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Sample records for membrane-proximal amino-terminal residues

  1. Major histocompatibility complex class I molecules bind natural peptide ligands lacking the amino-terminal binding residue in vivo.

    PubMed

    Yague, J; Marina, A; Vazquez, J; Lopez De Castro, J A

    2001-11-23

    Major histocompatibility complex (MHC) class I-peptide complexes are stabilized by multiple interactions, including those of the peptidic NH(2)-terminal group in the A pocket of the MHC molecule. In this study, the characterization of four natural HLA-B39 ligands lacking the amino-terminal binding residue is reported. These peptides were found in the endogenous peptide pool of one or more of the B*3901, B*3905, and B*3909 allotypes and sequenced by nanoelectrospray mass spectrometry. Control experiments ruled out that they resulted from exopeptidase trimming of their NH(2)-terminally extended counterparts: NAc-SHVAVENAL, EHGPNPIL, IHEPEPHIL, and EHAGVISVL, also present in the same peptide pools, during purification. HAGVISVL and HVAVENAL behaved similarly to the corresponding NH(2)-terminally extended peptides in their binding to B*3901 and B*3909 at the cell surface in vitro, and in cell surface stabilization of B*3901. This is, to our knowledge, the first demonstration that peptides lacking the amino-terminal binding residue bind in vivo to classical MHC class I molecules. The results indicate that canonical MHC-peptide interactions in the A pocket are not always necessary for endogenous peptide presentation.

  2. Amino-terminal cysteine residues differentially influence RGS4 protein plasma membrane targeting, intracellular trafficking, and function.

    PubMed

    Bastin, Guillaume; Singh, Kevin; Dissanayake, Kaveesh; Mighiu, Alexandra S; Nurmohamed, Aliya; Heximer, Scott P

    2012-08-17

    Regulator of G-protein signaling (RGS) proteins are potent inhibitors of heterotrimeric G-protein signaling. RGS4 attenuates G-protein activity in several tissues. Previous work demonstrated that cysteine palmitoylation on residues in the amino-terminal (Cys-2 and Cys-12) and core domains (Cys-95) of RGS4 is important for protein stability, plasma membrane targeting, and GTPase activating function. To date Cys-2 has been the priority target for RGS4 regulation by palmitoylation based on its putative role in stabilizing the RGS4 protein. Here, we investigate differences in the contribution of Cys-2 and Cys-12 to the intracellular localization and function of RGS4. Inhibition of RGS4 palmitoylation with 2-bromopalmitate dramatically reduced its localization to the plasma membrane. Similarly, mutation of the RGS4 amphipathic helix (L23D) prevented membrane localization and its G(q) inhibitory function. Together, these data suggest that both RGS4 palmitoylation and the amphipathic helix domain are required for optimal plasma membrane targeting and function of RGS4. Mutation of Cys-12 decreased RGS4 membrane targeting to a similar extent as 2-bromopalmitate, resulting in complete loss of its G(q) inhibitory function. Mutation of Cys-2 did not impair plasma membrane targeting but did partially impair its function as a G(q) inhibitor. Comparison of the endosomal distribution pattern of wild type and mutant RGS4 proteins with TGN38 indicated that palmitoylation of these two cysteines contributes differentially to the intracellular trafficking of RGS4. These data show for the first time that Cys-2 and Cys-12 play markedly different roles in the regulation of RGS4 membrane localization, intracellular trafficking, and G(q) inhibitory function via mechanisms that are unrelated to RGS4 protein stabilization.

  3. Spatial approximations between residues 6 and 12 in the amino-terminal region of glucagon-like peptide 1 and its receptor: a region critical for biological activity.

    PubMed

    Chen, Quan; Pinon, Delia I; Miller, Laurence J; Dong, Maoqing

    2010-08-06

    Understanding the molecular basis of natural ligand binding and activation of the glucagon-like peptide 1 (GLP1) receptor may facilitate the development of agonist drugs useful for the management of type 2 diabetes mellitus. We previously reported molecular approximations between carboxyl-terminal residues 24 and 35 within GLP1 and its receptor. In this work, we have focused on the amino-terminal region of GLP1, known to be critical for receptor activation. We developed two high-affinity, full agonist photolabile GLP1 probes having sites of covalent attachment in positions 6 and 12 of the 30-residue peptide (GLP1(7-36)). Both probes bound to the receptor specifically and covalently labeled single distinct sites. Chemical and protease cleavage of the labeled receptor identified the juxtamembrane region of its amino-terminal domain as the region of covalent attachment of the position 12 probe, whereas the region of labeling by the position 6 probe was localized to the first extracellular loop. Radiochemical sequencing identified receptor residue Tyr(145), adjacent to the first transmembrane segment, as the site of labeling by the position 12 probe, and receptor residue Tyr(205), within the first extracellular loop, as the site of labeling by the position 6 probe. These data provide support for a common mechanism for natural ligand binding and activation of family B G protein-coupled receptors. This region of interaction of peptide amino-terminal domains with the receptor may provide a pocket that can be targeted by small molecule agonists.

  4. N-terminal residues of an HIV-1 gp41 membrane-proximal external region antigen influence broadly neutralizing 2F5-like antibodies.

    PubMed

    Li, Dezhi; Liu, Jie; Zhang, Li; Xu, Tianshu; Chen, Junheng; Wang, Liping; Zhao, Qi

    2015-12-01

    The Human immunodeficiency virus type 1 (HIV-1) gp41 membrane proximal external region (MPER) is targeted by broadly neutralizing antibodies (e.g. 2F5, 4E10, Z13e and m66.6), which makes this region a promising target for vaccine design. One strategy to elicit neutralizing antibodies against the MPER epitope is to design peptide immunogens mimicking neutralization structures. To probe 2F5-like neutralizing antibodies, two yeast-displayed antibody libraries from peripheral blood mononuclear cells from a HIV-1 patient were screened against the 2F5 epitope peptide SP62. Two 2F5-like antibodies were identified that specifically recognized SP62. However, these antibodies only weakly neutralized HIV-1 primary isolates. The epitopes recognized by these two 2F5-like antibodies include not only the 2F5 epitope (amino acids (aa) 662-667 in the MPER) but also several other residues (aa 652-655) locating at the N-terminus in SP62. Experimental results suggest that residues of SP62 adjacent to the 2F5 epitope influence the response of broadly neutralizing 2F5-like antibodies in vaccination. Our findings may aid the design of vaccine immunogens and development of therapeutics against HIV-1 infection.

  5. A mutational analysis of the cytosolic domain of the tomato Cf-9 disease-resistance protein shows that membrane-proximal residues are important for Avr9-dependent necrosis.

    PubMed

    Chakrabarti, Apratim; Velusamy, Thilaga; Tee, Choon Yang; Jones, David A

    2016-05-01

    The tomato Cf-9 gene encodes a membrane-anchored glycoprotein that imparts race-specific resistance against the tomato leaf mould fungus Cladosporium fulvum in response to the avirulence protein Avr9. Although the N-terminal half of the extracellular leucine-rich repeat (eLRR) domain of the Cf-9 protein determines its specificity for Avr9, the C-terminal half, including its small cytosolic domain, is postulated to be involved in signalling. The cytosolic domain of Cf-9 carries several residues that are potential sites for ubiquitinylation or phosphorylation, or signals for endocytic uptake. A targeted mutagenesis approach was employed to investigate the roles of these residues and cellular processes in Avr9-dependent necrosis triggered by Cf-9. Our results indicate that the membrane-proximal region of the cytosolic domain of Cf-9 plays an important role in Cf-9-mediated necrosis, and two amino acids within this region, a threonine (T835) and a proline (P838), are particularly important for Cf-9 function. An alanine mutation of T835 had no effect on Cf-9 function, but an aspartic acid mutation, which mimics phosphorylation, reduced Cf-9 function. We therefore postulate that phosphorylation/de-phosphorylation of T835 could act as a molecular switch to determine whether Cf-9 is in a primed or inactive state. Yeast two-hybrid analysis was used to show that the cytosolic domain of Cf-9 interacts with the cytosolic domain of tomato VAP27. This interaction could be disrupted by an alanine mutation of P838, whereas interaction with CITRX remained unaffected. We therefore postulate that a proline-induced kink in the membrane-proximal region of the cytosolic domain of Cf-9 may be important for interaction with VAP27, which may, in turn, be important for Cf-9 function.

  6. Deletion of the carboxyl-terminal residue disrupts the amino-terminal folding, self-association, and thermal stability of an amphipathic antimicrobial peptide.

    PubMed

    Lee, Chang-Shin; Tung, Wei-Cheng; Lin, Yu-Hsin

    2014-06-01

    Understanding the complex relationship between amino acid sequence and protein behaviors, such as folding and self-association, is a major goal of protein research. In the present work, we examined the effects of deleting a C-terminal residue on the intrinsic properties of an amphapathic α-helix of mastoparan-B (MP-B), an antimicrobial peptide with the sequence LKLKSIVSWAKKVL-NH2. We used circular dichroism and nuclear magnetic resonance to demonstrate that the peptide MP-B([1-13]) displayed significant unwinding at the N-terminal helix compared with the parent peptide of MP-B, as the temperature increased when the residue at position 14 was deleted. Pulsed-field gradient nuclear magnetic resonance data revealed that MP-B forms a larger diffusion unit than MP-B([1-13]) at all experimental temperatures and continuously dissociates as the temperature increases. In contrast, the size of the diffusion unit of MP-B([1-13]) is almost independent of temperature. These findings suggest that deleting the flexible, hydrophobic amino acid from the C-terminus of MP-B is sufficient to change the intrinsic helical thermal stability and self-association. This effect is most likely because of the modulation of enthalpic interactions and conformational freedom that are specified by this residue. Our results implicate terminal residues in the biological function of an antimicrobial peptide. Copyright © 2014 European Peptide Society and John Wiley & Sons, Ltd.

  7. The role of glycine (residue 89) in the central helix of EF-hand protein troponin-C exposed following amino-terminal alpha-helix deletion.

    PubMed Central

    Ding, X. L.; Akella, A. B.; Su, H.; Gulati, J.

    1994-01-01

    Because an N-terminal alpha-helical (N-helix) arm and a KGK-triplet (residues 88KGK90) in the central helix of troponin-C (TnC) are missing in calmodulin, several recent studies have attempted to elucidate the structure-function correlations of these units. Presently, with a family of genetically manipulated derivatives especially developed for this study and tested on permeabilized isolated single skeletal muscle fiber segments, we explored the specificities of the amino acid residues within the N-helix and the KGK-triplet in TnC. Noticeably, the amino acid compositions vary between the N-helices of the cardiac and skeletal TnC isoforms. On the other hand, the KGK-triplet is located similarly in both TnC isoforms. We previously indicated that deletion of the N-helix (mutant delta Nt) diminishes the tension obtained on activation with maximal calcium, but the contractile function is revived by the superimposed deletion of the 88KGK90-triplet (mutant delta Nt delta KGK; see Gulati J, Babu A, Su H, Zhang YF, 1993, J Biol Chem 268:11685-11690). Using this functional test, we find that replacement of Gly-89 with a Leu or an Ala could also overcome the contractile defect associated with N-helix deletion. On the other hand, replacement of the skeletal TnC N-helix with cardiac type N-helix was unable to restore contractile function. The findings indicate a destabilizing influence of Gly-89 residue in skeletal TnC and suggest that the N-terminal arm in normal TnC serves to moderate this effect. Moreover, specificity of the N-helix between cardiac and skeletal TnCs raises the possibility that resultant structural disparities are also important for the functional distinctions of the TnC isoforms. PMID:7703855

  8. The amino-terminal residue of Sindbis virus glycoprotein E2 influences virus maturation, specific infectivity for BHK cells, and virulence in mice.

