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Sample records for ae-rich n-terminal trunk

  1. 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...

  2. 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.

  3. Trunk Stability, Trunk Strength and Sport Performance Level in Judo

    Science.gov (United States)

    Barbado, David; Lopez-Valenciano, Alejandro; Juan-Recio, Casto; Montero-Carretero, Carlos; van Dieën, Jaap H.; Vera-Garcia, Francisco J.

    2016-01-01

    Although trunk muscle function has been suggested to be a determinant of judo performance, its contribution to high-level performance in this sport has been poorly studied. Therefore, several tests were used to assess the differences in trunk muscle function between 11 international and 14 national level judo practitioners (judokas). Trunk strength and endurance were assessed using isokinetic tests and core stability was assessed using two protocols: 1) sudden loading, to assess trunk responses to unexpected external perturbations; 2) stable and unstable sitting, to assess the participants’ ability to control trunk balance. No differences between groups were found for trunk flexor isokinetic strength, trunk responses against lateral and posterior loading and trunk control while sitting. However, international level judokas showed significantly higher trunk extensor isokinetic strength (p <0.05) and lower trunk angular displacement after anterior trunk loading (p <0.05) than national level judokas. Few and low (r < 0.512) significant correlations were found between strength, endurance and stability parameters, which suggests that trunk strength and endurance are not limiting factors for trunk stability in competitive judokas. These results support the importance of trunk extensor strength and trunk stability against forward perturbations in elite judo performance. PMID:27232602

  4. Trunk Stability, Trunk Strength and Sport Performance Level in Judo.

    Directory of Open Access Journals (Sweden)

    David Barbado

    Full Text Available Although trunk muscle function has been suggested to be a determinant of judo performance, its contribution to high-level performance in this sport has been poorly studied. Therefore, several tests were used to assess the differences in trunk muscle function between 11 international and 14 national level judo practitioners (judokas. Trunk strength and endurance were assessed using isokinetic tests and core stability was assessed using two protocols: 1 sudden loading, to assess trunk responses to unexpected external perturbations; 2 stable and unstable sitting, to assess the participants' ability to control trunk balance. No differences between groups were found for trunk flexor isokinetic strength, trunk responses against lateral and posterior loading and trunk control while sitting. However, international level judokas showed significantly higher trunk extensor isokinetic strength (p <0.05 and lower trunk angular displacement after anterior trunk loading (p <0.05 than national level judokas. Few and low (r < 0.512 significant correlations were found between strength, endurance and stability parameters, which suggests that trunk strength and endurance are not limiting factors for trunk stability in competitive judokas. These results support the importance of trunk extensor strength and trunk stability against forward perturbations in elite judo performance.

  5. Trunk Stability, Trunk Strength and Sport Performance Level in Judo.

    Science.gov (United States)

    Barbado, David; Lopez-Valenciano, Alejandro; Juan-Recio, Casto; Montero-Carretero, Carlos; van Dieën, Jaap H; Vera-Garcia, Francisco J

    2016-01-01

    Although trunk muscle function has been suggested to be a determinant of judo performance, its contribution to high-level performance in this sport has been poorly studied. Therefore, several tests were used to assess the differences in trunk muscle function between 11 international and 14 national level judo practitioners (judokas). Trunk strength and endurance were assessed using isokinetic tests and core stability was assessed using two protocols: 1) sudden loading, to assess trunk responses to unexpected external perturbations; 2) stable and unstable sitting, to assess the participants' ability to control trunk balance. No differences between groups were found for trunk flexor isokinetic strength, trunk responses against lateral and posterior loading and trunk control while sitting. However, international level judokas showed significantly higher trunk extensor isokinetic strength (p judo performance. PMID:27232602

  6. N-terminal protein processing: A comparative proteogenomic analysis

    Energy Technology Data Exchange (ETDEWEB)

    Bonissone, Stefano; Gupta, Nitin; Romine, Margaret F.; Bradshaw, Ralph A.; Pevzner, Pavel A.

    2013-01-01

    N-Terminal Methionine Excision (NME) is a universally conserved mechanism with the same specificity across all life forms that removes the first Methionine in proteins when the second residue is Gly, Ala, Ser, Cys, Thr, Pro, or Val. In spite of its necessity for proper cell functioning, the functional role of NME remains unclear. In 1988, Arfin and Bradshaw connected NME with the N-end protein degradation rule and postulated that the role of NME is to expose the stabilizing residues with the goal to resist protein degradation. While this explanation (that treats 7 stabilizing residues in the same manner) has become the de facto dogma of NME, comparative proteogenomics analysis of NME tells a different story. We suggest that the primary role of NME is to expose only two (rather than seven) amino acids Ala and Ser for post-translational modifications (e.g., acetylation) rather than to regulate protein degradation. We argue that, contrary to the existing view, NME is not crucially important for proteins with 5 other stabilizing residue at the 2nd positions that are merely bystanders (their function is not affected by NME) that become exposed to NME because their sizes are comparable or smaller than the size of Ala and Ser.

  7. 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: ...

  8. Function of the N-terminal segment of the RecA-dependent nuclease Ref.

    Science.gov (United States)

    Gruber, Angela J; Olsen, Tayla M; Dvorak, Rachel H; Cox, Michael M

    2015-02-18

    The bacteriophage P1 Ref (recombination enhancement function) protein is a RecA-dependent, HNH endonuclease. It can be directed to create targeted double-strand breaks within a displacement loop formed by RecA. The 76 amino acid N-terminal region of Ref is positively charged (25/76 amino acid residues) and inherently unstructured in solution. Our investigation of N-terminal truncation variants shows this region is required for DNA binding, contains a Cys involved in incidental dimerization and is necessary for efficient Ref-mediated DNA cleavage. Specifically, Ref N-terminal truncation variants lacking between 21 and 47 amino acids are more effective RecA-mediated targeting nucleases. We propose a more refined set of options for the Ref-mediated cleavage mechanism, featuring the N-terminal region as an anchor for at least one of the DNA strand cleavage events.

  9. 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.

  10. N-terminal acetylation inhibits protein targeting to the endoplasmic reticulum.

    Directory of Open Access Journals (Sweden)

    Gabriella M A Forte

    2011-05-01

    Full Text Available Amino-terminal acetylation is probably the most common protein modification in eukaryotes with as many as 50%-80% of proteins reportedly altered in this way. Here we report a systematic analysis of the predicted N-terminal processing of cytosolic proteins versus those destined to be sorted to the secretory pathway. While cytosolic proteins were profoundly biased in favour of processing, we found an equal and opposite bias against such modification for secretory proteins. Mutations in secretory signal sequences that led to their acetylation resulted in mis-sorting to the cytosol in a manner that was dependent upon the N-terminal processing machinery. Hence N-terminal acetylation represents an early determining step in the cellular sorting of nascent polypeptides that appears to be conserved across a wide range of species.

  11. Secretin N-terminal hexapeptide potentiates insulin release in mouse islets

    DEFF Research Database (Denmark)

    Kofod, Hans

    1986-01-01

    Peptides representing the N-terminal part of secretin were synthesized and their effects tested on column-perifused isolated mouse pancreatic islets. Insulin release induced by D-glucose was potentiated by the two peptides His-Ser-Asp-Gly-Thr-Phe-OMe (S1-6) and Ser-Asp-Gly-Thr-Phe-OMe (S2......-6). The consecutive smaller N-terminal peptides Asp-Gly-Thr-Phe-OMe (S3-6) and Gly-Thr-Phe-OMe (S4-6) had no effects while the dipeptide ester Thr-Phe-OMe (S5-6) also potentiated the release of insulin. The results suggest that the N-terminal part of secretin may be involved in the marked in vitro glucose......-dependent insulin release induced by secretin....

  12. Expression and Biochemical Characterization of the Human Enzyme N-Terminal Asparagine Amidohydrolase (hNTAN1)

    OpenAIRE

    Cantor, Jason R.; Stone, Everett M.; Georgiou, George

    2011-01-01

    The enzymatic deamidation of N-terminal L-Asn by N-terminal asparagine amidohydrolase (NTAN1) is a feature of the ubiquitin-dependent N-end rule pathway of protein degradation, which relates the in vivo half-life of a protein to the identity of its N-terminal residue. Herein we report the bacterial expression, purification, and biochemical characterization of the human NTAN1 (hNTAN1). We show here that hNTAN1 is highly selective for the hydrolysis of N-terminal peptidyl L-Asn, but fails to de...

  13. Localization of the N-terminal domain of cauliflower mosaic virus coat protein precursor

    International Nuclear Information System (INIS)

    Cauliflower mosaic virus (CaMV) open reading frame (ORF) IV encodes a coat protein precursor (pre-CP) harboring an N-terminal extension that is cleaved off by the CaMV-encoded protease. In transfected cells, pre-CP is present in the cytoplasm, while the processed form (p44) of CP is targeted to the nucleus, suggesting that the N-terminal extension might be involved in keeping the pre-CP in the cytoplasm for viral assembly. This study reports for the first time the intracellular localization of the N-terminal extension during CaMV infection in Brassica rapa. Immunogold-labeling electron microscopy using polyclonal antibodies directed to the N-terminal extension of the pre-CP revealed that this region is closely associated with viral particles present in small aggregates, which we called small bodies, adjacent to the main inclusion bodies typical of CaMV infection. Based on these results, we propose a model for viral assembly of CaMV

  14. Supramolecular hydrogelators of N-terminated dipeptides selectively inhibit cancer cells.

    Science.gov (United States)

    Kuang, Yi; Gao, Yuan; Xu, Bing

    2011-12-21

    Consisting of N-terminated diphenylalanine, a new type of supramolecular hydrogelators forms hydrogels within a narrow pH window (pH 5.0 to 6.0) and selectively inhibits growth of HeLa cells, which provides important and useful insights for designing molecular nanofibers as potential nanomedicines. PMID:22037699

  15. Supramolecular hydrogelators of N-terminated dipeptides selectively inhibit cancer cells

    OpenAIRE

    Kuang, Yi; Gao, Yuan; Xu, Bing

    2011-01-01

    Consisting of N-terminated diphenylalanine, a new type of supramolecular hydrogelators forms hydrogels within a narrow pH window (pH 5.0 to 6.0) and selectively inhibits growth of HeLa cells, which provides important and useful insights for designing molecular nanofibers as potential nanomedicines.

  16. Selecting protein N-terminal peptides by combined fractional diagonal chromatography.

    Science.gov (United States)

    Staes, An; Impens, Francis; Van Damme, Petra; Ruttens, Bart; Goethals, Marc; Demol, Hans; Timmerman, Evy; Vandekerckhove, Joël; Gevaert, Kris

    2011-07-14

    In recent years, procedures for selecting the N-terminal peptides of proteins with analysis by mass spectrometry have been established to characterize protease-mediated cleavage and protein α-N-acetylation on a proteomic level. As a pioneering technology, N-terminal combined fractional diagonal chromatography (COFRADIC) has been used in numerous studies in which these protein modifications were investigated. Derivatization of primary amines--which can include stable isotope labeling--occurs before trypsin digestion so that cleavage occurs after arginine residues. Strong cation exchange (SCX) chromatography results in the removal of most of the internal peptides. Diagonal, reversed-phase peptide chromatography, in which the two runs are separated by reaction with 2,4,6-trinitrobenzenesulfonic acid, results in the removal of the C-terminal peptides and remaining internal peptides and the fractionation of the sample. We describe here the fully matured N-terminal COFRADIC protocol as it is currently routinely used, including the most substantial improvements (including treatment with glutamine cyclotransferase and pyroglutamyl aminopeptidase to remove pyroglutamate before SCX, and a sample pooling scheme to reduce the overall number of liquid chromatography-tandem mass spectrometry analyses) that were made since its original publication. Completion of the N-terminal COFRADIC procedure takes ~5 d.

  17. c-Jun N-terminal kinase mediates constitutive human eosinophil apoptosis

    OpenAIRE

    Hasala, Hannele; Zhang, Xianzhi; Saarelainen, Seppo; Moilanen, Eeva; Kankaanranta, Hannu

    2007-01-01

    c-Jun N-terminal kinase mediates constitutive human eosinophil apoptosis correspondence: Corresponding author. Tel.: +358335517318; fax: +358335518082. (Kankaanranta, Hannu) (Kankaanranta, Hannu) The Immunopharmacology Research Group--> , Medical School--> , University of Tampere--> , Tampere--> - FINLAND (Hasala, Hannele) The Immunopharmacology Research Group--> , Medical School--...

  18. 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.

  19. 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; Gøtze, 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 ev...

  20. Microwave sensing of tree trunks

    Science.gov (United States)

    Jezova, Jana; Mertens, Laurence; Lambot, Sebastien

    2015-04-01

    The main subject of this research is the observation of the inner part of living tree trunks using ground-penetrating radar (GPR). Trees are everyday part of human life and therefore it is important to pay attention to the tree conditions. The most obvious consequence of the poor tree condition is dead or injury caused by falling tree. The trunk internal structure is divided into three main parts: heartwood, sapwood and bark, which make this medium highly anisotropic and heterogeneous. Furthermore, the properties of the wood are not only specie-dependent but also depend on genetic and on environmental conditions. In urban areas the main problem for the stability of the trees relies in the apparition of decays provoked by fungi, insect or birds. This results in cavities or decreasing of the support capacity of the tree. GPR has proved itself to be a very powerful electromagnetic tool for non-destructive detection of buried objects. Since the beginning of the 20th century it has been used in several different areas (archaeology, landmine detection, civil engineering, ...). GPR uses the principle of the scattering of the electromagnetic waves that are radiated from a transmitting antenna. Then the waves propagate through the medium and are reflected from the object and then they are received by a receiving antenna. The velocity of the scattered signal is determined primarily by the permittivity of the material. The optimal functionality of the GPR was investigated using the numerical simulation tool gprMax2D. This tool is based on a Finite-Difference Time-Domain (FDTD) numerical model. Subsequently, the GPR functionality was tested using the laboratory model of a decayed tree trunk. Afterwards, the results and lessons learnt in the simplified tests will be used in the processing of the real data and will help to achieve deeper understanding of them. The laboratory model of the tree trunk was made by plastic or carton pipes and filled by sand. Space inside the model

  1. Plasma N-terminal pro-B-type natriuretic peptide and mortality in type 2 diabetes

    DEFF Research Database (Denmark)

    Tarnow, L; Gall, M-A; Hansen, B V;

    2006-01-01

    AIMS/HYPOTHESIS: Raised N-terminal pro-B-type natriuretic peptide (NT-proBNP) is associated with a poor cardiac outcome in non-diabetic populations. Elevated NT-proBNP predicts excess morbidity and mortality in diabetic patients with an elevated urinary albumin excretion rate. This study investig......AIMS/HYPOTHESIS: Raised N-terminal pro-B-type natriuretic peptide (NT-proBNP) is associated with a poor cardiac outcome in non-diabetic populations. Elevated NT-proBNP predicts excess morbidity and mortality in diabetic patients with an elevated urinary albumin excretion rate. This study...... investigated the prognostic value of NT-proBNP in a cohort of type 2 diabetic patients. SUBJECTS, MATERIALS AND METHODS: In a prospective observational follow-up study, 315 type 2 diabetic patients with normoalbuminuria (n=188), microalbuminuria (n=80) and macroalbuminuria (n=47) at baseline were followed...

  2. PRINT: A Protein Bioconjugation Method with Exquisite N-terminal Specificity

    Science.gov (United States)

    Sur, Surojit; Qiao, Yuan; Fries, Anja; O'Meally, Robert N.; Cole, Robert N.; Kinzler, Kenneth W.; Vogelstein, Bert; Zhou, Shibin

    2015-12-01

    Chemical conjugation is commonly used to enhance the pharmacokinetics, biodistribution, and potency of protein therapeutics, but often leads to non-specific modification or loss of bioactivity. Here, we present a simple, versatile and widely applicable method that allows exquisite N-terminal specific modification of proteins. Combining reversible side-chain blocking and protease mediated cleavage of a commonly used HIS tag appended to a protein, we generate with high yield and purity exquisitely site specific and selective bio-conjugates of TNF-α by using amine reactive NHS ester chemistry. We confirm the N terminal selectivity and specificity using mass spectral analyses and show near complete retention of the biological activity of our model protein both in vitro and in vivo murine models. We believe that this methodology would be applicable to a variety of potentially therapeutic proteins and the specificity afforded by this technique would allow for rapid generation of novel biologics.

  3. N-terminal-pro-B-type natriuretic peptide during pharmacological heart rate reduction in hyperthyroidism

    DEFF Research Database (Denmark)

    Schultz, M; Kistorp, C; Corell, P;

    2009-01-01

    ; resting heart rate [from mean 97 to 80 beats per min (17.5%), p...-index decreased from median 319 to 315 arbitrary units (p=0.039) and free triiodothyronine-index increased from 8.6 to 9.9 arbitrary units (p=0.010). No changes in echocardiographic parameters were observed. A decrease in resting heart rate in untreated hyperthyroidism due to verapamil treatment did not result......We hypothesized that elevated N-terminal-pro-B-type natriuretic peptide levels in hyperthyroidism are mainly driven by increased metabolism due to excess thyroid hormones. Therefore, serum levels of N-terminal-pro-B-type natriuretic peptide were studied during reduced cardiac work load by means...

  4. Resin-assisted Enrichment of N-terminal Peptides for Characterizing Proteolytic Processing

    Energy Technology Data Exchange (ETDEWEB)

    Kim, Jong Seo; Dai, Ziyu; Aryal, Uma K.; Moore, Ronald J.; Camp, David G.; Baker, Scott E.; Smith, Richard D.; Qian, Weijun

    2013-06-17

    Proteolytic processing is a ubiquitous, irreversible posttranslational modification that plays an important role in cellular regulation in all living organisms. Herein we report a resin-assisted positive selection method for specifically enriching protein N-terminal peptides to facilitate the characterization of proteolytic processing events by liquid chromatography-tandem mass spectrometry. In this approach, proteins are initially reduced and alkylated and their lysine residues are converted to homoarginines. Then, protein N-termini are selectively converted to reactive thiol groups. We demonstrate that these sequential reactions were achieved with nearly quantitative efficiencies. Thiol-containing N-terminal peptides are then captured (>98% efficiency) by a thiol-affinity resin, a significant improvement over the traditional avidin/biotin enrichment. Application to cell lysates of Aspergillus niger, a filamentous fungus of interest for biomass degradation, enabled the identification of 1672 unique protein N-termini and proteolytic cleavage sites from 690 unique proteins.

  5. Label-Free Quantitative Proteomics and N-terminal Analysis of Human Metastatic Lung Cancer Cells

    OpenAIRE

    Min, Hophil; Han, Dohyun; Kim, Yikwon; Cho, Jee Yeon; Jin, Jonghwa; Kim, Youngsoo

    2014-01-01

    Proteomic analysis is helpful in identifying cancer-associated proteins that are differentially expressed and fragmented that can be annotated as dysregulated networks and pathways during metastasis. To examine meta-static process in lung cancer, we performed a proteomics study by label-free quantitative analysis and N-terminal analysis in 2 human non-small-cell lung cancer cell lines with disparate metastatic potentials—NCI-H1703 (primary cell, stage I) and NCI-H1755 (metastatic cell, stage ...

  6. HEPATIC APOPTOSIS POST-BURN IS MEDIATED BY C-JUN N-TERMINAL KINASE-2

    OpenAIRE

    Marshall, Alexandra H; Brooks, Natasha C; Hiyama, Yaeko; Qa’aty, Nour; Al-mousawi, Ahmed; Finnerty, Celeste C.; Jeschke, Marc G.

    2013-01-01

    The trauma of a severe burn injury induces a hypermetabolic response that increases morbidity and mortality. Previously, our group showed that insulin resistance post-burn injury is associated with endoplasmic reticulum (ER) stress. Evidence suggests that c-jun N-terminal kinase (JNK) -2 may be involved in ER stress-induced apoptosis. Here, we hypothesized that JNK2 contributes to the apoptotic response after burn injury downstream of ER stress. To test this, we compared JNK2 knockout mice (−...

  7. The large N-terminal region of the Brr2 RNA helicase guides productive spliceosome activation.

    Science.gov (United States)

    Absmeier, Eva; Wollenhaupt, Jan; Mozaffari-Jovin, Sina; Becke, Christian; Lee, Chung-Tien; Preussner, Marco; Heyd, Florian; Urlaub, Henning; Lührmann, Reinhard; Santos, Karine F; Wahl, Markus C

    2015-12-15

    The Brr2 helicase provides the key remodeling activity for spliceosome catalytic activation, during which it disrupts the U4/U6 di-snRNP (small nuclear RNA protein), and its activity has to be tightly regulated. Brr2 exhibits an unusual architecture, including an ∼ 500-residue N-terminal region, whose functions and molecular mechanisms are presently unknown, followed by a tandem array of structurally similar helicase units (cassettes), only the first of which is catalytically active. Here, we show by crystal structure analysis of full-length Brr2 in complex with a regulatory Jab1/MPN domain of the Prp8 protein and by cross-linking/mass spectrometry of isolated Brr2 that the Brr2 N-terminal region encompasses two folded domains and adjacent linear elements that clamp and interconnect the helicase cassettes. Stepwise N-terminal truncations led to yeast growth and splicing defects, reduced Brr2 association with U4/U6•U5 tri-snRNPs, and increased ATP-dependent disruption of the tri-snRNP, yielding U4/U6 di-snRNP and U5 snRNP. Trends in the RNA-binding, ATPase, and helicase activities of the Brr2 truncation variants are fully rationalized by the crystal structure, demonstrating that the N-terminal region autoinhibits Brr2 via substrate competition and conformational clamping. Our results reveal molecular mechanisms that prevent premature and unproductive tri-snRNP disruption and suggest novel principles of Brr2-dependent splicing regulation. PMID:26637280

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

    Energy Technology Data Exchange (ETDEWEB)

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

    1986-04-08

    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.

  9. 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\

  10. Expanding the Phenotype Associated with NAA10-Related N-Terminal Acetylation Deficiency.

    Science.gov (United States)

    Saunier, Chloé; Støve, Svein Isungset; Popp, Bernt; Gérard, Bénédicte; Blenski, Marina; AhMew, Nicholas; de Bie, Charlotte; Goldenberg, Paula; Isidor, Bertrand; Keren, Boris; Leheup, Bruno; Lampert, Laetitia; Mignot, Cyril; Tezcan, Kamer; Mancini, Grazia M S; Nava, Caroline; Wasserstein, Melissa; Bruel, Ange-Line; Thevenon, Julien; Masurel, Alice; Duffourd, Yannis; Kuentz, Paul; Huet, Frédéric; Rivière, Jean-Baptiste; van Slegtenhorst, Marjon; Faivre, Laurence; Piton, Amélie; Reis, André; Arnesen, Thomas; Thauvin-Robinet, Christel; Zweier, Christiane

    2016-08-01

    N-terminal acetylation is a common protein modification in eukaryotes associated with numerous cellular processes. Inherited mutations in NAA10, encoding the catalytic subunit of the major N-terminal acetylation complex NatA have been associated with diverse, syndromic X-linked recessive disorders, whereas de novo missense mutations have been reported in one male and one female individual with severe intellectual disability but otherwise unspecific phenotypes. Thus, the full genetic and clinical spectrum of NAA10 deficiency is yet to be delineated. We identified three different novel and one known missense mutation in NAA10, de novo in 11 females, and due to maternal germ line mosaicism in another girl and her more severely affected and deceased brother. In vitro enzymatic assays for the novel, recurrent mutations p.(Arg83Cys) and p.(Phe128Leu) revealed reduced catalytic activity. X-inactivation was random in five females. The core phenotype of X-linked NAA10-related N-terminal-acetyltransferase deficiency in both males and females includes developmental delay, severe intellectual disability, postnatal growth failure with severe microcephaly, and skeletal or cardiac anomalies. Genotype-phenotype correlations within and between both genders are complex and may include various factors such as location and nature of mutations, enzymatic stability and activity, and X-inactivation in females. PMID:27094817

  11. N-terminal of L protein of vesicular stomatitis virus contains a new signal sequence

    Institute of Scientific and Technical Information of China (English)

    NIE Yuchun; KE Yeyan; WANG Zai; YU Xiang; DENG Hongkui; DING Mingxiao

    2003-01-01

    The L protein (241 kD) of vesicular stomatitis virus (VSV) is the mostimportant subunit of the replication complex. The existence of specific localization signal in the L protein was investigated by making recombinant constructs expressing truncated mutants of the L protein fused to green fluorescent protein(GFP) in transient transfection assays. The chimeric genes encoding varied N-terminal of L and GFP gene were put under the control of T7 promoter or CMV promoter. The fusion proteins were transiently expressed in BHK-21, COS-7, CHO or Hep G2 cells. When more than 120 residues were deleted or only 96 residues were kepton the N-terminal, the fusion proteins were shown to be distributed throughout the cells, cytoplasm and nucleus under the confocal microscope. However, other chimeric proteins with 120 or more amino acids were dotted and distributed in theperinuclear regions. And the fusion protein with 96-120 aa has the similar distribution. A thirteen-residue peptide QGYSFLHEVDKEA (108-120) was identified as localization signal, whose function would be absolutely distributed with the deficiency of D or V. Our results show that there is an independent localizing signal in N-terminal domain of L protein of VSV and this functional signal is conserved in different cell lines.

  12. 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.

  13. Effect of gait retraining for reducing ambulatory knee load on trunk biomechanics and trunk muscle activity.

    Science.gov (United States)

    Nüesch, Corina; Laffer, Dominik; Netzer, Cordula; Pagenstert, Geert; Mündermann, Annegret

    2016-06-01

    The purpose of this study was to test the hypothesis that walking with increased medio-lateral trunk sway is associated with lower external knee adduction moment and lower extremity muscle activation, and higher external ipsilateral trunk moment and trunk muscle activity than walking with normal trunk sway in healthy participants. Fifteen participants performed walking trials with normal and increased medio-lateral trunk sway. Maximum trunk sway, first maximum knee adduction moment, lateral trunk bending moment, and bilateral vastus medialis, vastus lateralis, gluteus medius, rectus abdominis, external oblique and erector spinae muscle activity were computed. Walking with increased trunk sway was associated with lower maximum knee adduction moment (95% confidence interval (CI): 0.50-0.62Nm/kg vs. 0.62-0.76Nm/kg; P<.001) and ipsilateral gluteus medius (-17%; P=.014) and erector spinae muscle activity (-24%; P=.004) and greater maximum lateral trunk bending moment (+34%; P<.001) and contralateral external oblique muscle activity (+60%; P=.009). In all participants, maximum knee adduction moment was negatively correlated and maximum trunk moment was positively correlated with maximum trunk sway. The results of this study suggest that walking with increased trunk sway not only reduces the external knee adduction moment but also alters and possibly increases the load on the trunk. Hence, load-altering biomechanical interventions should always be evaluated not only regarding their effects on the index joint but on other load-bearing joints such as the spine. PMID:27264398

  14. Two N-Terminal Acetyltransferases Antagonistically Regulate the Stability of a Nod-Like Receptor in Arabidopsis

    Science.gov (United States)

    Li, Lin; Gannon, Patrick; Linster, Eric; Huber, Monika; Kapos, Paul; Bienvenut, Willy; Giglione, Carmela; Zhang, Yuelin; Chen, She

    2015-01-01

    Nod-like receptors (NLRs) serve as immune receptors in plants and animals. The stability of NLRs is tightly regulated, though its mechanism is not well understood. Here, we show the crucial impact of N-terminal acetylation on the turnover of one plant NLR, Suppressor of NPR1, Constitutive 1 (SNC1), in Arabidopsis thaliana. Genetic and biochemical analyses of SNC1 uncovered its multilayered regulation by different N-terminal acetyltransferase (Nat) complexes. SNC1 exhibits a few distinct N-terminal isoforms generated through alternative initiation and N-terminal acetylation. Its first Met is acetylated by N-terminal acetyltransferase complex A (NatA), while the second Met is acetylated by N-terminal acetyltransferase complex B (NatB). Unexpectedly, the NatA-mediated acetylation serves as a degradation signal, while NatB-mediated acetylation stabilizes the NLR protein, thus revealing antagonistic N-terminal acetylation of a single protein substrate. Moreover, NatA also contributes to the turnover of another NLR, RESISTANCE TO P. syringae pv maculicola 1. The intricate regulation of protein stability by Nats is speculated to provide flexibility for the target protein in maintaining its homeostasis. PMID:25966763

  15. N-terminal amino acid sequences and some characteristics of fibrinolytic/hemorrhagic metalloproteinases purified from Bothrops jararaca venom.

    Science.gov (United States)

    Maruyama, Masugi; Sugiki, Masahiko; Anai, Keita; Yoshida, Etsuo

    2002-08-01

    We determined the N-terminal amino acid sequences of the fibrinolytic/hemorrhagic metalloproteinases (jararafibrases I, III and IV) purified from Bothrops jararaca venom. The N-terminal amino acid sequences of jararafibrase I and its degradation products were identical to those of jararhagin, another hemorrhagic metalloproteinase purified from the same snake venom. Together with enzymatic and immunological properties, we concluded that those two enzymes are identical. The N-terminal amino acid sequence of jararafibrase III was quite similar to C-type lectin isolated from Crotalus atrox, and the protein had a hemagglutinating activity on intact rat red blood cells. PMID:12165326

  16. Fully Automatic Trunk Packing with Free Placements

    CERN Document Server

    Althaus, Ernst

    2010-01-01

    We present a new algorithm to compute the volume of a trunk according to the SAE J1100 standard. Our new algorithm uses state-of-the-art methods from computational geometry and from combinatorial optimization. It finds better solutions than previous approaches for small trunks.

  17. Celiac trunk coverage in endovascular aneurysm repair

    DEFF Research Database (Denmark)

    Delle, Martin; Lönn, Lars; Henrikson, O;

    2010-01-01

    This retrospective study was undertaken to examine the risks associated with obstruction of the coeliac trunk in the process of treating aneurysms with endografting.......This retrospective study was undertaken to examine the risks associated with obstruction of the coeliac trunk in the process of treating aneurysms with endografting....

  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......-terminal cyclizations, cyclization of N-terminal glutamine and S-carbamoylmethylcysteine, it is dependent on pH instead of [NH(4)(+)]. The data set from our recent study on large-scale N(α)-modified peptides revealed a sequence requirement for the cyclization event similar to the well-known deamidation of Asn to iso......Asp and Asp. Detailed analysis using synthetic peptides confirmed that the cyclization forms between the N-terminus and its neighboring Asn residue, and the reaction shares the same succinimide intermediate with the Asn deamidation event. As a result, we, here, propose a molecular mechanism for this specific...

  19. Relationship between N-terminal pro-B-type natriuretic peptide levels and metabolic syndrome

    OpenAIRE

    Bao, Yuanyuan; Shang, Xiliang; Zhou, Linuo; Hu, Renming; Li, Yiming; Ding, Wei

    2011-01-01

    Introduction Previous studies have shown that obese individuals have reduced natriuretic peptide levels. But conflicting data exist on the relation of natriuretic peptide levels to other metabolic risk factors. Material and methods We investigated the relationship between plasma N-terminal pro-B-type natriuretic peptide levels (NT-proBNP) and metabolic syndrome (MetS) and metabolic risk factors in 469 patients free of heart failure. Two hundred thirty diagnosed MetS cases and 239 non-MetS cas...

  20. Activation of c-Jun N-terminal Kinases by Ribotoxic Stresses

    Institute of Scientific and Technical Information of China (English)

    Dong-Yun Ouyang; Yuan-Yuan Wang; Yong-Tang Zheng

    2005-01-01

    The c-Jun N-terminal kinases (JNKs) are classic stress-activated protein kinases. Many cellular stresses have been shown to stimulate JNK activation. In this review, we focus on ribotoxic stresses based on their multiple biological potencies including anti-HIV-1 activity. Some of the functions of ribotoxins and the signaling transduction pathway that mediated are mentioned. Different from other stimulators, ribotoxic stresses act on special motifs of 28S rRNA in translationally active mammal ribosomes. Binding and damaging on the motif leads to JNK activation and subsequently biological response to the signal initiator, which is named ribotoxic stress response.

  1. 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.

  2. Design, synthesis and aphicidal activity of N-terminal modified insect kinin analogs.

    Science.gov (United States)

    Zhang, Chuanliang; Qu, Yanyan; Wu, Xiaoqing; Song, Dunlun; Ling, Yun; Yang, Xinling

    2015-06-01

    The insect kinins are a class of multifunctional insect neuropeptides present in a diverse variety of insects. Insect kinin analogs showed multiple bioactivities, especially, the aphicidal activity. To find a biostable and bioactive insecticide candidate with simplified structure, a series of N-terminal modified insect kinin analogs was designed and synthesized based on the lead compound [Aib]-Phe-Phe-[Aib]-Trp-Gly-NH2. Their aphicidal activity against the soybean aphid Aphis glycines was evaluated. The results showed that all the analogs maintained the aphicidal activity. In particular, the aphicidal activity of the pentapeptide analog X Phe-Phe-[Aib]-Trp-Gly-NH2 (LC50=0.045mmol/L) was similar to the lead compound (LC50=0.048mmol/L). This indicated that the N-terminal protective group may not play an important role in the activity and the analogs structure could be simplified to pentapeptide analogs while retaining good aphicidal activity. The core pentapeptide analog X can be used as the lead compound for further chemical modifications to discover potential insecticides. PMID:25116632

  3. Plasma biomarker screening for liver fibrosis with the N-terminal isotope tagging strategy.

    Science.gov (United States)

    Li, ShuLong; Liu, Xin; Wei, Lai; Wang, HuiFen; Zhang, JiYang; Wei, HanDong; Qian, XiaoHong; Jiang, Ying; He, FuChu

    2011-05-01

    A non-invasive diagnostic approach is crucial for the evaluation of severity of liver disease, treatment decisions, and assessing drug efficacy. This study evaluated plasma proteomic profiling via an N-terminal isotope tagging strategy coupled with liquid chromatography/Fourier transform ion cyclotron resonance mass spectrometry measurement to detect liver fibrosis staging. Pooled plasma from different liver fibrosis stages, which were assessed in advance by the current gold-standard of liver biopsy, was quantitatively analyzed. A total of 72 plasma proteins were found to be dysregulated during the fibrogenesis process, and this finding constituted a valuable candidate plasma biomarker bank for follow-up analysis. Validation results of fibronectin by Western blotting reconfirmed the mass-based data. Ingenuity Pathways Analysis showed four types of metabolic networks for the functional effect of liver fibrosis disease in chronic hepatitis B patients. Consequently, quantitative proteomics via the N-terminal acetyl isotope labeling technique provides an effective and useful tool for screening plasma candidate biomarkers for liver fibrosis. We quantitatively monitored the fibrogenesis process in CHB patients. We discovered many new valuable candidate biomarkers for the diagnosis of liver fibrosis and also partly identified the mechanism involved in liver fibrosis disease. These results provide a clearer understanding of liver fibrosis pathophysiology and will also hopefully lead to improvement of clinical diagnosis and treatment.

  4. Recombinant N-Terminal Slit2 Inhibits TGF-β-Induced Fibroblast Activation and Renal Fibrosis.

    Science.gov (United States)

    Yuen, Darren A; Huang, Yi-Wei; Liu, Guang-Ying; Patel, Sajedabanu; Fang, Fei; Zhou, Joyce; Thai, Kerri; Sidiqi, Ahmad; Szeto, Stephen G; Chan, Lauren; Lu, Mingliang; He, Xiaolin; John, Rohan; Gilbert, Richard E; Scholey, James W; Robinson, Lisa A

    2016-09-01

    Fibrosis and inflammation are closely intertwined injury pathways present in nearly all forms of CKD for which few safe and effective therapies exist. Slit glycoproteins signaling through Roundabout (Robo) receptors have been described to have anti-inflammatory effects through regulation of leukocyte cytoskeletal organization. Notably, cytoskeletal reorganization is also required for fibroblast responses to TGF-β Here, we examined whether Slit2 also controls TGF-β-induced renal fibrosis. In cultured renal fibroblasts, which we found to express Slit2 and Robo-1, the bioactive N-terminal fragment of Slit2 inhibited TGF-β-induced collagen synthesis, actin cytoskeletal reorganization, and Smad2/3 transcriptional activity, but the inactive C-terminal fragment of Slit2 did not. In mouse models of postischemic renal fibrosis and obstructive uropathy, treatment with N-terminal Slit2 before or after injury inhibited the development of renal fibrosis and preserved renal function, whereas the C-terminal Slit2 had no effect. Our data suggest that administration of recombinant Slit2 may be a new treatment strategy to arrest chronic injury progression after ischemic and obstructive renal insults by not only attenuating inflammation but also, directly inhibiting renal fibrosis.

  5. Effect of N-Terminal Acylation on the Activity of Myostatin Inhibitory Peptides.

    Science.gov (United States)

    Takayama, Kentaro; Nakamura, Akari; Rentier, Cédric; Mino, Yusaku; Asari, Tomo; Saga, Yusuke; Taguchi, Akihiro; Yakushiji, Fumika; Hayashi, Yoshio

    2016-04-19

    Inhibition of myostatin, which negatively regulates skeletal muscle growth, is a promising strategy for the treatment of muscle atrophic disorders, such as muscular dystrophy, cachexia and sarcopenia. Recently, we identified peptide A (H-WRQNTRYSRIEAIKIQILSKLRL-NH2 ), the 23-amino-acid minimum myostatin inhibitory peptide derived from mouse myostatin prodomain, and highlighted the importance of its N-terminal tryptophan residue for the effective inhibition. In this study, we synthesized a series of acylated peptide derivatives focused on the tryptophan residue to develop potent myostatin inhibitors. As a result of the investigation, a more potent derivative of peptide A was successfully identified in which the N-terminal tryptophan residue is replaced with a 2-naphthyloxyacetyl moiety to give an inhibitory peptide three times (1.19±0.11 μm) more potent than parent peptide A (3.53±0.25 μm). This peptide could prove useful as a new starting point for the development of improved inhibitory peptides. PMID:26954624

  6. N-terminal palmitoylation is required for Toxoplasma gondii HSP20 inner membrane complex localization.

    Science.gov (United States)

    De Napoli, M G; de Miguel, N; Lebrun, M; Moreno, S N J; Angel, S O; Corvi, M M

    2013-06-01

    Toxoplasma gondii is an obligate intracellular parasite and the causative agent of toxoplasmosis. Protein palmitoylation is known to play roles in signal transduction and in enhancing the hydrophobicity of proteins thus contributing to their membrane association. Global inhibition of protein palmitoylation has been shown to affect T. gondii physiology and invasion of the host cell. However, the proteins affected by this modification have been understudied. This paper shows that the small heat shock protein 20 from T. gondii (TgHSP20) is synthesized as a mature protein in the cytosol and is palmitoylated in three cysteine residues. However, its localization at the inner membrane complex (IMC) is dependent only on N-terminal palmitoylation. Absence or incomplete N-terminal palmitoylation causes TgHSP20 to partially accumulate in a membranous structure. Interestingly, TgHSP20 palmitoylation is not responsible for its interaction with the daughter cells IMCs. Together, our data describe the importance of palmitoylation in protein targeting to the IMC in T. gondii.

  7. Role of the N-terminal seven residues of surfactant protein B (SP-B.

    Directory of Open Access Journals (Sweden)

    Mahzad Sharifahmadian

    Full Text Available Breathing is enabled by lung surfactant, a mixture of proteins and lipids that forms a surface-active layer and reduces surface tension at the air-water interface in lungs. Surfactant protein B (SP-B is an essential component of lung surfactant. In this study we probe the mechanism underlying the important functional contributions made by the N-terminal 7 residues of SP-B, a region sometimes called the "insertion sequence". These studies employed a construct of SP-B, SP-B (1-25,63-78, also called Super Mini-B, which is a 41-residue peptide with internal disulfide bonds comprising the N-terminal 7-residue insertion sequence and the N- and C-terminal helices of SP-B. Circular dichroism, solution NMR, and solid state (2H NMR were used to study the structure of SP-B (1-25,63-78 and its interactions with phospholipid bilayers. Comparison of results for SP-B (8-25,63-78 and SP-B (1-25,63-78 demonstrates that the presence of the 7-residue insertion sequence induces substantial disorder near the centre of the lipid bilayer, but without a major disruption of the overall mechanical orientation of the bilayers. This observation suggests the insertion sequence is unlikely to penetrate deeply into the bilayer. The 7-residue insertion sequence substantially increases the solution NMR linewidths, most likely due to an increase in global dynamics.

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

    International Nuclear Information System (INIS)

    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 35S-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

  9. Directed evolution of the TALE N-terminal domain for recognition of all 5' bases.

    Science.gov (United States)

    Lamb, Brian M; Mercer, Andrew C; Barbas, Carlos F

    2013-11-01

    Transcription activator-like effector (TALE) proteins can be designed to bind virtually any DNA sequence. General guidelines for design of TALE DNA-binding domains suggest that the 5'-most base of the DNA sequence bound by the TALE (the N0 base) should be a thymine. We quantified the N0 requirement by analysis of the activities of TALE transcription factors (TALE-TF), TALE recombinases (TALE-R) and TALE nucleases (TALENs) with each DNA base at this position. In the absence of a 5' T, we observed decreases in TALE activity up to >1000-fold in TALE-TF activity, up to 100-fold in TALE-R activity and up to 10-fold reduction in TALEN activity compared with target sequences containing a 5' T. To develop TALE architectures that recognize all possible N0 bases, we used structure-guided library design coupled with TALE-R activity selections to evolve novel TALE N-terminal domains to accommodate any N0 base. A G-selective domain and broadly reactive domains were isolated and characterized. The engineered TALE domains selected in the TALE-R format demonstrated modularity and were active in TALE-TF and TALEN architectures. Evolved N-terminal domains provide effective and unconstrained TALE-based targeting of any DNA sequence as TALE binding proteins and designer enzymes.

  10. Arm position influences the activation patterns of trunk muscles during trunk range-of-motion movements.

    Science.gov (United States)

    Siu, Aaron; Schinkel-Ivy, Alison; Drake, Janessa Dm

    2016-10-01

    To understand the activation patterns of the trunk musculature, it is also important to consider the implications of adjacent structures such as the upper limbs, and the muscles that act to move the arms. This study investigated the effects of arm positions on the activation patterns and co-activation of the trunk musculature and muscles that move the arm during trunk range-of-motion movements (maximum trunk axial twist, flexion, and lateral bend). Fifteen males and fifteen females, asymptomatic for low back pain, performed maximum trunk range-of-motion movements, with three arm positions for axial twist (loose, crossed, abducted) and two positions for flexion and lateral bend (loose, crossed). Electromyographical data were collected for eight muscles bilaterally, and activation signals were cross-correlated between trunk muscles and the muscles that move the arms (upper trapezius, latissimus dorsi). Results revealed consistently greater muscle co-activation (higher cross-correlation coefficients) between the trunk muscles and upper trapezius for the abducted arm position during maximum trunk axial twist, while results for the latissimus dorsi-trunk pairings were more dependent on the specific trunk muscles (either abdominal or back) and latissimus dorsi muscle (either right or left side), as well as the range-of-motion movement. The findings of this study contribute to the understanding of interactions between the upper limbs and trunk, and highlight the influence of arm positions on the trunk musculature. In addition, the comparison of the present results to those of individuals with back or shoulder conditions may ultimately aid in elucidating underlying mechanisms or contributing factors to those conditions.

  11. N-Terminal methionine processing by the zinc-activated Plasmodium falciparum methionine aminopeptidase 1b.

    Science.gov (United States)

    Calcagno, Sarah; Klein, Christian D

    2016-08-01

    The methionine aminopeptidase 1b from Plasmodium falciparum (PfMetAP 1b) was cloned, expressed in Escherichia coli and characterized. Surprisingly, and in contrast to other methionine aminopeptidases (MetAPs) that require heavy-metal cofactors such as cobalt, the enzyme is reliably activated by zinc ions. Immobilization of the enzyme is possible by His-tag metal chelation to iminodiacetic acid-agarose and by covalent binding to chloroacetamido-hexyl-agarose. The covalently immobilized enzyme shows long-term stability, allowing a continuous, heterogenous processing of N-terminal methionines, for example, in recombinant proteins. Activation by zinc, instead of cobalt as for other MetAPs, avoids the introduction of heavy metals with toxicological liabilities and oxidative potential into biotechnological processes. The PfMetAP 1b therefore represents a useful tool for the enzymatic, posttranslational processing of recombinant proteins. PMID:27023914

  12. Structural polymorphism in the N-terminal oligomerization domain of NPM1.

    Science.gov (United States)

    Mitrea, Diana M; Grace, Christy R; Buljan, Marija; Yun, Mi-Kyung; Pytel, Nicholas J; Satumba, John; Nourse, Amanda; Park, Cheon-Gil; Madan Babu, M; White, Stephen W; Kriwacki, Richard W

    2014-03-25

    Nucleophosmin (NPM1) is a multifunctional phospho-protein with critical roles in ribosome biogenesis, tumor suppression, and nucleolar stress response. Here we show that the N-terminal oligomerization domain of NPM1 (Npm-N) exhibits structural polymorphism by populating conformational states ranging from a highly ordered, folded pentamer to a highly disordered monomer. The monomer-pentamer equilibrium is modulated by posttranslational modification and protein binding. Phosphorylation drives the equilibrium in favor of monomeric forms, and this effect can be reversed by Npm-N binding to its interaction partners. We have identified a short, arginine-rich linear motif in NPM1 binding partners that mediates Npm-N oligomerization. We propose that the diverse functional repertoire associated with NPM1 is controlled through a regulated unfolding mechanism signaled through posttranslational modifications and intermolecular interactions.

  13. Partial N-terminal sequence analysis of human class II molecules expressing the DQw3 determinant.

    Science.gov (United States)

    Obata, F; Endo, T; Yoshii, M; Otani, F; Igarashi, M; Takenouchi, T; Ikeda, H; Ogasawara, K; Kasahara, M; Wakisaka, A

    1985-09-01

    HLA-DQ molecules were isolated from DRw9-homozygous and DR4-homozygous cell lines by using a monoclonal antibody HU-18, which recognizes class II molecules carrying the conventional DQw3 determinant. The partial N-terminal sequence analysis of the DQw3 molecules revealed that they have sequences homologous to those of murine I-A molecules. Within the limits of our sequence analysis, the DQw3 molecules from the two cell lines are identical to each other in both the alpha and beta chains. The DQ alpha as well as DQ beta chains were found to have amino acid substitutions when compared to other I-A-like molecules whose sequences have been reported. These differences may contribute to the DQw supertypic specificity. The polymorphic nature of DQ molecules is in marked contrast to that of DR molecules where DR alpha chains are highly conserved while DR beta chains have easily detectable amino acid substitutions. PMID:2411700

  14. Structure of the N-terminal domain of the metalloprotease PrtV from Vibrio cholerae.

    Science.gov (United States)

    Edwin, Aaron; Persson, Cecilia; Mayzel, Maxim; Wai, Sun Nyunt; Öhman, Anders; Karlsson, B Göran; Sauer-Eriksson, A Elisabeth

    2015-12-01

    The metalloprotease PrtV from Vibrio cholerae serves an important function for the ability of bacteria to invade the mammalian host cell. The protein belongs to the family of M6 proteases, with a characteristic zinc ion in the catalytic active site. PrtV constitutes a 918 amino acids (102 kDa) multidomain pre-pro-protein that undergoes several N- and C-terminal modifications to form a catalytically active protease. We report here the NMR structure of the PrtV N-terminal domain (residues 23-103) that contains two short α-helices in a coiled coil motif. The helices are held together by a cluster of hydrophobic residues. Approximately 30 residues at the C-terminal end, which were predicted to form a third helical structure, are disordered. These residues are highly conserved within the genus Vibrio, which suggests that they might be functionally important.

  15. Copper binding triggers compaction in N-terminal tail of human copper pump ATP7B.

    Science.gov (United States)

    Mondol, Tanumoy; Åden, Jörgen; Wittung-Stafshede, Pernilla

    2016-02-12

    Protein conformational changes are fundamental to biological reactions. For copper ion transport, the multi-domain protein ATP7B in the Golgi network receives copper from the cytoplasmic copper chaperone Atox1 and, with energy from ATP hydrolysis, moves the metal to the lumen for loading of copper-dependent enzymes. Although anticipated, conformational changes involved in ATP7B's functional cycle remain elusive. Using spectroscopic methods we here demonstrate that the four most N-terminal metal-binding domains in ATP7B, upon stoichiometric copper addition, adopt a more compact arrangement which has a higher thermal stability than in the absence of copper. In contrast to previous reports, no stable complex was found in solution between the metal-binding domains and the nucleotide-binding domain of ATP7B. Metal-dependent movement of the first four metal-binding domains in ATP7B may be a trigger that initiates the overall catalytic cycle.

  16. N-Terminal Truncation of TACO Inhibits PMA-Induced U937 Cell Adhesion

    Institute of Scientific and Technical Information of China (English)

    LIU Changzhen; SUI Senfang

    2005-01-01

    The effect of TACO1-299, the N-terminal truncation of TACO, on phorbol 12-myristate 13-acetate (PMA)-induced U937 cell adhesion was investigated. Full-length TACO and several truncations were overexpressed in U937 cells. The effects of the expressed proteins on U937 cell adhesion mediated by PMA-induced differentiation were observed by fluorescence microscopy. The results show that the overexpression of TACO1-299 inhibits cell adhesion while overexpressions of the other proteins do not have this effect. The actin-binding capability of TACO1-299 was investigated and the results show that TACO1-299 lacks the ability of TACO to bind F-actin. The inhibitive effect of TACO1-299, the functional domain of TACO, suggests that TACO may play a role in cell differentiation mediating adhesion of monoblastic leukemia cells.

  17. 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

    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......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......) 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...

  18. Site-Specific N-Terminal Labeling of Peptides and Proteins using Butelase 1 and Thiodepsipeptide.

    Science.gov (United States)

    Nguyen, Giang K T; Cao, Yuan; Wang, Wei; Liu, Chuan Fa; Tam, James P

    2015-12-21

    An efficient ligase with exquisite site-specificity is highly desirable for protein modification. Recently, we discovered the fastest known ligase called butelase 1 from Clitoria ternatea for intramolecular cyclization. For intermolecular ligation, butelase 1 requires an excess amount of a substrate to suppress the reverse reaction, a feature similar to other ligases. Herein, we describe the use of thiodepsipeptide substrates with a thiol as a leaving group and an unacceptable nucleophile to render the butelase-mediated ligation reactions irreversible and in high yields. Butelase 1 also accepted depsipeptides as substrates, but unlike a thiodesipeptide, the desipeptide ligation was partially reversible as butelase 1 can tolerate an alcohol group as a poor nucleophile. The thiodesipeptide method was successfully applied in N-terminal labeling of ubiquitin and green fluorescent protein using substrates with or without a biotin group in high yields. PMID:26563575

  19. Analysis of the secondary structure of a protein's N-terminal

    Science.gov (United States)

    Floare, C. G.; Bogdan, M.; Horovitz, O.; Mocanu, A.; Tomoaia-Cotisel, M.

    2009-08-01

    The major protein component from aleurone cells of barley (Hordeum vulgare L.), PACB, is related to 7S globulins present in other cereals and to the vicilin-type 7S globulins of legumes and cotton seed. It contains 4 subunits of about 20, 25, 40 and 50 kDa molecular weights. The N-terminal sequence of 16 amino acids (over 260 atoms) in the protein was previously determined, and our aim is the prediction of its secondary structure. The empirical Chou-Fasman method was applied in an improved version as well as the empirical DSC method (discrimination of protein secondary structure class) with quite similar results. A molecular dynamics simulation was also performed, using the FF99SB forcefield within AMBER version 9.0. Solvation effects were incorporated using the Born model. The results are compared and a 3D model is proposed.

  20. Analysis of the secondary structure of a protein's N-terminal

    Energy Technology Data Exchange (ETDEWEB)

    Floare, C G; Bogdan, M [National Institute for R and D of Isotopic and Molecular Technologies, 65-103 Donath, 400293 Cluj-Napoca (Romania); Horovitz, O; Mocanu, A; Tomoaia-Cotisel, M, E-mail: calin.floare@itim-cj.r [Babes-Bolyai University, Faculty of Chemistry and Chemical Engineering, Department of Physical Chemistry, 11 Arany Janos, 400028 Cluj-Napoca (Romania)

    2009-08-01

    The major protein component from aleurone cells of barley (Hordeum vulgare L.), PACB, is related to 7S globulins present in other cereals and to the vicilin-type 7S globulins of legumes and cotton seed. It contains 4 subunits of about 20, 25, 40 and 50 kDa molecular weights. The N-terminal sequence of 16 amino acids (over 260 atoms) in the protein was previously determined, and our aim is the prediction of its secondary structure. The empirical Chou-Fasman method was applied in an improved version as well as the empirical DSC method (discrimination of protein secondary structure class) with quite similar results. A molecular dynamics simulation was also performed, using the FF99SB forcefield within AMBER version 9.0. Solvation effects were incorporated using the Born model. The results are compared and a 3D model is proposed.

  1. 157 nm Photodissociation of Dipeptide Ions Containing N-Terminal Arginine

    Science.gov (United States)

    Webber, Nathaniel; He, Yi; Reilly, James P.

    2013-12-01

    Twenty singly-charged dipeptide ions with N-terminal arginine were photodissociated using 157 nm light in both a linear ion-trap mass spectrometer and a MALDI-TOF-TOF mass spectrometer. Analogous to previous work on dipeptides containing C-terminal arginine, this set of samples enabled insights into the photofragmentation propensities associated with individual residues. In addition to familiar products such as a-, d-, and immonium ions, m2 and m2+13 ions were also observed. Certain side chains tended to cleave between their β and γ carbons without necessarily forming d- or w-type ions, and a few other ions were produced by the high-energy fragmentation of multiple bonds.

  2. 47 CFR 69.112 - Direct-trunked transport.

    Science.gov (United States)

    2010-10-01

    ... 47 Telecommunication 3 2010-10-01 2010-10-01 false Direct-trunked transport. 69.112 Section 69.112... Computation of Charges § 69.112 Direct-trunked transport. (a) A flat-rated direct-trunked transport charge... other persons that use telephone company direct-trunked transport facilities. (b)(1) For...

  3. Reaction of the N-terminal methionine residues in cyanase with diethylpyrocarbonate.

    Science.gov (United States)

    Anderson, P M; Korte, J J; Holcomb, T A

    1994-11-29

    Cyanase is an inducible enzyme in Escherichia coli that catalyzes the reaction of cyanate with bicarbonate to give ammonia and carbon dioxide. The enzyme is a decamer of identical subunits (M(r) = 17,000). Previous studies have shown that modification of either the single cysteine residue or the single histidine residue in each subunit gives an active decameric derivative that dissociates reversibly to inactive dimer derivative, indicating that decameric structure is required for activity and that the SH and imidazole groups are not required for catalytic activity [Anderson, P. M., Korte, J. J., Holcomb, T. A., Cho, Y.-G., Son, C.-M., & Sung, Y.-C. (1994) J. Biol. Chem. 269, 15036-15045]. Here the effects of reaction of the reagent diethylpyrocarbonate (DEPC) with cyanase or mutant cyanases are reported. DEPC reacts stoichiometrically with the histidine residue and at one additional site in each subunit when the enzyme is in the inactive dimer form, preventing reactivation. DEPC reacts stoichiometrically (with the same result on reactivation) at only one site per subunit with the inactive dimer form of cyanase mutants in which the single histidine residue has been replaced by one of several different amino acids by site-directed mutagenesis; the site of the reaction was identified as the amino group of the N-terminal methionine. DEPC does not react with the histidine residue of the active decameric form of wild-type cyanase and does not affect activity of the active decameric form of wild-type or mutant cyanases. Reaction with the N-terminal amino group of methionine apparently prevents reactivation of the mutant enzymes by blocking association to decamer.(ABSTRACT TRUNCATED AT 250 WORDS)

  4. 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.

  5. 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.

  6. N-Terminal Presequence-Independent Import of Phosphofructokinase into Hydrogenosomes of Trichomonas vaginalis.

    Science.gov (United States)

    Rada, Petr; Makki, Abhijith Radhakrishna; Zimorski, Verena; Garg, Sriram; Hampl, Vladimír; Hrdý, Ivan; Gould, Sven B; Tachezy, Jan

    2015-12-01

    Mitochondrial evolution entailed the origin of protein import machinery that allows nuclear-encoded proteins to be targeted to the organelle, as well as the origin of cleavable N-terminal targeting sequences (NTS) that allow efficient sorting and import of matrix proteins. In hydrogenosomes and mitosomes, reduced forms of mitochondria with reduced proteomes, NTS-independent targeting of matrix proteins is known. Here, we studied the cellular localization of two glycolytic enzymes in the anaerobic pathogen Trichomonas vaginalis: PPi-dependent phosphofructokinase (TvPPi-PFK), which is the main glycolytic PFK activity of the protist, and ATP-dependent PFK (TvATP-PFK), the function of which is less clear. TvPPi-PFK was detected predominantly in the cytosol, as expected, while all four TvATP-PFK paralogues were imported into T. vaginalis hydrogenosomes, although none of them possesses an NTS. The heterologous expression of TvATP-PFK in Saccharomyces cerevisiae revealed an intrinsic capability of the protein to be recognized and imported into yeast mitochondria, whereas yeast ATP-PFK resides in the cytosol. TvATP-PFK consists of only a catalytic domain, similarly to "short" bacterial enzymes, while ScATP-PFK includes an N-terminal extension, a catalytic domain, and a C-terminal regulatory domain. Expression of the catalytic domain of ScATP-PFK and short Escherichia coli ATP-PFK in T. vaginalis resulted in their partial delivery to hydrogenosomes. These results indicate that TvATP-PFK and the homologous ATP-PFKs possess internal structural targeting information that is recognized by the hydrogenosomal import machinery. From an evolutionary perspective, the predisposition of ancient ATP-PFK to be recognized and imported into hydrogenosomes might be a relict from the early phases of organelle evolution.

  7. Conservation and antigenicity of N-terminal sequences of GP185 from different Plasmodium falciparum isolates.

    Science.gov (United States)

    Howard, R F; Ardeshir, F; Reese, R T

    1986-01-01

    Complementary DNA (cDNA) clones for GP185, a major antigenically diverse glycoprotein of Plasmodium falciparum, were isolated from a cDNA library of the Honduras I/CDC (Honduras I) isolate, and 1052 bp were sequenced. The expression of cDNA fragments in Escherichia coli using the vector pCQV2 allowed verification of the reading frame. This GP185 cDNA sequence, like the cDNA sequence for a homologous gene of the K1 isolate [Hall et al., Nature 311 (1984) 379-382], codes for a polypeptide which is truncated due to multiple, in-frame stop codons. This polypeptide corresponds to the N-terminal 15% of the proposed coding region of the GP185 gene [Holder et al., Nature 317 (1985) 270-273]. Comparison of the nucleotide sequences for the GP185 gene of Honduras I and five other isolates indicated that there are two areas of conserved DNA sequence, one of 310 bp (beginning 181 bp upstream from the proposed initiation codon) and the other of greater than or equal to 360 bp (located entirely within the coding region), separated by a region encoding isolate-specific tandem amino acid repeats. Rat antiserum was raised to a fusion protein derived from the conserved regions and the intervening repeat region of this Honduras I protein. This antiserum bound GP185 on immunoblots of the homologous Honduras I isolate and the heterologous K1 isolate, which has different tandem repeats. Serum from owl monkeys and humans previously infected with P. falciparum reacted with the fusion protein on immunoblots demonstrating that determinants in the N-terminal 15% of GP185 were immunogenic in infected individuals and suggesting that some of these sites are conserved among isolates.(ABSTRACT TRUNCATED AT 250 WORDS)

  8. 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.

