subclinical brain damage on magnetic resonance (MR) images in community-dwelling middle-aged and elderly subjects without dementia and without a clinical diagnosis of heart disease. Materials and Methods This prospective population-based cohort study was approved by a medical ethics committee overseen by the national government, and all participants gave written informed consent. Serum levels of NT-proBNP were measured in 2397 participants without dementia or stroke (mean age, 56.6 years; age range, 45.7-87.3 years) and without clinical diagnosis of heart disease who were drawn from the population-based Rotterdam Study. All participants were examined with a 1.5-T MR imager. Multivariable linear and logistic regression analyses were used to investigate the association between NT-proBNP level and MR imaging markers of subclinical brain damage, including volumetric, focal, and microstructural markers. Results A higher NT-proBNP level was associated with smaller total brain volume (mean difference in z score per standard deviation increase in NT-proBNP level, -0.021; 95% confidence interval [CI]: -0.034, -0.007; P = .003) and was predominantly driven by gray matter volume (mean difference in z score per standard deviation increase in NT-proBNP level, -0.037; 95% CI: -0.057, -0.017; P < .001). Higher NT-proBNP level was associated with larger white matter lesion volume (mean difference in z score per standard deviation increase in NT-proBNP level, 0.090; 95% CI: 0.051, 0.129; P < .001), with lower fractional anisotropy (mean difference in z score per standard deviation increase in NT-proBNP level, -0.048; 95% CI: -0.088, -0.008; P = .019) and higher mean diffusivity (mean difference in z score per standard deviation increase in NT-proBNP level, 0.054; 95% CI: 0.018, 0.091; P = .004) of normal-appearing white matter

  11. Effects of prion protein devoid of the N-terminal residues 25-50 on prion pathogenesis in mice.

    Science.gov (United States)

    Das, Nandita Rani; Miyata, Hironori; Hara, Hideyuki; Uchiyama, Keiji; Chida, Junji; Yano, Masashi; Watanabe, Hitomi; Kondoh, Gen; Sakaguchi, Suehiro

    2017-07-01

    The N-terminal polybasic region of the normal prion protein, PrP C , which encompasses residues 23-31, is important for prion pathogenesis by affecting conversion of PrP C into the pathogenic isoform, PrP Sc . We previously reported transgenic mice expressing PrP with residues 25-50 deleted in the PrP-null background, designated as Tg(PrP∆preOR)/Prnp 0/0 mice. Here, we produced two new lines of Tg(PrP∆preOR)/Prnp 0/0 mice, each expressing the mutant protein, PrP∆preOR, 1.1 and 1.6 times more than PrP C in wild-type mice, and subsequently intracerebrally inoculated RML and 22L prions into them. The lower expresser showed slightly reduced susceptibility to RML prions but not to 22L prions. The higher expresser exhibited enhanced susceptibility to both prions. No prion transmission barrier was created in Tg(PrP∆preOR)/Prnp 0/0 mice against full-length PrP Sc . PrP Sc ∆preOR accumulated in the brains of infected Tg(PrP∆preOR)/Prnp 0/0 mice less than PrP Sc in control wild-type mice, although lower in RML-infected Tg(PrP∆preOR)/Prnp 0/0 mice than in 22L-infected mice. Prion infectivity in infected Tg(PrP∆preOR)/Prnp 0/0 mice was also lower than that in wild-type mice. These results indicate that deletion of residues 25-50 only slightly affects prion susceptibility, the conversion of PrP C into PrP Sc , and prion infectivity in a strain-specific way. PrP∆preOR retains residues 23-24 and lacks residues 25-31 in the polybasic region. It is thus conceivable that residues 23-24 rather than 25-31 are important for the polybasic region to support prion pathogenesis. However, other investigators have reported that residues 27-31 not 23-24 are important to support prion pathogenesis. Taken together, the polybasic region might support prion pathogenesis through multiple sites including residues 23-24 and 27-31.

  12. Hospitalization and medical cost of patients with elevated serum N-terminal pro-brain natriuretic peptide levels.

    Directory of Open Access Journals (Sweden)

    Toshiro Kitagawa

    Full Text Available Patients with heart failure (HF are reportedly at high risk for 'all-cause' re-hospitalization. A biomarker for HF, N-terminal pro-brain natriuretic peptide (NT-proBNP, enables to simply detect patients with possible HF (pHF. We examined the hospitalization and medical cost of Japanese patients detected by an elevated serum NT-proBNP, and also evaluated the effects of institutional team approaches for HF on their all-cause hospitalizations.We retrospectively extracted all adult patients with serum NT-proBNP ≥400 pg/ml measured between January and March 2012 in Hiroshima University Hospital as pHF-positive patients. We studied their all-cause hospitalization records during the past 3-year period. We also extracted all pHF-negative patients with NT-proBNP <400 pg/ml and studied as well. In the pHF-positive patients followed for 3 years after starting interprofessional team approaches to prevent the onset and exacerbation of HF in the hospital, we compared the hospitalization and medical cost between the 3-year periods before and after the start of the team approaches.We enrolled 432 pHF-positive and 485 pHF-negative patients with one or more hospitalization records. Compared to the pHF-negative patients, the pHF-positive patients had longer total hospitalization days (median [interquartile range], 30 [13-58] versus. 18 [8-39], p <0.0001 and higher total medical cost for hospitalizations (2.42 [1.07-5.08] versus. 1.80 [0.79-3.65] million yen, p <0.0001. A subset of 303 pHF-positive patients was followed for 3 years after starting the team approaches, and we found that both total hospitalization days (30 [13-57] to 8 [0-31] and medical cost for hospitalizations (2.59 [1.37-5.05] to 0.76 [0-2.38] million yen showed marked reduction in them.Patients with an elevated serum NT-proBNP have longer hospitalizations and higher costs for all-cause hospitalizations than those without. Institutional team approaches for HF may reduce them.

  13. Serine phosphorylation of syndecan-2 proteoglycan cytoplasmic domain

    DEFF Research Database (Denmark)

    Oh, E S; Couchman, J R; Woods, A

    1997-01-01

    sequence. We investigated phosphorylation of syndecan-2 cytoplasmic domain by PKC, using purified GST-syndecan-2 fusion proteins and synthetic peptides corresponding to regions of the cytoplasmic domain. A synthetic peptide encompassing the entire cytoplasmic domain of syndecan-2 was phosphorylated by PKC...

  14. Cytoplasmic expression of a thermostable invertase from Thermotoga maritima in Lactococcus lactis.

    Science.gov (United States)

    Pek, Han Bin; Lim, Pei Yu; Liu, Chengcheng; Lee, Dong-Yup; Bi, Xuezhi; Wong, Fong Tian; Ow, Dave Siak-Wei

    2017-05-01

    To evaluate the secretory and cytoplasmic expression of a thermostable Thermogata maritima invertase in Lactococcus lactis. The thermostable invertase from T. maritima was cloned with and without the USP45 secretory peptide into the pNZ8148 vector for nisin-inducible expression in L. lactis. The introduction of an USP45 secretion peptide at the N-terminal of the enzyme led to a loss of protein solubility. Computational homology modeling and hydrophobicity studies indicated that the USP45 peptide exposes a stretch of hydrophobic amino acids on the protein surface resulting in lower solubility. Removal of the USP45 secretion peptide allowed a soluble and functional invertase to be expressed intracellularly in L. lactis. Immobilized metal affinity chromatography purification of the cell lysate with nickel-NTA gave a single protein band on SDS-PAGE, while E. coli-expressed invertase consistently co-purified with an additional band. The yields of the purified invertase from E. coli and L. lactis were 14.1 and 6.3 mg/l respectively. Invertase can be expressed in L. lactis and purified in a functional form. L. lactis is a suitable host for the production of food-grade invertase for use in the food and biotechnology industries.

  15. Solution Structure and Membrane Interaction of the Cytoplasmic Tail of HIV-1 gp41 Protein.

    Science.gov (United States)

    Murphy, R Elliot; Samal, Alexandra B; Vlach, Jiri; Saad, Jamil S

    2017-11-07

    The cytoplasmic tail of gp41 (gp41CT) remains the last HIV-1 domain with an unknown structure. It plays important roles in HIV-1 replication such as mediating envelope (Env) intracellular trafficking and incorporation into assembling virions, mechanisms of which are poorly understood. Here, we present the solution structure of gp41CT in a micellar environment and characterize its interaction with the membrane. We show that the N-terminal 45 residues are unstructured and not associated with the membrane. However, the C-terminal 105 residues form three membrane-bound amphipathic α helices with distinctive structural features such as variable degree of membrane penetration, hydrophobic and basic surfaces, clusters of aromatic residues, and a network of cation-π interactions. This work fills a major gap by providing the structure of the last segment of HIV-1 Env, which will provide insights into the mechanisms of Gag-mediated Env incorporation as well as the overall Env mobility and conformation on the virion surface. Copyright © 2017 Elsevier Ltd. All rights reserved.

  16. Granule-stored MUC5B mucins are packed by the non-covalent formation of N-terminal head-to-head tetramers.

    Science.gov (United States)

    Trillo-Muyo, Sergio; Nilsson, Harriet E; Recktenwald, Christian V; Ermund, Anna; Ridley, Caroline; Meiss, Lauren N; Bähr, Andrea; Klymiuk, Nikolai; Wine, Jeffrey J; Koeck, Philip J B; Thornton, David J; Hebert, Hans; Hansson, Gunnar C

    2018-04-13

    Most MUC5B mucin polymers in the upper airways of humans and pigs are produced by submucosal glands. MUC5B forms N-terminal covalent dimers that are further packed into larger assemblies because of low pH and high Ca 2+ in the secretory granule of the mucin-producing cell. We purified the recombinant MUC5B N-terminal covalent dimer and used single-particle electron microscopy to study its structure under intracellular conditions. We found that, at intragranular pH, the dimeric MUC5B organized into head-to-head noncovalent tetramers where the von Willebrand D1-D2 domains hooked into each other. These N-terminal tetramers further formed long linear complexes from which, we suggest, the mucin domains and their C termini project radially outwards. Using conventional and video microscopy, we observed that, upon secretion into the submucosal gland ducts, a flow of bicarbonate-rich fluid passes the mucin-secreting cells. We suggest that this unfolds and pulls out the MUC5B assemblies into long linear threads. These further assemble into thicker mucin bundles in the glandular ducts before emerging at the gland duct opening. We conclude that the combination of intracellular packing of the MUC5B mucin and the submucosal gland morphology creates an efficient machine for producing linear mucin bundles. © 2018 by The American Society for Biochemistry and Molecular Biology, Inc.

  17. Addition of N-terminal pro-B-type natriuretic peptide levels to electrocardiography criteria for detection of left ventricular hypertrophy: the ARIRANG study.

    Science.gov (United States)

    Ahn, Min-Soo; Yoo, Byung-Su; Lee, Ji Hyun; Lee, Jun-Won; Youn, Young Jin; Ahn, Sung Gyun; Kim, Jang-Young; Lee, Seung-Hwan; Yoon, Junghan; Park, Jong-Ku; Ahn, Song Vogue; Choi, Eunhee

    2015-04-01

    The utility of electrocardiography (ECG) in screening for left ventricular hypertrophy (LVH) in general populations is limited mainly because its low sensitivity. B-type natriuretic peptide (BNP) is released due to the remodeling processes of LVH and could improve the diagnostic accuracy for the ECG criteria for LVH. We hypothesized that addition of BNP levels to ECG criteria could aid LVH detection compared with ECG alone in a general population. We enrolled consecutive 343 subjects from a community-based cohort. LVH was defined as LV mass index > 95 g/m(2) for females and > 115 g/m(2) for males according to echocardiography. The area under the receiver operator characteristic (ROC) curve to detect LVH was 0.55 (95% confidence interval [CI], 0.50-0.61) in Sokolow-Lyon criteria and 0.53 (0.47-0.59) in the Cornell voltage criteria. After addition of N-terminal-proBNP levels to the model, the corresponding areas under the ROC were 0.63 (0.58-0.69) and 0.64 (0.59-0.69), respectively. P values for the comparison in areas under the ROC for models with and without N-terminal-proBNP levels were < 0.001. These data suggest that addition of N-terminal-proBNP levels to ECG criteria could significantly improve the diagnostic accuracy of LVH in general populations.

  18. Structural Basis for Recognition of H3T3ph and Smac/DIABLO N-terminal Peptides by Human Survivin

    Energy Technology Data Exchange (ETDEWEB)

    Du, Jiamu; Kelly, Alexander E.; Funabiki, Hironori; Patel, Dinshaw J. (MSKCC); (Rockefeller)

    2012-03-02

    Survivin is an inhibitor of apoptosis family protein implicated in apoptosis and mitosis. In apoptosis, it has been shown to recognize the Smac/DIABLO protein. It is also a component of the chromosomal passenger complex, a key player during mitosis. Recently, Survivin was identified in vitro and in vivo as the direct binding partner for phosphorylated Thr3 on histone H3 (H3T3ph). We have undertaken structural and binding studies to investigate the molecular basis underlying recognition of H3T3ph and Smac/DIABLO N-terminal peptides by Survivin. Our crystallographic studies establish recognition of N-terminal Ala in both complexes and identify intermolecular hydrogen-bonding interactions in the Survivin phosphate-binding pocket that contribute to H3T3ph mark recognition. In addition, our calorimetric data establish that Survivin binds tighter to the H3T3ph-containing peptide relative to the N-terminal Smac/DIABLO peptide, and this preference can be reversed through structure-guided mutations that increase the hydrophobicity of the phosphate-binding pocket.

  19. Modeling of the N-terminal Section and the Lumenal Loop of Trimeric Light Harvesting Complex II (LHCII) by Using EPR.

    Science.gov (United States)

    Fehr, Niklas; Dietz, Carsten; Polyhach, Yevhen; von Hagens, Tona; Jeschke, Gunnar; Paulsen, Harald

    2015-10-23

    The major light harvesting complex II (LHCII) of green plants plays a key role in the absorption of sunlight, the regulation of photosynthesis, and in preventing photodamage by excess light. The latter two functions are thought to involve the lumenal loop and the N-terminal domain. Their structure and mobility in an aqueous environment are only partially known. Electron paramagnetic resonance (EPR) has been used to measure the structure of these hydrophilic protein domains in detergent-solubilized LHCII. A new technique is introduced to prepare LHCII trimers in which only one monomer is spin-labeled. These heterogeneous trimers allow to measure intra-molecular distances within one LHCII monomer in the context of a trimer by using double electron-electron resonance (DEER). These data together with data from electron spin echo envelope modulation (ESEEM) allowed to model the N-terminal protein section, which has not been resolved in current crystal structures, and the lumenal loop domain. The N-terminal domain covers only a restricted area above the superhelix in LHCII, which is consistent with the "Velcro" hypothesis to explain thylakoid grana stacking (Standfuss, J., van Terwisscha Scheltinga, A. C., Lamborghini, M., and Kühlbrandt, W. (2005) EMBO J. 24, 919-928). The conformation of the lumenal loop domain is surprisingly different between LHCII monomers and trimers but not between complexes with and without neoxanthin bound. © 2015 by The American Society for Biochemistry and Molecular Biology, Inc.

  20. Modeling of the N-terminal Section and the Lumenal Loop of Trimeric Light Harvesting Complex II (LHCII) by Using EPR*

    Science.gov (United States)

    Fehr, Niklas; Dietz, Carsten; Polyhach, Yevhen; von Hagens, Tona; Jeschke, Gunnar; Paulsen, Harald

    2015-01-01

    The major light harvesting complex II (LHCII) of green plants plays a key role in the absorption of sunlight, the regulation of photosynthesis, and in preventing photodamage by excess light. The latter two functions are thought to involve the lumenal loop and the N-terminal domain. Their structure and mobility in an aqueous environment are only partially known. Electron paramagnetic resonance (EPR) has been used to measure the structure of these hydrophilic protein domains in detergent-solubilized LHCII. A new technique is introduced to prepare LHCII trimers in which only one monomer is spin-labeled. These heterogeneous trimers allow to measure intra-molecular distances within one LHCII monomer in the context of a trimer by using double electron-electron resonance (DEER). These data together with data from electron spin echo envelope modulation (ESEEM) allowed to model the N-terminal protein section, which has not been resolved in current crystal structures, and the lumenal loop domain. The N-terminal domain covers only a restricted area above the superhelix in LHCII, which is consistent with the “Velcro” hypothesis to explain thylakoid grana stacking (Standfuss, J., van Terwisscha Scheltinga, A. C., Lamborghini, M., and Kühlbrandt, W. (2005) EMBO J. 24, 919–928). The conformation of the lumenal loop domain is surprisingly different between LHCII monomers and trimers but not between complexes with and without neoxanthin bound. PMID:26316535

  1. Structural transitions in full-length human prion protein detected by xenon as probe and spin labeling of the N-terminal domain.

    Science.gov (United States)

    Narayanan, Sunilkumar Puthenpurackal; Nair, Divya Gopalakrishnan; Schaal, Daniel; Barbosa de Aguiar, Marisa; Wenzel, Sabine; Kremer, Werner; Schwarzinger, Stephan; Kalbitzer, Hans Robert

    2016-06-24

    Fatal neurodegenerative disorders termed transmissible spongiform encephalopathies (TSEs) are associated with the accumulation of fibrils of misfolded prion protein PrP. The noble gas xenon accommodates into four transiently enlarged hydrophobic cavities located in the well-folded core of human PrP(23-230) as detected by [(1)H, (15)N]-HSQC spectroscopy. In thermal equilibrium a fifth xenon binding site is formed transiently by amino acids A120 to L125 of the presumably disordered N-terminal domain and by amino acids K185 to T193 of the well-folded domain. Xenon bound PrP was modelled by restraint molecular dynamics. The individual microscopic and macroscopic dissociation constants could be derived by fitting the data to a model including a dynamic opening and closing of the cavities. As observed earlier by high pressure NMR spectroscopy xenon binding influences also other amino acids all over the N-terminal domain including residues of the AGAAAAGA motif indicating a structural coupling between the N-terminal domain and the core domain. This is in agreement with spin labelling experiments at positions 93 or 107 that show a transient interaction between the N-terminus and the start of helix 2 and the end of helix 3 of the core domain similar to that observed earlier by Zn(2+)-binding to the octarepeat motif.

  2. NMR and biophysical elucidation of structural effects on extra N-terminal methionine residue of recombinant amphibian RNases from Rana catesbeiana.

    Science.gov (United States)

    Hsu, Chun-Hua; Pan, Yun-Ru; Liao, You-Di; Wu, Shih-Hsiung; Chen, Chinpan

    2010-08-01

    The stability, structures and steric hindrances of recombinant RNases 2 and 4 expressed in bacteria were studied by circular dichroism (CD) and NMR techniques, and the results were compared with those of their authentic RNases extracted from oocytes of Rana catesbeiana. Although the overall structures of the recombinant and authentic proteins are almost identical, the extra N-terminal Met residue of the recombinant protein remarkably affects catalytic activity and stability. NMR chemical shift comparison of recombinant RNases and the authentic proteins indicated that the structural differences are mainly confined to the N-terminal helical and S2 anti-parallel beta-sheet regions. Significant shift changes for the residues located on the S2 region indicate that the major influences on the structure around the N terminus is due to the loss of the hydrogen bond between Pyr(1) and Val(95(96)) in recombinant RNases 2 and 4. We concluded the apparent steric hindrances of the extra Met to the binding pocket. As well, the affected conformational changes of active residues are attributed to the reduced activities of recombinant RNases. The structural integrity exerted by the N-terminal Pyr(1) residue may be crucial for amphibian RNases and the greatest structural differences occur on the network of the Pyr(1) residue and S2 beta-sheet region.

  3. Cytoplasm-to-myonucleus ratios following microgravity.

    Science.gov (United States)

    Kasper, C E; Xun, L

    1996-10-01

    The cytoplasmic volume-to-myonucleus ratio in the tibialis anterior and gastrocnemius muscles of juvenile rats after 5.4 days of microgravity was studied. Three groups of rats (n = 8 each) were used. The experimental group (space rats) was flown aboard the space shuttle Discovery (NASA, STS-48), while two ground-based groups, one hindlimb suspended (suspended rats), one non-suspended (control), served as controls. Single fibre analysis revealed a significant decrease in cross-sectional area (microns2) in the gastrocnemius for both the space and the suspended rats; in the tibialis anterior only the suspended rats showed a significant decrease. Myonuclei counts (myonuclei per mm) in both the tibialis anterior and gastrocnemius were significantly increased in the space rats but not in the suspended rats. The mean myonuclear volume (individual nuclei: microns3) in tibialis anterior fibres from the space rats, and in gastrocnemius fibres from both the space and the suspended rats, was significantly lower than that in the respective control group. Estimation of the total myonuclear volume (microns3 per.mm), however, revealed no significant differences between the three groups in either the tibialis anterior or gastrocnemius. The described changes in the cross-sectional area and myonuclei numbers resulted in significant decreases in the cytoplasmic volume-to-myonucleus ratio (microns3 x 10(3)) in both muscles and for both space and suspended rats (tibialis anterior; 15.6 +/- 0.6 (space), 17.2 +/- 1.0 (suspended), 20.8 +/- 0.9 (control): gastrocnemius; 13.4 +/- 0.4 (space) and 14.9 +/- 1.1 (suspended) versus 18.1 +/- 1.1 (control)). These results indicate that even short periods of unweighting due to microgravity or limb suspension result in changes in skeletal muscle fibres which lead to significant decreases in the cytoplasmic volume-to-myonucleus ratio.