    PubMed Central

    Heidner, H W; Johnston, R E

    1994-01-01

    The E2 glycoprotein of Sindbis virus is synthesized as a precursor, PE2, which is cleaved by furin or a furin-like host cell protease at a late stage of maturation. The four-residue PE2 cleavage signal conforms to the basic amino acid-X-basic-basic motif which is present in many other viral and cellular glycoproteins which are processed by the cellular enzyme(s). In this report, we present evidence that the amino acid which immediately follows the signal, the N-terminal residue of E2, can influence protease recognition, binding, and/or cleavage of PE2. Constructs encoding nine different amino acids at E2 position 1 (E2 1) were produced by site-directed mutagenesis of the full-length cDNA clone of our laboratory strain of Sindbis virus AR339 (pTRSB). Viruses derived from clones encoding Arg (TRSB), Asp, Ser, Phe, His, and Asn in a nonglycosylated form at E2 1 contained predominantly E2. Viruses encoding Ile, Leu, or Val at E2 1 contained the uncleaved form of PE2. The specific infectivity of TRSB (E2 Arg-1) for baby hamster kidney (BHK-21) cells was from 5- to greater than 100-fold higher than those of isogenic constructs with other residues at E2 1, suggesting that E2 Arg-1 represents a BHK-21 cell adaptive mutation in our laboratory strain. In newborn CD-1 mice, TRSB was more virulent than the PE2-containing viruses but less virulent than other PE2-cleaving viruses with alternative amino acids at E2 1. These results indicate that in TRSB, E2 Arg-1 increased the efficiency of virus-cell interactions in cultured BHK-21 cells but simultaneously decreased the ability of virus to mediate in vivo virus-cell interactions critical for the induction of disease. This suggests that the N terminus of E2 may participate in or be associated with virion domains which mediate these viral functions. Images PMID:7966596

  9. TonB/TolA amino-terminal domain modeling.

    PubMed

    Keller, Kimberly L; Brinkman, Kerry K; Larsen, Ray A

    2007-01-01

    TonB and TolA proteins are energy transducers that couple the ion electrochemical gradient of the cytoplasmic membrane to support energy-dependent processes in the outer membrane of gram-negative bacteria. Energization of these proteins involves specific interactions with multiprotein cytoplasmic membrane energy harvesting complexes. The specific mechanisms by which these energy transfers occur remain unclear, but the evidence to date indicates that the amino-terminally located signal anchors of TonB and TolA play essential roles in the process. Mutant hunts have identified one motif in this region, common to both TonB and TolA, as important for energization. Because TonB and TolA each have a "preferred" energy-harvesting complex, it is clear that additional motifs, not shared between TonB and TolA, are involved in interactions with energy harvesting complexes. We have adopted a strategy of examining derivatives with multiple-residue substitutions to identify such regions. This involves the characterization of specific TonB derivatives generated by two similar approaches: the block substitutions in TonB by alanyl residues and the exchange of short regions between TonB and TolA. The methods by which these derivatives are generated are described, with an illustrative example for each.

  10. Analysis of amino-terminal variants of amyloid-β peptides by capillary isoelectric focusing immunoassay.

    PubMed

    Haußmann, Ute; Jahn, Olaf; Linning, Philipp; Janßen, Christin; Liepold, Thomas; Portelius, Erik; Zetterberg, Henrik; Bauer, Chris; Schuchhardt, Johannes; Knölker, Hans-Joachim; Klafki, Hans; Wiltfang, Jens

    2013-09-03

    Here we present a novel assay for the separation and detection of amino-terminal amyloid-β (Aβ) peptide variants by capillary isoelectric focusing (CIEF) immunoassay. Specific amino-terminally truncated Aβ peptides appear to be generated by β-secretase (BACE1)-independent mechanisms and have previously been observed in cerebrospinal fluid (CSF) after BACE1 inhibitor treatment in an animal model. CIEF immunoassay sensitivity is sufficient to detect total Aβ in CSF without preconcentration. To analyze low-abundance amino-terminally truncated Aβ peptides from cell culture supernatants, we developed a CIEF-compatible immunoprecipitation protocol, allowing for selective elution of Aβ peptides with very low background. CIEF immunoassay and immunoprecipitation mass spectrometry analysis identified peptides starting at residue Arg(5) as the main amino-terminal Aβ variants produced in the presence of tripartite BACE1 inhibitor in our cell culture model. The CIEF immunoassay allows for robust relative quantification of Aβ peptide patterns in biological samples. To assess the future possibility of absolute quantification, we have prepared the Aβ peptides Aβ(x-10), Aβ(x-16), and Aβ(5-38(D23S)) by using solid phase peptide synthesis as internal standards for the CIEF immunoassay.

  11. Porphyromonas (Bacteroides) gingivalis fimbrillin: size, amino-terminal sequence, and antigenic heterogeneity.

    PubMed Central

    Lee, J Y; Sojar, H T; Bedi, G S; Genco, R J

    1991-01-01

    Bacterial fimbriae mediate cell adhesion and are important in colonization. Fimbrial proteins from strains of Porphyromonas (Bacteroides) gingivalis isolated from different individuals were compared for their size, amino-terminal sequence, and antigenic diversity. Two major protein components of the crude fimbrial preparations differed in apparent molecular mass, ranging from 41 to 49 kDa for the fimbrillin monomer and from 61 to 78 kDa for the other major protein. The amino-terminal sequence of the antigenically related group of proteins of the fimbrillin monomer in the 41- to 49-kDa range showed significant homology; however, minor sequence heterogeneity was observed, mainly in residues 4 to 6. One of the observed amino-terminal sequences, AFGVGDDESKVAKLTVMVYNG, resembled the deduced sequence of P. gingivalis 381 (D.P. Dickinson, M. K. Kubiniec, F. Yoshimura, and R.J. Genco, J. Bacteriol. 170:1658-1665, 1988). Fimbriae from all the strains of P. gingivalis showing this sequence contained a fimbrillin monomer of 43 kDa and showed a strong reaction with both polyclonal and monoclonal antibodies directed to the fimbriae from P. gingivalis 2561 (381). Fimbriae from strains showing amino-terminal sequence variations in residues 4 to 6 (i.e., substitution of VGD with either E or NAG) were more diverse in their molecular sizes. Most of these variant fimbriae showed weak reactions with the polyclonal antibodies and no reaction with the monoclonal antibodies induced to the fimbriae of strain 2561. No correlation could be established between the molecular size and immunological reactivity of the fimbrillin monomer of P. gingivalis strains. Strains 9-14K-1 and HG 564 not only showed markedly different sequences from the other three amino-terminal sequences but also did not react with either polyclonal or monoclonal antibodies to the fimbriae of strain 2561. Strains W50, W83, and AJW 5 failed to show any immunological reactivity with the antibodies to fimbrillin or fimbriae

  12. Identification of amino-terminal region of adiponectin as a physiologically functional domain.

    PubMed

    Ujiie, Hidetoshi; Oritani, Kenji; Kato, Hisashi; Yokota, Takafumi; Takahashi, Isao; Maeda, Tetsuo; Masaie, Hiroaki; Ichii, Michiko; Kamada, Yoshihiro; Tamura, Shinji; Kihara, Shinji; Funahashi, Tohru; Tomiyama, Yoshiaki; Kanakura, Yuzuru

    2006-05-01

    Adiponectin is an abundant adipose-specific protein, which acts as an anti-diabetic, anti-atherogenic, and anti-inflammatory adipokine. Although recent advances in the field of adiponectin have been made by the identification of adiponectin receptors and by the understanding about relationship between its multimerization and functions, detailed molecular background remains unclear. Our established anti-human adiponectin antibodies, ANOC 9103 and ANOC 9104, blocked some adiponectin functions such as the growth inhibition of B-lymphocytes on stromal cells and the inhibition of acetylated LDL uptake in macrophages, suggesting that they may recognize important functional regions of adiponectin. As a result of epitope mapping based on the ability to bind to the deleted adiponectin mutants, we identified that these antibodies recognize amino-terminal region of adiponectin before the beginning of the collagen-like domain. Notably, a peptide fragment (DQETTTQGPGVLLPLPKGACTGWMA) corresponding to amino acid residues 17-41 of human adiponectin could bind to restricted types of cells and block adiponectin-induced cyclooxygenase-2 gene expression and prostaglandin E2 production in MS-5 stromal cells. Moreover, the deletion of its amino-terminal region reduced the abilities to inhibit not only collagen-induced platelet aggregation but also diet-induced hepatic steatosis. These data indicate that amino-terminal region of adiponectin is a physiologically functional domain and that a novel receptor, which recognizes amino-terminal region of adiponectin, may exist on some types of cells. Further investigations will contribute to the understanding of molecular mechanisms about adiponectin functions as well as to the designing of novel strategies for the treatment of patients with insulin-resistance, vascular dysfunction, and chronic inflammation.

  13. Amino-terminated diamond surfaces: Photoelectron emission and photocatalytic properties

    NASA Astrophysics Data System (ADS)

    Zhu, Di; Bandy, Jason A.; Li, Shuo; Hamers, Robert J.

    2016-08-01

    We report a new approach to making stable negative electron-affinity diamond surfaces by terminating diamond with amino groups (also known as amine groups, -NH2). Previous studies have shown that negative electron affinity can be induced by terminating diamond surfaces with hydrogen, creating a surface dipole favorable toward electron emission. Here, we demonstrate that covalent tethering of positive charges in the form of protonated amino groups, -NH3+, also leads to negative electron affinity (NEA) and facile electron emission into vacuum and into water. Amino-terminated diamond was prepared using a very mild plasma discharge. Valence-band photoemission studies of the amino-terminated diamond samples show a characteristic ;NEA; peak, demonstrating that the amino-terminated surface has NEA. Diamond's ability to emit electrons into water was evaluated using photochemical conversion of N2 to NH3. Time-resolved surface photovoltage studies were used to characterize charge separation at the diamond interface, and Mott-Schottky measurements were performed to characterize band-bending at the diamond-water interface. XPS studies show that the amino-terminated surfaces provide increased chemical resistance to oxidation compared with H-terminated diamond when illuminated with ultraviolet light.

  14. Isolation of a cDNA clone encoding the amino-terminal region of human apolipoprotein B

    SciTech Connect

    Protter, A.A.; Hardman, D.A.; Schilling, J.W.; Miller, J.; Appleby, V.; Chen, G.C.; Kirsher, S.W.; McEnroe, G.; Kane, J.P.

    1986-03-01

    A partial cDNA clone for the B-26 region of apolipoprotein B was isolated from an adult human liver DNA library by screening with an oligonucleotide probe derived from amino-terminal protein sequence obtained from purified B-26 peptide. Antisera against a synthetic 17-residue peptide whose amino acid sequence was encoded by the clone cross-reacts with apolipoproteins B-26, B-100, and B-48, but not with B-74. The nucleotide sequence immediately upstream from the amino terminus of B-26 codes for an apparent signal sequence, implying that the B-26 moiety is in an amino-terminal locus in the B-100 protein. That this sequence represents a 5' end region is further supported by primer extension analysis using a fragment of the cDNA clone and by S1 nuclease protection experiments using the corresponding region in a genomic clone.

  15. The role of a conserved membrane proximal cysteine in altering αPS2CβPS integrin diffusion

    NASA Astrophysics Data System (ADS)

    Syed, Aleem; Arora, Neha; Bunch, Thomas A.; Smith, Emily A.

    2016-12-01

    Cysteine residues (Cys) in the membrane proximal region are common post-translational modification (PTM) sites in transmembrane proteins. Herein, the effects of a highly conserved membrane proximal α-subunit Cys1368 on the diffusion properties of αPS2CβPS integrins are reported. Sequence alignment shows that this cysteine is palmitoylated in human α3 and α6 integrin subunits. Replacing Cys1368 in wild-type integrins with valine (Val1368) putatively blocks a PTM site and alters integrins’ ligand binding and diffusion characteristics. Both fluorescence recovery after photobleaching (FRAP) and single particle tracking (SPT) diffusion measurements show Val1368 integrins are more mobile compared to wild-type integrins. Approximately 33% and 8% more Val1368 integrins are mobile as measured by FRAP and SPT, respectively. The mobile Val1368 integrins also exhibit less time-dependent diffusion, as measured by FRAP. Tandem mass spectrometry data suggest that Cys1368 contains a redox or palmitoylation PTM in αPS2CβPS integrins. This membrane proximal Cys may play an important role in the diffusion of other alpha subunits that contain this conserved residue.

  16. Secondary Structure of Huntingtin Amino-Terminal Region

    SciTech Connect

    Kim, Mee Whi; Chelliah, Yogarany; Kim, Sang Woo; Otwinowski, Zbyszek; Bezprozvanny, Ilya

    2010-09-21

    Huntington's disease is a genetic neurodegenerative disorder resulting from polyglutamine (polyQ) expansion (>36Q) within the first exon of Huntingtin (Htt) protein. We applied X-ray crystallography to determine the secondary structure of the first exon (EX1) of Htt17Q. The structure of Htt17Q-EX1 consists of an amino-terminal {alpha} helix, poly17Q region, and polyproline helix formed by the proline-rich region. The poly17Q region adopts multiple conformations in the structure, including {alpha} helix, random coil, and extended loop. The conformation of the poly17Q region is influenced by the conformation of neighboring protein regions, demonstrating the importance of the native protein context. We propose that the conformational flexibility of the polyQ region observed in our structure is a common characteristic of many amyloidogenic proteins. We further propose that the pathogenic polyQ expansion in the Htt protein increases the length of the random coil, which promotes aggregation and facilitates abnormal interactions with other proteins in cells.