  9. Sorting signals, N-terminal modifications and abundance of the chloroplast proteome.

    Directory of Open Access Journals (Sweden)

    Boris Zybailov

    Full Text Available Characterization of the chloroplast proteome is needed to understand the essential contribution of the chloroplast to plant growth and development. Here we present a large scale analysis by nanoLC-Q-TOF and nanoLC-LTQ-Orbitrap mass spectrometry (MS of ten independent chloroplast preparations from Arabidopsis thaliana which unambiguously identified 1325 proteins. Novel proteins include various kinases and putative nucleotide binding proteins. Based on repeated and independent MS based protein identifications requiring multiple matched peptide sequences, as well as literature, 916 nuclear-encoded proteins were assigned with high confidence to the plastid, of which 86% had a predicted chloroplast transit peptide (cTP. The protein abundance of soluble stromal proteins was calculated from normalized spectral counts from LTQ-Obitrap analysis and was found to cover four orders of magnitude. Comparison to gel-based quantification demonstrates that 'spectral counting' can provide large scale protein quantification for Arabidopsis. This quantitative information was used to determine possible biases for protein targeting prediction by TargetP and also to understand the significance of protein contaminants. The abundance data for 550 stromal proteins was used to understand abundance of metabolic pathways and chloroplast processes. We highlight the abundance of 48 stromal proteins involved in post-translational proteome homeostasis (including aminopeptidases, proteases, deformylases, chaperones, protein sorting components and discuss the biological implications. N-terminal modifications were identified for a subset of nuclear- and chloroplast-encoded proteins and a novel N-terminal acetylation motif was discovered. Analysis of cTPs and their cleavage sites of Arabidopsis chloroplast proteins, as well as their predicted rice homologues, identified new species-dependent features, which will facilitate improved subcellular localization prediction. No evidence

  10. Immobilization of the N-terminal helix stabilizes prefusion paramyxovirus fusion proteins.

    Science.gov (United States)

    Song, Albert S; Poor, Taylor A; Abriata, Luciano A; Jardetzky, Theodore S; Dal Peraro, Matteo; Lamb, Robert A

    2016-07-01

    Parainfluenza virus 5 (PIV5) is an enveloped, single-stranded, negative-sense RNA virus of the Paramyxoviridae family. PIV5 fusion and entry are mediated by the coordinated action of the receptor-binding protein, hemagglutinin-neuraminidase (HN), and the fusion protein (F). Upon triggering by HN, F undergoes an irreversible ATP- and pH-independent conformational change, going down an energy gradient from a metastable prefusion state to a highly stable postfusion state. Previous studies have highlighted key conformational changes in the F-protein refolding pathway, but a detailed understanding of prefusion F-protein metastability remains elusive. Here, using two previously described F-protein mutations (S443D or P22L), we examine the capacity to modulate PIV5 F stability and the mechanisms by which these point mutants act. The S443D mutation destabilizes prefusion F proteins by disrupting a hydrogen bond network at the base of the F-protein globular head. The introduction of a P22L mutation robustly rescues destabilized F proteins through a local hydrophobic interaction between the N-terminal helix and a hydrophobic pocket. Prefusion stabilization conferred by a P22L-homologous mutation is demonstrated in the F protein of Newcastle disease virus, a paramyxovirus of a different genus, suggesting a conserved stabilizing structural element within the paramyxovirus family. Taken together, the available data suggest that movement of the N-terminal helix is a necessary early step for paramyxovirus F-protein refolding and presents a novel target for structure-based drug design. PMID:27335462

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

    Institute of Scientific and Technical Information of China (English)

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

    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.

  12. 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.

  13. Heritability assessment of cartilage metabolism. A twin study on circulating procollagen IIA N-terminal propeptide (PIIANP)

    DEFF Research Database (Denmark)

    Munk, H L; Svendsen, A J; Hjelmborg, J V B;

    2014-01-01

    OBJECTIVE: The aim of this investigation was to estimate the heritability of circulating collagen IIA N-terminal propeptide (PIIANP) by studying mono- and dizygotic healthy twin pairs at different age and both genders. DESIGN: 598 monozygotic (MZ) and dizygotic (DZ) twin individuals aged 18...... the collagen IIA synthesis as assessed by the collagen IIA N-terminal propeptide in serum is attributable to genetic effectors while individual and shared environment account for 24% and 31% respectively. The heritability does not differ between genders or according to age....

  14. Arthropods vector grapevine trunk disease pathogens.

    Science.gov (United States)

    Moyo, P; Allsopp, E; Roets, F; Mostert, L; Halleen, F

    2014-10-01

    Arthropod-mediated dispersal of pathogens is known in many cropping systems but has never been demonstrated for grapevine trunk disease pathogens. Arthropods from vineyards were screened for the presence of pathogens associated with Petri disease and esca using cultural and molecular techniques. The ability of the most abundant pathogen-carrying species to inoculate healthy grapevine vascular tissues was also determined. Millipedes and ants were allowed to associate with a DsRed- Express-transformed Phaeomoniella chlamydospora, after which they were exposed to freshly pruned healthy grapevines under controlled conditions and wounds were monitored for subsequent infection. In addition, the possibility of millipede excreta, commonly found on pruning wounds in the field, to act as inoculum source was determined. A diverse arthropod fauna was associated with declining grapevines and many of these carried trunk disease pathogens. However, spiders, the ant Crematogaster peringueyi, and the millipede Ommattoiulus moreleti were the most abundant pathogen carriers. The ant and millipede species fed on pruning wound sap and effectively transmitted trunk disease pathogens. Millipede excreta contained viable spores of Phaeomoniella chlamydospora and may serve as an inoculum source. Numerous arthropods, including beneficial predators, are potential vectors of grapevine trunk disease pathogens. Our results highlight the need for an integrated approach, including targeted management of ants and millipedes at the time of pruning, to limit the spread of grapevine trunk diseases. PMID:24624953

  15. [Variations of the celiac trunk branches in the fetus].

    Science.gov (United States)

    Bordei, P; Antohe, D S

    2002-09-01

    The study was performed on 60 human foetuses, aged between 4 to 9 months, using as methods dissection and plastic and contrast substances injection. We studied the celiac trunk in what concerns the division into its terminal branches, insisting on the possible morphological variations, some rare collateral branches starting from the common arterial trunk, the dimensional relations between the branches at their origin and the level of the celiac trunk origin from the aorta, in relation with the vertebral column, the diaphragmatic passage of the aorta and with the superior mesenteric artery. We also assessed the dimensional relations (calibers at origin) between the branches of the celiac trunk. Ass possible variations of the division of the celiac trunk, we assessed: gastro-hepatic trunk, with the splenic artery directly from the aorta or from the hepatic artery; gastro-splenic trunk, with the hepatic artery originating from the aorta; hepato-splenic trunk, with origin of the left gastric artery either directly from the aorta or from the hepatic artery. Rare variations: celiaco-mesenteric trunk; two arterial trunks, hepato-splenic and hepato-gastric; separate aortic origin for all three "classic" branches of the celiac trunk; two hepatic arteries, one from the celiac trunk and the other from the aorta or superior mesenteric artery; celiac trunk that divides into several terminal branches; one or two suprarenal arteries originating from the celiac trunk. PMID:12572348

  16. BETA-N-TERMINAL GLYCOHEMOGLOBINS IN SUBJECTS WITH COMMON HEMOGLOBINOPATHIES - RELATION WITH FRUCTOSAMINE AND MEAN ERYTHROCYTE AGE

    NARCIS (Netherlands)

    MARTINA, WV; MARTIJN, EG; VANDERMOLEN, M; SCHERMER, JG; MUSKIET, FAJ

    1993-01-01

    Amounts of beta-N-terminal glycohemoglobins (HbX1c), serum fructosamine, and erythrocyte polyamines were determined in nondiabetic adults with HbAA, HbAC, HbAS, HbCC, HbSC, HbSS, and HbS/hereditary persistent HbF (HPFH). The groups did not differ in fructosamine concentrations. Mean (95% confidence

  17. 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...

  18. Dimeric structure of the N-terminal domain of PriB protein from Thermoanaerobacter tengcongensis solved ab initio.

    Science.gov (United States)

    Liebschner, Dorothee; Brzezinski, Krzysztof; Dauter, Miroslawa; Dauter, Zbigniew; Nowak, Marta; Kur, Józef; Olszewski, Marcin

    2012-12-01

    PriB is one of the components of the bacterial primosome, which catalyzes the reactivation of stalled replication forks at sites of DNA damage. The N-terminal domain of the PriB protein from the thermophilic bacterium Thermoanaerobacter tengcongensis (TtePriB) was expressed and its crystal structure was solved at the atomic resolution of 1.09 Å by direct methods. The protein chain, which encompasses the first 104 residues of the full 220-residue protein, adopts the characteristic oligonucleotide/oligosaccharide-binding (OB) structure consisting of a five-stranded β-barrel filled with hydrophobic residues and equipped with four loops extending from the barrel. In the crystal two protomers dimerize, forming a six-stranded antiparallel β-sheet. The structure of the N-terminal OB domain of T. tengcongensis shows significant differences compared with mesophile PriBs. While in all other known structures of PriB a dimer is formed by two identical OB domains in separate chains, TtePriB contains two consecutive OB domains in one chain. However, sequence comparison of both the N-terminal and the C-terminal domains of TtePriB suggests that they have analogous structures and that the natural protein possesses a structure similar to a dimer of two N-terminal domains.

  19. Fine tuning of the catalytic activity of colicin e7 nuclease domain by systematic n-terminal mutations

    DEFF Research Database (Denmark)

    Németh, Eszter; Körtvélyesi, Tamás; Thulstrup, Peter W.;

    2014-01-01

    The nuclease domain of colicin E7 (NColE7) promotes the nonspecific cleavage of nucleic acids at its C-terminal HNH motif. Interestingly, the deletion of four N-terminal residues (446–449NColE75KRNK) resulted in complete loss of the enzyme activity. R447A mutation was reported to decrease the nuc...

  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. 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...

  2. 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 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...

  3. 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.

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

    DEFF Research Database (Denmark)

    Feng, Tao; Nyffenegger, Christian; Højrup, Peter;

    2014-01-01

    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...

  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......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 inserted and is therefore a candidate motif to participate in interactions with other bilayers or monolayers. In the present work, we have detected intrinsic ability of a peptide based on the sequence of the N-terminal segment of SP-C to interact and insert spontaneously into preformed zwitterionic....... These results demonstrate that the sequence of the SP-C N-terminal region has intrinsic ability to interact with, insert into, and perturb the structure of zwitterionic and anionic phospholipid films, even in the absence of the palmitic chains attached to this segment in the native protein. This effect has been...

  6. Improved recovery of proteome-informative, protein N-terminal peptides by combined fractional diagonal chromatography (COFRADIC).

    Science.gov (United States)

    Staes, An; Van Damme, Petra; Helsens, Kenny; Demol, Hans; Vandekerckhove, Joël; Gevaert, Kris

    2008-04-01

    We previously described a proteome-wide, peptide-centric procedure for sorting protein N-terminal peptides and used these peptides as readouts for protease degradome and xenoproteome studies. This procedure is part of a repertoire of gel-free techniques known as COmbined FRActional DIagonal Chromatography (COFRADIC) and highly enriches for alpha-amino-blocked peptides, including alpha-amino-acetylated protein N-terminal peptides. Here, we introduce two additional steps that significantly increase the fraction of such proteome-informative, N-terminal peptides: strong cation exchange (SCX) segregation of alpha-amino-blocked and alpha-amino-free peptides and an enzymatic step liberating pyroglutamyl peptides for 2,4,6-trinitrobenzenesulphonic acid (TNBS) modification and thus COFRADIC sorting. The SCX step reduces the complexity of the analyte mixture by enriching N-terminal peptides and depleting alpha-amino-free internal peptides as well as proline-starting peptides prior to COFRADIC. The action of pyroglutamyl aminopeptidases prior to the first COFRADIC peptide separation results in greatly diminishing numbers of contaminating pyroglutamyl peptides in peptide maps. We further show that now close to 95% of all COFRADIC-sorted peptides are alpha-amino-acetylated and, using the same amount of starting material, our novel procedure leads to an increased number of protein identifications.

  7. Functional characterization of a special thermophilic multifunctional amylase OPMA-N and its N-terminal domain

    Institute of Scientific and Technical Information of China (English)

    Fan Li; Xuejun Zhu; Yanfei Li; Hao Cao; Yingjiu Zhang

    2011-01-01

    A gene encoding a special thermophilic muitifunetional amylase OPMA-N was cloned from Bacillus sp. ZW25311. OPMA-N has an additional 124-residue N-terminal domain compared with typical amylases and forms a relatively independent domain with a IS-pleated sheet and random coil structure. Here we reported an unusual substrate and product specificities of OPMA-N and the impact of the additional N-terminal domain (1-124 aa) on the function and properties of OPMA-N. Both OPMAN (12.82 U/mg) and its N-terminal domain-truncated AOPMA-N (12.55 U/mg) only degraded starch to produce oligosaccharides including maltose, maltotriose, isomaitotriose, and isomaitotetraose, but not to produce glucose. Therefore, the N-terminal domain did not determine its substrate and product specificities that were probably regulated by its C-terminal IS-pleated sheet structure. However, the N-terminal domain of OPMA-N seemed to modulate its catalytic feature, leading to the production of more isomaitotriose and less maltose, and it seemed to contribute to OPMA-N's thermostability since OPMA-N showed higher activity than AOPMA-N in a temperature range from 40 to 80~C and the halflife (tl) was 5 h for OPMA-N and 2 h for AOPMA-N at 60~C. Both OPMA-N and AOPMA-N were Ca-independent, but their activities could be influenced by Cu2+, Niz+, Zn2+, EDTA, SDS (1 mM), or Triton-X100 (1%). Kinetic analysis and starch-adsorption assay indicated that the N-terminal domain of OPMA-N could increase the OPMA-N-starch binding and subsequently increase the catalytic efficiency of OPMA-N for starch. In particular, the N-terminal domain of OPMA-N did not determine its oligomerization, because both OPMA-N and AOPMA-N could exist in the forms of monomer, homodimer, and homooligomer at the same time.

  8. PrP N-terminal domain triggers PrPSc-like aggregation of Dpl

    International Nuclear Information System (INIS)

    Transmissible spongiform encephalopathies are fatal neurodegenerative disorders thought to be transmitted by self-perpetuating conformational conversion of a neuronal membrane glycoprotein (PrPC, for 'cellular prion protein') into an abnormal state (PrPSc, for 'scrapie prion protein'). Doppel (Dpl) is a protein that shares significant biochemical and structural homology with PrPC. In contrast to its homologue PrPC, Dpl is unable to participate in prion disease progression or to achieve an abnormal PrPSc-like state. We have constructed a chimeric mouse protein, composed of the N-terminal domain of PrPC (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 α-helical monomer forms soluble β-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

  9. N-terminal peptides from unprocessed prion proteins enter cells by macropinocytosis

    International Nuclear Information System (INIS)

    A peptide derived from the N-terminus of the unprocessed bovine prion protein (bPrPp), incorporating the hydrophobic signal sequence (residues 1-24) and a basic domain (KKRPKP, residues 25-30), internalizes into mammalian cells, even when coupled to a sizeable cargo, and therefore functions as a cell-penetrating peptide (CPP). Confocal microscopy and co-localization studies indicate that the internalization of bPrPp is mainly through macropinocytosis, a fluid-phase endocytosis process, initiated by binding to cell-surface proteoglycans. Electron microscopy studies show internalized bPrPp-DNA-gold complexes residing in endosomal vesicles. bPrPp induces expression of a complexed luciferase-encoding DNA plasmid, demonstrating the peptide's ability to transport the cargo across the endosomal membrane and into the cytosol and nucleus. The novel CPP activity of the unprocessed N-terminal domain of PrP could be important for the retrotranslocation of partly processed PrP and for PrP trafficking inside or between cells, with implications for the infectivity associated with prion diseases

  10. Jun N-Terminal Protein Kinase Enhances Middle Ear Mucosal Proliferation during Bacterial Otitis Media▿

    Science.gov (United States)

    Furukawa, Masayuki; Ebmeyer, 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 paralleled mucosal hyperplasia in this in vivo rat model. Nuclear JNK phosphorylation was observed in many cells of both the mucosal epithelium and stroma by immunohistochemistry. In an in vitro model of primary rat middle ear mucosal explants, bacterially induced mucosal growth was blocked by the Rac/Cdc42 inhibitor Clostridium difficile toxin B, the mixed-lineage kinase inhibitor CEP11004, and the JNK inhibitor SP600125. Finally, the JNK inhibitor SP600125 significantly inhibited mucosal hyperplasia during in vivo bacterial otitis media in guinea pigs. Inhibition of JNK in vivo resulted in a diminished proliferative response, as shown by a local decrease in proliferating cell nuclear antigen protein expression by immunohistochemistry. We conclude that activation of JNK is a critical pathway for bacterially induced mucosal hyperplasia during otitis media, influencing tissue proliferation. PMID:17325051

  11. The N-terminal 33 amino acid domain of Siva-1 is sufficient for nuclear localization

    International Nuclear Information System (INIS)

    Siva-1 induces apoptosis in multiple pathological processes and plays an important role in the suppression of tumor metastasis, protein degradation, and other functions. Although many studies have demonstrated that Siva-1 functions in the cytoplasm, a few have found that Siva-1 can relocate to the nucleus. In this study, we found that the first 33 amino acid residues of Siva-1 are required for its nuclear localization. Further study demonstrated that the green fluorescent protein can be imported into the nucleus after fusion with these 33 amino acid residues. Other Siva-1 regions and domains showed less effect on Siva-1 nuclear localization. By site-mutagenesis of all of these 33 amino acid residues, we found that mutants of the first 1-18 amino acids affected Siva-1 nuclear compartmentalization but could not complete this localization independently. In summary, we demonstrated that the N-terminal 33 amino acid residues were sufficient for Siva-1 nuclear localization, but the mechanism of this translocation needs additional investigation

  12. Transcription-dependent nuclear localization of DAZAP1 requires an N-terminal signal

    Energy Technology Data Exchange (ETDEWEB)

    Lin, Yi-Tzu; Wen, Wan-Ching [Institute of Biomedical Sciences, Academia Sinica, Taipei 11529, Taiwan (China); Yen, Pauline H., E-mail: pyen@ibms.sinica.edu.tw [Institute of Biomedical Sciences, Academia Sinica, Taipei 11529, Taiwan (China)

    2012-11-23

    Highlights: Black-Right-Pointing-Pointer DAZAP1 shuttles between the nucleus and the cytoplasm. Black-Right-Pointing-Pointer DAZAP1 accumulates in the cytoplasm when the nuclear transcription is inhibited. Black-Right-Pointing-Pointer DAZAP1's transcription-dependent nuclear localization requires N-terminal N42. Black-Right-Pointing-Pointer SLIRP binds to N42 and may be involved in the process. -- Abstract: Deleted in Azoospermia Associated Protein 1 (DAZAP1) is a ubiquitous hnRNP protein required for normal development and spermatogenesis. It resides predominantly in the nucleus and moves between the nucleus and the cytoplasm via a ZNS shuttling signal at its C-terminus. DAZAP1 accumulates in the cytoplasm when RNA polymerase II activity is inhibited by actinomycin D. Here we report the mapping of a 42-amino acid segment (N42) at the N-terminus of DAZAP1 that is both necessary and sufficient for its transcription-dependent nuclear localization. In addition, using a yeast two-hybrid system, we have identified SLIRP as a N42-binding protein which may regulate DAZAP1 subcellular localization.

  13. 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

    2016-04-13

    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.

  14. 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.

  15. Serum type III procollagen N-terminal peptide in coal miners.

    Science.gov (United States)

    Janssen, Y M; Engelen, J J; Giancola, M S; Low, R B; Vacek, P; Borm, P J

    1992-01-01

    Health surveillance of workers exposed to fibrogenic agents ideally should identify individuals at risk or detect pulmonary fibrosis in preclinical stages. We investigated serum procollagen type III N-terminal peptide (PIIIP) in several groups of active miners and in a nondust-exposed control group. The purpose of this study was to determine the applicability of PIIIP as an early noninvasive marker of pulmonary fibrosis in workers exposed to coal mine dust. PIIIP levels were significantly elevated in miners without radiological signs of coal workers pneumoconiosis (CWP) as compared with the nonexposed controls. However, in coal miners with CWP beyond ILO classification 1/0, PIIIP levels were not significantly different from nondust-exposed controls. Trend analysis within the miners group indicated a decrease in PIIIP levels with progression of the fibrosis. Our data suggest that detection of early lung fibrosis by measuring serum PIIIP values may be more sensitive than radiological diagnosis of CWP. However, follow-up of the control miners with respect to serum PIIIP and chest radiography is essential to validate PIIIP as a biological marker for CWP. PMID:1572317

  16. PLC-δ1-Lf, a novel N-terminal extended phospholipase C-δ1.

    Science.gov (United States)

    Kim, Na Young; Ahn, Sang Jung; Kim, Moo-Sang; Seo, Jung Soo; Kim, Bo Seong; Bak, Hye Jin; Lee, Jin Young; Park, Myoung-Ae; Park, Ju Hyeon; Lee, Hyung Ho; Chung, Joon Ki

    2013-10-10

    Phospholipase C-δ (PLC-δ), a key enzyme in phosphoinositide turnover, is involved in a variety of physiological functions. The widely expressed PLC-δ1 isoform is the best characterized and the most well understood phospholipase family member. However, the functional and molecular mechanisms of PLC-δ1 remain obscure. Here, we identified that the N-terminal region of mouse PLC-δ1 gene has two variants, a novel alternative splicing form, named as long form (mPLC-δ1-Lf) and the previously reported short form (mPLC-δ1-Sf), having exon 2 and exon 1, respectively, while both the gene variants share exons 3-16 for RNA transcription. Furthermore, the expression, identification and enzymatic characterization of the two types of PLC-δ1 genes were compared. Expression of mPLC-δ1-Lf was found to be tissue specific, whereas mPLC-δ1-Sf was widely distributed. The recombinant mPLC-δ1-Sf protein exhibited higher activity than recombinant mPLC-δ1-Lf protein. Although, the general catalytic and regulatory properties of mPLC-δ1-Lf are similar to those of PLC-δ1-Sf isozyme, the mPLC-δ1-Lf showed some distinct regulatory properties, such as tissue-specific expression and lipid binding specificity, particularly for phosphatidylserine.

  17. The N-terminal 33 amino acid domain of Siva-1 is sufficient for nuclear localization

    Energy Technology Data Exchange (ETDEWEB)

    Chen, J.Y.; Yang, L.X. [Institute of Human Virology, Zhongshan School of Medicine, Sun Yat-sen University, Guangzhou (China); Huang, Z.F. [Institute of Human Virology, Zhongshan School of Medicine, Sun Yat-sen University, Guangzhou (China); Department of Biochemistry, Zhongshan School of Medicine, Sun Yat-sen University, Guangzhou (China); Key Laboratory of Tropical Diseases Control, Sun Yat-sen University, Ministry of Education in China, Guangzhou (China)

    2013-12-02

    Siva-1 induces apoptosis in multiple pathological processes and plays an important role in the suppression of tumor metastasis, protein degradation, and other functions. Although many studies have demonstrated that Siva-1 functions in the cytoplasm, a few have found that Siva-1 can relocate to the nucleus. In this study, we found that the first 33 amino acid residues of Siva-1 are required for its nuclear localization. Further study demonstrated that the green fluorescent protein can be imported into the nucleus after fusion with these 33 amino acid residues. Other Siva-1 regions and domains showed less effect on Siva-1 nuclear localization. By site-mutagenesis of all of these 33 amino acid residues, we found that mutants of the first 1-18 amino acids affected Siva-1 nuclear compartmentalization but could not complete this localization independently. In summary, we demonstrated that the N-terminal 33 amino acid residues were sufficient for Siva-1 nuclear localization, but the mechanism of this translocation needs additional investigation.

  18. 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.

    2016-01-01

    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. PMID:27072897

  19. Structure and function of the N-terminal domain of the human mitochondrial calcium uniporter.

    Science.gov (United States)

    Lee, Youngjin; Min, Choon Kee; Kim, Tae Gyun; Song, Hong Ki; Lim, Yunki; Kim, Dongwook; Shin, Kahee; Kang, Moonkyung; Kang, Jung Youn; Youn, Hyung-Seop; Lee, Jung-Gyu; An, Jun Yop; Park, Kyoung Ryoung; Lim, Jia Jia; Kim, Ji Hun; Kim, Ji Hye; Park, Zee Yong; Kim, Yeon-Soo; Wang, Jimin; Kim, Do Han; Eom, Soo Hyun

    2015-10-01

    The mitochondrial calcium uniporter (MCU) is responsible for mitochondrial calcium uptake and homeostasis. It is also a target for the regulation of cellular anti-/pro-apoptosis and necrosis by several oncogenes and tumour suppressors. Herein, we report the crystal structure of the MCU N-terminal domain (NTD) at a resolution of 1.50 Å in a novel fold and the S92A MCU mutant at 2.75 Å resolution; the residue S92 is a predicted CaMKII phosphorylation site. The assembly of the mitochondrial calcium uniporter complex (uniplex) and the interaction with the MCU regulators such as the mitochondrial calcium uptake-1 and mitochondrial calcium uptake-2 proteins (MICU1 and MICU2) are not affected by the deletion of MCU NTD. However, the expression of the S92A mutant or a NTD deletion mutant failed to restore mitochondrial Ca(2+) uptake in a stable MCU knockdown HeLa cell line and exerted dominant-negative effects in the wild-type MCU-expressing cell line. These results suggest that the NTD of MCU is essential for the modulation of MCU function, although it does not affect the uniplex formation.

  20. NMR assignments of the N-terminal domain of Ogataea polymorpha telomerase reverse transcriptase.

    Science.gov (United States)

    Polshakov, Vladimir I; Petrova, Olga A; Parfenova, Yulia Yu; Efimov, Sergey V; Klochkov, Vladimir V; Zvereva, Maria I; Dontsova, Olga A

    2016-04-01

    Telomerase is a ribonucleoprotein enzyme that adds telomeric DNA fragments to the ends of chromosomes. This enzyme is the focus of substantial attention, both because its structure and mechanism of action are still poorly studied, and because of its pivotal roles in aging and cellular proliferation. The use of telomerase as a potential target for the design of new anticancer drugs is also of great interest. The catalytic protein subunit of telomerase (TERT) contains an N-terminal domain (TEN) that is essential for activity and processivity. Elucidation of the structure and dynamics of TEN in solution is important for understanding the molecular mechanism of telomerase activity and for the design of new telomerase inhibitors. To approach this problem, in this study we report the (1)H, (13)C, and (15)N chemical shift assignments of TEN from Ogataea polymorpha. Analysis of the assigned chemical shifts allowed us to identify secondary structures and protein regions potentially involved in interaction with other participants of the telomerase catalytic cycle. PMID:26721464

  1. 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.

  2. Cdc13 N-Terminal Dimerization DNA Binding and Telomere Length Regulation

    Energy Technology Data Exchange (ETDEWEB)

    M Mitchell; J Smith; M Mason; S Harper; D Speicher; F Johnson; E Skordalakes

    2011-12-31

    The essential yeast protein Cdc13 facilitates chromosome end replication by recruiting telomerase to telomeres, and together with its interacting partners Stn1 and Ten1, it protects chromosome ends from nucleolytic attack, thus contributing to genome integrity. Although Cdc13 has been studied extensively, the precise role of its N-terminal domain (Cdc13N) in telomere length regulation remains unclear. Here we present a structural, biochemical, and functional characterization of Cdc13N. The structure reveals that this domain comprises an oligonucleotide/oligosaccharide binding (OB) fold and is involved in Cdc13 dimerization. Biochemical data show that Cdc13N weakly binds long, single-stranded, telomeric DNA in a fashion that is directly dependent on domain oligomerization. When introduced into full-length Cdc13 in vivo, point mutations that prevented Cdc13N dimerization or DNA binding caused telomere shortening or lengthening, respectively. The multiple DNA binding domains and dimeric nature of Cdc13 offer unique insights into how it coordinates the recruitment and regulation of telomerase access to the telomeres.

  3. 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.

  4. 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

    2016-01-01

    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. PMID:27072897

  5. 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.

  6. Structural insights into the human RyR2 N-terminal region involved in cardiac arrhythmias

    Energy Technology Data Exchange (ETDEWEB)

    Borko, Ľubomír; Bauerová-Hlinková, Vladena, E-mail: vladena.bauerova@savba.sk; Hostinová, Eva; Gašperík, Juraj [Institute of Molecular Biology, Slovak Academy of Sciences, Dúbravská cesta 21, 845 51 Bratislava (Slovakia); Beck, Konrad [Cardiff University School of Dentistry, Heath Park, Cardiff CF14 4XY Wales (United Kingdom); Lai, F. Anthony [Cardiff University School of Medicine, Cardiff CF14 4XN Wales (United Kingdom); Zahradníková, Alexandra, E-mail: vladena.bauerova@savba.sk [Institute of Molecular Biology, Slovak Academy of Sciences, Dúbravská cesta 21, 845 51 Bratislava (Slovakia); Institute of Molecular Physiology and Genetics, Slovak Academy of Sciences, Vlárska 5, 833 34 Bratislava (Slovakia); Ševčík, Jozef, E-mail: vladena.bauerova@savba.sk [Institute of Molecular Biology, Slovak Academy of Sciences, Dúbravská cesta 21, 845 51 Bratislava (Slovakia)

    2014-11-01

    X-ray and solution structures of the human RyR2 N-terminal region were obtained under near-physiological conditions. The structure exhibits a unique network of interactions between its three domains, revealing an important stabilizing role of the central helix. Human ryanodine receptor 2 (hRyR2) mediates calcium release from the sarcoplasmic reticulum, enabling cardiomyocyte contraction. The N-terminal region of hRyR2 (amino acids 1–606) is the target of >30 arrhythmogenic mutations and contains a binding site for phosphoprotein phosphatase 1. Here, the solution and crystal structures determined under near-physiological conditions, as well as a homology model of the hRyR2 N-terminal region, are presented. The N-terminus is held together by a unique network of interactions among its three domains, A, B and C, in which the central helix (amino acids 410–437) plays a prominent stabilizing role. Importantly, the anion-binding site reported for the mouse RyR2 N-terminal region is notably absent from the human RyR2. The structure concurs with the differential stability of arrhythmogenic mutations in the central helix (R420W, I419F and I419F/R420W) which are owing to disparities in the propensity of mutated residues to form energetically favourable or unfavourable contacts. In solution, the N-terminus adopts a globular shape with a prominent tail that is likely to involve residues 545–606, which are unresolved in the crystal structure. Docking the N-terminal domains into cryo-electron microscopy maps of the closed and open RyR1 conformations reveals C{sup α} atom movements of up to 8 Å upon channel gating, and predicts the location of the leucine–isoleucine zipper segment and the interaction site for spinophilin and phosphoprotein phosphatase 1 on the RyR surface.

  7. Dimeric structure of the N-terminal domain of PriB protein from Thermoanaerobacter tengcongensis solved ab initio

    Energy Technology Data Exchange (ETDEWEB)

    Liebschner, Dorothee [National Cancer Institute, Argonne National Laboratory, Argonne, IL 60439 (United States); Brzezinski, Krzysztof [National Cancer Institute, Argonne National Laboratory, Argonne, IL 60439 (United States); University of Bialystok, 15-399 Bialystok (Poland); Dauter, Miroslawa [Argonne National Laboratory, Argonne, IL 60439 (United States); Dauter, Zbigniew, E-mail: dauter@anl.gov [National Cancer Institute, Argonne National Laboratory, Argonne, IL 60439 (United States); Nowak, Marta; Kur, Józef; Olszewski, Marcin, E-mail: dauter@anl.gov [Gdansk University of Technology, 80-952 Gdansk (Poland); National Cancer Institute, Argonne National Laboratory, Argonne, IL 60439 (United States)

    2012-12-01

    The N-terminal domain of the PriB protein from the thermophilic bacterium T. tengcongensis (TtePriB) was expressed and its crystal structure has been solved at the atomic resolution of 1.09 Å by direct methods. PriB is one of the components of the bacterial primosome, which catalyzes the reactivation of stalled replication forks at sites of DNA damage. The N-terminal domain of the PriB protein from the thermophilic bacterium Thermoanaerobacter tengcongensis (TtePriB) was expressed and its crystal structure was solved at the atomic resolution of 1.09 Å by direct methods. The protein chain, which encompasses the first 104 residues of the full 220-residue protein, adopts the characteristic oligonucleotide/oligosaccharide-binding (OB) structure consisting of a five-stranded β-barrel filled with hydrophobic residues and equipped with four loops extending from the barrel. In the crystal two protomers dimerize, forming a six-stranded antiparallel β-sheet. The structure of the N-terminal OB domain of T. tengcongensis shows significant differences compared with mesophile PriBs. While in all other known structures of PriB a dimer is formed by two identical OB domains in separate chains, TtePriB contains two consecutive OB domains in one chain. However, sequence comparison of both the N-terminal and the C-terminal domains of TtePriB suggests that they have analogous structures and that the natural protein possesses a structure similar to a dimer of two N-terminal domains.

  8. Production and applications of an N-terminally-truncated recombinant beta-haemolysin from Staphylococcus aureus.

    Science.gov (United States)

    Singh, M; Singh, A; Sharma, A

    2014-07-01

    The beta-haemolysin of Staphylococcus aureus (SA-hlb) is a secreted neutral sphingomyelinase (nSMase) implicated in the pathogenesis of infection and responsible for the characteristic in vitro 'hot-cold' haemolytic ability of the bacterium. Here, we describe the production of a biologically active N-terminally-truncated recombinant SA-hlb protein for use in in vitro assays and as a research tool. Using local isolates of S. aureus, we PCR-amplified an SA-hlb DNA sequence of 891 nucleotides, 99 nucleotides shorter than the full-length molecule, before cloning and sequencing (GenBank accession no. JN580071). The pQE.TriSystem vector (Qiagen, Germany) was used to express recombinant SA-hlb (r-SA-hlb) with a C-terminal 8xHis tag in Escherichia coli JM107 cells. Both JM107 lysate and the purified r-SA-hlb possessed hot-cold lytic activity against sheep and buffalo erythrocytes, which was abolished by incubation at ≥90 °C for 30 min or exposure to dithiothreitol, and could be neutralized by bovine immune sera. Purified r-SA-hlb was also cytotoxic to buffalo mononuclear cells and was effective as a coating antigen for indirect ELISA to screen for reactive sera. Importantly, the r-SA-hlb was suitable for use as a β-toxin in the modified CAMP test. We conclude that the r-SA-hlb protein produced was functionally active and has numerous potential applications.

  9. Contributions of the RAD51 N-terminal domain to BRCA2-RAD51 interaction.

    Science.gov (United States)

    Subramanyam, Shyamal; Jones, William T; Spies, Maria; Spies, M Ashley

    2013-10-01

    RAD51 DNA strand exchange protein catalyzes the central step in homologous recombination, a cellular process fundamentally important for accurate repair of damaged chromosomes, preservation of the genetic integrity, restart of collapsed replication forks and telomere maintenance. BRCA2 protein, a product of the breast cancer susceptibility gene, is a key recombination mediator that interacts with RAD51 and facilitates RAD51 nucleoprotein filament formation on single-stranded DNA generated at the sites of DNA damage. An accurate atomistic level description of this interaction, however, is limited to a partial crystal structure of the RAD51 core fused to BRC4 peptide. Here, by integrating homology modeling and molecular dynamics, we generated a structure of the full-length RAD51 in complex with BRC4 peptide. Our model predicted previously unknown hydrogen bonding patterns involving the N-terminal domain (NTD) of RAD51. These interactions guide positioning of the BRC4 peptide within a cavity between the core and the NTDs; the peptide binding separates the two domains and restricts internal dynamics of RAD51 protomers. The model's depiction of the RAD51-BRC4 complex was validated by free energy calculations and in vitro functional analysis of rationally designed mutants. All generated mutants, RAD51(E42A), RAD51(E59A), RAD51(E237A), RAD51(E59A/E237A) and RAD51(E42A/E59A/E237A) maintained basic biochemical activities of the wild-type RAD51, but displayed reduced affinities for the BRC4 peptide. Strong correlation between the calculated and experimental binding energies confirmed the predicted structure of the RAD51-BRC4 complex and highlighted the importance of RAD51 NTD in RAD51-BRCA2 interaction. PMID:23935068

  10. HEPATIC APOPTOSIS POST-BURN IS MEDIATED BY C-JUN N-TERMINAL KINASE-2

    Science.gov (United States)

    Marshall, Alexandra H.; Brooks, Natasha C.; Hiyama, Yaeko; Qa’aty, Nour; Al-mousawi, Ahmed; Finnerty, Celeste C.; Jeschke, Marc G.

    2013-01-01

    The trauma of a severe burn injury induces a hypermetabolic response that increases morbidity and mortality. Previously, our group showed that insulin resistance post-burn injury is associated with endoplasmic reticulum (ER) stress. Evidence suggests that c-jun N-terminal kinase (JNK) -2 may be involved in ER stress-induced apoptosis. Here, we hypothesized that JNK2 contributes to the apoptotic response after burn injury downstream of ER stress. To test this, we compared JNK2 knockout mice (−/−) to wildtype mice after inducing a 30% total body surface area thermal injury. Animals were sacrificed after 1, 3 and 5 days. Inflammatory cytokines in the blood were measured by multiplex analysis. Hepatic ER stress and insulin signaling were assessed by Western Blotting and insulin resistance was measured by a peritoneal glucose tolerance test. Apoptosis in the liver was quantified by TUNEL staining. Liver function was quantified by AST and ALT activity assays. ER stress increased after burn in both JNK2−/− and wildtype mice, indicating that JNK2 activation is downstream of ER stress. Knockout of JNK2 did not affect serum inflammatory cytokines; however, the increase in IL-6 mRNA expression was prevented in the knockouts. Serum insulin did not significantly increase in the JNK2−/− group. On the other hand, insulin signaling (PI3K/Akt pathway) and glucose tolerance tests did not improve in JNK2−/−. As expected, apoptosis in the liver increased after burn injury in wildtype mice but not in JNK2−/−. AST/ALT activity revealed that liver function recovered more quickly in JNK2−/−. This study indicates that JNK2 is a central mediator of hepatic apoptosis after a severe burn. PMID:23324888

  11. Membrane binding of prion protein N-terminal peptides characterised by neutron reflectometry

    International Nuclear Information System (INIS)

    The prion protein (PrP) is widely recognised to mis-fold into the causative agent of the transmissible spongiform encephalopathies, known as Creutzfeldt–Jakob disease (CJD) in humans, scrappie in sheep or Bovine spongiform encephalopathy in cows (BSE, “mad cow disease”). PrP has previously been shown to bind to lipid membranes with binding influenced by both membrane composition and pH. Aside from the mis-folding events associated with prion pathogenesis, PrP can undergo various post-translational modifications, including internal cleavage events. Alpha and beta-cleavage of PrP produces two N-terminal fragments, N1 and N2 respectively, which interact specifically with negatively charged phospholipids at low pH. Previous work probing N1 and N2 interactions with supported bilayers raised the possibility that the peptides could insert deeply with minimal disruption [1]. This work aimed to refine the binding parameters of these peptides with lipid bilayers. To this end, neutron reflectometry was used to define the structural details of the interactions in combination with quartz crystal microbalance interrogation and calcein release assays. Neutron reflectometry confirmed that peptides equivalent to N1 and N2 insert into the interstitial space between the phospholipid headgroups but do not penetrate into the acyl tail region. In accord with previous studies, interactions were stronger for the N1 fragment than for the N2, with more peptide bound per lipid. Neutron reflectometry analysis also detected lengthening of the lipid acyl tails, with a concurrent decrease in lipid area. Overall, the data shows that the N1 and N2 peptides interact with the anionic phospholipid headgroups of supported lipid bilayers, inducing lipid ordering in the absence of significant penetration into the acyl tails or permeation of the membrane.

  12. 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.

  13. Structural modeling of the N-terminal signal–receiving domain of IκBα

    Directory of Open Access Journals (Sweden)

    Samira eYazdi

    2015-06-01

    Full Text Available The transcription factor nuclear factor-κB (NF-κB exerts essential roles in many biological processes including cell growth, apoptosis and innate and adaptive immunity. The NF-kB inhibitor (IκBα retains NF-κB in the cytoplasm and thus inhibits nuclear localization of NF-κB and its association with DNA. Recent protein crystal structures of the C-terminal part of IκBα in complex with NF-κB provided insights into the protein-protein interactions but could not reveal structural details about the N-terminal signal receiving domain (SRD. The SRD of IκBα contains a degron, formed following phosphorylation by IκB kinases (IKK. In current protein X-ray structures, however, the SRD is not resolved and assumed to be disordered. Here, we combined secondary structure annotation and domain threading followed by long molecular dynamics (MD simulations and showed that the SRD possesses well-defined secondary structure elements. We show that the SRD contains 3 additional stable α-helices supplementing the six ARDs present in crystallized IκBα. The IκBα/NF-κB protein-protein complex remained intact and stable during the entire simulations. Also in solution, free IκBα retains its structural integrity. Differences in structural topology and dynamics were observed by comparing the structures of NF-κB free and NF-κB bound IκBα-complex. This study paves the way for investigating the signaling properties of the SRD in the IκBα degron. A detailed atomic scale understanding of molecular mechanism of NF-κB activation, regulation and the protein-protein interactions may assist to design and develop novel chronic inflammation modulators.

  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. 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.

  16. Endogenous N-terminal Domain Cleavage Modulates α1D-Adrenergic Receptor Pharmacodynamics.

    Science.gov (United States)

    Kountz, Timothy S; Lee, Kyung-Soon; Aggarwal-Howarth, Stacey; Curran, Elizabeth; Park, Ji-Min; Harris, Dorathy-Ann; Stewart, Aaron; Hendrickson, Joseph; Camp, Nathan D; Wolf-Yadlin, Alejandro; Wang, Edith H; Scott, John D; Hague, Chris

    2016-08-26

    The α1D-adrenergic receptor (ADRA1D) is a key regulator of cardiovascular, prostate, and central nervous system functions. This clinically relevant G protein-coupled receptor has proven difficult to study, as it must form an obligate modular homodimer containing the PDZ proteins scribble and syntrophin or become retained in the endoplasmic reticulum as non-functional protein. We previously determined that targeted removal of the N-terminal (NT) 79 amino acids facilitates ADRA1D plasma membrane expression and agonist-stimulated functional responses. However, whether such an event occurs in physiological contexts was unknown. Herein, we report the ADRA1D is subjected to innate NT processing in cultured human cells. SNAP near-infrared imaging and tandem-affinity purification revealed the ADRA1D is expressed as both full-length and NT truncated forms in multiple human cell lines. Serial truncation mapping identified the cleavage site as Leu(90)/Val(91) in the 95-amino acid ADRA1D NT domain, suggesting human cells express a Δ1-91 ADRA1D species. Tandem-affinity purification MS/MS and co-immunoprecipitation analysis indicate NT processing of ADRA1D is not required to form scribble-syntrophin macromolecular complexes. Yet, label-free dynamic mass redistribution signaling assays demonstrate that Δ1-91 ADRA1D agonist responses were greater than WT ADRA1D. Mutagenesis of the cleavage site nullified the processing event, resulting in ADRA1D agonist responses less than the WT receptor. Thus, we propose that processing of the ADRA1D NT domain is a physiological mechanism employed by cells to generate a functional ADRA1D isoform with optimal pharmacodynamic properties. PMID:27382054

  17. The Pilin N-terminal Domain Maintains Neisseria gonorrhoeae Transformation Competence during Pilus Phase Variation.

    Science.gov (United States)

    Obergfell, Kyle P; Seifert, H Steven

    2016-05-01

    The obligate human pathogen Neisseria gonorrhoeae is the sole aetiologic agent of the sexually transmitted infection, gonorrhea. Required for gonococcal infection, Type IV pili (Tfp) mediate many functions including adherence, twitching motility, defense against neutrophil killing, and natural transformation. Critical for immune escape, the gonococcal Tfp undergoes antigenic variation, a recombination event at the pilE locus that varies the surface exposed residues of the major pilus subunit PilE (pilin) in the pilus fiber. This programmed recombination system has the potential to produce thousands of pilin variants and can produce strains with unproductive pilin molecules that are completely unable to form Tfp. Saturating mutagenesis of the 3' third of the pilE gene identified 68 unique single nucleotide mutations that each resulted in an underpiliated colony morphology. Notably, all isolates, including those with undetectable levels of pilin protein and no observable surface-exposed pili, retained an intermediate level of transformation competence not exhibited in ΔpilE strains. Site-directed, nonsense mutations revealed that only the first 38 amino acids of the mature pilin N-terminus (the N-terminal domain or Ntd) are required for transformation competence, and microscopy, ELISAs and pilus purification demonstrate that extended Tfp are not required for competence. Transformation in strains producing only the pilin Ntd has the same genetic determinants as wild-type transformation. The Ntd corresponds to the alternative product of S-pilin cleavage, a specific proteolysis unique to pathogenic Neisseria. Mutation of the S-pilin cleavage site demonstrated that S-pilin cleavage mediated release of the Ntd is required for competence when a strain produces unproductive pilin molecules that cannot assemble into a Tfp through mutation or antigenic variation. We conclude that S-pilin cleavage evolved as a mechanism to maintain competence in nonpiliated antigenic variants

  18. The Pilin N-terminal Domain Maintains Neisseria gonorrhoeae Transformation Competence during Pilus Phase Variation

    Science.gov (United States)

    2016-01-01

    The obligate human pathogen Neisseria gonorrhoeae is the sole aetiologic agent of the sexually transmitted infection, gonorrhea. Required for gonococcal infection, Type IV pili (Tfp) mediate many functions including adherence, twitching motility, defense against neutrophil killing, and natural transformation. Critical for immune escape, the gonococcal Tfp undergoes antigenic variation, a recombination event at the pilE locus that varies the surface exposed residues of the major pilus subunit PilE (pilin) in the pilus fiber. This programmed recombination system has the potential to produce thousands of pilin variants and can produce strains with unproductive pilin molecules that are completely unable to form Tfp. Saturating mutagenesis of the 3’ third of the pilE gene identified 68 unique single nucleotide mutations that each resulted in an underpiliated colony morphology. Notably, all isolates, including those with undetectable levels of pilin protein and no observable surface-exposed pili, retained an intermediate level of transformation competence not exhibited in ΔpilE strains. Site-directed, nonsense mutations revealed that only the first 38 amino acids of the mature pilin N-terminus (the N-terminal domain or Ntd) are required for transformation competence, and microscopy, ELISAs and pilus purification demonstrate that extended Tfp are not required for competence. Transformation in strains producing only the pilin Ntd has the same genetic determinants as wild-type transformation. The Ntd corresponds to the alternative product of S-pilin cleavage, a specific proteolysis unique to pathogenic Neisseria. Mutation of the S-pilin cleavage site demonstrated that S-pilin cleavage mediated release of the Ntd is required for competence when a strain produces unproductive pilin molecules that cannot assemble into a Tfp through mutation or antigenic variation. We conclude that S-pilin cleavage evolved as a mechanism to maintain competence in nonpiliated antigenic

  19. The Pilin N-terminal Domain Maintains Neisseria gonorrhoeae Transformation Competence during Pilus Phase Variation.

    Directory of Open Access Journals (Sweden)

    Kyle P Obergfell

    2016-05-01

    Full Text Available The obligate human pathogen Neisseria gonorrhoeae is the sole aetiologic agent of the sexually transmitted infection, gonorrhea. Required for gonococcal infection, Type IV pili (Tfp mediate many functions including adherence, twitching motility, defense against neutrophil killing, and natural transformation. Critical for immune escape, the gonococcal Tfp undergoes antigenic variation, a recombination event at the pilE locus that varies the surface exposed residues of the major pilus subunit PilE (pilin in the pilus fiber. This programmed recombination system has the potential to produce thousands of pilin variants and can produce strains with unproductive pilin molecules that are completely unable to form Tfp. Saturating mutagenesis of the 3' third of the pilE gene identified 68 unique single nucleotide mutations that each resulted in an underpiliated colony morphology. Notably, all isolates, including those with undetectable levels of pilin protein and no observable surface-exposed pili, retained an intermediate level of transformation competence not exhibited in ΔpilE strains. Site-directed, nonsense mutations revealed that only the first 38 amino acids of the mature pilin N-terminus (the N-terminal domain or Ntd are required for transformation competence, and microscopy, ELISAs and pilus purification demonstrate that extended Tfp are not required for competence. Transformation in strains producing only the pilin Ntd has the same genetic determinants as wild-type transformation. The Ntd corresponds to the alternative product of S-pilin cleavage, a specific proteolysis unique to pathogenic Neisseria. Mutation of the S-pilin cleavage site demonstrated that S-pilin cleavage mediated release of the Ntd is required for competence when a strain produces unproductive pilin molecules that cannot assemble into a Tfp through mutation or antigenic variation. We conclude that S-pilin cleavage evolved as a mechanism to maintain competence in nonpiliated

  20. Promoter-dependent activity on androgen receptor N-terminal domain mutations in androgen insensitivity syndrome.

    Science.gov (United States)

    Tadokoro-Cuccaro, Rieko; Davies, John; Mongan, Nigel P; Bunch, Trevor; Brown, Rosalind S; Audi, Laura; Watt, Kate; McEwan, Iain J; Hughes, Ieuan A

    2014-01-01

    Androgen receptor (AR) mutations are associated with androgen insensitivity syndrome (AIS). Missense mutations identified in the AR-N-terminal domain (AR-NTD) are rare, and clinical phenotypes are typically mild. We investigated 7 missense mutations and 2 insertion/deletions located in the AR-NTD. This study aimed to elucidate the pathogenic role of AR-NTD mutants in AIS and to use this knowledge to further define AR-NTD function. AR-NTD mutations (Q120E, A159T, G216R, N235K, G248V, L272F, and P380R) were introduced into AR-expression plasmids. Stably expressing cell lines were established for del57L and ins58L. Transactivation was measured using luciferase reporter constructs under the control of GRE and Pem promoters. Intrinsic fluorescence spectroscopy and partial proteolysis studies were performed for mutations which showed reduced activities by using a purified AR-AF1 protein. Pem-luciferase reporter activation was reduced for A159T, N235K, and G248V but not the GRE-luciferase reporter. Protein structure analysis detected no significant change in the AR-AF1 region for these mutations. Reduced cellular expression and transactivation activity were observed for ins58L. The mutations Q120E, G216R, L272F, P380R, and del57L showed small or no detectable changes in function. Thus, clinical and experimental analyses have identified novel AR-signalling defects associated with mutations in the structurally disordered AR-NTD domain in patients with AIS.

  1. N-terminal and C-terminal cytosine deaminase domain of APOBEC3G inhibit hepatitis B virus replication

    Institute of Scientific and Technical Information of China (English)

    Yan-Chang Lei; Dong-Liang Yang; Yong-Jun Tian; Hong-Hui Ding; Bao-Ju Wang; Yan Yang; You-Hua Hao; Xi-Ping Zhao; Meng-Ji Lu; Fei-Li Gong

    2006-01-01

    AIM: To investigate the effect of human apolipoprotein B mRNA-editing enzyme catalytic-polypeptide 3G(APOBEC3G) and its N-terminal or C-terminal cytosine deaminase domain-mediated antiviral activity against hepatitis B virus (HBV) in vitro and in vivo.METHODS: The mammalian hepatoma cells HepG2 and HuH7 were cotransfected with APOBEC3G and its N-terminal or C-terminal cytosine deaminase domain expression vector and 1.3-fold-overlength HBV DNA as well as the linear monomeric HBV of genotype B and C. For in vivo study, an HBV vector-based mouse model was used in which APOBEC3G and its N-terminal or C-terminal cytosine deaminase domain expression vectors were co-delivered with 1.3-fold-overlength HBV DNA via high-volume tail vein injection. Levels of hepatitis B virus surface antigen (HBsAg) and hepatitis B virus e antigen (HBeAg) in the media of the transfected cells and in the sera of mice were determined by ELISA.The expression of hepatitis B virus core antigen (HBcAg)in the transfected cells was determined by Western blot analysis. Core-associated HBV DNA was examined by Southern blot analysis. Levels of HBV DNA in the sera of mice as well as HBV core-associated RNA in the liver of mice were determined by quantitative PCR and quantitative RT-PCR analysis, respectively.RESULTS: Human APOBEC3G exerted an anti-HBV activity in a dose-dependent manner in HepG2 cells,and comparable suppressive effects were observed on genotype B and C as that of genotype A. Interestingly,the N-terminal or C-terminal cytosine deaminase domain alone could also inhibit HBV replication in HepG2 cells as well as Huh7 cells. Consistent with in vitro results, the levels of HBsAg in the sera of mice were dramatically decreased, with more than 50 times decrease in the levels of serum HBV DNA and core-associated RNA in the liver of mice treated with APOBEC3G and its N-terminal or C-terminal cytosine deaminase domain as compared to the controls.CONCLUSION: Our findings provide probably the first

  2. 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.

  3. 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.

  4. ELKS controls the pool of readily releasable vesicles at excitatory synapses through its N-terminal coiled-coil domains.

    Science.gov (United States)

    Held, Richard G; Liu, Changliang; Kaeser, Pascal S

    2016-06-02

    In a presynaptic nerve terminal, synaptic strength is determined by the pool of readily releasable vesicles (RRP) and the probability of release (P) of each RRP vesicle. These parameters are controlled at the active zone and vary across synapses, but how such synapse specific control is achieved is not understood. ELKS proteins are enriched at vertebrate active zones and enhance P at inhibitory hippocampal synapses, but ELKS functions at excitatory synapses are not known. Studying conditional knockout mice for ELKS, we find that ELKS enhances the RRP at excitatory synapses without affecting P. Surprisingly, ELKS C-terminal sequences, which interact with RIM, are dispensable for RRP enhancement. Instead, the N-terminal ELKS coiled-coil domains that bind to Liprin-α and Bassoon are necessary to control RRP. Thus, ELKS removal has differential, synapse-specific effects on RRP and P, and our findings establish important roles for ELKS N-terminal domains in synaptic vesicle priming.

  5. 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...... the relationship between N-terminal pro-BNP (NT-pro-BNP) levels and long-term mortality from all causes in a large cohort of patients with stable coronary heart disease. METHODS: NT-pro-BNP was measured in baseline serum samples from 1034 patients referred for angiography because of symptoms or signs of coronary...... of myocardial infarction, angina, hypertension, diabetes, or chronic heart failure; creatinine clearance rate; body-mass index; smoking status; plasma lipid levels; LVEF; and the presence or absence of clinically significant coronary artery disease on angiography. CONCLUSIONS: NT-pro-BNP is a marker of long...

  6. [Chemical synthesis of lactococcin B and functional evaluation of the N-terminal domain using a truncated synthetic analogue].

    Science.gov (United States)

    Lasta, S; Fajloun, Z; Mansuelle, P; Sabatier, J M; Boudabous, A; Sampieri, F

    2008-01-01

    The lactococcin B (LnB) is a hydrophobic, positively charged bacteriocin, produced by Lactococcus lactis ssp. cremoris 9B4. It consists of a peptidic chain made up of 47 amino acid residues, and inhibits Lactococcus exclusively. In order to study its biological activity a synthetic lactococcin B (LnBs) was obtained by solid-phase chemical synthesis using a Fmoc strategy. LnBs was shown to be indistinguishable from the natural peptide. In addition, a synthetic (7-47) LnBst analogue was obtained by withdrawal of peptidyl-resin after the 41 cycle of LnBs peptide chain assembly. The synthetic N-terminal truncated (7-47) LnBst analogue was found to be inactive on indicator strains. Our results strongly suggest that the first six N-terminal amino acid residues are involved in the bactericidal activity of LnB.

  7. Electrical Imaging of Roots and Trunks

    Science.gov (United States)

    Al Hagrey, S.; Werban, U.; Meissner, R.; Ismaeil, A.; Rabbel, W.

    2005-05-01

    We applied geoelectric and GPR techniques to analyze problems of botanical structures and even processes, e.g., mapping root zones, internal structure of trunks, and water uptake by roots. The dielectric nature of root zones and trunks is generally a consequence of relatively high moisture content. The electric method, applied to root zones, can discriminate between old, thick, isolated roots (high resistivity) and the network of young, active, and hydraulically conductive zones (low resistivity). Both types of roots show low radar velocity and a strong attenuation caused by the dominant effect of moisture (high dielectric constant) on the electromagnetic wave propagation. Single root branches could be observed in radargrams by their reflection and diffraction parabolas. We have perfected the inversion method for perfect and imperfect cylindrical objects, such as trunks, and developed a new multielectrodes (needle or gel) ring array for fast applications on living trees and discs. Using synthetic models we tested the technique successfully and analyzed it as a function of total electrode number and configuration. Measurements at a trunk show a well established inverse relationship between the imaged resistivity and the moisture content determined from cores. The central resistivity maximum of healthy trees strongly decreases toward the rim. This agrees with the moisture decrease to the outside where active sap flow processes take place. Branching, growth anomalies (new or old shoots) and meteorological effects (sunshine and wind direction) lead to deviations of the concentric electric structure. The strongest anomalies are related to infections causing wet, rotting spots or cavities. The heartwood resistivity is highest in olive and oak trunks, intermediate in young fruit trees and lowest in cork oak trunks that are considered to be anomalously wet. Compared to acoustic tomography our electric technique shows a better resolution in imaging internal ring structures

  8. The N-terminal domain of the tomato immune protein Prf contains multiple homotypic and Pto kinase interaction sites.

    Science.gov (United States)

    Saur, Isabel Marie-Luise; Conlan, Brendon Francis; Rathjen, John Paul

    2015-05-01

    Resistance to Pseudomonas syringae bacteria in tomato (Solanum lycopersicum) is conferred by the Prf recognition complex, composed of the nucleotide-binding leucine-rich repeats protein Prf and the protein kinase Pto. The complex is activated by recognition of the P. syringae effectors AvrPto and AvrPtoB. The N-terminal domain is responsible for Prf homodimerization, which brings two Pto kinases into close proximity and holds them in inactive conformation in the absence of either effector. Negative regulation is lost by effector binding to the catalytic cleft of Pto, leading to disruption of its P+1 loop within the activation segment. This change is translated through Prf to a second Pto molecule in the complex. Here we describe a schematic model of the unique Prf N-terminal domain dimer and its interaction with the effector binding determinant Pto. Using heterologous expression in Nicotiana benthamiana, we define multiple sites of N domain homotypic interaction and infer that it forms a parallel dimer folded centrally to enable contact between the N and C termini. Furthermore, we found independent binding sites for Pto at either end of the N-terminal domain. Using the constitutively active mutant ptoL205D, we identify a potential repression site for Pto in the first ∼100 amino acids of Prf. Finally, we find that the Prf leucine-rich repeats domain also binds the N-terminal region, highlighting a possible mechanism for transfer of the effector binding signal to the NB-LRR regulatory unit (consisting of a central nucleotide binding and C-terminal leucine-rich repeats).

  9. Regulation of Apoptotic c-Jun N-Terminal Kinase Signaling by a Stabilization-Based Feed-Forward Loop†

    OpenAIRE

    Xu, Zhiheng; Kukekov, Nikolay V.; Greene, Lloyd A.

    2005-01-01

    A sequential kinase cascade culminating in activation of c-Jun N-terminal kinases (JNKs) plays a fundamental role in promoting apoptotic death in many cellular contexts. The mechanisms by which this pathway is engaged in response to apoptotic stimuli and suppressed in viable cells are largely unknown. Here, we show that apoptotic stimuli increase endogenous cellular levels of pathway components, including POSH, mixed lineage kinases (MLKs), and JNK interacting protein 1, and that this effect ...

  10. The metalloid arsenite induces nuclear export of Id3 possibly via binding to the N-terminal cysteine residues

    Energy Technology Data Exchange (ETDEWEB)

    Kurooka, Hisanori, E-mail: hkurooka@u-fukui.ac.jp [Division of Molecular Genetics, Department of Biochemistry and Bioinformative Sciences, School of Medicine, Faculty of Medical Sciences, University of Fukui, Fukui (Japan); Research and Education Program for Life Science, University of Fukui, Fukui (Japan); Sugai, Manabu [Department of Experimental Therapeutics, Translational Research Center, Kyoto University Hospital, Kyoto (Japan); Mori, Kentaro [Division of Molecular Genetics, Department of Biochemistry and Bioinformative Sciences, School of Medicine, Faculty of Medical Sciences, University of Fukui, Fukui (Japan); Yokota, Yoshifumi, E-mail: yokota@u-fukui.ac.jp [Division of Molecular Genetics, Department of Biochemistry and Bioinformative Sciences, School of Medicine, Faculty of Medical Sciences, University of Fukui, Fukui (Japan); Research and Education Program for Life Science, University of Fukui, Fukui (Japan)

    2013-04-19

    Highlights: •Sodium arsenite induces cytoplasmic accumulation of Id3. •Arsenite binds to closely spaced N-terminal cysteine residues of Id3. •N-terminal cysteines are essential for arsenite-induced nuclear export of Id3. •Nuclear export of Id3 counteracts its transcriptional repression activity. -- Abstract: Ids are versatile transcriptional repressors that regulate cell proliferation and differentiation, and appropriate subcellular localization of the Id proteins is important for their functions. We previously identified distinct functional nuclear export signals (NESs) in Id1 and Id2, but no active NES has been reported in Id3. In this study, we found that treatment with the stress-inducing metalloid arsenite led to the accumulation of GFP-tagged Id3 in the cytoplasm. Cytoplasmic accumulation was impaired by a mutation in the Id3 NES-like sequence resembling the Id1 NES, located at the end of the HLH domain. It was also blocked by co-treatment with the CRM1-specific nuclear export inhibitor leptomycin B (LMB), but not with the inhibitors for mitogen-activated protein kinases (MAPKs). Importantly, we showed that the closely spaced N-terminal cysteine residues of Id3 interacted with the arsenic derivative phenylarsine oxide (PAO) and were essential for the arsenite-induced cytoplasmic accumulation, suggesting that arsenite induces the CRM1-dependent nuclear export of Id3 via binding to the N-terminal cysteines. Finally, we demonstrated that Id3 significantly repressed arsenite-stimulated transcription of the immediate-early gene Egr-1 and that this repression activity was inversely correlated with the arsenite-induced nuclear export. Our results imply that Id3 may be involved in the biological action of arsenite.