  4. Tumorigenic fragments of APC cause dominant defects in directional cell migration in multiple model systems

    Directory of Open Access Journals (Sweden)

    Scott A. Nelson

    2012-11-01

    Nonsense mutations that result in the expression of truncated, N-terminal, fragments of the adenomatous polyposis coli (APC tumour suppressor protein are found in most sporadic and some hereditary colorectal cancers. These mutations can cause tumorigenesis by eliminating β-catenin-binding sites from APC, which leads to upregulation of β-catenin and thereby results in the induction of oncogenes such as MYC. Here we show that, in three distinct experimental model systems, expression of an N-terminal fragment of APC (N-APC results in loss of directionality, but not speed, of cell motility independently of changes in β-catenin regulation. We developed a system to culture and fluorescently label live pieces of gut tissue to record high-resolution three-dimensional time-lapse movies of cells in situ. This revealed an unexpected complexity of normal gut cell migration, a key process in gut epithelial maintenance, with cells moving with spatial and temporal discontinuity. Quantitative comparison of gut tissue from wild-type mice and APC heterozygotes (APCMin/+; multiple intestinal neoplasia model demonstrated that cells in precancerous epithelia lack directional preference when moving along the crypt-villus axis. This effect was reproduced in diverse experimental systems: in developing chicken embryos, mesoderm cells expressing N-APC failed to migrate normally; in amoeboid Dictyostelium, which lack endogenous APC, expressing an N-APC fragment maintained cell motility, but the cells failed to perform directional chemotaxis; and multicellular Dictyostelium slug aggregates similarly failed to perform phototaxis. We propose that N-terminal fragments of APC represent a gain-of-function mutation that causes cells within tissue to fail to migrate directionally in response to relevant guidance cues. Consistent with this idea, crypts in histologically normal tissues of APCMin/+ intestines are overpopulated with cells, suggesting that a lack of migration might cause cell

  5. Antifungal properties of durancins isolated from Enterococcus durans A5-11 and of its synthetic fragments.

    Science.gov (United States)

    Belguesmia, Y; Choiset, Y; Rabesona, H; Baudy-Floc'h, M; Le Blay, G; Haertlé, T; Chobert, J-M

    2013-04-01

    The aim of this work was to study the antifungal properties of durancins isolated from Enterococcus durans A5-11 and of their chemically synthesized fragments. Enterococcus durans A5-11 is a lactic acid bacteria strain isolated from traditional Mongolian airag cheese. This strain inhibits the growth of several fungi including Fusarium culmorum, Penicillium roqueforti and Debaryomyces hansenii. It produces two bacteriocins: durancin A5-11a and durancin A5-11b, which have similar antimicrobial properties. The whole durancins A5-11a and A5-11b, as well as their N- and C-terminal fragments were synthesized, and their antifungal properties were studied. C-terminal fragments of both durancins showed stronger antifungal activities than other tested peptides. Treatment of D. hansenii LMSA2.11.003 strain with 2 mmol l(-1) of the synthetic peptides led to the loss of the membrane integrity and to several changes in the ultra-structure of the yeast cells. Chemically synthesized durancins and their synthetic fragments showed different antimicrobial properties from each other. N-terminal peptides show activities against both bacterial and fungal strains tested. C-terminal peptides have specific activities against tested fungal strain and do not show antibacterial activity. However, the C-terminal fragment enhances the activity of the N-terminal fragment in the whole bacteriocins against bacteria. © 2012 The Society for Applied Microbiology.

  6. [Prediction of the risk of coronary arterial lesions in Kawasaki disease by N-terminal pro-brain natriuretic peptide].

    Science.gov (United States)

    Huiling, Lu; Yaping, Liu; Xiufen, Hu

    2015-04-01

    To detect plasma N-terminal pro-brain natriuretic peptide (NT-proBNP) in acute Kawasaki disease (KD) and analyze the relationship between NT-proBNP and other bio-markers in order to evaluate if NT-proBNP could be as a useful diagnostic marker to predict the risk of coronary arterial lesions in acute KD. Totally 106 patients with KD were recruited from January 2012 to April 2014 at Department of Pediatrics of Tongji Hospital Affiliated to Tongji Medical College, Huazhong University of Science and Technology,64 were boys and 42 were girls, their age ranged from 2 months to 8 years and 4 months. Of the 106 cases, 48 had typical KD(TKD) and 58 incomplete KD(IKD). They were divided into two groups according to echocardiography results: coronary arterial lesions (KD-CAL, n = 33) and non coronary arterial lesions (KD-nCAL, n = 73). Forty children whose age and gender matched with respiratory tract infection were selected as control group, 22 were boys and 18 were girls, age range from 7 months to 7 years and 11 months. Plasma NT-proBNP levels were measured by using the enzyme-linked fluorescence analysis (ELFA) at the day of admission, meanwhile blood routine tests, liver function tests, determination of C-reactive protein (CEP), erythrocyte sedimentation rate (ESR), electrolytes were performed in these patients. Pearson's correlation analysis was used to evaluate the association. The ROC curve analysis was done to identify the threshold of coronary 'arterial lesions. The levels of NT-proBNP were (1 037 271) ng/L in TKD group and (1,325 ± 264) ng/L in IKD group. The levels of NT-proBNP in control group was (125 ± 22) ng/L. Both the levels of NT-proBNP in TKD and IKD group were significantly higher than that of control group (t = 3.360, 3.590; P blood cell count, neutrophil percentage, platelet count, CRP and ESR of KD-CAL group were significantly higher than those of the control group, however there was no significant difference between KD-CAL group and KD-nCAL group

  7. Superficial disposition of the N-terminal region of the surfactant protein SP-C and the absence of specific SP-B-SP-C interactions in phospholipid bilayers

    DEFF Research Database (Denmark)

    Plasencia, I; Cruz, A; Casals, C

    2001-01-01

    A dansylated form of porcine surfactant-associated protein C (Dns-SP-C), bearing a single dansyl group at its N-terminal end, has been used to characterize the lipid-protein and protein-protein interactions of SP-C reconstituted in phospholipid bilayers, using fluorescence spectroscopy...... of the N-terminal segment of the protein into less polar environments that originate during protein lateral segregation. This suggests that conformation and interactions of the N-terminal segment of SP-C could be important in regulating the lateral distribution of the protein in surfactant bilayers...

  8. Secretion of bacterial lipoproteins: through the cytoplasmic membrane, the periplasm and beyond.

    Science.gov (United States)

    Zückert, Wolfram R

    2014-08-01

    Bacterial lipoproteins are peripherally anchored membrane proteins that play a variety of roles in bacterial physiology and virulence in monoderm (single membrane-enveloped, e.g., gram-positive) and diderm (double membrane-enveloped, e.g., gram-negative) bacteria. After export of prolipoproteins through the cytoplasmic membrane, which occurs predominantly but not exclusively via the general secretory or Sec pathway, the proteins are lipid-modified at the cytoplasmic membrane in a multistep process that involves sequential modification of a cysteine residue and cleavage of the signal peptide by the signal II peptidase Lsp. In both monoderms and diderms, signal peptide processing is preceded by acylation with a diacylglycerol through preprolipoprotein diacylglycerol transferase (Lgt). In diderms but also some monoderms, lipoproteins are further modified with a third acyl chain through lipoprotein N-acyl transferase (Lnt). Fully modified lipoproteins that are destined to be anchored in the inner leaflet of the outer membrane (OM) are selected, transported and inserted by the Lol (lipoprotein outer membrane localization) pathway machinery, which consists of the inner-membrane (IM) ABC transporter-like LolCDE complex, the periplasmic LolA chaperone and the OM LolB lipoprotein receptor. Retention of lipoproteins in the cytoplasmic membrane results from Lol avoidance signals that were originally described as the "+2 rule". Surface localization of lipoproteins in diderms is rare in most bacteria, with the exception of several spirochetal species. Type 2 (T2SS) and type 5 (T5SS) secretion systems are involved in secretion of specific surface lipoproteins of γ-proteobacteria. In the model spirochete Borrelia burgdorferi, surface lipoprotein secretion does not follow established sorting rules, but remains dependent on N-terminal peptide sequences. Secretion through the outer membrane requires maintenance of lipoproteins in a translocation-competent unfolded conformation

  9. Ofloxacin induces cytoplasmic respiration-deficient mutants in yeast Saccharomyces cerevisiae.

    Science.gov (United States)

    Obernauerová, M; Subík, J; Ebringer, L

    1992-05-01

    Ofloxacin, a new quinolone with potent antibacterial activity, was also found to be effective against yeast. At relatively high concentrations, and at mild alkaline pH, ofloxacin inhibited the growth of yeast cells in medium containing glucose, and prevented growth on glycerol, as carbon and energy source. The cells growing in the presence of ofloxacin exhibited abberrantly budded forms, lost their viability and many of them converted to cytoplasmic respiration-deficient mutants. Induction of mutants was also observed under non-growing conditions. The petite clones analysed exhibited suppressiveness and contained different fragments of the wild-type mitochondrial genome.

  10. The N-end rule pathway counteracts cell death by destroying proapoptotic protein fragments.

    Science.gov (United States)

    Piatkov, Konstantin I; Brower, Christopher S; Varshavsky, Alexander

    2012-07-03

    In the course of apoptosis, activated caspases cleave ∼500 to ∼1,000 different proteins in a mammalian cell. The dynamics of apoptosis involve a number of previously identified, caspase-generated proapoptotic protein fragments, defined as those that increase the probability of apoptosis. In contrast to activated caspases, which can be counteracted by inhibitor of apoptosis proteins, there is little understanding of antiapoptotic responses to proapoptotic protein fragments. One possibility is the regulation of proapoptotic fragments through their selective degradation. The previously identified proapoptotic fragments Cys-RIPK1, Cys-TRAF1, Asp-BRCA1, Leu-LIMK1, Tyr-NEDD9, Arg-BID, Asp-BCL(XL), Arg-BIM(EL), Asp-EPHA4, and Tyr-MET bear destabilizing N-terminal residues. Tellingly, the destabilizing nature (but not necessarily the actual identity) of N-terminal residues of proapoptotic fragments was invariably conserved in evolution. Here, we show that these proapoptotic fragments are short-lived substrates of the Arg/N-end rule pathway. Metabolic stabilization of at least one such fragment, Cys-RIPK1, greatly augmented the activation of the apoptosis-inducing effector caspase-3. In agreement with this understanding, even a partial ablation of the Arg/N-end rule pathway in two specific N-end rule mutants is shown to sensitize cells to apoptosis. We also found that caspases can inactivate components of the Arg/N-end rule pathway, suggesting a mutual suppression between this pathway and proapoptotic signaling. Together, these results identify a mechanistically specific and functionally broad antiapoptotic role of the Arg/N-end rule pathway. In conjunction with other apoptosis-suppressing circuits, the Arg/N-end rule pathway contributes to thresholds that prevent a transient or otherwise weak proapoptotic signal from reaching the point of commitment to apoptosis.

  11. The cytoplasmic 60 kDa progesterone receptor isoform predominates in the human amniochorion and placenta at term

    Directory of Open Access Journals (Sweden)

    Bell Stephen C

    2009-03-01

    Full Text Available Abstract Background The mechanism that initiates human parturition has been proposed to be 'functional progesterone withdrawal' whereby the 116 kDa B-isoform of the progesterone receptor (PR-B switches in favour of the 94 kDa A-isoform (PR-A in reproductive tissues. Recently, other PR isoforms, PR-S, PR-C and PR-M generated from the same gene have been identified and partially characterised. Methods and Results Using immunohistochemical, western blotting and RT-PCR techniques, evidence is provided that indicates the major PR isoform present in human term fetal membranes (amnion and chorion and syncytiotrophoblast of the placenta is neither of the classical nuclear PR-B or PR-A isoforms but is the N-terminally truncated 60 kDa PR-C isoform. Evidence is also provided that this 60 kDa isoform resides in the cytoplasm of the expressing cell types. Data are also presented to show that PR-B, PR-A and PR-S isoforms are essentially absent from the amnion and chorion, whereas PR isoforms A, B, C and S are all present in the decidua, with PR-A being the major isoform. The syncytiotrophoblast of the placenta contains the cytoplasmic 60 kDa isoform, but not isoforms PR-A, PR-B or PR-S. Conclusion The major PR isoform in the amnion, chorion and placenta is a 60 kDa protein that could be PR-C, suggesting that the cytoplasmic isoform has a specific role in extra-embryonic tissues and may be involved in the regulation of human parturition.

  12. Inborn errors of cytoplasmic triglyceride metabolism.

    Science.gov (United States)

    Wu, Jiang Wei; Yang, Hao; Wang, Shu Pei; Soni, Krishnakant G; Brunel-Guitton, Catherine; Mitchell, Grant A

    2015-01-01

    Triglyceride (TG) synthesis, storage, and degradation together constitute cytoplasmic TG metabolism (CTGM). CTGM is mostly studied in adipocytes, where starting from glycerol-3-phosphate and fatty acyl (FA)-coenzyme A (CoA), TGs are synthesized then stored in cytoplasmic lipid droplets. TG hydrolysis proceeds sequentially, producing FAs and glycerol. Several reactions of CTGM can be catalyzed by more than one enzyme, creating great potential for complex tissue-specific physiology. In adipose tissue, CTGM provides FA as a systemic energy source during fasting and is related to obesity. Inborn errors and mouse models have demonstrated the importance of CTGM for non-adipose tissues, including skeletal muscle, myocardium and liver, because steatosis and dysfunction can occur. We discuss known inborn errors of CTGM, including deficiencies of: AGPAT2 (a form of generalized lipodystrophy), LPIN1 (childhood rhabdomyolysis), LPIN2 (an inflammatory condition, Majeed syndrome, described elsewhere in this issue), DGAT1 (protein loosing enteropathy), perilipin 1 (partial lipodystrophy), CGI-58 (gene ABHD5, neutral lipid storage disease (NLSD) with ichthyosis and "Jordan's anomaly" of vacuolated polymorphonuclear leukocytes), adipose triglyceride lipase (ATGL, gene PNPLA2, NLSD with myopathy, cardiomyopathy and Jordan's anomaly), hormone-sensitive lipase (HSL, gene LIPE, hypertriglyceridemia, and insulin resistance). Two inborn errors of glycerol metabolism are known: glycerol kinase (GK, causing pseudohypertriglyceridemia) and glycerol-3-phosphate dehydrogenase (GPD1, childhood hepatic steatosis). Mouse models often resemble human phenotypes but may diverge markedly. Inborn errors have been described for less than one-third of CTGM enzymes, and new phenotypes may yet be identified.

  13. Successful Pregnancy in a Couple with Severe Male Factor Infertility after Selection of Sperm with Cytoplasmic Droplets

    Directory of Open Access Journals (Sweden)

    Jenna Bellish

    2015-01-01

    Full Text Available We present live births resulting from two separate IVF cycles in a couple in which ICSI was performed with sperm specifically selected for presence of small cytoplasmic droplets. These cycles followed previous cycles using standard sperm selection methods in which very poor embryo development and no pregnancies ensued. The male partner was diagnosed with severe male factor infertility including elevated DNA fragmentation.

  14. Fission fragment angular momentum

    International Nuclear Information System (INIS)

    Frenne, D. De

    1991-01-01

    Most of the energy released in fission is converted into translational kinetic energy of the fragments. The remaining excitation energy will be distributed among neutrons and gammas. An important parameter characterizing the scission configuration is the primary angular momentum of the nascent fragments. Neutron emission is not expected to decrease the spin of the fragments by more than one unit of angular momentum and is as such of less importance in the determination of the initial fragment spins. Gamma emission is a suitable tool in studying initial fragment spins because the emission time, number, energy, and multipolarity of the gammas strongly depend on the value of the primary angular momentum. The main conclusions of experiments on gamma emission were that the initial angular momentum of the fragments is large compared to the ground state spin and oriented perpendicular to the fission axis. Most of the recent information concerning initial fragment spin distributions comes from the measurement of isomeric ratios for isomeric pairs produced in fission. Although in nearly every mass chain isomers are known, only a small number are suitable for initial fission fragment spin studies. Yield and half-life considerations strongly limit the number of candidates. This has the advantage that the behavior of a specific isomeric pair can be investigated for a number of fissioning systems at different excitation energies of the fragments and fissioning nuclei. Because most of the recent information on primary angular momenta comes from measurements of isomeric ratios, the global deexcitation process of the fragments and the calculation of the initial fragment spin distribution from measured isomeric ratios are discussed here. The most important results on primary angular momentum determinations are reviewed and some theoretical approaches are given. 45 refs., 7 figs., 2 tabs

  15. Structural Insight into the Critical Role of the N-Terminal Region in the Catalytic Activity of Dual-Specificity Phosphatase 26.

    Directory of Open Access Journals (Sweden)

    Eun-Young Won

    Full Text Available Human dual-specificity phosphatase 26 (DUSP26 is a novel target for anticancer therapy because its dephosphorylation of the p53 tumor suppressor regulates the apoptosis of cancer cells. DUSP26 inhibition results in neuroblastoma cell cytotoxicity through p53-mediated apoptosis. Despite the previous structural studies of DUSP26 catalytic domain (residues 61-211, DUSP26-C, the high-resolution structure of its catalytically active form has not been resolved. In this study, we determined the crystal structure of a catalytically active form of DUSP26 (residues 39-211, DUSP26-N with an additional N-terminal region at 2.0 Å resolution. Unlike the C-terminal domain-swapped dimeric structure of DUSP26-C, the DUSP26-N (C152S monomer adopts a fold-back conformation of the C-terminal α8-helix and has an additional α1-helix in the N-terminal region. Consistent with the canonically active conformation of its protein tyrosine phosphate-binding loop (PTP loop observed in the structure, the phosphatase assay results demonstrated that DUSP26-N has significantly higher catalytic activity than DUSP26-C. Furthermore, size exclusion chromatography-multiangle laser scattering (SEC-MALS measurements showed that DUSP26-N (C152S exists as a monomer in solution. Notably, the crystal structure of DUSP26-N (C152S revealed that the N-terminal region of DUSP26-N (C152S serves a scaffolding role by positioning the surrounding α7-α8 loop for interaction with the PTP-loop through formation of an extensive hydrogen bond network, which seems to be critical in making the PTP-loop conformation competent for phosphatase activity. Our study provides the first high-resolution structure of a catalytically active form of DUSP26, which will contribute to the structure-based rational design of novel DUSP26-targeting anticancer therapeutics.

  16. Associations of N-terminal pro-B-type natriuretic peptide with kidney function decline in persons without clinical heart failure in the Heart and Soul Study.

    Science.gov (United States)

    Park, Meyeon; Vittinghoff, Eric; Shlipak, Michael G; Mishra, Rakesh; Whooley, Mary; Bansal, Nisha

    2014-12-01

    Subclinical volume overload in the absence of diagnosed heart failure (HF) may be an underrecognized contributor to kidney function decline in coronary artery disease (CAD) patients. We evaluated associations of circulating N-terminal pro-B-type natriuretic peptide (NT-proBNP), a marker of ventricular stretch, with change in estimated glomerular filtration rate (eGFR). We evaluated 535 patients with stable CAD and no history of HF, who were enrolled in the Heart and Soul Study and followed for 5 years. N-terminal pro-B-type natriuretic peptide was measured at baseline. We evaluated the associations of NT-proBNP with change in kidney function over 5 years: (a) annual percent change in eGFR, (b) rapid kidney function loss (> 3% per year for 5 years), and (c) incident eGFR 280.9 pg/mL) had a greater odds of rapid kidney function loss after full adjustment (odds ratio 2.95; 95% CI 1-8.65; P = .0492). Associations with incident eGFR < 60 mL/min per 1.73 m2 were also significant (adjusted odds ratio 4.23; 95% CI 1.05-16.98; P = .0422). Results were similar when analyzed using BNP as the predictor. N-terminal pro-B-type natriuretic peptide and BNP are strongly and independently associated with accelerated kidney function loss, even in the absence of clinical HF. These findings suggest that subclinical cardiovascular dysfunction may contribute to elevated kidney disease risk in persons with CAD. Copyright © 2014 Elsevier Inc. All rights reserved.

  17. The DAF-16 FOXO transcription factor regulates natc-1 to modulate stress resistance in Caenorhabditis elegans, linking insulin/IGF-1 signaling to protein N-terminal acetylation.

    Science.gov (United States)

    Warnhoff, Kurt; Murphy, John T; Kumar, Sandeep; Schneider, Daniel L; Peterson, Michelle; Hsu, Simon; Guthrie, James; Robertson, J David; Kornfeld, Kerry

    2014-10-01

    The insulin/IGF-1 signaling pathway plays a critical role in stress resistance and longevity, but the mechanisms are not fully characterized. To identify genes that mediate stress resistance, we screened for C. elegans mutants that can tolerate high levels of dietary zinc. We identified natc-1, which encodes an evolutionarily conserved subunit of the N-terminal acetyltransferase C (NAT) complex. N-terminal acetylation is a widespread modification of eukaryotic proteins; however, relatively little is known about the biological functions of NATs. We demonstrated that loss-of-function mutations in natc-1 cause resistance to a broad-spectrum of physiologic stressors, including multiple metals, heat, and oxidation. The C. elegans FOXO transcription factor DAF-16 is a critical target of the insulin/IGF-1 signaling pathway that mediates stress resistance, and DAF-16 is predicted to directly bind the natc-1 promoter. To characterize the regulation of natc-1 by DAF-16 and the function of natc-1 in insulin/IGF-1 signaling, we analyzed molecular and genetic interactions with key components of the insulin/IGF-1 pathway. natc-1 mRNA levels were repressed by DAF-16 activity, indicating natc-1 is a physiological target of DAF-16. Genetic studies suggested that natc-1 functions downstream of daf-16 to mediate stress resistance and dauer formation. Based on these findings, we hypothesize that natc-1 is directly regulated by the DAF-16 transcription factor, and natc-1 is a physiologically significant effector of the insulin/IGF-1 signaling pathway that mediates stress resistance and dauer formation. These studies identify a novel biological function for natc-1 as a modulator of stress resistance and dauer formation and define a functionally significant downstream effector of the insulin/IGF-1 signaling pathway. Protein N-terminal acetylation mediated by the NatC complex may play an evolutionarily conserved role in regulating stress resistance.