  17. The primary structure of skeletal muscle myosin heavy chain: I. Sequence of the amino-terminal 23 kDa fragment.

    PubMed

    Hayashida, M; Maita, T; Matsuda, G

    1991-07-01

    Subfragment-1 was prepared from adult chicken pectoralis myosin by limited digestion with alpha-chymotrypsin, and an amino-terminal 23 kDa fragment of the heavy chain was obtained by digesting the subfragment-1 with trypsin. The 205-residue sequence of the fragment was determined by sequencing its cyanogen bromide, tryptic, and chymotryptic peptides. The amino-terminal alpha-amino group of the fragment was acetylated, and two methylated lysines; epsilon-N-monomethyllysine and epsilon-N-trimethyllysine were recognized at the 35th and 130th positions, respectively, as in rabbit skeletal myosin. Comparing the 205-residue sequence of the skeletal myosin with those of cardiac, and gizzard myosins from chicken, considerable differences are recognized, especially in the amino-terminal region, but strong homologies are observed around the reactive lysine residue, around the epsilon-N-trimethyllysine residue, and around the consensus sequence of GXXGXGKT for nucleotide-binding proteins. On the other hand, only 12 amino acid substitutions are recognized between adult and embryonic skeletal myosins, allowing for the post-translational methylation.

  18. Amino Acid Changes in the HIV-1 gp41 Membrane Proximal Region Control Virus Neutralization Sensitivity.

    PubMed

    Bradley, Todd; Trama, Ashley; Tumba, Nancy; Gray, Elin; Lu, Xiaozhi; Madani, Navid; Jahanbakhsh, Fatemeh; Eaton, Amanda; Xia, Shi-Mao; Parks, Robert; Lloyd, Krissey E; Sutherland, Laura L; Scearce, Richard M; Bowman, Cindy M; Barnett, Susan; Abdool-Karim, Salim S; Boyd, Scott D; Melillo, Bruno; Smith, Amos B; Sodroski, Joseph; Kepler, Thomas B; Alam, S Munir; Gao, Feng; Bonsignori, Mattia; Liao, Hua-Xin; Moody, M Anthony; Montefiori, David; Santra, Sampa; Morris, Lynn; Haynes, Barton F

    2016-10-01

    Most HIV-1 vaccines elicit neutralizing antibodies that are active against highly sensitive (tier-1) viruses or rare cases of vaccine-matched neutralization-resistant (tier-2) viruses, but no vaccine has induced antibodies that can broadly neutralize heterologous tier-2 viruses. In this study, we isolated antibodies from an HIV-1-infected individual that targeted the gp41 membrane-proximal external region (MPER) that may have selected single-residue changes in viral variants in the MPER that resulted in neutralization sensitivity to antibodies targeting distal epitopes on the HIV-1 Env. Similarly, a single change in the MPER in a second virus from another infected-individual also conferred enhanced neutralization sensitivity. These gp41 single-residue changes thus transformed tier-2 viruses into tier-1 viruses that were sensitive to vaccine-elicited tier-1 neutralizing antibodies. These data demonstrate that Env amino acid changes within the MPER bnAb epitope of naturally-selected escape viruses can increase neutralization sensitivity to multiple types of neutralizing antibodies, and underscore the critical importance of the MPER for maintaining the integrity of the tier-2 HIV-1 trimer.

  19. nti glucocorticoid receptor transcripts lack sequences encoding the amino-terminal transcriptional modulatory domain.

    PubMed Central

    Dieken, E S; Meese, E U; Miesfeld, R L

    1990-01-01

    Glucocorticoid induction of cell death (apoptosis) in mouse lymphoma S49 cells has long been studied as a molecular genetic model of steroid hormone action. To better understand the transcriptional control of glucocorticoid-induced S49 cell death, we isolated and characterized glucocorticoid receptor (GR) cDNA from two steroid-resistant nti S49 mutant cell lines (S49.55R and S49.143R) and the wild-type parental line (S49.A2). Our data reveal that nti GR transcripts encode intact steroid- and DNA-binding domains but lack 404 amino-terminal residues as a result of aberrant RNA splicing between exons 1 and 3. Results from transient cotransfection experiments into CV1 cells using nti receptor expression plasmids and a glucocorticoid-responsive reporter gene demonstrated that the truncated nti receptor exhibits a reduced transcriptional regulatory activity. Gene fusions containing portions of both the wild-type and the nti GR-coding sequences were constructed and used to functionally map the nti receptor mutation. We found that the loss of the modulatory domain alone is sufficient to cause the observed defect in nti transcriptional transactivation. These results support the proposal that glucocorticoid-induced S49 cell death requires GR sequences which have previously been shown to be required for transcriptional regulation, suggesting that steroid-regulated apoptosis is controlled at the level of gene expression. Images PMID:2388618

  20. Stability of the mitochondrial genome requires an amino-terminal domain of yeast mitochondrial RNA polymerase

    PubMed Central

    Wang, Yuanhong; Shadel, Gerald S.

    1999-01-01

    Mitochondrial RNA (mtRNA) polymerases are related to bacteriophage RNA polymerases, but contain a unique amino-terminal extension of unknown origin and function. In addition to harboring mitochondrial targeting information, we show here that the amino-terminal extension of yeast mtRNA polymerase is required for a mtDNA maintenance function that is separable from the known RNA polymerization activity of the enzyme. Deletion of 185 N-terminal amino acids from the enzyme results in a temperature-sensitive mitochondrial petite phenotype, characterized by increased instability and eventual loss of the mitochondrial genome. Mitochondrial transcription initiation in vivo is largely unaffected by this mutation and expression of just the amino-terminal portion of the protein in trans partially suppresses the mitochondrial defect, indicating that the amino-terminal extension of the enzyme harbors an independent functional domain that is required for mtDNA replication and/or stability. These results suggest that amino-terminal extensions present in mtRNA polymerases comprise functional domains that couple additional activities to the transcription process in mitochondria. PMID:10393945

  1. Different structures in the amino-terminal domain of the ornithine transcarbamylase leader peptide are involved in mitochondrial import and carboxyl-terminal cleavage.

    PubMed

    Kraus, J P; Novotný, J; Kalousek, F; Swaroop, M; Rosenberg, L E

    1988-12-01

    The cytoplasmic precursor of mitochondrial ornithine transcarbamylase (carbamoyl-phosphate:L-ornithine carbamoyltransferase, EC 2.1.3.2) contains an amino-terminal leader peptide of 32 amino acids. Secondary structure and helical-wheel analyses predict that the extreme amino-terminal domain (residues 1-15) forms an alpha-helix. To test this thesis, leucine residues at positions 2, 5, 8, and 9 were systematically replaced by either helix-breaking glycine residues or by helix-preserving alanine residues. Triple substitutions of glycine for leucine in positions 2, 5, and 9 or 5, 8, and 9 abolished the uptake of the rat precursor by intact mitochondria, whereas similar alanine substitutions had much less effect. Theoretical computations predicted that the decreased helical stability of the Gly-5,8,9 substitution could be significantly increased by replacing a serine in position with phenylalanine. The introduction of Phe-3, indeed, restored the mitochondrial uptake of the mutant precursor. These results lend strong support to the hypothesis that an alpha-helix is present at the leader's amino terminus during the import of the precursor by mitochondria. Although the precursors with the triply-substituted leaders were impaired with respect to import, they were still cleaved readily by a protease found in a mitochondrial matrix fraction. Substitution of glycine or alanine for all four leucine residues, however, rendered the leader uncleavable at the carboxyl-terminal cleavage site. These results suggest that the structure of the amino-terminal domain is important for recognition of the carboxyl-terminal cleavage sites by the matrix proteases.

  2. Mechanism of HIV-1 neutralization by antibodies targeting a membrane-proximal region of gp41.

    PubMed

    Chen, Jia; Frey, Gary; Peng, Hanqin; Rits-Volloch, Sophia; Garrity, Jetta; Seaman, Michael S; Chen, Bing

    2014-01-01

    Induction of broadly neutralizing antibodies (bNAbs) is an important goal for HIV-1 vaccine development. Two autoreactive bNAbs, 2F5 and 4E10, recognize a conserved region on the HIV-1 envelope glycoprotein gp41 adjacent to the viral membrane known as the membrane-proximal external region (MPER). They block viral infection by targeting a fusion-intermediate conformation of gp41, assisted by an additional interaction with the viral membrane. Another MPER-specific antibody, 10E8, has recently been reported to neutralize HIV-1 with potency and breadth much greater than those of 2F5 or 4E10, but it appeared not to bind phospholipids and might target the untriggered envelope spikes, raising the hope that the MPER could be harnessed for vaccine design without major immunological concerns. Here, we show by three independent approaches that 10E8 indeed binds lipid bilayers through two hydrophobic residues in its CDR H3 (third heavy-chain complementarity-determining region). Its weak affinity for membranes in general and preference for cholesterol-rich membranes may account for its great neutralization potency, as it is less likely than other MPER-specific antibodies to bind cellular membranes nonspecifically. 10E8 binds with high affinity to a construct mimicking the fusion intermediate of gp41 but fails to recognize the envelope trimers representing the untriggered conformation. Moreover, we can improve the potency of 4E10 without affecting its binding to gp41 by a modification of its lipid-interacting CDR H3. These results reveal a general mechanism of HIV-1 neutralization by MPER-specific antibodies that involves interactions with viral lipids.

  3. Germline variant FGFR4  p.G388R exposes a membrane-proximal STAT3 binding site.

    PubMed

    Ulaganathan, Vijay K; Sperl, Bianca; Rapp, Ulf R; Ullrich, Axel

    2015-12-24

    Variant rs351855-G/A is a commonly occurring single-nucleotide polymorphism of coding regions in exon 9 of the fibroblast growth factor receptor FGFR4 (CD334) gene (c.1162G>A). It results in an amino-acid change at codon 388 from glycine to arginine (p.Gly388Arg) in the transmembrane domain of the receptor. Despite compelling genetic evidence for the association of this common variant with cancers of the bone, breast, colon, prostate, skin, lung, head and neck, as well as soft-tissue sarcomas and non-Hodgkin lymphoma, the underlying biological mechanism has remained elusive. Here we show that substitution of the conserved glycine 388 residue to a charged arginine residue alters the transmembrane spanning segment and exposes a membrane-proximal cytoplasmic signal transducer and activator of transcription 3 (STAT3) binding site Y(390)-(P)XXQ(393). We demonstrate that such membrane-proximal STAT3 binding motifs in the germline of type I membrane receptors enhance STAT3 tyrosine phosphorylation by recruiting STAT3 proteins to the inner cell membrane. Remarkably, such germline variants frequently co-localize with somatic mutations in the Catalogue of Somatic Mutations in Cancer (COSMIC) database. Using Fgfr4 single nucleotide polymorphism knock-in mice and transgenic mouse models for breast and lung cancers, we validate the enhanced STAT3 signalling induced by the FGFR4 Arg388-variant in vivo. Thus, our findings elucidate the molecular mechanism behind the genetic association of rs351855 with accelerated cancer progression and suggest that germline variants of cell-surface molecules that recruit STAT3 to the inner cell membrane are a significant risk for cancer prognosis and disease progression.

  4. A membrane-proximal, C-terminal α-helix is required for plasma membrane localization and function of the G Protein-coupled receptor (GPCR) TGR5.

    PubMed

    Spomer, Lina; Gertzen, Christoph G W; Schmitz, Birte; Häussinger, Dieter; Gohlke, Holger; Keitel, Verena

    2014-02-07

    The C terminus of G protein-coupled receptors (GPCRs) is important for G protein-coupling and activation; in addition, sorting motifs have been identified in the C termini of several GPCRs that facilitate correct trafficking from the endoplasmic reticulum to the plasma membrane. The C terminus of the GPCR TGR5 lacks any known sorting motif such that other factors must determine its trafficking. Here, we investigate deletion and substitution variants of the membrane-proximal C terminus of TGR5 with respect to plasma membrane localization and function using immunofluorescence staining, flow cytometry, and luciferase assays. Peptides of the membrane-proximal C-terminal variants are subjected to molecular dynamics simulations and analyzed with respect to their secondary structure. Our results reveal that TGR5 plasma membrane localization and responsiveness to extracellular ligands is fostered by a long (≥ 9 residues) α-helical stretch at the C terminus, whereas the presence of β-strands or only a short α-helical stretch leads to retention in the endoplasmic reticulum and a loss of function. As a proof-of-principle, chimeras of TGR5 containing the membrane-proximal amino acids of the β2 adrenergic receptor (β2AR), the sphingosine 1-phosphate receptor-1 (S1P1), or the κ-type opioid receptor (κOR) were generated. These TGR5β2AR, TGR5S1P1, or TGR5κOR chimeras were correctly sorted to the plasma membrane. As the exchanged amino acids of the β2AR, the S1P1, or the κOR form α-helices in crystal structures but lack significant sequence identity to the respective TGR5 sequence, we conclude that the secondary structure of the TGR5 membrane-proximal C terminus is the determining factor for plasma membrane localization and responsiveness towards extracellular ligands.

  5. Resonance assignment of an engineered amino-terminal domain of a major ampullate spider silk with neutralized charge cluster.