  11. The metalloid arsenite induces nuclear export of Id3 possibly via binding to the N-terminal cysteine residues

    International Nuclear Information System (INIS)

    Highlights: •Sodium arsenite induces cytoplasmic accumulation of Id3. •Arsenite binds to closely spaced N-terminal cysteine residues of Id3. •N-terminal cysteines are essential for arsenite-induced nuclear export of Id3. •Nuclear export of Id3 counteracts its transcriptional repression activity. -- Abstract: Ids are versatile transcriptional repressors that regulate cell proliferation and differentiation, and appropriate subcellular localization of the Id proteins is important for their functions. We previously identified distinct functional nuclear export signals (NESs) in Id1 and Id2, but no active NES has been reported in Id3. In this study, we found that treatment with the stress-inducing metalloid arsenite led to the accumulation of GFP-tagged Id3 in the cytoplasm. Cytoplasmic accumulation was impaired by a mutation in the Id3 NES-like sequence resembling the Id1 NES, located at the end of the HLH domain. It was also blocked by co-treatment with the CRM1-specific nuclear export inhibitor leptomycin B (LMB), but not with the inhibitors for mitogen-activated protein kinases (MAPKs). Importantly, we showed that the closely spaced N-terminal cysteine residues of Id3 interacted with the arsenic derivative phenylarsine oxide (PAO) and were essential for the arsenite-induced cytoplasmic accumulation, suggesting that arsenite induces the CRM1-dependent nuclear export of Id3 via binding to the N-terminal cysteines. Finally, we demonstrated that Id3 significantly repressed arsenite-stimulated transcription of the immediate-early gene Egr-1 and that this repression activity was inversely correlated with the arsenite-induced nuclear export. Our results imply that Id3 may be involved in the biological action of arsenite

  12. Crystallization and preliminary X-ray crystallographic analysis of yeast prion protein Ure2p with shortened N-terminal

    Institute of Scientific and Technical Information of China (English)

    2001-01-01

    An orthorhombic crystal form of a recombinant yeast prion protein with shortened N-terminal, 90Ure2p, has been obtained. Crystals were grown by the vapordiffusion technique against a mother liquor containing imidazole. Crystals belong to the primitive orthorhombic lattice with the cell parameters a = 54.5 ?, b = 74.7 ?, c = 131.0 ?. The crystals diffract to beyond 3.0 ? resolution at a synchrotron beamline.

  13. c-Jun N-terminal Kinase (JNK) signaling as a therapeutic target for Alzheimer’s disease

    OpenAIRE

    Yarza, Ramon; Vela, Silvia; Solas, Maite; Ramirez, Maria J

    2016-01-01

    c-Jun N-terminal kinases (JNKs) are a family of protein kinases that play a central role in stress signaling pathways implicated in gene expression, neuronal plasticity, regeneration, cell death, and regulation of cellular senescence. It has been shown that there is a JNK pathway activation after exposure to different stressing factors, including cytokines, growth factors, oxidative stress, unfolded protein response signals or Aβ peptides. Altogether, JNKs have become a focus of screening str...

  14. Exercise Dependence of N-Terminal Pro-Brain Natriuretic Peptide in Patients with Precapillary Pulmonary Hypertension

    OpenAIRE

    Grachtrup, Sabine; Brügel, Mathias; Pankau, Hans; Halank, Michael; Wirtz, Hubert; Seyfarth, Hans-Jürgen

    2014-01-01

    Background: N-terminal pro-brain natriuretic peptide (NT-proBNP) is secreted by cardiac ventricular myocytes upon pressure and volume overload and is a prognostic marker to monitor the severity of precapillary pulmonary hypertension and the extent of right heart failure. Objectives: The impact of physical exercise on NT-proBNP levels in patients with left heart disease was demonstrated previously. No data regarding patients with isolated right heart failure and the influence of acute exer...

  15. 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......); sensitivity/specificity, 85%/86%), and LV ejection fraction (powerful marker for LV dimensions and systolic function in patients with heart failure and discriminates well between...

  16. 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. PMID:26632841

  17. N-terminal aromatic residues closely impact the cytolytic activity of cupiennin 1a, a major spider venom peptide.

    Science.gov (United States)

    Kuhn-Nentwig, Lucia; Sheynis, Tania; Kolusheva, Sofiya; Nentwig, Wolfgang; Jelinek, Raz

    2013-12-01

    Cupiennins are small cationic α-helical peptides from the venom of the ctenid spider Cupiennius salei which are characterized by high bactericidal as well as hemolytic activities. To gain insight into the determinants responsible for the broad cytolytic activities, two analogues of cupiennin 1a with different N-terminal hydrophobicities were designed. The insecticidal, bactericidal and hemolytic activities of these analogues were assayed and compared to the native peptide. Specifically, substitution of two N-terminal Phe residues by Ala results in less pronounced insecticidal and cytolytic activity, whereas a substitution by Lys reduces strongly its bactericidal activity and completely diminishes its hemolytic activity up to very high tested concentrations. Biophysical analyses of peptide/bilayer membrane interactions point to distinct interactions of the analogues with lipid bilayers, and dependence upon membrane surface charge. Indeed, we find that lower hemolytic activity was correlated with less surface association of the analogues. In contrast, our data indicate that the reduced bactericidal activity of the two cupiennin 1a analogues likely correspond to greater bilayer-surface localization of the peptides. Overall, ultimate insertion and destruction of the host cell membrane is highly dependent on the presence of Phe-2 and Phe-6 (Cu 1a) or Leu-6 (Cu 2a) in the N-terminal sequences of native cupiennins.

  18. 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.

  19. Protective epitopes of the Plasmodium falciparum SERA5 malaria vaccine reside in intrinsically unstructured N-terminal repetitive sequences.

    Directory of Open Access Journals (Sweden)

    Masanori Yagi

    Full Text Available The malaria vaccine candidate antigen, SE36, is based on the N-terminal 47 kDa domain of Plasmodium falciparum serine repeat antigen 5 (SERA5. In epidemiological studies, we have previously shown the inhibitory effects of SE36 specific antibodies on in vitro parasite growth and the negative correlation between antibody level and malaria symptoms. A phase 1 b trial of the BK-SE36 vaccine in Uganda elicited 72% protective efficacy against symptomatic malaria in children aged 6-20 years during the follow-up period 130-365 days post-second vaccination. Here, we performed epitope mapping with synthetic peptides covering the whole sequence of SE36 to identify and map dominant epitopes in Ugandan adult serum presumed to have clinical immunity to P. falciparum malaria. High titer sera from the Ugandan adults predominantly reacted with peptides corresponding to two successive N-terminal regions of SERA5 containing octamer repeats and serine rich sequences, regions of SERA5 that were previously reported to have limited polymorphism. Affinity purified antibodies specifically recognizing the octamer repeats and serine rich sequences exhibited a high antibody-dependent cellular inhibition (ADCI activity that inhibited parasite growth. Furthermore, protein structure predictions and structural analysis of SE36 using spectroscopic methods indicated that N-terminal regions possessing inhibitory epitopes are intrinsically unstructured. Collectively, these results suggest that strict tertiary structure of SE36 epitopes is not required to elicit protective antibodies in naturally immune Ugandan adults.

  20. Protective epitopes of the Plasmodium falciparum SERA5 malaria vaccine reside in intrinsically unstructured N-terminal repetitive sequences.

    Science.gov (United States)

    Yagi, Masanori; Bang, Gilles; Tougan, Takahiro; Palacpac, Nirianne M Q; Arisue, Nobuko; Aoshi, Taiki; Matsumoto, Yoshitsugu; Ishii, Ken J; Egwang, Thomas G; Druilhe, Pierre; Horii, Toshihiro

    2014-01-01

    The malaria vaccine candidate antigen, SE36, is based on the N-terminal 47 kDa domain of Plasmodium falciparum serine repeat antigen 5 (SERA5). In epidemiological studies, we have previously shown the inhibitory effects of SE36 specific antibodies on in vitro parasite growth and the negative correlation between antibody level and malaria symptoms. A phase 1 b trial of the BK-SE36 vaccine in Uganda elicited 72% protective efficacy against symptomatic malaria in children aged 6-20 years during the follow-up period 130-365 days post-second vaccination. Here, we performed epitope mapping with synthetic peptides covering the whole sequence of SE36 to identify and map dominant epitopes in Ugandan adult serum presumed to have clinical immunity to P. falciparum malaria. High titer sera from the Ugandan adults predominantly reacted with peptides corresponding to two successive N-terminal regions of SERA5 containing octamer repeats and serine rich sequences, regions of SERA5 that were previously reported to have limited polymorphism. Affinity purified antibodies specifically recognizing the octamer repeats and serine rich sequences exhibited a high antibody-dependent cellular inhibition (ADCI) activity that inhibited parasite growth. Furthermore, protein structure predictions and structural analysis of SE36 using spectroscopic methods indicated that N-terminal regions possessing inhibitory epitopes are intrinsically unstructured. Collectively, these results suggest that strict tertiary structure of SE36 epitopes is not required to elicit protective antibodies in naturally immune Ugandan adults. PMID:24886718

  1. Insights into the Functional Roles of N-Terminal and C-Terminal Domains of Helicobacter pylori DprA.

    Directory of Open Access Journals (Sweden)

    Gajendradhar R Dwivedi

    Full Text Available DNA processing protein A (DprA plays a crucial role in the process of natural transformation. This is accomplished through binding and subsequent protection of incoming foreign DNA during the process of internalization. DprA along with Single stranded DNA binding protein A (SsbA acts as an accessory factor for RecA mediated DNA strand exchange. H. pylori DprA (HpDprA is divided into an N-terminal domain and a C- terminal domain. In the present study, individual domains of HpDprA have been characterized for their ability to bind single stranded (ssDNA and double stranded DNA (dsDNA. Oligomeric studies revealed that HpDprA possesses two sites for dimerization which enables HpDprA to form large and tightly packed complexes with ss and dsDNA. While the N-terminal domain was found to be sufficient for binding with ss or ds DNA, C-terminal domain has an important role in the assembly of poly-nucleoprotein complex. Using site directed mutagenesis approach, we show that a pocket comprising positively charged amino acids in the N-terminal domain has an important role in the binding of ss and dsDNA. Together, a functional cross talk between the two domains of HpDprA facilitating the binding and formation of higher order complex with DNA is discussed.

  2. Insights into the Functional Roles of N-Terminal and C-Terminal Domains of Helicobacter pylori DprA.

    Science.gov (United States)

    Dwivedi, Gajendradhar R; Srikanth, Kolluru D; Anand, Praveen; Naikoo, Javed; Srilatha, N S; Rao, Desirazu N

    2015-01-01

    DNA processing protein A (DprA) plays a crucial role in the process of natural transformation. This is accomplished through binding and subsequent protection of incoming foreign DNA during the process of internalization. DprA along with Single stranded DNA binding protein A (SsbA) acts as an accessory factor for RecA mediated DNA strand exchange. H. pylori DprA (HpDprA) is divided into an N-terminal domain and a C- terminal domain. In the present study, individual domains of HpDprA have been characterized for their ability to bind single stranded (ssDNA) and double stranded DNA (dsDNA). Oligomeric studies revealed that HpDprA possesses two sites for dimerization which enables HpDprA to form large and tightly packed complexes with ss and dsDNA. While the N-terminal domain was found to be sufficient for binding with ss or ds DNA, C-terminal domain has an important role in the assembly of poly-nucleoprotein complex. Using site directed mutagenesis approach, we show that a pocket comprising positively charged amino acids in the N-terminal domain has an important role in the binding of ss and dsDNA. Together, a functional cross talk between the two domains of HpDprA facilitating the binding and formation of higher order complex with DNA is discussed. PMID:26135134

  3. Kadar N-Terminal Pro-Brain Natriuretic Peptide sebagai Prediktor Luaran Klinis Sindrom Koroner Akut

    Directory of Open Access Journals (Sweden)

    Florencia Idajanti Tandhana

    2012-06-01

    Full Text Available Plasma levels of N-terminal pro-brain natriuretic peptide (NT-proBNP levels may reflect the severity of ischemia, although there is no necrosis. A transient ischemia which can increase the heart wall stretch would induces BNP synthesis and release. Synthesis and release of BNP are comparable with the severity of ischemia. The aim of this study was to analyze whether NT-proBNP levels in patients with acute coronary syndrome (ACS can be used as a predictor for clinical outcome. Studies was held since January to March 2010. Subject were patients with ACS who came to emergency room Dr. Hasan Sadikin Hospital Bandung and were clinically diagnosed according to World Health Organization criteria. Subjects which were suited with the inclusion criteria, stored until assayed. NT-pro BNP concentration was examined by electrochemiluminescence immunoassay method along with creatine kinase muscle brain (enzymatic method and cardiac troponin T (quantitative method. Statistical analysis was performed using the one-sample Kolmogorov-Smirnov test for verifying normality, normally distributed data were analyzed using parametric analysis and abnormal distributed data was assayed using multiple logistic regression analysis to determine the parameters which can be used as predictor for clinical outcome in patients with ACS. Multiple logistic regression analysis on 83 subjects showed predictive value of NT-proBNP levels with OR=1.00, which mean there was no different likelihood in patients with high and low concentration of NT-proBNP to have longer hospitality duration. NT-proBNP β coefficient of 0.001 states that every addition of 1,000 pg/mL of NT-proBNP concentration will increase the length of hospitality duration for one day. On convalesce subjects, the most significant predictive value for predicting clinical outcome cTnT was more better than NT-proBNP concentration in patients with ACS (OR=32.53, 95%CI; 0.58–1,819.26. In conclusions, NT-proBNP is not a major

  4. Direct clipping of large basilar trunk aneurysm.

    Science.gov (United States)

    Kimura, Toshikazu; Nakagawa, Daichi; Kawai, Kensuke

    2015-01-01

    A large basilar trunk aneurysm was incidentally found in a 77-year-old woman in examination for headache. Though it was asymptomatic, high signal intensity was noticed in the brainstem around the aneurysm on FLAIR image of MRI. As she was otherwise healthy, surgical clipping was performed through anterior temporal approach. The video can be found here: http://youtu.be/0soWM8meCW8 . PMID:25554839

  5. Trunk and knee performance of rowers

    OpenAIRE

    Grosdent, Stéphanie; Demoulin, Christophe; Gauthier, Stéphanie; Croisier, Jean-Louis; Vanderthommen, Marc; Crielaard, Jean-Michel

    2012-01-01

    This study showed some difference in trunk and knee extensors strength between rowers and control subjects as well as between rowers and non-rowing athletes. The increased performances of extensor muscles in elite rowers appear to be probably related to the specificity of this sport. In the drive phase of rowing, rowers sequentially push with the legs and then pull with the arms and lower back, requiring both muscular strength and endurance. However, no difference was shown between groups reg...

  6. The relationship between N-terminal prosomatostatin, all-cause and cardiovascular mortality in patients with type 2 diabetes mellitus (ZODIAC-35)

    NARCIS (Netherlands)

    van Dijk, Peter R.; Landman, Gijs W. D.; van Essen, Larissa; Struck, Joachim; Groenier, Klaas H.; Bilo, Henk J. G.; Bakker, Stephan J. L.; Kleefstra, Nanne

    2015-01-01

    Background: The hormone somatostatin inhibits growth hormone release from the pituitary gland and is theoretically linked to diabetes and diabetes related complications. This study aimed to investigate the relationship between levels of the stable somatostatin precursor, N-terminal prosomatostatin (

  7. Intracellular trafficking of the human Wilson protein: the role of the six N-terminal metal-binding sites.

    Science.gov (United States)

    Cater, Michael A; Forbes, John; La Fontaine, Sharon; Cox, Diane; Mercer, Julian F B

    2004-01-01

    The Wilson protein (ATP7B) is a copper-transporting CPx-type ATPase defective in the copper toxicity disorder Wilson disease. In hepatocytes, ATP7B delivers copper to apo-ceruloplasmin and mediates the excretion of excess copper into bile. These distinct functions require the protein to localize at two different subcellular compartments. At the trans-Golgi network, ATP7B transports copper for incorporation into apo-ceruloplasmin. When intracellular copper levels are increased, ATP7B traffics to post-Golgi vesicles in close proximity to the canalicular membrane to facilitate biliary copper excretion. In the present study, we investigated the role of the six N-terminal MBSs (metal-binding sites) in the trafficking process. Using site-directed mutagenesis, we mutated or deleted various combinations of the MBSs and assessed the effect of these changes on the localization and trafficking of ATP7B. Results show that the MBSs required for trafficking are the same as those previously found essential for the copper transport function. Either MBS 5 or MBS 6 alone was sufficient to support the redistribution of ATP7B to vesicular compartments. The first three N-terminal motifs were not required for copper-dependent intracellular trafficking and could not functionally replace sites 4-6 when placed in the same sequence position. Furthermore, the N-terminal region encompassing MBSs 1-5 (amino acids 64-540) was not essential for trafficking, with only one MBS close to the membrane channel, necessary and sufficient to support trafficking. Our findings were similar to those obtained for the closely related ATP7A protein, suggesting similar mechanisms for trafficking between copper-transporting CPx-type ATPases. PMID:14998371

  8. 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.

  9. Thermodynamics of Calcium binding to the Calmodulin N-terminal domain to evaluate site-specific affinity constants and cooperativity.

    Science.gov (United States)

    Beccia, Maria Rosa; Sauge-Merle, Sandrine; Lemaire, David; Brémond, Nicolas; Pardoux, Romain; Blangy, Stéphanie; Guilbaud, Philippe; Berthomieu, Catherine

    2015-07-01

    Calmodulin (CaM) is an essential Ca(II)-dependent regulator of cell physiology. To understand its interaction with Ca(II) at a molecular level, it is essential to examine Ca(II) binding at each site of the protein, even if it is challenging to estimate the site-specific binding properties of the interdependent CaM-binding sites. In this study, we evaluated the site-specific Ca(II)-binding affinity of sites I and II of the N-terminal domain by combining site-directed mutagenesis and spectrofluorimetry. The mutations had very low impact on the protein structure and stability. We used these binding constants to evaluate the inter-site cooperativity energy and compared it with its lower limit value usually reported in the literature. We found that site I affinity for Ca(II) was 1.5 times that of site II and that cooperativity induced an approximately tenfold higher affinity for the second Ca(II)-binding event, as compared to the first one. We further showed that insertion of a tryptophan at position 7 of site II binding loop significantly increased site II affinity for Ca(II) and the intra-domain cooperativity. ΔH and ΔS parameters were studied by isothermal titration calorimetry for Ca(II) binding to site I, site II and to the entire N-terminal domain. They showed that calcium binding is mainly entropy driven for the first and second binding events. These findings provide molecular information on the structure-affinity relationship of the individual sites of the CaM N-terminal domain and new perspectives for the optimization of metal ion binding by mutating the EF-hand loops sequences.

  10. N-terminal PDZ-like domain of chromatin organizer SATB1 contributes towards its function as transcription regulator

    Indian Academy of Sciences (India)

    Dimple Notani; Praveena L Ramanujam; P Pavan Kumar; Kamalvishnu P Gottimukkala; Chandan Kumar-Sinha; Sanjeev Galande

    2011-08-01

    The special AT-rich DNA-binding protein 1 (SATB1) is a matrix attachment region (MAR)-binding protein that acts 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 are critical for its in vivo function as a global repressor. We reasoned that since the N-terminal PDZ-like domain (amino acid residues 1–204) lacks DNA binding activity, it would fail to recruit the interacting partners of SATB1 to its genomic binding sites and hence would not repress the SATB1-regulated genes. Indeed, in vivo MAR-linked luciferase reporter assay revealed that overexpression of the PDZ-like domain resulted in de-repression, indicating that the PDZ-like domain exerts a dominant negative effect on genes regulated by SATB1. Next, we developed a stable dominant negative model in human embryonic kidney (HEK) 293T cells that conditionally expressed the N-terminal 1–204 region harbouring the PDZ-like domain of SATB1. To monitor the effect of sequestration of the interaction partners on the global gene regulation by SATB1, transcripts from the induced and uninduced clones were subjected to gene expression profiling. Clustering of expression data revealed that 600 out of 19000 genes analysed were significantly upregulated upon overexpression of the PDZ-like domain. Induced genes were found to be involved in important signalling cascades and cellular functions. These studies clearly demonstrated the role of PDZ domain of SATB1 in global gene regulation presumably through its interaction with other cellular proteins.

  11. c-Jun N-terminal kinase phosphorylates DCP1a to control formation of P bodies

    OpenAIRE

    Rzeczkowski, Katharina; Beuerlein, Knut; Müller, Helmut; Dittrich-Breiholz, Oliver; Schneider, Heike; Kettner-Buhrow, Daniela; Holtmann, Helmut; Kracht, Michael

    2011-01-01

    Cytokines and stress-inducing stimuli signal through c-Jun N-terminal kinase (JNK) using a diverse and only partially defined set of downstream effectors. In this paper, the decapping complex subunit DCP1a was identified as a novel JNK target. JNK phosphorylated DCP1a at residue S315 in vivo and in vitro and coimmunoprecipitated and colocalized with DCP1a in processing bodies (P bodies). Sustained JNK activation by several different inducers led to DCP1a dispersion from P bodies, whereas IL-1...

  12. 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...

  13. Solution NMR characterization of WT CXCL8 monomer and dimer binding to CXCR1 N-terminal domain

    OpenAIRE

    Joseph, Prem Raj B.; Rajarathnam, Krishna

    2014-01-01

    Chemokine CXCL8 and its receptor CXCR1 are key mediators in combating infection and have also been implicated in the pathophysiology of various diseases including chronic obstructive pulmonary disease (COPD) and cancer. CXCL8 exists as monomers and dimers but monomer alone binds CXCR1 with high affinity. CXCL8 function involves binding two distinct CXCR1 sites – the N-terminal domain (Site-I) and the extracellular/transmembrane domain (Site-II). Therefore, higher monomer affinity could be due...

  14. Uncoupling Intramolecular Processing and Substrate Hydrolysis in the N-terminal Nucleophile Hydrolase hASRGL1 by Circular Permutation

    OpenAIRE

    Li, Wenzong; Cantor, Jason R.; Yogesha, S.D.; Yang, Shirley; Chantranupong, Lynne; Liu, June Qingxia; Agnello, Giulia; Georgiou, George; Stone, Everett M.; Yan ZHANG

    2012-01-01

    The human asparaginase-like protein 1 (hASRGL1) catalyzes the hydrolysis of l-asparagine and isoaspartyl-dipeptides. As an N-terminal nucleophile (Ntn) hydrolase superfamily member, the active form of hASRGL1 is generated by an intramolecular cleavage step with Thr168 as the catalytic residue. However, in vitro, autoprocessing is incomplete (~50 %), fettering the biophysical characterization of hASRGL1. We circumvented this obstacle by constructing a circularly permuted hASRGL1 that uncoupled...

  15. Melanocortin-4 receptor activation inhibits c-Jun N-terminal kinase activity and promotes insulin signaling

    OpenAIRE

    Chai, Biaoxin; Li, Ji-Yao; Zhang, Weizhen; Wang, Hui; Mulholland, Michael W.

    2009-01-01

    The melanocortin system is crucial to regulation of energy homeostasis. The melanocortin receptor type 4 (MC4R) modulates insulin signaling via effects on c-Jun N-terminal kinase (JNK). The melanocortin agonist NDP-MSH dose-dependently inhibited JNK activity in HEK293 cells stably expressing the human MC4R; effects were reversed by melanocortin receptor antagonist. NDP-MSH time- and dose-dependently inhibited IRS-1ser307 phosphorylation, effects also reversed by a specific melanocortin recept...

  16. Insights into the Functional Roles of N-Terminal and C-Terminal Domains of Helicobacter pylori DprA

    OpenAIRE

    Dwivedi, Gajendradhar R.; Kolluru D Srikanth; Praveen Anand; Javed Naikoo; Srilatha, N. S.; Rao, Desirazu N.

    2015-01-01

    DNA processing protein A (DprA) plays a crucial role in the process of natural transformation. This is accomplished through binding and subsequent protection of incoming foreign DNA during the process of internalization. DprA along with Single stranded DNA binding protein A (SsbA) acts as an accessory factor for RecA mediated DNA strand exchange. H. pylori DprA (HpDprA) is divided into an N-terminal domain and a C- terminal domain. In the present study, individual domains of HpDprA have been ...

  17. 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......, thus binding the N-terminus tightly to domain 2. We propose that this interaction is needed for aminoacyl-tRNA binding, and also for completing the structural rearrangement, which takes place when the factor switches from its GDP to its GTP form. Udgivelsesdato: 1997-Aug...

  18. 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

    .041) was closely associated with NT-pro-BNP levels above the median (1,381 pg/ml) after adjustment for traditional confounders (left ventricular ejection fraction, age, body mass index, atrial fibrillation, chronic kidney disease). In an adjusted Cox proportional hazard model, the 2 parameters were associated......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

  19. 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...... with spontaneous lipid/peptide interactions. Both canine and porcine peptides were able to form monolayers at air-liquid interfaces, the canine peptides occupying lower area/molecule and being compressible to higher pressures than the porcine sequences. The peptides also shifted the isotherms and perturbed...

  20. 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

    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...

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

    DEFF Research Database (Denmark)

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

    2013-01-01

    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....

  2. Enhancement of humoral immune responses to HBsAg by heat shock protein gp96 and its N-terminal fragment in mice

    Institute of Scientific and Technical Information of China (English)

    Hong-Tao Li; Jia-Bin Yan; Jing Li; Ming-Hai Zhou; Xiao-Dong Zhu; Yu-Xia Zhang; Po Tien

    2005-01-01

    AIM: Most studies on the immune effect of gp96 were focused on its enhancement of CTLs. It is interesting to know whether gp96 could influence the humoral immune response, and whether the recombinant N-terminal fragment of gp96 could substitute native gp96 to stimulate the immune system.METHODS: gp96 isolated from livers of normal mice and its N-terminal fragment (amino acid 22-355) expressed in E coli were used for immunization of BALb/c mice. Eight groups of mice received one of the following regiments subcutaneously in 100 μL phosphate buffered saline (PBS)at an interval of 3 wk. Group 1: PBS only; group 2:gp96 only; group 3: N-terminal fragment only; group 4: HBsAg only; group 5: HBsAg+gp96; group 6: HBsAg+N-terminalfragment; group 7: HBsAg+incomplete Freud's adjuvant; group 8: HBsAg+N-terminal fragment (95 ℃ heated for 30 min). Serum anti-HBsAg antibody levels were assayed by ELISA. CTL responses in splenocytes were analyzed by ELISPOT after the last vaccination.RESULTS: The average titer of serum anti-HBsAg antibodyin the mice immunized with HBsAg together with gp96 or its N-terminal fragment were much higher than those immunized with HBsAg alone detected by ELISA. The cellular immune response of the mice immunized with HBsAg together with gp96 or its N-terminal fragment was not different with those immunized with HBsAg alone measured by ELISPOT assay.CONCLUSION: gp96 or its N-terminal fragment greatly improved humoral immune response induced by HBsAg, but failed to enhance the CTL response, which demonstrated the potential of using gp96 or its N-terminal fragment as a possible adjuvant to augment humoral immune response against HBV infection.

  3. Regulatory Light Chain Phosphorylation and N-Terminal Extension Increase Cross-Bridge Binding and Power Output in Drosophila at In Vivo Myofilament Lattice Spacing

    OpenAIRE

    Miller, Mark S.; Farman, Gerrie P.; Braddock, Joan M.; Soto-Adames, Felipe N.; Irving, Thomas C.; Vigoreaux, Jim O.; Maughan, David W.

    2011-01-01

    The N-terminal extension and phosphorylation of the myosin regulatory light chain (RLC) independently improve Drosophila melanogaster flight performance. Here we examine the functional and structural role of the RLC in chemically skinned fibers at various thick and thin filament lattice spacings from four transgenic Drosophila lines: rescued null or control (Dmlc2+), truncated N-terminal extension (Dmlc2Δ2-46), disrupted myosin light chain kinase phosphorylation sites (Dmlc2S66A,S67A), and du...

  4. Trunk fatigue profile and low back pain in tennis players

    OpenAIRE

    Correia, José Pedro

    2013-01-01

    Mestrado em Ciências da Fisioterapia Introduction The trunk plays an important role in tennis strokes. Its asymmetric muscle activation, coupled with the high repeatability of the sport, places tennis players at risk for injuries such as low back pain. Objectives This study aimed to present a trunk fatigue profile in tennis players and verify its association with low back pain (LBP). Material and Methods 35 tennis players completed an isometric trunk endurance protocol co...

  5. Trunk robot rehabilitation training with active stepping reorganizes and enriches trunk motor cortex representations in spinal transected rats.

    Science.gov (United States)

    Oza, Chintan S; Giszter, Simon F

    2015-05-01

    Trunk motor control is crucial for postural stability and propulsion after low thoracic spinal cord injury (SCI) in animals and humans. Robotic rehabilitation aimed at trunk shows promise in SCI animal models and patients. However, little is known about the effect of SCI and robot rehabilitation of trunk on cortical motor representations. We previously showed reorganization of trunk motor cortex after adult SCI. Non-stepping training also exacerbated some SCI-driven plastic changes. Here we examine effects of robot rehabilitation that promotes recovery of hindlimb weight support functions on trunk motor cortex representations. Adult rats spinal transected as neonates (NTX rats) at the T9/10 level significantly improve function with our robot rehabilitation paradigm, whereas treadmill-only trained do not. We used intracortical microstimulation to map motor cortex in two NTX groups: (1) treadmill trained (control group); and (2) robot-assisted treadmill trained (improved function group). We found significant robot rehabilitation-driven changes in motor cortex: (1) caudal trunk motor areas expanded; (2) trunk coactivation at cortex sites increased; (3) richness of trunk cortex motor representations, as examined by cumulative entropy and mutual information for different trunk representations, increased; (4) trunk motor representations in the cortex moved toward more normal topography; and (5) trunk and forelimb motor representations that SCI-driven plasticity and compensations had caused to overlap were segregated. We conclude that effective robot rehabilitation training induces significant reorganization of trunk motor cortex and partially reverses some plastic changes that may be adaptive in non-stepping paraplegia after SCI.

  6. Calpain-Mediated Processing of Adenylate Cyclase Toxin Generates a Cytosolic Soluble Catalytically Active N-Terminal Domain.

    Directory of Open Access Journals (Sweden)

    Kepa B Uribe

    Full Text Available Bordetella pertussis, the whooping cough pathogen, secretes several virulence factors among which adenylate cyclase toxin (ACT is essential for establishment of the disease in the respiratory tract. ACT weakens host defenses by suppressing important bactericidal activities of the phagocytic cells. Up to now, it was believed that cell intoxication by ACT was a consequence of the accumulation of abnormally high levels of cAMP, generated exclusively beneath the host plasma membrane by the toxin N-terminal catalytic adenylate cyclase (AC domain, upon its direct translocation across the lipid bilayer. Here we show that host calpain, a calcium-dependent Cys-protease, is activated into the phagocytes by a toxin-triggered calcium rise, resulting in the proteolytic cleavage of the toxin N-terminal domain that releases a catalytically active "soluble AC". The calpain-mediated ACT processing allows trafficking of the "soluble AC" domain into subcellular organella. At least two strategic advantages arise from this singular toxin cleavage, enhancing the specificity of action, and simultaneously preventing an indiscriminate activation of cAMP effectors throughout the cell. The present study provides novel insights into the toxin mechanism of action, as the calpain-mediated toxin processing would confer ACT the capacity for a space- and time-coordinated production of different cAMP "pools", which would play different roles in the cell pathophysiology.

  7. Ubiquitin proteasome-dependent degradation of the transcriptional coactivator PGC-1{alpha} via the N-terminal pathway.

    Science.gov (United States)

    Trausch-Azar, Julie; Leone, Teresa C; Kelly, Daniel P; Schwartz, Alan L

    2010-12-17

    PGC-1α is a potent, inducible transcriptional coactivator that exerts control on mitochondrial biogenesis and multiple cellular energy metabolic pathways. PGC-1α levels are controlled in a highly dynamic manner reflecting regulation at both transcriptional and post-transcriptional levels. Here, we demonstrate that PGC-1α is rapidly degraded in the nucleus (t(½ 0.3 h) via the ubiquitin proteasome system. An N-terminal deletion mutant of 182 residues, PGC182, as well as a lysine-less mutant form, are nuclear and rapidly degraded (t(½) 0.5 h), consistent with degradation via the N terminus-dependent ubiquitin subpathway. Both PGC-1α and PGC182 degradation rates are increased in cells under low serum conditions. However, a naturally occurring N-terminal splice variant of 270 residues, NT-PGC-1α is cytoplasmic and stable (t(½>7 h), providing additional evidence that PGC-1α is degraded in the nucleus. These results strongly suggest that the nuclear N terminus-dependent ubiquitin proteasome pathway governs PGC-1α cellular degradation. In contrast, the cellular localization of NT-PCG-1α results in a longer-half-life and possible distinct temporal and potentially biological actions.

  8. Direct interaction of the N-terminal domain of ribosomal protein S1 with protein S2 in Escherichia coli.

    Science.gov (United States)

    Byrgazov, Konstantin; Manoharadas, Salim; Kaberdina, Anna C; Vesper, Oliver; Moll, Isabella

    2012-01-01

    Despite of the high resolution structure available for the E. coli ribosome, hitherto the structure and localization of the essential ribosomal protein S1 on the 30 S subunit still remains to be elucidated. It was previously reported that protein S1 binds to the ribosome via protein-protein interaction at the two N-terminal domains. Moreover, protein S2 was shown to be required for binding of protein S1 to the ribosome. Here, we present evidence that the N-terminal domain of S1 (amino acids 1-106; S1(106)) is necessary and sufficient for the interaction with protein S2 as well as for ribosome binding. We show that over production of protein S1(106) affects E. coli growth by displacing native protein S1 from its binding pocket on the ribosome. In addition, our data reveal that the coiled-coil domain of protein S2 (S2α(2)) is sufficient to allow protein S1 to bind to the ribosome. Taken together, these data uncover the crucial elements required for the S1/S2 interaction, which is pivotal for translation initiation on canonical mRNAs in gram-negative bacteria. The results are discussed in terms of a model wherein the S1/S2 interaction surface could represent a possible target to modulate the selectivity of the translational machinery and thereby alter the translational program under distinct conditions.

  9. N-terminal tetrapeptide T/SPLH motifs contribute to multimodal activation of human TRPA1 channel

    Science.gov (United States)

    Hynkova, Anna; Marsakova, Lenka; Vaskova, Jana; Vlachova, Viktorie

    2016-06-01

    Human transient receptor potential ankyrin channel 1 (TRPA1) is a polymodal sensor implicated in pain, inflammation and itching. An important locus for TRPA1 regulation is the cytoplasmic N-terminal domain, through which various exogenous electrophilic compounds such as allyl-isothiocyanate from mustard oil or cinnamaldehyde from cinnamon activate primary afferent nociceptors. This major region is comprised of a tandem set of 17 ankyrin repeats (AR1-AR17), five of them contain a strictly conserved T/SPLH tetrapeptide motif, a hallmark of an important and evolutionarily conserved contribution to conformational stability. Here, we characterize the functional consequences of putatively stabilizing and destabilizing mutations in these important structural units and identify AR2, AR6, and AR11-13 to be distinctly involved in the allosteric activation of TRPA1 by chemical irritants, cytoplasmic calcium, and membrane voltage. Considering the potential involvement of the T/SP motifs as putative phosphorylation sites, we also show that proline-directed Ser/Thr kinase CDK5 modulates the activity of TRPA1, and that T673 outside the AR-domain is its only possible target. Our data suggest that the most strictly conserved N-terminal ARs define the energetics of the TRPA1 channel gate and contribute to chemical-, calcium- and voltage-dependence.

  10. Basic amino acid residues located in the N-terminal region of BEND3 are essential for its nuclear localization

    International Nuclear Information System (INIS)

    BEN domain-containing protein 3 (BEND3) has recently been reported to function as a heterochromatin-associated protein in transcriptional repression in the nucleus. BEND3 should have nuclear localization signals (NLSs) to localize to the nucleus in light of its molecular weight, which is higher than that allowed to pass through nuclear pore complexes. We here analyzed the subcellular localization of deletion/site-directed mutants of human BEND3 by an immunofluorescence assay in an attempt to identify the amino acids essential for its nuclear localization. We found that three basic amino acid residues located in the N-terminal region of BEND3 (BEND356–58, KRK) are essential, suggesting that these residues play a role as a functional NLS. These results provide valuable information for progressing research on BEND3. - Highlights: • BEND3 localizes to the nucleus. • The N-terminal 60 amino acids region of BEND3 contains NLS. • Amino acids located between 56 and 58 of BEND3 (KRK) are part of NLS. • KRK motif is highly conserved among BEND3 homologs

  11. A new general pathway for synthesis of reference compounds of N-terminal valine-isocyanate adducts.

    Science.gov (United States)

    Davies, Ronnie; Rydberg, Per; Westberg, Emelie; Motwani, Hitesh V; Johnstone, Erik; Törnqvist, Margareta

    2010-03-15

    Adducts to Hb could be used as biomarkers to monitor exposure to isocyanates. Particularly useful is the measurement of carbamoylation of N-terminal valines in Hb, after detachment as hydantoins. The synthesis of references from the reactive isocyanates, especially diisocyanates, has been problematic due to side reactions and polymerization of the isocyanate starting material. A simpler, safer, and more general method for the synthesis of valine adducts of isocyanates has been developed using N-[(4-nitrophenyl)carbamate]valine methylamide (NPCVMA) as the key precursor to adducts of various mono- and diisocyanates of interest. By reacting NPCVMA with a range of isocyanate-related amines, carbamoylated valines are formed without the use of the reactive isocyanates. The carbamoylated products synthesized here were cyclized with good yields of the formed hydantoins. The carbamoylated derivative from phenyl isocyanate also showed quantitative yield in a test with cyclization under the conditions used in blood. This new pathway for the preparation of N-carbamoylated model compounds overcomes the above-mentioned problems in the synthesis and is a general and simplified approach, which could make such reference compounds of adducts to N-terminal valine from isocyanates accessible for biomonitoring purposes. The synthesized hydantoins corresponding to adducts from isocyanic acid, methyl isocyanate, phenyl isocyanate, and 2,6-toluene diisocyanate were characterized by LC-MS analysis. The background level of the hydantoin from isocyanic acid in human blood was analyzed with the LC-MS conditions developed.

  12. The N-terminal Part of Arabidopsis thaliana Starch Synthase 4 Determines the Localization and Activity of the Enzyme.

    Science.gov (United States)

    Raynaud, Sandy; Ragel, Paula; Rojas, Tomás; Mérida, Ángel

    2016-05-13

    Starch synthase 4 (SS4) plays a specific role in starch synthesis because it controls the number of starch granules synthesized in the chloroplast and is involved in the initiation of the starch granule. We showed previously that SS4 interacts with fibrillins 1 and is associated with plastoglobules, suborganelle compartments physically attached to the thylakoid membrane in chloroplasts. Both SS4 localization and its interaction with fibrillins 1 were mediated by the N-terminal part of SS4. Here we show that the coiled-coil region within the N-terminal portion of SS4 is involved in both processes. Elimination of this region prevents SS4 from binding to fibrillins 1 and alters SS4 localization in the chloroplast. We also show that SS4 forms dimers, which depends on a region located between the coiled-coil region and the glycosyltransferase domain of SS4. This region is highly conserved between all SS4 enzymes sequenced to date. We show that the dimerization seems to be necessary for the activity of the enzyme. Both dimerization and the functionality of the coiled-coil region are conserved among SS4 proteins from phylogenetically distant species, such as Arabidopsis and Brachypodium This finding suggests that the mechanism of action of SS4 is conserved among different plant species. PMID:26969163

  13. N-terminal tetrapeptide T/SPLH motifs contribute to multimodal activation of human TRPA1 channel.

    Science.gov (United States)

    Hynkova, Anna; Marsakova, Lenka; Vaskova, Jana; Vlachova, Viktorie

    2016-01-01

    Human transient receptor potential ankyrin channel 1 (TRPA1) is a polymodal sensor implicated in pain, inflammation and itching. An important locus for TRPA1 regulation is the cytoplasmic N-terminal domain, through which various exogenous electrophilic compounds such as allyl-isothiocyanate from mustard oil or cinnamaldehyde from cinnamon activate primary afferent nociceptors. This major region is comprised of a tandem set of 17 ankyrin repeats (AR1-AR17), five of them contain a strictly conserved T/SPLH tetrapeptide motif, a hallmark of an important and evolutionarily conserved contribution to conformational stability. Here, we characterize the functional consequences of putatively stabilizing and destabilizing mutations in these important structural units and identify AR2, AR6, and AR11-13 to be distinctly involved in the allosteric activation of TRPA1 by chemical irritants, cytoplasmic calcium, and membrane voltage. Considering the potential involvement of the T/SP motifs as putative phosphorylation sites, we also show that proline-directed Ser/Thr kinase CDK5 modulates the activity of TRPA1, and that T673 outside the AR-domain is its only possible target. Our data suggest that the most strictly conserved N-terminal ARs define the energetics of the TRPA1 channel gate and contribute to chemical-, calcium- and voltage-dependence. PMID:27345869

  14. The N-terminal tropomyosin- and actin-binding sites are important for leiomodin 2's function.

    Science.gov (United States)

    Ly, Thu; Moroz, Natalia; Pappas, Christopher T; Novak, Stefanie M; Tolkatchev, Dmitri; Wooldridge, Dayton; Mayfield, Rachel M; Helms, Gregory; Gregorio, Carol C; Kostyukova, Alla S

    2016-08-15

    Leiomodin is a potent actin nucleator related to tropomodulin, a capping protein localized at the pointed end of the thin filaments. Mutations in leiomodin-3 are associated with lethal nemaline myopathy in humans, and leiomodin-2-knockout mice present with dilated cardiomyopathy. The arrangement of the N-terminal actin- and tropomyosin-binding sites in leiomodin is contradictory and functionally not well understood. Using one-dimensional nuclear magnetic resonance and the pointed-end actin polymerization assay, we find that leiomodin-2, a major cardiac isoform, has an N-terminal actin-binding site located within residues 43-90. Moreover, for the first time, we obtain evidence that there are additional interactions with actin within residues 124-201. Here we establish that leiomodin interacts with only one tropomyosin molecule, and this is the only site of interaction between leiomodin and tropomyosin. Introduction of mutations in both actin- and tropomyosin-binding sites of leiomodin affected its localization at the pointed ends of the thin filaments in cardiomyocytes. On the basis of our new findings, we propose a model in which leiomodin regulates actin poly-merization dynamics in myocytes by acting as a leaky cap at thin filament pointed ends.

  15. 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. PMID:21571015

  16. c-Jun N-Terminal Phosphorylation: Biomarker for Cellular Stress Rather than Cell Death in the Injured Cochlea.

    Science.gov (United States)

    Anttonen, Tommi; Herranen, Anni; Virkkala, Jussi; Kirjavainen, Anna; Elomaa, Pinja; Laos, Maarja; Liang, Xingqun; Ylikoski, Jukka; Behrens, Axel; Pirvola, Ulla

    2016-01-01

    Prevention of auditory hair cell death offers therapeutic potential to rescue hearing. Pharmacological blockade of JNK/c-Jun signaling attenuates injury-induced hair cell loss, but with unsolved mechanisms. We have characterized the c-Jun stress response in the mouse cochlea challenged with acoustic overstimulation and ototoxins, by studying the dynamics of c-Jun N-terminal phosphorylation. It occurred acutely in glial-like supporting cells, inner hair cells, and the cells of the cochlear ion trafficking route, and was rapidly downregulated after exposures. Notably, death-prone outer hair cells lacked c-Jun phosphorylation. As phosphorylation was triggered also by nontraumatic noise levels and none of the cells showing this activation were lost, c-Jun phosphorylation is a biomarker for cochlear stress rather than an indicator of a death-prone fate of hair cells. Preconditioning with a mild noise exposure before a stronger traumatizing noise exposure attenuated the cochlear c-Jun stress response, suggesting that the known protective effect of sound preconditioning on hearing is linked to suppression of c-Jun activation. Finally, mice with mutations in the c-Jun N-terminal phosphoacceptor sites showed partial, but significant, hair cell protection. These data identify the c-Jun stress response as a paracrine mechanism that mediates outer hair cell death. PMID:27257624

  17. Human IgG responses against the N-terminal region of Merozoite Surface Protein 1 of Plasmodium vivax

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    Hernando Antonio Del Portillo

    1992-01-01

    Full Text Available The complete primary structure of the gene encoding the Merozoite Surface Protein 1 of Plasmodium vivax (PvMSP-1 revealed the existence of interspecies conserved regions among the analogous proteins of other Plasmodia species. Here, three DNA recombinant clones expressing 50, 200 and 500 amino acids from the N-terminal region of the PvMSP-1 protein were used on ELISA and protein immunoblotting assays to look at the IgG antibody responses of malaria patients from the Brasilian amazon region of Rondônia. The results showed the existance of P. vivax and P. falciparum IgG antibodies directed against PvMSP-1 antigenic determinants expressed in the clones containing the first 200 and the following 500 amino acids of the molecule, but not within the one expressing the most N-terminal 50 amino acids. Interestingly, there was no correlation between the levels of these IgG antibodies and the previous number of malaria infections.

  18. Solution structure and membrane-binding property of the N-terminal tail domain of human annexin I.

    Science.gov (United States)

    Yoon, M K; Park, S H; Won, H S; Na, D S; Lee, B J

    2000-11-10

    The conformational preferences of AnxI(N26), a peptide corresponding to residues 2-26 of human annexin I, were investigated using CD and NMR spectroscopy. CD results showed that AnxI(N26) adopts a mainly alpha-helical conformation in membrane-mimetic environments, TFE/water and SDS micelles, while a predominantly random structure with slight helical propensity in aqueous buffer. The helical region of AnxI(N26) showed a nearly identical conformation between in TFE/water and in SDS micelles, except for the orientation of the Trp-12 side-chain, which was quite different between the two. The N-terminal region of the AnxI(N26) helix showed a typical amphipathic nature, which could be stabilized by the neighboring hydrophobic cluster. The helical stability of the peptide in SDS micelles was increased by addition of calcium ions. These results suggest that the N-terminal tail domain of human annexin I interacts with biological membranes in a partially calcium-dependent manner.

  19. Interaction between GInB and the N-terminal domain of NifA in Azospirillum brasilense

    Institute of Scientific and Technical Information of China (English)

    ZHOU XiaoYu; ZOU XiaoXiao; LI JiLun

    2008-01-01

    Azospirillum brasilense is a diazotroph associated with many important agricultural crops and shows potential as a biofertilizer. NifA, the transcriptional activator of nitrogen fixation (nif) genes, and GInB, one of P,, signal transduction family protein, are key proteins in the regulation of nitrogen fixation in A. brasilense. It was previously reported that the regulation of NifA activity in A. brasilense depends on GInB. We report here that GInB was found to interact directly with the N-terminal domain of NifA in vivo under nitrogen-free conditions and the N-terminal mutant of NifA in which the Tyr residues at position 18 and 53 were replaced by Phe (NifA-N-Y18/53F) strengthened the interaction with GInB. Moreover, we also found that the amino acid residues 66-88 and 165-176 in N-terminus of NifA are responsible for the interaction with GInB.

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

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    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.

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

    Science.gov (United States)

    Doritchamou, Justin; Sabbagh, Audrey; Jespersen, Jakob S; Renard, Emmanuelle; Salanti, Ali; Nielsen, Morten A; Deloron, Philippe; Tuikue Ndam, Nicaise

    2015-01-01

    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. PMID:26393516

  2. The effect of superficial trunk muscle exercise and deep trunk muscle exercise on the foot pressure of healthy adults

    OpenAIRE

    Kim, Suzy; Shim, JeMyung; Kim, SungJoong; Namkoong, Seung; Kim, Hwanhee

    2015-01-01

    [Purpose] The purpose of this study was to analyze the effect of superficial trunk muscle exercise and deep trunk muscle exercise on the foot pressure of healthy adults. [Subjects] The subjects were 30 healthy females and males who agreed to participate in this study. There were two groups, a superficial trunk muscle exercise group and a deep trunk muscle exercise group, with 15 participants in each. [Methods] The exercises were conducted 5 times a week for 4 weeks for both groups. A gait ana...

  3. Trunk exercises performed on an unstable surface improve trunk muscle activation, postural control, and gait speed in patients with stroke.

    Science.gov (United States)

    Jung, Kyoung-Sim; Cho, Hwi-Young; In, Tae-Sung

    2016-03-01

    [Purpose] This study examined the effects of trunk exercises performed on an unstable surface on trunk muscle activation, postural control, and gait speed in stroke patients. [Subjects] Twenty-four participants with stroke were recruited in this study and randomly distributed into experimental (n = 12) and control groups (n = 12). [Methods] Subjects in the experimental group participated in trunk exercises on the balance pad for 30 min, five times a week for 4 weeks; those in the control group performed trunk exercises on a stable surface for 30 min, five times a week for 4 weeks. Trunk muscle activation was measured by using surface electromyography, and trunk control was evaluated with the Trunk Impairment Scale (TIS). Gait speed was measured with the 10-Meter Walk Test. [Results] Activity of the external and internal oblique muscles in the experimental group was significantly higher than that in the control group. The TIS score of the experimental group showed significantly greater improvement than did that of the control group. The 10-Meter Walk Test (10MWT) score also significantly improved in the experimental group. [Conclusion] Trunk exercises on an unstable surface improve trunk muscle activation, postural control, and gait speed in patients with hemiparetic stroke.

  4. Identification and analysis of the acetylated status of poplar proteins reveals analogous N-terminal protein processing mechanisms with other eukaryotes.

    Directory of Open Access Journals (Sweden)

    Chang-Cai Liu

    Full Text Available BACKGROUND: The N-terminal protein processing mechanism (NPM including N-terminal Met excision (NME and N-terminal acetylation (N(α-acetylation represents a common protein co-translational process of some eukaryotes. However, this NPM occurred in woody plants yet remains unknown. METHODOLOGY/PRINCIPAL FINDINGS: To reveal the NPM in poplar, we investigated the N(α-acetylation status of poplar proteins during dormancy by combining tandem mass spectrometry with TiO2 enrichment of acetylated peptides. We identified 58 N-terminally acetylated (N(α-acetylated proteins. Most proteins (47, >81% are subjected to N(α-acetylation following the N-terminal removal of Met, indicating that N(α-acetylation and NME represent a common NPM of poplar proteins. Furthermore, we confirm that poplar shares the analogous NME and N(α-acetylation (NPM to other eukaryotes according to analysis of N-terminal features of these acetylated proteins combined with genome-wide identification of the involving methionine aminopeptidases (MAPs and N-terminal acetyltransferase (Nat enzymes in poplar. The N(α-acetylated reactions and the involving enzymes of these poplar proteins are also identified based on those of yeast and human, as well as the subcellular location information of these poplar proteins. CONCLUSIONS/SIGNIFICANCE: This study represents the first extensive investigation of N(α-acetylation events in woody plants, the results of which will provide useful resources for future unraveling the regulatory mechanisms of N(α-acetylation of proteins in poplar.

  5. Trunk Flexibility Improvement in Response to Powered Assisted Exercise

    Directory of Open Access Journals (Sweden)

    B.S. Bains

    2016-07-01

    Full Text Available Background study: Flexibility in human spine has always plays an important role in dexterity and seamless ambulatory activities. When optimum range is not maintained by the trunk column, due to lack of flexibility, the posture gets affected resulting in reduce trunk rotation flexibility and mobility hence loss of complete trunk rotation. Objective: The purpose of this study is to determine the effect of Shapemaster Power Assisted Exercise Equipment (SPAEE on trunk flexibility. Methodology: Twenty healthy individual ages between 40 to 60 years were randomly divided into control and exercise groups. Shapemaster exercise program performed two times per week for 5 weeks and 45 minutes per session. Before and after 10 sessions of Shapemaster exercise protocol, Seated trunk rotation test was used to measure trunk flexibility. Results: Repeated measurement ANOVA were used to analysis data between groups. The results of this study illustrated that after 10th sessions trunk flexibility significantly improved (F (1.0, 18 = 11.732, p < 0.003. Conclusion: In conclusion results were determined that SPAEE is safe and it did effectively enhance flexibility among individual healthy adults.Keyword: Shapemaster Power Assisted Exercise Equipment (SPAEE, Trunk Flexibility, Healthy individual

  6. Mechanical trunk in pine wood for cattle

    Directory of Open Access Journals (Sweden)

    Antonio Orlando da Luz Freire Neto

    2012-12-01

    Full Text Available The timber reforestation, mainly by Eucalyptus and Pinus sp., has low power processing, strength, good natural durability and, most importantly, provide reduce pressure on native forests. The concern with native forests and the high price of some of these woods force the market to replace those species by other, more abundant and available at most competitive prices. Anything that involves the handling of animals in its various phases has a direct dependency of husbandry facilities, pastures and actions of the people involved (best practices. With the segment of the production and export of meat increasingly competitive and globalized world, the adoption of best practices and animal welfare criteria are striking and decision makers for the acceptance of Brazilian beef in the world market, especially the European market. The use of appropriate animal husbandry facilities is critical to the proper rational management ("action with knowledge" of animals and increased productivity. The trunk restraint carries important role in the implementation and conduct of good animal welfare as having desirable features strength, durability, ability to contain cattle of various sizes, as well as easy to manipulate when the animal inside. Available on the market in the form of different models and costs, is an installation manufactured in wood and iron or galvanized, and may or may not be coupled with an analytical balance or digital, still and mechanical and other systems or electronics. The concern in this installation is perceived improvement in the number of patents filed and recorded and the constant evolution of their functions, with various companies operating in this segment. However, the development and validation of containment trunks with alternative materials, reflecting mainly the reduction of the final cost are poorly studied. In this first phase of the project will be considered the construction of trunk restraint coupled with analytical balance

  7. Structure and dynamics of the N-terminal domain of the Cu(I) binding protein CusB.

    Science.gov (United States)

    Ucisik, Melek N; Chakravorty, Dhruva K; Merz, Kenneth M

    2013-10-01

    CusCFBA is one of the metal efflux systems in Escherichia coli that is highly specific for its substrates, Cu(I) and Ag(I). It serves to protect the bacteria in environments that have lethal concentrations of these metals. The membrane fusion protein CusB is the periplasmic piece of CusCFBA, which has not been fully characterized by crystallography because of its extremely disordered N-terminal region. This region has both structural and functional importance because it has been experimentally proven to transfer the metal by itself from the metallochaperone CusF and to induce a structural change in the rest of CusB to increase Cu(I)/Ag(I) resistance. Understanding metal uptake from the periplasm is critical to gain insight into the mechanism of the whole CusCFBA pump, which makes resolving a structure for the N-terminal region necessary because it contains the metal binding site. We ran extensive molecular dynamics simulations to reveal the structural and dynamic properties of both the apo and Cu(I)-bound versions of the CusB N-terminal region. In contrast to its functional companion CusF, Cu(I) binding to the N-terminus of CusB causes only a slight, local stabilization around the metal site. The trajectories were analyzed in detail, revealing extensive structural disorder in both the apo and holo forms of the protein. CusB was further analyzed by breaking the protein up into three subdomains according to the extent of the observed disorder: the N- and C-terminal tails, the central beta strand motif, and the M21-M36 loop connecting the two metal-coordinating methionine residues. Most of the observed disorder was traced back to the tail regions, leading us to hypothesize that the latter two subdomains (residues 13-45) may form a functionally competent metal-binding domain because the tail regions appear to play no role in metal binding. PMID:23988152

  8. ANOMALOUS BRANCHING PATTERN OF COELIAC TRUNK – A CASE REPORT

    Directory of Open Access Journals (Sweden)

    Deena Usha

    2015-08-01

    Full Text Available While doing a routine dissection in an 85 years old cadaver, it was observed that the coeliac trunk arised as gastro splenic trunk and the common hepatic artery arised from the superior mesenteric artery instea d of the coeliac trunk. Knowledge of anatomical variants of coeliac trunk, hepatic vascular structures is of great importance in general surgery, especially hepatic surgery. As coeliac trunk supplies the foregut, the anomaly and morphometric variation may lead to abnormalities of the foregut and its derivatives. This leads to anatomical, surgical and clinical complications. This knowledge is also of great importance with regard to liver transplantations, laparoscopic surgeries, radiological procedures, and the treatment of penetrating injuries involving the peri - hepatic area

  9. All joint moments significantly contribute to trunk angular acceleration.

    Science.gov (United States)

    Nott, Cameron R; Zajac, Felix E; Neptune, Richard R; Kautz, Steven A

    2010-09-17

    Computationally advanced biomechanical analyses of gait demonstrate the often counter-intuitive roles of joint moments on various aspects of gait such as propulsion, swing initiation, and balance. Each joint moment can produce linear and angular acceleration of all body segments (including those on which the moment does not directly act) due to the dynamic coupling inherent in the interconnected musculoskeletal system. This study presents quantitative relationships between individual joint moments and trunk control with respect to balance during gait to show that the ankle, knee, and hip joint moments all affect the angular acceleration of the trunk. We show that trunk angular acceleration is affected by all joints in the leg with varying degrees of dependence during the gait cycle. Furthermore, it is shown that inter-planar coupling exists and a two-dimensional analysis of trunk balance neglects important out-of-plane joint moments that affect trunk angular acceleration.

  10. The N-terminal domain plays a crucial role in the structure of a full-length human mitochondrial Lon protease

    Science.gov (United States)

    Kereïche, Sami; Kováčik, Lubomír; Bednár, Jan; Pevala, Vladimír; Kunová, Nina; Ondrovičová, Gabriela; Bauer, Jacob; Ambro, Ľuboš; Bellová, Jana; Kutejová, Eva; Raška, Ivan

    2016-01-01

    Lon is an essential, multitasking AAA+ protease regulating many cellular processes in species across all kingdoms of life. Altered expression levels of the human mitochondrial Lon protease (hLon) are linked to serious diseases including myopathies, paraplegia, and cancer. Here, we present the first 3D structure of full-length hLon using cryo-electron microscopy. hLon has a unique three-dimensional structure, in which the proteolytic and ATP-binding domains (AP-domain) form a hexameric chamber, while the N-terminal domain is arranged as a trimer of dimers. These two domains are linked by a narrow trimeric channel composed likely of coiled-coil helices. In the presence of AMP-PNP, the AP-domain has a closed-ring conformation and its N-terminal entry gate appears closed, but in ADP binding, it switches to a lock-washer conformation and its N-terminal gate opens, which is accompanied by a rearrangement of the N-terminal domain. We have also found that both the enzymatic activities and the 3D structure of a hLon mutant lacking the first 156 amino acids are severely disturbed, showing that hLon’s N-terminal domains are crucial for the overall structure of the hLon, maintaining a conformation allowing its proper functioning. PMID:27632940

  11. The solution structure of the N-terminal domain of human tubulin binding cofactor C reveals a platform for tubulin interaction.

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    Ma Flor Garcia-Mayoral

    Full Text Available Human Tubulin Binding Cofactor C (TBCC is a post-chaperonin involved in the folding and assembly of α- and β-tubulin monomers leading to the release of productive tubulin heterodimers ready to polymerize into microtubules. In this process it collaborates with other cofactors (TBC's A, B, D, and E and forms a supercomplex with TBCD, β-tubulin, TBCE and α-tubulin. Here, we demonstrate that TBCC depletion results in multipolar spindles and mitotic failure. Accordingly, TBCC is found at the centrosome and is implicated in bipolar spindle formation. We also determine by NMR the structure of the N-terminal domain of TBCC. The TBCC N-terminal domain adopts a spectrin-like fold topology composed of a left-handed 3-stranded α-helix bundle. Remarkably, the 30-residue N-terminal segment of the TBCC N-terminal domain is flexible and disordered in solution. This unstructured region is involved in the interaction with tubulin. Our data lead us to propose a testable model for TBCC N-terminal domain/tubulin recognition in which the highly charged N-terminus as well as residues from the three helices and the loops interact with the acidic hypervariable regions of tubulin monomers.