  18. The biological variation of N-terminal pro-brain natriuretic peptide in postmenopausal women with type 2 diabetes: a case control study.

    Science.gov (United States)

    González, Susana; Kilpatrick, Eric S; Atkin, Stephen L

    2012-01-01

    The incidence of heart failure in type 2 diabetes is high and it has poorer prognosis when compared with patients without diabetes. Access to echocardiography is limited and alternative methods to identify early heart failure such as the measurement of natriuretic peptides levels have been proposed. However, their wide biological variation could limit their clinical utility. Our aim was to determine if the intrinsic biological variation of one of these peptides, N-terminal proBNP, is as wide in type 2 diabetes as it is in health and to calculate the critical difference values that could be utilised in clinical practice to ensure changes observed between two samples are due to intervention rather than to its biological variability. 12 postmenopausal women with diet controlled type 2 diabetes and without heart failure were compared with 11 control postmenopausal women without diabetes. N-terminal proBNP levels were measured on 10 occasions. The biological variation was calculated according to Fraser's methods. The mean NT-proBNP level was similar in both groups (mean ± standard deviation; type 2 diabetes, 10.7 pmol/L± 8.5 versus 8.49±6.0 pmol/L, p = 0.42). The biological variation was also similarly wide. The critical difference in patients with type 2 diabetes was between -70% and ±236%. Type 2 diabetes does not appear to significantly influence the marked biological variation of N-terminal proBNP in postmenopausal women. The critical difference values reported in this study could be used to titrate therapy or monitor response to interventions although the change required in between samples is wide and this might limit its utility.

  19. The biological variation of N-terminal pro-brain natriuretic peptide in postmenopausal women with type 2 diabetes: a case control study.

    Directory of Open Access Journals (Sweden)

    Susana González

    Full Text Available BACKGROUND: The incidence of heart failure in type 2 diabetes is high and it has poorer prognosis when compared with patients without diabetes. Access to echocardiography is limited and alternative methods to identify early heart failure such as the measurement of natriuretic peptides levels have been proposed. However, their wide biological variation could limit their clinical utility. Our aim was to determine if the intrinsic biological variation of one of these peptides, N-terminal proBNP, is as wide in type 2 diabetes as it is in health and to calculate the critical difference values that could be utilised in clinical practice to ensure changes observed between two samples are due to intervention rather than to its biological variability. METHODOLOGY/PRINCIPAL FINDINGS: 12 postmenopausal women with diet controlled type 2 diabetes and without heart failure were compared with 11 control postmenopausal women without diabetes. N-terminal proBNP levels were measured on 10 occasions. The biological variation was calculated according to Fraser's methods. The mean NT-proBNP level was similar in both groups (mean ± standard deviation; type 2 diabetes, 10.7 pmol/L± 8.5 versus 8.49±6.0 pmol/L, p = 0.42. The biological variation was also similarly wide. The critical difference in patients with type 2 diabetes was between -70% and ±236%. CONCLUSIONS: Type 2 diabetes does not appear to significantly influence the marked biological variation of N-terminal proBNP in postmenopausal women. The critical difference values reported in this study could be used to titrate therapy or monitor response to interventions although the change required in between samples is wide and this might limit its utility.

  20. The N-terminal domain of the thermo-regulated surface protein PrpA of Enterococcus faecium binds to fibrinogen, fibronectin and platelets.

    Science.gov (United States)

    Guzmán Prieto, Ana M; Urbanus, Rolf T; Zhang, Xinglin; Bierschenk, Damien; Koekman, C Arnold; van Luit-Asbroek, Miranda; Ouwerkerk, Janneke P; Pape, Marieke; Paganelli, Fernanda L; Wobser, Dominique; Huebner, Johannes; Hendrickx, Antoni P A; Bonten, Marc J M; Willems, Rob J L; van Schaik, Willem

    2015-12-17

    Enterococcus faecium is a commensal of the mammalian gastrointestinal tract, but is also found in non-enteric environments where it can grow between 10 °C and 45 °C. E. faecium has recently emerged as a multi-drug resistant nosocomial pathogen. We hypothesized that genes involved in the colonization and infection of mammals exhibit temperature-regulated expression control and we therefore performed a transcriptome analysis of the clinical isolate E. faecium E1162, during mid-exponential growth at 25 °C and 37 °C. One of the genes that exhibited differential expression between 25 °C and 37 °C, was predicted to encode a peptidoglycan-anchored surface protein. The N-terminal domain of this protein is unique to E. faecium and closely related enterococci, while the C-terminal domain is homologous to the Streptococcus agalactiae surface protein BibA. This region of the protein contains proline-rich repeats, leading us to name the protein PrpA for proline-rich protein A. We found that PrpA is a surface-exposed protein which is most abundant during exponential growth at 37 °C in E. faecium E1162. The heterologously expressed and purified N-terminal domain of PrpA was able to bind to the extracellular matrix proteins fibrinogen and fibronectin. In addition, the N-terminal domain of PrpA interacted with both non-activated and activated platelets.

  1. Left-handed helical preference in an achiral peptide chain is induced by an L-amino acid in an N-terminal type II β-turn.

    Science.gov (United States)

    De Poli, Matteo; De Zotti, Marta; Raftery, James; Aguilar, Juan A; Morris, Gareth A; Clayden, Jonathan

    2013-03-15

    Oligomers of the achiral amino acid Aib adopt helical conformations in which the screw-sense may be controlled by a single N-terminal residue. Using crystallographic and NMR techniques, we show that the left- or right-handed sense of helical induction arises from the nature of the β-turn at the N terminus: the tertiary amino acid L-Val induces a left-handed type II β-turn in both the solid state and in solution, while the corresponding quaternary amino acid L-α-methylvaline induces a right-handed type III β-turn.

  2. N-terminal diproline and charge group effects on the stabilization of helical conformation in alanine-based short peptides: CD studies with water and methanol as solvent.

    Science.gov (United States)

    Goyal, Bhupesh; Srivastava, Kinshuk Raj; Durani, Susheel

    2017-06-01

    Protein folding problem remains a formidable challenge as main chain, side chain and solvent interactions remain entangled and have been difficult to resolve. Alanine-based short peptides are promising models to dissect protein folding initiation and propagation structurally as well as energetically. The effect of N-terminal diproline and charged side chains is assessed on the stabilization of helical conformation in alanine-based short peptides using circular dichroism (CD) with water and methanol as solvent. A1 (Ac-Pro-Pro-Ala-Lys-Ala-Lys-Ala-Lys-Ala-NH 2 ) is designed to assess the effect of N-terminal homochiral diproline and lysine side chains to induce helical conformation. A2 (Ac-Pro-Pro-Glu-Glu-Ala-Ala-Lys-Lys-Ala-NH 2 ) and A3 (Ac-dPro-Pro-Glu-Glu-Ala-Ala-Lys-Lys-Ala-NH 2 ) with N-terminal homochiral and heterochiral diproline, respectively, are designed to assess the effect of Glu...Lys (i, i + 4) salt bridge interactions on the stabilization of helical conformation. The CD spectra of A1, A2 and A3 in water manifest different amplitudes of the observed polyproline II (PPII) signals, which indicate different conformational distributions of the polypeptide structure. The strong effect of solvent substitution from water to methanol is observed for the peptides, and CD spectra in methanol evidence A2 and A3 as helical folds. Temperature-dependent CD spectra of A1 and A2 in water depict an isodichroic point reflecting coexistence of two conformations, PPII and β-strand conformation, which is consistent with the previous studies. The results illuminate the effect of N-terminal diproline and charged side chains in dictating the preferences for extended-β, semi-extended PPII and helical conformation in alanine-based short peptides. The results of the present study will enhance our understanding on stabilization of helical conformation in short peptides and hence aid in the design of novel peptides with helical structures. Copyright © 2017 European Peptide

  3. Nickel Ligation of the N-Terminal Amine of HypA Is Required for Urease Maturation in Helicobacter pylori

    Energy Technology Data Exchange (ETDEWEB)

    Hu, Heidi Q. [Department; Johnson, Ryan C. [Microbiology; Merrell, D. Scott [Microbiology; Maroney, Michael J. [Department

    2017-02-17

    The human pathogen Helicobacter pylori requires nickel for colonization of the acidic environment of the stomach. HypA, a Ni metallochaperone that is typically associated with hydrogenase maturation, is also required for urease maturation and acid survival of H. pylori. There are two proposed Ni site structures for HypA; one is a paramagnetic six-coordinate site characterized by X-ray absorption spectroscopy (XAS) in unmodified HypA, while another is a diamagnetic four-coordinate planar site characterized by solution nuclear magnetic resonance in an N-terminally modified HypA construct. To determine the role of the N-terminal amine in Ni binding of HypA, an N-terminal extension variant, L2*-HypA, in which a leucine residue was inserted into the second position of the amino acid sequence in the proposed Ni-binding motif, was characterized in vitro and in vivo. Structural characterization of the Ni site using XAS showed a coordination change from six-coordinate in wild-type HypA (WT-HypA) to five-coordinate pyramidal in L2*-HypA, which was accompanied by the loss of two N/O donor protein ligands and the addition of an exogenous bromide ligand from the buffer. The magnetic properties of the Ni sites in WT-HypA compared to those of the Ni sites in L2*-HypA confirmed that a spin-state change from high to low spin accompanied this change in structure. The L2*-HypA H. pylori strain was shown to be acid sensitive and deficient in urease activity in vivo. In vitro characterization showed that L2*-HypA did not disrupt the HypA–UreE interaction that is essential for urease maturation but was at least 20-fold weaker in Ni binding than WT-HypA. Characterization of the L2*-HypA variant clearly demonstrates that the N-terminal amine of HypA is involved in proper Ni coordination and is necessary for urease activity and acid survival.

  4. Prognostic Value of N-Terminal Pro-B-Type Natriuretic Peptide Levels in Heart Failure Patients With and Without Atrial Fibrillation

    DEFF Research Database (Denmark)

    Kristensen, Søren Lund; Jhund, Pardeep S; Mogensen, Ulrik M

    2017-01-01

    BACKGROUND: Patients with heart failure (HF) and atrial fibrillation (AF) have higher circulating levels of NT-proBNP (N-terminal pro-B-type natriuretic peptide) than HF patients without AF. There is uncertainty about the prognostic importance of a given concentration of NT-proBNP in HF patients...... patients with AF had higher NT-proBNP than those without AF. However, above a concentration of 400 pg/mL (representing most patients in each group), NT-proBNP had similar predictive value for adverse cardiovascular outcomes, irrespective of AF status. CLINICAL TRIAL REGISTRATION: URL: https...

  5. Investigation of functional aspects of the N-terminal region of elongation factor Tu from Escherichia coli using a protein engineering approach

    DEFF Research Database (Denmark)

    Laurberg, M; Mansilla, Francisco; Clark, Brian F. C.

    1998-01-01

    The function of the N-terminal region of elongation factor Tu is still unexplained. Until recently, it has not been visible in electron density maps from x-ray crystallography studies, but the presence of several well conserved basic residues suggest that this part of the molecule is of structural...... importance for the factor to function properly. In this study, two lysines at positions 4 and 9 were mutated separately to alanine or glutamate. The resulting four point mutants were expressed and purified using the pGEX system. The untagged products were characterized with regard to guanine...

  6. Yeast aminopeptidase I is post-translationally sorted from the cytosol to the vacuole by a mechanism mediated by its bipartite N-terminal extension.

    OpenAIRE

    Seguí-Real, B; Martinez, M; Sandoval, I V

    1995-01-01

    Transport of aminopeptidase I (API) to the vacuole appears to be insensitive to blockage of the secretory pathway. Here we show that the N-terminal extension of the 61 kDa precursor of API (pAPI) is proteolytically processed in two sequential steps. The first step involves proteinase A (PrA) and produces a 55 kDa unstable intermediate (iAPI). The second step involves proteinase B (PrB) and converts iAPI into the 50 kDa stable, mature enzyme (mAPI). Reversion of the cup1 growth phenotype by a ...

  7. Binding Mechanism of the N-Terminal SH3 Domain of CrkII and Proline-Rich Motifs in cAbl

    OpenAIRE

    Bhatt, Veer��S.; Zeng, Danyun; Krieger, Inna; Sacchettini, James��C.; Cho, Jae-Hyun

    2016-01-01

    The N-terminal Src homology 3 (nSH3) domain of a signaling adaptor protein, CT-10 regulator of kinase II (CrkII), recognizes proline-rich motifs (PRMs) of binding partners, such as cAbl kinase. The interaction between CrkII and cAbl kinase is��involved in the regulation of cell spreading, microbial pathogenesis, and cancer metastasis. Here, we report the detailed biophysical characterizations of the interactions between the nSH3 domain of CrkII and PRMs in cAbl. We identified that the nSH3 do...

  8. The molecular mechanism and physiological role of cytoplasmic streaming.

    Science.gov (United States)

    Tominaga, Motoki; Ito, Kohji

    2015-10-01

    Cytoplasmic streaming occurs widely in plants ranging from algae to angiosperms. However, the molecular mechanism and physiological role of cytoplasmic streaming have long remained unelucidated. Recent molecular genetic approaches have identified specific myosin members (XI-2 and XI-K as major and XI-1, XI-B, and XI-I as minor motive forces) for the generation of cytoplasmic streaming among 13 myosin XIs in Arabidopsis thaliana. Simultaneous knockout of these myosin XI members led to a reduced velocity of cytoplasmic streaming and marked defects of plant development. Furthermore, the artificial modifications of myosin XI-2 velocity changed plant and cell sizes along with the velocity of cytoplasmic streaming. Therefore, we assume that cytoplasmic streaming is one of the key regulators in determining plant size. Copyright © 2015 The Authors. Published by Elsevier Ltd.. All rights reserved.

  9. N-terminal proB-type natriuretic peptide (NT-proBNP) concentrations in elite rugby players at rest and after active and passive recovery following strenuous training sessions.

    Science.gov (United States)

    Banfi, Giuseppe; D'Eril, Gianlodovico Melzi; Barassi, Alessandra; Lippi, Giuseppe

    2008-01-01

    The serum biomarker N-terminal proB-type natriuretic peptide (NT-proBNP), a cleaved fragment of the brain natriuretic peptide (BNP) precursor (amino acids 1-76), is accepted as a standard marker for evaluating and monitoring cardiac injury characterized by myocardial wall stress. Strenuous exercise may generate transitory ischemia, myocardial stress and diastolic left ventricular dysfunction, possibly inducing increased concentrations of NT-proBNP. A purported caveat to prolonged strenuous exercise is based on evidence for biochemical and structural signs of heart dysfunction in recreational athletes after continuous exertion. We compared NT-proBNP levels in three groups of physically fit subjects: top-level rugby players, professional soccer players and healthy controls. NT-proBNP concentrations were measured at rest and after an intensive training session followed by two different recovery strategies (passive or active). A comparison of the three samples showed that NT-proBNP concentrations in the rugby players were lower than those in controls at rest and were similar to those in professional soccer players. Elevated post-training NT-proBNP levels were unaffected by the type of recovery. The relatively high NT-proBNP levels after active recovery when psychophysical stress is higher, because of cycling and cold water immersion, suggest that not only endurance exercise, but also strenuous, stressful short exercise can induce an increase in NT-proBNP concentrations. In this sample of professional athletes, NT-proBNP was low at rest, and the increase after physical exercise was physiological.

  10. The Green Tea Component (-)-Epigallocatechin-3-Gallate Sensitizes Primary Endothelial Cells to Arsenite-Induced Apoptosis by Decreasing c-Jun N-Terminal Kinase-Mediated Catalase Activity.

    Science.gov (United States)

    Kim, Jee-Youn; Choi, Ji-Young; Lee, Hyeon-Ju; Byun, Catherine Jeonghae; Park, Jung-Hyun; Park, Jae Hoon; Cho, Ho-Seong; Cho, Sung-Jin; Jo, Sangmee Ahn; Jo, Inho

    2015-01-01

    The green tea component (-)-epigallocatechin-3-gallate (EGCG) has been shown to sensitize many different types of cancer cells to anticancer drug-induced apoptosis, although it protects against non-cancerous primary cells against toxicity from certain conditions such as exposure to arsenic (As) or ultraviolet irradiation. Here, we found that EGCG promotes As-induced toxicity of primary-cultured bovine aortic endothelial cells (BAEC) at doses in which treatment with each chemical alone had no such effect. Increased cell toxicity was accompanied by an increased condensed chromatin pattern and fragmented nuclei, cleaved poly(ADP-ribose) polymerase (PARP), activity of the pro-apoptotic enzymes caspases 3, 8 and 9, and Bax translocation into mitochondria, suggesting the involvement of an apoptotic signaling pathway. Fluorescence activated cell sorting analysis revealed that compared with EGCG or As alone, combined EGCG and As (EGCG/As) treatment significantly induced production of reactive oxygen species (ROS), which was accompanied by decreased catalase activity and increased lipid peroxidation. Pretreatment with N-acetyl-L-cysteine or catalase reversed EGCG/As-induced caspase activation and EC toxicity. EGCG/As also increased the phosphorylation of c-Jun N-terminal kinase (JNK), which was not reversed by catalase. However, pretreatment with the JNK inhibitor SP600125 reversed all of the observed effects of EGCG/As, suggesting that JNK may be the most upstream protein examined in this study. Finally, we also found that all the observed effects by EGCG/As are true for other types of EC tested. In conclusion, this is firstly to show that EGCG sensitizes non-cancerous EC to As-induced toxicity through ROS-mediated apoptosis, which was attributed at least in part to a JNK-activated decrease in catalase activity.

  11. The Green Tea Component (--Epigallocatechin-3-Gallate Sensitizes Primary Endothelial Cells to Arsenite-Induced Apoptosis by Decreasing c-Jun N-Terminal Kinase-Mediated Catalase Activity.

    Directory of Open Access Journals (Sweden)

    Jee-Youn Kim

    Full Text Available The green tea component (--epigallocatechin-3-gallate (EGCG has been shown to sensitize many different types of cancer cells to anticancer drug-induced apoptosis, although it protects against non-cancerous primary cells against toxicity from certain conditions such as exposure to arsenic (As or ultraviolet irradiation. Here, we found that EGCG promotes As-induced toxicity of primary-cultured bovine aortic endothelial cells (BAEC at doses in which treatment with each chemical alone had no such effect. Increased cell toxicity was accompanied by an increased condensed chromatin pattern and fragmented nuclei, cleaved poly(ADP-ribose polymerase (PARP, activity of the pro-apoptotic enzymes caspases 3, 8 and 9, and Bax translocation into mitochondria, suggesting the involvement of an apoptotic signaling pathway. Fluorescence activated cell sorting analysis revealed that compared with EGCG or As alone, combined EGCG and As (EGCG/As treatment significantly induced production of reactive oxygen species (ROS, which was accompanied by decreased catalase activity and increased lipid peroxidation. Pretreatment with N-acetyl-L-cysteine or catalase reversed EGCG/As-induced caspase activation and EC toxicity. EGCG/As also increased the phosphorylation of c-Jun N-terminal kinase (JNK, which was not reversed by catalase. However, pretreatment with the JNK inhibitor SP600125 reversed all of the observed effects of EGCG/As, suggesting that JNK may be the most upstream protein examined in this study. Finally, we also found that all the observed effects by EGCG/As are true for other types of EC tested. In conclusion, this is firstly to show that EGCG sensitizes non-cancerous EC to As-induced toxicity through ROS-mediated apoptosis, which was attributed at least in part to a JNK-activated decrease in catalase activity.

  12. Inhibiting c-Jun N-terminal kinase partially attenuates caffeine-dependent cell death without alleviating the caffeine-induced reduction in mitochondrial respiration in C2C12 skeletal myotubes.