    PubMed

    Schaal, Daniel; Bauer, Joschka; Schweimer, Kristian; Scheibel, Thomas; Rösch, Paul; Schwarzinger, Stephan

    2016-04-01

    Spider dragline fibers are predominantly made out of the major ampullate spidroins (MaSp) 1 and 2. The assembly of dissolved spidroin into a stable fiber is highly controlled for example by dimerization of its amino-terminal domain (NRN) upon acidification, as well as removal of sodium chloride along the spinning duct. Clustered residues D39, E76 and E81 are the most highly conserved residues of the five-helix bundle, and they are hypothesized to be key residues for switching between a monomeric and a dimeric conformation. Simultaneous replacement of these residues by their non-titratable analogues results in variant D39N/E76Q/E81Q, which is supposed to fold into an intermediate conformation between that of the monomeric and the dimeric state at neutral pH. Here we report the resonance assignment of Latrodectus hesperus NRN variant D39N/E76Q/E81Q at pH 7.2 obtained by high-resolution triple resonance NMR spectroscopy.

  6. Amino-terminal truncations of the ribulose-bisphosphate carboxylase small subunit influence catalysis and subunit interactions.

    PubMed Central

    Paul, K; Morell, M K; Andrews, T J

    1993-01-01

    The first 20 residues at the amino terminus of the small subunit of spinach ribulose-1,5-bisphosphate carboxylase form an irregular arm that makes extensive contacts with the large subunit and also with another small subunit (S. Knight, I. Andersson, and C.-I. Brändén [1990] J Mol Biol 215: 113-160). The influence of these contacts on subunit binding and, indirectly, on catalysis was investigated by constructing truncations from the amino terminus of the small subunit of the highly homologous enzyme from Synechococcus PCC 6301 expressed in Escherichia coli. Removal of the first six residues (and thus the region of contact with a neighboring small subunit) affected neither the affinity with which the small subunits bound to the large subunits nor the catalytic properties of the assembled holoenzyme. Extending the truncation to include the first 12 residues (which encroaches into a highly conserved region that interacts with the large subunit) also did not weaken intersubunit binding appreciably, but it reduced the catalytic activity of the holoenzyme nearly 5-fold. Removal of an additional single residue (i.e. removal of a total of 13 residues) weakened intersubunit binding approximately 80-fold. Paradoxically, this partially restored catalytic activity to approximately 40% of that of the wild-type holoenzyme. None of these truncations materially affected the Km values for ribulose-1,5-bisphosphate or CO2. Removal of all 20 residues of the irregular arm (thereby deleting the conserved region of contact with large subunits) totally abolished the small subunit's ability to bind to large subunits to form a stable holoenzyme. However, this truncated small subunit was still synthesized by the E. coli cells. These data are interpreted in terms of the role of the amino-terminal arm of the small subunit in maintaining the structure of the holoenzyme. PMID:8278544

  7. The hydrophilic amino-terminal arm of reovirus core shell protein lambda1 is dispensable for particle assembly.

    PubMed

    Kim, Jonghwa; Zhang, Xing; Centonze, Victoria E; Bowman, Valorie D; Noble, Simon; Baker, Timothy S; Nibert, Max L

    2002-12-01

    The reovirus core particle is a molecular machine that mediates synthesis, capping, and export of the viral plus strand RNA transcripts. Its assembly and structure-function relationships remain to be well understood. Following the lead of previous studies with other Reoviridae family members, most notably orbiviruses and rotaviruses, we used recombinant baculoviruses to coexpress reovirus core proteins lambda1, lambda2, and sigma2 in insect cells. The resulting core-like particles (CLPs) were purified and characterized. They were found to be similar to cores with regard to their sizes, morphologies, and protein compositions. Like cores, they could also be coated in vitro with the two major outer-capsid proteins, micro 1 and sigma3, to produce virion-like particles. Coexpression of core shell protein lambda1 and core nodule protein sigma2 was sufficient to yield CLPs that could withstand purification, whereas expression of lambda1 alone was not, indicating a required role for sigma2 as a previous study also suggested. In addition, CLPs that lacked lambda2 (formed from lambda1 and sigma2 only) could not be coated with micro 1 and sigma3, indicating a required role for lambda2 in the assembly of these outer-capsid proteins into particles. To extend the use of this system for understanding the core and its assembly, we addressed the hypothesis that the hydrophilic amino-terminal region of lambda1, which adopts an extended arm-like conformation around each threefold axis in the reovirus core crystal structure, plays an important role in assembling the core shell. Using a series of lambda1 deletion mutants, we showed that the amino-terminal 230 residues of lambda1, including its zinc finger, are dispensable for CLP assembly. Residues in the 231-to-259 region of lambda1, however, were required. The core crystal structure suggests that residues in the 231-to-259 region are necessary because they affect the interaction of lambda1 with the threefold and/or fivefold copies of

  8. A Study of the α-Helical Intermediate Preceding the Aggregation of the Amino-Terminal Fragment of the β Amyloid Peptide (Aβ1–28)

    PubMed Central

    Rojas, Ana V.; Liwo, Adam; Scheraga, Harold A.

    2011-01-01

    The β amyloid (Aβ) peptide aggregates to form β-rich structures that are known to trigger Alzheimer’s disease. Experiments suggest that an α-helical intermediate precedes the formation of these aggregates. However, a description at the molecular level of the α-to-β transition has not been obtained. Because it has been proposed that the transition might be initiated in the amino-terminal region of Aβ, we studied the aggregation of the 28-residue amino-terminal fragment of Aβ (Aβ1–28) using molecular dynamics and a coarse-grained force field. Simulations starting from extended and helical conformations showed that oligomerization is initiated by formation of intermolecular β -sheets between the residues in the N-terminal regions. In simulations starting from the α-helical conformation, forcing residues 17–21 to remain in the initial (helical) conformation prevents aggregation but allows for the formation of dimers, indicating that oligomerization, initiated along the non-helical N-terminal regions, cannot progress without the α-to-β transition propagating along the chains. PMID:21939202

  9. Amino-terminal arginylation as a degradation signal for selective autophagy.

    PubMed

    Cha-Molstad, Hyunjoo; Kwon, Yong Tae; Kim, Bo Yeon

    2015-09-01

    The ubiquitin-proteasome system and the autophagy lysosome system are the two major protein degradation machineries in eukaryotic cells. These two systems coordinate the removal of unwanted intracellular materials, but the mechanism by which they achieve this synchronization is largely unknown. The ubiquitination of substrates serves as a universal degradation signal for both systems. Our study revealed that the amino-terminal Arg, a canonical N-degron in the ubiquitin-proteasome system, also acts as a degradation signal in autophagy. We showed that many ER residents, such as BiP, contain evolutionally conserved arginylation permissive pro-N-degrons, and that certain inducers like dsDNA or proteasome inhibitors cause their translocation into the cytoplasm where they bind misfolded proteins and undergo amino-terminal arginylation by arginyl transferase 1 (ATE1). The amino-terminal Arg of BiP binds p62, which triggers p62 oligomerization and enhances p62-LC3 interaction, thereby stimulating autophagic delivery and degradation of misfolded proteins, promoting cell survival. This study reveals a novel ubiquitin-independent mechanism for the selective autophagy pathway, and provides an insight into how these two major protein degradation pathways communicate in cells to dispose the unwanted proteins.

  10. Surface expression of influenza virus neuraminidase, an amino-terminally anchored viral membrane glycoprotein, in polarized epithelial cells.

    PubMed Central

    Jones, L V; Compans, R W; Davis, A R; Bos, T J; Nayak, D P

    1985-01-01

    We have investigated the site of surface expression of the neuraminidase (NA) glycoprotein of influenza A virus, which, in contrast to the hemagglutinin, is bound to membranes by hydrophobic residues near the NH2-terminus. Madin-Darby canine kidney or primary African green monkey kidney cells infected with influenza A/WSN/33 virus and subsequently labeled with monoclonal antibody to the NA and then with a colloidal gold- or ferritin-conjugated second antibody exhibited specific labeling of apical surfaces. Using simian virus 40 late expression vectors, we also studied the surface expression of the complete NA gene (SNC) and a truncated NA gene (SN10) in either primary or a polarized continuous line (MA104) of African green monkey kidney cells. The polypeptides encoded by the cloned NA cDNAs were expressed on the surface of both cell types. Analysis of [3H]mannose-labeled polypeptides from recombinant virus-infected MA104 cells showed that the products of cloned NA cDNA comigrated with glycosylated NA from influenza virus-infected cells. Both the complete and the truncated glycoproteins were found to be preferentially expressed on apical plasma membranes, as detected by immunogold labeling. These results indicate that the NA polypeptide contains structural features capable of directing the transport of the protein to apical cell surfaces and the first 10 amino-terminal residues of the NA polypeptide are not involved in this process. Images PMID:3016520

  11. Mutagenesis of Paramyxovirus Hemagglutinin-Neuraminidase Membrane-Proximal Stalk Region Influences Stability, Receptor Binding, and Neuraminidase Activity

    PubMed Central

    Adu-Gyamfi, Emmanuel; Kim, Lori S.; Jardetzky, Theodore S.

    2016-01-01

    ABSTRACT Paramyxoviridae consist of a large family of enveloped, negative-sense, nonsegmented single-stranded RNA viruses that account for a significant number of human and animal diseases. The fusion process for nearly all paramyxoviruses involves the mixing of the host cell plasma membrane and the virus envelope in a pH-independent fashion. Fusion is orchestrated via the concerted action of two surface glycoproteins: an attachment protein called hemagglutinin-neuraminidase (HN [also called H or G depending on virus type and substrate]), which acts as a receptor binding protein, and a fusion (F) protein, which undergoes a major irreversible refolding process to merge the two membranes. Recent biochemical evidence suggests that receptor binding by HN is dispensable for cell-cell fusion. However, factors that influence the stability and/or conformation of the HN 4-helix bundle (4HB) stalk have not been studied. Here, we used oxidative cross-linking as well as functional assays to investigate the role of the structurally unresolved membrane-proximal stalk region (MPSR) (residues 37 to 58) of HN in the context of headless and full-length HN membrane fusion promotion. Our data suggest that the receptor binding head serves to stabilize the stalk to regulate fusion. Moreover, we found that the MPSR of HN modulates receptor binding and neuraminidase activity without a corresponding regulation of F triggering. IMPORTANCE Paramyxoviruses require two viral membrane glycoproteins, the attachment protein variously called HN, H, or G and the fusion protein (F), to couple host receptor recognition to virus-cell fusion. The HN protein has a globular head that is attached to a membrane-anchored flexible stalk of ∼80 residues and has three activities: receptor binding, neuraminidase, and fusion activation. In this report, we have identified the functional significance of the membrane-proximal stalk region (MPSR) (HN, residues 37 to 56) of the paramyxovirus parainfluenza virus

  12. Membrane-proximal binding of STAT3 revealed by cancer-associated receptor variants.

    PubMed

    Ulaganathan, Vijay K; Ullrich, Axel

    2016-05-01

    In cancer biology, somatic mutations in the extracellular (ligand binding) and cytosolic (functional/catalytic) domains are pursued with great interest. However, in our recent publication we report that germline mutations in the membrane-proximal region of type I receptors are able to modulate the amplitude of signal transducer and activator of transcription 3 (STAT3) signaling in cells. This unexpected finding has implications for the prognosis of heritable cancer.

  13. 4E10-Resistant HIV-1 Isolated from Four Subjects with Rare Membrane-Proximal External Region Polymorphisms

    PubMed Central

    Nakamura, Kyle J.; Gach, Johannes S.; Jones, Laura; Semrau, Katherine; Walter, Jan; Bibollet-Ruche, Frederic; Decker, Julie M.; Heath, Laura; Decker, William D.; Sinkala, Moses; Kankasa, Chipepo; Thea, Donald; Mullins, James; Kuhn, Louise; Zwick, Michael B.; Aldrovandi, Grace M.

    2010-01-01

    Human antibody 4E10 targets the highly conserved membrane-proximal external region (MPER) of the HIV-1 transmembrane glycoprotein, gp41, and has extraordinarily broad neutralizing activity. It is considered by many to be a prototype for vaccine development. In this study, we describe four subjects infected with viruses carrying rare MPER polymorphisms associated with resistance to 4E10 neutralization. In one case resistant virus carrying a W680G substitution was transmitted from mother to infant. We used site-directed mutagenesis to demonstrate that the W680G substitution is necessary for conferring the 4E10-resistant phenotype, but that it is not sufficient to transfer the phenotype to a 4E10-sensitive Env. Our third subject carried Envs with a W680R substitution causing variable resistance to 4E10, indicating that residues outside the MPER are required to confer the phenotype. A fourth subject possessed a F673L substitution previously associated with 4E10 resistance. For all three subjects with W680 polymorphisms, we observed additional residues in the MPER that co-varied with position 680 and preserved charged distributions across this region. Our data provide important caveats for vaccine development targeting the MPER. Naturally occurring Env variants described in our study also represent unique tools for probing the structure-function of HIV-1 envelope. PMID:20352106

  14. The membrane proximal region of the cannabinoid receptor CB1 N-terminus can allosterically modulate ligand affinity.

    PubMed

    Fay, Jonathan F; Farrens, David L

    2013-11-19

    The human cannabinoid receptor, CB1, a G protein-coupled receptor (GPCR), contains a relatively long (∼110 a.a.) amino terminus, whose function is still not defined. Here we explore a potential role for the CB1 N-terminus in modulating ligand binding to the receptor. Although most of the CB1 N-terminus is not necessary for ligand binding, previous studies have found that mutations introduced into its conserved membrane proximal region (MPR) do impair the receptors ability to bind ligand. Moreover, within the highly conserved MPR (∼ residues 90-110) lie two cysteine residues that are invariant in all CB1 receptors. We find these two cysteines (C98 and C107) form a disulfide in heterologously expressed human CB1, and this C98-C107 disulfide is much more accessible to reducing agents than the previously known disulfide in extracellular loop 2 (EL2). Interestingly, the presence of the C98-C107 disulfide modulates ligand binding to the receptor in a way that can be quantitatively analyzed by an allosteric model. The C98-C107 disulfide also alters the effects of allosteric ligands for CB1, Org 27569 and PSNCBAM-1. Together, these results provide new insights into how the N-terminal MPR and EL2 act together to influence the high-affinity orthosteric ligand binding site in CB1 and suggest that the CB1 N-terminal MPR may be an area through which allosteric modulators can act.