  12. The N-terminal domain plays a crucial role in the structure of a full-length human mitochondrial Lon protease.

    Science.gov (United States)

    Kereïche, Sami; Kováčik, Lubomír; Bednár, Jan; Pevala, Vladimír; Kunová, Nina; Ondrovičová, Gabriela; Bauer, Jacob; Ambro, Ľuboš; Bellová, Jana; Kutejová, Eva; Raška, Ivan

    2016-01-01

    Lon is an essential, multitasking AAA(+) protease regulating many cellular processes in species across all kingdoms of life. Altered expression levels of the human mitochondrial Lon protease (hLon) are linked to serious diseases including myopathies, paraplegia, and cancer. Here, we present the first 3D structure of full-length hLon using cryo-electron microscopy. hLon has a unique three-dimensional structure, in which the proteolytic and ATP-binding domains (AP-domain) form a hexameric chamber, while the N-terminal domain is arranged as a trimer of dimers. These two domains are linked by a narrow trimeric channel composed likely of coiled-coil helices. In the presence of AMP-PNP, the AP-domain has a closed-ring conformation and its N-terminal entry gate appears closed, but in ADP binding, it switches to a lock-washer conformation and its N-terminal gate opens, which is accompanied by a rearrangement of the N-terminal domain. We have also found that both the enzymatic activities and the 3D structure of a hLon mutant lacking the first 156 amino acids are severely disturbed, showing that hLon's N-terminal domains are crucial for the overall structure of the hLon, maintaining a conformation allowing its proper functioning. PMID:27632940

  13. Effect of Jun N-terminal kinase 1 and 2 on the replication of Penicillium marneffei in human macrophages.

    Science.gov (United States)

    Chen, Renqiong; Xi, Liyan; Huang, Xiaowen; Ma, Tuan; Ren, Hong; Ji, Guangquan

    2015-05-01

    Penicillium marneffei (P. marneffei) is a human pathogen which persists in macrophages and threatens the immunocompromised patients. To clarify the mechanisms involved, we evaluated the effect of c-Jun N-terminal kinase 1 and 2 (JNK1/2) on cytokine expression, phagosomal maturation and multiplication of P. marneffei in P. marneffei-stimulated human macrophages. P. marneffei induced the rapid phosphorylation of JNK1/2. Using the specific inhibitor of JNK1/2 (SP600125), we found that the inhibition of JNK1/2 suppressed P. marneffei-induced tumor necrosis factor-α and IL-10 production. In addition, the presence of SP600125 increased phagosomal acidification and maturation and decreased intracellular replication. These data suggest that JNK1/2 may play an important role in promoting the replication of P. marneffei. Our findings further indicate that the pathogen through the JNK1/2 pathway may attenuate the immune response and macrophage antifungal function.

  14. 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.

  15. Heart murmur and N-terminal pro-brain natriuretic peptide as predictors of death in 2977 consecutive hospitalized patients

    DEFF Research Database (Denmark)

    Iversen, Kasper; Nielsen, O.W.; Kirk, V.;

    2008-01-01

    -pro-BNP, discovery of valvular heart disease by echocardiography yielded no additional prognostic information. Conclusions: Detection of a cardiac murmur during routine medical examination of hospitalized patients is associated with increased risk of death within a year. A blood test for NT-pro-BNP gives significant...... valvular heart disease. We wanted to test whether murmur predicts mortality in unselected patients admitted to the hospital and whether NT-pro-BNP is capable of distinguishing between innocent and significant murmurs. Methods: Consecutive patients (n = 2977) older than 40 years admitted to a local hospital......Background: Little is known about the prognostic importance of murmur in unselected patients. It is difficult to distinguish between innocent and significant murmurs. N-terminal pro-brain natriuretic peptide (NT-pro-BNP) and BNP have recently been shown to be useful in small series of patients with...

  16. Regulation of apoptotic c-Jun N-terminal kinase signaling by a stabilization-based feed-forward loop.

    Science.gov (United States)

    Xu, Zhiheng; Kukekov, Nikolay V; Greene, Lloyd A

    2005-11-01

    A sequential kinase cascade culminating in activation of c-Jun N-terminal kinases (JNKs) plays a fundamental role in promoting apoptotic death in many cellular contexts. The mechanisms by which this pathway is engaged in response to apoptotic stimuli and suppressed in viable cells are largely unknown. Here, we show that apoptotic stimuli increase endogenous cellular levels of pathway components, including POSH, mixed lineage kinases (MLKs), and JNK interacting protein 1, and that this effect occurs through protein stabilization and requires the presence of POSH as well as activation of MLKs and JNKs. Our findings suggest a self-amplifying, feed-forward loop mechanism by which apoptotic stimuli promote the stabilization of JNK pathway components, thereby contributing to cell death. PMID:16260609

  17. Regulation of Apoptotic c-Jun N-Terminal Kinase Signaling by a Stabilization-Based Feed-Forward Loop†

    Science.gov (United States)

    Xu, Zhiheng; Kukekov, Nikolay V.; Greene, Lloyd A.

    2005-01-01

    A sequential kinase cascade culminating in activation of c-Jun N-terminal kinases (JNKs) plays a fundamental role in promoting apoptotic death in many cellular contexts. The mechanisms by which this pathway is engaged in response to apoptotic stimuli and suppressed in viable cells are largely unknown. Here, we show that apoptotic stimuli increase endogenous cellular levels of pathway components, including POSH, mixed lineage kinases (MLKs), and JNK interacting protein 1, and that this effect occurs through protein stabilization and requires the presence of POSH as well as activation of MLKs and JNKs. Our findings suggest a self-amplifying, feed-forward loop mechanism by which apoptotic stimuli promote the stabilization of JNK pathway components, thereby contributing to cell death. PMID:16260609

  18. FS23 binds to the N-terminal domain of human Hsp90:A novel small inhibitor for Hsp90

    Institute of Scientific and Technical Information of China (English)

    李健; 石峰; 陈丹琦; 曹慧玲; 熊兵; 沈竞康; 何建华

    2015-01-01

    The N-terminal domain of heat shock protein 90 (Hsp90N) is responsible for the catalytic activity of Hsp90. The reported inhibitors of Hsp90 bind to this domain and would inhibit tumor growth and progression. Here, we synthesized FS23, a small molecule inhibitor of hsp90 and collected X-ray diffraction data of the complex crystal of Hsp90-FS23. High resolution X-ray crystallography shows that FS23 interacted with Hsp90N at the nucleotide binding cleft, and this suggests that FS23 may complete with nucleotides to bind to Hsp90N. The crystal structure and the interaction between Hsp90N and FS23 suggest a rational basis for the design of novel antitumor drugs.

  19. 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...

  20. The influence of anaemia on stroke prognosis and its relation to N-terminal pro-brain natriuretic peptide

    DEFF Research Database (Denmark)

    Nybo, M; Kristensen, S R; Mickley, H;

    2007-01-01

    Anaemia is a negative prognostic factor for patients with heart failure and impaired renal function, but its role in stroke patients is unknown. Furthermore, anaemia has been shown to influence the level of N-terminal pro-brain natriuretic peptide (NT-proBNP), but this is only investigated...... in patients with heart failure, not in stroke patients. Two-hundred-and-fifty consecutive, well-defined ischemic stroke patients were investigated. Mortality was recorded at 6 months follow-up. Anaemia was diagnosed in 37 patients (15%) in whom stroke severity was worse than in the non-anaemic group, whilst...... the prevalence of renal affection, smoking and heart failure was lower. At 6 months follow-up, 23 patients were dead, and anaemia had an odds ratio of 4.7 when adjusted for age, Scandinavian Stroke Scale and a combined variable of heart and/or renal failure and/or elevation of troponin T using logistic...

  1. The cyanobacterial cell division factor Ftn6 contains an N-terminal DnaD-like domain

    Directory of Open Access Journals (Sweden)

    Saguez Cyril

    2009-08-01

    Full Text Available Abstract Background DNA replication and cell cycle as well as their relationship have been extensively studied in the two model organisms E. coli and B. subtilis. By contrast, little is known about these processes in cyanobacteria, even though they are crucial to the biosphere, in utilizing solar energy to renew the oxygenic atmosphere and in producing the biomass for the food chain. Recent studies have allowed the identification of several cell division factors that are specifics to cyanobacteria. Among them, Ftn6 has been proposed to function in the recruitment of the crucial FtsZ proteins to the septum or the subsequent Z-ring assembly and possibly in chromosome segregation. Results In this study, we identified an as yet undescribed domain located in the conserved N-terminal region of Ftn6. This 77 amino-acids-long domain, designated here as FND (Ftn6 N-Terminal Domain, exhibits striking sequence and structural similarities with the DNA-interacting module, listed in the PFAM database as the DnaD-like domain (pfam04271. We took advantage of the sequence similarities between FND and the DnaD-like domains to construct a homology 3D-model of the Ftn6 FND domain from the model cyanobacterium Synechocystis PCC6803. Mapping of the conserved residues exposed onto the FND surface allowed us to identify a highly conserved area that could be engaged in Ftn6-specific interactions. Conclusion Overall, similarities between FND and DnaD-like domains as well as previously reported observations on Ftn6 suggest that FND may function as a DNA-interacting module thereby providing an as yet missing link between DNA replication and cell division in cyanobacteria. Consistently, we also showed that Ftn6 is involved in tolerance to DNA damages generated by UV rays.

  2. 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

    2016-07-01

    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.

  3. Copper complex species within a fragment of the N-terminal repeat region in opossum PrP protein.

    Science.gov (United States)

    Vagliasindi, Laura I; Arena, Giuseppe; Bonomo, Raffaele P; Pappalardo, Giuseppe; Tabbì, Giovanni

    2011-03-21

    A spectroscopic (UV-Vis, CD and EPR), thermodynamic and voltammetric study of the copper(ii) complexes with the Ac-PHPGGSNWGQ-NH(2) polypeptide (L), a fragment of the opossum PrP protein N-terminal four-repeat region, was carried out in aqueous solution. It suggests the formation of a highly distorted [Cu(L)H(-2)] complex species in the neutral region, the stereochemistry of which is ascribable to a square base pyramid and a CuN(3)O(2) chromophore, resulting from the coordination of a histidine imidazole and two peptide nitrogen atoms and probably oxygen atoms from water molecules. At basic pH values a [Cu(L)H(-3)](-) species with a pseudo-octahedral geometry was also obtained, with four nitrogen donor atoms in its equatorial plane, coming from the histidine residue and from peptidic nitrogen atoms. Interestingly, at pH values relatively higher than the neutrality, the coordination sphere of the copper complex in the [Cu(L)H(-2)] species changes its stereochemistry towards a pseudo-octahedron, as suggested by the change in the parallel copper hyperfine coupling constant of the EPR spectra at low temperature. A slight difference in the redox potentials between this two-faced [Cu(L)H(-2)] complex species seems to confirm this behaviour. Both potentiometric and spectroscopic data were compared with the analogous species obtained with the Ac-PHGGGWGQ-NH(2) peptide, belonging to the octarepeat domain of the human prion protein (hPrP) N-terminal region. The [Cu(L)H(-2)] species formed by the Ac-PHPGGSNWGQ-NH(2) decapeptide, having a slightly lower stability, turned out to be less abundant and to exist within a narrow pH range. PMID:21283898

  4. Characterization of the N-terminal domain of BteA: a Bordetella type III secreted cytotoxic effector.

    Directory of Open Access Journals (Sweden)

    Chen Guttman

    Full Text Available BteA, a 69-kDa cytotoxic protein, is a type III secretion system (T3SS effector in the classical Bordetella, the etiological agents of pertussis and related mammalian respiratory diseases. Currently there is limited information regarding the structure of BteA or its subdomains, and no insight as to the identity of its eukaryotic partners(s and their modes of interaction with BteA. The mechanisms that lead to BteA dependent cell death also remain elusive. The N-terminal domain of BteA is multifunctional, acting as a docking platform for its cognate chaperone (BtcA in the bacterium, and targeting the protein to lipid raft microdomains within the eukaryotic host cell. In this study we describe the biochemical and biophysical characteristics of this domain (BteA287 and determine its architecture. We characterize BteA287 as being a soluble and highly stable domain which is rich in alpha helical content. Nuclear magnetic resonance (NMR experiments combined with size exclusion and analytical ultracentrifugation measurements confirm these observations and reveal BteA287 to be monomeric in nature with a tendency to oligomerize at concentrations above 200 µM. Furthermore, diffusion-NMR demonstrated that the first 31 residues of BteA287 are responsible for the apparent aggregation behavior of BteA287. Light scattering analyses and small angle X-ray scattering experiments reveal a prolate ellipsoidal bi-pyramidal dumb-bell shape. Thus, our biophysical characterization is a first step towards structure determination of the BteA N-terminal domain.

  5. Zinc(II) interactions with brain-derived neurotrophic factor N-terminal peptide fragments: inorganic features and biological perspectives.

    Science.gov (United States)

    Travaglia, Alessio; La Mendola, Diego; Magrì, Antonio; Pietropaolo, Adriana; Nicoletti, Vincenzo G; Grasso, Giuseppe; Malgieri, Gaetano; Fattorusso, Roberto; Isernia, Carla; Rizzarelli, Enrico

    2013-10-01

    Brain-derived neurotrophic factor (BDNF) is a neurotrophin essential for neuronal differentiation, growth, and survival; it is involved in memory formation and higher cognitive functions. The N-terminal domain of BDNF is crucial for the binding selectivity and activation of its specific TrkB receptor. Zn(2+) ion binding may influence BDNF activity. Zn(2+) complexes with the peptide fragment BDNF(1-12) encompassing the sequence 1-12 of the N-terminal domain of BDNF were studied by means of potentiometry, electrospray mass spectrometry, NMR, and density functional theory (DFT) approaches. The predominant Zn(2+) complex species, at physiological pH, is [ZnL] in which the metal ion is bound to an amino, an imidazole, and two water molecules (NH2, N(Im), and 2O(water)) in a tetrahedral environment. DFT-based geometry optimization of the zinc coordination environment showed a hydrogen bond between the carboxylate and a water molecule bound to zinc in [ZnL]. The coordination features of the acetylated form [AcBDNF(1-12)] and of a single mutated peptide [BDNF(1-12)D3N] were also characterized, highlighting the role of the imidazole side chain as the first anchoring site and ruling out the direct involvement of the aspartate residue in the metal binding. Zn(2+) addition to the cell culture medium induces an increase in the proliferative activity of the BDNF(1-12) peptide and of the whole protein on the SHSY5Y neuroblastoma cell line. The effect of Zn(2+) is opposite to that previously observed for Cu(2+) addition, which determines a decrease in the proliferative activity for both peptide and protein, suggesting that these metals might discriminate and modulate differently the activity of BDNF.

  6. PKC phosphorylates residues in the N-terminal of the DA transporter to regulate amphetamine-induced DA efflux.

    Science.gov (United States)

    Wang, Qiang; Bubula, Nancy; Brown, Jason; Wang, Yunliang; Kondev, Veronika; Vezina, Paul

    2016-05-27

    The DA transporter (DAT), a phosphoprotein, controls extracellular dopamine (DA) levels in the central nervous system through transport or reverse transport (efflux). Multiple lines of evidence support the claim that PKC significantly contributes to amphetamine-induced DA efflux. Other signaling pathways, involving CaMKII and ERK, have also been shown to regulate DAT mediated efflux. Here we assessed the contribution of putative PKC residues (S4, S7, S13) in the N-terminal of the DAT to amphetamine-induced DA efflux by transfecting DATs containing different serine to alanine (S-A) point mutations into DA pre-loaded HEK-293 cells and incubating these cells in amphetamine (2μM). The effects of a S-A mutation at the non-PKC residue S12 and a threonine to alanine (T-A) mutation at the ERK T53 residue were also assessed for comparison. WT-DATs were used as controls. In an initial experiment, we confirmed that inhibiting PKC with Go6976 (130nM) significantly reduced amphetamine-induced DA efflux. In subsequent experiments, cells transfected with the S4A, S12A, S13A, T53A and S4,7,13A mutants showed a reduction in amphetamine-induced DA efflux similar to that observed with Go6976. Interestingly, cells transfected with the S7A mutant, identified by some as a PKC-PKA residue, showed unperturbed WT-DAT levels of amphetamine-induced DA efflux. These results indicate that phosphorylation by PKC of select residues in the DAT N-terminal can regulate amphetamine-induced efflux. PKC can act either independently or in concert with other kinases such as ERK to produce this effect.

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

    Science.gov (United States)

    Koç Öztürk, L; Yarat, A; Akyuz, S; Furuncuoglu, H

    2016-01-01

    ABSTRACT 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.

  8. Role of the N-terminal signal peptide in the membrane insertion of Aquifex aeolicus F1F0 ATP synthase c-subunit.

    Science.gov (United States)

    Zhang, Chunli; Marcia, Marco; Langer, Julian D; Peng, Guohong; Michel, Hartmut

    2013-07-01

    Rotary ATPases are membrane protein complexes that couple ATP hydrolysis to ion translocation across the membrane. Overall, they are evolutionarily well conserved, but the N-terminal segments of their rotary subunits (c-subunits) possess different lengths and levels of hydrophobicity across species. By analyzing the N-terminal variability, we distinguish four phylogenetic groups of c-subunits (groups 1-4). We characterize a member of group 2, the c-subunit from Aquifex aeolicus F1F0 ATP synthase, both in native cells and in a heterologous expression system. We demonstrate that its N-terminal segment forms a signal peptide with signal recognition particle (SRP) recognition features and is obligatorily required for membrane insertion. Based on our study and on previous characterizations of c-subunits from other organisms, we propose that c-subunits follow different membrane insertion pathways. PMID:23663226

  9. 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

    detected a 29 kDa component in immunoblots of Raji and AL-34 cell plasma membrane proteins separated by SDS gel electrophoresis. The binding of either N-terminal peptide antiserum was selectively inhibited only by the peptide used as antigen. Indirect immunofluorescence analysis by flow cytofluorometry......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...... chains of HLA-DR and -DQ have been prepared by the preparation by the production of antibodies against the N-terminal sequences of each polypeptide....

  10. The effect of removing the N-terminal extension of the Drosophila myosin regulatory light chain upon flight ability and the contractile dynamics of indirect flight muscle.

    OpenAIRE

    Moore, J R; Dickinson, M H; Vigoreaux, J O; Maughan, D W

    2000-01-01

    The Drosophila myosin regulatory light chain (DMLC2) is homologous to MLC2s of vertebrate organisms, except for the presence of a unique 46-amino acid N-terminal extension. To study the role of the DMLC2 N-terminal extension in Drosophila flight muscle, we constructed a truncated form of the Dmlc2 gene lacking amino acids 2-46 (Dmlc2(Delta2-46)). The mutant gene was expressed in vivo, with no wild-type Dmlc2 gene expression, via P-element-mediated germline transformation. Expression of the tr...

  11. N-terminal guanidinylation of TIPP (Tyr-Tic-Phe-Phe) peptides results in major changes of the opioid activity profile

    OpenAIRE

    Weltrowska, Grazyna; Nguyen, Thi M.-D.; Chung, Nga N.; Wilkes, Brian C.; Schiller, Peter W.

    2013-01-01

    Derivatives of peptides of the TIPP (Tyr-Tic-Phe-Phe; Tic = 1,2,3,4- tetrahydroisoquinoline-3-carboxylic acid) family containing a guanidino (Guan) function in place of the N-terminal amino group were synthesized in an effort to improve their blood-brain barrier permeability. Unexpectedly, N-terminal amidination significantly altered the in vitro opioid activity profiles. Guan-analogues of TIPP-related δ opioid antagonists showed δ partial agonist or mixed δ partial agonist/μ partial agonist ...

  12. The methylated N-terminal tail of RCC1 is required for stabilisation of its interaction with chromatin by Ran in live cells

    Directory of Open Access Journals (Sweden)

    Sanderson Helen S

    2010-06-01

    Full Text Available Abstract Background Regulator of chromosome condensation 1 (RCC1 is the guanine nucleotide exchange factor for Ran GTPase. Localised generation of Ran-GTP by RCC1 on chromatin is critical for nucleocytoplasmic transport, mitotic spindle assembly and nuclear envelope formation. Both the N-terminal tail of RCC1 and its association with Ran are important for its interaction with chromatin in cells. In vitro, the association of Ran with RCC1 induces a conformational change in the N-terminal tail that promotes its interaction with DNA. Results We have investigated the mechanism of the dynamic interaction of the α isoform of human RCC1 (RCC1α with chromatin in live cells using fluorescence recovery after photobleaching (FRAP of green fluorescent protein (GFP fusions. We show that the N-terminal tail stabilises the interaction of RCC1α with chromatin and this function can be partially replaced by another lysine-rich nuclear localisation signal. Removal of the tail prevents the interaction of RCC1α with chromatin from being stabilised by RanT24N, a mutant that binds stably to RCC1α. The interaction of RCC1α with chromatin is destabilised by mutation of lysine 4 (K4Q, which abolishes α-N-terminal methylation, and this interaction is no longer stabilised by RanT24N. However, α-N-terminal methylation of RCC1α is not regulated by the binding of RanT24N. Conversely, the association of Ran with precipitated RCC1α does not require the N-terminal tail of RCC1α or its methylation. The mobility of RCC1α on chromatin is increased by mutation of aspartate 182 (D182A, which inhibits guanine-nucleotide exchange activity, but RCC1αD182A can still bind nucleotide-free Ran and its interaction with chromatin is stabilised by RanT24N. Conclusions These results show that the stabilisation of the dynamic interaction of RCC1α with chromatin by Ran in live cells requires the N-terminal tail of RCC1α. α-N-methylation is not regulated by formation of the binary

  13. 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

    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...... parameters (Mr, pI and N-terminal sequence). This work provides a preliminary basis for a future and progressive compilation of a genome-linked database of chlamydial proteins....

  14. Guideline of stereotactic radiotherapy of body trunk

    International Nuclear Information System (INIS)

    The guideline is issued for safe and effective practice of the stereotactic radiotherapy of body trunk by giving appropriate methodologies and their theoretical backgrounds to radiological stuff concerned, such as doctors, technologists, physicists, quality assurance (QA)/quality control (QC) personnel, and nurses. The issue is motivated by the recent and expected increase of facilities conducting the therapy popularized from its approval by health insurance authorities in 2004, is based on the drafts by the Study Group for improving prognosis of the accurate 3-D radiotherapy organized in the MHLW and by Jap. 3-D Conformal External Beam Radiotherapy Group and, after edition by QA committee of Jap. Soc. Ther. Radiol. Oncol., is herein published by the Society. The guideline is composed mainly from 4 chapters of Introduction, Clinical practice, Physics and technology, and QA/QC of equipments and systems. The second chapter contains, concerning the therapy, its definition, contraindication, application to health insurance (applicable diseases and requirement), target setting, radiation dose and fractionation, risk organs (serial and parallel ones) and dose limits, and progress observation post therapy. The third chapter, its definition and methods, therapeutic planning, and actual performance, and the forth, the principle, essential concept, items particularly needed (dosimetry and mechanical/geometrical accuracy of equipments, equipments for therapy planning, and QA/QC of the system). The guideline is to be revised within 2-3 years hereafter. (R.T.)

  15. EXTENSIVE NECROTIZING FASCIITIS OF TRUNK FOLLOWING APPENDICECTOMY

    Directory of Open Access Journals (Sweden)

    Siddharth

    2015-03-01

    Full Text Available Acute appendicitis is usually diagnosed and managed easily with a low mortality and morbidity. However, in 20.74% patients, acute appendicitis may occasionally become extraordinarily complicated and life threatening with significant morbidity and mortality . [1] We report a case of 44 year old male, a known case of Diabetes Mellitus since 4 years (on irregular treatment who was brought to our hospital with severe pain in right lower quadrant of abdomen since 4 days. On examination, patient had tachycardia, tender ness and guarding in the right iliac fossa. Laboratory results revealed leucocytosis and raised blood glucose levels. Measures for glycemic control were initiated. At exploratory laparotomy, an inflamed and retrocaecal appendix ruptured at the base with sm all local abscess was found. Abscess was drained and appendectomy done. Initially patient did well but at the end of second post - operative week, the patient started having pain and swelling over posterior chest wall, flank, sacral and occipital regions sug gestive of inflammation. CT abdomen and pelvis showed extensive necrotizing fasciitis of trunk, occiput and sacrum. Repeated multiple fasciotomies were performed over the back. The patient finally recovered fully at the end of 8 th post - operative week

  16. Observing of tree trunks and other cylindrical objects using GPR

    Science.gov (United States)

    Jezova, Jana; Lambot, Sebastien

    2016-04-01

    Trees are a part of our everyday life, hence it is important to prevent their collapse to protect people and urban infrastructures. It is also important to characterize tree wood properties for usages in construction. In order to investigate internal parts of tree trunks non-invasively, ground-penetrating radar (GPR), or in this case, ultra-wideband microwave radar as a general tool, appears to be a very promising technology. Nevertheless, tree trunk tomography using microwave radar is a complicated task due to the circular shape of the trunk and the very complex (heterogeneous and anisotropic) internal structures of the trunk. Microwave sensing of tree trunks is also complicated due to the electromagnetic properties of living wood, which strongly depend on water content, density and temperature of wood. The objective of this study is to describe tree trunk radar cross sections including specific features originating from the particular circumferential data acquisition geometry. In that respect, three experiments were performed: (1) numerical simulations using a finite-difference time-domain software, namely, gprMax 2D, (2) measurements on a simplified laboratory trunk model including plastic and cardboard pipes, sand and air, and (3) measurements over a real tree trunk. The analysis was further deepened by considering: (1) common zero-offset reflection imaging, (2) imaging with a planar perfect electrical conductor (PEC) at the opposite side of the trunk, and (3) imaging with a PEC arc at the opposite side of the trunk. Furthermore, the shape of the reflection curve of a cylindrical target was analytically derived based on the straight-ray propagation approximation. Subsequently, the total internal reflection (TIR) phenomenon occurring in cylindrical objects was observed and analytically described. Both the straight-ray reflection curve and TIR were well observed on the simulated and laboratory radar data. A comparison between all experiments and radar

  17. Calmodulin activation of an endoplasmic reticulum-located calcium pump involves an interaction with the N-terminal autoinhibitory domain

    Science.gov (United States)

    Hwang, I.; Harper, J. F.; Liang, F.; Sze, H.

    2000-01-01

    To investigate how calmodulin regulates a unique subfamily of Ca(2+) pumps found in plants, we examined the kinetic properties of isoform ACA2 identified in Arabidopsis. A recombinant ACA2 was expressed in a yeast K616 mutant deficient in two endogenous Ca(2+) pumps. Orthovanadate-sensitive (45)Ca(2+) transport into vesicles isolated from transformants demonstrated that ACA2 is a Ca(2+) pump. Ca(2+) pumping by the full-length protein (ACA2-1) was 4- to 10-fold lower than that of the N-terminal truncated ACA2-2 (Delta2-80), indicating that the N-terminal domain normally acts to inhibit the pump. An inhibitory sequence (IC(50) = 4 microM) was localized to a region within valine-20 to leucine-44, because a peptide corresponding to this sequence lowered the V(max) and increased the K(m) for Ca(2+) of the constitutively active ACA2-2 to values comparable to the full-length pump. The peptide also blocked the activity (IC(50) = 7 microM) of a Ca(2+) pump (AtECA1) belonging to a second family of Ca(2+) pumps. This inhibitory sequence appears to overlap with a calmodulin-binding site in ACA2, previously mapped between aspartate-19 and arginine-36 (J.F. Harper, B. Hong, I. Hwang, H.Q. Guo, R. Stoddard, J.F. Huang, M.G. Palmgren, H. Sze inverted question mark1998 J Biol Chem 273: 1099-1106). These results support a model in which the pump is kept "unactivated" by an intramolecular interaction between an autoinhibitory sequence located between residues 20 and 44 and a site in the Ca(2+) pump core that is highly conserved between different Ca(2+) pump families. Results further support a model in which activation occurs as a result of Ca(2+)-induced binding of calmodulin to a site overlapping or immediately adjacent to the autoinhibitory sequence.

  18. 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

  19. The Scavenger Receptor SSc5D Physically Interacts with Bacteria through the SRCR-Containing N-Terminal Domain

    Science.gov (United States)

    Bessa Pereira, Catarina; Bocková, Markéta; Santos, Rita F.; Santos, Ana Mafalda; Martins de Araújo, Mafalda; Oliveira, Liliana; Homola, Jiří; Carmo, Alexandre M.

    2016-01-01

    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. Toward 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 Escherichia 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 N-SSc5D compares with Spα in the capacity to bind to E. coli and Listeria 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

  20. Corticospinal Excitability of Trunk Muscles during Different Postural Tasks.

    Science.gov (United States)

    Chiou, Shin-Yi; Gottardi, Sam E A; Hodges, Paul W; Strutton, Paul H

    2016-01-01

    Evidence suggests that the primary motor cortex (M1) is involved in both voluntary, goal-directed movements and in postural control. Trunk muscles are involved in both tasks, however, the extent to which M1 controls these muscles in trunk flexion/extension (voluntary movement) and in rapid shoulder flexion (postural control) remains unclear. The purpose of this study was to investigate this question by examining excitability of corticospinal inputs to trunk muscles during voluntary and postural tasks. Twenty healthy adults participated. Transcranial magnetic stimulation was delivered to the M1 to examine motor evoked potentials (MEPs) in the trunk muscles (erector spinae (ES) and rectus abdominis (RA)) during dynamic shoulder flexion (DSF), static shoulder flexion (SSF), and static trunk extension (STE). The level of background muscle activity in the ES muscles was matched across tasks. MEP amplitudes in ES were significantly larger in DSF than in SSF or in STE; however, this was not observed for RA. Further, there were no differences in levels of muscle activity in RA between tasks. Our findings reveal that corticospinal excitability of the ES muscles appears greater during dynamic anticipatory posture-related adjustments than during static tasks requiring postural (SSF) and goal-directed voluntary (STE) activity. These results suggest that task-oriented rehabilitation of trunk muscles should be considered for optimal transfer of therapeutic effect to function.

  1. The Mechanistic Connection Between the Trunk, Knee, and Anterior Cruciate Ligament Injury

    OpenAIRE

    Hewett, Timothy E.; Myer, Gregory D.

    2011-01-01

    Neuromuscular control of the trunk and knee predicts anterior cruciate ligament injury risk with high sensitivity and specificity. These predictors are linked, as lateral trunk positioning creates high knee abduction torque (load). The hypotheses explored are that lateral trunk motion increases load and that neuromuscular training that increases trunk control will decrease load.

  2. Fear of Movement Is Related to Trunk Stiffness in Low Back Pain.

    NARCIS (Netherlands)

    Karayannis, N.V.; Smeets, R.J.P.; Hoorn, W. van den; Hodges, P.W.

    2013-01-01

    BACKGROUND: Psychological features have been related to trunk muscle activation patterns in low back pain (LBP). We hypothesised higher pain-related fear would relate to changes in trunk mechanical properties, such as higher trunk stiffness. OBJECTIVES: To evaluate the relationship between trunk mec

  3. Comparison of Midregional Pro-A-Type Natriuretic Peptide and the N-Terminal Pro-B-Type Natriuretic Peptide for Predicting Mortality and Cardiovascular Events

    NARCIS (Netherlands)

    van Hateren, Kornelis J. J.; Alkhalaf, Alaa; Kleefstra, Nanne; Groenier, Klaas H.; de Jong, Paul E.; de Zeeuw, Dick; Gans, Rijk O. B.; Struck, Joachim; Bilo, Henk J. G.; Gansevoort, Ron T.; Bakker, Stephan J. L.

    2012-01-01

    BACKGROUND: N-terminal pro-B-type natriuretic peptide (NT-proBNP) provides prognostic information on mortality and future cardiovascular events for individuals from the general population. A novel immunoassay was recently developed that measures a midregional fragment of pro-A-type natriuretic pepti

  4. Activation of G Protein-Coupled Receptor Kinase 1 Involves Interactions between Its N-Terminal Region and Its Kinase Domain

    Energy Technology Data Exchange (ETDEWEB)

    Huang, Chih-chin; Orban, Tivadar; Jastrzebska, Beata; Palczewski, Krzysztof; Tesmer, John J.G. (Case Western); (Michigan)

    2012-03-16

    G protein-coupled receptor kinases (GRKs) phosphorylate activated G protein-coupled receptors (GPCRs) to initiate receptor desensitization. In addition to the canonical phosphoacceptor site of the kinase domain, activated receptors bind to a distinct docking site that confers higher affinity and activates GRKs allosterically. Recent mutagenesis and structural studies support a model in which receptor docking activates a GRK by stabilizing the interaction of its 20-amino acid N-terminal region with the kinase domain. This interaction in turn stabilizes a closed, more active conformation of the enzyme. To investigate the importance of this interaction for the process of GRK activation, we first validated the functionality of the N-terminal region in rhodopsin kinase (GRK1) by site-directed mutagenesis and then introduced a disulfide bond to cross-link the N-terminal region of GRK1 with its specific binding site on the kinase domain. Characterization of the kinetic and biophysical properties of the cross-linked protein showed that disulfide bond formation greatly enhances the catalytic efficiency of the peptide phosphorylation, but receptor-dependent phosphorylation, Meta II stabilization, and inhibition of transducin activation were unaffected. These data indicate that the interaction of the N-terminal region with the kinase domain is important for GRK activation but does not dictate the affinity of GRKs for activated receptors.

  5. 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 electrophoret

  6. 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 formald

  7. 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.

  8. A novel method to isolate protein N-terminal peptides from proteome samples using sulfydryl tagging and gold-nanoparticle-based depletion.

    Science.gov (United States)

    Li, Lanting; Wu, Runqing; Yan, Guoquan; Gao, Mingxia; Deng, Chunhui; Zhang, Xiangmin

    2016-01-01

    A novel method to isolate global N-termini using sulfydryl tagging and gold-nanoparticle-based depletion (STagAu method) is presented. The N-terminal and lysine amino groups were first completely dimethylated at the protein level, after which the proteins were digested. The newly generated internal peptides were tagged with sulfydryl by Traut's reagent through digested N-terminal amines in yields of 96%. The resulting sulfydryl peptides were depleted through binding onto nano gold composite materials. The Au-S bond is stable and widely used in materials science. Nano gold composite materials showed nearly complete depletion of sulfydryl peptides. A set of the acetylated and dimethylated N-terminal peptides were analyzed by liquid chromatography-tandem mass spectrometry. This method was demonstrated to be an efficient N-terminus enrichment method because of the use of an effective derivatization reaction, in combination with robust and relative easy to implement Au-S coupling. We identified 632 N-terminal peptides from 386 proteins in a mouse liver sample. The STagAu approach presented is therefore a facile and efficient method for mass-spectrometry-based analysis of proteome N-termini or protease-generated cleavage products.

  9. Solution structure of N-terminal SH3 domain of Vav and the recognition site for Grb2 C-terminal SH3 domain

    International Nuclear Information System (INIS)

    The three-dimensional structure of the N-terminal SH3 domain (residues 583-660) of murine Vav, which contains a tetra-proline sequence (Pro 607-Pro 610), was determined by NMR. The solution structure of the SH3 domain shows a typical SH3 fold, but it exists in two conformations due to cis-trans isomerization at the Gly614-Pro615 bond. The NMR structure of the P615G mutant, where Pro615 is replaced by glycine, reveals that the tetra-proline region is inserted into the RT-loop and binds to its own SH3 structure. The C-terminal SH3 domain of Grb2 specifically binds to the trans form of the N-terminal SH3 domain of Vav. The surface of Vav N-terminal SH3 which binds to Grb2 C-terminal SH3 was elucidated by chemical shift mapping experiments using NMR. The surface does not involve the tetra-proline region but involves the region comprising the n-src loop, the N-terminal and the C-terminal regions. This surface is located opposite to the tetra-proline containing region, consistent with that of our previous mutagenesis studies

  10. Prognostic assessment of elderly patients with symptoms of heart failure by combining high-sensitivity troponin T and N-terminal pro-B-type natriuretic peptide measurements

    DEFF Research Database (Denmark)

    Alehagen, Urban; Dahlström, Ulf; Rehfeld, Jens F.;

    2010-01-01

    N-terminal pro-B-type natriuretic peptide (NT-proBNP) is a useful biomarker in heart failure assessment, whereas measurement of cardiac troponin is central in the diagnosis of patients with acute coronary syndromes. This report examined the prognostic use of combining high-sensitivity cardiac...

  11. Ultrafast resonance energy transfer from a site-specifically attached fluorescent chromophore reveals the folding of the N-terminal domain of CP29

    NARCIS (Netherlands)

    Oort, van B.F.; Murali, S.; Wientjes, E.; Koehorst, R.B.M.; Spruijt, R.B.; Hoek, van A.; Croce, R.; Amerongen, van H.

    2009-01-01

    The photosynthetic minor antenna complex CP29 of higher plants was singly mutated, overexpressed in Escherichia coli, selectively labeled with the fluorescent dye TAMRA at three positions in the N-terminal domain, and reconstituted with its natural pigments. Picosecond fluorescence experiments revea

  12. 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 microg...

  13. An Experimental Investigation of the Evolution of Chirality in a Potential Dynamic Peptide System: N-Terminal Epimerization and Degradation into Diketopiperazine

    Science.gov (United States)

    Danger, Grégoire; Plasson, Raphaël; Pascal, Robert

    2010-08-01

    The APED model (activation-polymerization-epimerization-depolymerization) is a unique example of a chemical system that allows symmetry breaking through a dynamic process involving indirect network autocatalysis. In its simplest version, the autocatalytic behavior of this model partly relies on the reproduction of local chiral centers in dipeptides through an epimerization process, with a thermodynamic preference for homochiral chains. We studied the reactivity of di- and tripeptides, containing a N-terminal phenylglycine (Phg) residue, as model compounds for the experimental determination of the kinetic and thermodynamic parameters related to the N-terminal epimerization process. Although the N-terminal residue is prone to spontaneous epimerization, catalysis was required for the epimerization to reach the equilibrium state in reasonable time. Unexpectedly, the observed equilibrium diastereoisomeric excesses have shown a general tendency for more stable heterochiral peptides, especially strong in the case of dipeptides. In parallel to this process, a stereoselective peptide cleavage through diketopiperazine formation was observed. Contrary to the N-terminal epimerization of peptides, the diketopiperazine formation did not need any catalyst, and heterochiral peptides were shown to be dynamically unstabilized, as they were cleaved faster than homochiral peptides. The validity of the extrapolation of these results to other residues and longer peptide chains is discussed, and some directions for future developments of the theoretical model are given.

  14. Plasma Pro-C3 (N-terminal type III collagen propeptide) predicts fibrosis progression in patients with chronic hepatitis C

    DEFF Research Database (Denmark)

    Nielsen, Mette J.; Veidal, Sanne S.; Karsdal, Morten A.;

    2015-01-01

    BACKGROUND & AIMS: Fibrogenesis results in release of certain extracellular matrix protein fragments into the circulation. We evaluated the diagnostic and prognostic performance of two novel serological markers, the precisely cleaved N-terminal propeptide of type III collagen (Pro-C3) and a peptide...

  15. 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...

  16. On the structure of the N-terminal domain of the MscL channel: helical bundle or membrane interface.

    Science.gov (United States)

    Iscla, Irene; Wray, Robin; Blount, Paul

    2008-09-01

    The mechanosensitive channel of large conductance, MscL, serves as a biological emergency release valve protecting bacteria from acute osmotic downshock and is to date the best characterized mechanosensitive channel. A well-recognized and supported model for Escherichia coli MscL gating proposes that the N-terminal 11 amino acids of this protein form a bundle of amphipathic helices in the closed state that functionally serves as a cytoplasmic second gate. However, a recently reexamined crystal structure of a closed state of the Mycobacterium tuberculosis MscL shows these helices running along the cytoplasmic surface of the membrane. Thus, it is unclear if one structural model is correct or if they both reflect valid closed states. Here, we have systematically reevaluated this region utilizing cysteine-scanning, in vivo functional characterization, in vivo SCAM, electrophysiological studies, and disulfide-trapping experiments. The disulfide-trapping pattern and functional studies do not support the helical bundle and second-gate hypothesis but correlate well with the proposed structure for M. tuberculosis MscL. We propose a functional model that is consistent with the collective data. PMID:18515388

  17. The N-terminal domain is a transcriptional activation domain required for Nanog to maintain ES cell self-renewal

    Institute of Scientific and Technical Information of China (English)

    GUO YunQian; ZHANG Juan; YE Li; CHEN Mo; YAO Dong; PAN GuangJin; ZHANG JieQiong; PEI DuanQing

    2009-01-01

    Nanog is a transcription factor identified by its ability to maintain the self-renewal of ES cells in the absence of leukemia inhibitory factor (LIF). Nanog protein contains an N-terminal domain (ND),a DNA-binding homeobox domain (HD) and a C-terminal domain (CD). We previously reported that the CD in Nanog is a transcriptional activation domain essential for the in vivo function of Nanog. Here we demonstrated that the ND in Nanog is also functionally important. Deletion of the ND reduces the transcriptional activity of Nanog on either artificial reporters or native Nanog promoters. This truncated Nanog is also less effective in regulating the endogenous Nanog target genes. Furthermore,the ND truncation disrupted the ability of Nanog to maintain ES cell self-renewal as well. We found that the ND Is not required for the nuclear localization of Nanog. These results suggest that the regulation of endogenous pluripotent genes such as oct3/4 and rex-1 is required for the in vivo function of Nanog.

  18. Recombinant Expression of Trichoderma reesei Cel61A in Pichia pastoris: Optimizing Yield and N-terminal Processing.

    Science.gov (United States)

    Tanghe, Magali; Danneels, Barbara; Camattari, Andrea; Glieder, Anton; Vandenberghe, Isabel; Devreese, Bart; Stals, Ingeborg; Desmet, Tom

    2015-12-01

    The auxiliary activity family 9 (AA9, formerly GH61) harbors a recently discovered group of oxidative enzymes that boost cellulose degradation. Indeed, these lytic polysaccharide monooxygenases (LPMOs) are able to disrupt the crystalline structure of cellulose, thereby facilitating the work of hydrolytic enzymes involved in biomass degradation. Since these enzymes require an N-terminal histidine residue for activity, their recombinant production as secreted protein is not straightforward. We here report the expression optimization of Trichoderma reesei Cel61A (TrCel61A) in the host Pichia pastoris. The use of the native TrCel61A secretion signal instead of the alpha-mating factor from Saccharomyces cerevisiae was found to be crucial, not only to obtain high protein yields (>400 mg/L during fermentation) but also to enable the correct processing of the N-terminus. Furthermore, the LPMO activity of the enzyme is demonstrated here for the first time, based on its degradation profile of a cellulosic substrate.

  19. DNA replication checkpoint signaling depends on a Rad53-Dbf4 N-terminal interaction in Saccharomyces cerevisiae.

    Science.gov (United States)

    Chen, Ying-Chou; Kenworthy, Jessica; Gabrielse, Carrie; Hänni, Christine; Zegerman, Philip; Weinreich, Michael

    2013-06-01

    Dbf4-dependent kinase (DDK) and cyclin-dependent kinase (CDK) are essential to initiate DNA replication at individual origins. During replication stress, the S-phase checkpoint inhibits the DDK- and CDK-dependent activation of late replication origins. Rad53 kinase is a central effector of the replication checkpoint and both binds to and phosphorylates Dbf4 to prevent late-origin firing. The molecular basis for the Rad53-Dbf4 physical interaction is not clear but occurs through the Dbf4 N terminus. Here we found that both Rad53 FHA1 and FHA2 domains, which specifically recognize phospho-threonine (pT), interacted with Dbf4 through an N-terminal sequence and an adjacent BRCT domain. Purified Rad53 FHA1 domain (but not FHA2) bound to a pT Dbf4 peptide in vitro, suggesting a possible phospho-threonine-dependent interaction between FHA1 and Dbf4. The Dbf4-Rad53 interaction is governed by multiple contacts that are separable from the Cdc5- and Msa1-binding sites in the Dbf4 N terminus. Importantly, abrogation of the Rad53-Dbf4 physical interaction blocked Dbf4 phosphorylation and allowed late-origin firing during replication checkpoint activation. This indicated that Rad53 must stably bind to Dbf4 to regulate its activity.

  20. c-Jun-N-terminal kinase 1 is necessary for nicotine-induced enhancement of contextual fear conditioning.

    Science.gov (United States)

    Leach, Prescott T; Kenney, Justin W; Gould, Thomas J

    2016-08-01

    Acute nicotine enhances hippocampus-dependent learning. Identifying how acute nicotine improves learning will aid in understanding how nicotine facilitates the development of maladaptive memories that contribute to drug-seeking behaviors, help development of medications to treat disorders associated with cognitive decline, and advance understanding of the neurobiology of learning and memory. The effects of nicotine on learning may involve recruitment of signaling through the c-Jun N-terminal kinase family (JNK 1-3). Learning in the presence of acute nicotine increases the transcription of mitogen-activated protein kinase 8 (MAPK8, also known as JNK1), likely through a CREB-dependent mechanism. The functional significance of JNK1 in the effects of acute nicotine on learning, however, is unknown. The current studies undertook a backward genetic approach to determine the functional contribution JNK1 protein makes to nicotine-enhanced contextual fear conditioning. JNK1 wildtype (WT) and knockout (KO) mice were administered acute nicotine prior to contextual and cued fear conditioning. 24h later, mice were evaluated for hippocampus-dependent (contextual fear conditioning) and hippocampus-independent (cued fear conditioning) memory. Nicotine selectively enhanced contextual conditioning in WT mice, but not in KO mice. Nicotine had no effect on hippocampus-independent learning in either genotype. JNK1 KO and WT mice given saline showed similar levels of learning. These data suggest that JNK1 may be recruited by nicotine and is functionally necessary for the acute effects of nicotine on learning and memory. PMID:27235579

  1. PredSL: A Tool for the N-terminal Sequence-based Prediction of Protein Subcellular Localization

    Institute of Scientific and Technical Information of China (English)

    Evangelia I. Petsalaki; Pantelis G. Bagos; Zoi I. Litou; Stavros J. Hamodrakas

    2006-01-01

    The ability to predict the subcellular localization of a protein from its sequence is of great importance, as it provides information about the protein's function.We present a computational tool, PredSL, which utilizes neural networks, Markov chains, profile hidden Markov models, and scoring matrices for the prediction of the subcellular localization of proteins in eukaryotic cells from the N-terminal amino acid sequence. It aims to classify proteins into five groups: chloroplast,thylakoid, mitochondrion, secretory pathway, and "other". When tested in a fivefold cross-validation procedure, PredSL demonstrates 86.7% and 87.1% overall accuracy for the plant and non-plant datasets, respectively. Compared with TargetP, which is the most widely used method to date, and LumenP, the results of PredSL are comparable in most cases. When tested on the experimentally verified proteins of the Saccharomyces cerevisiae genome, PredSL performs comparably if not better than any available algorithm for the same task. Furthermore, PredSL is the only method capable for the prediction of these subcellular localizations that is available as a stand-alone application through the URL:http://bioinformatics.biol.uoa.gr/PredSL/.

  2. C-jun N-terminal Kinase-mediated Signaling Is Essential for Staphylococcus Aureus-induced U937 Apoptosis

    Institute of Scientific and Technical Information of China (English)

    Jia-he Wang; Bo Yu; Hui-yan Niu; Hui Li; Yi Zhang; Xin Wang; Ping He

    2009-01-01

    Objective To investigate the effect of SP600125, a specific c-jun N-terminal protein kinase (JNK) inhibitor, on Staphylococcus aureus (S. aureus)-induced U937 cell death and the underlying mechanism. Methods The human monocytic U937 cells were treated with S. aureus at different time with or without SP600125. Cell apoptosis was analyzed by flow cytometry. JNK, Bax, and caspase-3 activities were detected by Western blotting. Results S. aureus induced apoptosis in cultured U937 cells in a time-dependent manner. Expression of Bax and phospho-JNK significantly increased in S. aureus-treated U937 cells, and the level of activated caspase-3 also increased in a time-dependent manner. Inhibition of JNK with SP600125 significantly inhibited S. aureus-induced apoptosis in U937 cells. Conclusions S. aureus can induce apoptosis in U937 cells by phosphorylation of JNK and activation of Bax and caspase-3. SP600125 protects U937 cells from apoptosis induced by S. aureus via inhibiting the activity of JNK.

  3. Different Roles of N-Terminal and C-Terminal Domains in Calmodulin for Activation of Bacillus anthracis Edema Factor

    Directory of Open Access Journals (Sweden)

    Carolin Lübker

    2015-07-01

    Full Text Available Bacillus anthracis adenylyl cyclase toxin edema factor (EF is one component of the anthrax toxin and is essential for establishing anthrax disease. EF activation by the eukaryotic Ca2+-sensor calmodulin (CaM leads to massive cAMP production resulting in edema. cAMP also inhibits the nicotinamide adenine dinucleotide phosphate (NADPH-oxidase, thus reducing production of reactive oxygen species (ROS used for host defense in activated neutrophils and thereby facilitating bacterial growth. Methionine (Met residues in CaM, important for interactions between CaM and its binding partners, can be oxidized by ROS. We investigated the impact of site-specific oxidation of Met in CaM on EF activation using thirteen CaM-mutants (CaM-mut with Met to leucine (Leu substitutions. EF activation shows high resistance to oxidative modifications in CaM. An intact structure in the C-terminal region of oxidized CaM is sufficient for major EF activation despite altered secondary structure in the N-terminal region associated with Met oxidation. The secondary structures of CaM-mut were determined and described in previous studies from our group. Thus, excess cAMP production and the associated impairment of host defence may be afforded even under oxidative conditions in activated neutrophils.

  4. Identification of a novel family of laminin N-terminal alternate splice isoforms: structural and functional characterization.

    Science.gov (United States)

    Hamill, Kevin J; Langbein, Lutz; Jones, Jonathan C R; McLean, W H Irwin

    2009-12-18

    The laminins are a family of heterotrimeric basement membrane proteins that play roles in cellular adhesion, migration, and tissue morphogenesis. Through in silico analysis of the laminin-encoding genes, we identified a novel family of alternate splice isoforms derived from the 5'-end of the LAMA3 and LAMA5 genes. These isoforms resemble the netrins in that they contain a laminin N-terminal domain followed by a short stretch of laminin-type epidermal growth factor-like repeats. We suggest the terms LaNt (laminin N terminus) alpha3 and LaNt alpha5, for the predicted protein products of these mRNAs. RT-PCR confirmed the presence of these transcripts at the mRNA level. Moreover, they exhibit differential, tissue-specific, expression profiles. To confirm the existence of LaNt alpha3 protein, we generated an antibody to a unique domain within the putative polypeptide. This antibody recognizes a protein at the predicted molecular mass of 64 kDa by immunoblotting. Furthermore, immunofluorescence analyses revealed a basement membrane staining in epithelial tissue for LaNt alpha3 and LaNt alpha3 localized along the substratum-associated surface of cultured keratinocytes. We have also tested the functionality LaNt alpha3 through RNAi-mediated knockdown. Keratinocytes exhibiting specific knockdown of LaNt alpha3 displayed impaired adhesion, stress resistance, and reduced ability to close scratch wounds in vitro. PMID:19773554

  5. 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

  6. Analyses of Compact Trichinella Kinomes Reveal a MOS-Like Protein Kinase with a Unique N-Terminal Domain

    Directory of Open Access Journals (Sweden)

    Andreas J. Stroehlein

    2016-09-01

    Full Text Available 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.

  7. Plasminogen N-terminal activation peptide modulates the activity of angiostatin-related peptides on endothelial cell proliferation and migration.

    Science.gov (United States)

    Hayashi, Moyuru; Tamura, Yosuke; Dohmae, Naoshi; Kojima, Soichi; Shimonaka, Motoyuki

    2008-05-01

    Angiostatin, a potent inhibitor of angiogenesis, is derived from the fibrinolytic proenzyme, plasminogen, by enzymatic processing. Plasminogen N-terminal activation peptide (PAP) is one of the products concomitantly released aside from angiostatin (kringles 1-4) and mini-plasminogen (kringle 5 plus the catalytic domain) when plasminogen is processed. To determine whether PAP alone or together with the angiostatin-related peptides derived from the processing of plasminogen modulate the proliferation and motility of endothelial cells, we have generated a recombinant PAP and used it to study its effects on endothelial cells in the presence and absence of the angiostatin-related peptides. Our results showed that PAP alone slightly increased the migration but not the proliferation of endothelial cells. However, in the presence of the angiostatin-related peptides, PAP attenuated the inhibitory activity of the angiostatin-related peptides on the proliferation and migration of endothelial cells. The inhibitory effect of PAP on the angiostatin-related peptides could be due to its binding to the kringle domains of the latter peptides. PMID:18294956

  8. Plasminogen N-terminal activation peptide modulates the activity of angiostatin-related peptides on endothelial cell proliferation and migration

    International Nuclear Information System (INIS)

    Angiostatin, a potent inhibitor of angiogenesis, is derived from the fibrinolytic proenzyme, plasminogen, by enzymatic processing. Plasminogen N-terminal activation peptide (PAP) is one of the products concomitantly released aside from angiostatin (kringles 1-4) and mini-plasminogen (kringle 5 plus the catalytic domain) when plasminogen is processed. To determine whether PAP alone or together with the angiostatin-related peptides derived from the processing of plasminogen modulate the proliferation and motility of endothelial cells, we have generated a recombinant PAP and used it to study its effects on endothelial cells in the presence and absence of the angiostatin-related peptides. Our results showed that PAP alone slightly increased the migration but not the proliferation of endothelial cells. However, in the presence of the angiostatin-related peptides, PAP attenuated the inhibitory activity of the angiostatin-related peptides on the proliferation and migration of endothelial cells. The inhibitory effect of PAP on the angiostatin-related peptides could be due to its binding to the kringle domains of the latter peptides

  9. Crystal Structure of the N-Terminal RNA Recognition Motif of mRNA Decay Regulator AUF1

    Directory of Open Access Journals (Sweden)

    Young Jun Choi

    2016-01-01

    Full Text Available AU-rich element binding/degradation factor 1 (AUF1 plays a role in destabilizing mRNAs by forming complexes with AU-rich elements (ARE in the 3′-untranslated regions. Multiple AUF1-ARE complexes regulate the translation of encoded products related to the cell cycle, apoptosis, and inflammation. AUF1 contains two tandem RNA recognition motifs (RRM and a Gln- (Q- rich domain in their C-terminal region. To observe how the two RRMs are involved in recognizing ARE, we obtained the AUF1-p37 protein covering the two RRMs. However, only N-terminal RRM (RRM1 was crystallized and its structure was determined at 1.7 Å resolution. It appears that the RRM1 and RRM2 separated before crystallization. To demonstrate which factors affect the separate RRM1-2, we performed limited proteolysis using trypsin. The results indicated that the intact proteins were cleaved by unknown proteases that were associated with them prior to crystallization. In comparison with each of the monomers, the conformations of the β2-β3 loops were highly variable. Furthermore, a comparison with the RRM1-2 structures of HuR and hnRNP A1 revealed that a dimer of RRM1 could be one of the possible conformations of RRM1-2. Our data may provide a guidance for further structural investigations of AUF1 tandem RRM repeat and its mode of ARE binding.

  10. The N-terminal part of Als1 protein from Candida albicans specifically binds fucose-containing glycans.

    Science.gov (United States)

    Donohue, Dagmara S; Ielasi, Francesco S; Goossens, Katty V Y; Willaert, Ronnie G

    2011-06-01

    The opportunistic pathogen Candida albicans expresses on its surface Als (Agglutinin like sequence) proteins, which play an important role in the adhesion to host cells and in the development of candidiasis. The binding specificity of these proteins is broad, as they can bind to various mammalian proteins, such as extracellular matrix proteins, and N- and E-cadherins. The N-terminal part of Als proteins constitutes the substrate-specific binding domain and is responsible for attachment to epithelial and endothelial cells. We have used glycan array screening to identify possible glycan receptors for the binding domain of Als1p-N. Under those conditions, Als1p-N binds specifically to fucose-containing glycans, which adds a lectin function to the functional diversity of the Als1 protein. The binding between Als1p-N and BSA-fucose glycoconjugate was quantitatively characterized using surface plasmon resonance, which demonstrated a weak millimolar affinity between Als1p-N and fucose. Furthermore, we have also quantified the affinity of Als1p-N to the extracellular matrix proteins proteins fibronectin and laminin, which is situated in the micromolar range. Surface plasmon resonance characterization of Als1p-N-Als1p-N interaction was in the micromolar affinity range.

  11. NAA10 mutation causing a novel intellectual disability syndrome with Long QT due to N-terminal acetyltransferase impairment

    Science.gov (United States)

    Casey, Jillian P.; Støve, Svein I.; McGorrian, Catherine; Galvin, Joseph; Blenski, Marina; Dunne, Aimee; Ennis, Sean; Brett, Francesca; King, Mary D.; Arnesen, Thomas; Lynch, Sally Ann

    2015-01-01

    We report two brothers from a non-consanguineous Irish family presenting with a novel syndrome characterised by intellectual disability, facial dysmorphism, scoliosis and long QT. Their mother has a milder phenotype including long QT. X-linked inheritance was suspected. Whole exome sequencing identified a novel missense variant (c.128 A > C; p.Tyr43Ser) in NAA10 (X chromosome) as the cause of the family’s disorder. Sanger sequencing confirmed that the mutation arose de novo in the carrier mother. NAA10 encodes the catalytic subunit of the major human N-terminal acetylation complex NatA. In vitro assays for the p.Tyr43Ser mutant enzyme showed a significant decrease in catalytic activity and reduced stability compared to wild-type Naa10 protein. NAA10 has previously been associated with Ogden syndrome, Lenz microphthalmia syndrome and non-syndromic developmental delay. Our findings expand the clinical spectrum of NAA10 and suggest that the proposed correlation between mutant Naa10 enzyme activity and phenotype severity is more complex than anticipated; the p.Tyr43Ser mutant enzyme has less catalytic activity than the p.Ser37Pro mutant associated with lethal Ogden syndrome but results in a milder phenotype. Importantly, we highlight the need for cardiac assessment in males and females with NAA10 variants as both patients and carriers can have long QT. PMID:26522270

  12. The c-Jun N-terminal Kinase 2 Plays a Dominant Role in Human Epidermal Neoplasia

    Science.gov (United States)

    Ke, Hengning; Harris, Rebecca; Coloff, Jonathan; Jin, Jane Y.; Leshin, Benjamin; de Marval, Paula Miliani; Tao, Shiying; Rathmell, Jeffrey C; Hall, Russell P.; Zhang, Jennifer Y.

    2010-01-01

    The c-Jun N-terminal Kinase (JNK) signaling cascade has been implicated in a wide range of diseases, including cancer. It is unclear how different JNK proteins contribute to human cancer. Here, we report that JNK2 is activated in over 70% of human squamous cell carcinoma (SCC) samples and that inhibition of JNK2 pharmacologically or genetically impairs tumorigenesis of human SCC cells. Most importantly, JNK2, but not JNK1, is sufficient to couple with oncogenic Ras to transform primary human epidermal cells into malignancy with features of SCC. JNK2 prevents Ras-induced cell senescence and growth arrest by reducing the expression levels of the cell cycle inhibitor p16 and NF-κB activation. On the other hand, JNK, along with PI3K, is essential for Ras-induced glycolysis, an energy producing process known to benefit cancer growth. These data indicate that JNK2 collaborates with other oncogenes, such as Ras, at multiple molecular levels to promote tumorigenesis and hence represents a promising therapeutic target for cancer. PMID:20354187

  13. Regulation of Telomere Length Requires a Conserved N-Terminal Domain of Rif2 in Saccharomyces cerevisiae.

    Science.gov (United States)

    Kaizer, Hannah; Connelly, Carla J; Bettridge, Kelsey; Viggiani, Christopher; Greider, Carol W

    2015-10-01

    The regulation of telomere length equilibrium is essential for cell growth and survival since critically short telomeres signal DNA damage and cell cycle arrest. While the broad principles of length regulation are well established, the molecular mechanism of how these steps occur is not fully understood. We mutagenized the RIF2 gene in Saccharomyces cerevisiae to understand how this protein blocks excess telomere elongation. We identified an N-terminal domain in Rif2 that is essential for length regulation, which we have termed BAT domain for Blocks Addition of Telomeres. Tethering this BAT domain to Rap1 blocked telomere elongation not only in rif2Δ mutants but also in rif1Δ and rap1C-terminal deletion mutants. Mutation of a single amino acid in the BAT domain, phenylalanine at position 8 to alanine, recapitulated the rif2Δ mutant phenotype. Substitution of F8 with tryptophan mimicked the wild-type phenylalanine, suggesting the aromatic amino acid represents a protein interaction site that is essential for telomere length regulation.