    Science.gov (United States)

    Downs, R M; Hughes, M A; Kinsey, S T; Johnson, M C; Baumgarner, B L

    2016-11-04

    Caffeine is a widely consumed stimulant that has previously been shown to promote cytotoxic stress and even cell death in numerous mammalian cell lines. Thus far there is little information available regarding the toxicity of caffeine in skeletal muscle cells. Our preliminary data revealed that treating C2C12 myotubes with 5 mM caffeine for 6 h increased nuclear fragmentation and reduced basal and maximal oxygen consumption rate (OCR) in skeletal myotubes. The purpose of this study was to further elucidate the pathways by which caffeine increased cell death and reduced mitochondrial respiration. We specifically examined the role of c-Jun N-terminal kinase (JNK), which has previously been shown to simultaneously increase caspase-dependent cell death and reduce mitochondrial respiration in other mammalian cell lines. We found that caffeine promoted a dose-dependent increase in cell death in multinucleated myotubes but did not in mononucleated myoblasts. The addition of 10 μM Z-DEVD-FMK, a specific inhibitor of executioner caspases, completely inhibited caffeine-dependent cell death. Further, the addition of 400 μM dantrolene, a specific ryanodine receptor (RYR) inhibitor, prevented the caffeine-dependent increase in cell death and the reduction in basal and maximal OCR. We also discovered that caffeine treatment significantly increased the phosphorylation of JNK and that the addition of 30 μM SP600125 (JNKi), a specific JNK inhibitor, partially attenuated caffeine-induced cell death without preventing the caffeine-dependent reduction in basal and maximal OCR. Our results suggest that JNK partially mediates the increase in caspase-dependent cell death but does not contribute to reduced mitochondrial respiration in caffeine-treated skeletal muscle cells. We conclude that caffeine increased cell death and reduced mitochondrial respiration in a calcium-dependent manner by activating the RYR and promoting reticular calcium release. Copyright © 2016 Elsevier Inc

  13. Hydrogen rearrangement to and from radical z fragments in electron capture dissociation of peptides

    DEFF Research Database (Denmark)

    Savitski, Mikhail M; Kjeldsen, Frank; Nielsen, Michael L

    2007-01-01

    Hydrogen rearrangement is an important process in radical chemistry. A high degree of H. rearrangement to and from z. ionic fragments (combined occurrence frequency 47% compared with that of z.) is confirmed in analysis of 15,000 tandem mass spectra of tryptic peptides obtained with electron...... capture dissociation (ECD), including previously unreported double H. losses. Consistent with the radical character of H. abstraction, the residue determining the formation rate of z' = z. + H. species is found to be the N-terminal residue in z. species. The size of the complementary c(m)' fragment turned...... that other factors than thermochemistry are responsible for directing the site of ECD cleavage. Understanding hydrogen attachment to and loss from ECD fragments should facilitate automatic interpretation ECD mass spectra in protein identification and characterization, including de novo sequencing....

  14. Self-cytoplasmic DNA upregulates the mutator enzyme APOBEC3A leading to chromosomal DNA damage.

    Science.gov (United States)

    Suspène, Rodolphe; Mussil, Bianka; Laude, Hélène; Caval, Vincent; Berry, Noémie; Bouzidi, Mohamed S; Thiers, Valérie; Wain-Hobson, Simon; Vartanian, Jean-Pierre

    2017-04-07

    Foreign and self-cytoplasmic DNA are recognized by numerous DNA sensor molecules leading to the production of type I interferons. Such DNA agonists should be degraded otherwise cells would be chronically stressed. Most human APOBEC3 cytidine deaminases can initiate catabolism of cytoplasmic mitochondrial DNA. Using the human myeloid cell line THP-1 with an interferon inducible APOBEC3A gene, we show that cytoplasmic DNA triggers interferon α and β production through the RNA polymerase III transcription/RIG-I pathway leading to massive upregulation of APOBEC3A. By catalyzing C→U editing in single stranded DNA fragments, the enzyme prevents them from re-annealing so attenuating the danger signal. The price to pay is chromosomal DNA damage in the form of CG→TA mutations and double stranded DNA breaks which, in the context of chronic inflammation, could drive cells down the path toward cancer. © The Author(s) 2017. Published by Oxford University Press on behalf of Nucleic Acids Research.

  15. The 133-kDa N-terminal domain enables myosin 15 to maintain mechanotransducing stereocilia and is essential for hearing

    Science.gov (United States)

    Fang, Qing; Indzhykulian, Artur A; Mustapha, Mirna; Riordan, Gavin P; Dolan, David F; Friedman, Thomas B; Belyantseva, Inna A; Frolenkov, Gregory I; Camper, Sally A; Bird, Jonathan E

    2015-01-01

    The precise assembly of inner ear hair cell stereocilia into rows of increasing height is critical for mechanotransduction and the sense of hearing. Yet, how the lengths of actin-based stereocilia are regulated remains poorly understood. Mutations of the molecular motor myosin 15 stunt stereocilia growth and cause deafness. We found that hair cells express two isoforms of myosin 15 that differ by inclusion of an 133-kDa N-terminal domain, and that these isoforms can selectively traffic to different stereocilia rows. Using an isoform-specific knockout mouse, we show that hair cells expressing only the small isoform remarkably develop normal stereocilia bundles. However, a critical subset of stereocilia with active mechanotransducer channels subsequently retracts. The larger isoform with the 133-kDa N-terminal domain traffics to these specialized stereocilia and prevents disassembly of their actin core. Our results show that myosin 15 isoforms can navigate between functionally distinct classes of stereocilia, and are independently required to assemble and then maintain the intricate hair bundle architecture. DOI: http://dx.doi.org/10.7554/eLife.08627.001 PMID:26302205

  16. The structure of the BIR3 domain of cIAP1 in complex with the N-terminal peptides of SMAC and caspase-9

    Energy Technology Data Exchange (ETDEWEB)

    Kulathila, Raviraj; Vash, Brian; Sage, David; Cornell-Kennon, Susan; Wright, Kirk; Koehn, James; Stams, Travis; Clark, Kirk; Price, Allen ((Novartis)); ((Emmanuel))

    2009-06-24

    The inhibitor of apoptosis protein (IAP) family of molecules inhibit apoptosis through the suppression of caspase activity. It is known that the XIAP protein regulates both caspase-3 and caspase-9 through direct protein-protein interactions. Specifically, the BIR3 domain of XIAP binds to caspase-9 via a 'hotspot' interaction in which the N-terminal residues of caspase-9 bind in a shallow groove on the surface of XIAP. This interaction is regulated via SMAC, the N-terminus of which binds in the same groove, thus displacing caspase-9. The mechanism of suppression of apoptosis by cIAP1 is less clear. The structure of the BIR3 domain of cIAP1 (cIAP1-BIR3) in complex with N-terminal peptides from both SMAC and caspase-9 has been determined. The binding constants of these peptides to cIAP1-BIR3 have also been determined using the surface plasmon resonance technique. The structures show that the peptides interact with cIAP1 in the same way that they interact with XIAP: both peptides bind in a similar shallow groove in the BIR3 surface, anchored at the N-terminus by a charge-stabilized hydrogen bond. The binding data show that the SMAC and caspase-9 peptides bind with comparable affinities (85 and 48 nM, respectively).

  17. Pushing the limits of sulfur SAD phasing: de novo structure solution of the N-terminal domain of the ectodomain of HCV E1

    Energy Technology Data Exchange (ETDEWEB)

    El Omari, Kamel; Iourin, Oleg; Kadlec, Jan [University of Oxford, Oxford OX3 7BN (United Kingdom); Fearn, Richard; Hall, David R. [Diamond Light Source Ltd, Diamond House, Harwell Science and Innovation Campus, Didcot OX11 0DE (United Kingdom); Harlos, Karl [University of Oxford, Oxford OX3 7BN (United Kingdom); Grimes, Jonathan M.; Stuart, David I., E-mail: dave@strubi.ox.ac.uk [University of Oxford, Oxford OX3 7BN (United Kingdom); Diamond Light Source Ltd, Diamond House, Harwell Science and Innovation Campus, Didcot OX11 0DE (United Kingdom)

    2014-08-01

    The sulfur SAD phasing method was successfully used to determine the structure of the N-terminal domain of HCV E1 from low-resolution diffracting crystals by combining data from 32 crystals. Single-wavelength anomalous dispersion of S atoms (S-SAD) is an elegant phasing method to determine crystal structures that does not require heavy-atom incorporation or selenomethionine derivatization. Nevertheless, this technique has been limited by the paucity of the signal at the usual X-ray wavelengths, requiring very accurate measurement of the anomalous differences. Here, the data collection and structure solution of the N-terminal domain of the ectodomain of HCV E1 from crystals that diffracted very weakly is reported. By combining the data from 32 crystals, it was possible to solve the sulfur substructure and calculate initial maps at 7 Å resolution, and after density modication and phase extension using a higher resolution native data set to 3.5 Å resolution model building was achievable.

  18. Expression, crystallization and preliminary X-ray diffraction analysis of the N-terminal domain of nsp2 from avian infectious bronchitis virus

    International Nuclear Information System (INIS)

    Yang, Anqi; Wei, Lei; Zhao, Weiran; Xu, Yuanyuan; Rao, Zihe

    2009-01-01

    The N-terminal domain of nsp2 from avian infectious bronchitis virus has been purified and crystallized. The crystals diffracted to 2.5 Å resolution. Avian infectious bronchitis virus (IBV) is a prototype of the group III coronaviruses and encodes 15 nonstructural proteins which make up the transcription/replication machinery. The nsp2 protein from IBV has a unique and novel sequence and has no experimentally confirmed function in replication, whereas it has been proposed to be crucial for early viral infection and may inhibit the early host immune response. The gene that encodes a double-mutant IBV nsp2 N-terminal domain (residues 9–393 of the polyprotein, with mutations Q132L and L270F) was cloned and expressed in Escherichia coli and the protein was subjected to crystallization trials. The crystals diffracted to 2.5 Å resolution and belonged to space group P6 2 or P6 4 , with unit-cell parameters a = b = 114.2, c = 61.0 Å, α = β = 90, γ = 120°. Each asymmetric unit contained one molecule

  19. Glycosylation of the N-terminal potential N-glycosylation sites in the human α1,3-fucosyltransferase V and -VI (hFucTV and -VI)

    DEFF Research Database (Denmark)

    Christensen, Lise Lotte; Bross, Peter Gerd; Ørntoft, Torben Falck

    2000-01-01

    Human alpha1,3-fucosyltransferase V and -VI (hFucTV and -VI) each contain four potential N-glycosylation sites (hFucTV: Asn60, Asn105, Asn167 and Asn198 and hFucTVI: Asn46, Asn91, Asn153 and Asn184). Glycosylation of the two N-terminal potential N-glycosylation sites (hFucTV: Asn60, Asn105 and h......FucTVI: Asn46 and Asn91) have never been studied in detail. In the present study, we have analysed the glycosylation of these potential N-glycosylation sites. Initially, we compared the molecular mass of hFucTV and -VI expressed in COS-7 cells treated with tunicamycin with the mass of the proteins...... in untreated cells. The difference in molecular mass between the proteins in treated and untreated cells corresponded to the presence of at least three N-linked glycans. We then made a series of mutants, in which the asparagine residues in the N-terminal potential N-glycosylation sites were replaced...

  20. Characterization of the N-Terminal Catalytic Domain of Lytµ1/6, an Endolysin from Streptomyces aureofaciens Phage µ1/6.

    Science.gov (United States)

    Farkašovská, Jarmila; Godány, Andrej

    2016-10-01

    Previous characterization of Lytµ1/6, an endolysin from Streptomyces aureofaciens phage µ1/6, suggested that the N-terminal domain is responsible for the catalytic activity of Lytµ1/6. Mutational analyses (deletions and site-directed mutagenesis) demonstrated that lytic activity of Lytµ1/6 relies on the N-terminal part of about 200 amino acid residues. Various C-terminally truncated versions of Lytµ1/6 failed to cause lysis, indicating the necessity of the CBD for full enzyme activity. Functional analysis of the point mutants suggested that the residues K27, H31, E109, H176, and D184 were essential for lytic activity of the µ1/6 endolysin. Further characterization of the purified Lytµ1/6 revealed that this endolysin is an N-acetylmuramoyl-L-alanine amidase which seems to be unrelated to any of the known conserved catalytic domains of phage endolysins or bacterial autolysins.

  1. Local helix content and RNA-binding activity of the N-terminal leucine-repeat region of hepatitis delta antigen

    Energy Technology Data Exchange (ETDEWEB)

    Cheng Jyawei; Lin Ijin; Lou Yuanchou; Pai Mingtao [National Tsing Hua University, Department of Life Science (China); Wu Hueynan [Academia Sinica, Institute of Molecular Biology (China)

    1998-07-15

    Hepatitis delta virus (HDV) is a satellite virus of the hepatitis B virus (HBV) which provides the surface antigen for the viral coat. Our results show that the N-terminal leucine-repeat region of hepatitis delta antigen (HDAg), encompassing residues 24-50, binds to the autolytic domain of HDV genomic RNA and attenuates its autolytic activity. The solution conformation of a synthetic peptide corresponding to residues 24-50 of HDAg as determined by two-dimensional {sup 1}H NMR and circular dichroism techniques is found to be an {alpha}-helix. The local helix content of this peptide was analyzed by NOEs and coupling constants. Mutagenesis studies indicate that Lys{sup 38}, Lys{sup 39}, and Lys{sup 40} within this {alpha}-helical peptide may be directly involved in RNA binding. A structural knowledge of the N-terminal leucine-repeat region of HDAg thus provides a molecular basis for understanding its role in the interaction with RNA.

  2. The N-terminal domain of APJ, a CNS-based coreceptor for HIV-1, is essential for its receptor function and coreceptor activity

    International Nuclear Information System (INIS)

    Zhou Naiming; Zhang Xiaoling; Fan Xuejun; Argyris, Elias; Fang Jianhua; Acheampong, Edward; DuBois, Garrett C.; Pomerantz, Roger J.

    2003-01-01

    The human APJ, a G protein-coupled seven-transmembrane receptor, has been found to be dramatically expressed in the human central nervous system (CNS) and also to serve as a coreceptor for the entry of human immunodeficiency virus type 1 (HIV-1) and simian immunodeficiency virus (SIV). Studies with animal models suggested that APJ and its natural ligand, apelin, play an important role in the central control of body fluid homeostasis, and in regulation of blood pressure and cardiac contractility. In this study, we characterize the structural and functional determinants of the N-terminal domain of APJ in interactions with its natural ligand and HIV-1 envelope glycoprotein. We demonstrate that the second 10 residues of the N-terminal domain of APJ are critical for association with apelin, while the first 20 amino acids play an important role in supporting cell-cell fusion mediated by HIV-1 gp120. With site-directed mutagenesis, we have identified that the negatively charged amino acid residues Glu20 and Asp23 are involved in receptor and coreceptor functions, but residues Tyr10 and Tyr11 substantially contribute to coreceptor function for both T-tropic (CXCR4) and dual-tropic (CXCR4 and CCR5) HIV-1 isolates. Thus, this study provides potentially important information for further characterizing APJ-apelin functions in vitro and in vivo and designing small molecules for treatment of HIV-1 infection in the CNS

  3. The relationship between N-terminal pro-brain natriuretic peptide and risk for hospitalization and mortality is curvilinear in patients with chronic heart failure

    DEFF Research Database (Denmark)

    Schou, Morten; Gustafsson, Finn; Corell, Pernille

    2007-01-01

    BACKGROUND: N-terminal pro-brain natriuretic peptide (NT-proBNP) carries prognostic information in patients with chronic heart failure and predicts risk for mortality and cardiovascular events. It is unknown whether NT-proBNP predicts risk for hospitalization for any cause. Furthermore, a clinica......BACKGROUND: N-terminal pro-brain natriuretic peptide (NT-proBNP) carries prognostic information in patients with chronic heart failure and predicts risk for mortality and cardiovascular events. It is unknown whether NT-proBNP predicts risk for hospitalization for any cause. Furthermore....... METHODS: Data from 345 patients with chronic heart failure were collected prospectively in our heart failure clinic, and the patients were followed for 28 months (median). Seventy patients died, and 201 patients were hospitalized. Cox proportional hazard models for mortality and hospitalization were...... constructed with NT-proBNP as a dichotomous (median 1381 pg/mL) and a continuous variable (log2 NT-proBNP). RESULTS: Patients with supramedian levels of NT-proBNP had a 2.40-fold (95% CI 1.40-4.10) increased risk for mortality and 1.71-fold (95% CI 1.24-2.36) increased risk for hospitalization. The effect...

  4. Depletion of histone N-terminal-acetyltransferase Naa40 induces p53-independent apoptosis in colorectal cancer cells via the mitochondrial pathway.

    Science.gov (United States)

    Pavlou, Demetria; Kirmizis, Antonis

    2016-03-01

    Protein N-terminal acetylation is an abundant post-translational modification in eukaryotes implicated in various fundamental cellular and biochemical processes. This modification is catalysed by evolutionarily conserved N-terminal acetyltransferases (NATs) whose deregulation has been linked to cancer development and thus, are emerging as useful diagnostic and therapeutic targets. Naa40 is a highly selective NAT that acetylates the amino-termini of histones H4 and H2A and acts as a sensor of cell growth in yeast. In the present study, we examine the role of Naa40 in cancer cell survival. We demonstrate that depletion of Naa40 in HCT116 and HT-29 colorectal cancer cells decreases cell survival by enhancing apoptosis, whereas Naa40 reduction in non-cancerous mouse embryonic fibroblasts has no effect on cell viability. Specifically, Naa40 knockdown in colon cancer cells activates the mitochondrial caspase-9-mediated apoptotic cascade. Consistent with this, we show that caspase-9 activation is required for the induced apoptosis because treatment of cells with an irreversible caspase-9 inhibitor impedes apoptosis when Naa40 is depleted. Furthermore, the effect of Naa40-depletion on cell-death is mediated through a p53-independent mechanism since p53-null HCT116 cells still undergo apoptosis upon reduction of the acetyltransferase. Altogether, these findings reveal an anti-apoptotic role for Naa40 and exhibit its potential as a therapeutic target in colorectal cancers.

  5. Plasma N-terminal pro-brain natriuretic peptide as a major risk marker for cardiovascular disease in patients with type 2 diabetes and microalbuminuria

    DEFF Research Database (Denmark)

    Gaede, P; Hildebrandt, P; Hess, G

    2005-01-01

    AIMS/HYPOTHESIS: We examined whether plasma N-terminal probrain natriuretic peptide (NT-proBNP) predicts cardiovascular outcome in patients with type 2 diabetes. METHODS: A total of 160 microalbuminuric type 2 diabetic patients (mean age 55.1 years [SD 7.2], 119 men) were enrolled in the Steno-2 ....../INTERPRETATION: We conclude that high plasma NT-proBNP is a major risk marker for cardiovascular disease in patients with type 2 diabetes and microalbuminuria.......AIMS/HYPOTHESIS: We examined whether plasma N-terminal probrain natriuretic peptide (NT-proBNP) predicts cardiovascular outcome in patients with type 2 diabetes. METHODS: A total of 160 microalbuminuric type 2 diabetic patients (mean age 55.1 years [SD 7.2], 119 men) were enrolled in the Steno-2......, myocardial infarction, stroke, revascularisation procedures in the heart or legs, and amputations. RESULTS: In the whole group, plasma NT-proBNP being above the median was associated with an increased risk of cardiovascular disease during follow-up, with an unadjusted hazard ratio of 4.4 (95% CI 2.3-8.4; p

  6. The C-Terminal SynMuv/DdDUF926 Domain Regulates the Function of the N-Terminal Domain of DdNKAP.

    Directory of Open Access Journals (Sweden)

    Bhagyashri D Burgute

    Full Text Available NKAP (NF-κB activating protein is a highly conserved SR (serine/arginine-rich protein involved in transcriptional control and splicing in mammals. We identified DdNKAP, the Dictyostelium discoideum ortholog of mammalian NKAP, as interacting partner of the nuclear envelope protein SUN-1. DdNKAP harbors a number of basic RDR/RDRS repeats in its N-terminal domain and the SynMuv/DUF926 domain at its C-terminus. We describe a novel and direct interaction between DdNKAP and Prp19 (Pre mRNA processing factor 19 which might be relevant for the observed DdNKAP ubiquitination. Genome wide analysis using cross-linking immunoprecipitation-high-throughput sequencing (CLIP-seq revealed DdNKAP association with intergenic regions, exons, introns and non-coding RNAs. Ectopic expression of DdNKAP and its domains affects several developmental aspects like stream formation, aggregation, and chemotaxis. We conclude that DdNKAP is a multifunctional protein, which might influence Dictyostelium development through its interaction with RNA and RNA binding proteins. Mutants overexpressing full length DdNKAP and the N-terminal domain alone (DdN-NKAP showed opposite phenotypes in development and opposite expression profiles of several genes and rRNAs. The observed interaction between DdN-NKAP and the DdDUF926 domain indicates that the DdDUF926 domain acts as negative regulator of the N-terminus.

  7. BtcA, A class IA type III chaperone, interacts with the BteA N-terminal domain through a globular/non-globular mechanism.

    Directory of Open Access Journals (Sweden)

    Chen Guttman

    Full Text Available Bordetella pertussis, the etiological agent of "whooping cough" disease, utilizes the type III secretion system (T3SS to deliver a 69 kDa cytotoxic effector protein, BteA, directly into the host cells. As with other T3SS effectors, prior to its secretion BteA binds BtcA, a 13.9 kDa protein predicted to act as a T3SS class IA chaperone. While this interaction had been characterized for such effector-chaperone pairs in other pathogens, it has yet to be fully investigated in Bordetella. Here we provide the first biochemical proof that BtcA is indeed a class IA chaperone, responsible for the binding of BteA's N-terminal domain. We bring forth extensive evidence that BtcA binds its substrate effector through a dual-interface binding mechanism comprising of non-globular and bi-globular interactions at a moderate micromolar level binding affinity. We demonstrate that the non-globular interactions involve the first 31 N-terminal residues of BteA287 and their removal leads to destabilization of the effector-chaperone complex and lower binding affinities to BtcA. These findings represent an important first step towards a molecular understanding of BteA secretion and cell entry.

  8. Improved procedures for N-terminal sulfonation of peptides for matrix-assisted laser desorption/ionization post-source decay peptide sequencing.