  15. Structure of the HIV-1 gp41 Membrane-Proximal Ectodomain Region in a Putative Prefusion Conformation

    SciTech Connect

    Liu, J.; Deng, Y; Dey, A; Moore, J; Lu, M

    2009-01-01

    The conserved membrane-proximal external region (MPER) of the HIV-1 gp41 envelope protein is the established target for very rare but broadly neutralizing monoclonal antibodies (NAbs) elicited during natural human infection. Nevertheless, attempts to generate an HIV-1 neutralizing antibody response with immunogens bearing MPER epitopes have met with limited success. Here we show that the MPER peptide (residues 662-683) forms a labile ?-helical trimer in aqueous solution and report the crystal structure of this autonomous folding subdomain stabilized by addition of a C-terminal isoleucine zipper motif. The structure reveals a parallel triple-stranded coiled coil in which the neutralization epitope residues are buried within the interface between the associating MPER helices. Accordingly, both the 2F5 and 4E10 NAbs recognize the isolated MPER peptide but fail to bind the trimeric MPER subdomain. We propose that the trimeric MPER structure represents the prefusion conformation of gp41, preceding the putative prehairpin intermediate and the postfusion trimer-of-hairpins structure. As such, the MPER trimer should inform the design of new HIV-1 immunogens to elicit broadly neutralizing antibodies.

  16. Functional Evidence for the Critical Amino-Terminal Conserved Domain and Key Amino Acids of Arabidopsis 4-HYDROXY-3-METHYLBUT-2-ENYL DIPHOSPHATE REDUCTASE1[W][OPEN

    PubMed Central

    Hsieh, Wei-Yu; Sung, Tzu-Ying; Wang, Hsin-Tzu; Hsieh, Ming-Hsiun

    2014-01-01

    The plant 4-HYDROXY-3-METHYLBUT-2-ENYL DIPHOSPHATE REDUCTASE (HDR) catalyzes the last step of the methylerythritol phosphate pathway to synthesize isopentenyl diphosphate and its allyl isomer dimethylallyl diphosphate, which are common precursors for the synthesis of plastid isoprenoids. The Arabidopsis (Arabidopsis thaliana) genomic HDR transgene-induced gene-silencing lines are albino, variegated, or pale green, confirming that HDR is essential for plants. We used Escherichia coli isoprenoid synthesis H (Protein Data Bank code 3F7T) as a template for homology modeling to identify key amino acids of Arabidopsis HDR. The predicted model reveals that cysteine (Cys)-122, Cys-213, and Cys-350 are involved in iron-sulfur cluster formation and that histidine (His)-152, His-241, glutamate (Glu)-242, Glu-243, threonine (Thr)-244, Thr-312, serine-379, and asparagine-381 are related to substrate binding or catalysis. Glu-242 and Thr-244 are conserved only in cyanobacteria, green algae, and land plants, whereas the other key amino acids are absolutely conserved from bacteria to plants. We used site-directed mutagenesis and complementation assay to confirm that these amino acids, except His-152 and His-241, were critical for Arabidopsis HDR function. Furthermore, the Arabidopsis HDR contains an extra amino-terminal domain following the transit peptide that is highly conserved from cyanobacteria, and green algae to land plants but not existing in the other bacteria. We demonstrated that the amino-terminal conserved domain was essential for Arabidopsis and cyanobacterial HDR function. Further analysis of conserved amino acids in the amino-terminal conserved domain revealed that the tyrosine-72 residue was critical for Arabidopsis HDR. These results suggest that the structure and reaction mechanism of HDR evolution have become specific for oxygen-evolving photosynthesis organisms and that HDR probably evolved independently in cyanobacteria versus other prokaryotes. PMID:25037211

  17. Biophysical Characterization of a Vaccine Candidate against HIV-1: The Transmembrane and Membrane Proximal Domains of HIV-1 gp41 as a Maltose Binding Protein Fusion

    PubMed Central

    Gong, Zhen; Martin-Garcia, Jose M.; Daskalova, Sasha M.; Craciunescu, Felicia M.; Song, Lusheng; Dörner, Katerina; Hansen, Debra T.; Yang, Jay-How; LaBaer, Joshua; Hogue, Brenda G.; Mor, Tsafrir S.; Fromme, Petra

    2015-01-01

    The membrane proximal region (MPR, residues 649–683) and transmembrane domain (TMD, residues 684–705) of the gp41 subunit of HIV-1’s envelope protein are highly conserved and are important in viral mucosal transmission, virus attachment and membrane fusion with target cells. Several structures of the trimeric membrane proximal external region (residues 662–683) of MPR have been reported at the atomic level; however, the atomic structure of the TMD still remains unknown. To elucidate the structure of both MPR and TMD, we expressed the region spanning both domains, MPR-TM (residues 649–705), in Escherichia coli as a fusion protein with maltose binding protein (MBP). MPR-TM was initially fused to the C-terminus of MBP via a 42 aa-long linker containing a TEV protease recognition site (MBP-linker-MPR-TM). Biophysical characterization indicated that the purified MBP-linker-MPR-TM protein was a monodisperse and stable candidate for crystallization. However, crystals of the MBP-linker-MPR-TM protein could not be obtained in extensive crystallization screens. It is possible that the 42 residue-long linker between MBP and MPR-TM was interfering with crystal formation. To test this hypothesis, the 42 residue-long linker was replaced with three alanine residues. The fusion protein, MBP-AAA-MPR-TM, was similarly purified and characterized. Significantly, both the MBP-linker-MPR-TM and MBP-AAA-MPR-TM proteins strongly interacted with broadly neutralizing monoclonal antibodies 2F5 and 4E10. With epitopes accessible to the broadly neutralizing antibodies, these MBP/MPR-TM recombinant proteins may be in immunologically relevant conformations that mimic a pre-hairpin intermediate of gp41. PMID:26295457

  18. Biophysical Characterization of a Vaccine Candidate against HIV-1: The Transmembrane and Membrane Proximal Domains of HIV-1 gp41 as a Maltose Binding Protein Fusion.

    PubMed

    Gong, Zhen; Martin-Garcia, Jose M; Daskalova, Sasha M; Craciunescu, Felicia M; Song, Lusheng; Dörner, Katerina; Hansen, Debra T; Yang, Jay-How; LaBaer, Joshua; Hogue, Brenda G; Mor, Tsafrir S; Fromme, Petra

    2015-01-01

    The membrane proximal region (MPR, residues 649-683) and transmembrane domain (TMD, residues 684-705) of the gp41 subunit of HIV-1's envelope protein are highly conserved and are important in viral mucosal transmission, virus attachment and membrane fusion with target cells. Several structures of the trimeric membrane proximal external region (residues 662-683) of MPR have been reported at the atomic level; however, the atomic structure of the TMD still remains unknown. To elucidate the structure of both MPR and TMD, we expressed the region spanning both domains, MPR-TM (residues 649-705), in Escherichia coli as a fusion protein with maltose binding protein (MBP). MPR-TM was initially fused to the C-terminus of MBP via a 42 aa-long linker containing a TEV protease recognition site (MBP-linker-MPR-TM). Biophysical characterization indicated that the purified MBP-linker-MPR-TM protein was a monodisperse and stable candidate for crystallization. However, crystals of the MBP-linker-MPR-TM protein could not be obtained in extensive crystallization screens. It is possible that the 42 residue-long linker between MBP and MPR-TM was interfering with crystal formation. To test this hypothesis, the 42 residue-long linker was replaced with three alanine residues. The fusion protein, MBP-AAA-MPR-TM, was similarly purified and characterized. Significantly, both the MBP-linker-MPR-TM and MBP-AAA-MPR-TM proteins strongly interacted with broadly neutralizing monoclonal antibodies 2F5 and 4E10. With epitopes accessible to the broadly neutralizing antibodies, these MBP/MPR-TM recombinant proteins may be in immunologically relevant conformations that mimic a pre-hairpin intermediate of gp41.

  19. Multifunctional cholesterol-based peroxide for modification of amino-terminated surfaces: Synthesis, structure and characterization of grafted layer

    NASA Astrophysics Data System (ADS)

    Stetsyshyn, Y.; Kostruba, A.; Harhay, K.; Donchak, V.; Ohar, H.; Savaryn, V.; Kulyk, B.; Ripak, L.; Nastishin, Yu. A.

    2015-08-01

    A multifunctional cholesterol-based peroxide modifier - monoperoxided monocholesteryl pyromellitate (PChP) with residual acid chloride groups has been synthesized. The structure of the peroxide modifier was confirmed using IR and 1H NMR spectroscopies. Grafted PChP coating was successfully fabricated onto amino-terminated glass surfaces. Thickness and refractive index of the coated layer, its morphology, wettability, and alignment of elongated PChP molecules, attached to the surface at different grafting times were characterized by means of ellipsometry, AFM, measurements of wetting contact angle and testing alignment of a nematic liquid crystal on the coatings. In a flat cell assembled of a pair of glass substrates coated with the PChP layer nematic molecules align preferentially tilted with respect to the cell normal, though in some place one finds homeotropic alignment, where nematic molecules are perpendicular to the surface. Liquid crystal textures visualize inhomogeneities in surface profile of the coated layer. Homeotropic alignment is observed in places where roughness of the layer is completely randomized in agreement with AFM data, providing visualization of evolution of the surface profile. Concentration of grafted molecules per area of the surface deduced from ellipsometry data suggests that PChP molecules are grafted to the surface rather densely already in about ten minutes.

  20. Differential effects of amino-terminal distal and proximal domains in the regulation of human erg K(+) channel gating.

    PubMed Central

    Viloria, C G; Barros, F; Giráldez, T; Gómez-Varela, D; de la Peña, P

    2000-01-01

    The participation of amino-terminal domains in human ether-a-go-go (eag)-related gene (HERG) K(+) channel gating was studied using deleted channel variants expressed in Xenopus oocytes. Selective deletion of the HERG-specific sequence (HERG Delta138-373) located between the conserved initial amino terminus (the eag or PAS domain) and the first transmembrane helix accelerates channel activation and shifts its voltage dependence to hyperpolarized values. However, deactivation time constants from fully activated states and channel inactivation remain almost unaltered after the deletion. The deletion effects are equally manifested in channel variants lacking inactivation. The characteristics of constructs lacking only about half of the HERG-specific domain (Delta223-373) or a short stretch of 19 residues (Delta355-373) suggest that the role of this domain is not related exclusively to its length, but also to the presence of specific sequences near the channel core. Deletion-induced effects are partially reversed by the additional elimination of the eag domain. Thus the particular combination of HERG-specific and eag domains determines two important HERG features: the slow activation essential for neuronal spike-frequency adaptation and maintenance of the cardiac action potential plateau, and the slow deactivation contributing to HERG inward rectification. PMID:10866950

  1. Cooperative DNA Binding and Sequence-Selective Recognition Conferred by the STAT Amino-Terminal Domain

    NASA Astrophysics Data System (ADS)

    Xu, Xiang; Sun, Ya-Lin; Hoey, Timothy

    1996-08-01

    STAT proteins (signal transducers and activators of transcription) activate distinct target genes despite having similar DNA binding preferences. The transcriptional specificity of STAT proteins was investigated on natural STAT binding sites near the interferon-gamma gene. These sites are arranged in multiple copies and required cooperative interactions for STAT binding. The conserved amino-terminal domain of STAT proteins was required for cooperative DNA binding, although this domain was not essential for dimerization or binding to a single site. Cooperative binding interactions enabled the STAT proteins to recognize variations of the consensus site. These sites can be specific for the different STAT proteins and may function to direct selective transcriptional activation.

  2. Assessment of the PrPc Amino-Terminal Domain in Prion Species Barriers

    PubMed Central

    Davenport, Kristen A.; Henderson, Davin M.; Mathiason, Candace K.