  14. 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.

  15. Tarantula myosin free head regulatory light chain phosphorylation stiffens N-terminal extension, releasing it and blocking its docking back.

    Science.gov (United States)

    Alamo, Lorenzo; Li, Xiaochuan Edward; Espinoza-Fonseca, L Michel; Pinto, Antonio; Thomas, David D; Lehman, William; Padrón, Raúl

    2015-08-01

    Molecular dynamics simulations of smooth and striated muscle myosin regulatory light chain (RLC) N-terminal extension (NTE) showed that diphosphorylation induces a disorder-to-order transition. Our goal here was to further explore the effects of mono- and diphosphorylation on the straightening and rigidification of the tarantula myosin RLC NTE. For that we used MD simulations followed by persistence length analysis to explore the consequences of secondary and tertiary structure changes occurring on RLC NTE following phosphorylation. Static and dynamic persistence length analysis of tarantula RLC NTE peptides suggest that diphosphorylation produces an important 24-fold straightening and a 16-fold rigidification of the RLC NTE, while monophosphorylation has a less profound effect. This new information on myosin structural mechanics, not fully revealed by previous EM and MD studies, add support to a cooperative phosphorylation-dependent activation mechanism as proposed for the tarantula thick filament. Our results suggest that the RLC NTE straightening and rigidification after Ser45 phosphorylation leads to a release of the constitutively Ser35 monophosphorylated free head swaying away from the thick filament shaft. This is so because the stiffened diphosphorylated RLC NTE would hinder the docking back of the free head after swaying away, becoming released and mobile and unable to recover its original interacting position on activation. PMID:26038302

  16. Role of N-terminal domain of HMW 1Dx5 in the functional and structural properties of wheat dough.

    Science.gov (United States)

    Wang, Jing Jing; Liu, Guang; Huang, Yan-Bo; Zeng, Qiao-Hui; Song, Guo-Sheng; Hou, Yi; Li, Lin; Hu, Song-Qing

    2016-12-15

    Effects of N-terminal domain of high molecular weight glutenin subunit (HMW-GS) 1Dx5 (1Dx5-N) on functional and structural properties of wheat dough were determined by farinographic and rheological analysis, size exclusion chromatography, non-reducing/reducing SDS-PAGE, total free sulfhydryl determination, scanning electron microscopy and Fourier transform infrared spectroscopy. Results showed that 1Dx5-N improved the quality of dough with the increased water absorption, dough stability time, elastic and viscous modulus, and the decreased degree of softening, loss tangent. These improvements could be attributed to the formation of the macro-molecular weight aggregates and massive protein networks, which were favored by 1Dx5-N through disulfide bonds and hydrophobic interactions. Additionally, 1Dx5-N drove the transition of α-helix and random coil conformations to β-sheet and β-turn conformations, further demonstrating the formation of HMW-GS polymers and the enhancement of dough strength. Moreover, all the positive effects of 1Dx5-N were reinforced by edible salt NaCl. PMID:27451235

  17. The Aquaporin Splice Variant NbXIP1;1α Is Permeable to Boric Acid and Is Phosphorylated in the N-terminal Domain

    Science.gov (United States)

    Ampah-Korsah, Henry; Anderberg, Hanna I.; Engfors, Angelica; Kirscht, Andreas; Norden, Kristina; Kjellstrom, Sven; Kjellbom, Per; Johanson, Urban

    2016-01-01

    Aquaporins (AQPs) are membrane channel proteins that transport water and uncharged solutes across different membranes in organisms in all kingdoms of life. In plants, the AQPs can be divided into seven different subfamilies and five of these are present in higher plants. The most recently characterized of these subfamilies is the XIP subfamily, which is found in most dicots but not in monocots. In this article, we present data on two different splice variants (α and β) of NbXIP1;1 from Nicotiana benthamiana. We describe the heterologous expression of NbXIP1;1α and β in the yeast Pichia pastoris, the subcellular localization of the protein in this system and the purification of the NbXIP1;1α protein. Furthermore, we investigated the functionality and the substrate specificity of the protein by stopped-flow spectrometry in P. pastoris spheroplasts and with the protein reconstituted in proteoliposomes. The phosphorylation status of the protein and localization of the phosphorylated amino acids were verified by mass spectrometry. Our results show that NbXIP1;1α is located in the plasma membrane when expressed in P. pastoris, that it is not permeable to water but to boric acid and that the protein is phosphorylated at several amino acids in the N-terminal cytoplasmic domain of the protein. A growth assay showed that the yeast cells expressing the N-terminally His-tagged NbXIP1;1α were more sensitive to boric acid as compared to the cells expressing the C-terminally His-tagged isoform. This might suggest that the N-terminal His-tag functionally mimics the phosphorylation of the N-terminal domain and that the N-terminal domain is involved in gating of the channel. PMID:27379142

  18. The Aquaporin Splice Variant NbXIP1;1α Is Permeable to Boric Acid and Is Phosphorylated in the N-terminal Domain.

    Science.gov (United States)

    Ampah-Korsah, Henry; Anderberg, Hanna I; Engfors, Angelica; Kirscht, Andreas; Norden, Kristina; Kjellstrom, Sven; Kjellbom, Per; Johanson, Urban

    2016-01-01

    Aquaporins (AQPs) are membrane channel proteins that transport water and uncharged solutes across different membranes in organisms in all kingdoms of life. In plants, the AQPs can be divided into seven different subfamilies and five of these are present in higher plants. The most recently characterized of these subfamilies is the XIP subfamily, which is found in most dicots but not in monocots. In this article, we present data on two different splice variants (α and β) of NbXIP1;1 from Nicotiana benthamiana. We describe the heterologous expression of NbXIP1;1α and β in the yeast Pichia pastoris, the subcellular localization of the protein in this system and the purification of the NbXIP1;1α protein. Furthermore, we investigated the functionality and the substrate specificity of the protein by stopped-flow spectrometry in P. pastoris spheroplasts and with the protein reconstituted in proteoliposomes. The phosphorylation status of the protein and localization of the phosphorylated amino acids were verified by mass spectrometry. Our results show that NbXIP1;1α is located in the plasma membrane when expressed in P. pastoris, that it is not permeable to water but to boric acid and that the protein is phosphorylated at several amino acids in the N-terminal cytoplasmic domain of the protein. A growth assay showed that the yeast cells expressing the N-terminally His-tagged NbXIP1;1α were more sensitive to boric acid as compared to the cells expressing the C-terminally His-tagged isoform. This might suggest that the N-terminal His-tag functionally mimics the phosphorylation of the N-terminal domain and that the N-terminal domain is involved in gating of the channel. PMID:27379142

  19. Calcium Occupancy of N-terminal Sites within Calmodulin Induces Inhibition of the Ryanodine Receptor Calcium Release Channel

    Energy Technology Data Exchange (ETDEWEB)

    Boschek, Curt B; Jones, Terry E; Squier, Thomas C; Bigelow, Diana J

    2007-08-01

    Calmodulin (CaM) regulates calcium release from intracellular stores in skeletal muscle through its association with the ryanodine receptor (RyR1) calcium release channel, where CaM association enhances channel opening at resting calcium levels and its closing at micromolar calcium levels associated with muscle contraction. A high-affinity CaM-binding sequence (RyRp) has been identified in RyR1, which corresponds to a 30-residue sequence (i.e., K3614 – N3643) located within the central portion of the primary sequence. However, it is currently unclear whether the identified CaM-binding sequence a) senses calcium over the physiological range of calcium-concentrations associated with RyR1 regulation or b) plays a structural role unrelated to the calcium-dependent modulation of RyR1 function. Therefore, we have measured the calcium-dependent activation of the individual domains of CaM in association with RyRp and their relationship to the CaM-dependent regulation of RyR1. These measurements utilize an engineered CaM, permitting the site-specific incorporation of N-(1-pyrene) maleimide at either T34C (PyN-CaM) or T110C (PyC-CaM) in the N- and C-domains, respectively. Consistent with prior measurements, we observe a high-affinity association between both apo- and calcium-activated CaM and RyRp. Upon association with RyRp, fluorescence changes in PyN-CaM or PyC-CaM permit the measurement of the calcium-activation of these individual domains. Fluorescence changes upon calcium-activation of PyC-CaM in association with RyRp are indicative of high-affinity calcium-dependent activation of the C-terminal domain of CaM bound to RyRp at resting calcium levels and the activation of the N-terminal domain at levels of calcium associated cellular activation. In comparison, occupancy of calcium-binding sites in the N-domain of CaM mirrors the calcium-dependence of RyR1 inhibition observed at activating calcium levels, where [Ca]1/2 = 4.3 0.4 μM, suggesting a direct regulation of Ry

  20. High circulating N-terminal pro-brain natriuretic peptide and tumor necrosis factor-α in mixed cryoglobulinemia

    Institute of Scientific and Technical Information of China (English)

    Alessandro Antonelli; Clodoveo Ferri; Silvia Martina Ferrari; Fabio Galetta; Ferdinando Franzoni; Gino Santoro; Salvatore De Marco; Emiliano Ghiri; Poupak Fallahi

    2009-01-01

    AIM: To evaluate serum levels of N-terminal pro-brain natriuretic peptide (NTproBNP) and tumor necrosis factor α (TNF-α) in a large series of patients with hepatitis C associated with mixed cryoglobulinemia (MC+HCV).METHODS: Serum NTproBNP and TNF-α levels were assayed in 50 patients with MC+HCV, and in 50 sex-and age-matched controls.RESULTS: Cryoglobulinemic patients showed significantly higher mean NTproBNP and TNF-α levels than controls ( P < 0.001; Mann-Whitney U test). By defining high NTproBNP level as a value higher than 125 pg/mL (the single cut-off point for outpatients under 75 years of age), 30% of MC+HCV and 6% of controls had high NTproBNP (χ~2, P < 0.01). With a cut-off point of 300 pg/mL (used to rule out heart failure (HF) in patients under 75 years of age), 8% of MC+HCV and 0 controls had high NTproBNP (χ~2, P < 0.04). With a cut-off point of 900 pg/mL (used for ruling in HF in patients aged 50-75 years; such as the patients of our study), 6% of MC+HCV and 0 controls had high NTproBNP (χ~2, P = 0.08).CONCLUSION: The study demonstrates high levels of circulating NTproBNP and TNF-α in MC+HCV patients.The increase of NTproBNP may indicate the presence of a subclinical cardiac dysfunction.

  1. N-Terminal Domain of Feline Calicivirus (FCV) Proteinase-Polymerase Contributes to the Inhibition of Host Cell Transcription.

    Science.gov (United States)

    Wu, Hongxia; Zu, Shaopo; Sun, Xue; Liu, Yongxiang; Tian, Jin; Qu, Liandong

    2016-01-01

    Feline Calicivirus (FCV) infection results in the inhibition of host protein synthesis, known as "shut-off". However, the precise mechanism of shut-off remains unknown. Here, we found that the FCV strain 2280 proteinase-polymerase (PP) protein can suppress luciferase reporter gene expression driven by endogenous and exogenous promoters. Furthermore, we found that the N-terminal 263 aa of PP (PPN-263) determined its shut-off activity using the expression of truncated proteins. However, the same domain of the FCV strain F9 PP protein failed to inhibit gene expression. A comparison between strains 2280 and F9 indicated that Val27, Ala96 and Ala98 were key sites for the inhibition of host gene expression by strain 2280 PPN-263, and PPN-263 exhibited the ability to shut off host gene expression as long as it contained any two of the three amino acids. Because the N-terminus of the PP protein is required for its proteinase and shut-off activities, we investigated the ability of norovirus 3C-like proteins (3CLP) from the GII.4-1987 and -2012 isolates to interfere with host gene expression. The results showed that 3CLP from both isolates was able to shut off host gene expression, but 3CLP from GII.4-2012 had a stronger inhibitory activity than that from GII.4-1987. Finally, we found that 2280 PP and 3CLP significantly repressed reporter gene transcription but did not affect mRNA translation. Our results provide new insight into the mechanism of the FCV-mediated inhibition of host gene expression. PMID:27447663

  2. Type I Collagen Synthesis Marker Procollagen I N-Terminal Peptide (PINP) in Prostate Cancer Patients Undergoing Intermittent Androgen Suppression

    International Nuclear Information System (INIS)

    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. Molecular clone and characterization of c-Jun N-terminal kinases 2 from orange-spotted grouper, Epinephelus coioides.

    Science.gov (United States)

    Guo, Minglan; Wei, Jingguang; Zhou, Yongcan; Qin, Qiwei

    2016-02-01

    c-Jun N-terminal kinase 2 (JNK2) is a multifunctional mitogen-activated protein kinases involving in cell differentiation and proliferation, apoptosis, immune response and inflammatory conditions. In this study, we reported a new JNK2 (Ec-JNK2) derived from orange-spotted grouper, Epinephelus coioides. The full-length cDNA of Ec-JNK2 was 1920 bp in size, containing a 174 bp 5'-untranslated region (UTR), 483 bp 3'-UTR, and a 1263 bp open reading frame (ORF), which encoded a putative protein of 420 amino acids. The deduced protein sequence of Ec-JNK2 contained a conserved Thr-Pro-Tyr (TPY) motif in the domain of serine/threonine protein kinase (S-TKc). Ec-JNK2 has been found to involve in the immune response to pathogen challenges in vivo, and the infection of Singapore grouper iridovirus (SGIV) in vitro. Immunofluorescence staining showed that Ec-JNK2 was localized in the cytoplasm of grouper spleen (GS) cells, and moved to the nucleus after infecting with SGIV. Ec-JNK2 distributed in all immune-related tissues examined. After challenging with lipopolysaccharide (LPS), SGIV and polyriboinosinic polyribocytidylic acid (poly I:C), the mRNA expression of Ec-JNK2 was significantly (P orange-spotted grouper. Over-expressing Ec-JNK2 in fathead minnow (FHM) cells increased the SGIV infection and replication, while over-expressing the dominant-negative Ec-JNK2Δ181-183 mutant decreased it. These results indicated that Ec-JNK2 could be an important molecule in the successful infection and evasion of SGIV.

  4. 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.

  5. C-Jun N-terminal kinase controls TDP-43 accumulation in stress granules induced by oxidative stress

    Directory of Open Access Journals (Sweden)

    Masters Colin L

    2011-08-01

    Full Text Available Abstract Background TDP-43 proteinopathies are characterized by loss of nuclear TDP-43 expression and formation of C-terminal TDP-43 fragmentation and accumulation in the cytoplasm. Recent studies have shown that TDP-43 can accumulate in RNA stress granules (SGs in response to cell stresses and this could be associated with subsequent formation of TDP-43 ubiquinated protein aggregates. However, the initial mechanisms controlling endogenous TDP-43 accumulation in SGs during chronic disease are not understood. In this study we investigated the mechanism of TDP-43 processing and accumulation in SGs in SH-SY5Y neuronal-like cells exposed to chronic oxidative stress. Cell cultures were treated overnight with the mitochondrial inhibitor paraquat and examined for TDP-43 and SG processing. Results We found that mild stress induced by paraquat led to formation of TDP-43 and HuR-positive SGs, a proportion of which were ubiquitinated. The co-localization of TDP-43 with SGs could be fully prevented by inhibition of c-Jun N-terminal kinase (JNK. JNK inhibition did not prevent formation of HuR-positive SGs and did not prevent diffuse TDP-43 accumulation in the cytosol. In contrast, ERK or p38 inhibition prevented formation of both TDP-43 and HuR-positive SGs. JNK inhibition also inhibited TDP-43 SG localization in cells acutely treated with sodium arsenite and reduced the number of aggregates per cell in cultures transfected with C-terminal TDP-43 162-414 and 219-414 constructs. Conclusions Our studies are the first to demonstrate a critical role for kinase control of TDP-43 accumulation in SGs and may have important implications for development of treatments for FTD and ALS, targeting cell signal pathway control of TDP-43 aggregation.

  6. 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.

  7. N-terminal additions to the WE14 peptide of chromogranin A create strong autoantigen agonists in type 1 diabetes.

    Science.gov (United States)

    Jin, Niyun; Wang, Yang; Crawford, Frances; White, Janice; Marrack, Philippa; Dai, Shaodong; Kappler, John W

    2015-10-27

    Chromogranin A (ChgA) is an autoantigen for CD4(+) T cells in the nonobese diabetic (NOD) mouse model of type 1 diabetes (T1D). The natural ChgA-processed peptide, WE14, is a weak agonist for the prototypical T cell, BDC-2.5, and other ChgA-specific T-cell clones. Mimotope peptides with much higher activity share a C-terminal motif, WXRM(D/E), that is predicted to lie in the p5 to p9 position in the mouse MHC class II, IA(g7) binding groove. This motif is also present in WE14 (WSRMD), but at its N terminus. Therefore, to place the WE14 motif into the same position as seen in the mimotopes, we added the amino acids RLGL to its N terminus. Like the other mimotopes, RLGL-WE14, is much more potent than WE14 in T-cell stimulation and activates a diverse population of CD4(+) T cells, which also respond to WE14 as well as islets from WT, but not ChgA(-/-) mice. The crystal structure of the IA(g7)-RLGL-WE14 complex confirmed the predicted placement of the peptide within the IA(g7) groove. Fluorescent IA(g7)-RLGL-WE14 tetramers bind to ChgA-specific T-cell clones and easily detect ChgA-specific T cells in the pancreas and pancreatic lymph nodes of NOD mice. The prediction that many different N-terminal amino acid extensions to the WXRM(D/E) motif are sufficient to greatly improve T-cell stimulation leads us to propose that such a posttranslational modification may occur uniquely in the pancreas or pancreatic lymph nodes, perhaps via the mechanism of transpeptidation. This modification could account for the escape of these T cells from thymic negative selection. PMID:26453556

  8. Mutation of androgen receptor N-terminal phosphorylation site Tyr-267 leads to inhibition of nuclear translocation and DNA binding.

    Science.gov (United States)

    Karaca, Mehmet; Liu, Yuanbo; Zhang, Zhentao; De Silva, Dinuka; Parker, Joel S; Earp, H Shelton; Whang, Young E

    2015-01-01

    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.

  9. Relationship of the plasma urotensin Ⅱ with proadrenomedullin N-terminal 20 peptide in patients with congestive heart failure

    Institute of Scientific and Technical Information of China (English)

    钟萍; 李志樑; 吴宏超; 唐朝枢; 陆青

    2003-01-01

    Objective: To understand the role of urotensin Ⅱ(UⅡ) and proadrenomedullin N-terminal 20 peptide (PAMP), a fragment of proadrenomedullin (proADM) possessing biological activity, in the pathophysiological process of congestive heart failure (CHF) by observing the variation of their plasma levels and exploring their interrelations. Methods: Plasma UⅡ and PAMP levels were measured by radioimmunoassay in 52 patients with CHF and 14 healthy subjects. Left ventricular ejection fraction (LVEF) and the ratio of E/A were determined by echocardiography. Results: The plasma UⅡ level was significantly lower in patients with CHF than the healthy subjects (1.5±1.0 pg/ml vs 4.3±1.2 pg/ml, P<0.05), while plasma PAMP level was significantly higher in the former group (30.6±5.8 pg/ml vs 21.0±6.6 pg/ml P<0.05). The levels of UⅡ and PAMP were parallel with the severity of CHF, and significant correlation of plasma levels of UⅡ with LVEF (r=0.530, P=0.000) and the ratio of E/A (r=0.618, P=0.000) was noted. LVEF and ratio of E/A were found to be inversely correlated with plasma PAMP levels in the patients (r=-0.568, P=0.000; r=-0.350, P=0.004). Also found was the significant correlation between plasma UⅡ and PAMP levels (r=-0.528, P=0.000). The treatment of the patients resulted in increased plasma UⅡ levels and lowered PAMP levels. Conclusion: The variations of plasma levels of UⅡ and PAMP are parallel with the severity of CHF, suggesting their cooperative actions in the pathophysiology of CHF.

  10. Elevated N-terminal pro-brain natriuretic peptide is associated with mortality in tobacco smokers independent of airflow obstruction.

    Directory of Open Access Journals (Sweden)

    Jason A Stamm

    Full Text Available BACKGROUND: Tobacco use is associated with an increased prevalence of cardiovascular disease. N-terminal pro-brain natiuretic peptide (NT-proBNP, a widely available biomarker that is associated with cardiovascular outcomes in other conditions, has not been investigated as a predictor of mortality in tobacco smokers. We hypothesized that NT-proBNP would be an independent prognostic marker in a cohort of well-characterized tobacco smokers without known cardiovascular disease. METHODS: Clinical data from 796 subjects enrolled in two prospective tobacco exposed cohorts was assessed to determine factors associated with elevated NT-proBNP and the relationship of these factors and NT-proBNP with mortality. RESULTS: Subjects were followed for a median of 562 (IQR 252-826 days. Characteristics associated with a NT-proBNP above the median (≥49 pg/mL were increased age, female gender, and decreased body mass index. By time-to-event analysis, an NT-proBNP above the median (≥49 pg/mL was a significant predictor of mortality (log rank p = 0.02. By proportional hazard analysis controlling for age, gender, cohort, and severity of airflow obstruction, an elevated NT-proBNP level (≥49 pg/mL remained an independent predictor of mortality (HR = 2.19, 95% CI 1.07-4.46, p = 0.031. CONCLUSIONS: Elevated NT-proBNP is an independent predictor of mortality in tobacco smokers without known cardiovascular disease, conferring a 2.2 fold increased risk of death. Future studies should assess the ability of this biomarker to guide further diagnostic testing and to direct specific cardiovascular risk reduction inventions that may positively impact quality of life and survival.

  11. Two Distinctive Binding Modes of Endonuclease Inhibitors to the N-Terminal Region of Influenza Virus Polymerase Acidic Subunit.

    Science.gov (United States)

    Fudo, Satoshi; Yamamoto, Norio; Nukaga, Michiyoshi; Odagiri, Takato; Tashiro, Masato; Hoshino, Tyuji

    2016-05-10

    Influenza viruses are global threat to humans, and the development of new antiviral agents are still demanded to prepare for pandemics and to overcome the emerging resistance to the current drugs. Influenza polymerase acidic protein N-terminal domain (PAN) has endonuclease activity and is one of the appropriate targets for novel antiviral agents. First, we performed X-ray cocrystal analysis on the complex structures of PAN with two endonuclease inhibitors. The protein crystallization and the inhibitor soaking were done at pH 5.8. The binding modes of the two inhibitors were different from a common binding mode previously reported for the other influenza virus endonuclease inhibitors. We additionally clarified the complex structures of PAN with the same two endonuclease inhibitors at pH 7.0. In one of the crystal structures, an additional inhibitor molecule, which chelated to the two metal ions in the active site, was observed. On the basis of the crystal structures at pH 7.0, we carried out 100 ns molecular dynamics (MD) simulations for both of the complexes. The analysis of simulation results suggested that the binding mode of each inhibitor to PAN was stable in spite of the partial deviation of the simulation structure from the crystal one. Furthermore, crystal structure analysis and MD simulation were performed for PAN in complex with an inhibitor, which was already reported to have a high compound potency for comparison. The findings on the presence of multiple binding sites at around the PAN substrate-binding pocket will provide a hint for enhancing the binding affinity of inhibitors. PMID:27088785

  12. Arabidopsis MKS1 is involved in basal immunity and requires an intact N-terminal domain for proper function.

    Directory of Open Access Journals (Sweden)

    Klaus Petersen

    Full Text Available BACKGROUND: Innate immune signaling pathways in animals and plants are regulated by mitogen-activated protein kinase (MAPK cascades. MAP kinase 4 (MPK4 functions downstream of innate immune receptors via a nuclear substrate MKS1 to regulate the activity of the WRKY33 transcription factor, which in turn controls the production of anti-microbial phytoalexins. METHODOLOGY/PRINCIPAL FINDINGS: We investigate the role of MKS1 in basal resistance and the importance of its N- and C-terminal domains for MKS1 function. We used the information that mks1 loss-of-function partially suppresses the mpk4 loss-of-function phenotype, and that transgenic expression of functional MKS1 in mpk4/mks1 double mutants reverted the mpk4 dwarf phenotype. Transformation of mks1/mpk4 with mutant versions of MKS1 constructs showed that a single amino acid substitution in a putative MAP kinase docking domain, MKS1-L32A, or a truncated MKS1 version unable to interact with WRKY33, were deficient in reverting the double mutant to the mpk4 phenotype. These results demonstrate functional requirement in MKS1 for the interaction with MPK4 and WRKY33. In addition, nuclear localization of MKS1 was shown to depend on an intact N-terminal domain. Furthermore, loss-of-function mks1 mutants exhibited increased susceptibility to strains of Pseudomonas syringae and Hyaloperonospora arabidopsidis, indicating that MKS1 plays a role in basal defense responses. CONCLUSIONS: Taken together, our results indicate that MKS1 function and subcellular location requires an intact N-terminus important for both MPK4 and WRKY33 interactions.

  13. Runx2-I isoform contributes to fetal bone formation even in the absence of specific N-terminal amino acids.

    Directory of Open Access Journals (Sweden)

    Hideaki Okura

    Full Text Available The Runt-related transcription factor 2 (Runx2 gene encodes the transcription factor Runx2, which is the master regulator of osteoblast development; insufficiency of this protein causes disorders of bone development such as cleidocranial dysplasia. Runx2 has two isoforms, Runx2-II and Runx2-I, and production of each isoform is controlled by a unique promoter: a distal promoter (P1 and a proximal promoter (P2, respectively. Although several studies have focused on differences and similarities between the two Runx2 isoforms, their individual roles in bone formation have not yet been determined conclusively, partly because a Runx2-I-targeted mouse model is not available. In this study, we established a novel Runx2-manipulated mouse model in which the first ATG of Runx2-I was replaced with TGA (a stop codon, and a neomycin-resistant gene (neo cassette was inserted at the first intron of Runx2-I. Homozygous Runx2-Ineo/neo mice showed severely reduced expression of Runx2-I, whereas Runx2-II expression was largely retained. Runx2-Ineo/neo mice showed neonatal lethality, and in these mice, intramembranous ossification was more severely defective than endochondral ossification, presumably because of the greater involvement of Runx2-I, compared with that of Runx2-II in intramembranous ossification. Interestingly, the depletion of neo rescued the above-described phenotypes, indicating that the isoform-specific N-terminal region of Runx2-I is not functionally essential for bone development. Taken together, our results provide a novel clue leading to a better understanding of the roles of Runx2 isoforms in osteoblast development.

  14. High efficiency adenovirus-mediated expression of truncated N-terminal huntingtin fragment (htt552) in primary rat astrocytes

    Institute of Scientific and Technical Information of China (English)

    Linhui Wang; Fang Lin; Junchao Wu; Zhenghong Qin

    2009-01-01

    Huntington's disease (HD) is caused by an expansion of polyglutamine tract in N-terminus of huntingtin (htt).The mutation of htt leads to dysfunction and premature death of striatal and cortical neurons. However, the effects of htt mutation on glia remain largely unknown.This study aimed to establish a glia HD model using an adenoviral vector to express wild-type and mutant N-terminal huntingtin fragment 1-552 amino acids (htt552) in rat primary cortical astrocytes. We have eval-uated optimal conditions for the infection of astrocytes with adenovirai vectors, and the kinetics of the expression of htt552 in astrocytes. The majority of astroeytes expressed the transgene after infection. At 24 h post-infection, the highest rate of infection was 89 + 3% for the wild-type (htt552-18Q) with a multiplicity of infection (m.o.i.) of 80, and the highest rate of infection was 91 +4% for the mutant type (htt552-100Q) with the same viral dose. The duration of expression of htt552 lasted for about 7 days with a relatively high level from 1 to 4 days post-infection. Mutant huntingtin (htt552-100Q) pro-duced the characteristic HD pathology after 3 days by the appearance of cytoplasmic aggregates and intranue-lear inclusions. The result of MTT (3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazoliu mbromide)assay showed that the inhibition of viability by virus on astrocytes was also dose-dependent. To obtain high infection rate and low toxicity, the viral dose with an m.o.i, of 40 was optimal to our cell model. The present study demonstrates that adenovirai-mediated expression of mutant htt provides an advantageous system for his-tological and biochemical analysis of HD pathogenesis in primary cortical astrocyte cultures.

  15. Myocardial protective effects of a c-Jun N-terminal kinase inhibitor in rats with brain death.

    Science.gov (United States)

    Guo, Wenzhi; Cao, Shengli; Yan, Bing; Zhang, Gong; Li, Jie; Zhao, Yongfu; Zhang, Shuijun

    2016-07-01

    To investigate whether the mitochondrial apoptotic pathway mediates myocardial cell injuries in rats under brain death (BD), and observe the effects and mechanisms of the c-Jun N-terminal kinase (JNK) inhibitor SP600125 on cell death in the heart. Forty healthy male Sprague-Dawley (SD) rats were randomized into four groups: sham group (dural external catheter with no BD); BD group (maintain the induced BD state for 6 hrs); BD + SP600125 group (intraperitoneal injection of SP600125 10 mg/kg 1 hr before inducing BD, and maintain BD for 6 hrs); and BD + Dimethyl Sulphoxide (DMSO) group (intraperitoneal injection of DMSO 1 hr before inducing BD, and maintain BD for 6 hrs). Real-time quantitative PCR was used to evaluate mRNA levels of Cyt-c and caspase-3. Western blot analysis was performed to examine the levels of mitochondrial apoptosis-related proteins p-JNK, Bcl-2, Bax, Cyt-c and Caspase-3. TUNEL assay was employed to evaluate myocardial apoptosis. Compared with the sham group, the BD group exhibited increased mitochondrial apoptosis-related gene expression, accompanied by the elevation of p-JNK expression and myocardial apoptosis. As the vehicle control, DMSO had no treatment effects. The BD + SP600125 group had decreased p-JNK expression, and reduced mitochondrial apoptosis-related gene expression. Furthermore, the apoptosis rate of myocardial cells was reduced. The JNK inhibitor SP600125 could protect myocardial cells under BD through the inhibition of mitochondrial apoptosis-related pathways. PMID:27072084

  16. Lumbopelvic flexibility modulates neuromuscular responses during trunk flexion-extension.

    Science.gov (United States)

    Sánchez-Zuriaga, Daniel; Artacho-Pérez, Carla; Biviá-Roig, Gemma

    2016-06-01

    Various stimuli such as the flexibility of lumbopelvic structures influence the neuromuscular responses of the trunk musculature, leading to different load sharing strategies and reflex muscle responses from the afferents of lumbopelvic mechanoreceptors. This link between flexibility and neuromuscular response has been poorly studied. The aim of this study was to investigate the relationship between lumbopelvic flexibility and neuromuscular responses of the erector spinae, hamstring and abdominal muscles during trunk flexion-extension. Lumbopelvic movement patterns were measured in 29 healthy women, who were separated into two groups according to their flexibility during trunk flexion-extension. The electromyographic responses of erector spinae, rectus abdominis and biceps femoris were also recorded. Subjects with greater lumbar flexibility had significantly less pelvic flexibility and vice versa. Subjects with greater pelvic flexibility had a higher rate of relaxation and lower levels of hamstring activation during maximal trunk flexion. The neuromuscular response patterns of the hamstrings seem partially modulated by pelvic flexibility. Not so with the lumbar erector spinae and lumbar flexibility, despite the assertions of some previous studies. The results of this study improve our knowledge of the relationships between trunk joint flexibility and neuromuscular responses, a relationship which may play a role in low back pain. PMID:27155332

  17. Ring-shaped variation of the coeliac trunk branches.

    Science.gov (United States)

    Raikos, A; Pynadath, N; Anguswamy, N; Vallath, S; Kordali, P; Stirling, A

    2015-01-01

    Aberrant arterial variations in the branching pattern of the coeliac trunk are of great interest to surgeons and radiologists. We report on a rare arterial variation found in a 79-year-old cadaver during educational dissection. Specifically, the coeliac axis formed a unique incomplete trunk termed the hepato-hepatic trunk. The splenic artery arose separately from the anterior aspect of the abdominal aorta. On the right side, there was a right hepatic artery giving rise to a gastroduodenal but an absence of the left hepatic. On the left side, there was a branch coursing towards the porta hepatis; the left hepatic artery, dividing into the left gastric, an accessory left gastric, and a branch to the distal oesophagus. The hepato-hepatic trunk formed a ring-shaped vascular structure around the caudate lobe of the liver. Precise mapping and observation of the extrahepatic arteries and bile duct branches is essential in a variety of hepato-biliary laparoscopic procedures of the liver and gallbladder. Other operative procedures requiring, a comprehensive kno-wledge of the varied coeliac trunk patterns are liver transplantation and arterial embolism for hepatic tumour therapy. PMID:26620519

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

    Science.gov (United States)

    Nagel, Claus-Henning; Binz, Anne; Sodeik, Beate; Bauerfeind, Rudolf; Bailer, Susanne M.

    2015-01-01

    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 envelopment. PMID:26083367

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

    Directory of Open Access Journals (Sweden)

    Kaare eBjerregaard-Andersen

    2013-11-01

    Full Text Available The sodium dependent bicarbonate transporter NCBE/NBCn2 is predominantly expressed in the central nervous system (CNS. The highest protein abundance is found in the choroid plexus. The primary function of this integral plasma membrane transport protein is to regulate intracellular neuronal pH and probably to maintain the pH homeostasis across the blood-cerebrospinal fluid barrier (CSFB. NCBE has a transmembrane region consisting of 10 predicted α-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 cytoplasmic N-terminal domain is likely involved in bicarbonate recognition and transport and contains key areas of regulation through pH sensing and protein - protein interactions (PPIs. Intrinsic disordered proteins (IDPs and regions (IDPRs are defined as not having any rigid three-dimensional structure under physiological conditions and are believed to be involved in signaling networks in which specific, though with low affinity, PPIs play an important role in the signaling event. We show that NCBE and other SLC4 family members have a high level of predicted intrinsic disorder prevalent in the cytoplasmic regions. To provide biophysical evidence for the IDPR predicted in Nt-NCBE, we isolated recombinant NCBE from E. coli and purified it to >99 % purity and used it to perform differential scanning fluorescence spectroscopy (DSF, in the search for small molecules that induce secondary or tertiary structure. This will promote the current need to develop selective drugs for individual SLC4 family members. We have also determined a low resolution X-ray crystal structure of the N-terminal core domain at 4.0 Å resolution. The N-terminal cytoplasmic domain of AE1 (cdb3 shares a similar fold with the N-terminal core domain of NCBE. The crystal conditions for the full-length N-terminal domain have been explored, however, only the core domain forms diffracting

  20. 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

  1. Studi Perencanaan Migrasi Sistem Digital Oleh Penyelenggara Radio Trunking di Indonesia

    Directory of Open Access Journals (Sweden)

    Awangga Febian Surya Admaja

    2013-06-01

    Full Text Available Sistem Radio Trunking merupakan sistem radio yang berbasis repeater untuk satu atau lebih menara dengan menggunakan lebih dari satu frekuensi, dimana pengguna secara semi-privat dapat memiliki kanal tersendiri untuk melakukan pembicaraan secara grup. Di Indonesia, alokasi pada pita frekuensi radio trunking analog direncanakan akan digunakan untuk trunking digital, dimana aplikasi sistem radio trunking  yang baru harus menggunakan teknologi trunking digital dan sistem analog yang ada disyaratkan untuk beralih ke teknologi digital. Studi ini bertujuan untuk melihat seberapa besar kesiapan dari penyelenggara radio trunking di Indonesia dalam melakukan migrasi radio trunking digital. Studi ini menggunakan pendekatan kualitatif melalui wawancara mendalam dan didukung dengan data kuantitatif untuk menunjukkan nilai indeks kesiapan dari penyelenggara radio trunking. Studi ini menghasilkan nilai indeks kesiapan dari sampel penyelenggara radio trunking dengan skema perencanaan migrasi sesuai dengan nilai indeks kesiapan.

  2. Common celiaco-mesenterico-phrenic trunk and renal vascular variations.

    Science.gov (United States)

    Nayak, Satheesha

    2006-12-01

    The knowledge of vascular variations like other anatomical variations, is important during the operative, diagnostic, and endovascular procedures in abdomen. This report describes multiple variations in the upper abdominal vessels as found during the routine dissection in a 60-year-old male cadaver. The variations found were; presence of a celiaco-mesenterico-phrenic trunk, a common inferior phrenic trunk, 2 right renal arteries originating from abdominal aorta, 2 suprarenal arteries originating from the lower right renal artery, 3 right renal veins opening separately into inferior vena cava, and termination of right testicular vein into the lowest vein among the 3 right renal veins. The existence of a celiaco-mesenterico-phrenic trunk has not been reported yet. Although, other variations reported in this case exist as individual variations, a concomitant variation of them has not been reported yet. The knowledge of such variations is quite useful in planning any upper abdominal surgery. PMID:17143371

  3. Evolution of the head-trunk interface in tetrapod vertebrates.

    Science.gov (United States)

    Sefton, Elizabeth M; Bhullar, Bhart-Anjan S; Mohaddes, Zahra; Hanken, James

    2016-01-01

    Vertebrate neck musculature spans the transition zone between head and trunk. The extent to which the cucullaris muscle is a cranial muscle allied with the gill levators of anamniotes or is instead a trunk muscle is an ongoing debate. Novel computed tomography datasets reveal broad conservation of the cucullaris in gnathostomes, including coelacanth and caecilian, two sarcopterygians previously thought to lack it. In chicken, lateral plate mesoderm (LPM) adjacent to occipital somites is a recently identified embryonic source of cervical musculature. We fate-map this mesoderm in the axolotl (Ambystoma mexicanum), which retains external gills, and demonstrate its contribution to posterior gill-levator muscles and the cucullaris. Accordingly, LPM adjacent to the occipital somites should be regarded as posterior cranial mesoderm. The axial position of the head-trunk border in axolotl is congruent between LPM and somitic mesoderm, unlike in chicken and possibly other amniotes. PMID:27090084

  4. Postoperative Trunk Shift in Lenke 1C Scoliosis

    DEFF Research Database (Denmark)

    Wang, Yu; Bünger, Cody Eric; Wu, Chunsen;

    2012-01-01

    STUDY DESIGN: A risk factor analysis study. OBJECTIVE: To identify the causative factors for postoperative trunk shift in Lenke 1C scoliosis and investigate how to prevent it. SUMMARY OF BACKGROUND DATA: When selective thoracic fusion is performed, postoperative trunk shift is a significant problem...... in the management of Lenke 1C scoliosis. It is often accompanied by unsatisfactory clinical outcomes and a risk of reoperation. METHODS: We reviewed all the patients with adolescent idiopathic scoliosis (AIS) surgically treated in our institution from 2002 through 2008. Inclusion criteria were as follows: (1...... of MT: TL/L Cobb angle) - 3.9 (preoperative LIV-LEV difference). The model R2 = 0.55. CONCLUSION: Both LIV selection and ratio of MT: TL/L curve were found to be highly correlated with the onset of postoperative trunk shift in Lenke 1C scoliosis. Amount of correction obtained by surgery, however, did...

  5. Evolution of the head-trunk interface in tetrapod vertebrates.

    Science.gov (United States)

    Sefton, Elizabeth M; Bhullar, Bhart-Anjan S; Mohaddes, Zahra; Hanken, James

    2016-04-19

    Vertebrate neck musculature spans the transition zone between head and trunk. The extent to which the cucullaris muscle is a cranial muscle allied with the gill levators of anamniotes or is instead a trunk muscle is an ongoing debate. Novel computed tomography datasets reveal broad conservation of the cucullaris in gnathostomes, including coelacanth and caecilian, two sarcopterygians previously thought to lack it. In chicken, lateral plate mesoderm (LPM) adjacent to occipital somites is a recently identified embryonic source of cervical musculature. We fate-map this mesoderm in the axolotl (Ambystoma mexicanum), which retains external gills, and demonstrate its contribution to posterior gill-levator muscles and the cucullaris. Accordingly, LPM adjacent to the occipital somites should be regarded as posterior cranial mesoderm. The axial position of the head-trunk border in axolotl is congruent between LPM and somitic mesoderm, unlike in chicken and possibly other amniotes.

  6. Crystallization and preliminary X-ray crystallographic analysis of a 40 kDa N-terminal fragment of the yeast prion-remodeling factor Hsp104

    International Nuclear Information System (INIS)

    An N-terminal fragment of S. cerevisiae Hsp104 has been crystallized. This is the first report of the crystallization of a eukaryotic member of the Hsp100 family of molecular chaperones. A 40 kDa N-terminal fragment of Saccharomyces cerevisiae Hsp104 was crystallized in two different crystal forms. Native 1 diffracted to 2.6 Å resolution and belonged to space group P212121, with unit-cell parameters a = 66.6, b = 75.8, c = 235.7 Å. Native 2 diffracted to 2.9 Å resolution and belonged to space group P6122 or P6522, with unit-cell parameters a = 179.1, b = 179.1, c = 69.7 Å. This is the first report of the crystallization of a eukaryotic member of the Hsp100 family of molecular chaperones

  7. The N-terminal domain of NifA determines the temperature sensitivity of Nif A in Klebsiella pneumoniae and Enterobacter cloacae

    Institute of Scientific and Technical Information of China (English)

    顾剑颖; 俞冠翘; 朱家璧; 沈善炯

    2000-01-01

    The NifA protein is the central regulator of the nitrogen fixation genes. It activates transcription of nif genes by an alternative holoenzyme form of RNA polymerase containing the σ54 factor. The NifA protein from Klebsiella pneumoniae consists of the N-terminal domain of unknown function, the central catalytic domain with ATPase activity and the C-terminal DNA-binding domain. The Kp NifA protein is sensitive to temperature, while the Enterobacter cloacae NifA protein is less sensitive to temperature than Kp NifA. Our results show that the N-terminal domain of NifA plays the decisive role in the temperature sensitivity of the protein.

  8. A highly conserved N-terminal sequence for teleost vitellogenin with potential value to the biochemistry, molecular biology and pathology of vitellogenesis

    Science.gov (United States)

    Folmar, L.D.; Denslow, N.D.; Wallace, R.A.; LaFleur, G.; Gross, T.S.; Bonomelli, S.; Sullivan, C.V.

    1995-01-01

    N-terminal amino acid sequences for vitellogenin (Vtg) from six species of teleost fish (striped bass, mummichog, pinfish, brown bullhead, medaka, yellow perch and the sturgeon) are compared with published N-terminal Vtg sequences for the lamprey, clawed frog and domestic chicken. Striped bass and mummichog had 100% identical amino acids between positions 7 and 21, while pinfish, brown bullhead, sturgeon, lamprey, Xenopus and chicken had 87%, 93%, 60%, 47%, 47-60%) for four transcripts and had 40% identical, respectively, with striped bass for the same positions. Partial sequences obtained for medaka and yellow perch were 100% identical between positions 5 to 10. The potential utility of this conserved sequence for studies on the biochemistry, molecular biology and pathology of vitellogenesis is discussed.

  9. The N-terminal cellulose-binding domain of EGXA increases thermal stability of xylanase and changes its specific activities on different substrates

    Institute of Scientific and Technical Information of China (English)

    Ming Ding; Yigang Teng; Qiuyu Yin; Jie Zhao; Fukun Zhao

    2008-01-01

    A full-length EGXA enzyme from a mollusk, Ampullaria crossean, was cloned into pFastBac vector and then heterogeneously expressed in insect Tn5 cells. Its natural N-terminal signal peptide worked well in the insect Tn5 cells.The recombinant EGXA was a 63 kDa protein and had active endo-β-1,4-glucanase (EC 3.2.1.4) and endo-β-1,4-xylanase (EC 3.2.1.8). The specific activity of endo-β-1,4-xylanase was higher than in the EGX, which was purified from the stomach tissues of Ampullaria crossen. The N-terminal cellulosebinding domain of EGXA made it bind to cellulose and xylan more efficiently. This cellulose-binding domain also increased the thermal stability of this recombinant enzyme and decreased the recombinant EGXA's specific activities on p-nitrophenyi-β-D-cellobioside and sodium carboxymethyl cellulose.

  10. The N-terminal domain of NifA determines the temperature sensitivity of NifA in Klebsiella pneumoniae and Enterobacter cloacae

    Institute of Scientific and Technical Information of China (English)

    2000-01-01

    The NifA protein is the central regulator of the nitrogen fixation genes.It activates transcription of nif genes by an alternative holoenzyme form of RNA polymerase containing the σ54 factor.The NifA protein from Klebsiella pneumoniae consists of the N-terminal domain of unknown function,the central catalytic domain with ATPase activity and the C-terminal DNA-binding domain.The Kp NifA protein is sensitive to temperature,while the Enterobacter cloacae NifA protein is less sensitive to temperature than Kp NifA.Our results show that the N-terminal domain of NifA plays the decisive role in the temperature sensitivity of the protein.

  11. Test-retest reliability of trunk accelerometric gait analysis

    DEFF Research Database (Denmark)

    Henriksen, Marius; Lund, Hans; Moe-Nilssen, R;

    2004-01-01

    The purpose of this study was to determine the test-retest reliability of a trunk accelerometric gait analysis in healthy subjects. Accelerations were measured during walking using a triaxial accelerometer mounted on the lumbar spine of the subjects. Six men and 14 women (mean age 35.2; range 18-...... a definite potential in clinical gait analysis.......The purpose of this study was to determine the test-retest reliability of a trunk accelerometric gait analysis in healthy subjects. Accelerations were measured during walking using a triaxial accelerometer mounted on the lumbar spine of the subjects. Six men and 14 women (mean age 35.2; range 18...

  12. Bilateral lingual-facial trunk: anatomic and clinical implications.

    Science.gov (United States)

    Troupis, T; Michalinos, A; Kakisis, J; Natsis, K; Sofidis, G; Skandalakis, P

    2015-01-01

    Common origin of lingual and facial artery is a relatively frequent anatomic varia-tion. Instead, bilateral lingual-facial trunk has been described only sparsely in the literature. In this report authors describe and analyse a case of bilateral common lingual-facial trunk in the context of its anatomical, clinical and embryological implications. We also describe possible consequences in performance of elective and emergent surgical operations and modification in surgical techniques that should be considered. We believe that surgeons should be suspicious for this variation's existence and keep alternative solutions in their armentarium.

  13. The effect of active sitting on trunk motion

    OpenAIRE

    Henry Wang; Kaitlyn J. Weiss; Mason C. Haggerty; Jacqueline E Heath

    2014-01-01

    Background: Prolonged sitting is a risk factor for low-back pain. The primary purpose of this study is to determine if prolonged active sitting will result in increased trunk motion. Methods: Fifteen healthy female participants volunteered to sit for 30 min on each of three surfaces including an air-cushion, a stability ball, and a hard surface. Trunk motion was monitored using a Vicon motion capture system, and foot center of pressure was collected with two AMTI force plates. Results: ...

  14. Role of N-terminal extension of Bacillus stearothermophilus RNase H2 and C-terminal extension of Thermotoga maritima RNase H2.

    Science.gov (United States)

    Permanasari, Etin-Diah; Angkawidjaja, Clement; Koga, Yuichi; Kanaya, Shigenori

    2013-10-01

    Bacillus stearothermophilus RNase H2 (BstRNH2) and Thermotoga maritima RNase H2 (TmaRNH2) have N-terminal and C-terminal extensions, respectively, as compared with Aquifex aeolicus RNase H2 (AaeRNH2). To analyze the role of these extensions, BstRNH2 and TmaRNH2 without these extensions were constructed, and their biochemical properties were compared with those of their intact partners and AaeRNH2. The far-UV CD spectra of all proteins were similar, suggesting that the protein structure is not significantly altered by removal of these extensions. However, both the junction ribonuclease and RNase H activities of BstRNH2 and TmaRNH2, as well as their substrate-binding affinities, were considerably decreased by removal of these extensions. The stability of BstRNH2 and TmaRNH2 was also decreased by removal of these extensions. The activity, substrate binding affinity and stability of TmaRNH2 without the C-terminal 46 residues were partly restored by the attachment of the N-terminal extension of BstRNH2. These results suggest that the N-terminal extension of BstRNH2 functions as a substrate-binding domain and stabilizes the RNase H domain. Because the C-terminal extension of TmaRNH2 assumes a helix hairpin structure and does not make direct contact with the substrate, this extension is probably required to make the conformation of the substrate-binding site functional. AaeRNH2 showed comparable junction ribonuclease activity to those of BstRNH2 and TmaRNH2, and was more stable than these proteins, indicating that bacterial RNases H2 do not always require an N-terminal or C-terminal extension to increase activity, substrate-binding affinity, and/or stability. PMID:23937561

  15. Distribution of distances between the tryptophan and the N-terminal residue of melittin in its complex with calmodulin, troponin C, and phospholipids.

    OpenAIRE

    Lakowicz, J.R.; Gryczynski, I.; Laczko, G; Wiczk, W; Johnson, M.L.

    1994-01-01

    We used frequency-domain measurements of fluorescence resonance energy transfer to measure the distribution of distances between Trp-19 of melittin and a 1-dimethylamino-5-sulfonylnaphthalene (dansyl) residue on the N-terminal-alpha-amino group. Distance distributions were obtained for melittin free in solution and when complexed with calmodulin (CaM), troponin C (TnC), or palmitoyloleoyl-L-alpha-phosphatidylcholine (POPC) vesicles. A wide range of donor (Trp-19)-to-acceptor (dansyl) distance...

  16. Chimeric RXFP1 and RXFP2 Receptors Highlight the Similar Mechanism of Activation Utilizing Their N-Terminal Low-Density Lipoprotein Class A Modules

    OpenAIRE

    Bruell, Shoni; Kong, Roy C. K.; Petrie, Emma J.; Hoare, Brad; John D Wade; Scott, Daniel J.; Gooley, Paul R.; Bathgate, Ross A. D.

    2013-01-01

    Relaxin family peptide (RXFP) receptors 1 and 2 are unique G-protein coupled receptors in that they contain an N-terminal low-density lipoprotein type A (LDLa) module which is necessary for receptor activation. The current hypothesis suggests that upon ligand binding the LDLa module interacts with the transmembrane (TM) domain of a homodimer partner receptor to induce the active receptor conformations. We recently demonstrated that three residues in the N-terminus of the RXFP1 LDLa module are...

  17. Chimeric RXFP1 and RXFP2 receptors highlight the similar mechanism of activation utilizing their N-terminal low density lipoprotein class A modules

    OpenAIRE

    RossBathgate; EmmaJunePetrie; JohnDWade

    2013-01-01

    Relaxin family peptide (RXFP) receptors 1 and 2 are unique G-protein coupled receptors in that they contain an N-terminal low density lipoprotein type A (LDLa) module which is necessary for receptor activation. The current hypothesis suggests that upon ligand binding the LDLa module interacts with the transmembrane (TM) domain of a homodimer partner receptor to induce the active receptor conformations. We recently demonstrated that three residues in the N-terminus of the RXFP1 LDLa module are...

  18. Evaluation of Serum Levels of N-terminal Pro Brain Natriuretic Peptide and Atrial Natriuretic Peptide in Neonates with Respiratory Distress

    OpenAIRE

    Noor Mohammad Noori; Sima Savadkoohi; Alireza Teimouri; Fatemeh Alizadeh

    2016-01-01

    Background: Acute respiratory distress (ARD) is a critical respiratory failure due to lung injury of neonates leading to the clinical appearance of poor lung compliance. The aimed of the study was to evaluate the diagnostic values in differentiating respiratory from heart diseases with using of N-terminal pro brain natriuretic peptide (NT-pro BNP) and Atrial natriuretic peptide(ANP) in neonates. Material and Methods: Ninety  neonates  randomly collected from those who hospitalized in the neon...

  19. Opposing actions of intact and N-terminal fragments of the human prolactin/growth hormone family members on angiogenesis: An efficient mechanism for the regulation of angiogenesis

    OpenAIRE

    Struman, Ingrid; Bentzien, Frauke; Lee, Hsinyu; Mainfroid, Véronique; D’Angelo, Gisela; Goffin, Vincent; Weiner, Richard I.; Martial, Joseph A.

    1999-01-01

    Angiogenesis, the process of development of a new microvasculature, is regulated by a balance of positive and negative factors. We show both in vivo and in vitro that the members of the human prolactin/growth hormone family, i.e., human prolactin, human growth hormone, human placental lactogen, and human growth hormone variant are angiogenic whereas their respective 16-kDa N-terminal fragments are antiangiogenic. The opposite actions are regulated in part via activ...

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

    International Nuclear Information System (INIS)

    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 15N-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

  1. 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.

  2. Simple Screening Method for Autoantigen Proteins Using the N-Terminal Biotinylated Protein Library Produced by Wheat Cell-Free Synthesis

    OpenAIRE

    Matsuoka, Kazuhiro; Komori, Hiroaki; Nose, Masato; Endo, Yaeta; Sawasaki, Tatsuya

    2010-01-01

    Autoimmune diseases are a heterogeneous group of diseases characterized by immune reactions against either a major or a limited number of the bodies own autoantigens, causing inflammation and damage to tissues and organs. Thus, identification of autoantigens is an important first step to understanding autoimmune diseases. Here we demonstrate a simple screening method for identification of autoantigens reacting with patient serum antibodies by combination of an N-terminal biotinylated protein ...

  3. New approaches for synthesis and analysis of adducts to N-terminal valine in hemoglobin from isocyanates, aldehydes, methyl vinyl ketone and diepoxybutane

    OpenAIRE

    Davies, Ronnie

    2009-01-01

    Human exposure to harmful compounds in the environment, from intake via food, occupational exposures or other sources, could have health implications. Exposure to reactive compounds/metabolites can be identified and quantified as hemoglobin (Hb) adducts by mass spectrometry. This thesis aimed at improved synthetic pathways for reference standards, and improved analytical methods for adducts to N-terminal valine in Hb from a range of reactive compounds; isocyanates, aldehydes, methyl vinyl ket...

  4. Thrombospondin-1-N-Terminal Domain Induces a Phagocytic State and Thrombospondin-1-C-Terminal Domain Induces a Tolerizing Phenotype in Dendritic Cells

    OpenAIRE

    Tabib, Adi; Krispin, Alon; Trahtemberg, Uriel; Verbovetski, Inna; Lebendiker, Mario; Danieli, Tsafi; Mevorach, Dror

    2009-01-01

    In our previous study, we have found that thrombospondin-1 (TSP-1) is synthesized de novo upon monocyte and neutrophil apoptosis, leading to a phagocytic and tolerizing phenotype of dendritic cells (DC), even prior to DC-apoptotic cell interaction. Interestingly, we were able to show that heparin binding domain (HBD), the N-terminal portion of TSP-1, was cleaved and secreted simultaneously in a caspase- and serine protease- dependent manner. In the current study we were interested to examine ...

  5. Intracellular membrane association of the N-terminal domain of classical swine fever virus NS4B determines viral genome replication and virulence.

    Science.gov (United States)

    Tamura, Tomokazu; Ruggli, Nicolas; Nagashima, Naofumi; Okamatsu, Masatoshi; Igarashi, Manabu; Mine, Junki; Hofmann, Martin A; Liniger, Matthias; Summerfield, Artur; Kida, Hiroshi; Sakoda, Yoshihiro

    2015-09-01

    Classical swine fever virus (CSFV) causes a highly contagious disease in pigs that can range from a severe haemorrhagic fever to a nearly unapparent disease, depending on the virulence of the virus strain. Little is known about the viral molecular determinants of CSFV virulence. The nonstructural protein NS4B is essential for viral replication. However, the roles of CSFV NS4B in viral genome replication and pathogenesis have not yet been elucidated. NS4B of the GPE-  vaccine strain and of the highly virulent Eystrup strain differ by a total of seven amino acid residues, two of which are located in the predicted trans-membrane domains of NS4B and were described previously to relate to virulence, and five residues clustering in the N-terminal part. In the present study, we examined the potential role of these five amino acids in modulating genome replication and determining pathogenicity in pigs. A chimeric low virulent GPE- -derived virus carrying the complete Eystrup NS4B showed enhanced pathogenicity in pigs. The in vitro replication efficiency of the NS4B chimeric GPE-  replicon was significantly higher than that of the replicon carrying only the two Eystrup-specific amino acids in NS4B. In silico and in vitro data suggest that the N-terminal part of NS4B forms an amphipathic α-helix structure. The N-terminal NS4B with these five amino acid residues is associated with the intracellular membranes. Taken together, this is the first gain-of-function study showing that the N-terminal domain of NS4B can determine CSFV genome replication in cell culture and viral pathogenicity in pigs. PMID:26018962

  6. Structural and functional characterization of the Geobacillus copper nitrite reductase: involvement of the unique N-terminal region in the interprotein electron transfer with its redox partner.

    Science.gov (United States)

    Fukuda, Yohta; Koteishi, Hiroyasu; Yoneda, Ryohei; Tamada, Taro; Takami, Hideto; Inoue, Tsuyoshi; Nojiri, Masaki

    2014-03-01

    The crystal structures of copper-containing nitrite reductase (CuNiR) from the thermophilic Gram-positive bacterium Geobacillus kaustophilus HTA426 and the amino (N)-terminal 68 residue-deleted mutant were determined at resolutions of 1.3Å and 1.8Å, respectively. Both structures show a striking resemblance with the overall structure of the well-known CuNiRs composed of two Greek key β-barrel domains; however, a remarkable structural difference was found in the N-terminal region. The unique region has one β-strand and one α-helix extended to the northern surface of the type-1 copper site. The superposition of the Geobacillus CuNiR model on the electron-transfer complex structure of CuNiR with the redox partner cytochrome c551 in other denitrifier system led us to infer that this region contributes to the transient binding with the partner protein during the interprotein electron transfer reaction in the Geobacillus system. Furthermore, electron-transfer kinetics experiments using N-terminal residue-deleted mutant and the redox partner, Geobacillus cytochrome c551, were carried out. These structural and kinetics studies demonstrate that the region is directly involved in the specific partner recognition.

  7. 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

    2016-01-01

    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 [1H, 15N]-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 Zn2+-binding to the octarepeat motif. PMID:27341298

  8. 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.

  9. 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.

  10. Roles of N-terminal fatty acid acylations in membrane compartment partitioning: Arabidopsis h-type thioredoxins as a case study.

    Science.gov (United States)

    Traverso, José A; Micalella, Chiara; Martinez, Aude; Brown, Spencer C; Satiat-Jeunemaître, Béatrice; Meinnel, Thierry; Giglione, Carmela

    2013-03-01

    N-terminal fatty acylations (N-myristoylation [MYR] and S-palmitoylation [PAL]) are crucial modifications affecting 2 to 4% of eukaryotic proteins. The role of these modifications is to target proteins to membranes. Predictive tools have revealed unexpected targets of these acylations in Arabidopsis thaliana and other plants. However, little is known about how N-terminal lipidation governs membrane compartmentalization of proteins in plants. We show here that h-type thioredoxins (h-TRXs) cluster in four evolutionary subgroups displaying strictly conserved N-terminal modifications. It was predicted that one subgroup undergoes only MYR and another undergoes both MYR and PAL. We used plant TRXs as a model protein family to explore the effect of MYR alone or MYR and PAL in the same family of proteins. We used a high-throughput biochemical strategy to assess MYR of specific TRXs. Moreover, various TRX-green fluorescent protein fusions revealed that MYR localized protein to the endomembrane system and that partitioning between this membrane compartment and the cytosol correlated with the catalytic efficiency of the N-myristoyltransferase acting at the N terminus of the TRXs. Generalization of these results was obtained using several randomly selected Arabidopsis proteins displaying a MYR site only. Finally, we demonstrated that a palmitoylatable Cys residue flanking the MYR site is crucial to localize proteins to micropatching zones of the plasma membrane.