    Science.gov (United States)

    Wang, Dongxia; Kalb, Suzanne R; Cotter, Robert J

    2004-01-01

    Post source decay (PSD) analysis of precursor ions generated from matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry is a powerful tool for amino acid sequencing and primary structure analysis of proteins. N-Terminal sulfonation has become an effective derivatization strategy in facilitating de novo peptide sequencing by the formation of predominate y-type ion series in MALDI PSD spectra. Recently, an effective and inexpensive N-terminal derivatization method has been reported using 4-sulfophenyl isothiocyanate (SPITC) as the derivatization reagent (J. Mass. Spectrom. 2003; 38: 373-377). In this paper, we report an improvement in the derivatization procedure with this reagent that involves replacing an organic co-reagent with other chemicals and eliminating the use of organic solvent. The method is demonstrated on a model peptide and on tryptic digests of two proteins. The results indicate that the improved sulfonation reaction can be implemented with high efficiency under aqueous conditions and that the sensitivity of mass detection can be increased considerably. Copyright 2003 John Wiley & Sons, Ltd.

  9. The N-terminal leucine-zipper motif in PTRF/cavin-1 is essential and sufficient for its caveolae-association

    International Nuclear Information System (INIS)

    Wei, Zhuang; Zou, Xinle; Wang, Hongzhong; Lei, Jigang; Wu, Yuan; Liao, Kan

    2015-01-01

    Highlight: • The N-terminal leucine-zipper motif in PTRF/cavin-1 determines caveolar association. • Different cellular localization of PTRF/cavin-1 influences its serine 389 and 391 phosphorylation state. • PTRF/cavin-1 regulates cell motility via its caveolar association. - Abstract: PTRF/cavin-1 is a protein of two lives. Its reported functions in ribosomal RNA synthesis and in caveolae formation happen in two different cellular locations: nucleus vs. plasma membrane. Here, we identified that the N-terminal leucine-zipper motif in PTRF/cavin-1 was essential for the protein to be associated with caveolae in plasma membrane. It could counteract the effect of nuclear localization sequence in the molecule (AA 235–251). Deletion of this leucine-zipper motif from PTRF/cavin-1 caused the mutant to be exclusively localized in nuclei. The fusion of this leucine-zipper motif with histone 2A, which is a nuclear protein, could induce the fusion protein to be exported from nucleus. Cell migration was greatly inhibited in PTRF/cavin-1 −/− mouse embryonic fibroblasts (MEFs). The inhibited cell motility could only be rescued by exogenous cavin-1 but not the leucine-zipper motif deleted cavin-1 mutant. Plasma membrane dynamics is an important factor in cell motility control. Our results suggested that the membrane dynamics in cell migration is affected by caveolae associated PTRF/cavin-1

  10. A Linear Epitope in the N-Terminal Domain of CCR5 and Its Interaction with Antibody.

    Directory of Open Access Journals (Sweden)

    Benny Chain

    Full Text Available The CCR5 receptor plays a role in several key physiological and pathological processes and is an important therapeutic target. Inhibition of the CCR5 axis by passive or active immunisation offers one very selective strategy for intervention. In this study we define a new linear epitope within the extracellular domain of CCR5 recognised by two independently produced monoclonal antibodies. A short peptide encoding the linear epitope can induce antibodies which recognise the intact receptor when administered colinear with a tetanus toxoid helper T cell epitope. The monoclonal antibody RoAb 13 is shown to bind to both cells and peptide with moderate to high affinity (6x10^8 and 1.2x107 M-1 respectively, and binding to the peptide is enhanced by sulfation of tyrosines at positions 10 and 14. RoAb13, which has previously been shown to block HIV infection, also blocks migration of monocytes in response to CCR5 binding chemokines and to inflammatory macrophage conditioned medium. A Fab fragment of RoAb13 has been crystallised and a structure of the antibody is reported to 2.1 angstrom resolution.

  11. Cytoplasmic crystalloids in irradiated rat parotid glands

    International Nuclear Information System (INIS)

    Sholley, M.M.; Pratt, N.E.; Sodicoff, M.

    1981-01-01

    Cytoplasmic crystalloids were found in paroid acinar cells of rats given a large (6400 R) single exposure of X-rays to the head and neck. The crystalloids were first observed 1 day after irradiation and became numerous at 3-4 days. They were associated with autophagic vacuoles, which were seen in acinar cells as early as 3-6 h. Crystalloids sometimes appeared to be forming within autophagic vacuoles, which also contained membranous residues and apparently degenerating secretory material. They were bounded by a single, smooth membrane and had a substructure consisting of dense, parallel longitudinal striations. The crystalloids were also seen in macrophages associated with the basal surfaces of acinar cells. At 3-4 days macrophages were numerous and many contained crystalloids, degenerated secretory droplets, and other cellular debris, which they apparently had phagocytosed. By 6-8 days crystalloids and macrophages were seen infrequently. Regarding mode of formation, removal by macrophages, and ultrastructure, the crystalloids resembled those described by others after ethionine intoxication. Ethionine-induced crystalloids have cytochemical characteristics consistent with a lysosomal identity. The crystalloids in irradiated parotid glands probably reflect a variant type of lysosome, which is a nonspecific manifestation of severe cellular injury and can be elicited by a variety of injurious agents. (author)

  12. A physical perspective on cytoplasmic streaming.

    Science.gov (United States)

    Goldstein, Raymond E; van de Meent, Jan-Willem

    2015-08-06

    Organisms show a remarkable range of sizes, yet the dimensions of a single cell rarely exceed 100 µm. While the physical and biological origins of this constraint remain poorly understood, exceptions to this rule give valuable insights. A well-known counterexample is the aquatic plant Chara, whose cells can exceed 10 cm in length and 1 mm in diameter. Two spiralling bands of molecular motors at the cell periphery drive the cellular fluid up and down at speeds up to 100 µm s(-1), motion that has been hypothesized to mitigate the slowness of metabolite transport on these scales and to aid in homeostasis. This is the most organized instance of a broad class of continuous motions known as 'cytoplasmic streaming', found in a wide range of eukaryotic organisms-algae, plants, amoebae, nematodes and flies-often in unusually large cells. In this overview of the physics of this phenomenon, we examine the interplay between streaming, transport and cell size and discuss the possible role of self-organization phenomena in establishing the observed patterns of streaming.

  13. Cytoplasmic Dynein Promotes HIV-1 Uncoating

    Directory of Open Access Journals (Sweden)

    Paulina Pawlica

    2014-11-01

    Full Text Available Retroviral capsid (CA cores undergo uncoating during their retrograde transport (toward the nucleus, and/or after reaching the nuclear membrane. However, whether HIV-1 CA core uncoating is dependent upon its transport is not understood. There is some evidence that HIV-1 cores retrograde transport involves cytoplasmic dynein complexes translocating on microtubules. Here we investigate the role of dynein-dependent transport in HIV-1 uncoating. To interfere with dynein function, we depleted dynein heavy chain (DHC using RNA interference, and we over-expressed p50/dynamitin. In immunofluorescence microscopy experiments, DHC depletion caused an accumulation of CA foci in HIV-1 infected cells. Using a biochemical assay to monitor HIV-1 CA core disassembly in infected cells, we observed an increase in amounts of intact (pelletable CA cores upon DHC depletion or p50 over-expression. Results from these two complementary assays suggest that inhibiting dynein-mediated transport interferes with HIV-1 uncoating in infected cells, indicating the existence of a functional link between HIV-1 transport and uncoating.

  14. Brain Metabolic DNA in Rat Cytoplasm.

    Science.gov (United States)

    Giuditta, Antonio; Rutigliano, Bruno

    2018-02-09

    Brain metabolic DNA (BMD) is not involved in cell division or DNA repair but is modulated by memory acquisition, sleep processing, and circadian oscillations. Using routine methods of subcellular fractionation, newly synthesized BMD from male rats is shown to be localized in crude nuclear, mitochondrial, and microsomal fractions and in two fractions of purified nuclei. Sub-fractionation of the mitochondrial fraction indicates the prevalent localization of BMD in free mitochondria and to a lesser degree in synaptosomes and myelin. Cesium density profiles of homogenate, subcellular fractions, and purified nuclei obtained after incorporation periods from 30 min to 4 h indicate that BMD synthesis takes place by reverse transcription in cytoplasmic organelles. Following the acquisition of the double-stranded structure, BMD is transferred to nuclei. Kinetic analyses lasting several weeks highlight the massive BMD turnover in subcellular fractions and purified nuclei and its dependence on age. Data are in agreement with the role of BMD as a temporary information store of cell responses of potential use in comparable forthcoming experiences.

  15. Antineutrophil cytoplasm antibody: positivity and clinical correlation.

    Science.gov (United States)

    Martínez Téllez, Goitybell; Torres Rives, Bárbara; Rangel Velázquez, Suchiquil; Sánchez Rodríguez, Vicky; Ramos Ríos, María Antonia; Fuentes Smith, Lisset Evelyn

    2015-01-01

    To determine positivity and clinical correlation of anti-neutrophil cytoplasmic antibodies (ANCA), taking into account the interference of antinuclear antibodies (ANA). A prospective study was conducted in the Laboratory of Immunology of the National Cuban Center of Medical Genetic during one year. Two hounded sixty-seven patients with indication for ANCA determination were included. ANCA and ANA determinations with different cut off points and assays were determined by indirect immunofluorescense. Anti proteinase 3 and antimyeloperoxidase antibodies were determined by ELISA. Most positivity for ANCA was seen in patients with ANCA associated, primary small-vessel vasculitides, rheumatoid arthritis and systemic lupus erythematosus. Presence of ANCA without positivity for proteinase 3 and myeloperoxidase was higher in patients with ANA and little relation was observed between the perinuclear pattern confirmed in formalin and specificity by myeloperoxidase. Highest sensibility and specificity values for vasculitides diagnostic were achieved by ANCA determination using indirect immunofluorescense with a cut off 1/80 and confirming antigenic specificities with ELISA. ANCA can be present in a great number of chronic inflammatory or autoimmune disorders in the population studied. This determination using indirect immunofluorescence and following by ELISA had a great value for vasculitis diagnosis. Anti mieloperoxidasa assay has a higher utility than the formalin assay when ANA is present. Copyright © 2013 Elsevier España, S.L.U. All rights reserved.

  16. Antibody-mediated enzyme replacement therapy targeting both lysosomal and cytoplasmic glycogen in Pompe disease.

    Science.gov (United States)

    Yi, Haiqing; Sun, Tao; Armstrong, Dustin; Borneman, Scott; Yang, Chunyu; Austin, Stephanie; Kishnani, Priya S; Sun, Baodong

    2017-05-01

    Pompe disease is characterized by accumulation of both lysosomal and cytoplasmic glycogen primarily in skeletal and cardiac muscles. Mannose-6-phosphate receptor-mediated enzyme replacement therapy (ERT) with recombinant human acid α-glucosidase (rhGAA) targets the enzyme to lysosomes and thus is unable to digest cytoplasmic glycogen. Studies have shown that anti-DNA antibody 3E10 penetrates living cells and delivers "cargo" proteins to the cytosol or nucleus via equilibrative nucleoside transporter ENT2. We speculate that 3E10-mediated ERT with GAA will target both lysosomal and cytoplasmic glycogen in Pompe disease. A fusion protein (FabGAA) containing a humanized Fab fragment derived from the murine 3E10 antibody and the 110 kDa human GAA precursor was constructed and produced in CHO cells. Immunostaining with an anti-Fab antibody revealed that the Fab signals did not co-localize with the lysosomal marker LAMP2 in cultured L6 myoblasts or Pompe patient fibroblasts after incubation with FabGAA. Western blot with an anti-GAA antibody showed presence of the 150 kDa full-length FabGAA in the cell lysates, in addition to the 95- and 76 kDa processed forms of GAA that were also seen in the rhGAA-treated cells. Blocking of mannose-6-phosphate receptor with mannose-6-phosphate markedly reduced the 95- and the 76 kDa forms but not the 150 kDa form. In GAA-KO mice, FabGAA achieved similar treatment efficacy as rhGAA at an equal molar dose in reducing tissue glycogen contents. Our data suggest that FabGAA retains the ability of rhGAA to treat lysosomal glycogen accumulation and has the beneficial potential over rhGAA to reduce cytoplasmic glycogen storage in Pompe disease. FabGAA can be delivered to both the cytoplasm and lysosomes in cultured cells. FabGAA equally reduced lysosomal glycogen accumulation as rhGAA in GAA-KO mice. FabGAA has the beneficial potential over rhGAA to clear cytoplasmic glycogen. This study suggests a novel antibody-enzyme fusion protein therapy

  17. Embedded Fragments Registry (EFR)

    Data.gov (United States)

    Department of Veterans Affairs — In 2009, the Department of Defense estimated that approximately 40,000 service members who served in OEF/OIF may have embedded fragment wounds as the result of small...

  18. DNA fragmentation in spermatozoa

    DEFF Research Database (Denmark)

    Rex, A S; Aagaard, J.; Fedder, J

    2017-01-01

    Sperm DNA Fragmentation has been extensively studied for more than a decade. In the 1940s the uniqueness of the spermatozoa protein complex which stabilizes the DNA was discovered. In the fifties and sixties, the association between unstable chromatin structure and subfertility was investigated....... In the seventies, the impact of induced DNA damage was investigated. In the 1980s the concept of sperm DNA fragmentation as related to infertility was introduced as well as the first DNA fragmentation test: the Sperm Chromatin Structure Assay (SCSA). The terminal deoxynucleotidyl transferase nick end labelling...... (TUNEL) test followed by others was introduced in the nineties. The association between DNA fragmentation in spermatozoa and pregnancy loss has been extensively investigated spurring the need for a therapeutic tool for these patients. This gave rise to an increased interest in the aetiology of DNA damage...

  19. Fragmented Work Stories

    DEFF Research Database (Denmark)

    Humle, Didde Maria; Reff Pedersen, Anne

    2015-01-01

    , edited and performed by the storyteller in an ongoing process allowing tensions, discontinuities and editing between failures and achievements, between dreams and work realities and between home and work life. We argue that by including different types of fragmentation, we offer a new type......Following a strand of narrative studies pointing to the living conditions of storytelling and the micro-level implications of working within fragmented narrative perspectives, this article contributes to narrative research on work stories by focusing on how meaning is created from fragmented...... stories. We argue that meaning by story making is not always created by coherence and causality; meaning is created by different types of fragmentation: discontinuities, tensions and editing. The objective of this article is to develop and advance antenarrative practice analysis of work stories...

  20. Fragmentation Main Model

    Data.gov (United States)

    Earth Data Analysis Center, University of New Mexico — The fragmentation model combines patch size and patch continuity with diversity of vegetation types per patch and rarity of vegetation types per patch. A patch was...

  1. Physics of projectile fragments

    International Nuclear Information System (INIS)

    Minamisono, Tadanori

    1982-01-01

    This is a study report on the polarization phenomena of the projectile fragments produced by heavy ion reactions, and the beta decay of fragments. The experimental project by using heavy ions with the energy from 50 MeV/amu to 250 MeV/amu was designed. Construction of an angle-dispersion spectrograph for projectile fragments was proposed. This is a two-stage spectrograph. The first stage is a QQDQQ type separator, and the second stage is QDQD type. Estimation shows that Co-66 may be separated from the nuclei with mass of 65 and 67. The orientation of fragments can be measured by detecting beta-ray. The apparatus consists of a uniform field magnet, an energy absorber, a stopper, a RF coil and a beta-ray hodoscope. This system can be used for not only this purpose but also for the measurement of hyperfine structure. (Kato, T.)

  2. Fragment Impact Toolkit (FIT)

    Energy Technology Data Exchange (ETDEWEB)

    Shevitz, Daniel Wolf [Los Alamos National Lab. (LANL), Los Alamos, NM (United States); Key, Brian P. [Los Alamos National Lab. (LANL), Los Alamos, NM (United States); Garcia, Daniel B. [Los Alamos National Lab. (LANL), Los Alamos, NM (United States)

    2017-09-05

    The Fragment Impact Toolkit (FIT) is a software package used for probabilistic consequence evaluation of fragmenting sources. The typical use case for FIT is to simulate an exploding shell and evaluate the consequence on nearby objects. FIT is written in the programming language Python and is designed as a collection of interacting software modules. Each module has a function that interacts with the other modules to produce desired results.

  3. Interactions between the Cytoplasmic Domains of PspB and PspC Silence the Yersinia enterocolitica Phage Shock Protein Response.

    Science.gov (United States)

    Flores-Kim, Josué; Darwin, Andrew J

    2016-12-15

    The phage shock protein (Psp) system is a widely conserved cell envelope stress response that is essential for the virulence of some bacteria, including Yersinia enterocolitica Recruitment of PspA by the inner membrane PspB-PspC complex characterizes the activated state of this response. The PspB-PspC complex has been proposed to be a stress-responsive switch, changing from an OFF to an ON state in response to an inducing stimulus. In the OFF state, PspA cannot access its binding site in the C-terminal cytoplasmic domain of PspC (PspC CT ), because this site is bound to PspB. PspC has another cytoplasmic domain at its N-terminal end (PspC NT ), which has been thought to play a role in maintaining the OFF state, because its removal causes constitutive activation. However, until now, this role has proved recalcitrant to experimental investigation. Here, we developed a combination of approaches to investigate the role of PspC NT in Y. enterocolitica Pulldown assays provided evidence that PspC NT mediates the interaction of PspC with the C-terminal cytoplasmic domain of PspB (PspB CT ) in vitro Furthermore, site-specific oxidative cross-linking suggested that a PspC NT -PspB CT interaction occurs only under noninducing conditions in vivo Additional experiments indicated that mutations in pspC might cause constitutive activation by compromising this PspC NT binding site or by causing a conformational disturbance that repositions PspC NT in vivo These findings have provided the first insight into the regulatory function of the N-terminal cytoplasmic domain of PspC, revealing that its ability to participate in an inhibitory complex is essential to silencing the Psp response. The phage shock protein (Psp) response has generated widespread interest because it is linked to important phenotypes, including antibiotic resistance, biofilm formation, and virulence in a diverse group of bacteria. Therefore, achieving a comprehensive understanding of how this response is controlled

  4. Cytoplasmic regionalization in starfish oocyte occurrence and localization of cytoplasmic determinants responsible for the formation of archenteron and primary mesenchyme in starfish ( asterias amurensis) oocytes

    Science.gov (United States)

    Zhang, Shicui; Wu, Xianhan; Zhou, Jing; Wang, Renxue; Wu, Shangqin

    1990-09-01

    Starfish oocytes with intact germinal vesicles (GVs) were cut along desired planes with glass needles or ligated using silk thread loops into two parts and allowed to mature in vitro, and inseminated. The experimental results showed that (1) only the parts with GVs or partial GV contents (PGVCs) cleaved, those without any GV materials did not; but nucleated and non-nucleated fragments cut from mature eggs were able to divide; (2) the development of animal parts of oocytes containing GVs or PGVCs was like that of animal fragments of matured oocytes with female pronuclei; most of them gave rise to permanent blastulae, and just a few formed ectodermal vesicles with a little primary mesenchyme; (3) a large part of vegetal fragments with GVs or PGVCs, and the vegetal parts of mature eggs without female pronuclei developed into small but normal embryos; (4) the fragments containing GVs or PGVCs obtained from the oocytes along a plane parallel to the animal-vegetal (A-V) axis developed as normally as the halves (with or without female pronuclei) severed from mature eggs along the same axis. Based on the data above, it was concluded that (1) the non-chromatin materials in the oocyte GVs are indispensable for successful fertilization and cleavage of starfish eggs; (2) some factor (s) located asymmetrically in the vegetal hemispheres of starfish oocytes is (are) responsible for formation of the archenteron and primary mesenchyme. It is evident from the above findings that the oocyte cytoplasm of the starfish had already regionalized before the GV break-down.

  5. Antineutrophil Cytoplasmic Antibodies, Autoimmune Neutropenia, and Vasculitis

    Science.gov (United States)

    Grayson, Peter C.; Sloan, J. Mark; Niles, John L.; Monach, Paul A.; Merkel, Peter A.

    2011-01-01

    Objectives Reports of an association between antineutrophil cytoplasmic antibodies (ANCA) and autoimmune neutropenia have rarely included cases of proven vasculitis. A case of ANCA-associated vasculitis (AAV) with recurrent neutropenia is described and relevant literature on the association between ANCA, neutropenia, and vasculitis is reviewed. Methods Longitudinal clinical assessments and laboratory findings are described in a patient with AAV and recurrent episodes of profound neutropenia from December 2008 – October 2010. A PubMed database search of the medical literature was performed for papers published from 1960 through October 2010 to identify all reported cases of ANCA and neutropenia. Results A 49 year-old man developed recurrent neutropenia, periodic fevers, arthritis, biopsy-proven cutaneous vasculitis, sensorineural hearing loss, epididymitis, and positive tests for ANCA with specificity for antibodies to both proteinase 3 and myeloperoxidase. Antineutrophil membrane antibodies were detected during an acute neutropenic phase and were not detectable in a post-recovery sample, whereas ANCA titers did not seem to correlate with neutropenia. An association between ANCA and neutropenia has been reported in 74 cases from 24 studies in the context of drug/toxin exposure, underlying autoimmune disease, or chronic neutropenia without underlying autoimmune disease. In these cases, the presence of atypical ANCA patterns and other antibodies were common; however, vasculitis was uncommon and when it occurred was usually limited to the skin and in cases of underlying toxin exposure. Conclusions ANCA is associated with autoimmune neutropenia, but systemic vasculitis rarely occurs in association with ANCA and neutropenia. The interaction between neutrophils and ANCA may provide insight into understanding both autoimmune neutropenia and AAV. PMID:21507463

  6. Deletion of N-terminal amino acids from human lecithin:cholesterol acyltransferase differentially affects enzyme activity toward alpha- and beta-substrate lipoproteins.