    2016-01-01

    ABSTRACT Chronic wasting disease (CWD) in cervids and bovine spongiform encephalopathy (BSE) in cattle are prion diseases that are caused by the same protein-misfolding mechanism, but they appear to pose different risks to humans. We are interested in understanding the differences between the species barriers of CWD and BSE. We used real-time, quaking-induced conversion (RT-QuIC) to model the central molecular event in prion disease, the templated misfolding of the normal prion protein, PrPc, to a pathogenic, amyloid isoform, scrapie prion protein, PrPSc. We examined the role of the PrPc amino-terminal domain (N-terminal domain [NTD], amino acids [aa] 23 to 90) in cross-species conversion by comparing the conversion efficiency of various prion seeds in either full-length (aa 23 to 231) or truncated (aa 90 to 231) PrPc. We demonstrate that the presence of white-tailed deer and bovine NTDs hindered seeded conversion of PrPc, but human and bank vole NTDs did the opposite. Additionally, full-length human and bank vole PrPcs were more likely to be converted to amyloid by CWD prions than were their truncated forms. A chimera with replacement of the human NTD by the bovine NTD resembled human PrPc. The requirement for an NTD, but not for the specific human sequence, suggests that the NTD interacts with other regions of the human PrPc to increase promiscuity. These data contribute to the evidence that, in addition to primary sequence, prion species barriers are controlled by interactions of the substrate NTD with the rest of the substrate PrPc molecule. IMPORTANCE We demonstrate that the amino-terminal domain of the normal prion protein, PrPc, hinders seeded conversion of bovine and white-tailed deer PrPcs to the prion forms, but it facilitates conversion of the human and bank vole PrPcs to the prion forms. Additionally, we demonstrate that the amino-terminal domain of human and bank vole PrPcs requires interaction with the rest of the molecule to facilitate conversion by

  3. Torque Generation in F1-ATPase Devoid of the Entire Amino-Terminal Helix of the Rotor That Fills Half of the Stator Orifice

    PubMed Central

    Kohori, Ayako; Chiwata, Ryohei; Hossain, Mohammad Delawar; Furuike, Shou; Shiroguchi, Katsuyuki; Adachi, Kengo; Yoshida, Masasuke; Kinosita, Kazuhiko

    2011-01-01

    F1-ATPase is an ATP-driven rotary molecular motor in which the central γ-subunit rotates inside a cylinder made of α3β3 subunits. The amino and carboxyl termini of the γ rotor form a coiled coil of α-helices that penetrates the stator cylinder to serve as an axle. Crystal structures indicate that the axle is supported by the stator at two positions, at the orifice and by the hydrophobic sleeve surrounding the axle tip. The sleeve contacts are almost exclusively to the longer carboxyl-terminal helix, whereas nearly half the orifice contacts are to the amino-terminal helix. Here, we truncated the amino-terminal helix stepwise up to 50 residues, removing one half of the axle all the way up and far beyond the orifice. The half-sliced axle still rotated with an unloaded speed a quarter of the wild-type speed, with torque nearly half the wild-type torque. The truncations were made in a construct where the rotor tip was connected to a β-subunit via a short peptide linker. Linking alone did not change the rotational characteristics significantly. These and previous results show that nearly half the normal torque is generated if rotor-stator interactions either at the orifice or at the sleeve are preserved, suggesting that the make of the motor is quite robust. PMID:21723829

  4. Torque generation in F1-ATPase devoid of the entire amino-terminal helix of the rotor that fills half of the stator orifice.

    PubMed

    Kohori, Ayako; Chiwata, Ryohei; Hossain, Mohammad Delawar; Furuike, Shou; Shiroguchi, Katsuyuki; Adachi, Kengo; Yoshida, Masasuke; Kinosita, Kazuhiko

    2011-07-06

    F(1)-ATPase is an ATP-driven rotary molecular motor in which the central γ-subunit rotates inside a cylinder made of α(3)β(3) subunits. The amino and carboxyl termini of the γ rotor form a coiled coil of α-helices that penetrates the stator cylinder to serve as an axle. Crystal structures indicate that the axle is supported by the stator at two positions, at the orifice and by the hydrophobic sleeve surrounding the axle tip. The sleeve contacts are almost exclusively to the longer carboxyl-terminal helix, whereas nearly half the orifice contacts are to the amino-terminal helix. Here, we truncated the amino-terminal helix stepwise up to 50 residues, removing one half of the axle all the way up and far beyond the orifice. The half-sliced axle still rotated with an unloaded speed a quarter of the wild-type speed, with torque nearly half the wild-type torque. The truncations were made in a construct where the rotor tip was connected to a β-subunit via a short peptide linker. Linking alone did not change the rotational characteristics significantly. These and previous results show that nearly half the normal torque is generated if rotor-stator interactions either at the orifice or at the sleeve are preserved, suggesting that the make of the motor is quite robust. Copyright © 2011 Biophysical Society. Published by Elsevier Inc. All rights reserved.

  5. How the other half lives, the amino-terminal domain of the retinoblastoma tumor suppressor protein.

    PubMed

    Goodrich, David W

    2003-11-01

    The retinoblastoma tumor suppressor gene (RB1) is currently the only known gene whose mutation is necessary and sufficient for the development of a human cancer. Mutation or deregulation of RB1 is observed so frequently in other tumor types that compromising RB1 function may be a prerequisite for malignant transformation. Identifying the molecular mechanisms that provide the basis for RB1-mediated tumor suppression has become an important goal in the quest to understand and treat cancer. The lion's share of research on these mechanisms has focused on the carboxy-terminal half of the RB1 encoded protein (pRB). This focus is with good reason since this part of the protein, now called the "large pocket," is required for most of its known activities identified in vitro and in vivo. Large pocket mediated mechanisms alone, however, cannot account for all observed properties of pRB. The thesis presented here is that the relatively uncharacterized amino-terminal half of the protein makes important contributions to pRB-mediated tumor suppression. The goals of this review are to summarize evidence indicating that an amino-terminal structural domain is important for pRB function and to suggest a general hypothesis as to how this domain can be integrated with current models of pRB function.

  6. Expression, Purification, and Refolding of Recombinant Collagen α1(XI) Amino Terminal Domain Splice Variants

    PubMed Central

    Warner, Lisa R.; Blasick, Christina M.; Brown, Raquel J.; Oxford, Julia Thom

    2009-01-01

    The amino terminal domain of collagen type XI α1 chain is a noncollagenous structure that is essential for the regulation of fibrillogenesis in developing cartilage. The amino terminal domain is alternatively spliced at the mRNA level, resulting in proteins expressed as splice variants. These splice variants, or isoforms, have unique distribution in growing tissues, alluding to distinct roles in development. We report here a rapid and straightforward method for expression, purification and in vitro folding of recombinant collagen XI isoforms α1(XI) NTD[p7] and α1(XI) NTD[p6b+7]. The recombinant isoforms were expressed in Escherichia coli as bacterial inclusion bodies. Unfolded carboxy terminal polyhistidine tagged proteins were purified via nickel affinity chromatography and refolded with specific protocols optimized for each isoform. Purity was assessed by SDS-PAGE and correct secondary structure by a comparison of circular dichroism data with that obtained for Npp. Protein expression and purification of the recombinant collagen XI splice variants will allow further studies to elucidate the structure and molecular interactions with components of the extracellular matrix. This research will clarify the mechanism of collagen XI mediated regulation of collagen fibrillogenesis. PMID:17166742

  7. Immunolocalization of an amino-terminal fragment of apolipoprotein E in the Pick's disease brain.

    PubMed

    Rohn, Troy T; Day, Ryan J; Catlin, Lindsey W; Brown, Raquel J; Rajic, Alexander J; Poon, Wayne W

    2013-01-01

    Although the risk factor for apolipoprotein E (apoE) polymorphism in Alzheimer's disease (AD) has been well described, the role that apoE plays in other neurodegenerative diseases, including Pick's disease, is not well established. To examine a possible role of apoE in Pick's disease, an immunohistochemical analysis was performed utilizing a novel site-directed antibody that is specific for an amino-terminal fragment of apoE. Application of this antibody, termed the amino-terminal apoE cleavage fragment (nApoECF) antibody, consistently labeled Pick bodies within area CA1 of the hippocampus in 4 of the 5 cases examined. Co-localization of the nApoECF antibody with PHF-1, a general marker for Pick bodies, as well as with an antibody to caspase-cleaved tau (TauC3) was evident within the hippocampus. While staining of the nApoECF antibody was robust in area CA1, little co-localization with PHF-1 in Pick bodies within the dentate gyrus was observed. A quantitative analysis indicated that approximately 86% of the Pick bodies identified in area CA1 labeled with the nApoECF antibody. The presence of truncated apoE within Pick bodies suggests a broader role of apoE beyond AD and raises the question as to whether this protein contributes to pathogenesis associated with Pick's disease.

  8. Immunogenic properties of a trimeric gp41-based immunogen containing an exposed membrane-proximal external region

    PubMed Central

    Habte, Habtom H.; Banerjee, Saikat; Shi, Heliang; Qin, Yali; Cho, Michael W

    2015-01-01

    The membrane-proximal external region (MPER) of HIV-1 gp41 is an attractive target for vaccine development. Thus, better understanding of its immunogenic properties in various structural contexts is important. We previously described the crystal structure of a trimeric protein complex named gp41-HR1-54Q, which consists of the heptad repeat regions 1 and 2 and the MPER. The protein was efficiently recognized by broadly neutralizing antibodies. Here, we describe its immunogenic properties in rabbits. The protein was highly immunogenic, especially the C-terminal end of the MPER containing 4E10 and 10E8 epitopes (671NWFDITNWLWYIK683). Although antibodies exhibited strong competition activity against 4E10 and 10E8, neutralizing activity was not detected. Detailed mapping analyses indicated that amino acid residues critical for recognition resided on faces of the alpha helix that are either opposite of or perpendicular to the epitopes recognized by 4E10 and 10E8. These results provide critical information for designing the next generation of MPER-based immunogens. PMID:26454663

  9. Immunogenic properties of a trimeric gp41-based immunogen containing an exposed membrane-proximal external region.

    PubMed

    Habte, Habtom H; Banerjee, Saikat; Shi, Heliang; Qin, Yali; Cho, Michael W

    2015-12-01

    The membrane-proximal external region (MPER) of HIV-1 gp41 is an attractive target for vaccine development. Thus, better understanding of its immunogenic properties in various structural contexts is important. We previously described the crystal structure of a trimeric protein complex named gp41-HR1-54Q, which consists of the heptad repeat regions 1 and 2 and the MPER. The protein was efficiently recognized by broadly neutralizing antibodies. Here, we describe its immunogenic properties in rabbits. The protein was highly immunogenic, especially the C-terminal end of the MPER containing 4E10 and 10E8 epitopes ((671)NWFDITNWLWYIK(683)). Although antibodies exhibited strong competition activity against 4E10 and 10E8, neutralizing activity was not detected. Detailed mapping analyses indicated that amino acid residues critical for recognition resided on faces of the alpha helix that are either opposite of or perpendicular to the epitopes recognized by 4E10 and 10E8. These results provide critical information for designing the next generation of MPER-based immunogens.

  10. Membrane-Proximal External HIV-1 gp41 Motif Adapted for Destabilizing the Highly Rigid Viral Envelope

    PubMed Central

    Apellániz, Beatriz; Ivankin, Andrey; Nir, Shlomo; Gidalevitz, David; Nieva, José L.

    2011-01-01

    Electron microscopy structural determinations suggest that the membrane-proximal external region (MPER) of glycoprotein 41 (gp41) may associate with the HIV-1 membrane interface. It is further proposed that MPER-induced disruption and/or deformation of the lipid bilayer ensue during viral fusion. However, it is predicted that the cholesterol content of this membrane (∼45 mol %) will act against MPER binding and restructuring activity, in agreement with alternative structural models proposing that the MPER constitutes a gp41 ectodomain component that does not insert into the viral membrane. Here, using MPER-based peptides, we test the hypothesis that cholesterol impedes the membrane association and destabilizing activities of this gp41 domain. To that end, partitioning and leakage assays carried out in lipid vesicles were combined with x-ray reflectivity and grazing-incidence diffraction studies of monolayers. CpreTM, a peptide combining the carboxyterminal MPER sequence with aminoterminal residues of the transmembrane domain, bound and destabilized effectively cholesterol-enriched membranes. Accordingly, virion incubation with this peptide inhibited cell infection potently but nonspecifically. Thus, CpreTM seems to mimic the envelope-perturbing function of the MPER domain and displays antiviral activity. As such, we infer that CpreTM bound to cholesterol-enriched membranes would represent a relevant target for anti-HIV-1 immunogen and inhibitor development. PMID:22098741

  11. Direct Contacts Between Extracellular Membrane-Proximal Domains are Required for VEGF Receptor Activation and Cell Signaling

    SciTech Connect

    Yang, Y.; Xie, P; Opatowsky, Y; Schlessinger, J

    2010-01-01

    Structural analyses of the extracellular region of stem cell factor (SCF) receptor (also designated KIT) in complex with SCF revealed a sequence motif in a loop in the fourth Ig-like domain (D4) that is responsible for forming homotypic receptor contacts and for ligand-induced KIT activation and cell signaling. An identical motif was identified in the most membrane-proximal seventh Ig-like domain (D7) of vascular endothelial growth factor receptor 1 (VEGFR1), VEGFR2, and VEGFR3. In this report we demonstrate that ligand-induced tyrosine autophosphorylation and cell signaling via VEGFR1 or VEGFR2 harboring mutations in critical residues (Arg726 or Asp731) in D7 are strongly impaired. We also describe the crystal structure of D7 of VEGFR2 to a resolution of 2.7 {angstrom}. The structure shows that homotypic D7 contacts are mediated by salt bridges and van der Waals contacts formed between Arg726 of one protomer and Asp731 of the other protomer. The structure of D7 dimer is very similar to the structure of D4 dimers seen in the crystal structure of KIT extracellular region in complex with SCF. The high similarity between VEGFR D7 and KIT D4 in both structure and function provides further evidence for common ancestral origins of type III and type V RTKs. It also reveals a conserved mechanism for RTK activation and a novel target for pharmacological intervention of pathologically activated RTKs.