  11. Roles of N-Terminal Fatty Acid Acylations in Membrane Compartment Partitioning: Arabidopsis h-Type Thioredoxins as a Case Study[C][W

    Science.gov (United States)

    Traverso, José A.; Micalella, Chiara; Martinez, Aude; Brown, Spencer C.; Satiat-Jeunemaître, Béatrice; Meinnel, Thierry; Giglione, Carmela

    2013-01-01

    N-terminal fatty acylations (N-myristoylation [MYR] and S-palmitoylation [PAL]) are crucial modifications affecting 2 to 4% of eukaryotic proteins. The role of these modifications is to target proteins to membranes. Predictive tools have revealed unexpected targets of these acylations in Arabidopsis thaliana and other plants. However, little is known about how N-terminal lipidation governs membrane compartmentalization of proteins in plants. We show here that h-type thioredoxins (h-TRXs) cluster in four evolutionary subgroups displaying strictly conserved N-terminal modifications. It was predicted that one subgroup undergoes only MYR and another undergoes both MYR and PAL. We used plant TRXs as a model protein family to explore the effect of MYR alone or MYR and PAL in the same family of proteins. We used a high-throughput biochemical strategy to assess MYR of specific TRXs. Moreover, various TRX–green fluorescent protein fusions revealed that MYR localized protein to the endomembrane system and that partitioning between this membrane compartment and the cytosol correlated with the catalytic efficiency of the N-myristoyltransferase acting at the N terminus of the TRXs. Generalization of these results was obtained using several randomly selected Arabidopsis proteins displaying a MYR site only. Finally, we demonstrated that a palmitoylatable Cys residue flanking the MYR site is crucial to localize proteins to micropatching zones of the plasma membrane. PMID:23543785

  12. The N-terminal domains of Vps3 and Vps8 are critical for localization and function of the CORVET tethering complex on endosomes.

    Directory of Open Access Journals (Sweden)

    Nadine Epp

    Full Text Available Endosomal biogenesis depends on multiple fusion and fission events. For fusion, the heterohexameric CORVET complex as an effector of the endosomal Rab5/Vps21 GTPase has a central function in the initial tethering event. Here, we show that the CORVET-specific Vps3 and Vps8 subunits, which interact with Rab5/Vps21, require their N-terminal domains for localization and function. Surprisingly, CORVET may lack either one of the two N-terminal domains, but not both, to promote protein sorting via the endosome. The dually truncated complex mislocalizes to the cytosol and is impaired in endocytic protein sorting, but not in assembly. Furthermore, the endosomal localization can be rescued by overexpression of Vps21 or one of the truncated CORVET subunits, even though CORVET assembly is not impaired by loss of the N-terminal domains or in strains lacking all endosomal Rab5s and Ypt7. We thus conclude that CORVET requires only its C-terminal domains for assembly and has beyond its putative β-propeller domains additional binding sites for endosomes, which could be important to bind Vps21 and other endosome-specific factors for efficient endosome tethering.

  13. Functional analysis of the extended N-terminal region in PLC-δ1 (MlPLC-δ1) from the mud loach, Misgurnus mizolepis.

    Science.gov (United States)

    Kim, Na Young; Ahn, Sang Jung; Kim, Moo-Sang; Seo, Jung Soo; Jung, Se Hwan; Park, Sung Hwan; Lee, Hyung Ho; Chung, Joon Ki

    2014-01-01

    Mud loach phospholipase C-δ1 (MlPLC-δ1) contains all the characteristic domains found in mammalian PLC-δ isozymes (pleckstrin homology domain, EF-hands, X–Y catalytic region, and C2 domain) as well as an extended 26-amino acid (aa)-long N-terminal region that is an alternative splice form of PLC-δ1 and is novel to vertebrate PLC-δ. In the present structure-function analysis, deletion of the extended N-terminal region caused complete loss of phosphatidylinositol (PI)- and phosphatidylinositol 4,5-bisphosphate (PIP2)-hydrolyzing activity in MlPLC-δ1. Additionally, recombinant full-length MlPLC-δ1 PLC activity was reduced in a dose-dependent manner by coincubation with the 26-aa protein fragment. Using a protein-lipid overlay assay, both full-length MlPLC-δ1 and the 26-aa protein fragment had substantial affinity for PIP2, whereas deletion of the 26-aa region from MlPLC-δ1 (MlPLC-δ1-deletion) resulted in lower affinity for PIP2. These results suggest that the novel N-terminal exon of MlPLC-δ1 could play an important role in the regulation of PLC-δ1.

  14. The N-terminal repeat and the ligand binding domain A of SdrI protein is involved in hydrophobicity of S. saprophyticus.

    Science.gov (United States)

    Kleine, Britta; Ali, Liaqat; Wobser, Dominique; Sakιnç, Türkân

    2015-03-01

    Staphylococcus saprophyticus is an important cause of urinary tract infection, and its cell surface hydrophobicity may contribute to virulence by facilitating adherence of the organism to uroepithelia. S. saprophyticus expresses the surface protein SdrI, a member of the serine-aspartate repeat (SD) protein family, which has multifunctional properties. The SdrI knock out mutant has a reduced hydrophobicity index (HPI) of 25%, and expressed in the non-hydrophobic Staphylococcus carnosus strain TM300 causes hydrophobicity. Using hydrophobic interaction chromatography (HIC), we confined the hydrophobic site of SdrI to the N-terminal repeat region. S. saprophyticus strains carrying different plasmid constructs lacking either the N-terminal repeats, both B or SD-repeats were less hydrophobic than wild type and fully complemented SdrI mutant (HPI: 51%). The surface hydrophobicity and HPI of both wild type and the complemented strain were also influenced by calcium (Ca(2+)) and were reduced from 81.3% and 82.4% to 10.9% and 12.3%, respectively. This study confirms that the SdrI protein of S. saprophyticus is a crucial factor for surface hydrophobicity and also gives a first significant functional description of the N-terminal repeats, which in conjunction with the B-repeats form an optimal hydrophobic conformation.

  15. The N-terminal repeat and the ligand binding domain A of SdrI protein is involved in hydrophobicity of S. saprophyticus.

    Science.gov (United States)

    Kleine, Britta; Ali, Liaqat; Wobser, Dominique; Sakιnç, Türkân

    2015-03-01

    Staphylococcus saprophyticus is an important cause of urinary tract infection, and its cell surface hydrophobicity may contribute to virulence by facilitating adherence of the organism to uroepithelia. S. saprophyticus expresses the surface protein SdrI, a member of the serine-aspartate repeat (SD) protein family, which has multifunctional properties. The SdrI knock out mutant has a reduced hydrophobicity index (HPI) of 25%, and expressed in the non-hydrophobic Staphylococcus carnosus strain TM300 causes hydrophobicity. Using hydrophobic interaction chromatography (HIC), we confined the hydrophobic site of SdrI to the N-terminal repeat region. S. saprophyticus strains carrying different plasmid constructs lacking either the N-terminal repeats, both B or SD-repeats were less hydrophobic than wild type and fully complemented SdrI mutant (HPI: 51%). The surface hydrophobicity and HPI of both wild type and the complemented strain were also influenced by calcium (Ca(2+)) and were reduced from 81.3% and 82.4% to 10.9% and 12.3%, respectively. This study confirms that the SdrI protein of S. saprophyticus is a crucial factor for surface hydrophobicity and also gives a first significant functional description of the N-terminal repeats, which in conjunction with the B-repeats form an optimal hydrophobic conformation. PMID:25497915

  16. Dissecting functions of the N-terminal domain and GAS-site recognition in STAT3 nuclear trafficking.

    Science.gov (United States)

    Martincuks, Antons; Fahrenkamp, Dirk; Haan, Serge; Herrmann, Andreas; Küster, Andrea; Müller-Newen, Gerhard

    2016-08-01

    Signal transducer and activator of transcription 3 (STAT3) is a ubiquitous transcription factor involved in many biological processes, including hematopoiesis, inflammation and cancer progression. Cytokine-induced gene transcription greatly depends on tyrosine phosphorylation of STAT3 on a single tyrosine residue with subsequent nuclear accumulation and specific DNA sequence (GAS) recognition. In this study, we analyzed the roles of the conserved STAT3 N-terminal domain (NTD) and GAS-element binding ability of STAT3 in nucleocytoplasmic trafficking. Our results demonstrate the nonessential role of GAS-element recognition for both cytokine-induced and basal nuclear import of STAT3. Substitution of five key amino acids within the DNA-binding domain rendered STAT3 unable to bind to GAS-elements while still maintaining the ability for nuclear localization. In turn, deletion of the NTD markedly decreased nuclear accumulation upon IL-6 treatment resulting in a prolonged accumulation of phosphorylated dimers in the cytoplasm, at the same time preserving specific DNA recognition ability of the truncation mutant. Observed defect in nuclear localization could not be explained by flawed importin-α binding, since both wild-type and NTD deletion mutant of STAT3 could precipitate both full-length and autoinhibitory domain (∆IBB) deletion mutants of importin-α5, as well as ∆IBB-α3 and ∆IBB-α7 isoforms independently of IL-6 stimulation. Despite its inability to translocate to the nucleus upon IL-6 stimulation, the NTD lacking mutant still showed nuclear accumulation in resting cells similar to wild-type upon inhibition of nuclear export by leptomycin B. At the same time, blocking the nuclear export pathway could not rescue cytoplasmic trapping of phosphorylated STAT3 molecules without NTD. Moreover, STAT3 mutant with dysfunctional SH2 domain (R609Q) also localized in the nucleus of unstimulated cells after nuclear export blocking, while upon cytokine treatment the

  17. N-Terminal Prosomatostatin as a Risk Marker for Cardiovascular Disease and Diabetes in a General Population

    Science.gov (United States)

    Almgren, Peter; Nilsson, Peter M.; Melander, Olle

    2016-01-01

    Context: Somatostatin inhibits a range of hormones, including GH, insulin, and glucagon, but little is known about its role in the development of cardiometabolic disease. Objective: The objective of the study was to investigate whether fasting plasma concentration of N-terminal prosomatostatin (NT-proSST) is associated with the development of diabetes, coronary artery disease (CAD), and mortality. Design, Setting, and Participants: NT-proSST was measured in plasma from 5389 fasting participants of the population-based study Malmö Preventive Project, with a mean baseline age of 69.4 ± 6.2 years. Cox proportional hazards models adjusted for traditional cardiovascular risk factors were used to investigate the relationships between baseline NT-proSST and end points, with a mean follow-up of 5.6 ± 1.4 years. Main Outcome Measures: CAD, diabetes, and mortality were measured. Results: Overall, NT-proSST (hazard ratio [HR] per SD increment of log transformed NT-proSST) was unrelated to the risk of incident diabetes (220 events; HR 1.05; 95% confidence interval [CI] 0.91–1.20; P = .531) but was related to the risk of incident CAD (370 events; HR 1.17; 95% CI 1.06–1.30; P = .003), all-cause mortality (756 events; HR 1.24; 95% CI 1.15–1.33; P < .001), and cardiovascular mortality (283 events; HR 1.33; 95% CI 1.19–1.43; P < .001). The relationships were not linear, with most of the excess risk observed in subjects with high values of NT-proSST. Subjects in the top vs bottom decile had a severely increased risk of incident CAD (HR 2.41; 95% CI 1.45–4.01; P < .001), all-cause mortality (HR 1.84; 95% CI 1.33–2.53; P < .001), and cardiovascular mortality (HR 2.44; 95% CI 1.39–4.27; P < .001). Conclusion: NT-proSST was significantly and independently associated with the development of CAD, all-cause mortality, and cardiovascular mortality. PMID:27399347

  18. Correlation of N-terminal pro-B-type natriuretic peptide with clinical parameters in patients with hypertension

    Directory of Open Access Journals (Sweden)

    Pejović Janko

    2013-01-01

    Full Text Available Background/Aim. Identification of patients with arterial hypertension and a possible onset of heart failure by determining the concentration of N-terminal pro-B-type natriuretic peptide (NT-proBNP enables timely intensification of treatment and allows clinicians to prescribe and implement optimal and appropriate care. The aim of this study was to evaluate NT-proBNP in patients with longstanding hypertension and in patients with signs of hypertensive cardiomyopathy. Methods. The study involved 3 groups, with 50 subjects each: “healthy” persons (control group, patients with hypertension and normal left ventricular systolic function (group 1 and patients with longstanding hypertension and signs of hypertensive cardiomyopathy with impaired left ventricular systolic function (group 2. We measured levels of NT-proBNP, Creactive protein and creatinine according to the manufacturer’s instructions. All the patients were clinically examined including physical examination of the heart with blood pressure, pulse rate, electrocardiogram (ECG and echocardiogram. Results. Our results showed that the determined parameters generally differed significantly (Student’s t-test among the groups. The mean (± SD values of NT-proBNP in the control group, group 1 and group 2 were: 2.794 (± 1.515 pmol/L, 9.575 (± 5.449 pmol/L and 204.60 (84,93 pmol/L, respectively. NTproBNP correlated significantly with the determined parameters both in the group 1 and the group 2. In the group 1, the highest correlation was obtained with Creactive protein (r = 0.8424. In the group 2, the highest correlation was obtained with ejection fraction (r = - 0.9111. NT-proBNP showed progressive increase in proportion to the New York Heart Association (NYHA classification. The patients in thegroup 2 who belonged to the II and III NYHA class had significantly higher levels of NTproBNP than those in the NYHA class I (ANOVA test, p = 0.001. Conclusion. The obtained results suggest that

  19. 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.

  20. Vestibular and proprioceptive influences on trunk movements during quiet standing.

    NARCIS (Netherlands)

    Horlings, C.G.; Kung, U.M.; Honegger, F.; Engelen, B.G.M. van; Alfen, N. van; Bloem, B.R.; Allum, J.H.J.

    2009-01-01

    We characterized upper trunk and pelvis motion in normal subjects and in subjects with vestibular or proprioceptive loss, to document upper body movement modes in the pitch and roll planes during quiet stance. Six bilateral vestibular loss (VL), six bilateral lower-leg proprioceptive loss (PL) and 2

  1. Diverticulum of the brachiocephalic trunk - angiography and embryological explanation

    Energy Technology Data Exchange (ETDEWEB)

    Pfefferkorn, J.R.; Lunkenheimer, A.; Loeser, H.; Hilgenberg, F.

    1983-03-01

    A diverticulum of the brachiocephalic trunk is described in 5 children with unilateral absence of the pulmonary artery, in a child with tetralogy of Fallot, and in an other child with Bland-White-Garland syndrome. The diverticulum is a remnant of a contralateral ductus arteriosus, which closed after birth and represents the origin of the distal part of the sixth aortic arch.

  2. MUSCLE POWER, SITTING UNSUPPORTED AND TRUNK ROTATION IN PRETERM INFANTS

    NARCIS (Netherlands)

    DEGROOT, L; HOPKINS, B; TOUWEN, B

    1995-01-01

    Discrepancies between active and passive muscle power are often seen in pre-term infants over the first year. Generally of a transient nature, they are most obvious in the extensor muscles of the trunk where there is a high active muscle power relative to the passive component. While high active mus

  3. Preventing Trunk Diseases in the Vineyard: Choosing the Best Practices

    Science.gov (United States)

    Over years of research on control of grapevine trunk diseases, field trials identified cultural and chemical practices that prevent and limit infections of pruning wounds by the spores. These practices include delayed pruning, double pruning, and applications of pruning-wound protectants (e.g., thio...

  4. Trunk recruitment during spoon use in tetraparetic cerebral palsy

    NARCIS (Netherlands)

    Roon, D.B. van; Steenbergen, B.; Meulenbroek, R.G.J.

    2004-01-01

    In the present study we investigated the extent to which individuals suffering from spastic tetraparesis as a consequence of cerebral palsy tune their trunk involvement to accuracy demands in a spoon-handling task. Twenty-two participants (ten adolescents with spastic tetraparesis and 12 control par

  5. Comparison of trunk activity during gait initiation and walking in humans.

    Directory of Open Access Journals (Sweden)

    Jean-Charles Ceccato

    Full Text Available To understand the role of trunk muscles in maintenance of dynamic postural equilibrium we investigate trunk movements during gait initiation and walking, performing trunk kinematics analysis, Erector spinae muscle (ES recordings and dynamic analysis. ES muscle expressed a metachronal descending pattern of activity during walking and gait initiation. In the frontal and horizontal planes, lateroflexion and rotation occur before in the upper trunk and after in the lower trunk. Comparison of ES muscle EMGs and trunk kinematics showed that trunk muscle activity precedes corresponding kinematics activity, indicating that the ES drive trunk movement during locomotion and thereby allowing a better pelvis mobilization. EMG data showed that ES activity anticipates propulsive phases in walking with a repetitive pattern, suggesting a programmed control by a central pattern generator. Our findings also suggest that the programs for gait initiation and walking overlap with the latter beginning before the first has ended.

  6. Comparison of Trunk Activity during Gait Initiation and Walking in Humans

    Science.gov (United States)

    Azevedo, Christine; Cazalets, Jean-René

    2009-01-01

    To understand the role of trunk muscles in maintenance of dynamic postural equilibrium we investigate trunk movements during gait initiation and walking, performing trunk kinematics analysis, Erector spinae muscle (ES) recordings and dynamic analysis. ES muscle expressed a metachronal descending pattern of activity during walking and gait initiation. In the frontal and horizontal planes, lateroflexion and rotation occur before in the upper trunk and after in the lower trunk. Comparison of ES muscle EMGs and trunk kinematics showed that trunk muscle activity precedes corresponding kinematics activity, indicating that the ES drive trunk movement during locomotion and thereby allowing a better pelvis mobilization. EMG data showed that ES activity anticipates propulsive phases in walking with a repetitive pattern, suggesting a programmed control by a central pattern generator. Our findings also suggest that the programs for gait initiation and walking overlap with the latter beginning before the first has ended. PMID:19997606

  7. Surgical treatment of celiomesenteric trunk aneurysm-7 case report.

    Science.gov (United States)

    Wang, Chunxi; Cai, Xiangjun; Liang, Faqi; Chu, Futao; Chen, Gang; Duan, Zhiquan

    2014-01-01

    The celiomesenteric trunk is a rare anomaly characterized by a common origin of the celiac axis and superior mesenteric artery from the aorta, which accounts for less than 1% of all celiac artery anomalies, so the aneurysm occurred in such trunk is even rarer. There have been few reports on how to diagnose and deal with such malformed celiomesenteric trunk aneurysms till now. This paper tries to summarize the experience of how to expose and excise such kind of aneurysm according to the seven cases' data. The clinic data were collected retrospectively. There were seven cases with celiomesenteric trunk aneurysm from February 2000 to February 2013, including 5 males and 2 females aged 35~62. The operations were done including aneurysm resection and vascular reconstruction under general anesthesia. The operated patients were followed-up at the sixth month and each year post operation. The vascular stomas were detected or examined by Color Doppler Sonography, spiral Computed Tomography angiography (SCTA). The seven operated patients were cured and discharged from hospital, and they were followed up for 3~10 years (mean time 5 years), with four patients being followed up longer than 5 years. No sign of intestinal ischemia or hepatic ischemia or splenic ischemia was found, and no image of anastomosis stricture or stenosis was found during the follow-up. Five patients are alive now while two patients were dead, with one dying of large area myocardial infarction unexpectedly at 6 years post operation and the other dying of cerebral infarction abruptly at 4 years post operation. It is an effective and safe method to treat the celiomesenteric trunk aneurysm by using by-pass operation with artificial blood vessels, originating from inferior kidney aorta to visceral arteries including hepatic artery, splenic artery and superior mesenteric artery. Its short-term and middle-term effects are relatively better. PMID:25227061

  8. Fear of Movement Is Related to Trunk Stiffness in Low Back Pain.

    OpenAIRE

    Karayannis, Nicholas V.; Smeets, Rob J. E. M.; Wolbert van den Hoorn; Paul W Hodges

    2013-01-01

    BACKGROUND: Psychological features have been related to trunk muscle activation patterns in low back pain (LBP). We hypothesised higher pain-related fear would relate to changes in trunk mechanical properties, such as higher trunk stiffness. OBJECTIVES: To evaluate the relationship between trunk mechanical properties and psychological features in people with recurrent LBP. METHODS: The relationship between pain-related fear (Tampa Scale for Kinesiophobia, TSK; Photograph Series of Daily Activ...

  9. Structure of the recombinant N-terminal lobe of human lactoferrin at 2.0 A resolution.

    Science.gov (United States)

    Day, C L; Anderson, B F; Tweedie, J W; Baker, E N

    1993-08-20

    The three-dimensional structure of the N-terminal half-molecule of human lactoferrin, LfN, prepared by recombinant DNA methods, has been determined by X-ray crystallography at 2.0 A resolution. The protein is in its iron-bound form and is deglycosylated. X-ray diffraction data were obtained by diffractometry to 3.2 A resolution and synchrotron data collection, using Weissenberg photography with imaging plates, to 1.8 A resolution. The structure was solved by molecular replacement, using the N-lobe of native diferric human lactoferrin (Lf) as search model. Restrained least squares refinement (program TNT) has resulted in a model structure with an R-factor of 0.184 for all data 34,180 (reflections) in the resolution range 8.0 to 2.0 A. The model comprises 2490 protein atoms (residues 4 to 327), 1 Fe3+, 1 CO3(2-) and 180 solvent molecules, all regarded as water. The structure of LfN is essentially the same as that of the N-lobe of intact Lf, being folded into two similar alpha/beta domains, with the Fe3+ and CO3(2-) bound in a specific site in the interdomain cleft. These details are not affected by either deglycosylation or expression in a non-native system. At the C terminus, however, the conformation of residues 321 to 333 is changed. Whereas in Lf residues 321 to 332 form a helix crossing between the domains at the back of the iron site, in LfN residues 321 to 326 have an extended conformation, forming a third interdomain beta-strand, and residues 328 to 333 appear disordered. The conformational change is attributed to the loss of stabilizing interactions from the C-lobe and is mediated by two Gly residues, at positions 321 and 323. It is further proposed that the conformational change is responsible for the more facile iron release properties of LfN, by its effect on the hinge mechanism and increased solvent exposure of residues near the back of the iron site. Other details of the polypeptide chain conformation and the binding site have also been analysed. Two

  10. c-Jun N-terminal kinase is required for thermotherapy-induced apoptosis in human gastric cancer cells

    Institute of Scientific and Technical Information of China (English)

    Feng Xiao; Bin Liu; Qing-Xian Zhu

    2012-01-01

    AIM:To investigate the role of c-Jun N-terminal kinase (JNK) in thermotherapy-induced apoptosis in human gastric cancer SGC-7901 cells.METHODS:Human gastric cancer SGC-7901 cells were cultured in vitro.Following thermotherapy at 43 ℃ 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.RESULTS: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-7901 cells in G0/G1 phase,but a reduced population in S phase following therrnotherapy for 1 or 2 h,compared to untreated cells (P < 0.05).The increased number of SGC-7901 cells in G0/G1 phase was consistent with induced apoptosis (flow cytometry) following 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 < 0.01),respectively.This was supported by the TUNEL assay (48.2% ± 0.4%,40.1% ± 0.2%,61.2% ± 0.29% and 52.0% ± 0.42% vs 12.2% ± 0.22%,P < 0.01) respectively.More importantly,the expression of p-JNK protein and JNK mRNA levels were significantly higher at 0.5 h than at 0 h post-treatment (P < 0.01),and peaked at 2 h.A similar pattem was detected for Bax and caspase-3 proteins.Bcl-2 increased at 0.5 h,peaked at 1 h,and then decreased

  11. File list: DNS.Emb.20.AllAg.Embryonic_trunk [Chip-atlas[Archive

    Lifescience Database Archive (English)

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    Lifescience Database Archive (English)

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  13. File list: His.Emb.50.AllAg.Embryonic_trunk [Chip-atlas[Archive

    Lifescience Database Archive (English)

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  14. File list: InP.Emb.10.AllAg.Embryonic_trunk [Chip-atlas[Archive

    Lifescience Database Archive (English)

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    Lifescience Database Archive (English)

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  16. File list: ALL.Emb.20.AllAg.Embryonic_trunk [Chip-atlas[Archive

    Lifescience Database Archive (English)

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  17. File list: DNS.Emb.50.AllAg.Embryonic_trunk [Chip-atlas[Archive

    Lifescience Database Archive (English)

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  18. File list: His.Emb.10.AllAg.Embryonic_trunk [Chip-atlas[Archive

    Lifescience Database Archive (English)

    Full Text Available His.Emb.10.AllAg.Embryonic_trunk mm9 Histone Embryo Embryonic trunk SRX093317,SRX09...3316 http://dbarchive.biosciencedbc.jp/kyushu-u/mm9/assembled/His.Emb.10.AllAg.Embryonic_trunk.bed ...

  19. File list: ALL.Emb.05.AllAg.Embryonic_trunk [Chip-atlas[Archive

    Lifescience Database Archive (English)

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  20. A review on assessment and treatment of the trunk in stroke A need or luxury

    Institute of Scientific and Technical Information of China (English)

    Suruliraj Karthikbabu; Mahabala Chakrapani; Sailakshmi Ganeshan; Kedambadi C Rakshith; Syed Nafeez; Venkatesan Prem

    2012-01-01

    Trunk function has been identified as an important early predictor of functional outcome after stroke and the same deteriorates on both contralateral and ipsilateral sides of the body following stroke. The primary contribution of the trunk muscles is to allow the body to remain upright, adjust weight shifts, and control movements against constant pull of gravity and is considered central key point of the body. Proximal stability of the trunk is a pre-requisite for distal limb mobility, balance, gait and functional activities and its positive correlation in hemiplegia has been demonstrated in a crosssectional study. Both isokinetic and handheld dynamometer muscle strength testing demonstratedthe weakness of bilateral trunk flexors, extensors and rotator muscles in both acute and chronic hemiplegic patients. This was confirmed by electromyography analysis which identified poor bilateral trunk muscles activity in patients with stroke. Trunk impairment scale is sensitive to evaluate the selective muscle control of upper and lower trunk, and it has been reported that lateral flexion of the trunk is easier than rotation of the trunk and the clinical observation concurs to the difficulty in lower trunk rotation of stroke patients. However, trunk exercises given early after stroke could produce enhanced balance performance post- stroke. This review attempts to report the evidence supporting the involvement of the trunk and its influence on balance and functional performance in post-stroke hemiplegia.

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    Lifescience Database Archive (English)

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    Lifescience Database Archive (English)

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  3. File list: His.Emb.20.AllAg.Embryonic_trunk [Chip-atlas[Archive

    Lifescience Database Archive (English)

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  5. File list: ALL.Emb.50.AllAg.Embryonic_trunk [Chip-atlas[Archive

    Lifescience Database Archive (English)

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  14. File list: NoD.Emb.05.AllAg.Embryonic_trunk [Chip-atlas[Archive

    Lifescience Database Archive (English)

    Full Text Available NoD.Emb.05.AllAg.Embryonic_trunk mm9 No description Embryo Embryonic trunk ERX40226...7,ERX402264 http://dbarchive.biosciencedbc.jp/kyushu-u/mm9/assembled/NoD.Emb.05.AllAg.Embryonic_trunk.bed ...

  15. File list: Oth.Emb.20.AllAg.Embryonic_trunk [Chip-atlas[Archive

    Lifescience Database Archive (English)

    Full Text Available Oth.Emb.20.AllAg.Embryonic_trunk mm9 TFs and others Embryo Embryonic trunk SRX17071...5,SRX170716,SRX170714 http://dbarchive.biosciencedbc.jp/kyushu-u/mm9/assembled/Oth.Emb.20.AllAg.Embryonic_trunk.bed ...

  16. Trunk muscle co-activation using functional electrical stimulation modifies center of pressure fluctuations during quiet sitting by increasing trunk stiffness

    OpenAIRE

    Milosevic, Matija; Masani, Kei; Wu, Noel; McConville, Kristiina M. V.; Popovic, Milos R.

    2015-01-01

    Background The purpose of this study was to examine the impact of functional electrical stimulation (FES) induced co-activation of trunk muscles during quiet sitting. We hypothesized that FES applied to the trunk muscles will increase trunk stiffness. The objectives of this study were to: 1) compare the center of pressure (COP) fluctuations during unsupported and FES-assisted quiet sitting - an experimental study and; 2) investigate how FES influences sitting balance - an analytical (simulati...

  17. 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...... phosphatidylglycerol (PG), the fluorescence of Dns-SP-C shows a 3-fold increase with respect to the fluorescence of phosphatidylcholine (PC), suggesting that electrostatic lipid-protein interactions induce important effects on the structure and disposition of the N-terminal segment of the protein in these membranes...

  18. The 5-amino acid N-terminal extension of non-sulfated drosulfakinin II is a unique target to generate novel agonists.

    Science.gov (United States)

    Leander, M; Heimonen, J; Brocke, T; Rasmussen, M; Bass, C; Palmer, G; Egle, J; Mispelon, M; Berry, K; Nichols, R

    2016-09-01

    The ability to design agonists that target peptide signaling is a strategy to delineate underlying mechanisms and influence biology. A sequence that uniquely characterizes a peptide provides a distinct site to generate novel agonists. Drosophila melanogaster sulfakinin encodes non-sulfated drosulfakinin I (nsDSK I; FDDYGHMRF-NH2) and nsDSK II (GGDDQFDDYGHMRF-NH2). Drosulfakinin is typical of sulfakinin precursors, which are conserved throughout invertebrates. Non-sulfated DSK II is structurally related to DSK I, however, it contains a unique 5-residue N-terminal extension; drosulfakinins signal through G-protein coupled receptors, DSK-R1 and DSK-R2. Drosulfakinin II distinctly influences adult and larval gut motility and larval locomotion; yet, its structure-activity relationship was unreported. We hypothesized substitution of an N-terminal extension residue may alter nsDSK II activity. By targeting the extension we identified, not unexpectedly, analogs mimicking nsDSK II, yet, surprisingly, we also discovered novel agonists with increased (super) and opposite (protean) effects. We determined [A3] nsDSK II increased larval gut contractility rather than, like nsDSK II, decrease it. [N4] nsDSK II impacted larval locomotion, although nsDSK II was inactive. In adult gut, [A1] nsDSK II, [A2] nsDSKII, and [A3] nsDSK II mimicked nsDSK II, and [A4] nsDSK II and [A5] nsDSK II were more potent; [N3] nsDSK II and [N4] nsDSK II mimicked nsDSK II. This study reports nsDSK II signals through DSK-R2 to influence gut motility and locomotion, identifying a novel role for the N-terminal extension in sulfakinin biology and receptor activation; it also led to the discovery of nsDSK II structural analogs that act as super and protean agonists. PMID:27397853

  19. 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.

  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. Immunization with the DNA-encoding N-terminal domain of proteophosphoglycan of Leishmania donovani generates Th1-type immunoprotective response against experimental visceral leishmaniasis.

    Science.gov (United States)

    Samant, Mukesh; Gupta, Reema; Kumari, Shraddha; Misra, Pragya; Khare, Prashant; Kushawaha, Pramod Kumar; Sahasrabuddhe, Amogh Anant; Dube, Anuradha

    2009-07-01

    Leishmania produce several types of mucin-like glycoproteins called proteophosphoglycans (PPGs) which exist as secretory as well as surface-bound forms in both promastigotes and amastigotes. The structure and function of PPGs have been reported to be species and stage specific as in the case of Leishmania major and Leishmania mexicana; there has been no such information available for Leishmania donovani. We have recently demonstrated that PPG is differentially expressed in sodium stibogluconate-sensitive and -resistant clinical isolates of L. donovani. To further elucidate the structure and function of the ppg gene of L. donovani, a partial sequence of its N-terminal domain of 1.6 kb containing the majority of antigenic determinants, was successfully cloned and expressed in prokaryotic as well as mammalian cells. We further evaluated the DNA-encoding N-terminal domain of the ppg gene as a vaccine in golden hamsters (Mesocricetus auratus) against the L. donovani challenge. The prophylactic efficacy to the tune of approximately 80% was observed in vaccinated hamsters and all of them could survive beyond 6 mo after challenge. The efficacy was supported by a surge in inducible NO synthase, IFN-gamma, TNF-alpha, and IL-12 mRNA levels along with extreme down-regulation of TGF-beta, IL-4, and IL-10. A rise in the level of Leishmania-specific IgG2 was also observed which was indicative of enhanced cellular immune response. The results suggest the N-terminal domain of L. donovani ppg as a potential DNA vaccine against visceral leishmaniasis.

  2. The N-terminal cleavage of chondromodulin-I in growth-plate cartilage at the hypertrophic and calcified zones during bone development.

    Directory of Open Access Journals (Sweden)

    Shigenori Miura

    Full Text Available Chondromodulin-I (ChM-I is a 20-25 kDa anti-angiogenic glycoprotein in cartilage matrix. In the present study, we identified a novel 14-kDa species of ChM-I by immunoblotting, and purified it by immunoprecipitation with a newly raised monoclonal antibody against ChM-I. The N-terminal amino acid sequencing indicated that it was an N-terminal truncated form of ChM-I generated by the proteolytic cleavage at Asp37-Asp38. This 14-kDa ChM-I was shown by the modified Boyden chamber assay to have very little inhibitory activity on the VEGF-A-induced migration of vascular endothelial cells in contrast to the intact 20-25 kDa form of ChM-I (ID50 = 8 nM. Immunohistochemistry suggested that 20-25 kDa ChM-I was exclusively localized in the avascular zones, i.e. the resting, proliferating, and prehypertrophic zones, of the cartilaginous molds of developing long bone, whereas the 14-kDa form of ChM-I was found in hypertrophic and calcified zones. Immunoblotting demonstrated that mature growth-plate chondrocytes isolated from rat costal cartilage actively secrete ChM-I almost exclusively as the intact 20-25 kDa form into the medium in primary culture. Taken together, our results suggest that intact 20-25 kDa ChM-I is stored as a component of extracellular matrix in the avascular cartilage zones, but it is inactivated by a single N-terminal proteolytic cleavage in the hypertrophic zone of growth-plate cartilage.

  3. Phosphorylation and the N-terminal extension of the regulatory light chain help orient and align the myosin heads in Drosophila flight muscle

    Energy Technology Data Exchange (ETDEWEB)

    Farman, Gerrie P.; Miller, Mark S.; Reedy, Mary C.; Soto-Adames, Felipe N.; Vigoreaux, Jim O.; Maughan, David W.; Irving, Thomas C.; (IIT); (Vermont); (Duke)

    2010-02-02

    X-ray diffraction of the indirect flight muscle (IFM) in living Drosophila at rest and electron microscopy of intact and glycerinated IFM was used to compare the effects of mutations in the regulatory light chain (RLC) on sarcomeric structure. Truncation of the RLC N-terminal extension (Dmlc2{sup {Delta}2-46}) or disruption of the phosphorylation sites by substituting alanines (Dmlc2{sup S66A, S67A}) decreased the equatorial intensity ratio (I{sub 20}/I{sub 10}), indicating decreased myosin mass associated with the thin filaments. Phosphorylation site disruption (Dmlc2{sup S66A, S67A}), but not N-terminal extension truncation (Dmlc2{sup {Delta}2-46}), decreased the 14.5 nm reflection intensity, indicating a spread of the axial distribution of the myosin heads. The arrangement of thick filaments and myosin heads in electron micrographs of the phosphorylation mutant (Dmlc2{sup S66A, S67A}) appeared normal in the relaxed and rigor states, but when calcium activated, fewer myosin heads formed cross-bridges. In transgenic flies with both alterations to the RLC (Dmlc2{sup {Delta}2-46; S66A, S67A}), the effects of the dual mutation were additive. The results suggest that the RLC N-terminal extension serves as a 'tether' to help pre-position the myosin heads for attachment to actin, while phosphorylation of the RLC promotes head orientations that allow optimal interactions with the thin filament.

  4. Identification of a c-Jun N-terminal kinase-2-dependent signal amplification cascade that regulates c-Myc levels in ras transformation

    DEFF Research Database (Denmark)

    Mathiasen, D.P.; Egebjerg, C.; Andersen, S.H.;

    2012-01-01

    Ras is one of the most frequently activated oncogenes in cancer. Two mitogen-activated protein kinases (MAPKs) are important for ras transformation: extracellular signal-regulated kinase (ERK) and c-Jun N-terminal kinase 2 (JNK2). Here we present a downstream signal amplification cascade that is...... essential for ras transformation. Previous studies show that ERK-mediated serine 62 phosphorylation protects c-Myc from proteasomal degradation. ERK is, however, not alone sufficient to stabilize c-Myc but requires the cooperation of cancerous inhibitor of protein phosphatase 2A (CIP2A), an oncogene that...

  5. Phosphorylation and the N-terminal Extension of the Regulatory Light Chain Help Orient and Align the Myosin Heads in Drosophila Flight Muscle

    OpenAIRE

    Farman, Gerrie P.; Miller, Mark S.; Reedy, Mary C.; Soto-Adames, Felipe N.; Vigoreaux, Jim O.; Maughan, David W.; Irving, Thomas C.

    2009-01-01

    X-ray diffraction of the indirect flight muscle (IFM) in living Drosophila at rest and electron microscopy of intact and glycerinated IFM was used to compare the effects of mutations in the regulatory light chain (RLC) on sarcomeric structure. Truncation of the RLC N-terminal extension (Dmlc2Δ2-46) or disruption of the phosphorylation sites by substituting alanines (Dmlc2S66A, S67A) decreased the equatorial intensity ratio (I20/I10), indicating decreased myosin mass associated with the thin f...

  6. Evidence for an Interaction between the SH3 Domain and the N-terminal Extension of the Essential Light Chain in Class II Myosins

    OpenAIRE

    Lowey, Susan; Saraswat, Lakshmi D.; Liu, HongJun; Volkmann, Niels; Hanein, Dorit

    2007-01-01

    The function of the src-homology 3 (SH3) domain in class II myosins, a distinct β-barrel structure, remains unknown. Here we provide evidence, using electron cryomicroscopy, in conjunction with light scattering, fluorescence and kinetic analyses, that the SH3 domain facilitates the binding of the N-terminal extension of the essential light chain isoform (ELC-1) to actin. The 41-residue extension contains four conserved lysines followed by a repeating sequence of seven Pro/Ala residues. It is ...

  7. Sepp1UF forms are N-terminal selenoprotein P truncations that have peroxidase activity when coupled with thioredoxin reductase-1

    OpenAIRE

    Kurokawa, Suguru; Eriksson, Sofi; Rose, Kristie L.; Wu, Sen; Motley, Amy K.; Hill, Salisha; Winfrey, Virginia P.; McDonald, W. Hayes; Capecchi, Mario R.; Atkins, John F; Arnér, Elias S. J.; Hill, Kristina E.; Raymond F Burk

    2014-01-01

    Mouse selenoprotein P (Sepp1) consists of an N-terminal domain (residues 1-239) that contains 1 selenocysteine (U) as residue 40 in a proposed redox-active motif (-UYLC-) and a C-terminal domain (residues 240-361) that contains 9 selenocysteines. Sepp1 transports selenium from the liver to other tissues by receptor-mediated endocytosis. It also reduces oxidative stress in vivo by an unknown mechanism. A previously uncharacterized plasma form of Sepp1 is filtered in the glomerulus and taken up...

  8. Enhancing the Secretion Efficiency and Thermostability of a Bacillus deramificans Pullulanase Mutant (D437H/D503Y) by N-Terminal Domain Truncation

    OpenAIRE

    Duan, Xuguo; WU, Jing

    2015-01-01

    Pullulanase (EC 3.2.1.41), an important enzyme in the production of starch syrup, catalyzes the hydrolysis of α-1,6 glycosidic bonds in complex carbohydrates. A double mutant (DM; D437H/D503Y) form of Bacillus deramificans pullulanase was recently constructed to enhance the thermostability and catalytic efficiency of the enzyme (X. Duan, J. Chen, and J. Wu, Appl Environ Microbiol 79:4072–4077, 2013, http://dx.doi.org/10.1128/AEM.00457-13). In the present study, three N-terminally truncated...

  9. The crystal structure of Z-Aib-Gly-Aib-Leu-Aib-OtBu, the synthetic, protected N-terminal pentapeptide of trichotoxin.

    Science.gov (United States)

    Gessmann, R; Brueckner, H; Kokkinidis, M

    1991-01-01

    Z-Aib-Gly-Aib-Leu-Aib-OtBu, the alpha-aminoisobutyric acid (Aib)-containing N-terminal pentapeptide of the antibiotic trichotoxin, has been studied by x-ray crystallography. The molecule forms a right-handed helix with a reversal of the sense of the helix at the C-terminus. Torsion angles and hydrogen bonding pattern are consistent with a mixed 3(10)-/alpha-helical conformation. In the crystal, continuous columns are formed by head-to-tail arrangement of hydrogen-bonded molecules along the helix axis. The helical columns associate via hydrogen bonds forming closely packed parallel pairs.

  10. 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......-binding module family 21 were traceable and were included in the present HvLD structure but were too flexible to be traced and included in the structures of the two HvLD-inhibitor complexes....

  11. Cationic liposomes enhance targeted delivery and expression of exogenous DNA mediated by N-terminal modified poly(L-lysine)-antibody conjugate in mouse lung endothelial cells.

    Science.gov (United States)

    Trubetskoy, V S; Torchilin, V P; Kennel, S; Huang, L

    1992-07-15

    A new and improved system for targeted gene delivery and expression is described. Transfection efficiency of N-terminal modified poly(L-lysine) (NPLL) conjugated with anti-thrombomodulin antibody 34A can be improved by adding to the system a lipophilic component, cationic liposomes. DNA, antibody conjugate and cationic liposomes form a ternary electrostatic complex which preserves the ability to bind specifically to the target cells. At the same time the addition of liposomes enhance the specific transfection efficiency of antibody-polylysine/DNA binary complex by 10 to 20-fold in mouse lung endothelial cells in culture.

  12. Use of N-terminal modified poly(L-lysine)-antibody conjugate as a carrier for targeted gene delivery in mouse lung endothelial cells.

    Science.gov (United States)

    Trubetskoy, V S; Torchilin, V P; Kennel, S J; Huang, L

    1992-01-01

    A DNA targeted delivery and expression system has been designed based on an N-terminal modified poly(L-lysine) (NPLL)-antibody conjugate, which readily forms a complex with plasmid DNA. Monoclonal antibodies against the cell-surface thrombomodulin conjugated with NPLL were used for targeted delivery of foreign plasmid DNA to an antigen-expressing mouse lung endothelial cell line in vitro and to mouse lungs in vivo. In both cases significant amounts of DNA can be specifically bound to the target cells or tissues. Specific gene expression was observed in the treated mouse lung endothelial cells.

  13. 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.

  14. Thrombosis of celiacomesenteric trunk: Report of a case

    Institute of Scientific and Technical Information of China (English)

    Federico Lovisetto; Gianbattista Finocchiaro De Lorenzi; Piera Stancampiano; Carmen Corradini; Fabio De Cesare; Orazio Geraci; Mario Manzi

    2012-01-01

    Here we present the case of a 79-year-old woman who complained of acute abdominal pain,vomiting and diarrhoea.Laboratory exams demonstrated a severe metabolic imbalance.Abdominal X-rays showed bowel overdistension and pneumatosis of the stomach wall.Abdominal tomography revealed infarction of the stomach,duodenum and small bowel due to thrombosis of the celiacomesenteric trunk.Exploratory laparotomy revealed ischemia of the liver,spleen infarction and necrosis of the gastro-intestinal tube (from the stomach up to the first third of the transverse colon).No further surgical procedures were performed.The patient died the following day.To our knowledge,this is the first reported case about severe gastro-intestinal ischemia due to thrombosis of the celiacomesenteric trunk,a rare anatomic variation of the gastrointestinal vascularisation.

  15. Quantifying coordination between the head and the trunk during locomotion

    Science.gov (United States)

    Mulavara, Ajitkumar P.

    This study developed unique measures of coordination between the head and the trunk during the combined tasks of locomotion and gaze fixation of visual targets. These measures will be used to determine the effects of long-duration space flight on sensorimotor function. This will enable evaluation of the efficacy of countermeasures and postflight rehabilitation programs. Indices were proposed as composite measures reflecting the functional aspects of the control system involved in gaze fixation during locomotion. The stiffness index (Nm/deg) was calculated as the ratio between the change in the magnitude of the net relative moments to the change in magnitude of the relative angular motion. The viscosity index (Nm-sec/deg) was calculated as the ratio between the change in the magnitude of the net relative moments to the change in magnitude of the relative angular velocity. These coordination measures were used to evaluate the normal dynamic pattern of coordination between the head and the trunk with respect to the events occurring in a gait cycle. The indices were evaluated for three discrete speeds of locomotion for the same gaze fixation task and for three discrete gaze fixation tasks at the same speed of locomotion. The indices were found to be repeatable measures reflecting inter-segmental coordination strategies while performing an activity of daily living. These indices showed that the coordination of the head with respect to the trunk was significantly different between the events of heel strike and swing phases during the gait cycle. These indices showed no significant differences between the different gaze fixation tasks. The speed of locomotion had a significant effect on the magnitude of these indices. The results indicate that the CNS dynamically modulates head motion with respect to the trunk dependent on the events occurring during the gait cycle. This modulation is appropriate for stabilizing gaze during locomotion. The results support the hypothesis

  16. Safety and pitfalls in frozen elephant trunk implantation

    OpenAIRE

    Damberg, Anneke; Schälte, Gereon; Autschbach, Rüdiger; Hoffman, Andras

    2013-01-01

    The frozen elephant trunk (FET) procedure, or open stent grafting, is a tool for the combined open and endovascular treatment via a median sternotomy of extensive aortic disease involving both aortic arch and descending thoracic aorta. The technique aims to stabilize the maximum extent of the thoracic aorta in one step, with the goal of either rendering a secondary intervention to the downstream aorta unnecessary or producing an easy landing zone for secondary thoracic endovascular aortic rep...

  17. Multiphasic strain differentiation of atypical mycobacteria from elephant trunk wash

    OpenAIRE

    Chan, Kok-Gan; Loke, Mun Fai; Ong, Bee Lee; Wong, Yan Ling; Hong, Kar Wai; Tan, Kian Hin; Kaur, Sargit; Ng, Hien Fuh; Abdul Razak, MFA; Ngeow, Yun Fong

    2015-01-01

    Background. Two non-tuberculous mycobacterial strains, UM_3 and UM_11, were isolated from the trunk wash of captive elephants in Malaysia. As they appeared to be identical phenotypes, they were investigated further by conventional and whole genome sequence-based methods of strain differentiation. Methods. Multiphasic investigations on the isolates included species identification with hsp65 PCR-sequencing, conventional biochemical tests, rapid biochemical profiling using API strips and the Bio...

  18. The measurement of blood speed in the pulmonary artery trunk

    International Nuclear Information System (INIS)

    The paper describes a non invasive methodology for the measurement of blood speed in the pulmonary artery trunk. The methodology has been tested with a moving radioactive tracer (nuclear medicine). An image processing technique is proposed, for detection and analysis of a moving object with variable shape and intensity over time (radioactive bolus). Experiments on the application of the technique in nuclear medicine are critically analysed. (authors)

  19. Geophysical imaging of root-zone, trunk, and moisture heterogeneity.

    Science.gov (United States)

    Attia Al Hagrey, Said

    2007-01-01

    The most significant biotic and abiotic stress agents of water extremity, salinity, and infection lead to wood decay and modifications of moisture and ion content, and density. This strongly influences the (di-)electrical and mechanical properties and justifies the application of geophysical imaging techniques. These are less invasive and have high resolution in contrast to classical methods of destructive, single-point measurements for inspecting stresses in trees and soils. This review presents some in situ and in vivo applications of electric, radar, and seismic methods for studying water status and movement in soils, roots, and tree trunks. The electrical properties of a root-zone are a consequence of their moisture content. Electrical imaging discriminates resistive, woody roots from conductive, soft roots. Both types are recognized by low radar velocities and high attenuation. Single roots can generate diffraction hyperbolas in radargrams. Pedophysical relationships of water content to electrical resistivity and radar velocity are established by diverse infiltration experiments in the field, laboratory, and in the full-scale 'GeoModel' at Kiel University. Subsurface moisture distributions are derived from geophysical attribute models. The ring electrode technique around trunks images the growth ring structure of concentric resistivity, which is inversely proportional to the fluid content. Healthy trees show a central high resistivity within the dry heartwood that strongly decreases towards the peripheral wet sapwood. Observed structural deviations are caused by infection, decay, shooting, or predominant light and/or wind directions. Seismic trunk tomography also differentiates between decayed and healthy woods. PMID:17229759

  20. Immediate Effects of Different Trunk Exercise Programs on Jump Performance.

    Science.gov (United States)

    Imai, A; Kaneoka, K; Okubo, Y; Shiraki, H

    2016-03-01

    The aim of this study was to investigate the immediate effects of trunk stabilization exercise (SE) and conventional trunk exercise (CE) programs on jump performance. 13 adolescent male soccer players performed 2 kinds of jump testing before and immediate after 3 experimental conditions: SE, CE, and non-exercise (NE). The SE program consisted of the elbow-toe, hand-knee, and back bridge, and the CE program consisted of the sit-up, sit-up with trunk rotation and back extension. Testing of a countermovement jump (CMJ) and rebound jump (RJ) were performed to assess jump performance. Jump height of the CMJ and RJ-index, contact time, and jump height of the RJ were analyzed. The RJ index was improved significantly only after SE (p=0.017). However, contact time and jump height did not improve significantly in the SE condition. Moreover, no significant interaction or main effects of time or group were observed in the CMJ. Consequently, this study showed the different immediate effect on the RJ between the SE and CE, and suggested the possibility that the SE used in this study is useful as a warm-up program to improve the explosive movements.

  1. Evolution of the head-trunk interface in tetrapod vertebrates

    Science.gov (United States)

    Sefton, Elizabeth M; Bhullar, Bhart-Anjan S; Mohaddes, Zahra; Hanken, James

    2016-01-01

    Vertebrate neck musculature spans the transition zone between head and trunk. The extent to which the cucullaris muscle is a cranial muscle allied with the gill levators of anamniotes or is instead a trunk muscle is an ongoing debate. Novel computed tomography datasets reveal broad conservation of the cucullaris in gnathostomes, including coelacanth and caecilian, two sarcopterygians previously thought to lack it. In chicken, lateral plate mesoderm (LPM) adjacent to occipital somites is a recently identified embryonic source of cervical musculature. We fate-map this mesoderm in the axolotl (Ambystoma mexicanum), which retains external gills, and demonstrate its contribution to posterior gill-levator muscles and the cucullaris. Accordingly, LPM adjacent to the occipital somites should be regarded as posterior cranial mesoderm. The axial position of the head-trunk border in axolotl is congruent between LPM and somitic mesoderm, unlike in chicken and possibly other amniotes. DOI: http://dx.doi.org/10.7554/eLife.09972.001 PMID:27090084

  2. 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.

  3. Specific amplification of gene encoding N-terminal region of catalase-peroxidase protein (KatG-N) for diagnosis of disseminated MAC disease in HIV patients.

    Science.gov (United States)

    Latawa, Romica; Singh, Krishna Kumar; Wanchu, Ajay; Sethi, Sunil; Sharma, Kusum; Sharma, Aman; Laal, Suman; Verma, Indu

    2014-10-01

    Disseminated Mycobacterium avium-intracellulare complex (MAC) infection is considered as severe complication of advanced HIV/AIDS disease. Currently available various laboratory investigations have not only limited ability to discriminate between MAC infection and tuberculosis but are also laborious and time consuming. The aim of this study was, therefore, to design a molecular-based strategy for specific detection of MAC and its differentiation from Mycobacterium tuberculosis (M. tb) isolated from the blood specimens of HIV patients. A simple PCR was developed based on the amplification of 120-bp katG-N gene corresponding to the first 40 amino acids of N-terminal catalase-peroxidase (KatG) protein of Mycobacterium avium that shows only ~13% sequence homology by clustal W alignment to N-terminal region of M. tb KatG protein. This assay allowed the accurate and rapid detection of MAC bacteremia, distinguishing it from M. tb in a single PCR reaction without any need for sequencing or hybridization protocol to be performed thereafter. This study produced enough evidence that a significant proportion of Indian HIV patients have disseminated MAC bacteremia, suggesting the utility of M. avium katG-N gene PCR for early detection of MAC disease in HIV patients.

  4. Structural analysis of the starfish SALMFamide neuropeptides S1 and S2: the N-terminal region of S2 facilitates self-association.

    Science.gov (United States)

    Otara, Claire B; Jones, Christopher E; Younan, Nadine D; Viles, John H; Elphick, Maurice R

    2014-02-01

    The neuropeptides S1 (GFNSALMFamide) and S2 (SGPYSFNSGLTFamide), which share sequence similarity, were discovered in the starfish Asterias rubens and are prototypical members of the SALMFamide family of neuropeptides in echinoderms. SALMFamide neuropeptides act as muscle relaxants and both S1 and S2 cause relaxation of cardiac stomach and tube foot preparations in vitro but S2 is an order of magnitude more potent than S1. Here we investigated a structural basis for this difference in potency using spectroscopic techniques. Circular dichroism spectroscopy showed that S1 does not have a defined structure in aqueous solution and this was supported by 2D nuclear magnetic resonance experiments. In contrast, we found that S2 has a well-defined conformation in aqueous solution. However, the conformation of S2 was concentration dependent, with increasing concentration inducing a transition from an unstructured to a structured conformation. Interestingly, this property of S2 was not observed in an N-terminally truncated analogue of S2 (short S2 or SS2; SFNSGLTFamide). Collectively, the data obtained indicate that the N-terminal region of S2 facilitates peptide self-association at high concentrations, which may have relevance to the biosynthesis and/or bioactivity of S2 in vivo.

  5. Recombinant expression of rat glycine N-methyltransferase and evidence for contribution of N-terminal acetylation to co-operative binding of S-adenosylmethionine.

    Science.gov (United States)

    Ogawa, H; Gomi, T; Takata, Y; Date, T; Fujioka, M

    1997-10-15

    An expression vector was constructed that produced rat glycine N-methyltransferase in Escherichia coli. Recombinant glycine N-methyltransferase was purified to homogeneity by DEAE-cellulose and gel-filtration chromatography, with a yield of more than 80 mg of pure enzyme from a 1 litre culture. HPLC of tryptic peptides and analysis of isolated peptides showed that the recombinant enzyme was structurally identical with the liver enzyme except for the absence of N-terminal blocking. The alpha-amino group of rat glycine N-methyltransferase is blocked by acetylation [Ogawa, Konishi, Takata, Nakashima and Fujioka (1987) Eur. J. Biochem. 168, 141-151]. In contrast with the liver enzyme, which shows sigmoidal kinetics toward S-adenosylmethionine at all pH values tested [Ogawa and Fujioka (1982) J. Biol. Chem. 257, 3447-3452], the recombinant enzyme exhibited hyperbolic kinetics at low pH and sigmoidal rate behaviour at high pH. The Hill coefficient increased with increasing pH and a pKa of 8.11 was obtained in this transition. The values of Vmax and Km for glycine were not different between the two enzymes. These results suggest that elimination of the positive charge at the N-terminal end either by acetylation or deprotonation is required for co-operative behaviour. PMID:9359408

  6. A TPR domain-containing N-terminal module of MPS1 is required for its kinetochore localization by Aurora B.

    Science.gov (United States)

    Nijenhuis, Wilco; von Castelmur, Eleonore; Littler, Dene; De Marco, Valeria; Tromer, Eelco; Vleugel, Mathijs; van Osch, Maria H J; Snel, Berend; Perrakis, Anastassis; Kops, Geert J P L

    2013-04-15

    The mitotic checkpoint ensures correct chromosome segregation by delaying cell cycle progression until all kinetochores have attached to the mitotic spindle. In this paper, we show that the mitotic checkpoint kinase MPS1 contains an N-terminal localization module, organized in an N-terminal extension (NTE) and a tetratricopeptide repeat (TPR) domain, for which we have determined the crystal structure. Although the module was necessary for kinetochore localization of MPS1 and essential for the mitotic checkpoint, the predominant kinetochore binding activity resided within the NTE. MPS1 localization further required HEC1 and Aurora B activity. We show that MPS1 localization to kinetochores depended on the calponin homology domain of HEC1 but not on Aurora B-dependent phosphorylation of the HEC1 tail. Rather, the TPR domain was the critical mediator of Aurora B control over MPS1 localization, as its deletion rendered MPS1 localization insensitive to Aurora B inhibition. These data are consistent with a model in which Aurora B activity relieves a TPR-dependent inhibitory constraint on MPS1 localization.

  7. N-terminal guanidinylation of TIPP (Tyr-Tic-Phe-Phe) peptides results in major changes of the opioid activity profile.

    Science.gov (United States)

    Weltrowska, Grazyna; Nguyen, Thi M-D; Chung, Nga N; Wilkes, Brian C; Schiller, Peter W

    2013-09-15

    Derivatives of peptides of the TIPP (Tyr-Tic-Phe-Phe; Tic=1,2,3,4-tetrahydroisoquinoline-3-carboxylic acid) family containing a guanidino (Guan) function in place of the N-terminal amino group were synthesized in an effort to improve their blood-brain barrier permeability. Unexpectedly, N-terminal amidination significantly altered the in vitro opioid activity profiles. Guan-analogues of TIPP-related δ opioid antagonists showed δ partial agonist or mixed δ partial agonist/μ partial agonist activity. Guanidinylation of the mixed μ agonist/δ antagonists H-Dmt-Tic-Phe-Phe-NH2 (DIPP-NH2) and H-Dmt-TicΨ[CH2NH]Phe-Phe-NH2 (DIPP-NH2[Ψ]) converted them to mixed μ agonist/δ agonists. A docking study revealed distinct positioning of DIPP-NH2 and Guan-DIPP-NH2 in the δ receptor binding site. Lys(3)-analogues of DIPP-NH2 and DIPP-NH2[Ψ] (guanidinylated or non-guanidinylated) turned out to be mixed μ/κ agonists with δ antagonist-, δ partial agonist- or δ full agonist activity. Compounds with some of the observed mixed opioid activity profiles have therapeutic potential as analgesics with reduced side effects or for treatment of cocaine addiction. PMID:23932788

  8. 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...

  9. A CaV2.1 N-terminal fragment relieves the dominant-negative inhibition by an Episodic ataxia 2 mutant.

    Science.gov (United States)

    Dahimene, Shehrazade; Page, Karen M; Nieto-Rostro, Manuela; Pratt, Wendy S; D'Arco, Marianna; Dolphin, Annette C

    2016-09-01

    Episodic ataxia 2 (EA2) is an autosomal dominant disorder caused by mutations in the gene CACNA1A that encodes the pore-forming CaV2.1 calcium channel subunit. The majority of EA2 mutations reported so far are nonsense or deletion/insertion mutations predicted to form truncated proteins. Heterologous expression of wild-type CaV2.1, together with truncated constructs that mimic EA2 mutants, significantly suppressed wild-type calcium channel function, indicating that the truncated protein produces a dominant-negative effect (Jouvenceau et al., 2001; Page et al., 2004). A similar finding has been shown for CaV2.2 (Raghib et al., 2001). We show here that a highly conserved sequence in the cytoplasmic N-terminus is involved in this process, for both CaV2.1 and CaV2.2 channels. Additionally, we were able to interfere with the suppressive effect of an EA2 construct by mutating key N-terminal residues within it. We postulate that the N-terminus of the truncated channel plays an essential part in its interaction with the full-length CaV2.1, which prevents the correct folding of the wild-type channel. In agreement with this, we were able to disrupt the interaction between EA2 and the full length channel by co-expressing a free N-terminal peptide. PMID:27260834

  10. Nuclear import of influenza B virus nucleoprotein: Involvement of an N-terminal nuclear localization signal and a cleavage-protection motif

    Energy Technology Data Exchange (ETDEWEB)

    Wanitchang, Asawin; Narkpuk, Jaraspim; Jongkaewwattana, Anan, E-mail: anan.jon@biotec.or.th

    2013-08-15

    The nucleoprotein of influenza B virus (BNP) shares several characteristics with its influenza A virus counterpart (ANP), including localization in the host's nucleus. However, while the nuclear localization signal(s) (NLS) of ANP are well characterized, little is known about those of BNP. In this study, we showed that the fusion protein bearing the BNP N-terminus fused with GFP (N70–GFP) is exclusively nuclear, and identified a highly conserved KRXR motif spanning residues 44–47 as a putative NLS. In addition, we demonstrated that residues 3–15 of BNP, though not an NLS, are also crucial for nuclear import. Results from mutational analyses of N70–GFP and the full-length BNP suggest that this region may be required for protection of the N-terminus from proteolytic cleavage. Altogether, we propose that the N-terminal region of BNP contains the NLS and cleavage-protection motif, which together drive its nuclear localization. - Highlights: • The N-terminal region of BNP is required for nuclear accumulation. • The conserved motif at position 44–47 is a putative nuclear localization signal. • The first 15 amino acids of BNP may function as a cleavage-protection motif. • BNP may get access to the nucleus via a mechanism distinct from ANP.