    Science.gov (United States)

    Vickaryous, Nicola K; Teh, Evelyn M; Stewart, Bruce; Dolphin, Peter J; Too, Catherine K L; McLeod, Roger S

    2003-03-21

    Lecithin:cholesterol acyltransferase (LCAT) is the enzyme responsible for generation of the majority of the cholesteryl esters (CE) in human plasma. Although most plasma cholesterol esterification occurs on high-density lipoprotein (HDL), via alpha-LCAT activity, esterification also occurs on low-density lipoprotein (LDL) via the beta-activity of the enzyme. Computer threading techniques have provided a three-dimensional model for use in the structure-function analysis of the core and catalytic site of the LCAT protein, but the model does not extend to the N-terminal region of the enzyme, which may mediate LCAT interaction with lipoprotein substrates. In the present study, we have examined the functional consequences of deletion of the highly conserved hydrophobic N-terminal amino acids (residues 1-5) of human LCAT. Western blot analysis showed that the mutant proteins (Delta 1-Delta 5) were synthesized and secreted from transfected COS-7 cells at levels approximately equivalent to those of wild-type hLCAT. The secreted proteins had apparent molecular weights of 67 kDa, indicating that they were correctly processed and glycosylated during cellular transit. However, deletion of the first residue of the mature LCAT protein (Delta 1 mutant) resulted in a dramatic loss of alpha-LCAT activity (5% of wild type using reconstituted HDL substrate, rHDL), although this mutant retained full beta-LCAT activity (108% of wild-type using human LDL substrate). Removal of residues 1 and 2 (Delta 2 mutant) abolished alpha-LCAT activity and reduced beta-LCAT activity to 12% of wild type. Nevertheless, LCAT Delta 1 and Delta 2 mutants retained their ability to bind to rHDL and LDL lipoprotein substrates. The dramatic loss of enzyme activity suggests that the N-terminal residues of LCAT may be involved in maintaining the conformation of the lid domain and influence activation by the alpha-LCAT cofactor apoA-I (in Delta 1) and/or loss of enzyme activity (in Delta 1-Delta 5). Since the

  7. Pediatric Inflammatory Bowel Disease with Cytoplasmic Staining of Antineutrophil Cytoplasmic Antibodies

    Directory of Open Access Journals (Sweden)

    Omar I. Saadah

    2013-01-01

    Full Text Available Background. It is unusual for the antineutrophil cytoplasmic antibody with cytoplasmic pattern (cANCA to present in patients with inflammatory bowel disease (IBD without vasculitis. The purpose of this study was to describe the occurrence and characteristics of pediatrics IBD with cANCA. Methods. A retrospective review of pediatric IBD associated with cANCA serology in patients from King Abdulaziz University Hospital, Saudi Arabia, between September 2002 and February 2012. Results. Out of 131 patients with IBD screened for cANCAs, cANCA was positive in 7 (5.3% patients of whom 4 had ulcerative colitis and 3 had Crohn's disease. The median age was 8.8 years (2–14.8 years. Six (86% were males. Of the 7 patients, 5 (71% were Saudi Arabians and 2 were of Indian ethnicity. The most common symptoms were diarrhea, abdominal pain, weight loss, and rectal bleeding. None had family history or clinical features suggestive of vasculitis involving renal and respiratory systems. No difference in the disease location or severity was observed between cANCA positive and cANCA negative patients apart from male preponderance in cANCA positive patients. Conclusion. The occurrence of cANCA in pediatric IBD is rare. Apart from male preponderance, there were no peculiar characteristics for the cANCA positive patients.

  8. Catalytic roles of lysines (K9, K27, K31) in the N-terminal domain in human adenylate kinase by random site-directed mutagenesis.

    Science.gov (United States)

    Ayabe, T; Park, S K; Takenaka, H; Sumida, M; Uesugi, S; Takenaka, O; Hamada, M

    1996-11-01

    To elucidate lysine residues in the N-terminal domain of human cytosolic adenylate kinase (hAK1, EC 2.7.4.3), random site-directed mutagenesis of K9, K27, and K31 residues was performed, and six mutants were analyzed by steady-state kinetics. K9 residue may play an important role in catalysis by interacting with AMP2-. K27 and K31 residues appear to play a functional role in catalysis by interacting with MgATP2-. In human AK, the epsilon-amino group in the side chain of these lysine residues would be essential for phosphoryl transfer between MgATP2- and AMP2- during transition state.

  9. Diagnostic Value of N Terminal Pro B Type Natriuretic Peptide (NT-pro BNP in Cardiac Involvement in Patients with Beta- Thalassemia

    Directory of Open Access Journals (Sweden)

    Noor Mohammad Noori

    2017-04-01

    Full Text Available Background Heart failure is a major cause of death in thalassemia. The study aimed to determine the diagnostic value of N Terminal Pro B Type Natriuretic Peptide (NT-pro BNP, to early diagnose the cardiac involvement in beta- thalassemia major patients. Materials and Methods  80 thalassemia patients aged 7 to 18 years old (patients group, and 80 healthy age and gender matched controls were enrolled in the case-control study. Patients were selected from those attending to the clinic of Aliasghar hospital, Zahedan-Iran. They were subjected to echo-Doppler tissue and conventional examination for both right and left heart function. Data were analysis using SPSS 18.0 software. Results  NT-pro BNP increased in patients compared the controls (P

  10. Enzyme-linked immunosorbent serum assays (ELISAs) for rat and human N-terminal pro-peptide of collagen type I (PINP) - Assessment of corresponding epitopes

    DEFF Research Database (Denmark)

    Leeming, Diana Julie; Larsen, D.V.; Zhang, C.

    2010-01-01

    Objectives: The present study describes two newly developed N-terminal pro-peptides of collagen type I (PINP) competitive enzyme-linked immunosorbent assays (ELISAs) for the assessment of corresponding PINP epitopes in the rat- and human species. Methods: Monoclonal antibodies were raised against...... corresponding rat and human PINP sequences and competitive assays were developed for each species. They were evaluated in relevant pre-clinical or clinical studies. Results: The antibody characterizations indicated that PINP indeed was recognized. Technical robust assays were obtained. Rat PINP and tALP showed...... similar patterns in the gold standard osteoporosis rat ovariectomized (OVX) model. No liver contribution was observed in the liver fibrosis rat bile duct ligation model (BDL). In an osteoporosis study, the human serum PINP levels were significantly decreased after ibandronate treatment compared to placebo...

  11. N-terminal pro-B-type natriuretic peptide for the prognostic prediction of severe enterovirus 71-associated hand, foot, and mouth disease.

    Science.gov (United States)

    Qiu, Jun; Lu, Xiulan; Liu, Pingping; Zhang, Xinping; Zuo, Chao; Xiao, Zhenghui

    2017-01-01

    The aim of this study was to determine whether N-terminal pro-B-type natriuretic peptide (NT-proBNP) can predict impending brainstem encephalitis, pulmonary edema, pulmonary hemorrhage, cardiopulmonary failure, and death in children with severe enterovirus 71 (EV71)-associated hand, foot, and mouth disease (HFMD). Plasma NT-proBNP levels of 282 children with severe EV71-associated HFMD were measured. NT-proBNP levels were significantly higher in patients with elevated blood glucose (>7.8 mmol/l) and increased white blood cell counts (>14×10 9 /l). HFMD patients who had no complications had significantly lower NT-proBNP values than patients who died or had complications (pdisease in the intensive care unit. Serum NT-proBNP values ≥1300pg/ml on admission could be indicative of circulatory failure and increased mortality. Copyright © 2016 The Author(s). Published by Elsevier Ltd.. All rights reserved.

  12. N-Terminal Pro-B Type Natriuretic Peptide as a Marker of Bronchopulmonary Dysplasia or Death in Very Preterm Neonates

    DEFF Research Database (Denmark)

    Sellmer, Anna; Hjortdal, Vibeke Elisabeth; Bjerre, Jesper Vandborg

    2015-01-01

    BACKGROUND: Bronchopulmonary dysplasia (BPD) is a serious complication of preterm birth. Plasma N-terminal pro-B type natriuretic peptide (NT-proBNP) has been suggested as a marker that may predict BPD within a few days after birth. OBJECTIVES: To investigate the association between NT-proBNP day...... three and bronchopulmonary dysplasia (BPD) or death and further to assess the impact of patent ductus arteriosus (PDA) on this association in neonates born before 32 gestational weeks. METHODS: A cohort study of 183 neonates born before 32 gestational weeks consecutively admitted to the Neonatal...... Intensive Care Unit, Aarhus University Hospital, Denmark. On day three plasma samples were collected and echocardiography carried out. NT-proBNP was measured by routine immunoassays. The combined outcome BPD or death was assessed at 36 weeks of postmenstrual age. Receiver operator characteristic (ROC...

  13. c-Jun N-terminal kinase 3 expression in the retina of ocular hypertension mice: a possible target to reduce ganglion cell apoptosis

    Directory of Open Access Journals (Sweden)

    Yue He

    2015-01-01

    Full Text Available Glaucoma, a type of optic neuropathy, is characterized by the loss of retinal ganglion cells. It remains controversial whether c-Jun N-terminal kinase (JNK participates in the apoptosis of retinal ganglion cells in glaucoma. This study sought to explore a possible mechanism of action of JNK signaling pathway in glaucoma-induced retinal optic nerve damage. We established a mouse model of chronic ocular hypertension by reducing the aqueous humor followed by photocoagulation using the laser ignition method. Results showed significant pathological changes in the ocular tissues after the injury. Apoptosis of retinal ganglion cells increased with increased intraocular pressure, as did JNK3 mRNA expression in the retina. These data indicated that the increased expression of JNK3 mRNA was strongly associated with the increase in intraocular pressure in the retina, and correlated positively with the apoptosis of retinal ganglion cells.

  14. N-terminal amino acid sequences of the major outer membrane proteins from a Neisseria meningitidis group B strain isolated in Brazil

    Directory of Open Access Journals (Sweden)

    Salvatore Giovanni De Simone

    1996-02-01

    Full Text Available The four dominant outer membrane proteins (46, 38, 33 and 28 kDa were detected by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE in a semi-purified preparation of vesicle membranes of a Neisseria meningitidis (N44/89, B:4:P1.15:P5.5,7 strain isolated in Brazil. The N-terminal amino acid sequence for the 46 kDa and 28 kDa proteins matched that reported by others for class 1 and 5 proteins respectively, whereas the sequence (25 amino acids for the 38 kDa (class 3 protein was similar to class 1 meningococcal proteins. The sequence for the 33 kDa (class 4 was unique and not homologous to any known protein.

  15. The N-Terminal GYPSY Motif Is Required for Pilin-Specific Sortase SrtC1 Functionality in Lactobacillus rhamnosus Strain GG.

    Directory of Open Access Journals (Sweden)

    François P Douillard

    Full Text Available Predominantly identified in pathogenic Gram-positive bacteria, sortase-dependent pili are also found in commensal species, such as the probiotic-marketed strain Lactobacillus rhamnosus strain GG. Pili are typically associated with host colonization, immune signalling and biofilm formation. Comparative analysis of the N-terminal domains of pilin-specific sortases from various piliated Gram-positive bacteria identified a conserved motif, called GYPSY, within the signal sequence. We investigated the function and role of the GYPSY residues by directed mutagenesis in homologous (rod-shaped and heterologous (coccoid-shaped expression systems for pilus formation. Substitutions of some of the GYPSY residues, and more specifically the proline residue, were found to have a direct impact on the degree of piliation of Lb. rhamnosus GG. The present findings uncover a new signalling element involved in the functionality of pilin-specific sortases controlling the pilus biogenesis of Lb. rhamnosus GG and related piliated Gram-positive species.

  16. The N-terminal cleavage site of PrPSc from BSE differs from that of PrPSc from scrapie.

    Science.gov (United States)

    Hayashi, Hiroko K; Yokoyama, Takashi; Takata, Masuhiro; Iwamaru, Yoshifumi; Imamura, Morikazu; Ushiki, Yuko K; Shinagawa, Morikazu

    2005-03-25

    Heterogeneity in transmissible spongiform encephalopathy is thought to have derived from conformational variation in an abnormal isoform of the prion protein (PrPSc). To characterize PrPSc in bovine spongiform encephalopathy (BSE) and scrapie, we analyzed the newly generated N-terminus of PrPSc isoforms by digestion with proteinase K (PK). With a lower concentration of PK, the terminal amino acid of BSE PrPSc converged at N96. Under the same conditions, however, the terminal amino acid of scrapie PrPSc was G81 or G85. Furthermore, with an increase of PK concentration, the N-terminal amino acid was shifted and converged at G89. The results suggest that the PK cleavage site of BSE PrPSc is uniform and is different from the cleavage site of scrapie PrPSc.

  17. Mapping of Chlamydia trachomatis proteins by immobiline-polyacrylamide two-dimensional electrophoresis: spot identification by N-terminal sequencing and immunoblotting

    DEFF Research Database (Denmark)

    Bini, L; Sanchez-Campillo, M; Santucci, A

    1996-01-01

    Proteins from purified elementary bodies of Chlamydia trachomatis were separated by two-dimensional gel electrophoresis on nonlinear wide-range immobilized pH gradients in the first dimension and polyacrylamide gradient gels in the second dimension. The maps obtained with this system are highly...... reproducible and resolve ca. 600 spots. By using immunoblot analysis with specific antibodies and/or N-terminal amino acid sequencing, we established the map positions of a number of described chlamydial proteins, such as the major outer membrane protein (MOMP) the 60 kDa cystein-rich outer membrane protein...... (OMP2), the DnaK-like, GroEL-like, and macrophage infectivity potentiator (MIP)-like proteins, the plasmid-encoded pgp3 protein, two ribosomal proteins (S1 and L7/L12), and the protein-elongation factor EF-Tu. Other proteins, for which gene assignment was not possible, have been identified by three...

  18. The N-Terminal GYPSY Motif Is Required for Pilin-Specific Sortase SrtC1 Functionality in Lactobacillus rhamnosus Strain GG.

    Science.gov (United States)

    Douillard, François P; Rasinkangas, Pia; Bhattacharjee, Arnab; Palva, Airi; de Vos, Willem M

    2016-01-01

    Predominantly identified in pathogenic Gram-positive bacteria, sortase-dependent pili are also found in commensal species, such as the probiotic-marketed strain Lactobacillus rhamnosus strain GG. Pili are typically associated with host colonization, immune signalling and biofilm formation. Comparative analysis of the N-terminal domains of pilin-specific sortases from various piliated Gram-positive bacteria identified a conserved motif, called GYPSY, within the signal sequence. We investigated the function and role of the GYPSY residues by directed mutagenesis in homologous (rod-shaped) and heterologous (coccoid-shaped) expression systems for pilus formation. Substitutions of some of the GYPSY residues, and more specifically the proline residue, were found to have a direct impact on the degree of piliation of Lb. rhamnosus GG. The present findings uncover a new signalling element involved in the functionality of pilin-specific sortases controlling the pilus biogenesis of Lb. rhamnosus GG and related piliated Gram-positive species.

  19. CTP synthase forms cytoophidia in the cytoplasm and nucleus

    Energy Technology Data Exchange (ETDEWEB)

    Gou, Ke-Mian [MRC Functional Genomics Unit, Department of Physiology, Anatomy and Genetics, University of Oxford, Oxford OX1 3PT (United Kingdom); State Key Laboratory for Agrobiotechnology, College of Biological Sciences, China Agricultural University, Beijing 100193 (China); Chang, Chia-Chun [Institute of Biotechnology, National Taiwan University, Taipei, Taiwan, ROC (China); Shen, Qing-Ji [MRC Functional Genomics Unit, Department of Physiology, Anatomy and Genetics, University of Oxford, Oxford OX1 3PT (United Kingdom); Sung, Li-Ying, E-mail: liyingsung@ntu.edu.tw [Institute of Biotechnology, National Taiwan University, Taipei, Taiwan, ROC (China); Agricultural Biotechnology Research Center, Academia Sinica, Taipei 115, Taiwan, ROC (China); Liu, Ji-Long, E-mail: jilong.liu@dpag.ox.ac.uk [MRC Functional Genomics Unit, Department of Physiology, Anatomy and Genetics, University of Oxford, Oxford OX1 3PT (United Kingdom)

    2014-04-15

    CTP synthase is an essential metabolic enzyme responsible for the de novo synthesis of CTP. Multiple studies have recently showed that CTP synthase protein molecules form filamentous structures termed cytoophidia or CTP synthase filaments in the cytoplasm of eukaryotic cells, as well as in bacteria. Here we report that CTP synthase can form cytoophidia not only in the cytoplasm, but also in the nucleus of eukaryotic cells. Both glutamine deprivation and glutamine analog treatment promote formation of cytoplasmic cytoophidia (C-cytoophidia) and nuclear cytoophidia (N-cytoophidia). N-cytoophidia are generally shorter and thinner than their cytoplasmic counterparts. In mammalian cells, both CTP synthase 1 and CTP synthase 2 can form cytoophidia. Using live imaging, we have observed that both C-cytoophidia and N-cytoophidia undergo multiple rounds of fusion upon glutamine analog treatment. Our study reveals the coexistence of cytoophidia in the cytoplasm and nucleus, therefore providing a good opportunity to investigate the intracellular compartmentation of CTP synthase. - Highlights: • CTP synthase forms cytoophidia not only in the cytoplasm but also in the nucleus. • Glutamine deprivation and Glutamine analogs promotes cytoophidium formation. • N-cytoophidia exhibit distinct morphology when compared to C-cytoophidia. • Both CTP synthase 1 and CTP synthase 2 form cytoophidia in mammalian cells. • Fusions of cytoophidia occur in the cytoplasm and nucleus.

  20. Intricate Effects of α-Amino and Lysine Modifications on Arginine Methylation of the N-Terminal Tail of Histone H4.

    Science.gov (United States)

    Fulton, Melody D; Zhang, Jing; He, Maomao; Ho, Meng-Chiao; Zheng, Y George

    2017-07-18

    Chemical modifications of the DNA and nucleosomal histones tightly control the gene transcription program in eukaryotic cells. The "histone code" hypothesis proposes that the frequency, combination, and location of post-translational modifications (PTMs) of the core histones compose a complex network of epigenetic regulation. Currently, there are at least 23 different types and >450 histone PTMs that have been discovered, and the PTMs of lysine and arginine residues account for a crucial part of the histone code. Although significant progress has been achieved in recent years, the molecular basis for the histone code is far from being fully understood. In this study, we investigated how naturally occurring N-terminal acetylation and PTMs of histone H4 lysine-5 (H4K5) affect arginine-3 methylation catalyzed by both type I and type II PRMTs at the biochemical level. Our studies found that acylations of H4K5 resulted in decreased levels of arginine methylation by PRMT1, PRMT3, and PRMT8. In contrast, PRMT5 exhibits an increased rate of arginine methylation upon H4K5 acetylation, propionylation, and crotonylation, but not upon H4K5 methylation, butyrylation, or 2-hydroxyisobutyrylation. Methylation of H4K5 did not affect arginine methylation by PRMT1 or PRMT5. There was a small increase in the rate of arginine methylation by PRMT8. Strikingly, a marked increase in the rate of arginine methylation was observed for PRMT3. Finally, N-terminal acetylation reduced the rate of arginine methylation by PRMT3 but had little influence on PRMT1, -5, and -8 activity. These results together highlight the underlying mechanistic differences in substrate recognition among different PRMTs and pave the way for the elucidation of the complex interplay of histone modifications.

  1. Phage display-mediated discovery of novel tyrosinase-targeting tetrapeptide inhibitors reveals the significance of N-terminal preference of cysteine residues and their functional sulfur atom.

    Science.gov (United States)

    Lee, Yu-Ching; Hsiao, Nai-Wan; Tseng, Tien-Sheng; Chen, Wang-Chuan; Lin, Hui-Hsiung; Leu, Sy-Jye; Yang, Ei-Wen; Tsai, Keng-Chang

    2015-02-01

    Tyrosinase, a key copper-containing enzyme involved in melanin biosynthesis, is closely associated with hyperpigmentation disorders, cancer, and neurodegenerative diseases, and as such, it is an essential target in medicine and cosmetics. Known tyrosinase inhibitors possess adverse side effects, and there are no safety regulations; therefore, it is necessary to develop new inhibitors with fewer side effects and less toxicity. Peptides are exquisitely specific to their in vivo targets, with high potencies and relatively few off-target side effects. Thus, we systematically and comprehensively investigated the tyrosinase-inhibitory abilities of N- and C-terminal cysteine/tyrosine-containing tetrapeptides by constructing a phage-display random tetrapeptide library and conducting computational molecular docking studies on novel tyrosinase tetrapeptide inhibitors. We found that N-terminal cysteine-containing tetrapeptides exhibited the most potent tyrosinase-inhibitory abilities. The positional preference of cysteine residues at the N terminus in the tetrapeptides significantly contributed to their tyrosinase-inhibitory function. The sulfur atom in cysteine moieties of N- and C-terminal cysteine-containing tetrapeptides coordinated with copper ions, which then tightly blocked substrate-binding sites. N- and C-terminal tyrosine-containing tetrapeptides functioned as competitive inhibitors against mushroom tyrosinase by using the phenol ring of tyrosine to stack with the imidazole ring of His263, thus competing for the substrate-binding site. The N-terminal cysteine-containing tetrapeptide CRVI exhibited the strongest tyrosinase-inhibitory potency (with an IC50 of 2.7 ± 0.5 μM), which was superior to those of the known tyrosinase inhibitors (arbutin and kojic acid) and outperformed kojic acid-tripeptides, mimosine-FFY, and short-sequence oligopeptides at inhibiting mushroom tyrosinase. Copyright © 2014 by The American Society for Pharmacology and Experimental

  2. Regulation of Nucleosome Stacking and Chromatin Compaction by the Histone H4 N-Terminal Tail-H2A Acidic Patch Interaction.