  12. Genetic perturbation of the putative cytoplasmic membrane-proximal salt bridge aberrantly activates α4 integrins

    PubMed Central

    Imai, Yoichi; Park, Eun Jeong; Peer, Dan; Peixoto, António; Cheng, Guiying; von Andrian, Ulrich H.; Carman, Christopher V.

    2008-01-01

    α4 integrins play a pivotal role in leukocyte migration and tissue-specific homing. The ability of integrins to bind ligand is dynamically regulated by activation-dependent conformational changes triggered in the cytoplasmic domain. An NMR solution structure defined a putative membrane-proximal salt bridge between the αIIbβ3 integrin cytoplasmic tails, which restrains integrins in their low-affinity state. However, the physiological importance of this salt bridge in α4 integrin regulation remains to be elucidated. To address this question, we disrupted the salt bridge in murine germ line by mutating the conserved cytoplasmic arginine RGFFKR in α4 integrins. In lymphocytes from knock-in mice (α4-R/AGFFKR), α4β1 and α4β7 integrins exhibited constitutively up-regulated ligand binding. However, transmigration of these cells across VCAM-1 and MAdCAM-1 substrates, or across endothelial monolayers, was reduced. Perturbed detachment of the tail appeared to cause the reduced cell migration of α4-R/AGFFKR lymphocytes. In vivo, α4-R/AGFFKR cells exhibited increased firm adhesion to Peyer patch venules but reduced homing to the gut. Our results demonstrate that the membrane-proximal salt bridge plays a critical role in supporting proper α4 integrin adhesive dynamics. Loss of this interaction destabilizes the nonadhesive conformation, and thereby perturbs the properly balanced cycles of adhesion and deadhesion required for efficient cell migration. PMID:18809756

  13. Immunological Functions of the Membrane Proximal Region of MHC Class II Molecules

    PubMed Central

    Harton, Jonathan; Jin, Lei; Hahn, Amy; Drake, Jim

    2016-01-01

    Major histocompatibility complex (MHC) class II molecules present exogenously derived antigen peptides to CD4 T cells, driving activation of naïve T cells and supporting CD4-driven immune functions. However, MHC class II molecules are not inert protein pedestals that simply bind and present peptides. These molecules also serve as multi-functional signaling molecules delivering activation, differentiation, or death signals (or a combination of these) to B cells, macrophages, as well as MHC class II-expressing T cells and tumor cells. Although multiple proteins are known to associate with MHC class II, interaction with STING (stimulator of interferon genes) and CD79 is essential for signaling. In addition, alternative transmembrane domain pairing between class II α and β chains influences association with membrane lipid sub-domains, impacting both signaling and antigen presentation. In contrast to the membrane-distal region of the class II molecule responsible for peptide binding and T-cell receptor engagement, the membrane-proximal region (composed of the connecting peptide, transmembrane domain, and cytoplasmic tail) mediates these “non-traditional” class II functions. Here, we review the literature on the function of the membrane-proximal region of the MHC class II molecule and discuss the impact of this aspect of class II immunobiology on immune regulation and human disease. PMID:27006762

  14. Amino terminal sequence of heavy and light chains from ratfish immunoglobulin.

    PubMed

    De Ioannes, A E; Aguila, H L

    1989-01-01

    The ratfish, Callorhinchus callorhinchus, a representative of the Holocephali, has a natural serum hemagglutinin (Mr 960,000), composed of heavy (Mr 71,000), light (Mr 22,500), and J (Mr 16,000) chains. To approach the mechanisms that generate diversity at this level of evolution, the amino terminal sequence of the heavy and light chains was determined by automated microsequencing. The chains are unblocked and have modest internal sequence heterogeneity. The heavy chains show sequence similarity with the terminal region of the heavy chain from the horned shark, Heterodontus francisci, and other species. In contrast to the heavy chain, the ratfish light chains display low sequence similarity with their shark kappa counterparts. However, their similarity with the variable region of the chicken lambda light chains is about 75%.

  15. Radioimmunoassay of Pro-. gamma. -melanotropin, the amino-terminal fragment of proopiolipomelanocortin. [Swine

    SciTech Connect

    Ekman, R.; Hakanson, R.; Larsson, I.; Sundler, F.; Thorell, J.I.

    1982-08-01

    A RIA has been developed for natural porcine pro-..gamma..-MSH, the 103-amino acid peptide that represents the amino-terminal part of proopiolipomelanocortin. Rabbits were immunized with the purified peptide polymerized with glutaraldehyde. The antiserum is directed against the amino-terminial end of the antigen and does not cross-react with corticotropin, ..beta..-lipotropin, ..beta..-endorphin, ..gamma../sub 3/MSH, or ..gamma../sub 2/MSH. The minimum detectable concentration is 0.15 ng/ml standard pro-..gamma..MSH (15 pg/tube). Pro-..gamma..MSH-like immunoreactivity was detected in plasma and extracts of the hypothalamus and pituitary of pigs. Gel chromatography of these extracts revealed at least three immunoreactive peaks in the anterior and neurointermediate lobes of the pituitary, wheras two immunoreactive peaks were found in extracts of the hypothalalmus. (Endocrinology 111:578,1982)

  16. The degradation sequence of adenovirus E1A consists of the amino-terminal tetrapeptide Met-Arg-His-Ile.

    PubMed Central

    Simon, R; Richter, J D

    1990-01-01

    The adenovirus E1A gene product is a potent transcriptional activator and nuclear oncoprotein. Like other regulatory proteins, E1A has a short half-life, in the range of 30 to 120 min. This short half-life, which was measured in cells synthesizing E1A, is not observed in cells injected with E1A protein made in bacteria or in vitro. In these cases, E1A is essentially refractory to degradation. In an attempt to reconcile this apparent paradox, we suggested that E1A was marked for degradation during its synthesis. Furthermore, we showed that a domain in the amino terminus of E1A was required for rapid degradation in cells translating E1A mRNA (J. M. Slavicek, N. C. Jones, and J. D. Richter, EMBO J. 7:3171-3180, 1988). In this study, we have used Xenopus laevis oocytes injected with mRNAs encoding altered E1A proteins to show that the amino-terminal tetrapeptide Met-Arg-His-Ile is required for E1A degradation. Even conservative amino acid substitutions in this degradation sequence render it nonfunctional. This degradation sequence can function as a transferable signal, since it induces instability when fused to another normally stable protein. Furthermore, the degradation sequence requires a proximity of no more than six residues from the amino terminus for activity. These data suggest that a trans-acting factor recognizes the amino terminus of E1A during the translation of its message to mark the protein for subsequent destruction. Images PMID:2146491

  17. Conformational flexibility in the apolipoprotein E amino-terminal domain structure determined from three new crystal forms: implications for lipid binding.

    PubMed Central

    Segelke, B. W.; Forstner, M.; Knapp, M.; Trakhanov, S. D.; Parkin, S.; Newhouse, Y. M.; Bellamy, H. D.; Weisgraber, K. H.; Rupp, B.

    2000-01-01

    An amino-terminal fragment of human apolipoprotein E3 (residues 1-165) has been expressed and crystallized in three different crystal forms under similar crystallization conditions. One crystal form has nearly identical cell dimensions to the previously reported orthorhombic (P2(1)2(1)2(1)) crystal form of the amino-terminal 22 kDa fragment of apolipoprotein E (residues 1-191). A second orthorhombic crystal form (P2(1)2(1)2(1) with cell dimensions differing from the first form) and a trigonal (P3(1)21) crystal form were also characterized. The structures of the first orthorhombic and the trigonal form were determined by seleno-methionine multiwavelength anomalous dispersion, and the structure of the second orthorhombic form was determined by molecular replacement using the structure from the trigonal form as a search model. A combination of modern experimental and computational techniques provided high-quality electron-density maps, which revealed new features of the apolipoprotein E structure, including an unambiguously traced loop connecting helices 2 and 3 in the four-helix bundle and a number of multiconformation side chains. The three crystal forms contain a common intermolecular, antiparallel packing arrangement. The electrostatic complimentarity observed in this antiparallel packing resembles the interaction of apolipoprotein E with the monoclonal antibody 2E8 and the low density lipoprotein receptor. Superposition of the model structures from all three crystal forms reveals flexibility and pronounced kinks in helices near one end of the four-helix bundle. This mobility at one end of the molecule provides new insights into the structural changes in apolipoprotein E that occur with lipid association. PMID:10850798

  18. Hydrophobic Residues in Helix 8 of Cannabinoid Receptor 1 Are Critical for Structural and Functional Properties†

    PubMed Central

    Ahn, Kwang H.; Nishiyama, Akiko; Mierke, Dale F.; Kendall, Debra A.

    2010-01-01

    In addition to the heptahelical transmembrane domain shared by all G protein-coupled receptors (GPCRs), many class A GPCRs adopt a helical domain, termed helix 8, in the membrane-proximal region of the C terminus. We investigated the role of residues in the hydrophobic and hydrophilic faces of amphiphilic helix 8 of human cannabinoid receptor 1 (CB1). To differentiate between a role for specific residues and global features, we made two key mutants: one involving replacement of the highly hydrophobic groups, Leu404, Phe408, and Phe412, all with alanine and the second involving substitution of the basic residues, Lys402, Arg405, and Arg409, all with the neutral glutamine. The former showed a very low Bmax based on binding isotherms, a minimal Emax based on GTPγS binding analysis, and defective localization relative to the wild-type CB1 receptor as revealed by confocal microscopy. However, the latter mutant and the wild-type receptors were indistinguishable. Circular dichroism spectroscopy of purified peptides with corresponding sequences indicated that the highly hydrophobic residues are critical for maintaining a strong helical structure in detergent, whereas the positively charged residues are not. Further investigation of mutant receptors revealed that CB1 localization requires a threshold level of hydrophobicity but not specific amino acids. Moreover, mutant receptors carrying two- to six-residue insertions amino-terminal to helix 8 revealed a graded decrease in Bmax values. Our results identify the key helix 8 components (including hydrophobicity of specific residues, structure, and location relative to TM7) determinant for receptor localization leading to robust ligand binding and G protein activation. PMID:20025243

  19. Modulating immunogenic properties of HIV-1 gp41 membrane-proximal external region by destabilizing six-helix bundle structure

    SciTech Connect

    Banerjee, Saikat; Shi, Heliang; Habte, Habtom H.; Qin, Yali; Cho, Michael W.

    2016-03-15

    The C-terminal alpha-helix of gp41 membrane-proximal external region (MPER; {sup 671}NWFDITNWLWYIK{sup 683}) encompassing 4E10/10E8 epitopes is an attractive target for HIV-1 vaccine development. We previously reported that gp41-HR1-54Q, a trimeric protein comprised of the MPER in the context of a stable six-helix bundle (6HB), induced strong immune responses against the helix, but antibodies were directed primarily against the non-neutralizing face of the helix. To better target 4E10/10E8 epitopes, we generated four putative fusion intermediates by introducing double point mutations or deletions in the heptad repeat region 1 (HR1) that destabilize 6HB in varying degrees. One variant, HR1-∆10-54K, elicited antibodies in rabbits that targeted W672, I675 and L679, which are critical for 4E10/10E8 recognition. Overall, the results demonstrated that altering structural parameters of 6HB can influence immunogenic properties of the MPER and antibody targeting. Further exploration of this strategy could allow development of immunogens that could lead to induction of 4E10/10E8-like antibodies. - Highlights: • Four gp41 MPER-based immunogens that resemble fusion intermediates were generated. • C-terminal region of MPER that contains 4E10/10E8 epitopes was highly immunogenic. • Altering 6HB structure can influence immunogenic properties of the MPER. • Induced antibodies targeted multiple residues critical for 4E10/10E8 binding. • Development of immunogens based on fusion intermediates is a promising strategy.