  11. Crystallization and X-ray analysis of the T = 4 particle of hepatitis B capsid protein with an N-terminal extension

    International Nuclear Information System (INIS)

    Hepatitis B virus capsids have significant potential as carriers for immunogenic peptides. The crystal structure of the T = 4 particle of hepatitis B core protein containing an N-terminal extension reveals that the fusion peptide is exposed on the exterior of the particle. Hepatitis B core (HBc) particles have been extensively exploited as carriers for foreign immunological epitopes in the development of multicomponent vaccines and diagnostic reagents. Crystals of the T = 4 HBc particle were grown in PEG 20 000, ammonium sulfate and various types of alcohols. A temperature jump from 277 or 283 to 290 K was found to enhance crystal growth. A crystal grown using MPD as a cryoprotectant diffracted X-rays to 7.7 Å resolution and data were collected to 99.6% completeness at 8.9 Å. The crystal belongs to space group P212121, with unit-cell parameters a = 352.3, b = 465.5, c = 645.0 Å. The electron-density map reveals a protrusion that is consistent with the N-terminus extending out from the surface of the capsid. The structure presented here supports the idea that N-terminal insertions can be exploited in the development of diagnostic reagents, multicomponent vaccines and delivery vehicles into mammalian cells

  12. Single amino-acid substitution in the N-terminal arm altered the tetramer stability of rat muscle lactate dehydrogenase A

    Institute of Scientific and Technical Information of China (English)

    袁翀; 胡红雨; 许根俊

    2001-01-01

    Lactate dehydrogenase A (LDHA) is a well-characterized tetrameric enzyme. Its N-terminal arm, comprised of an (-helix and a (-strand, was suggested to be essential for subunit interactions. To examine the critical amino acid residues in the N-terminus involved in the subunit association, two single-point mutants, Leu3Pro (L3P) and Ile8Glu (I8E), have been constructed. We compared the stability of WT-LDHA (WT) and its variants by unfolding experiments. For WT, a dimeric but inactive intermediate was observed by size-exclusion chromatography at 0.6-0.8 mol/L GdmCl. Leu3Pro exists in an active tetrameric structure in aqueous solution as WT does, but it dissociates into dimers under lower concentration of GdmCl (0.2 mol/L). In aqueous solution, the Ile8Glu variant exists predominantly in the dimeric form with increased KM and decreased kcat as compared with those of WT and L3P. However, the activity of Ile8Glu increases significantly in the presence of sodium sulfate. In conclusion, two mutants are less stable than WT in oligomer structure. Results also support the fact that some residues in the N-terminal arm, especially the Leu8 in the (-structure, contribute the important binding energies to the dimerization of dimers, which might affect the assembly of the enzyme as well as the catalytic function.

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

    International Nuclear Information System (INIS)

    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

  14. Solution structure of the N-terminal A domain of the human voltage-gated Ca2+channel beta4a subunit.

    Science.gov (United States)

    Vendel, Andrew C; Rithner, Christopher D; Lyons, Barbara A; Horne, William A

    2006-02-01

    Ca2+ channel beta subunits regulate trafficking and gating (opening and closing) of voltage-dependent Ca2+ channel alpha1 subunits. Based on primary sequence comparisons, they are thought to be modular structures composed of five domains (A-E) that are related to the large family of membrane associated guanylate-kinase (MAGUK) proteins. The crystal structures of the beta subunit core, B-D, domains have recently been reported; however, very little is known about the structures of the A and E domains. The N-terminal A domain is a hypervariable region that differs among the four subtypes of Ca2+ channel beta subunits (beta1-beta4). Furthermore, this domain undergoes alternative splicing to create multiple N-terminal structures within a given gene class that have distinct effects on gating. We have solved the solution structure of the A domain of the human beta4a subunit, a splice variant that we have shown previously to have alpha1 subunit subtype-specific effects on Ca2+ channel trafficking and gating. PMID:16385006

  15. 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.

  16. The N-terminal leucine-zipper motif in PTRF/cavin-1 is essential and sufficient for its caveolae-association

    Energy Technology Data Exchange (ETDEWEB)

    Wei, Zhuang [State Key Laboratory of Cell Biology, Institute of Biochemistry and Cell Biology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Shanghai 200031 (China); Laboratory of System Biology, Institute of Biochemistry and Cell Biology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Shanghai 200031 (China); Zou, Xinle [State Key Laboratory of Cell Biology, Institute of Biochemistry and Cell Biology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Shanghai 200031 (China); Wang, Hongzhong; Lei, Jigang; Wu, Yuan [State Key Laboratory of Cell Biology, Institute of Biochemistry and Cell Biology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Shanghai 200031 (China); Laboratory of System Biology, Institute of Biochemistry and Cell Biology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Shanghai 200031 (China); Liao, Kan, E-mail: kliao@sibs.ac.cn [State Key Laboratory of Cell Biology, Institute of Biochemistry and Cell Biology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Shanghai 200031 (China); Laboratory of System Biology, Institute of Biochemistry and Cell Biology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Shanghai 200031 (China)

    2015-01-16

    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{sup −/−} 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.

  17. Purification, N-terminal amino acid sequence, and some properties of Cu, Zn-superoxide dismutase from Japanese flounder (Paralichthys olivaceus) hepato-pancreas.

    Science.gov (United States)

    Osatomi, K; Masuda, Y; Hara, K; Ishihara, T

    2001-04-01

    Cu, Zn-superoxide dismutase (SOD) has been purified to homogeneity from Japanese flounder Paralichthys olivaceus hepato-pancreas. The purification of the enzyme was carried out by an ethanol/chloroform treatment and acetone precipitation, and then followed by column chromatographies on Q-Sepharose, S-Sepharose and Ultrogel AcA 54. On SDS-PAGE, the purified enzyme gave a single protein band with molecular mass of 17.8 kDa under reducing conditions, and showed approximately equal proportions of 17.8 and 36 kDa molecular mass under non-reducing conditions. Three bands were obtained when the purified enzyme was subjected to native-PAGE, both on protein and activity staining, but the electrophoretic mobility of the purified enzyme differed from that of bovine erythrocyte Cu, Zn-SOD. Isoelectric point values of 5.9, 6.0 and 6.2, respectively, were obtained for the three components. The N-terminal amino acid sequence of the purified enzyme was determined for 25 amino acid residues, and the sequence was compared with other Cu, Zn-SODs. The N-terminal alanine residue was unacetylated, as in the case of swordfish SOD. Above 60 degrees C, the thermostability of the enzyme was much lower than that of bovine Cu, Zn-SOD. PMID:11290457

  18. Characterization of an invertase with pH tolerance and truncation of its N-terminal to shift optimum activity toward neutral pH.

    Directory of Open Access Journals (Sweden)

    Liqin Du

    Full Text Available Most invertases identified to date have optimal activity at acidic pH, and are intolerant to neutral or alkaline environments. Here, an acid invertase named uninv2 is described. Uninv2 contained 586 amino acids, with a 100 amino acids N-terminal domain, a catalytic domain and a C-terminal domain. With sucrose as the substrate, uninv2 activity was optimal at pH 4.5 and at 45°C. Removal of N-terminal domain of uninv2 has shifted the optimum pH to 6.0 while retaining its optimum temperaure at 45°C. Both uninv2 and the truncated enzyme retained highly stable at neutral pH at 37°C, and they were stable at their optimum pH at 4°C for as long as 30 days. These characteristics make them far superior to invertase from Saccharomyces cerevisiae, which is mostly used as industrial enzyme.

  19. Small-angle X-ray scattering reveals compact domain-domain interactions in the N-terminal region of filamin C.

    Directory of Open Access Journals (Sweden)

    Ritika Sethi

    Full Text Available Filamins are multi-domain, actin cross-linking, and scaffolding proteins. In addition to the actin cross-linking function, filamins have a role in mechanosensor signaling. The mechanosensor function is mediated by domain-domain interaction in the C-terminal region of filamins. Recently, we have shown that there is a three-domain interaction module in the N-terminal region of filamins, where the neighboring domains stabilize the structure of the middle domain and thereby regulate its interaction with ligands. In this study, we have used small-angle X-ray scattering as a tool to screen for potential domain-domain interactions in the N-terminal region. We found evidence of four domain-domain interactions with varying flexibility. These results confirm our previous study showing that domains 3, 4, and 5 exist as a compact three domain module. In addition, we report interactions between domains 11-12 and 14-15, which are thus new candidate sites for mechanical regulation.

  20. Nuclear import of influenza B virus nucleoprotein: Involvement of an N-terminal nuclear localization signal and a cleavage-protection motif

    International Nuclear Information System (INIS)

    The nucleoprotein of influenza B virus (BNP) shares several characteristics with its influenza A virus counterpart (ANP), including localization in the host's nucleus. However, while the nuclear localization signal(s) (NLS) of ANP are well characterized, little is known about those of BNP. In this study, we showed that the fusion protein bearing the BNP N-terminus fused with GFP (N70–GFP) is exclusively nuclear, and identified a highly conserved KRXR motif spanning residues 44–47 as a putative NLS. In addition, we demonstrated that residues 3–15 of BNP, though not an NLS, are also crucial for nuclear import. Results from mutational analyses of N70–GFP and the full-length BNP suggest that this region may be required for protection of the N-terminus from proteolytic cleavage. Altogether, we propose that the N-terminal region of BNP contains the NLS and cleavage-protection motif, which together drive its nuclear localization. - Highlights: • The N-terminal region of BNP is required for nuclear accumulation. • The conserved motif at position 44–47 is a putative nuclear localization signal. • The first 15 amino acids of BNP may function as a cleavage-protection motif. • BNP may get access to the nucleus via a mechanism distinct from ANP

  1. The N-terminal cysteine-rich domain of tobacco class I chitinase is essential for chitin binding but not for catalytic or antifungal activity.

    Science.gov (United States)

    Iseli, B; Boller, T; Neuhaus, J M

    1993-09-01

    The vacuolar chitinases of class I possess an N-terminal cysteine-rich domain homologous to hevein and chitin-binding lectins such as wheat germ agglutinin and Urtica dioica lectin. To investigate the significance of this domain for the biochemical and functional characteristics of chitinase, chimeric genes encoding the basic chitinase A of tobacco (Nicotiana tabacum) with and without this domain were constructed and constitutively expressed in transgenic Nicotiana sylvestris. The chitinases were subsequently isolated and purified to homogeneity from the transgenic plants. Chromatography on colloidal chitin revealed that only the form with the N-terminal domain, and not the one without it, had chitin-binding properties, demonstrating directly that the domain is a chitin-binding domain (CBD). Under standard assay conditions with radioactive colloidal chitin, both forms of chitinase had approximately the same catalytic activity. However, kinetic analysis demonstrated that the enzyme without CBD had a considerably lower apparent affinity for its substrate. The pH and temperature optima of the two chitinases were similar, but the form with the CBD had an approximately 3-fold higher activation energy and retained a higher activity at low pH values. Both chitinases were capable of inhibiting growth of Trichoderma viride, although the form with the CBD was about three times more effective than the one without it. Thus, the CBD is not necessary for catalytic or antifungal activity of chitinase. PMID:8208848

  2. Sequential control signals determine arm and trunk contributions to hand transport during reaching in humans.

    Science.gov (United States)

    Rossi, Elena; Mitnitski, Arnold; Feldman, Anatol G

    2002-01-15

    When reaching towards objects placed outside the arm workspace, the trunk assumes an active role in transport of the hand by contributing to the extent of movement while simultaneously maintaining the direction of reach. We investigated the spatial-temporal aspects of the integration of the trunk motion into reaching. Specifically, we tested the hypothesis that the efficiency ('gain') of the arm-trunk co-ordination determining the contribution of the trunk to the extent of hand movement may vary substantially with the phase of reaching. Sitting subjects made fast pointing movements towards ipsi- and a contralateral targets placed beyond the reach of the right arm so that a forward trunk motion was required to assist in transporting the hand to the target. Sight of the arm and target was blocked before the movement onset. In randomly selected trials, the trunk motion was unexpectedly prevented by an electromagnet. Subjects were instructed to make stereotypical movements whether or not the trunk was arrested. In non-perturbed trials, most subjects began to move the hand and trunk simultaneously. In trunk-blocked trials, it was impossible for the hand to cover the whole pointing distance but the hand trajectory and velocity profile initially matched those from the trials in which the trunk motion was free, approximately until the hand reached its peak velocity. The arm inter-joint co-ordination substantially changed in response to the trunk arrest at a minimal latency of 40 ms after the perturbation onset. The results suggest that when the trunk was free, the influence of the trunk motion on the hand trajectory and velocity profile was initially neutralized by appropriate changes in the arm joint angles. Only after the hand had reached its peak velocity did the trunk contribute to the extent of pointing. Previous studies suggested that the central commands underlying the transport component of arm movements are completed when the hand reaches peak velocity. These

  3. Sequence-dependent nucleosome structural and dynamic polymorphism. Potential involvement of histone H2B N-terminal tail proximal domain.

    Science.gov (United States)

    Sivolob, Andrei; Lavelle, Christophe; Prunell, Ariel

    2003-02-01

    Relaxation of nucleosomes on an homologous series (pBR) of ca 350-370 bp DNA minicircles originating from plasmid pBR322 was recently used as a tool to study their structure and dynamics. These nucleosomes thermally fluctuated between three distinct DNA conformations within a histone N-terminal tail-modulated equilibrium: one conformation was canonical, with 1.75 turn wrapping and negatively crossed entering and exiting DNAs; another was also "closed", but with these DNAs positively crossed; and the third was "open", with a lower than 1.5 turn wrapping and uncrossed DNAs. In this work, a new minicircle series (5S) of similar size was used, which contained the 5S nucleosome positioning sequence. Results showed that DNA in pBR nucleosomes was untwisted by approximately 0.2 turn relative to 5S nucleosomes, which DNase I footprinting confirmed in revealing a approximately 1 bp untwisting at each of the two dyad-distal sites where H2B N-terminal tails pass between the two gyres. In contrast, both nucleosomes showed untwistings at the dyad-proximal sites, i.e. on the other gyre, which were also observed in the high-resolution crystal structure. 5S nucleosomes also differ with respect to their dynamics: they hardly accessed the positively crossed conformation, but had an easier access to the negatively crossed conformation. Simulation showed that such reverse effects on the conformational free energies could be simply achieved by slightly altering the trajectories of entering and exiting DNAs. We propose that this is accomplished by H2B tail untwisting at the distal sites through action at a distance ( approximately 20 bp) on H3-tail interactions with the small groove at the nucleosome entry-exit. These results may help to gain a first glimpse into the two perhaps most intriguing features of the high-resolution structure: the alignment of the grooves on the two gyres and the passage of H2B and H3 N-terminal tails between them. PMID:12547190

  4. Mechanical and neuromuscular changes with lateral trunk lean gait modifications.

    Science.gov (United States)

    Robbins, Shawn M; Teoli, Anthony; Preuss, Richard A

    2016-09-01

    Lateral trunk lean (LTL) is a proposed intervention for knee osteoarthritis but increased muscular demands have not been considered. The objective was to compare lower extremity and trunk muscle activation and joint mechanics between normal and increased LTL gait in healthy adults. Participants (n=20, mean age 22 years) were examined under two gait conditions: normal and increased LTL. A motion capture system and force plates sampled at 100 and 2000Hz respectively were used to determine joint angles and external moments including LTL angle and external knee adduction moment (KAM). Surface electromyography, sampled at 2000Hz, measured activation of six trunk/hip muscles bilaterally. Peak LTL angle, peak KAM, gait speed, and mean values from electromyography waveforms were compared between normal and LTL conditions using paired t-tests or 2-way analysis of variance. There was a significant (p<0.05) increase in peak LTL angle, decrease in first but not second peak KAM, and decrease in gait speed during LTL gait. There were significant (p<0.01) increases in external oblique and iliocostalis muscle activation during LTL gait. There was no change in activation for internal oblique, rectus abdominis, longissimus, and gluteus medius. LTL gait decreased early/mid-stance KAM demonstrating its ability to decrease medial compartment knee loading. Increases in external oblique and iliocostalis activation were present but small to moderate in size and unlikely to lead to short term injury. Longitudinal studies should evaluate the effectiveness of increased LTL for knee osteoarthritis and if the increase in muscular demands leads to negative long term side effects.

  5. Oct4 Is a Key Regulator of Vertebrate Trunk Length Diversity.

    Science.gov (United States)

    Aires, Rita; Jurberg, Arnon D; Leal, Francisca; Nóvoa, Ana; Cohn, Martin J; Mallo, Moisés

    2016-08-01

    Vertebrates exhibit a remarkably broad variation in trunk and tail lengths. However, the evolutionary and developmental origins of this diversity remain largely unknown. Posterior Hox genes were proposed to be major players in trunk length diversification in vertebrates, but functional studies have so far failed to support this view. Here we identify the pluripotency factor Oct4 as a key regulator of trunk length in vertebrate embryos. Maintaining high Oct4 levels in axial progenitors throughout development was sufficient to extend trunk length in mouse embryos. Oct4 also shifted posterior Hox gene-expression boundaries in the extended trunks, thus providing a link between activation of these genes and the transition to tail development. Furthermore, we show that the exceptionally long trunks of snakes are likely to result from heterochronic changes in Oct4 activity during body axis extension, which may have derived from differential genomic rearrangements at the Oct4 locus during vertebrate evolution. PMID:27453501

  6. A tutorial on queuing and trunking with applications to communications

    CERN Document Server

    Tranter, William H

    2012-01-01

    The motivation for developing this synthesis lecture was to provide a tutorial on queuing and trunking, with extensions to networks of queues, suitable for supplementing courses in communications, stochastic processes, and networking. An essential component of this lecture is MATLAB-based demonstrations and exercises, which can be easily modified to enable the student to observe and evaluate the impact of changing parameters, arrival and departure statistics, queuing disciplines, the number of servers, and other important aspects of the underlying system model. Much of the work in this lecture

  7. A CADAVERIC STUDY OF VARIATION IN BRANCHING PATTERN OF COELIAC TRUNK IN SOUTH INDIAN POPULATION

    Directory of Open Access Journals (Sweden)

    Jayamma

    2015-06-01

    Full Text Available BACKGROUND: purpose of present study was to describe the variations in the branching pattern of caeliac trunk. Caeliac trunk is one of the ventral branch of abdominal aorta.it arises from the abdominal aorta opposite to intervertebral disc of T12 & L1 vertebrae.it has short course of 1.5 cm after which it terminates by dividing in to three branches splenic artery , left ga stric artery and common hepatic artery. Here we report unusual branching pattern of caeliac trunk. Knowledge of such variations in branching pattern of caeliac trunk is very important for surgeons , anatomists and anaesthetists. METHODS : The present study is done on 30 cadavers irrespective of sex in department of Anatomy in KURNOOL MEDICAL COLLEGE , Kurnool. According to dissection guidelines of cunnighams practical manual of Anatomy. RESULTS: The present study was an attempt to study the occurrence of the branching pattern of the coeliac trunk. The available literature was reviewed. The coeliac trunk took origin from the ventral surface of the aorta in all the 30 specimens. The various patterns were normal hepatolienogastric trunk in 92% , lienogastric trunk in. In one Type I , 2% to Type II , 2% to Type III , 2% to Type IV. The most common pattern of branching of the Coeliac trunk was the Hepatogastrolienal type , which has been accepted as the normal pattern o f specimen , coeliac trunk divided into common hepati c & splenic arteries , the left gastric artery took origin from the splenic artery. CONCLUSION : In my study out of 30 specimens 92% belonged to Type I , 2% to Type II , 2% to Type III , 2% to Type IV. This classification is according to LIPSCHUTZ (1917. The most common pattern of branching of the Coeliac trunk was the Hepatogastrolienal type , which has been accepted as the normal pattern of branching of the Coeliac trunk.

  8. Using Program Package NSPCG to Analyze the Trunk Reservation Service Protection Method

    DEFF Research Database (Denmark)

    Barker, Vincent A.; Nielsen, Bo Friis

    1994-01-01

    Unlike certain service protection methods for mixed traffic streams, such as the class-limitation method, the trunk reservation scheme cannot be based on a product form property of a stationary probability distribution vector. Rather, the analysis of the trunk reservation scheme requires solving,...... Analysis at the University of Texas at Austin. In this paper we report our experience in applying the NSPCG package to a typical system arising from the trunk reservation scheme....

  9. Reliability of trunk shape measurements based on 3-D surface reconstructions

    OpenAIRE

    Pazos, Valérie; Cheriet, Farida; Danserau, Jean; Ronsky, Janet; Zernicke, Ronald F.; Labelle, Hubert

    2007-01-01

    This study aimed to estimate the reliability of 3-D trunk surface measurements for the characterization of external asymmetry associated with scoliosis. Repeated trunk surface acquisitions using the Inspeck system (Inspeck Inc., Montreal, Canada), with two different postures A (anatomical position) and B (‘‘clavicle’’ position), were obtained from patients attending a scoliosis clinic. For each acquisition, a 3-D model of the patient’s trunk was built and a series of measurements was computed...

  10. Natural insertions within the N-terminal region of the coat protein of Maize dwarf mosaic potyvirus (MDMV) have an effect on the RNA stability.

    Science.gov (United States)

    Petrik, Kathrin; Sebestyén, Endre; Gell, Gyöngyvér; Balázs, Ervin

    2010-02-01

    A 13 amino acid residue insertion was found in the N-terminal region of the coat protein of several Maize dwarf mosaic virus isolates (MDMV). These insertions seem to be the result of a direct duplication event, but differ in some positions. In order to evaluate the influence of the insertion on the RNA secondary structure and stability, the RNA secondary structures and minimum free energies (MFE) of all existing MDMV coat protein sequences were estimated using three different softwares, the Vienna RNA Package, NUPACK, and UNAFold, and compared to the secondary structure and MFE of various random sequence collections preserving the nucleotide distribution of MDMV. The bioinformatic analysis showed that the insertion stabilizes the RNA structure of the coat protein gene.

  11. Extended N-terminal region of the essential phosphorelay signaling protein Ypd1 from Cryptococcus neoformans contributes to structural stability, phosphostability and binding of calcium ions.

    Science.gov (United States)

    Kennedy, Emily N; Menon, Smita K; West, Ann H

    2016-09-01

    Rapid response to external stimuli is crucial for survival and proliferation of microorganisms. Pathogenic fungi employ histidine-to-aspartate multistep phosphorelay systems to respond to environmental stress, progress through developmental stages and to produce virulence factors. Because these His-to-Asp phosphorelay systems are not found in humans, they are potential targets for the development of new antifungal therapies. Here we report the characterization of the histidine phosphotransfer (HPt) protein Ypd1 from the human fungal pathogen Cryptococcus neoformans Results from this study demonstrate that CnYpd1 indeed functions as a phosphorelay protein in vitro, and that H138 is confirmed as the site of phosphorylation. We found that CnYpd1 exhibits unique characteristics in comparison to other histidine phosphotransfer proteins, such as an extended N-terminal amino acid sequence, which we find contributes to structural integrity, a longer phosphorylated life time and the ability to bind calcium ions. PMID:27549628

  12. The SWI/SNF Subunit INI1 Contains an N-Terminal Winged Helix DNA Binding Domain that Is a Target for Mutations in Schwannomatosis.

    Science.gov (United States)

    Allen, Mark D; Freund, Stefan M V; Zinzalla, Giovanna; Bycroft, Mark

    2015-07-01

    SWI/SNF complexes use the energy of ATP hydrolysis to remodel chromatin. In mammals they play a central role in regulating gene expression during differentiation and proliferation. Mutations in SWI/SNF subunits are among the most frequent gene alterations in cancer. The INI1/hSNF5/SMARCB1 subunit is mutated in both malignant rhabdoid tumor, a highly aggressive childhood cancer, and schwannomatosis, a tumor-predisposing syndrome characterized by mostly benign tumors of the CNS. Here, we show that mutations in INI1 that cause schwannomatosis target a hitherto unidentified N-terminal winged helix DNA binding domain that is also present in the BAF45a/PHF10 subunit of the SWI/SNF complex. The domain is structurally related to the SKI/SNO/DAC domain, which is found in a number of metazoan chromatin-associated proteins. PMID:26073604

  13. Effects of body mass index and age on N-terminal pro brain natriuretic peptide are associated with glomerular filtration rate in chronic heart failure patients

    DEFF Research Database (Denmark)

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

    2007-01-01

    BACKGROUND: Obesity is a state characterized by glomerular hyperfiltration and age-related decreases in glomerular filtration rate (GFR). Body mass index (BMI), age, and GFR are associated with plasma concentrations of N-terminal pro-brain natriuretic peptide (NT-proBNP) in chronic heart failure...... (CHF) patients. We hypothesized that the effects of BMI and age on plasma concentrations of NT-proBNP are associated with GFR. METHODS: We obtained clinical data and laboratory test results from 345 CHF patients at the baseline visit in our heart failure clinic and examined the hypothesis using...... multiple linear regression models. RESULTS: Age (P = 0.0184), BMI (P = 0.0098), hemoglobin (P = 0.0043), heart rhythm (P GFR estimated by the Cockcroft and Gault equation, the parameter...

  14. Combined measurement of copeptin, high-sensitivity troponin T, and N-terminal proBNP improves the identification of patients at risk of cardiovascular death

    DEFF Research Database (Denmark)

    Alehagen, Urban; Dahlström, Ulf; Carstensen, John;

    2012-01-01

    Objectives: A multimarker strategy for the handling of patients with heart failure has been suggested in the literature. Therefore, the potential prognostic relevance of combined copeptin, high-sensitivity troponin T (HS-TnT), and N-terminal proBNP (NT-proBNP) measurement in plasma from elderly...... and all mortality was registered. Cardiovascular mortality was evaluated using Kaplan–Meier plots and multivariate Cox proportional hazard regression analyses. Results: Copeptin, HS-TnT, and NT-proBNP measurements provided independent prognostic information in a multivariate setting over 5 years......-up period). In the 13-year follow-up, combined copeptin and HS-TnT measurement did not provide independent prognostic information. Conclusion: Combined copeptin, HS-TnT, and NT-proBNP plasma measurements provide prognostic information on cardiovascular mortality that is superior to single biomarker use....

  15. 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

    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......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......-nucleotide interaction, intrinsic GTPase activity, and binding of aminoacyl-tRNA (aa-tRNA). The results show that Lys9 is especially strongly involved in the association with guanine nucleotides and the binding of aa-tRNA. Also Lys4 plays a role in the association of GDP and GTP and is also of some importance in aa...

  16. Hydrokinetic activity of secretion and secretin analogues, modified in the N-terminal sequence, and of vasoactive intestinal peptide in the dog pancreas.

    Science.gov (United States)

    Lehnert, P; Forell, M M; Jaeger, E; Moroder, L; Wünsch, E

    1981-01-01

    In the dog pancreas in vivo, the biological activity of secretin and vasoactive intestinal peptide was compared to that of secretin analogues modified in their N-terminal hexapeptide and to X-secretion (alpha, beta-Asp3-secretin) and Y-secretin (a conversion product of X-secretin consisting of about 15% secretin and 85% beta-Asp3-secretin). Replacement of Asp3 by glutamic acid reduced secretin activity markedly. Replacement by neutral amino acids abolished the activity nearly completely. alpha, beta-Asp3-secretin and beta-Asp3-secretin appeared to be ineffective. The results indicate that the free beta-carboxy group of the side chain of the Asp3 residue of the secretin molecule is of decisive importance for hydrokinetic action. PMID:7274610

  17. N-terminal domain of PB1-F2 protein of influenza A virus can fold into amyloid-like oligomers and damage cholesterol and cardiolipid containing membranes.

    Science.gov (United States)

    Ajjaji, Dalila; Richard, Charles-Adrien; Mazerat, Sandra; Chevalier, Christophe; Vidic, Jasmina

    2016-08-12

    PB1-F2 protein is a factor of virulence of influenza A viruses which increases the mortality and morbidity associated with infection. Most seasonal H1N1 Influenza A viruses express nowadays a truncated version of PB1-F2. Here we show that truncation of PB1-F2 modified supramolecular organization of the protein in a membrane-mimicking environment. In addition, full-length PB1-F2(1-90) and C-terminal PB1-F2 domain (53-90), efficiently permeabilized various anionic liposomes while N-terminal domain PB1-F2(1-52) only lysed cholesterol and cardiolipin containing lipid bilayers. These findings suggest that the truncation of PB1-F2 may impact the pathogenicity of a given virus strain. PMID:27282484

  18. Solution behavior of the intrinsically disordered N-terminal domain of the Retinoid X Receptor alpha in the context of full-length protein

    Science.gov (United States)

    Peluso-Iltis, Carole; Kieffer, Bruno; Svergun, Dmitri I.; Rochel, Natacha

    2016-01-01

    Retinoid X receptors (RXRs) are transcription factors with important functions in embryonic development, metabolic processes, differentiation and apoptosis. A particular feature of RXRs is their ability to act as obligatory heterodimerisation partners of class II nuclear receptors. At the same time, these receptors are also able to form homodimers that bind to direct repeat (DR1) hormone response elements. Since the discovery of RXRs, most of the studies focused on its ligand binding and DNA-binding domains, while its N-terminal domain (NTD) harboring a ligand-independent activation function remained poorly characterized. Here, we investigated the solution properties of the NTD domain of RXRα alone and in the context of the full-length receptor using small-angle X-ray scattering (SAXS) and nuclear magnetic resonance (NMR) spectroscopy. We report the solution structure of the full-length homodimeric RXRα on DNA and show that the NTD remains highly flexible within this complex. PMID:26937780

  19. Stress-induced phosphorylation of c-Jun-N-terminal kinases and nuclear translocation of Hsp70 in the Wistar rat hippocampus

    Directory of Open Access Journals (Sweden)

    Adžić M.

    2009-01-01

    Full Text Available Glucocorticoids are key regulators of the neuroendocrine stress response in the hippocampus. Their action is partly mediated through the subfamily of MAPKs termed c-Jun-N-terminal kinases (JNKs,whose activation correlates with neurodegeneration. The stress response also involves activation of cell protective mechanisms through various heat shock proteins (HSPs that mediate neuroprotection. We followed both JNKs and Hsp70 signals in the cytoplasmic and nuclear compartments of the hippocampus of Wistar male rats exposed to acute, chronic, and combined stress. The activity of JNK1 was decreased in both compartments by all three types of stress, while the activity of cytoplasmic JNK2/3 was elevated in acute and unaltered or lowered in chronic and combined stress. Under all stress conditions, Hsp70 translocation to the nucleus was markedly increased. The results suggest that neurodegenerative signaling of JNKs may be counteracted by increase of nuclear Hsp70,especially under chronic stress.

  20. 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.

  1. c-Jun N-terminal kinase 3 expression in the retina of ocular hypertension mice:a possible target to reduce ganglion cell apoptosis

    Institute of Scientific and Technical Information of China (English)

    Yue He; Jie Chen; Shu-guang Zhang; Yuan-sheng Yuan; Yan Li; Hong-bin Lv; Jin-hua Gan

    2015-01-01

    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 pho-tocoagulation using the laser ignition method. Results showed signiifcant 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.

  2. A Lanthipeptide-like N-Terminal Leader Region Guides Peptide Epimerization by Radical SAM Epimerases: Implications for RiPP Evolution.

    Science.gov (United States)

    Fuchs, Sebastian W; Lackner, Gerald; Morinaka, Brandon I; Morishita, Yohei; Asai, Teigo; Riniker, Sereina; Piel, Jörn

    2016-09-26

    Ribosomally synthesized and posttranslationally modified peptide natural products (RiPPs) exhibit diverse structures and bioactivities and are classified into distinct biosynthetic families. A recently reported family is the proteusins, with the prototype members polytheonamides being generated by almost 50 maturation steps, including introduction of d-residues at multiple positions by an unusual radical SAM epimerase. A region in the protein-like N-terminal leader of proteusin precursors is identified that is crucial for epimerization. It resembles a precursor motif previously shown to mediate interaction in thioether bridge-formation in class I lanthipeptide biosynthesis. Beyond this region, similarities were identified between proteusin and further RiPP families, including class I lanthipeptides. The data suggest that common leader features guide distinct maturation types and that nitrile hydratase-like enzymes are ancestors of several RiPP classes.

  3. The N-terminal segment of pulmonary surfactant lipopeptide SP-C has intrinsic propensity to interact with and perturb phospholipid bilayers

    DEFF Research Database (Denmark)

    Plasencia, Inés; Rivas, Luis; Keough, Kevin M W;

    2004-01-01

    In the present study, 13-residue peptides with sequences corresponding to the native N-terminal segment of pulmonary SP-C (surfactant protein C) have been synthesized and their interaction with phospholipid bilayers characterized. The peptides are soluble in aqueous media but associate...... spontaneously with bilayers composed of either zwitterionic (phosphatidylcholine) or anionic (phosphatidylglycerol) phospholipids. The peptides show higher affinity for anionic than for zwitterionic membranes. Interaction of the peptides with both zwitterionic and anionic membranes promotes phospholipid vesicle...... aggregation, and leakage of the aqueous content of the vesicles. The lipid-peptide interaction includes a significant hydrophobic component for both zwitterionic and anionic membranes, although the interaction with phosphatidylglycerol bilayers is also electrostatic in nature. The effects of the SP-C N...

  4. Resveratrol alleviates diabetes-induced testicular dysfunction by inhibiting oxidative stress and c-Jun N-terminal kinase signaling in rats.

    Science.gov (United States)

    Faid, Iman; Al-Hussaini, Heba; Kilarkaje, Narayana

    2015-12-15

    Diabetes adversely affects reproductive functions in humans and animals. The present study investigated the effects of Resveratrol on diabetes-induced alterations in oxidative stress, c-Jun N-terminal kinase (JNK) signaling and apoptosis in the testis. Adult male Wistar rats (13-15 weeks; n=6/group) were segregated into 1) normal control, 2) Resveratrol-treated (5mg/kg; ip; given during last 3 weeks), 3) Streptozotocin-induced diabetic and, 4) Resveratrol-treated diabetic groups, and euthanized on day 42 after the confirmation of diabetes. Resveratrol did not normalize blood glucose levels in diabetic rats. Resveratrol supplementation recovered diabetes-induced decreases in reproductive organ weights, sperm count and motility, intra-testicular levels of superoxide dismutase, catalase, and glutathione peroxidase and an increase in 4-hydroxynonenal activities (Prats. These results suggest that Resveratrol supplementation may be a useful strategy to treat diabetes-induced testicular dysfunction. PMID:26499206

  5. Is N-terminal pro B-type natriuretic peptide (NT-proBNP) a useful screening test for angiographic findings in patients with stable coronary disease?

    DEFF Research Database (Denmark)

    Kragelund, Charlotte; Grønning, Bjørn; Omland, Torbjørn;

    2006-01-01

    BACKGROUND: Whether N-terminal pro B-type natriuretic peptide (NT-proBNP) is a useful screening tool for angiographic coronary artery disease in patients with angina is not known. Therefore, the purpose of this study was to assess the diagnostic test performance of NT-proBNP in detecting coronary...... atherosclerotic lesions, as assessed by coronary angiography. METHODS: We examined 1034 patients referred for diagnostic angiography because of symptoms or signs of coronary artery disease. The diagnostic value of NT-proBNP in predicting clinically significant coronary disease was assessed. RESULTS: In a multiple...... logistic regression model, NT-proBNP above the upper normal limit (125 pg/mL) predicted clinically significant coronary disease at angiography independently of traditional cardiovascular risk factors and invasive measurements of left ventricular function (odds ratio 2.1, 95% CI 1.3-3.2, P = .001...

  6. 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

    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.

  7. The Relaxin Receptor (RXFP1) Utilizes Hydrophobic Moieties on a Signaling Surface of Its N-terminal Low Density Lipoprotein Class A Module to Mediate Receptor Activation*

    Science.gov (United States)

    Kong, Roy C. K.; Petrie, Emma J.; Mohanty, Biswaranjan; Ling, Jason; Lee, Jeremy C. Y.; Gooley, Paul R.; Bathgate, Ross A. D.

    2013-01-01

    The peptide hormone relaxin is showing potential as a treatment for acute heart failure. Although it is known that relaxin mediates its actions through the G protein-coupled receptor relaxin family peptide receptor 1 (RXFP1), little is known about the molecular mechanisms by which relaxin binding results in receptor activation. Previous studies have highlighted that the unique N-terminal low density lipoprotein class A (LDLa) module of RXFP1 is essential for receptor activation, and it has been hypothesized that this module is the true “ligand” of the receptor that directs the conformational changes necessary for G protein coupling. In this study, we confirmed that an RXFP1 receptor lacking the LDLa module binds ligand normally but cannot signal through any characterized G protein-coupled receptor signaling pathway. Furthermore, we comprehensively examined the contributions of amino acids in the LDLa module to RXFP1 activity using both gain-of-function and loss-of-function mutational analysis together with NMR structural analysis of recombinant LDLa modules. Gain-of-function studies with an inactive RXFP1 chimera containing the LDLa module of the human LDL receptor (LB2) demonstrated two key N-terminal regions of the module that were able to rescue receptor signaling. Loss-of-function mutations of residues in these regions demonstrated that Leu-7, Tyr-9, and Lys-17 all contributed to the ability of the LDLa module to drive receptor activation, and judicious amino acid substitutions suggested this involves hydrophobic interactions. Our results demonstrate that these key residues contribute to interactions driving the active receptor conformation, providing further evidence of a unique mode of G protein-coupled receptor activation. PMID:23926099

  8. The relaxin receptor (RXFP1) utilizes hydrophobic moieties on a signaling surface of its N-terminal low density lipoprotein class A module to mediate receptor activation.

    Science.gov (United States)

    Kong, Roy C K; Petrie, Emma J; Mohanty, Biswaranjan; Ling, Jason; Lee, Jeremy C Y; Gooley, Paul R; Bathgate, Ross A D

    2013-09-27

    The peptide hormone relaxin is showing potential as a treatment for acute heart failure. Although it is known that relaxin mediates its actions through the G protein-coupled receptor relaxin family peptide receptor 1 (RXFP1), little is known about the molecular mechanisms by which relaxin binding results in receptor activation. Previous studies have highlighted that the unique N-terminal low density lipoprotein class A (LDLa) module of RXFP1 is essential for receptor activation, and it has been hypothesized that this module is the true "ligand" of the receptor that directs the conformational changes necessary for G protein coupling. In this study, we confirmed that an RXFP1 receptor lacking the LDLa module binds ligand normally but cannot signal through any characterized G protein-coupled receptor signaling pathway. Furthermore, we comprehensively examined the contributions of amino acids in the LDLa module to RXFP1 activity using both gain-of-function and loss-of-function mutational analysis together with NMR structural analysis of recombinant LDLa modules. Gain-of-function studies with an inactive RXFP1 chimera containing the LDLa module of the human LDL receptor (LB2) demonstrated two key N-terminal regions of the module that were able to rescue receptor signaling. Loss-of-function mutations of residues in these regions demonstrated that Leu-7, Tyr-9, and Lys-17 all contributed to the ability of the LDLa module to drive receptor activation, and judicious amino acid substitutions suggested this involves hydrophobic interactions. Our results demonstrate that these key residues contribute to interactions driving the active receptor conformation, providing further evidence of a unique mode of G protein-coupled receptor activation. PMID:23926099

  9. γ-Aminobutyric Acid Type A (GABAA) Receptor Subunits Play a Direct Structural Role in Synaptic Contact Formation via Their N-terminal Extracellular Domains.

    Science.gov (United States)

    Brown, Laura E; Nicholson, Martin W; Arama, Jessica E; Mercer, Audrey; Thomson, Alex M; Jovanovic, Jasmina N

    2016-07-01

    The establishment of cell-cell contacts between presynaptic GABAergic neurons and their postsynaptic targets initiates the process of GABAergic synapse formation. GABAA receptors (GABAARs), the main postsynaptic receptors for GABA, have been recently demonstrated to act as synaptogenic proteins that can single-handedly induce the formation and functional maturation of inhibitory synapses. To establish how the subunit composition of GABAARs influences their ability to induce synaptogenesis, a co-culture model system incorporating GABAergic medium spiny neurons and the HEK293 cells, stably expressing different combinations of receptor subunits, was developed. Analyses of HEK293 cell innervation by medium spiny neuron axons using immunocytochemistry, activity-dependent labeling, and electrophysiology have indicated that the γ2 subunit is required for the formation of active synapses and that its effects are influenced by the type of α/β subunits incorporated into the functional receptor. To further characterize this process, the large N-terminal extracellular domains (ECDs) of α1, α2, β2, and γ2 subunits were purified using the baculovirus/Sf9 cell system. When these proteins were applied to the co-cultures of MSNs and α1/β2/γ2-expressing HEK293 cells, the α1, β2, or γ2 ECD each caused a significant reduction in contact formation, in contrast to the α2 ECD, which had no effect. Together, our experiments indicate that the structural role of GABAARs in synaptic contact formation is determined by their subunit composition, with the N-terminal ECDs of each of the subunits directly participating in interactions between the presynaptic and postsynaptic elements, suggesting the these interactions are multivalent and specific. PMID:27129275

  10. 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

  11. Just one position-independent lysine residue can direct MelanA into proteasomal degradation following N-terminal fusion of ubiquitin.

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    Christian Setz

    Full Text Available N-terminal stable in frame fusion of ubiquitin (Ub has been shown to target the fusion protein for proteasomal degradation. This pathway, called the Ub fusion degradation (UFD, might also elevate MHC class I (MHC-I antigen presentation of specific antigens. The UFD, mainly studied on cytosolic proteins, has been described to be mediated by polyubiquitination of specific lysine residues within the fused Ub moiety. Using the well characterized melanoma-specific antigen MelanA as a model protein, we analyzed the requirements of the UFD for ubiquitination and proteasomal degradation of a transmembrane protein. Here we show that fusion of the non-cleavable Ub(G76V variant to the N-terminus of MelanA results in rapid proteasomal degradation via the endoplasmic reticulum-associated degradation (ERAD pathway and, consequently, leads to an increased MHC-I antigen presentation. While lysine residues within Ub are dispensable for these effects, the presence of one single lysine residue, irrespectively of its location along the fusion protein, is sufficient to induce degradation of MelanA. These results show that the ubiquitination, ER to cytosol relocation and proteasomal degradation of a transmembrane protein can be increased by N-terminal fusion of Ub at the presence of at least one, position independent lysine residue. These findings are in contrast to the conventional wisdom concerning the UFD and indicate a new concept to target a protein into the ubiquitin-proteasome system (UPS and thus for enhanced MHC-I antigen presentation, and might open up new possibilities in the development of tumor vaccines.

  12. Characterization of amino acid residues within the N-terminal region of Ubc9 that play a role in Ubc9 nuclear localization

    International Nuclear Information System (INIS)

    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

  13. LC-MS/MS screening strategy for unknown adducts to N-terminal valine in hemoglobin applied to smokers and nonsmokers.

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    Carlsson, Henrik; von Stedingk, Hans; Nilsson, Ulrika; Törnqvist, Margareta

    2014-12-15

    Electrophilically reactive compounds have the ability to form adducts with nucleophilic sites in DNA and proteins, constituting a risk for toxic effects. Mass spectrometric detection of adducts to N-terminal valine in hemoglobin (Hb) after detachment by modified Edman degradation procedures is one approach for in vivo monitoring of exposure to electrophilic compounds/metabolites. So far, applications have been limited to one or a few selected reactive species, such as acrylamide and its metabolite glycidamide. This article presents a novel screening strategy for unknown Hb adducts to be used as a basis for an adductomic approach. The method is based on a modified Edman procedure, FIRE, specifically developed for LC-MS/MS analysis of N-terminal valine adducts in Hb detached as fluorescein thiohydantoin (FTH) derivatives. The aim is to detect and identify a priori unknown Hb adducts in human blood samples. Screening of valine adducts was performed by stepwise scanning of precursor ions in small mass increments, monitoring four fragments common for the FTH derivative of valine with different N-substitutions in the multiple-reaction mode, covering a mass range of 135 Da (m/z 503-638). Samples from six smokers and six nonsmokers were analyzed. Control experiments were performed to compare these results with known adducts and to check for artifactual formation of adducts. In all samples of smokers and nonsmokers, seven adducts were identified, of which six have previously been studied. Nineteen unknown adducts were observed, and 14 of those exhibited fragmentation patterns similar to earlier studied FTH derivatives of adducts to valine. Identification of the unknown adducts will be the focus of future work. The presented methodology is a promising screening tool using Hb adducts to indicate exposure to potentially toxic electrophilic compounds and metabolites.

  14. The Tip of the Four N-Terminal α-Helices of Clostridium sordellii Lethal Toxin Contains the Interaction Site with Membrane Phosphatidylserine Facilitating Small GTPases Glucosylation

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    Carolina Varela Chavez

    2016-03-01

    Full Text Available Clostridium sordellii lethal toxin (TcsL is a powerful virulence factor responsible for severe toxic shock in man and animals. TcsL belongs to the large clostridial glucosylating toxin (LCGT family which inactivates small GTPases by glucosylation with uridine-diphosphate (UDP-glucose as a cofactor. Notably, TcsL modifies Rac and Ras GTPases, leading to drastic alteration of the actin cytoskeleton and cell viability. TcsL enters cells via receptor-mediated endocytosis and delivers the N-terminal glucosylating domain (TcsL-cat into the cytosol. TcsL-cat was found to preferentially bind to phosphatidylserine (PS-containing membranes and to increase the glucosylation of Rac anchored to the lipid membrane. We have previously reported that the N-terminal four helical bundle structure (1–93 domain recognizes a broad range of lipids, but that TcsL-cat specifically binds to PS and phosphatidic acid. Here, we show using mutagenesis that the PS binding site is localized on the tip of the four-helix bundle which is rich in positively-charged amino acids. Residues Y14, V15, F17, and R18 on loop 1, between helices 1 and 2, in coordination with R68 from loop 3, between helices 3 and 4, form a pocket which accommodates L-serine. The functional PS-binding site is required for TcsL-cat binding to the plasma membrane and subsequent cytotoxicity. TcsL-cat binding to PS facilitates a high enzymatic activity towards membrane-anchored Ras by about three orders of magnitude as compared to Ras in solution. The PS-binding site is conserved in LCGTs, which likely retain a common mechanism of binding to the membrane for their full activity towards membrane-bound GTPases.

  15. Site-specific conjugation of the quencher on peptide's N-terminal for the synthesis of a targeted non-spreading activatable optical probe.

    Science.gov (United States)

    Simard, Bryan; Mironov, Gleb G; Tomanek, Boguslaw; van Veggel, Frank C J M; Abulrob, Abedelnasser

    2016-06-01

    Optical imaging offers high sensitivity and portability at low cost. The design of 'smart' or 'activatable' probes can decrease the background noise and increase the specificity of the signal. By conjugating a fluorescent dye and a compatible quencher on each side of an enzyme's substrate, the signal remains in its 'off ' state until it reaches the area where a specific enzyme is expressed. However, the signal can leak from that area unless the dye is attached to a molecule able to bind to a specific target also presented in that area. The aim of this study was to (i) specifically conjugate the quencher on the α-amino group of the peptide's N-terminus, (ii) conjugate the dye on the ε-amino group of a lysine in C-terminus, and (iii) conjugate the carboxyl group of the peptide's C-terminus to an amino group present on an antibody, using carbodiimide chemistry. The use of protecting groups, such as Boc or Fmoc, to allow site-specific conjugation, presents several drawbacks including 'on beads labeling', additional steps required for deprotection and removal from the resin, decreased yield, and dye degradation. A method of preferential labeling of α-amino N-terminal group in slightly acidic solution, proposed by Selo et al. (1996) has partially solved the problem. The present study reports improvements of the method allowing to (i) avoid the homo-bilabeling, (ii) increase the yield of the N-terminal labeling by two folds, and (iii) decrease the cost by 44-fold. Copyright © 2016 European Peptide Society and John Wiley & Sons, Ltd. PMID:27282138

  16. 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.

  17. The N-terminal hybrid binding domain of RNase HI from Thermotoga maritima is important for substrate binding and Mg2+-dependent activity.

    Science.gov (United States)

    Jongruja, Nujarin; You, Dong-Ju; Kanaya, Eiko; Koga, Yuichi; Takano, Kazufumi; Kanaya, Shigenori

    2010-11-01

    Thermotoga maritima ribonuclease H (RNase H) I (Tma-RNase HI) contains a hybrid binding domain (HBD) at the N-terminal region. To analyze the role of this HBD, Tma-RNase HI, Tma-W22A with the single mutation at the HBD, the C-terminal RNase H domain (Tma-CD) and the N-terminal domain containing the HBD (Tma-ND) were overproduced in Escherichia coli, purified and biochemically characterized. Tma-RNase HI prefers Mg(2+) to Mn(2+) for activity, and specifically loses most of the Mg(2+)-dependent activity on removal of the HBD and 87% of it by the mutation at the HBD. Tma-CD lost the ability to suppress the RNase H deficiency of an E. coli rnhA mutant, indicating that the HBD is responsible for in vivo RNase H activity. The cleavage-site specificities of Tma-RNase HI are not significantly changed on removal of the HBD, regardless of the metal cofactor. Binding analyses of the proteins to the substrate using surface plasmon resonance indicate that the binding affinity of Tma-RNase HI is greatly reduced on removal of the HBD or the mutation. These results indicate that there is a correlation between Mg(2+)-dependent activity and substrate binding affinity. Tma-CD was as stable as Tma-RNase HI, indicating that the HBD is not important for stability. The HBD of Tma-RNase HI is important not only for substrate binding, but also for Mg(2+)-dependent activity, probably because the HBD affects the interaction between the substrate and enzyme at the active site, such that the scissile phosphate group of the substrate and the Mg(2+) ion are arranged ideally.

  18. An unusual N-terminal deletion of the laminin alpha3a isoform leads to the chronic granulation tissue disorder laryngo-onycho-cutaneous syndrome.

    Science.gov (United States)

    McLean, W H Irwin; Irvine, Alan D; Hamill, Kevin J; Whittock, Neil V; Coleman-Campbell, Carrie M; Mellerio, Jemima E; Ashton, Gabrielle S; Dopping-Hepenstal, Patricia J H; Eady, Robin A J; Jamil, Tanvir; Phillips, Roderic J; Shabbir, S Ghulam; Haroon, Tahir S; Khurshid, Khawar; Moore, Jonathan E; Page, Brian; Darling, Jonathan; Atherton, David J; Van Steensel, Maurice A M; Munro, Colin S; Smith, Frances J D; McGrath, John A; Phillips, Rodney J

    2003-09-15

    Laryngo-onycho-cutaneous (LOC or Shabbir) syndrome (OMIM 245660) is an autosomal recessive epithelial disorder confined to the Punjabi Muslim population. The condition is characterized by cutaneous erosions, nail dystrophy and exuberant vascular granulation tissue in certain epithelia, especially conjunctiva and larynx. Genome-wide homozygosity mapping localized the gene to a 2 Mb region on chromosome 18q11.2 with an LOD score of 19.8 at theta=0. This region includes the laminin alpha3 gene (LAMA3), in which loss-of-expression mutations cause the lethal skin blistering disorder Herlitz junctional epidermolysis bullosa. Detailed investigation showed that this gene possesses a further 38 exons (76 exons in total) spanning 318 kb of genomic DNA, and encodes three distinct proteins, designated laminin alpha3a, alpha3b1 and alpha3b2. The causative mutation in 15 families was a frameshift mutation 151insG predicting a stop codon 7 bp downstream in an exon that is specific to laminin alpha3a. This protein is secreted only by the basal keratinocytes of stratified epithelia, implying that LOC is caused by dysfunction of keratinocyte-mesenchymal communication. Surprisingly, the 151insG mutation does not result in nonsense-mediated mRNA decay due to rescue of the transcript by an alternative translation start site 6 exons downstream. The resultant N-terminal deletion of laminin alpha3a was confirmed by immunoprecipitation of secreted proteins from LOC keratinocytes. These studies show that the laminin alpha3a N-terminal domain is a key regulator of the granulation tissue response, with important implications not only in LOC but in a range of other clinical conditions associated with abnormal wound healing. PMID:12915477

  19. Simple screening method for autoantigen proteins using the N-terminal biotinylated protein library produced by wheat cell-free synthesis.

    Science.gov (United States)

    Matsuoka, Kazuhiro; Komori, Hiroaki; Nose, Masato; Endo, Yaeta; Sawasaki, Tatsuya

    2010-08-01

    Autoimmune diseases are a heterogeneous group of diseases characterized by immune reactions against either a major or a limited number of the bodies own autoantigens, causing inflammation and damage to tissues and organs. Thus, identification of autoantigens is an important first step to understanding autoimmune diseases. Here we demonstrate a simple screening method for identification of autoantigens reacting with patient serum antibodies by combination of an N-terminal biotinylated protein library (BPL), produced using a wheat cell-free protein production system, and a commercially available luminescence system. Optimization studies using well-characterized autoantigens showed specific interactions between N-terminal biotinylated proteins and antibody that were sensitively detected under homogeneous reaction conditions. In this optimized assay, 1 microL of the translation mixture expressing the biotinylated proteins produced significant luminescence signal by addition of diluted serum between 1:500 and 1:10 000 in 25 microL of reaction volume. For the BPL construction, 214 mouse genes, consisting of 103 well-known autoantigens and 111 genes in the mouse autoimmune susceptibility loci, and the sera of MRL/lpr mouse were used as an autoimmune model. By this screening method, 25 well-known autoantigens and 71 proteins in the loci were identified as autoantigen proteins specifically reacting with sera antibodies. Cross-referencing with the Gene Ontology Database, 26 and 38 of autoantigen proteins were predicted to have nuclear localization and identified as membrane and/or extracellular proteins. The immune reaction of six randomly selected proteins was confirmed by immunoprecipitation and/or immunoblot analyses. Interestingly, three autoantigen proteins were recognized by immunoprecipitation but not by immunoblot analysis. These results suggest that the BPL-based method could provide a simple system for screening of autoantigen proteins and would help with

  20. Enhancement of HCV polytope DNA vaccine efficacy by fusion to an N-terminal fragment of heat shock protein gp96.

    Science.gov (United States)

    Pishraft-Sabet, Leila; Kosinska, Anna D; Rafati, Sima; Bolhassani, Azam; Taheri, Tahereh; Memarnejadian, Arash; Alavian, Seyed-Moayed; Roggendorf, Michael; Samimi-Rad, Katayoun

    2015-01-01

    Induction of a strong hepatitis C virus (HCV)-specific immune response plays a key role in control and clearance of the virus. A polytope (PT) DNA vaccine containing B- and T-cell epitopes could be a promising vaccination strategy against HCV, but its efficacy needs to be improved. The N-terminal domain of heat shock protein gp96 (NT(gp96)) has been shown to be a potent adjuvant for enhancing immunity. We constructed a PT DNA vaccine encoding four HCV immunodominant cytotoxic T lymphocyte epitopes (two HLA-A2- and two H2-D(d)-specific motifs) from the Core, E2, NS3 and NS5B antigens in addition to a T-helper CD4+ epitope from NS3 and a B-cell epitope from E2. The NT(gp96) was fused to the C- or N-terminal end of the PT DNA (PT-NT(gp96) or NT(gp96)-PT), and their potency was compared. Cellular and humoral immune responses against the expressed peptides were evaluated in CB6F1 mice. Our results showed that immunization of mice with PT DNA vaccine fused to NT(gp96) induced significantly stronger T-cell and antibody responses than PT DNA alone. Furthermore, the adjuvant activity of NT(gp96) was more efficient in the induction of immune responses when fused to the C-terminal end of the HCV DNA polytope. In conclusion, the NT(gp96) improved the efficacy of the DNA vaccine, and this immunomodulatory effect was dependent on the position of the fusion.

  1. Association between resting heart rate and N-terminal pro-brain natriuretic peptide in a community-based population study in Beijing

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    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

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

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    Andrew T N Tebbenkamp

    Full Text Available BACKGROUND: 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. RESULTS: 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. CONCLUSIONS: 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.

  3. Studies on N-terminal glycation of peptides in hypoallergenic infant formulas: quantification of alpha-N-(2-furoylmethyl) amino acids.

    Science.gov (United States)

    Penndorf, Ilka; Biedermann, Daniela; Maurer, Sarah V; Henle, Thomas

    2007-02-01

    To obtain information about the extent of the early Maillard reaction between the N-termini of peptides and lactose, alpha-N-(2-furoylmethyl) amino acids (FMAAs) were quantified together with epsilon-N-(2-furoylmethyl)lysine (furosine) in acid hydrolyzates of hypoallergenic infant formulas, conventional infant formulas, and human milk samples using RP-HPLC with UV-detection. FMAAs are formed during acid hydrolysis of peptide-bound N-terminal Amadori products (APs), and furosine is formed from the Amadori products of peptide-bound lysine. Unambiguous identification was achieved by means of LC/MS and UV-spectroscopy using independently prepared reference material. The extent of acid-induced conversion of APs to FMAAs was studied by RP-HPLC with chemiluminescent nitrogen detection (CLND). Depending on the corresponding alpha-N-lactulosyl amino acid, between 6.0% and 18.1% of FMAAs were formed during hydrolysis for 23 h at 110 degrees C in 8 N HCl. From epsilon-N-lactulosyllysine, 50% furosine is formed under these conditions. Whereas furosine was detectable in all assayed samples, five different FMAAs, alpha-FM-Lys, alpha-FM-Ala, alpha-FM-Val, alpha-FM-Ile, and alpha-FM-Leu, were exclusively detected in acid hydrolyzates of hypoallergenic infant formulas in amounts ranging from 35 to 396 mumol/100 g protein. Taking the conversion factors into account, modification of N-terminal amino acids in peptides by reducing carbohydrates was between 0.3% and 8.4%. This has to be considered within the discussion concerning the nutritional quality of peptide-containing foods.

  4. High-resolution structure of the N-terminal endonuclease domain of the Lassa virus L polymerase in complex with magnesium ions.

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    Gregor D Wallat

    Full Text Available Lassa virus (LASV causes deadly hemorrhagic fever disease for which there are no vaccines and limited treatments. LASV-encoded L polymerase is required for viral RNA replication and transcription. The functional domains of L-a large protein of 2218 amino acid residues-are largely undefined, except for the centrally located RNA-dependent RNA polymerase (RdRP motif. Recent structural and functional analyses of the N-terminal region of the L protein from lymphocytic choriomeningitis virus (LCMV, which is in the same Arenaviridae family as LASV, have identified an endonuclease domain that presumably cleaves the cap structures of host mRNAs in order to initiate viral transcription. Here we present a high-resolution crystal structure of the N-terminal 173-aa region of the LASV L protein (LASV L173 in complex with magnesium ions at 1.72 Å. The structure is highly homologous to other known viral endonucleases of arena- (LCMV NL1, orthomyxo- (influenza virus PA, and bunyaviruses (La Crosse virus NL1. Although the catalytic residues (D89, E102 and K122 are highly conserved among the known viral endonucleases, LASV L endonuclease structure shows some notable differences. Our data collected from in vitro endonuclease assays and a reporter-based LASV minigenome transcriptional assay in mammalian cells confirm structural prediction of LASV L173 as an active endonuclease. The high-resolution structure of the LASV L endonuclease domain in complex with magnesium ions should aid the development of antivirals against lethal Lassa hemorrhagic fever.

  5. 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.

  6. The conserved residue Arg46 in the N-terminal heptad repeat domain of HIV-1 gp41 is critical for viral fusion and entry.

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    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.

  7. The N-terminal domain of Npro of classical swine fever virus determines its stability and regulates type I IFN production.