    Science.gov (United States)

    Chen, Qinming; Yang, Renliang; Korolev, Nikolay; Liu, Chuan Fa; Nordenskiöld, Lars

    2017-06-30

    Chromatin folding and dynamics are critically dependent on nucleosome-nucleosome interactions with important contributions from internucleosome binding of the histone H4 N-terminal tail K16-R23 domain to the surface of the H2A/H2B dimer. The H4 Lys16 plays a pivotal role in this regard. Using in vitro reconstituted 12-mer nucleosome arrays, we have investigated the mechanism of the H4 N-terminal tail in maintaining nucleosome-nucleosome stacking and mediating intra- and inter-array chromatin compaction, with emphasis on the role of K16 and the positive charge region, R17-R23. Analytical ultracentrifugation sedimentation velocity experiments and precipitation assays were employed to analyze effects on chromatin folding and self-association, respectively. Effects on chromatin folding caused by various mutations and modifications at position K16 in the H4 histone were studied. Additionally, using charge-quenching mutations, we characterized the importance of the interaction of the residues within the H4 positive charge region R17-R23 with the H2A acidic patch of the adjacent nucleosome. Furthermore, crosslinking experiments were conducted to establish the proximity of the basic tail region to the acidic patch. Our data indicate that the positive charge and length of the side chain of H4 K16 are important for its access to the adjacent nucleosome in the process of nucleosome-nucleosome stacking and array folding. The location and orientation of the H4 R17-R23 domain on the H2A/H2B dimer surface of the neighboring nucleosome core particle (NCP) in the compacted chromatin fiber were established. The dominance of electrostatic interactions in maintaining intra-array interaction was demonstrated. Copyright © 2017 Elsevier Ltd. All rights reserved.

  3. Structural diversity and evolution of the N-terminal isoform-specific region of ecdysone receptor-A and -B1 isoforms in insects

    Directory of Open Access Journals (Sweden)

    Kubo Takeo

    2010-02-01

    Full Text Available Abstract Background The ecdysone receptor (EcR regulates various cellular responses to ecdysteroids during insect development. Insects have multiple EcR isoforms with different N-terminal A/B domains that contain the isoform-specific activation function (AF-1 region. Although distinct physiologic functions of the EcR isoforms have been characterized in higher holometabolous insects, they remain unclear in basal direct-developing insects, in which only A isoform has been identified. To examine the structural basis of the EcR isoform-specific AF-1 regions, we performed a comprehensive structural comparison of the isoform-specific region of the EcR-A and -B1 isoforms in insects. Results The EcR isoforms were newly identified in 51 species of insects and non-insect arthropods, including direct-developing ametabolous and hemimetabolous insects. The comprehensive structural comparison revealed that the isoform-specific region of each EcR isoform contained evolutionally conserved microdomain structures and insect subgroup-specific structural modifications. The A isoform-specific region generally contained four conserved microdomains, including the SUMOylation motif and the nuclear localization signal, whereas the B1 isoform-specific region contained three conserved microdomains, including an acidic activator domain-like motif. In addition, the EcR-B1 isoform of holometabolous insects had a novel microdomain at the N-terminal end. Conclusions Given that the nuclear receptor AF-1 is involved in cofactor recruitment and transcriptional regulation, the microdomain structures identified in the isoform-specific A/B domains might function as signature motifs and/or as targets for cofactor proteins that play essential roles in the EcR isoform-specific AF-1 regions. Moreover, the novel microdomain in the isoform-specific region of the holometabolous insect EcR-B1 isoform suggests that the holometabolous insect EcR-B1 acquired additional transcriptional

  4. Structural diversity and evolution of the N-terminal isoform-specific region of ecdysone receptor-A and -B1 isoforms in insects.

    Science.gov (United States)

    Watanabe, Takayuki; Takeuchi, Hideaki; Kubo, Takeo

    2010-02-12

    The ecdysone receptor (EcR) regulates various cellular responses to ecdysteroids during insect development. Insects have multiple EcR isoforms with different N-terminal A/B domains that contain the isoform-specific activation function (AF)-1 region. Although distinct physiologic functions of the EcR isoforms have been characterized in higher holometabolous insects, they remain unclear in basal direct-developing insects, in which only A isoform has been identified. To examine the structural basis of the EcR isoform-specific AF-1 regions, we performed a comprehensive structural comparison of the isoform-specific region of the EcR-A and -B1 isoforms in insects. The EcR isoforms were newly identified in 51 species of insects and non-insect arthropods, including direct-developing ametabolous and hemimetabolous insects. The comprehensive structural comparison revealed that the isoform-specific region of each EcR isoform contained evolutionally conserved microdomain structures and insect subgroup-specific structural modifications. The A isoform-specific region generally contained four conserved microdomains, including the SUMOylation motif and the nuclear localization signal, whereas the B1 isoform-specific region contained three conserved microdomains, including an acidic activator domain-like motif. In addition, the EcR-B1 isoform of holometabolous insects had a novel microdomain at the N-terminal end. Given that the nuclear receptor AF-1 is involved in cofactor recruitment and transcriptional regulation, the microdomain structures identified in the isoform-specific A/B domains might function as signature motifs and/or as targets for cofactor proteins that play essential roles in the EcR isoform-specific AF-1 regions. Moreover, the novel microdomain in the isoform-specific region of the holometabolous insect EcR-B1 isoform suggests that the holometabolous insect EcR-B1 acquired additional transcriptional regulation mechanisms.

  5. The conserved residue Arg46 in the N-terminal heptad repeat domain of HIV-1 gp41 is critical for viral fusion and entry.

    Directory of Open Access Journals (Sweden)

    Xiaoyi Wang

    Full Text Available During the process of HIV-1 fusion with the target cell, the N-terminal heptad repeat (NHR of gp41 interacts with the C-terminal heptad repeat (CHR to form fusogenic six-helix bundle (6-HB core. We previously identified a crucial residue for 6-HB formation and virus entry--Lys63 (K63 in the C-terminal region of NHR (aa 54-70, which forms a hydrophobic cavity. It can form an important salt bridge with Asp121 (D121 in gp41 CHR. Here, we found another important conserved residue for virus fusion and entry, Arg46 (R46, in the N-terminal region of NHR (aa 35-53, which forms a hydrogen bond with a polar residue, Asn43 (N43, in NHR, as a part of the hydrogen-bond network. R46 can also form a salt bridge with a negatively charged residue, Glu137 (E137, in gp41 CHR. Substitution of R46 with the hydrophobic residue Ala (R46A or the negatively charged residue Glu (R46E resulted in disruption of the hydrogen bond network, breakage of the salt bridge and reduction of 6-HB's stability, leading to impairment of viral fusion and decreased inhibition of N36, an NHR peptide. Similarly, CHR peptide C34 with substitution of E137 for Ala (E137A or Arg (E137R also exhibited reduced inhibitory activity against HIV-1 infection and HIV-1-mediated cell-to-cell fusion. These results suggest that the positively charged residue R46 and its hydrogen bond network, together with the salt bridge between R46 and E137, are important for viral fusion and entry and may therefore serve as a target for designing novel HIV fusion/entry inhibitors.

  6. Feline tetherin is characterized by a short N-terminal region and is counteracted by the feline immunodeficiency virus envelope glycoprotein.

    Science.gov (United States)

    Celestino, Michele; Calistri, Arianna; Del Vecchio, Claudia; Salata, Cristiano; Chiuppesi, Flavia; Pistello, Mauro; Borsetti, Alessandra; Palù, Giorgio; Parolin, Cristina

    2012-06-01

    Tetherin (BST2) is the host cell factor that blocks the particle release of some enveloped viruses. Two putative feline tetherin proteins differing at the level of the N-terminal coding region have recently been described and tested for their antiviral activity. By cloning and comparing the two reported feline tetherins (called here cBST2(504) and cBST2*) and generating specific derivative mutants, this study provides evidence that feline tetherin has a shorter intracytoplasmic domain than those of other known homologues. The minimal tetherin promoter was identified and assayed for its ability to drive tetherin expression in an alpha interferon-inducible manner. We also demonstrated that cBST2(504) is able to dimerize, is localized at the cellular membrane, and impairs human immunodeficiency virus type 1 (HIV-1) particle release, regardless of the presence of the Vpu antagonist accessory protein. While cBST2(504) failed to restrict wild-type feline immunodeficiency virus (FIV) egress, FIV mutants, bearing a frameshift at the level of the envelope-encoding region, were potently blocked. The transient expression of the FIV envelope glycoprotein was able to rescue mutant particle release from feline tetherin-positive cells but did not antagonize human BST2 activity. Moreover, cBST2(504) was capable of specifically immunoprecipitating the FIV envelope glycoprotein. Finally, cBST2(504) also exerted its function on HIV-2 ROD10 and on the simian immunodeficiency virus SIVmac239. Taken together, these results show that feline tetherin does indeed have a short N-terminal region and that the FIV envelope glycoprotein is the predominant factor counteracting tetherin restriction.

  7. Characterization of amino acid residues within the N-terminal region of Ubc9 that play a role in Ubc9 nuclear localization

    Energy Technology Data Exchange (ETDEWEB)

    Sekhri, Palak [Department of Biological Sciences, Wayne State University, 5947 Gullen Mall, Detroit, MI 48202 (United States); Tao, Tao [School of Life Sciences, Xiamen University, Xiamen (China); Kaplan, Feige [Department of Human Genetics, McGill University, Montreal (Canada); Zhang, Xiang-Dong, E-mail: xzhang@wayne.edu [Department of Biological Sciences, Wayne State University, 5947 Gullen Mall, Detroit, MI 48202 (United States)

    2015-02-27

    As the sole E2 enzyme for SUMOylation, Ubc9 is predominantly nuclear. However, the underlying mechanisms of Ubc9 nuclear localization are still not well understood. Here we show that RNAi-depletion of Imp13, an importin known to mediate Ubc9 nuclear import, reduces both Ubc9 nuclear accumulation and global SUMOylation. Furthermore, Ubc9-R13A or Ubc9-H20D mutation previously shown to interrupt the interaction of Ubc9 with nucleus-enriched SUMOs reduces the nuclear enrichment of Ubc9, suggesting that the interaction of Ubc9 with the nuclear SUMOs may enhance Ubc9 nuclear retention. Moreover, Ubc9-R17E mutation, which is known to disrupt the interaction of Ubc9 with both SUMOs and Imp13, causes a greater decrease in Ubc9 nuclear accumulation than Ubc9-R13A or Ubc9-H20D mutation. Lastly, Ubc9-K74A/S89D mutations that perturb the interaction of Ubc9 with nucleus-enriched SUMOylation-consensus motifs has no effect on Ubc9 nuclear localization. Altogether, our results have elucidated that the amino acid residues within the N-terminal region of Ubc9 play a pivotal role in regulation of Ubc9 nuclear localization. - Highlights: • Imp13-mediated nuclear import of Ubc9 is critical for global SUMOylation. • Ubc9 mutations disrupting Ubc9-SUMO interaction decrease Ubc9 nuclear accumulation. • N-terminal amino acid residues of Ubc9 are critical for Ubc9 nuclear enrichment.

  8. N-terminal pro-brain natriuretic peptide can be an adjunctive diagnostic marker of hyper-acute phase of Kawasaki disease.

    Science.gov (United States)

    Kwon, Hyuksool; Lee, Jin Hee; Jung, Jae Yun; Kwak, Young Ho; Kim, Do Kyun; Jung, Jin Hee; Chang, Ikwan; Kim, Kyuseok

    2016-12-01

    The purpose of this study was to determine whether the serum N-terminal pro-brain natriuretic peptide (NT-proBNP) level could be a useful marker for Kawasaki disease in the pediatric emergency department (PED) and in the presence of fever duration of 4 days or less (hyper-acute phase of Kawasaki disease). Medical records of patients who were 1 month to 15 years old of age and presented at the PED with suspected Kawasaki disease from January 1, 2010, to December 31, 2014, were collected retrospectively. Two hundred thirty-nine patients with a history of fever for 4 days or less were diagnosed with Kawasaki disease, as well as 111 patients with other febrile diseases, and were enrolled. The NT-proBNP level was significantly higher in patients with Kawasaki disease (Kawasaki disease vs. other febrile disease group, 444.8 (189.7-951.5) vs. 153.4 (68.9-287.6) pg/mL; p Kawasaki disease was 0.763 (95 % CI 0.712-0.814). NT-proBNP might be an adjunctive laboratory marker for hyper-acute phase of Kawasaki disease in the PED. What is Known: • N-terminal pro-brain natriuretic peptide level has been reported as a useful marker for diagnosis in patients with the acute phase of Kawasaki disease. • But, in the cases of less than 5 days of fever, the appropriate level of NT-proBNP for differentiating Kawasaki disease in PED has not been yet evaluated. What is New: • NT-proBNP might be an adjunctive laboratory marker for hyper-acute phase of Kawasaki disease.

  9. Three-dimensional structure of N-terminal domain of DnaB helicase and helicase-primase interactions in Helicobacter pylori.

    Directory of Open Access Journals (Sweden)

    Tara Kashav

    2009-10-01

    Full Text Available Replication initiation is a crucial step in genome duplication and homohexameric DnaB helicase plays a central role in the replication initiation process by unwinding the duplex DNA and interacting with several other proteins during the process of replication. N-terminal domain of DnaB is critical for helicase activity and for DnaG primase interactions. We present here the crystal structure of the N-terminal domain (NTD of H. pylori DnaB (HpDnaB helicase at 2.2 A resolution and compare the structural differences among helicases and correlate with the functional differences. The structural details of NTD suggest that the linker region between NTD and C-terminal helicase domain plays a vital role in accurate assembly of NTD dimers. The sequence analysis of the linker regions from several helicases reveals that they should form four helix bundles. We also report the characterization of H. pylori DnaG primase and study the helicase-primase interactions, where HpDnaG primase stimulates DNA unwinding activity of HpDnaB suggesting presence of helicase-primase cohort at the replication fork. The protein-protein interaction study of C-terminal domain of primase and different deletion constructs of helicase suggests that linker is essential for proper conformation of NTD to interact strongly with HpDnaG. The surface charge distribution on the primase binding surface of NTDs of various helicases suggests that DnaB-DnaG interaction and stability of the complex is most probably charge dependent. Structure of the linker and helicase-primase interactions indicate that HpDnaB differs greatly from E.coli DnaB despite both belong to gram negative bacteria.

  10. Association between resting heart rate and N-terminal pro-brain natriuretic peptide in a community-based population study in Beijing

    Directory of Open Access Journals (Sweden)

    Cao R

    2014-12-01

    Full Text Available Ruihua Cao, Yongyi Bai, Ruyi Xu, Ping Ye Department of Geriatric Cardiology, Chinese PLA General Hospital, Beijing, People’s Republic of China Background: N-terminal pro-brain natriuretic peptide (NT-proBNP is associated with an increased risk of cardiac insufficiency, which possibly leads to heart failure. However, the relationship between resting heart rate and NT-proBNP is unclear.Objective: This study focuses on this relativity between resting heart rate and plasma NT-proBNP levels in a surveyed community-based population.Methods: We evaluated the relativity between resting heart rate and plasma levels of NT-proBNP in 1,567 participants (mean age 61.0 years, range 21–96 years from a community-based population in Beijing, People’s Republic of China.Results: In patients with high resting heart rate (≥75 beats/min, NT-proBNP was higher than in those having low resting heart rate (<75 beats/min. In multiple linear stepwise regression analysis, plasma NT-proBNP was associated with resting heart rate (partial correlation coefficient, 0.82; 95% confidence interval, 0.18–1.51; P=0.011. A subsequent subgroup analysis revealed that the association between resting heart rate and plasma NT-proBNP was strengthened in subjects over 60 years old (partial correlation coefficient 1.28; 95% confidence interval, 0.49–2.36; P=0.031; while the relativity between resting heart rate and plasma NT-proBNP was not emerged in the younger subgroup (<60 years old.Conclusions: Resting heart rate was associated with plasma NT-proBNP in the elderly, which indicated a relationship between resting heart rate and cardiac function damage. Keywords: resting heart rate, N-terminal pro-brain natriuretic peptide, epidemiology, cardiac function, relationship

  11. Structure of the N-terminal domain of the protein Expansion: an ‘Expansion’ to the Smad MH2 fold

    Energy Technology Data Exchange (ETDEWEB)

    Beich-Frandsen, Mads; Aragón, Eric [Institute for Research in Biomedicine (IRB Barcelona), Baldiri Reixac 10, 08028 Barcelona (Spain); Llimargas, Marta [Institut de Biologia Molecular de Barcelona, IBMB–CSIC, Baldiri Reixac 10, 08028 Barcelona (Spain); Benach, Jordi [ALBA Synchrotron, BP 1413, km 3.3, Cerdanyola del Vallès (Spain); Riera, Antoni [Institute for Research in Biomedicine (IRB Barcelona), Baldiri Reixac 10, 08028 Barcelona (Spain); Universitat de Barcelona, Martí i Franqués 1-11, 08028 Barcelona (Spain); Pous, Joan [Institute for Research in Biomedicine (IRB Barcelona), Baldiri Reixac 10, 08028 Barcelona (Spain); Platform of Crystallography IBMB–CSIC, Baldiri Reixac 10, 08028 Barcelona (Spain); Macias, Maria J., E-mail: maria.macias@irbbarcelona.org [Institute for Research in Biomedicine (IRB Barcelona), Baldiri Reixac 10, 08028 Barcelona (Spain); Catalan Institution for Research and Advanced Studies (ICREA), Passeig Lluís Companys 23, 08010 Barcelona (Spain)

    2015-04-01

    Expansion is a modular protein that is conserved in protostomes. The first structure of the N-terminal domain of Expansion has been determined at 1.6 Å resolution and the new Nα-MH2 domain was found to belong to the Smad/FHA superfamily of structures. Gene-expression changes observed in Drosophila embryos after inducing the transcription factor Tramtrack led to the identification of the protein Expansion. Expansion contains an N-terminal domain similar in sequence to the MH2 domain characteristic of Smad proteins, which are the central mediators of the effects of the TGF-β signalling pathway. Apart from Smads and Expansion, no other type of protein belonging to the known kingdoms of life contains MH2 domains. To compare the Expansion and Smad MH2 domains, the crystal structure of the Expansion domain was determined at 1.6 Å resolution, the first structure of a non-Smad MH2 domain to be characterized to date. The structure displays the main features of the canonical MH2 fold with two main differences: the addition of an α-helical region and the remodelling of a protein-interaction site that is conserved in the MH2 domain of Smads. Owing to these differences, to the new domain was referred to as Nα-MH2. Despite the presence of the Nα-MH2 domain, Expansion does not participate in TGF-β signalling; instead, it is required for other activities specific to the protostome phyla. Based on the structural similarities to the MH2 fold, it is proposed that the Nα-MH2 domain should be classified as a new member of the Smad/FHA superfamily.

  12. A series of N-terminal epitope tagged Hdh knock-in alleles expressing normal and mutant huntingtin: their application to understanding the effect of increasing the length of normal huntingtin’s polyglutamine stretch on CAG140 mouse model pathogenesis

    Directory of Open Access Journals (Sweden)

    Zheng Shuqiu

    2012-08-01

    Full Text Available Abstract Background Huntington’s disease (HD is an autosomal dominant neurodegenerative disease that is caused by the expansion of a polyglutamine (polyQ stretch within Huntingtin (htt, the protein product of the HD gene. Although studies in vitro have suggested that the mutant htt can act in a potentially dominant negative fashion by sequestering wild-type htt into insoluble protein aggregates, the role of the length of the normal htt polyQ stretch, and the adjacent proline-rich region (PRR in modulating HD mouse model pathogenesis is currently unknown. Results We describe the generation and characterization of a series of knock-in HD mouse models that express versions of the mouse HD gene (Hdh encoding N-terminal hemaglutinin (HA or 3xFlag epitope tagged full-length htt with different polyQ lengths (HA7Q-, 3xFlag7Q-, 3xFlag20Q-, and 3xFlag140Q-htt and substitution of the adjacent mouse PRR with the human PRR (3xFlag20Q- and 3xFlag140Q-htt. Using co-immunoprecipitation and immunohistochemistry analyses, we detect no significant interaction between soluble full-length normal 7Q- htt and mutant (140Q htt, but we do observe N-terminal fragments of epitope-tagged normal htt in mutant htt aggregates. When the sequences encoding normal mouse htt’s polyQ stretch and PRR are replaced with non-pathogenic human sequence in mice also expressing 140Q-htt, aggregation foci within the striatum, and the mean size of htt inclusions are increased, along with an increase in striatal lipofuscin and gliosis. Conclusion In mice, soluble full-length normal and mutant htt are predominantly monomeric. In heterozygous knock-in HD mouse models, substituting the normal mouse polyQ and PRR with normal human sequence can exacerbate some neuropathological phenotypes.

  13. Mapping of the antigenic and allergenic epitopes of Lol p VB using gene fragmentation.

    Science.gov (United States)

    Ong, E K; Knox, R B; Singh, M B

    1995-03-01

    The recombinant proteins of Lol p VA and Lol p VB expressed in E. coli reacted with IgE antibodies from sera of allergic patients and mAbs FMC A7 and PpV1. Cross-absorption analyses using these recombinant proteins showed that Lol p VA and Lol p VB possess both similar and unique IgE binding determinants. Gene fragmentation was utilized to localize the antigenic and allergenic determinants of Lol p VB. When full-length cDNA of Lol p VB was digested into three fragments and expressed as the fusions from the glutathione transferase of pGEX vectors, fragments Met1-Val196 and Asp197-Val339 bound IgE while fragment Met1-Pro96 did not. The data suggest that there are at least two IgE binding determinants in Lol p VB. In addition, only fragment Met1-Val196 reacted with mAb PpV1. The localization of these determinants was further resolved using random fragment expression libraries. The mAb PpV1 determinant was near the N-terminal region of Lol p VB molecule. The IgE binding determinants were distributed in the central region: region I (amino acids 111-195) and II (199-254). These IgE binding determinants are conserved in Lol p VA.

  14. Fragmentation of kidney stones

    International Nuclear Information System (INIS)

    Kovacs, K.; Kun, F.; Vertse, T.

    2005-01-01

    Complete text of publication follows. Fragmentation, i.e. the breaking of particulate materials into smaller pieces is abundant in nature and underlies several industrial processes, which attracted a continuous interest in scientific and engineering research over the past decades. In industrial applications, fragmentation processes are mostly used for the comminution of ores in various types of mills. Kidney stone is a well known human dis- ease which embitters the life of many people (in a country like the USA about 10 6 cases are registered yearly). In order to extract large kidney stones (diameter ≥ 1 cm) from the human body without operation, one of the most efficient treatment is the fragmentation of kidney stones by the so-called extracorporal shock wave lithography method: a shock wave penetrating the human body is generated by an electric pulse. The repeated application of the shock wave gradually fragments the stones into pieces of size ≤ 2 mm which then leave the body through the urine system. Recently, a novel type of lithographic method has been suggested by using widely focused shock waves which fragment the stones by a squeezing mechanism. Laboratory experiments showed that the widely focused squeezing waves achieve a higher fragmentation efficiency than the frequently used shock waves of sharp focus. Based on this method a novel medical treatment can be introduced which is less demanding for the patients. Before the application of the method in the clinical practice a detailed understanding of the fragmentation mechanism of kidney stones due to shock waves is required. Since analytic theoretical methods have serious limitations in this field, we develop a realistic model of the mechanical behavior of kidney stones and a simulation code which makes possible to study the mechanism of breakup under various external conditions. Computer simulations in two dimensions have revealed a peculiar way of crack formation, i.e. the crack which finally breaks

  15. On the evolution of cytoplasmic incompatibility in haplodiploid species

    NARCIS (Netherlands)

    Egas, C.J.M.; de Freitas Vala Salvador, F.; Breeuwer, J.A.J.

    2002-01-01

    The most enigmatic sexual manipulation by Wolbachia endosymbionts is cytoplasmic incompatibility (CI): infected mates are reproductively incompatible with uninfected females. In this paper, we extend the theory on population dynamics and evolution of CI, with emphasis on haplodiploid species. First,

  16. Cytoplasmic ATR Activation Promotes Vaccinia Virus Genome Replication

    Directory of Open Access Journals (Sweden)

    Antonio Postigo

    2017-05-01

    Full Text Available In contrast to most DNA viruses, poxviruses replicate their genomes in the cytoplasm without host involvement. We find that vaccinia virus induces cytoplasmic activation of ATR early during infection, before genome uncoating, which is unexpected because ATR plays a fundamental nuclear role in maintaining host genome integrity. ATR, RPA, INTS7, and Chk1 are recruited to cytoplasmic DNA viral factories, suggesting canonical ATR pathway activation. Consistent with this, pharmacological and RNAi-mediated inhibition of canonical ATR signaling suppresses genome replication. RPA and the sliding clamp PCNA interact with the viral polymerase E9 and are required for DNA replication. Moreover, the ATR activator TOPBP1 promotes genome replication and associates with the viral replisome component H5. Our study suggests that, in contrast to long-held beliefs, vaccinia recruits conserved components of the eukaryote DNA replication and repair machinery to amplify its genome in the host cytoplasm.

  17. Refractory disease in antineutrophil cytoplasmic antibodies associated vasculitis

    NARCIS (Netherlands)

    Rutgers, Abraham; Kallenberg, Cornelis

    Purpose of review Induction treatment of antineutrophil cytoplasmic antibodies (ANCA) associated vasculitis (AAV) is not always successful and nonresponding patients are considered refractory. Recent findings Refractory disease should be subdefined to the treatment that was received.

  18. ( Atp9) gene between cytoplasmic male sterile line and its ...

    African Journals Online (AJOL)

    Administrator

    2011-09-07

    , the atp9 gene in soybeans was cloned from a soybean cytoplasmic male sterile line NJCMS2A and its maintainer line NJCMS2B. Sequence alignment was performed, and protein structures were analyzed and compared ...

  19. Fragments of the Past

    OpenAIRE

    Peter Szende; Annie Holcombe

    2016-01-01

    With travel being made more accessible throughout the decades, the hospitality industry constantly evolved their practices as society and technology progressed. Hotels looked for news ways up service their customers, which led to the invention of the Servidor in 1918. Once revolutionary innovations have gone extinct, merely becoming fragments of the past.

  20. Picking Up (On) Fragments

    NARCIS (Netherlands)

    Ellis, Phil

    2015-01-01

    abstractThis article discusses the implications for archival and media archaeological research and reenactment artwork relating to a recent arts practice project: reenacttv: 30 lines / 60 seconds. It proposes that archival material is unstable but has traces and fragments that are full of creative

  1. Fragments of Time

    DEFF Research Database (Denmark)

    Christiansen, Steen Ledet

    Time travel films necessarily fragment linear narratives, as scenes are revisited with differences from the first time we saw it. Popular films such as Back to the Future mine comedy from these visitations, but there are many different approaches. One extreme is Chris Marker's La Jetée - a film...

  2. Wildlife habitat fragmentation.

    Science.gov (United States)

    John. Lehmkuhl

    2005-01-01

    A primary issue in forest wildlife management is habitat fragmentation and its effects on viability, which is the "bottom line" for plant and animal species of conservation concern. Population viability is the likelihood that a population will be able to maintain itself (remain viable) over a long period of time-usually 100 years or more. Though it is true...

  3. Fragments of the Past

    Directory of Open Access Journals (Sweden)

    Peter Szende

    2016-10-01

    Full Text Available With travel being made more accessible throughout the decades, the hospitality industry constantly evolved their practices as society and technology progressed. Hotels looked for news ways up service their customers, which led to the invention of the Servidor in 1918. Once revolutionary innovations have gone extinct, merely becoming fragments of the past.

  4. Stone fragmentation by ultrasound

    Indian Academy of Sciences (India)

    Some delicate nerves and fibres in the surrounding areas of the stones present in the kidney are also damaged by high ultrasonic intensity used in such systems. In the present work, enhancement of the kidney stone fragmentation by using ultrasound is studied. The cavitation bubbles are found to implode faster, with more ...

  5. Synthesis of arabinoxylan fragments

    DEFF Research Database (Denmark)

    Underlin, Emilie Nørmølle; Böhm, Maximilian F.; Madsen, Robert

    , or production of commercial chemicals which are mainly obtained from fossil fuels today.The arbinoxylan fragments have a backbone of β-1,4-linked xylans with α-L-arabinose units attached at specific positions. The synthesis ultilises an efficient synthetic route, where all the xylan units can be derived from D...

  6. N-Terminal Region of GbIspH1, Ginkgo biloba IspH Type 1, May Be Involved in the pH-Dependent Regulation of Enzyme Activity

    OpenAIRE

    Shin, Bok-Kyu; Ahn, Joong-Hoon; Han, Jaehong

    2015-01-01

    GbIspH1, IspH type 1 in Ginkgo biloba chloroplast, is the Fe/S enzyme catalyzing the reductive dehydroxylation of HMBPP to isopentenyl diphosphate (IPP) and dimethylallyl diphosphate (DMAPP) at the final step of methylerythritol phosphate pathway in chloroplast. Compared to the bacterial IspH, plant IspH, including GbIspH1, has an additional polypeptide chain at the N-terminus. Here, biochemical function of the N-terminal region of GbIspH1 was investigated with the N-terminal truncated GbIspH...

  7. Hexa-histidin tag position influences disulfide structure but not binding behavior of in vitro folded N-terminal domain of rat corticotropin-releasing factor receptor type 2a

    OpenAIRE

    Klose, Jana; Wendt, Norbert; Kubald, Sybille; Krause, Eberhard; Fechner, Klaus; Beyermann, Michael; Bienert, Michael; Rudolph, Rainer; Rothemund, Sven

    2004-01-01

    The oxidative folding, particularly the arrangement of disulfide bonds of recombinant extracellular N-terminal domains of the corticotropin-releasing factor receptor type 2a bearing five cysteines (C2 to C6), was investigated. Depending on the position of a His-tag, two types of disulfide patterns were found. In the case of an N-terminal His-tag, the disulfide bonds C2–C3 and C4–C6 were found, leaving C5 free, whereas the C-terminal position of the His-tag led to the disulfide pattern C2–C5 a...

  8. Age-dependent values of N-terminal pro-B-type natriuretic peptide are superior to a single cut-point for ruling out suspected systolic dysfunction in primary care

    DEFF Research Database (Denmark)

    Hildebrandt, Per; Collinson, Paul O; Doughty, Robert N

    2010-01-01

    The study evaluated the use of age-related decision limits for N-terminal pro-B-type natriuretic peptide (NT-proBNP), for ruling out suspected systolic dysfunction in symptomatic patients in primary care, compared with the present standards.......The study evaluated the use of age-related decision limits for N-terminal pro-B-type natriuretic peptide (NT-proBNP), for ruling out suspected systolic dysfunction in symptomatic patients in primary care, compared with the present standards....

  9. The 75-kilodalton cytoplasmic Chlamydia trachomatis L2 polypeptide is a DnaK-like protein

    DEFF Research Database (Denmark)

    Birkelund, Svend; Lundemose, AG; Christiansen, Gunna

    1990-01-01

    ,980-base-pair open reading frame revealed 94% homology with a 75-kilodalton protein from C. trachomatis serovar D and 57% homology with the DnaK proteins of E. coli and of Bacillus megaterium, while amino acid homology with human heat shock protein 70 (hsp70) was 42%. The promoter region was identified......The gene coding for the 75-kilodalton cytoplasmic Chlamydia trachomatis L2 polypeptide has been cloned in Escherichia coli, and the nucleotide sequence has been determined. The cloned DNA fragment contained the coding region as well as the putative promoter. The deduced amino acid sequence of the 1...... by computer search and by primer extension of mRNA synthesized in recombinant E. coli. The promoter region which differed from the putative promoter region in serovar D was shown to be a mixed promoter type in which the -10 region showed a regular TATA box configuration while the -35 region showed high...

  10. Microbial Disease Spectrum Linked to a Novel IL-12Rβ1 N-Terminal Signal Peptide Stop-Gain Homozygous Mutation with Paradoxical Receptor Cell-Surface Expression

    Science.gov (United States)

    Louvain de Souza, Thais; de Souza Campos Fernandes, Regina C.; Azevedo da Silva, Juliana; Gomes Alves Júnior, Vladimir; Gomes Coelho, Adelia; Souza Faria, Afonso C.; Moreira Salomão Simão, Nabia M.; Souto Filho, João T.; Deswarte, Caroline; Boisson-Dupuis, Stéphanie; Torgerson, Dara; Casanova, Jean-Laurent; Bustamante, Jacinta; Medina-Acosta, Enrique

    2017-01-01

    Patients with Mendelian Susceptibility to Mycobacterial Diseases (MSMD) exhibit variable vulnerability to infections by mycobacteria and other intramacrophagic bacteria (e.g., Salmonella and Klebsiella) and fungi (e.g., Histoplasma, Candida, Paracoccidioides, Coccidioides, and Cryptococcus). The hallmark of MSMD is the inherited impaired production of interferon gamma (IFN-γ) or the lack of response to it. Mutations in the interleukin (IL)-12 receptor subunit beta 1 (IL12RB1) gene accounts for 38% of cases of MSMD. Most IL12RB1 pathogenic allele mutations, including ten known stop-gain variants, cause IL-12Rβ1 complete deficiency (immunodeficiency-30, IMD30) by knocking out receptor cell-surface expression. IL12RB1 loss-of-function genotypes impair both IL-12 and IL-23 responses. Here, we assess the health effects of a rare, novel IL12RB1 stop-gain homozygous genotype with paradoxical IL-12Rβ1 cell-surface expression. We appraise four MSMD children from three unrelated Brazilian kindreds by clinical consultation, medical records, and genetic and immunologic studies. The clinical spectrum narrowed down to Bacillus Calmette-Guerin (BCG) vaccine-related suppurative adenitis in all patients with one death, and recrudescence in two, histoplasmosis, and recurrence in one patient, extraintestinal salmonellosis in one child, and cutaneous vasculitis in another. In three patients, we established the homozygous Trp7Ter predicted loss-of-function inherited genotype and inferred it from the heterozygote parents of the fourth case. The Trp7Ter mutation maps to the predicted IL-12Rβ1 N-terminal signal peptide sequence. BCG- or phytohemagglutinin-blasts from the three patients have reduced cell-surface expression of IL-12Rβ1 with impaired production of IFN-γ and IL-17A. Screening of 227 unrelated healthy subjects from the same geographic region revealed one heterozygous genotype (allele frequency 0.0022) vs. one in over 841,883 public genome/exomes. We also show that the

  11. Cordyceps militaris Fraction induces apoptosis and G2/M Arrest via c-Jun N-Terminal kinase signaling pathway in oral squamous carcinoma KB Cells

    Science.gov (United States)

    Xie, Wangshi; Zhang, Zhang; Song, Liyan; Huang, Chunhua; Guo, Zhongyi; Hu, Xianjing; Bi, Sixue; Yu, Rongmin

    2018-01-01

    Background: Cordyceps militaris fraction (CMF) has been shown to possess in vitro antitumor activity against human chronic myeloid leukemia K562 cells in our previous research. Materials and Methods: The in vitro inhibitory activities of CMF on the growth of KB cells were evaluated by viability assay. The apoptotic and cell cycle influences of CMF were detected by 4′,6-diamidino-2-phenylindole staining and flow cytometry assay. The expression of different apoptosis-associated proteins and cell cycle regulatory proteins was examined by Western blot assay. The nuclear localization of c-Jun was observed by fluorescence staining. Objective: The objective of this study was to investigate the antiproliferative effect of CMF as well as the mechanism underlying the apoptosis and cell cycle arrest it induces in KB cells. Results: CMF suppressed KB cells’ proliferation in a dose- and time-dependent manner. Flow cytometric analysis indicated that CMF induced G2/M cell cycle arrest and apoptosis. Western blot analysis revealed that CMF induced caspase-3, caspase-9, and PARP cleavages, and increased the Bax/Bcl-2 ratio. CMF also led to increased expression of p21, decreased expression of cyclin B1, mitotic phosphatase cdc25c, and mitotic kinase cdc2, as well as unchanged expression of p53. In addition, CMF stimulated c-Jun N-terminal kinases (JNK) protein phosphorylations, resulting in upregulated expression of c-Jun and nuclear localization of c-Jun. Pretreatment with JNK inhibitor SP600125 suppressed CMF-induced apoptosis and G2/M arrest. Conclusions: CMF is capable of modulating c-Jun caspase and Bcl-2 family proteins through JNK-dependent apoptosis, which results in G2/M phase arrest in KB cells. CMF could be developed as a promising candidate for the new antitumor agents. SUMMARY CMF exhibited strong anticancer activity against oral squamous carcinoma KB cellsCMF inhibited KB cells’ proliferation via induction of apoptosis and G2/M cell cycle arrestCMF activated JNK

  12. Dynamics of Galectin-3 in the Nucleus and Cytoplasm

    Science.gov (United States)

    Haudek, Kevin C.; Spronk, Kimberly J.; Voss, Patricia G.; Patterson, Ronald J.; Wang, John L.; Arnoys, Eric J.

    2009-01-01

    This review summarizes selected studies on galectin-3 (Gal3) as an example of the dynamic behavior of a carbohydrate-binding protein in the cytoplasm and nucleus of cells. Within the 15-member galectin family of proteins, Gal3 (Mr ~30,000) is the sole representative of the chimera subclass in which a proline- and glycine-rich NH2-terminal domain is fused onto a COOH-terminal carbohydrate recognition domain responsible for binding galactose-containing glycoconjugates. The protein shuttles between the cytoplasm and nucleus on the basis of targeting signals that are recognized by importin(s) for nuclear localization and exportin-1 (CRM1) for nuclear export. Depending on the cell type, specific experimental conditions in vitro, or tissue location, Gal3 has been reported to be exclusively cytoplasmic, predominantly nuclear, or distributed between the two compartments. The nuclear versus cytoplasmic distribution of the protein must reflect, then, some balance between nuclear import and export, as well as mechanisms of cytoplasmic anchorage or binding to a nuclear component. Indeed, a number of ligands have been reported for Gal3 in the cytoplasm and in the nucleus. Most of the ligands appear to bind Gal3, however, through protein-protein interactions rather than through protein-carbohydrate recognition. In the cytoplasm, for example, Gal3 interacts with the apoptosis repressor Bcl-2 and this interaction may be involved in Gal3’s anti-apoptotic activity. In the nucleus, Gal3 is a required pre-mRNA splicing factor; the protein is incorporated into spliceosomes via its association with the U1 small nuclear ribonucleoprotein (snRNP) complex. Although the majority of these interactions occur via the carbohydrate recognition domain of Gal3 and saccharide ligands such as lactose can perturb some of these interactions, the significance of the protein’s carbohydrate-binding activity, per se, remains a challenge for future investigations. PMID:19616076

  13. Subcloning of DNA fragments.

    Science.gov (United States)

    Struhl, K

    2001-05-01

    The essence of recombinant DNA technology is the joining of two or more separate segments of DNA to generate a single DNA molecule that is capable of autonomous replication in a given host. The simplest constructions of hybrid DNA molecules involve the cloning of insert sequences into plasmid or bacteriophage cloning vectors. The insert sequences can derive from essentially any organism, and they may be isolated directly from the genome, from mRNA, or from previously cloned DNA segments (in which case, the procedure is termed subcloning). Alternatively, insert DNAs can be created directly by DNA synthesis. This unit provides protocols for the subcloning of DNA fragments and ligation of DNA fragments in gels.

  14. The Serendipity of Fragmentation

    DEFF Research Database (Denmark)

    Leixnering, Stephan; Meyer, Renate E.

    , it was the central government’s task to coordinate, steer and control the newly emerged decentralized organizations. This raises questions about the overall design of the public sector at present. Our paper engages with the prevalent public governance phenomenon of fragmentation from a design perspective in order......Reform approaches in the public sector led to significant changes in the sector’s design. Especially NPM-inspired reform measures which had largely aimed at organizational disaggregation created pluriform landscapes of public sector organizations (PSOs). Following a core public governance principle...... form of organizing between networks and formal organization: lacking a single center and featuring multiplex and multifaceted relations within the politico-administrative apparatus and between government and PSOs, high fragmentation, local and robust action, but latent structures of significant formal...

  15. Nuclear Localization of PTEN by a Ran-dependent Mechanism Enhances Apoptosis: Involvement of an N-Terminal Nuclear Localization Domain and Multiple Nuclear Exclusion Motifs

    OpenAIRE

    Gil, Anabel; Andrés-Pons, Amparo; Fernández, Elena; Valiente, Miguel; Torres, Josema; Cervera, Javier; Pulido, Rafael

    2006-01-01

    The targeting of the tumor suppressor PTEN protein to distinct subcellular compartments is a major regulatory mechanism of PTEN function, by controlling its access to substrates and effector proteins. Here, we investigated the molecular basis and functional consequences of PTEN nuclear/cytoplasmic distribution. PTEN accumulated in the nucleus of cells treated with apoptotic stimuli. Nuclear accumulation of PTEN was enhanced by mutations targeting motifs in distinct PTEN domains, and it was de...

  16. Detection of titin fragments in urine in response to exercise-induced muscle damage.

    Directory of Open Access Journals (Sweden)

    Kazue Kanda

    Full Text Available Many studies have attempted to determine the associations between blood biomarkers and exercise-induced muscle damage. However, poor correlations between the changes in biomarker levels and the magnitude of muscle symptoms have been reported. Recent advances in proteomic tools offer a strategy for the comprehensive analysis of protein expression, which can be used to identify biomarkers. Here, we used a proteomic analysis to identify urinary proteins that appear in response to a calf-raise exercise, including repetitive eccentric muscle contractions, and found that a titin (also known as connectin N-terminal fragment molecule appears in the urine after eccentric exercise. We measured the titin fragment in urine samples from nine individuals before and after eccentric exercise using a newly-established enzyme-linked immunosorbent assay and found that the titin fragment excretion rate increased 96 h after the exercise (5.1 to 77.6 pg/min, p <0.01. The changes in the titin fragment excretion rate were correlated strongly with blood markers of muscle damage and with muscle symptoms. These findings suggest that the urinary titin fragment is potentially a noninvasive biomarker of muscle damage.

  17. Generation of biologically active endostatin fragments from human collagen XVIII by dist