    Science.gov (United States)

    Mine, Junki; Tamura, Tomokazu; Mitsuhashi, Kazuya; Okamatsu, Masatoshi; Parchariyanon, Sujira; Pinyochon, Wasana; Ruggli, Nicolas; Tratschin, Jon-Duri; Kida, Hiroshi; Sakoda, Yoshihiro

    2015-07-01

    The viral protein Npro is unique to the genus Pestivirus within the family Flaviviridae. After autocatalytic cleavage from the nascent polyprotein, Npro suppresses type I IFN (IFN-α/β) induction by mediating proteasomal degradation of IFN regulatory factor 3 (IRF-3). Previous studies found that the Npro-mediated IRF-3 degradation was dependent of a TRASH domain in the C-terminal half of Npro coordinating zinc by means of the amino acid residues C112, C134, D136 and C138. Interestingly, four classical swine fever virus (CSFV) isolates obtained from diseased pigs in Thailand in 1993 and 1998 did not suppress IFN-α/β induction despite the presence of an intact TRASH domain. Through systematic analyses, it was found that an amino acid mutation at position 40 or mutations at positions 17 and 61 in the N-terminal half of Npro of these four isolates were related to the lack of IRF-3-degrading activity. Restoring a histidine at position 40 or both a proline at position 17 and a lysine at position 61 based on the sequence of a functional Npro contributed to higher stability of the reconstructed Npro compared with the Npro from the Thai isolate. This led to enhanced interaction of Npro with IRF-3 along with its degradation by the proteasome. The results of the present study revealed that amino acid residues in the N-terminal domain of Npro are involved in the stability of Npro, in interaction of Npro with IRF-3 and subsequent degradation of IRF-3, leading to downregulation of IFN-α/β production. PMID:25809915

  8. 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. PMID:25403678

  9. Variation in susceptibility to wind along the trunk of an isolated Larix kaempferi (Pinaceae) tree.

    Science.gov (United States)

    Minamino, Ryoko; Tateno, Masaki

    2014-07-16

    • Premise of the study: The force of the wind is a significant hazard to the survival of trees and can affect tree morphology. However, the actual distribution of the stress that wind causes to a tree trunk is not well understood in spite of its expected importance to tree morphology. The uniform stress hypothesis (i.e., tree trunks take a form that equalizes the distribution of stress along the outer surface of the stem) has been tested indirectly as a model, placing high importance on the mechanical safety of a trunk, and rejected theoretically. But stress on a tree's trunk has not yet been measured directly.• Methods: Actual strains at the surface of the trunk of an isolated Larix kaempferi tree due to wind loads were measured at seven heights on the trunk for 1 yr.• Key results: During the measurement period, wind-induced stress was higher in the upper portions of the trunk than in the lower portions, regardless of wind speed, and the difference increased as wind speed increased. The deflection of the trunk recorded at each position was also larger in the upper portions than in the lower portions.• Conclusions: The results indicate that the upper portions of the trunk of an isolated Larix kaempferi tree are more susceptible to wind than are the lower portions. These results do not support the uniform stress hypothesis and suggest that another limitation (either mechanical or nonmechanical) acts on the morphology of the tree.

  10. Trunk orientation induces neglect-like lateral biases in covert attention.

    Science.gov (United States)

    Grubb, Jefferson D; Reed, Catherine L

    2002-11-01

    The purpose of this study was to resolve a paradox in the literature on the effects of body orientation on spatial attention. Neuropsychological studies have found that real or simulated trunk rotation relieves contralesional inattention in patients with unilateral neglect, suggesting that trunk orientation affects how attention is allocated to space. However in two previous studies, trunk orientation did not affect spatial attention in other populations. In this study we investigated the effects of trunk orientation on the performance of a covert attention task by neurologically intact adults. The covert attention task allowed the evaluation of the effects of trunk orientation on both the allocation of attention to space and the ability to shift that attention to new locations. As in previous research, trunk orientation did not affect participants' response times (RTs) to validly cued targets. However rotating participants' trunks to the left increased their RTs to invalidly cued targets on the right and decreased their RTs to invalidly cued targets on the left. These results indicate that trunk orientation induces directional biases in the ability to shift attention. Thus, for intact participants, trunk rotation created lateral biases in the covert attention task similar to those seen in neglect patients.

  11. Visual afference mediates head and trunk stability in vestibular hypofunction.

    Science.gov (United States)

    Wei, Shun-Hwa; Chen, Po-Yin; Chen, Hung-Ju; Kao, Chung-Lan; Schubert, Michael C

    2016-07-01

    Humans must maintain head and trunk stability while walking. The purpose of this study was to compare the kinematics of healthy controls and patients with vestibular hypofunction (VH) when walking and making head rotations of different frequencies in both light and dark conditions. We recruited eight individuals with VH and nine healthy control subjects to perform four tasks at their preferred gait speed, being normal walk, walking and making yaw head rotations at 1.5Hz and 2Hz, and walking in the dark and making yaw head rotations at 1.5Hz. Linear kinematics as well as head, trunk, and pelvis angular velocities were captured using the Vicon motion analysis system (Vicon Motion Systems, Oxford, UK). We found no difference in walking velocities for any of the four walking conditions across groups. The lateral displacement of the center of mass was increased in VH patients. In the dark, patients had more head instability in pitch (larger amplitudes and velocities) even though they were walking and making active yaw head rotations. Patients also had a smaller relative phase angle (mean 3.50±standard deviation 2.13°) than controls (mean 10.31±standard deviation 2.70°) (plight. PMID:26976344

  12. Muscular condition and trunk stability in judoka of national and international level

    Directory of Open Access Journals (Sweden)

    Casto Juan-Recio

    2013-12-01

    Full Text Available Background: It is theorized that the development of the ability to stabilize the trunk may improve the performance of a judoka because it improves body balance control and optimizes force transmission from the lower extremities to the upper limbs. However, there is a lack of scientific evidence to establish a clear relationship between trunk stability and performance in judo.Aim: The purpose of this study was to determine whether the quantification of trunk stability and muscular strength and endurance allowed differentiation between national level (n = 7 and international level judoka (n = 6. In addition, the relationship between trunk stability and muscular strength and endurance of the muscles involved in trunk stability control was analyzed.Method: To assess trunk stability, trunk responses to sudden loads applied by a pneumatic mechanism were analyzed, as well as trunk postural control through an unstable sitting paradigm. Muscular strength and endurance were assessed via a flexion and extension trunk test using an isokinetic dynamometer.Results/Conclusions: International level judokas showed lower CoP displacement in the most complex task in unstable seat (7.00 ± 1.19 vs 8.93 ± 1.45 mm, T = .025 and higher absolute and relative peak torque in extensor muscles (7.05 ± 0.87 vs 5.74 ± 0.72 Nm, T = .013 than national level judoka. According to these results, core stability and trunk muscular condition are important qualities in the physical training of elite judoka. Correlational analysis found no relation between the analyzed variables, thus muscular strength and endurance appear to have a non-significant effect on performance in the trunk stability tests.

  13. 49 CFR 571.401 - Standard No. 401; Interior trunk release.

    Science.gov (United States)

    2010-10-01

    ... Motor Vehicle Safety Standards § 571.401 Standard No. 401; Interior trunk release. S1. Purpose and scope... compartment. S2. Application. This standard applies to passenger cars that have a trunk compartment. This standard does not apply to passenger cars with a back door. S3. Definitions. Back door means a door or...

  14. Three-dimensional trunk kinematics in golf: between-club differences and relationships to clubhead speed.

    Science.gov (United States)

    Joyce, Christopher; Burnett, Angus; Cochrane, Jodie; Ball, Kevin

    2013-06-01

    The aims of this study were (i) to determine whether significant three-dimensional (3D) trunk kinematic differences existed between a driver and a five-iron during a golf swing; and (ii) to determine the anthropometric, physiological, and trunk kinematic variables associated with clubhead speed. Trunk range of motion and golf swing kinematic data were collected from 15 low-handicap male golfers (handicap = 2.5 +/- 1.9). Data were collected using a 10-camera motion capture system operating at 250 Hz. Data on clubhead speed and ball velocity were collected using a real-time launch monitor. Paired t-tests revealed nine significant (p golf swing kinematics, namely trunk and lower trunk flexion/extension and lower trunk axial rotation. Multiple regression analyses explained 33.7-66.7% of the variance in clubhead speed for the driver and five-iron, respectively, with both trunk and lower trunk variables showing associations with clubhead speed. Future studies should consider the role of the upper limbs and modifiable features of the golf club in developing clubhead speed for the driver in particular. PMID:23898684

  15. Postural control of the trunk during unstable sitting in Parkinson's disease.

    NARCIS (Netherlands)

    Burg, J. van der; Wegen, E. van; Rietberg, M.B.; Kwakkel, G.; Dieen, J.H. van

    2006-01-01

    Postural instability and falls, both common in Parkinson's disease (PD), have been related to altered trunk control. In this study, we investigated dynamic trunk control with subjects balancing on a seat mounted on a hemisphere, for up to 15s in five trials. We compared eight PD patients with a fall

  16. Changes in Trunk and Head Stability in Children with Cerebral Palsy after Hippotherapy: A Pilot Study

    Science.gov (United States)

    Shurtleff, Tim L.; Engsberg, Jack R.

    2010-01-01

    Hippotherapy (HPOT) is a therapy that uses horse movement. This pilot investigation objectively evaluated the efficacy of HPOT in improving head/trunk stability in children with cerebral palsy (CP). The participants were six children with spastic diplegia and six children without disability. Head and trunk stability was challenged by using a…

  17. The Performance Analysis of Traffic Channel Coding in Digital Trunking System

    Institute of Scientific and Technical Information of China (English)

    2001-01-01

    The encoding and decoding processes of traffic channel in digital trunking system are studied. On the basis of computer simulation, the BER (bit error ratio) with different RCPC decoding step is analyzed. As a result, the optimal RCPC decoding step is provided, which gives essential theoretical evidences for the implementation of digital trunking system.

  18. Trunk position modulates anterior cruciate ligament forces and strains during a single-leg squat

    NARCIS (Netherlands)

    Kulas, Anthony S.; Hortobagyi, Tibor; DeVita, Paul

    2012-01-01

    Background: Although the squat exercise and its variations are commonly prescribed for anterior cruciate ligament rehabilitation, whether trunk position affects these ligament forces and strains during the squat is unclear. Our purpose was to evaluate the effects of trunk position on anterior crucia

  19. Effect of short-term application of kinesio tape on the flexion-relaxation phenomenon, trunk postural control and trunk repositioning in healthy females.

    Science.gov (United States)

    Ruggiero, Sara A; Frost, Lydia R; Vallis, Lori Ann; Brown, Stephen H M

    2016-01-01

    This study was designed to investigate the potential effects of kinesio tape on the flexion-relaxation phenomenon, trunk postural control and trunk position sense when applied for a short period (30 min) to the low back of healthy female participants. Twenty-four participants were assigned to one of two groups: kinesio tape applied in either the recommended stretched or non-stretched (control) manner over the low back. Tests were performed at three time points (pre-tape, with tape, post-tape) to assess low-back muscle flexion-relaxation, position sense during active trunk repositioning and trunk postural control during seated balance. Results demonstrated that wearing kinesio tape did not affect the angle at which the erector spinae muscles became silent during trunk flexion (flexion-relaxation). Trunk repositioning error increased when wearing kinesio tape in both the stretched and non-stretched manner, and this increased error persisted after the tape was removed. Seated balance control improved when wearing kinesio tape in both the stretched and non-stretched manner, and these improvements persisted after the tape was removed. In conclusion, these findings do not support the general suggestions that short-term use of kinesio tape on the low-back region alter low-back muscle activation and enhance tasks related to proprioception, at least under these taping conditions in a group of healthy females.

  20. Safety and pitfalls in frozen elephant trunk implantation.

    Science.gov (United States)

    Damberg, Anneke; Schälte, Gereon; Autschbach, Rüdiger; Hoffman, Andras

    2013-09-01

    The frozen elephant trunk (FET) procedure, or open stent grafting, is a tool for the combined open and endovascular treatment via a median sternotomy of extensive aortic disease involving both aortic arch and descending thoracic aorta. The technique aims to stabilize the maximum extent of the thoracic aorta in one step, with the goal of either rendering a secondary intervention to the downstream aorta unnecessary or producing an easy landing zone for secondary thoracic endovascular aortic repair (TEVAR) or open surgery. Even though large case series have reported good results, we still have no conclusive evidence as to which patients and what kind of pathologies benefit from this technique. The surgical sequences described for total arch replacement with the FET procedure are just as varied as the associated devices and indications. This article focuses on important perioperative and surgical aspects, as well as potential complications during FET procedures. PMID:24109583

  1. Structural evolution and diversity of the caterpillar trunk

    DEFF Research Database (Denmark)

    Dupont, Steen Thorleif

    The thesis explores some major transformation series in the structure of the lepidopteran larval trunk, focusing partly on the initial events in the evolution of the order, partly on one of the more spectacular cases of subsequent biological diversification within ‘typical’/’higher’ Lepidoptera...... identify possible ground plan characteristics of the Lepidoptera and Amphiesmenoptera (MS1), 2) to describe and understand the evolution of the neolepidopteran caterpillar, and in particularly its crochet-bearing prolegs that are closely linked to walking on silken substrates and an external arboreal...... morphology in an attempt to link form and function (MS2-3). 4) to re-evaluate the previously indicated correlation between the cuticle thickness of lycaenid larvae and the degree of myrmecophily in a selection of species in this family, and through a comparative study to better understand the link between...

  2. Along the Grand Trunk Road: The Photography of Raghubir Singh

    Directory of Open Access Journals (Sweden)

    Chaya Chandrasekhar

    2013-05-01

    Full Text Available For more than two millennia, the historic Grand Trunk Road, the busy thoroughfare that extends some 1500 miles through north India and Pakistan served as the main artery of South Asia. It was also the gateway through which waves of immigrants, travelers, and invaders entered the subcontinent. As a result, a great deal of diversity and tolerance marks the road. Between 1988 and 1991, Raghubir Singh (1942-1999, one of India’s renowned documentary photographers, traveled and photographed the Indian stretch of the Road. Ninety-six photographs from his journeys appear in the publication, 'The Grand Trunk Road: A Passage Through India '(1995. Singh used the pictorial style of street photography that he is known for to capture everyday life along the path. Further, he emphasized the tremendous diversity he witnessed along the road through the selections he made for inclusion in the book and the specific manner in which he arranged many of them. By underscoring the heterogeneity, Singh provided a critical visual commentary of the political climate in India during the 1980s and early nineties. This period coincided with the rise of Hindu nationalism, which aimed to erase the subcontinent’s diverse past and promote instead the idea of a homogenous/Hindu India. By documenting the road in his uniquely pictorial style and arranging the photographs in his book to draw attention to the differences and tolerance witnessed along the path, Singh demonstrated that India was not a monolithic culture as the politics of the time claimed, but a rich interwoven fabric of many varied strands.

  3. The effect of kinesio taping on lower trunk range of motions.

    Science.gov (United States)

    Yoshida, Ayako; Kahanov, Leamor

    2007-01-01

    The purpose of the study was to determine the effects of kinesio taping (KT) on trunk flexion, extension, and lateral flexion. Thirty healthy subjects with no history of lower trunk or back issues participated in the study. Subjects performed two experimental measurements of range of motion (with and without the application of KT) in trunk flexion, extension, and right lateral flexion. A dependent t test was used to compare the range of motion measurements before and after the application of KT. Through evaluation of the sum of all scores, KT in flexion produced a gain of 17.8 cm compared with the non-kinesiotape group (t(29)=2.51, p0.05) or lateral flexion (3 cm; t(29)=-1.25, p>0.05). Based on the findings, we determined that KT applied over the lower trunk may increase active lower trunk flexion range of motion. Further investigation on the effects of KT is warranted. PMID:17578750

  4. Inter-joint coordination between hips and trunk during downswings: Effects on the clubhead speed.

    Science.gov (United States)

    Choi, Ahnryul; Lee, In-Kwang; Choi, Mun-Taek; Mun, Joung Hwan

    2016-10-01

    Understanding of the inter-joint coordination between rotational movement of each hip and trunk in golf would provide basic knowledge regarding how the neuromuscular system organises the related joints to perform a successful swing motion. In this study, we evaluated the inter-joint coordination characteristics between rotational movement of the hips and trunk during golf downswings. Twenty-one right-handed male professional golfers were recruited for this study. Infrared cameras were installed to capture the swing motion. The axial rotation angle, angular velocity and inter-joint coordination were calculated by the Euler angle, numerical difference method and continuous relative phase, respectively. A more typical inter-joint coordination demonstrated in the leading hip/trunk than trailing hip/trunk. Three coordination characteristics of the leading hip/trunk reported a significant relationship with clubhead speed at impact (r golf swing performance of unskilled golfers. PMID:26911704

  5. The effects of trunk kinesio taping on balance ability and gait function in stroke patients.

    Science.gov (United States)

    Lee, Yang Jin; Kim, Ji Young; Kim, Seong Yoel; Kim, Kyung Hoon

    2016-08-01

    [Purpose] Kinesio taping is a therapeutic method used in the treatment of various musculoskeletal and neuromuscular deficits, but there is limited evidence of the effects of trunk kinesio taping in neurologic patients. Therefore, this study aimed to determine the effects of trunk kinesio taping on balance ability and gait function in persons after a stroke. [Subjects and Methods] Twenty-five post-stroke patients were included in this study. Kinesio tape was applied to four trunk muscles. Before and after the taping, in all subjects, the balance ability was measured using the Wii Balance Board, and gait function was assessed using the GAITRite system. [Results] The difference in gait function before and after trunk taping was not statistically significant. However, a variability of balance ability showed statistically significant differences. [Conclusion] These results suggest that taping may be a helpful method during rehabilitation programs for stroke patients. Its application to the trunk muscles is especially useful for improving balance ability.

  6. The effects of trunk kinesio taping on balance ability and gait function in stroke patients

    Science.gov (United States)

    Lee, Yang Jin; Kim, Ji Young; Kim, Seong Yoel; Kim, Kyung Hoon

    2016-01-01

    [Purpose] Kinesio taping is a therapeutic method used in the treatment of various musculoskeletal and neuromuscular deficits, but there is limited evidence of the effects of trunk kinesio taping in neurologic patients. Therefore, this study aimed to determine the effects of trunk kinesio taping on balance ability and gait function in persons after a stroke. [Subjects and Methods] Twenty-five post-stroke patients were included in this study. Kinesio tape was applied to four trunk muscles. Before and after the taping, in all subjects, the balance ability was measured using the Wii Balance Board, and gait function was assessed using the GAITRite system. [Results] The difference in gait function before and after trunk taping was not statistically significant. However, a variability of balance ability showed statistically significant differences. [Conclusion] These results suggest that taping may be a helpful method during rehabilitation programs for stroke patients. Its application to the trunk muscles is especially useful for improving balance ability. PMID:27630439

  7. Three in-frame N-terminally different proteins are produced from the repressor locus of the Streptomyces bacteriophage phi C31.

    Science.gov (United States)

    Smith, M C; Owen, C E

    1991-11-01

    The sequence of the repressor locus, c, of the Streptomyces temperate phage, phi C31, was shown previously to contain an open reading frame encoding a 74 kDa protein. Further analysis of the transcriptional and translational products of the c gene shows a more complex pattern of expression. A nest of three in-frame N-terminally different, C-terminally identical proteins of 74, 54 and 42 kDa were found to be expressed from a corresponding nest of transcripts. The repressor proteins were produced in Escherichia coli and the 42 kDa protein was purified, verified by N-terminal sequencing, and used to raise antibody. The antibody cross-reacted in Western blots with the 74, 54 and 42 kDa proteins expressed in E. coli and Streptomyces lividans and from Streptomyces coelicolor phi C31 lysogens. Analysis of transcription of the c gene by S1 mapping and primer extension showed that the nest of transcripts encoding the repressor protein were induced after heat treatment of the cts locus (Sinclair and Bibb, 1989; this paper). Correspondingly, all three of the repressor proteins were induced. In addition to a promoter, cp1, which lies upstream of the 74 kDa open reading frame, the c locus contained at least one internal promoter, cp2, which transcribes DNA encoding the 54 and 42 kDa proteins. Transcripts initiating from cp3 were observed in RNA preparations from S. lividans containing the c gene deleted for cp1 and cp2, but gene fusions using DNA which should contain any putative promoting activity from this region transcriptionally fused to the xylE gene showed very low levels of expression of catechol 2,3 dioxygenase in S. lividans. The 74 kDa protein was not necessary for super-infection immunity. Data described here and current knowledge of the nature of other 'dual start' genes suggest a model for the regulation of lysis versus lysogeny in phi C31.

  8. Photoprotective effect of the N-terminal 5-mer peptide analog P165 of amyloid precursor protein in human dermal fibroblasts

    Institute of Scientific and Technical Information of China (English)

    Wang Ying; Chen Hui; Lin Yuying; Wang Wen; Wang Rong; Lian Shi; Zhu Wei

    2014-01-01

    Background We showed in our previous study that the N-terminal 17-mer peptide of amyloid precursor protein (APP17-mer peptide),an active peptide segment with trophic and antioxidative effects,protects skin fibroblasts against ultraviolet (UV) damage and downregulates matrix metalloproteinase 1 (MMP-1) expression.The aim of the current study was to explore the protective effects of P165,the N-terminal 5-mer peptide analog of amyloid precursor protein that is resistant to enzymolysis,on UVA-induced damage in human dermal fibroblasts (HDFs).Methods HDFs were cultured in Dulbecco's modified Eagle's medium without and with P165 (concentrations were 1,10,and 100 μJmol/L).Then,15 J/cm2 UVA irradiation was used to obtain the UV-irradiated model.Cell proliferation was analyzed using MTT kit.The collagen type Ⅰ and MMP-1 contents in cell lysate were determined by enzyme-linked immunosorbent assay (ELISA).Fluorometric assays were performed to detect the formation of intracellular reactive oxygen species (ROS) in the cells.Results P165 significantly protected the HDFs against UVA-induced cytotoxicity.Compared with the UVA-irradiated control,1,10,and 100 μmol/L P165 elevated cell proliferation by 14.98% (P<0.05),17.52% (P<0.01) and 28.34% (P<0.001),respectively.Simultaneously,10 and 100 μmol/L P165 increased collagen type Ⅰ content (both P<0.05).Moreover,P165 treatment (all concentrations) also markedly suppressed the UVA-induced MMP-1 expression (all P<0.001).P165 at 1,10,and 100 μmol/L also reduced UVA-induced ROS generation by 11.27%,13.69% (both P<0.05),and 25.48% (P<0.001),respectively.Conclusions P165 could protect the HDFs against UVA-induced photodamage,including cytotoxicity,and MMP-1 generation.Furthermore,it also increased the collagen type Ⅰ content in the cells.The inhibitory effect on intracellular ROS generation might be involved in these photoprotective effects.Thus,P165 may be a useful candidate in the prevention and

  9. Binding of the N-Terminal Domain of the Lactococcal Bacteriophage TP901-1 CI Repressor to Its Target DNA: A Crystallography, Small Angle Scattering, and Nuclear Magnetic Resonance Study

    DEFF Research Database (Denmark)

    Frandsen, Kristian Erik Høpfner; Rasmussen, Kim K.; Jensen, Malene Ringkjøbing;

    2013-01-01

    In most temperate bacteriophages, regulation of the choice of lysogenic or lytic life cycle is controlled by a CI repressor protein. Inhibition of transcription is dependent on a helix–turn–helix motif, often located in the N-terminal domain (NTD), which binds to specific DNA sequences (operator ...

  10. Ligand-controlled interaction of histone acetyltransferase binding to ORC-1 (HBO1) with the N-terminal transactivating domain of progesterone receptor induces steroid receptor coactivator 1-dependent coactivation of transcription

    NARCIS (Netherlands)

    M. Georgiakaki (Maria); L.J. Blok (Leen); R. Milgrom (Roni); M. Lombès (Marc); A. Guiochon-Mantel (Anne); H. Loosfelt (Hugues); N. Chabbert-Buffet (Nathalie); B. Dasen (Boris); G. Meduri (Geri); S. Wenk (Sandra); L. Rajhi (Leila); L. Amazit (Larbi); A. Chauchereau (Anne); C.W. Burger (Curt)

    2006-01-01

    textabstractModulators of cofactor recruitment by nuclear receptors are expected to play an important role in the coordination of hormone-induced transactivation processes. To identify such factors interacting with the N-terminal domain (NTD) of the progesterone receptor (PR), we used this domain as

  11. Joint detection of troponin T,high sensitivity C-reactive protein,N-terminal probrain natriuretic peptide applied in the diagnosis of acute coronary syndrome for elderly patients

    Institute of Scientific and Technical Information of China (English)

    赵月霞

    2012-01-01

    Objective To investigate the value of the joint detection of Troponin T(TnT) ,highsensitivity C-reactive protein (hs-CRP) and N-terminal probrain natriuretic peptide(NT-proBNP) for the clinical diagnosis of acute coronary syndrome(ACS) in elderly patients.

  12. The effects of virtual reality game training on trunk to pelvis coupling in a child with cerebral palsy

    OpenAIRE

    Barton Gabor J; Hawken Malcolm B; Foster Richard J; Holmes Gill; Butler Penny B

    2013-01-01

    Abstract Background Good control of trunk and pelvic movements is necessary for well controlled leg movements required to perform activities of daily living. The nature of movement coupling between the trunk and pelvis varies and depends on the type of activity. Children with cerebral palsy often have reduced ability to modulate coupling between the trunk and pelvis but movement patterns of the pelvis can be improved by training. The aim of this study was to examine how pelvis to trunk coupli...

  13. The Effect of Trunk Stabilization Exercise Using an Unstable Surface on the Abdominal Muscle Structure and Balance of Stroke Patients

    OpenAIRE

    Yoo, Junsang; Jeong, Juri; Lee, Wanhee

    2014-01-01

    [Purpose] This study investigated the effect of unstable surface trunk stabilization exercise on the abdominal muscle structure and balance of stroke patients. [Subjects] The subjects were divided into two groups: an unstable surface trunk stabilization exercise group (n=13), and a stable surface trunk stabilization exercise group (n=11). [Methods] Both groups performed trunk stabilization exercise for 30 minutes, 3 days per week for 6 weeks. Abdominal muscle thickness and the Berg Balance Sc...

  14. The Relationship between Performance and Trunk Movement During Change of Direction.

    Science.gov (United States)

    Sasaki, Shogo; Nagano, Yasuharu; Kaneko, Satoshi; Sakurai, Takakuni; Fukubayashi, Toru

    2011-01-01

    The purpose of this study was to obtain the trunk kinematics data during a change-of-direction task and to determine the relationship between trunk kinematics and the change-of-direction performance. The design of this investigation was a descriptive laboratory study. Twelve healthy male collegiate soccer players (age: 21.3 ± 1.0 yrs, body mass: 67. 7 ± 6.7 kg, and height: 1.75 ± 0.05 m) participated in this study. Participants performed a shuttle run cutting task with a 180 degree pivot as quickly as possible. The shuttle run cutting time, ground contact time during a change-of-direction, and trunk inclination angle were measured. The shuttle run cutting time tends to correlate positively with ground contact time. During the change- of-direction task, the trunk forward inclination angle gradually increased during the first 50% of the stance phase and decreased subsequently whereas the trunk flexed, maintaining a left inclination during the first 40% of the stance phase and changing exponentially in the opposite direction. Forward angular displacement of the trunk between foot-contact and maximum trunk inclination correlated positively with the shuttle run cutting time (r = 0.61, p < 0.05) and ground contact time (r = 0.65, p < 0.05). These findings suggest that the change-of-direction performance could be related to the small angular displacement of the trunk during a change of direction. Moreover, it was considered that there might be optimal inclination angles related to change-of-direction performance. Therefore, coaches in field sports should check body posture and trunk movements during changes of direction. Key pointsSmall forward angular displacement of the trunk during a direction change is related to the change-of-direction performance.Trunk stability during a change of direction is an important factor in the change-of-direction performance.There might be a range of optimal angles of trunk inclination during a change of direction.Coaches in field

  15. 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.

  16. Insights into the Function of the Unstructured N-Terminal Domain of Proteins 4.1R and 4.1G in Erythropoiesis

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    Wataru Nunomura

    2011-01-01

    Full Text Available Membrane skeletal protein 4.1R is the prototypical member of a family of four highly paralogous proteins that include 4.1G, 4.1N, and 4.1B. Two isoforms of 4.1R (4.1R135 and 4.1R80, as well as 4.1G, are expressed in erythroblasts during terminal differentiation, but only 4.1R80 is present in mature erythrocytes. One goal in the field is to better understand the complex regulation of cell type and isoform-specific expression of 4.1 proteins. To start answering these questions, we are studying in depth the important functions of 4.1 proteins in the organization and function of the membrane skeleton in erythrocytes. We have previously reported that the binding profiles of 4.1R80 and 4.1R135 to membrane proteins and calmodulin are very different despite the similar structure of the membrane-binding domain of 4.1G and 4.1R135. We have accumulated evidence for those differences being caused by the N-terminal 209 amino acids headpiece region (HP. Interestingly, the HP region is an unstructured domain. Here we present an overview of the differences and similarities between 4.1 isoforms and paralogs. We also discuss the biological significance of unstructured domains.

  17. Single-chain protein mimetics of the N-terminal heptad-repeat region of gp41 with potential as anti-HIV-1 drugs.

    Science.gov (United States)

    Crespillo, Sara; Cámara-Artigas, Ana; Casares, Salvador; Morel, Bertrand; Cobos, Eva S; Mateo, Pedro L; Mouz, Nicolas; Martin, Christophe E; Roger, Marie G; El Habib, Raphaelle; Su, Bin; Moog, Christiane; Conejero-Lara, Francisco

    2014-12-23

    During HIV-1 fusion to the host cell membrane, the N-terminal heptad repeat (NHR) and the C-terminal heptad repeat (CHR) of the envelope subunit gp41 become transiently exposed and accessible to fusion inhibitors or Abs. In this process, the NHR region adopts a trimeric coiled-coil conformation that can be a target for therapeutic intervention. Here, we present an approach to rationally design single-chain protein constructs that mimic the NHR coiled-coil surface. The proteins were built by connecting with short loops two parallel NHR helices and an antiparallel one with the inverse sequence followed by engineering of stabilizing interactions. The constructs were expressed in Escherichia coli, purified with high yield, and folded as highly stable helical coiled coils. The crystal structure of one of the constructs confirmed the predicted fold and its ability to accurately mimic an exposed gp41 NHR surface. These single-chain proteins bound to synthetic CHR peptides with very high affinity, and furthermore, they showed broad inhibitory activity of HIV-1 fusion on various pseudoviruses and primary isolates. PMID:25489108

  18. Single-chain protein mimetics of the N-terminal heptad-repeat region of gp41 with potential as anti–HIV-1 drugs

    Science.gov (United States)

    Crespillo, Sara; Cámara-Artigas, Ana; Casares, Salvador; Morel, Bertrand; Cobos, Eva S.; Mateo, Pedro L.; Mouz, Nicolas; Martin, Christophe E.; Roger, Marie G.; El Habib, Raphaelle; Su, Bin; Moog, Christiane; Conejero-Lara, Francisco

    2014-01-01

    During HIV-1 fusion to the host cell membrane, the N-terminal heptad repeat (NHR) and the C-terminal heptad repeat (CHR) of the envelope subunit gp41 become transiently exposed and accessible to fusion inhibitors or Abs. In this process, the NHR region adopts a trimeric coiled-coil conformation that can be a target for therapeutic intervention. Here, we present an approach to rationally design single-chain protein constructs that mimic the NHR coiled-coil surface. The proteins were built by connecting with short loops two parallel NHR helices and an antiparallel one with the inverse sequence followed by engineering of stabilizing interactions. The constructs were expressed in Escherichia coli, purified with high yield, and folded as highly stable helical coiled coils. The crystal structure of one of the constructs confirmed the predicted fold and its ability to accurately mimic an exposed gp41 NHR surface. These single-chain proteins bound to synthetic CHR peptides with very high affinity, and furthermore, they showed broad inhibitory activity of HIV-1 fusion on various pseudoviruses and primary isolates. PMID:25489108

  19. Long-lasting mnemotropic effect of substance P and its N-terminal fragment (SP1-7 on avoidance learning

    Directory of Open Access Journals (Sweden)

    C. Tomaz

    1997-02-01

    Full Text Available We investigated the long-lasting effect of peripheral injection of the neuropeptide substance P (SP and of some N- or C-terminal SP fragments (SPN and SPC, respectively on retention test performance of avoidance learning. Male Wistar rats (220 to 280 g were trained in an inhibitory step-down avoidance task and tested 24 h or 21 days later. Immediately after the training trial rats received an intraperitoneal injection of SP (50 µg/kg, SPN 1-7 (167 µg/kg or SPC 7-11 (134 µg/kg. Control groups were injected with vehicle or SP 5 h after the training trial. The immediate post-training administration of SP and SPN, but not SPC, facilitated avoidance behavior in rats tested 24 h or 21 days later, i.e., the retention test latencies of the SP and SPN groups were significantly longer (P<0.05, Mann-Whitney U-test during both training-test intervals. These observations suggest that the memory-enhancing effect of SP is long-lasting and that the amino acid sequence responsible for this effect is encoded by its N-terminal part

  20. Structural and functional analyses of five conserved positively charged residues in the L1 and N-terminal DNA binding motifs of archaeal RADA protein.

    Directory of Open Access Journals (Sweden)

    Li-Tzu Chen

    Full Text Available RecA family proteins engage in an ATP-dependent DNA strand exchange reaction that includes a ssDNA nucleoprotein helical filament and a homologous dsDNA sequence. In spite of more than 20 years of efforts, the molecular mechanism of homology pairing and strand exchange is still not fully understood. Here we report a crystal structure of Sulfolobus solfataricus RadA overwound right-handed filament with three monomers per helical pitch. This structure reveals conformational details of the first ssDNA binding disordered loop (denoted L1 motif and the dsDNA binding N-terminal domain (NTD. L1 and NTD together form an outwardly open palm structure on the outer surface of the helical filament. Inside this palm structure, five conserved basic amino acid residues (K27, K60, R117, R223 and R229 surround a 25 A pocket that is wide enough to accommodate anionic ssDNA, dsDNA or both. Biochemical analyses demonstrate that these five positively charged residues are essential for DNA binding and for RadA-catalyzed D-loop formation. We suggest that the overwound right-handed RadA filament represents a functional conformation in the homology search and pairing reaction. A new structural model is proposed for the homologous interactions between a RadA-ssDNA nucleoprotein filament and its dsDNA target.

  1. The Unstructured N-terminal Region of Arabidopsis Group 4 Late Embryogenesis Abundant (LEA) Proteins Is Required for Folding and for Chaperone-like Activity under Water Deficit.

    Science.gov (United States)

    Cuevas-Velazquez, Cesar L; Saab-Rincón, Gloria; Reyes, José Luis; Covarrubias, Alejandra A

    2016-05-13

    Late embryogenesis abundant (LEA) proteins are a conserved group of proteins widely distributed in the plant kingdom that participate in the tolerance to water deficit of different plant species. In silico analyses indicate that most LEA proteins are structurally disordered. The structural plasticity of these proteins opens the question of whether water deficit modulates their conformation and whether these possible changes are related to their function. In this work, we characterized the secondary structure of Arabidopsis group 4 LEA proteins. We found that they are disordered in aqueous solution, with high intrinsic potential to fold into α-helix. We demonstrate that complete dehydration is not required for these proteins to sample ordered structures because milder water deficit and macromolecular crowding induce high α-helix levels in vitro, suggesting that prevalent conditions under water deficit modulate their conformation. We also show that the N-terminal region, conserved across all group 4 LEA proteins, is necessary and sufficient for conformational transitions and that their protective function is confined to this region, suggesting that folding into α-helix is required for chaperone-like activity under water limitation. We propose that these proteins can exist as different conformers, favoring functional diversity, a moonlighting property arising from their structural dynamics. PMID:27006402

  2. Structural and functional insights into the role of the N-terminal Mps1 TPR domain in the SAC (spindle assembly checkpoint).

    Science.gov (United States)

    Thebault, Philippe; Chirgadze, Dimitri Y; Dou, Zhen; Blundell, Tom L; Elowe, Sabine; Bolanos-Garcia, Victor M

    2012-12-15

    The SAC (spindle assembly checkpoint) is a surveillance system that ensures the timely and accurate transmission of the genetic material to offspring. The process implies kinetochore targeting of the mitotic kinases Bub1 (budding uninhibited by benzamidine 1), BubR1 (Bub1 related) and Mps1 (monopolar spindle 1), which is mediated by the N-terminus of each kinase. In the present study we report the 1.8 Å (1 Å=0.1 nm) crystal structure of the TPR (tetratricopeptide repeat) domain in the N-terminal region of human Mps1. The structure reveals an overall high similarity to the TPR motif of the mitotic checkpoint kinases Bub1 and BubR1, and a number of unique features that include the absence of the binding site for the kinetochore structural component KNL1 (kinetochore-null 1; blinkin), and determinants of dimerization. Moreover, we show that a stretch of amino acids at the very N-terminus of Mps1 is required for dimer formation, and that interfering with dimerization results in mislocalization and misregulation of kinase activity. The results of the present study provide an important insight into the molecular details of the mitotic functions of Mps1 including features that dictate substrate selectivity and kinetochore docking.

  3. Crystal structures of the F and pSLT plasmid TraJ N-terminal regions reveal similar homodimeric PAS folds with functional interchangeability

    Energy Technology Data Exchange (ETDEWEB)

    Lu, Jun; Wu, Ruiying; Adkins, Joshua N.; Joachimiak, Andrzej; Glover, Mark

    2014-09-16

    In the F-family of conjugative plasmids, TraJ is an essential transcriptional activator of the tra operon that encodes most of the proteins required for conjugation. Here we report for the first time the X-ray crystal structures of the TraJ N-terminal regions from the prototypic F plasmid (TraJF11-130) and from the Salmonella virulence plasmid pSLT (TraJpSLT 1-128). Both proteins form similar homodimeric Per-ARNT-Sim (PAS) fold structures. Mutational analysis reveals that the observed dimeric interface is critical for TraJF transcriptional activation, indicating that dimerization of TraJ is required for its in vivo function. An artificial ligand (oxidized dithiothreitol) occupies a cavity in the TraJF dimer interface, while a smaller cavity in corresponding region of the TraJpSLT structure lacks a ligand. Gas chromatography/mass spectrometry-electron ionization analysis of dithiothreitol-free TraJF suggests indole may be the natural TraJ ligand; however, disruption of the indole biosynthetic pathway does not affect TraJF function. Heterologous PAS domains from pSLT and R100 TraJ can functionally replace the TraJF PAS domain, suggesting that TraJ allelic specificity is mediated by the region C-terminal to the PAS domain.

  4. NEW POSSIBILITIES FOR EVALUATION OF SEVERITY AND PROGNOSIS IN PATIENTS WITH CHRONIC HEART FAILURE BASED ON N-TERMINAL PRO-BRAIN NATRIURETIC PEPTIDE PLASMA LEVEL

    Directory of Open Access Journals (Sweden)

    A. S. Galjavich

    2016-01-01

    Full Text Available Aim. To study an importance of plasma N-terminal pro-brain natriuretic peptide (N-proBNP in evaluation of severity and prognosis in patients with chronic heart failure (CHF of ischemic genesis.Material and methods. 77 patients (60 men and 17 women; 59,4±10,7 y.o. with CHF of ischemic genesis were included in the study. All patients had sinus rhythm and history of Q wave myocardial infarction. Standard examination was performed to all patients. Besides N-proBNP plasma level and patients yearly survival were evaluated.Results. N-proBNP plasma level had direct correlation with clinical indices (exercise tolerance, blood pressure, heart rate and echocardiographic heart sizes. N-proBNP plasma level had relationship with prognosis of CHF patients. Baseline N-proBNP level was more than 2 times higher in died patients in comparison with survived patients. The yearly survival rate of CHF patients was 51,3% if N-proBNP level had been more than 400 fmol/ml (>15% of normal value. The clinico-laboratory index (based on N-proBNP plasma level of severity and prognosis in CHF patients was developed.Conclusion. The clinico-laboratory index based on N-proBNP plasma level is easy to use and can improve medical practice.

  5. NEW POSSIBILITIES FOR EVALUATION OF SEVERITY AND PROGNOSIS IN PATIENTS WITH CHRONIC HEART FAILURE BASED ON N-TERMINAL PRO-BRAIN NATRIURETIC PEPTIDE PLASMA LEVEL

    Directory of Open Access Journals (Sweden)

    A. S. Galjavich

    2009-01-01

    Full Text Available Aim. To study an importance of plasma N-terminal pro-brain natriuretic peptide (N-proBNP in evaluation of severity and prognosis in patients with chronic heart failure (CHF of ischemic genesis.Material and methods. 77 patients (60 men and 17 women; 59,4±10,7 y.o. with CHF of ischemic genesis were included in the study. All patients had sinus rhythm and history of Q wave myocardial infarction. Standard examination was performed to all patients. Besides N-proBNP plasma level and patients yearly survival were evaluated.Results. N-proBNP plasma level had direct correlation with clinical indices (exercise tolerance, blood pressure, heart rate and echocardiographic heart sizes. N-proBNP plasma level had relationship with prognosis of CHF patients. Baseline N-proBNP level was more than 2 times higher in died patients in comparison with survived patients. The yearly survival rate of CHF patients was 51,3% if N-proBNP level had been more than 400 fmol/ml (>15% of normal value. The clinico-laboratory index (based on N-proBNP plasma level of severity and prognosis in CHF patients was developed.Conclusion. The clinico-laboratory index based on N-proBNP plasma level is easy to use and can improve medical practice.

  6. Reference intervals for N-terminal pro-B-type natriuretic peptide in amniotic fluid between 10 and 34 weeks of gestation.

    Directory of Open Access Journals (Sweden)

    Waltraut M Merz

    Full Text Available BACKGROUND: In adult and pediatric cardiology, n-terminal pro-B-type natriuretic peptide (nt-proBNP serves as biomarker in the diagnosis and management of cardiovascular dysfunction. Elevated levels of circulating nt-proBNP are present in fetal conditions associated with myocardial pressure or volume load. Compared to fetal blood sampling, amniocentesis is technically easier and can be performed from early pregnancy onwards. We aimed to investigate amniotic fluid (AF nt-proBNP concentrations in normal pregnancies between 10 and 34 weeks of gestation. METHODS: Nt-proBNP and total protein (TP was measured in AF by chemiluminescence assay (photometry, respectively. To adjust for a potential dilutional effect, the AF-nt-proBNP/AF-TP ratio was analyzed. Reference intervals were constructed by regression modeling across gestational age. RESULTS: 132 samples were analyzed. A negative correlation between AF-nt-proBNP/AF-TP ratio and gestational age was observed. Curves for the mean and the 5% and 95% reference interval between 10 and 34 weeks of gestation were established. CONCLUSION: In normal pregnancy, nt-proBNP is present in AF and decreases during gestation. Our data provide the basis for research on AF-nt-proBNP as biomarker in fetal medicine.

  7. Multifunctional roles for the N-terminal basic motif of Alfalfa mosaic virus coat protein: nucleolar/cytoplasmic shuttling, modulation of RNA-binding activity, and virion formation.

    Science.gov (United States)

    Herranz, Mari Carmen; Pallas, Vicente; Aparicio, Frederic

    2012-08-01

    In addition to virion formation, the coat protein (CP) of Alfalfa mosaic virus (AMV) is involved in the regulation of replication and translation of viral RNAs, and in cell-to-cell and systemic movement of the virus. An intriguing feature of the AMV CP is its nuclear and nucleolar accumulation. Here, we identify an N-terminal lysine-rich nucleolar localization signal (NoLS) in the AMV CP required to both enter the nucleus and accumulate in the nucleolus of infected cells, and a C-terminal leucine-rich domain which might function as a nuclear export signal. Moreover, we demonstrate that AMV CP interacts with importin-α, a component of the classical nuclear import pathway. A mutant AMV RNA 3 unable to target the nucleolus exhibited reduced plus-strand RNA synthesis and cell-to-cell spread. Moreover, virion formation and systemic movement were completely abolished in plants infected with this mutant. In vitro analysis demonstrated that specific lysine residues within the NoLS are also involved in modulating CP-RNA binding and CP dimerization, suggesting that the NoLS represents a multifunctional domain within the AMV CP. The observation that nuclear and nucleolar import signals mask RNA-binding properties of AMV CP, essential for viral replication and translation, supports a model in which viral expression is carefully modulated by a cytoplasmic/nuclear balance of CP accumulation. PMID:22746826

  8. Immunodetection and N-terminal sequencing of DNA replication proteins of bacteriophage BFK20 - lytic phage of Brevibacterium flavum.

    Science.gov (United States)

    Bukovská, G; Halgašová, N; Hromadová, L; Koščová, H; Bukovský, M

    2014-01-01

    Phages are excellent models for studying the mechanism of DNA replication in prokaryotes. Identification of phage proteins involved in phage DNA replication is the first prerequisite for elucidation of the phage replication module. We focused on replication proteins gp41 (a putative helicase from SF2 superfamily), gp43 (a RepA-like protein), and gp44 (a putative DNA polymerase A) of phage BFK20 grown in Brevibacterium flavum. To identify them in the phage-host system, we prepared antibodies to these proteins which were cloned and expressed in Escherichia coli as his-tagged recombinant proteins. After purification to homogeneity the recombinant proteins served for raising specific polyclonal antibodies in mice. Using these antibodies in Western blot analysis the phage proteins gp41, gp43 and gp44 were detected during the phage growth cycle. The proteins gp41 and gp43, prepared from cell lysate by ammonium sulphate precipitation, were N-terminally sequenced and found to contain the sequences N-SVKPRELR-C and N-MLGSTML-C, respectively. This means that gp41 starts with serine but not with common methionine. We consider these findings an initial but important step towards more thorough characterization of replication proteins of phage BFK20. PMID:24957720

  9. Isolation of key amino acid residues at the N-terminal end of the core region Streptococcus downei glucansucrase, GTF-I.

    Science.gov (United States)

    Monchois, V; Vignon, M; Russell, R R

    1999-11-01

    Related streptococcal and Leuconostoc mesenteroides glucansucrases are enzymes of medical and biotechnological interest. Molecular modelling has suggested that the catalytic domain contains a circularly permuted version of the (beta/alpha)8 barrel structure found in the amylase superfamily, and site-directed mutagenesis has identified critical amino acids in this region. In this study, sequential N-terminal truncations of Streptococcus downei GTF-I showed that key amino acids are also present in the first one-third of the core domain. Mutations were introduced at Trp-344, Glu-349 and His-355, residues that are conserved in all glucansucrases and lie within a region which is a target for inhibitory antibodies. W344L, E349L and H355V substitutions were assayed for their effect on mutan synthesis and also on oligosaccharide synthesis with various acceptors. It appeared that Trp-344 and His-355 are involved in the action mechanism of GTF-I; His-355 may also play a role in a binding subsite necessary for oligosaccharide and glucan elongation. PMID:10570812

  10. The role of p38 MAP kinase and c-Jun N-terminal protein kinase signaling in the differentiation and apoptosis of immortalized neural stem cells

    International Nuclear Information System (INIS)

    The two distinct members of the mitogen-activated protein (MAP) kinase family c-Jun N-terminal protein kinase (JNK) and p38 MAP kinase, play an important role in central nervous system (CNS) development and differentiation. However, their role and functions are not completely understood in CNS. To facilitate in vitro study, we have established an immortal stem cell line using SV40 from fetal rat embryonic day 17. In these cells, MAP kinase inhibitors (SP600125, SB202190, and PD98059) were treated for 1, 24, 48, and 72 h to examine the roles of protein kinases. Early inhibition of JNK did not alter phenotypic or morphological changes of immortalized cells, however overexpression of Bax and decrease of phosphorylated AKT was observed. The prolonged inhibition of JNK induced polyploidization of immortalized cells, and resulted in differentiation and inhibition of cell proliferation. Moreover, JNK and p38 MAP kinase but not ERK1/2 was activated, and p21, p53, and Bax were overexpressed by prolonged inhibition of JNK. These results indicate that JNK and p38 MAP kinase could play dual roles on cell survival and apoptosis. Furthermore, this established cell line could facilitate study of the role of JNK and p38 MAP kinase on CNS development or differentiation/apoptosis

  11. Transient stability of the helical pattern of region F19-L22 of the N-terminal domain of p53: a molecular dynamics simulation study.

    Science.gov (United States)

    Espinoza-Fonseca, L Michel; Trujillo-Ferrara, José G

    2006-04-28

    Two molecular dynamics simulations of the region E17-N29 of p53 (p53(17-29)) at different temperatures were performed for a total time of 0.2 micros, to study the conformational landscape of this region. Previous studies have suggested that this region displays different structural motifs, such as helix of a double beta-turn, and that its secondary structure might be transiently stable. Interestingly, in this study it was found that the region F19-L25, and particularly its fragment F19-L22, display a stable, transient helical pattern at sub-microsecond periods. The region F19-L22, which contains one of the most important residues needed for the interaction of p53 with MDM2, seems to be formed and stabilized by the existence of one hydrophobic and one aromatic cluster. The main function of these clusters is to help their surrounding area to desolvate, to allow the hydrogen bond network, therefore favoring the formation of a stable helix. This preliminary study would be useful for a better understanding of the structure and function of the N-terminal domain of p53 and its implications for the control of different types of cancer. PMID:16530164

  12. Inhibition of Apoptosis in Prostate Cancer Cells by Androgens Is Mediated through Downregulation of c-Jun N-terminal Kinase Activation

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    Petra Isabel Lorenzo

    2008-05-01

    Full Text Available Androgen deprivation induces the regression of prostate tumors mainly due to an increase in the apoptosis rate; however, the molecular mechanisms underlying the antiapoptotic actions of androgens are not completely understood. We have studied the antiapoptotic effects of androgens in prostate cancer cells exposed to different proapoptotic stimuli. Terminal deoxynucleotidyl transferase-mediated nick-end labeling and nuclear fragmentation analyses demonstrated that androgens protect LNCaP prostate cancer cells from apoptosis induced by thapsigargin, the phorbol ester 12-O-tetradecanoyl-13-phorbol-acetate, or UV irradiation. These three stimuli require the activation of the c-Jun N-terminal kinase (JNK pathway to induce apoptosis and in all three cases, androgen treatment blocks JNK activation. Interestingly, okadaic acid, a phosphatase inhibitor that causes apoptosis in LNCaP cells, induces JNK activation that is also inhibited by androgens. Actinomycin D, the antiandrogen bicalutamide or specific androgen receptor (AR knockdown by small interfering RNA all blocked the inhibition of JNK activation mediated by androgens indicating that this activity requires AR-dependent transcriptional activation. These data suggest that the crosstalk between AR and JNK pathways may have important implications in prostate cancer progression and may provide targets for the development of new therapies.

  13. Fast closure of N-terminal long loops but slow formation of β strands precedes the folding transition state of Escherichia coli adenylate kinase.

    Science.gov (United States)

    Orevi, Tomer; Ben Ishay, Eldad; Gershanov, Sivan Levin; Dalak, Mayan Ben; Amir, Dan; Haas, Elisha

    2014-05-20

    The nature of the earliest steps of the initiation of the folding pathway of globular proteins is still controversial. To elucidate the role of early closure of long loop structures in the folding transition, we studied the folding kinetics of subdomain structures in Escherichia coli adenylate kinase (AK) using Förster type resonance excitation energy transfer (FRET)-based methods. The overall folding rate of the AK molecule and of several segments that form native β strands is 0.5 ± 0.3 s(-1), in sharp contrast to the 1000-fold faster closure of three long loop structures in the CORE domain. A FRET-based "double kinetics" analysis revealed complex transient changes in the initially closed N-terminal loop structure that then opens and closes again at the end of the folding pathway. The study of subdomain folding in situ suggests a hierarchic ordered folding mechanism, in which early and rapid cross-linking by hydrophobic loop closure provides structural stabilization at the initiation of the folding pathway. PMID:24787383

  14. Hexokinase I N-terminal based peptide prevents the VDAC1-SOD1 G93A interaction and re-establishes ALS cell viability

    Science.gov (United States)

    Magrì, Andrea; Belfiore, Ramona; Reina, Simona; Tomasello, Marianna Flora; Di Rosa, Maria Carmela; Guarino, Francesca; Leggio, Loredana; De Pinto, Vito; Messina, Angela

    2016-01-01

    Superoxide Dismutase 1 mutants associate with 20–25% of familial Amyotrophic Lateral Sclerosis (ALS) cases, producing toxic aggregates on mitochondria, notably in spinal cord. The Voltage Dependent Anion Channel isoform 1 (VDAC1) in the outer mitochondrial membrane is a docking site for SOD1 G93A mutant in ALS mice and the physiological receptor of Hexokinase I (HK1), which is poorly expressed in mouse spinal cord. Our results demonstrate that HK1 competes with SOD1 G93A for binding VDAC1, suggesting that in ALS spinal cord the available HK1-binding sites could be used by SOD1 mutants for docking mitochondria, producing thus organelle dysfunction. We tested this model by studying the action of a HK1-N-terminal based peptide (NHK1). This NHK1 peptide specifically interacts with VDAC1, inhibits the SOD1 G93A binding to mitochondria and restores the viability of ALS model NSC34 cells. Altogether, our results suggest that NHK1 peptide could be developed as a therapeutic tool in ALS, predicting an effective role also in other proteinopathies. PMID:27721436

  15. Identification of a human ABCC10 orthologue in Catharanthus roseus reveals a U12-type intron determinant for the N-terminal domain feature

    Indian Academy of Sciences (India)

    Taissir El-Guizani; Clotilde Guibert; Saïda Triki; Benoit St-Pierre; Eric Ducos

    2014-04-01

    ABC (ATP-binding cassette) transporters are members of a large superfamily of proteins that utilize ATP hydrolysis to translocate a wide range of substrates across biological membranes. In general, members of C subfamily (ABCC) are structurally characterized by an additional (N-terminal) transmembrane domain (TMD0). Phylogenetic analysis of plant ABCCs separates their protein sequences into three distinct clusters: I and II are plant specific whereas cluster III contains both human and plant ABCCs. Screening of the Plant Medicinal Genomics Resource database allowed us to identify 16 ABCCs partial sequences in Catharanthus roseus; two of which belong to the unique CrABCC1 transcript that we identified in cluster III. Genomic organization of CrABCC1 TMD0 coding sequence displays an AT-AC U12-type intron that is conserved in higher plant orthologues. We showed that CrABCC1, like its human orthologue ABCC10, produces alternative transcripts that encode protein sequences with a truncated form of TMD0 without the first transmembrane span (TM1). Subcellular localization of CrABCC1 TMD0 variants using yellow fluorescent protein fusions reveals that the TM1 is required for a correct routing of the TMD0 to the tonoplast. Finally, the specific repartition of CrABCC1 orthologues in some species suggests that this gene was lost several times during evolution and that its physiological function may, rely on a common feature of multicellular eukaryotes.

  16. Differential localization of processed fragments of Plasmodium falciparum serine repeat antigen and further processing of its N-terminal 47 kDa fragment.

    Science.gov (United States)

    Li, Jie; Mitamura, Toshihide; Fox, Barbara A; Bzik, David J; Horii, Toshihiro

    2002-12-01

    The serine repeat antigen (SERA) of Plasmodium falciparum is a blood stage malaria vaccine candidate. It has been shown that 120 kDa SERA was proteolytically processed into N-terminal 47 kDa fragment (P47), central 56 kDa fragment (P56) that was further converted to 50 kDa (P50), and C-terminal 18 kDa fragment (P18). Here, we have examined the processing of SERA and the localization of its processed fragments by using mouse antibodies directed against recombinant proteins corresponding to different domains of SERA. Western blot analysis showed that all the processing events occurred inside parasitized erythrocytes at the stage just prior to the schizont rupture, that P47 was further processed into two 25 kDa fragments and that the two fragments, which were linked to P18 through disulfide bonds, were associated with the merozoite. In contrast, P50 was completely shed into culture medium and absent from the merozoite. This observation was further supported by the results of indirect immunofluorescence assay. These results could account for the findings that antibodies against P47 were inhibitory to the parasite growth in vitro but those against P50 were not. Finally, we demonstrated that the further processing of P47 is allelic type-dependent. The results of the present study would help in vaccine designing based on SERA. PMID:12421632

  17. Identifying the activation motif in the N-terminal of rainbow trout and zebrafish melanocortin-2 receptor accessory protein 1 (MRAP1) orthologs.

    Science.gov (United States)

    Dores, Robert M; Liang, Liang; Hollmann, Rebecca E; Sandhu, Navdeep; Vijayan, Mathilakath M

    2016-08-01

    The activation of mammalian melanocortin-2 receptor (MC2R) orthologs is dependent on a four-amino acid activation motif (LDYL/I) located in the N-terminal of mammalian MRAP1 (melanocortin-2 receptor accessory protein). Previous alanine substitution analysis had shown that the Y residue in this motif appears to be the most important for mediating the activation of mammalian MC2R orthologs. Similar, but not identical amino acid motifs were detected in rainbow trout MRAP1 (YDYL) and zebrafish MRAP1 (YDYV). To determine the importance of these residues in the putative activation motifs, rainbow trout and zebrafish MRAP1 orthologs were individually co-expressed in CHO cells with rainbow trout MC2R, and the activation of this receptor with either the wild-type MRAP1 ortholog or alanine-substituted analogs of the two teleost MRAP1s was analyzed. Alanine substitutions at all four amino acid positions in rainbow trout MRAP1 blocked activation of the rainbow trout MC2R. Single alanine substitutions of the D and Y residues in rainbow trout and zebrafish MRAP1 indicate that these two residues play a significant role in the activation of rainbow trout MC2R. These observations indicate that there are subtle differences in the way that teleost and mammalian MRAPs are involved in the activation of their corresponding MC2R orthologs. PMID:26752246

  18. Molecular insight into the role of the N-terminal extension in the maturation, substrate recognition, and catalysis of a bacterial alginate lyase from polysaccharide lyase family 18.

    Science.gov (United States)

    Dong, Sheng; Wei, Tian-Di; Chen, Xiu-Lan; Li, Chun-Yang; Wang, Peng; Xie, Bin-Bin; Qin, Qi-Long; Zhang, Xi-Ying; Pang, Xiu-Hua; Zhou, Bai-Cheng; Zhang, Yu-Zhong

    2014-10-24

    Bacterial alginate lyases, which are members of several polysaccharide lyase (PL) families, have important biological roles and biotechnological applications. The mechanisms for maturation, substrate recognition, and catalysis of PL18 alginate lyases are still largely unknown. A PL18 alginate lyase, aly-SJ02, from Pseudoalteromonas sp. 0524 displays a β-jelly roll scaffold. Structural and biochemical analyses indicated that the N-terminal extension in the aly-SJ02 precursor may act as an intramolecular chaperone to mediate the correct folding of the catalytic domain. Molecular dynamics simulations and mutational assays suggested that the lid loops over the aly-SJ02 active center serve as a gate for substrate entry. Molecular docking and site-directed mutations revealed that certain conserved residues at the active center, especially those at subsites +1 and +2, are crucial for substrate recognition. Tyr(353) may function as both a catalytic base and acid. Based on our results, a model for the catalysis of aly-SJ02 in alginate depolymerization is proposed. Moreover, although bacterial alginate lyases from families PL5, 7, 15, and 18 adopt distinct scaffolds, they share the same conformation of catalytic residues, reflecting their convergent evolution. Our results provide the foremost insight into the mechanisms of maturation, substrate recognition, and catalysis of a PL18 alginate lyase.

  19. A specific isoform of poly(ADP-ribose) glycohydrolase is targeted to the mitochondrial matrix by a N-terminal mitochondrial targeting sequence

    Energy Technology Data Exchange (ETDEWEB)

    Whatcott, Clifford J. [Department of Pharmacology and Toxicology, College of Pharmacy, Arizona Cancer Center, University of Arizona, Tucson, AZ 85728 (United States); Meyer-Ficca, Mirella L.; Meyer, Ralph G. [Department of Animal Biology and Mari Lowe Center for Comparative Oncology, School of Veterinary Medicine, NBC Center for Animal Transgenesis and Germ Cell Research, University of Pennsylvania, Kennett Square, PA 19348 (United States); Jacobson, Myron K., E-mail: mjacobson@pharmacy.arizona.edu [Department of Pharmacology and Toxicology, College of Pharmacy, Arizona Cancer Center, University of Arizona, Tucson, AZ 85728 (United States)

    2009-12-10

    Poly(ADP-ribose) polymerases (PARPs) convert NAD to polymers of ADP-ribose that are converted to free ADP-ribose by poly(ADP-ribose) glycohydrolase (PARG). The activation of the nuclear enzyme PARP-1 following genotoxic stress has been linked to release of apoptosis inducing factor from the mitochondria, but the mechanisms by which signals are transmitted between nuclear and mitochondrial compartments are not well understood. The study reported here has examined the relationship between PARG and mitochondria in HeLa cells. Endogenous PARG associated with the mitochondrial fraction migrated in the range of 60 kDa. Transient transfection of cells with PARG expression constructs with amino acids encoded by exon 4 at the N-terminus was targeted to the mitochondria as demonstrated by subcellular fractionation and immunofluorescence microscopy of whole cells. Deletion and missense mutants allowed identification of a canonical N-terminal mitochondrial targeting sequence consisting of the first 16 amino acids encoded by PARG exon 4. Sub-mitochondrial localization experiments indicate that this mitochondrial PARG isoform is targeted to the mitochondrial matrix. The identification of a PARG isoform as a component of the mitochondrial matrix raises several interesting possibilities concerning mechanisms of nuclear-mitochondrial cross talk involved in regulation of cell death pathways.

  20. 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

    Full Text Available BACKGROUND: 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. PRINCIPAL FINDINGS: 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. CONCLUSIONS/SIGNIFICANCE: 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.