  20. Acid-base-induced association of amino-terminated polystyrenes. 1. Linear chains and ring formation

    SciTech Connect

    Merkle, G.; Burchard, W.

    1992-05-14

    The associating coupling process of teritiary-amino-terminated polystyrene (PS) chains with monofunctional and bifunctional dinitrophenol reagents has been studied by means of UV-vis spectroscopy and light-scattering measurements as a function of polymer concentration. The mechanism of coupling was first checked with monofunctional PS chains. The equilibrium constants of the two functional groups of the coupler were found to be slightly different from each other; but with the same average values as for the monofunctional phenol compound. The increase of molecular weight agreed well with predictions from Flory`s theory. Much smaller weight average molecular weights were found, however, for the coupling of telechelic PS chains, and the deviations are stronger for the short chains than for the longer ones. The effect is explained by ring formation and three approximations were applied. These are (a) a mean field approximation, (b) the Jacobson-Stockmayer (JS) approach, and (c) the JS approach including excluded volume effect and its influence on Gaussian statistics. Molecular weights were also calculated from the weight fraction of rings and fitted to those obtained by LS where the effect bond length b was chosen to be the adjustable parameter. In the original JS treatment the effective bond length increased with the chain length of the primary chain. In treatment c this could be eliminated by consideration of excluded volume, but absolute values of b are unrealistically low. 50 refs., 8 figs., 3 tabs.

  1. Adsorption behaviors of Hg(II) on chitosan functionalized by amino-terminated hyperbranched polyamidoamine polymers.

    PubMed

    Ma, Fang; Qu, Rongjun; Sun, Changmei; Wang, Chunhua; Ji, Chunnuan; Zhang, Ying; Yin, Ping

    2009-12-30

    The adsorption behaviors of Hg(II) on adsorbents, chitosan functionalized by generation 1.0-3.0 of amino-terminated hyperbranched polyamidoamine polymers (denoted as CTS-1.0, CTS-2.0 and CTS-3.0, respectively), were studied. The optimum pH corresponding to the maximum adsorption capacities was found to be 5.0 for the three adsorbents. The experimental equilibrium data of Hg(II) on the three adsorbents were fitted to the Freundlich and the Langmuir models, and it is found that the Langmuir isotherm was the best fitting model to describe the equilibrium adsorption. The kinetics data indicated that the adsorption process of Hg(II) ions on CTS-1.0, CTS-2.0 and CTS-3.0 were governed by the film diffusion and followed pseudo-second-order rate model. Thermodynamic analysis and FTIR analysis revealed that the adsorption behaviors of Hg(II) ions on the three adsorbents could be considered as spontaneous, endothermic and chemical sorption process, resulting in their higher adsorption capacities at higher temperature.

  2. Introduced Amino Terminal Epitopes Can Reduce Surface Expression of Neuronal Nicotinic Receptors

    PubMed Central

    Bracamontes, John R.; Akk, Gustav; Steinbach, Joe Henry

    2016-01-01

    Epitopes accessible on the surface of intact cells are extremely valuable in studies of membrane proteins, allowing quantification and determination of the distribution of proteins as well as identification of cells expressing large numbers of proteins. However for many membrane proteins there are no suitable antibodies to native sequences, due to lack of availability, low affinity or lack of specificity. In these cases the use of an introduced epitope at specific sites in the protein of interest can often provide a suitable tool for studies. However, the introduction of the epitope sequence has the potential to affect protein expression, the assembly of multisubunit proteins or transport to the surface membrane. We find that surface expression of heteromeric neuronal nicotinic receptors containing the α4 and β4 subunits can be affected by introduced epitopes when inserted near the amino terminus of a subunit. The FLAG epitope greatly reduces surface expression when introduced into either α4 or β4 subunits, the V5 epitope has little effect when placed in either, while the Myc epitope reduces expression more when inserted into β4 than α4. These results indicate that the extreme amino terminal region is important for assembly of these receptors, and demonstrate that some widely used introduced epitopes may severely reduce surface expression. PMID:26963253

  3. Amino-terminated biphenylthiol self-assembled monolayers as highly reactive molecular templates

    SciTech Connect

    Meyerbroeker, N.; Waske, P.; Zharnikov, M.

    2015-03-14

    Self-assembled monolayers (SAMs) with amino tail groups are of interest due to their ability of coupling further compounds. Such groups can be, in particular, created by electron irradiation of nitro- or nitrile-substituted aromatic SAMs, which provide a basis for chemical nanolithography and the fabrication of functionalized nanomembranes. An estimate of reactivity of the created amino groups requires a reference system of homogeneous, amino-terminated aromatic SAMs, which can also be used as a highly reactive molecular template. Here, we describe the synthesis of 4′-aminobiphenyl-4-thiol (ABPT) and SAMs prepared from this precursor on Au(111). The monolayers were characterized by X-ray photoelectron spectroscopy and near edge X-ray absorption fine structure spectroscopy, which revealed that they are well defined, chemically uniform, densely packed, and highly ordered. To examine the influence of electron irradiation on the reactivity of the terminal amino groups, ABPT SAMs were exposed to low energy (50 eV) electrons up to a dose of 40 mC/cm{sup 2} and, subsequently, immersed in either trifluoroacetic, pentafluoropropionic, or heptafluorobutyric anhydride. Analysing the amount of the attached anhydride species made it possible to determine the percentage of reactive amino groups as well as the effect of steric hindrance upon the coupling reaction. The above results are compared with those obtained for the well-established nitro-substituted biphenylthiol monolayers.

  4. Amino-terminal precursor sequence modulates canine distemper virus fusion protein function.

    PubMed

    von Messling, Veronika; Cattaneo, Roberto

    2002-05-01

    The fusion (F) proteins of most paramyxoviruses are classical type I glycoproteins with a short hydrophobic leader sequence closely following the translation initiation codon. The predicted reading frame of the canine distemper virus (CDV) F protein is more complex, with a short hydrophobic sequence beginning 115 codons downstream of the first AUG. To verify if the sequence between the first AUG and the hydrophobic region is translated, we produced a specific antiserum that indeed detected a short-lived F protein precursor that we named PreF(0). A peptide resulting from PreF(0) cleavage was identified and named Pre, and its half-life was measured to be about 30 min. PreF(0) cleavage was completed before proteolytic activation of F(0) into its F(1) and F(2) subunits by furin. To test the hypothesis that the Pre peptide may influence protein activity, we compared the function of F proteins synthesized with that peptide to that of F proteins synthesized with a shorter amino-terminal signal sequence. F proteins synthesized with the Pre peptide were more stable and less active. Thus, the Pre peptide modulates the function of the CDV F protein. Interestingly, a distinct two-hit activation process has been recently described for human respiratory syncytial virus, another paramyxovirus.

  5. Specific RNA binding by amino-terminal peptides of alfalfa mosaic virus coat protein.

    PubMed Central

    Baer, M L; Houser, F; Loesch-Fries, L S; Gehrke, L

    1994-01-01

    Specific RNA-protein interactions and ribonucleoprotein complexes are essential for many biological processes, but our understanding of how ribonucleoprotein particles form and accomplish their biological functions is rudimentary. This paper describes the interaction of alfalfa mosaic virus (A1MV) coat protein or peptides with viral RNA. A1MV coat protein is necessary both for virus particle formation and for the initiation of replication of the three genomic RNAs. We have examined protein determinants required for specific RNA binding and analyzed potential structural changes elicited by complex formation. The results indicate that the amino-terminus of the viral coat protein, which lacks primary sequence homology with recognized RNA binding motifs, is both necessary and sufficient for binding to RNA. Circular dichroism spectra and electrophoretic mobility shift experiments suggest that the RNA conformation is altered when amino-terminal coat protein peptides bind to the viral RNA. The peptide--RNA interaction is functionally significant because the peptides will substitute for A1MV coat protein in initiating RNA replication. The apparent conformational change that accompanies RNA--peptide complex formation may generate a structure which, unlike the viral RNA alone, can be recognized by the viral replicase. Images PMID:8313916

  6. Repression of HNF1α-mediated transcription by amino-terminal enhancer of split (AES)

    SciTech Connect

    Han, Eun Hee; Gorman, Amanda A.; Singh, Puja; Chi, Young-In

    2015-12-04

    HNF1α (Hepatocyte Nuclear Factor 1α) is one of the master regulators in pancreatic beta-cell development and function, and the mutations in Hnf1α are the most common monogenic causes of diabetes mellitus. As a member of the POU transcription factor family, HNF1α exerts its gene regulatory function through various molecular interactions; however, there is a paucity of knowledge in their functional complex formation. In this study, we identified the Groucho protein AES (Amino-terminal Enhancer of Split) as a HNF1α-specific physical binding partner and functional repressor of HNF1α-mediated transcription, which has a direct link to glucose-stimulated insulin secretion in beta-cells that is impaired in the HNF1α mutation-driven diabetes. - Highlights: • We identified AES as a transcriptional repressor for HNF1α in pancreatic beta-cell. • AES's repressive activity was HNF1α-specific and was not observed with HNF1β. • AES interacts with the transactivation domain of HNF1α. • Small molecules can be designed or discovered to disrupt this interaction and improve insulin secretion and glucose homeostasis.

  7. Herbal compound triptolide synergistically enhanced antitumor activity of amino-terminal fragment of urokinase

    PubMed Central

    2013-01-01

    Background Urokinase (uPA) and its receptor (uPAR) play an important role in tumour growth and metastasis, and overexpression of these molecules is strongly correlated with poor prognosis in a variety of malignant tumours. Targeting the excessive activation of this system as well as the proliferation of the tumour vascular endothelial cell would be expected to prevent tumour neovasculature and halt tumour development. The amino terminal fragment (ATF) of urokinase has been confirmed effective to inhibit the proliferation, migration and invasiveness of cancer cells via interrupting the interaction of uPA and uPAR. Triptolide (TPL) is a purified diterpenoid isolated from the Chinese herb Tripterygium wilfordii Hook F that has shown antitumor activities in various cancer cell types. However, its therapeutic application is limited by its toxicity in normal tissues and complications caused in patients. In this study, we attempted to investigate the synergistic anticancer activity of TPL and ATF in various solid tumour cells. Methods Using in vitro and in vivo experiments, we investigated the combined effect of TPL and ATF at a low dosage on cell proliferation, cell apoptosis, cell cycle distribution, cell migration, signalling pathways, xenograft tumour growth and angiogenesis. Results Our data showed that the sensitivity of a combined therapy using TPL and ATF was higher than that of TPL or ATF alone. Suppression of NF-κB transcriptional activity, activation of caspase-9/caspase-3, cell cycle arrest, and inhibition of uPAR-mediated signalling pathway contributed to the synergistic effects of this combination therapy. Furthermore, using a mouse xenograft model, we demonstrated that the combined treatment completely suppressed tumour growth by inhibiting angiogenesis as compared with ATF or TPL treatment alone. Conclusions Our study suggests that lower concentration of ATF and TPL used in combination may produce a synergistic anticancer efficacy that warrants further

  8. Neurosteroid binding to the amino terminal and glutamate binding domains of ionotropic glutamate receptors.

    PubMed

    Cameron, Krasnodara; Bartle, Emily; Roark, Ryan; Fanelli, David; Pham, Melissa; Pollard, Beth; Borkowski, Brian; Rhoads, Sarah; Kim, Joon; Rocha, Monica; Kahlson, Martha; Kangala, Melinda; Gentile, Lisa

    2012-06-01

    The endogenous neurosteroids, pregnenolone sulfate (PS) and 3α-hydroxy-5β-pregnan-20-one sulfate (PREGAS), have been shown to differentially regulate the ionotropic glutamate receptor (iGluR) family of ligand-gated ion channels. Upon binding to these receptors, PREGAS decreases current flow through the channels. Upon binding to non-NMDA or NMDA receptors containing an GluN2C or GluN2D subunit, PS also decreases current flow through the channels, however, upon binding to NMDA receptors containing an GluN2A or GluN2B subunit, flow through the channels increases. To begin to understand this differential regulation, we have cloned the S1S2 and amino terminal domains (ATD) of the NMDA GluN2B and GluN2D and AMPA GluA2 subunits. Here we present results that show that PS and PREGAS bind to different sites in the ATD of the GluA2 subunit, which when combined with previous results from our lab, now identifies two binding domains for each neurosteroid. We also show both neurosteroids bind only to the ATD of the GluN2D subunit, suggesting that this binding is distinct from that of the AMPA GluA2 subunit, with both leading to iGluR inhibition. Finally, we provide evidence that both PS and PREGAS bind to the S1S2 domain of the NMDA GluN2B subunit. Neurosteroid binding to the S1S2 domain of NMDA subunits responsible for potentiation of iGluRs and to the ATD of NMDA subunits responsible for inhibition of iGluRs, provides an interesting option for therapeutic design.

  9. The conserved amino-terminal domain of hSRP1 alpha is essential for nuclear protein import.

    PubMed Central

    Weis, K; Ryder, U; Lamond, A I

    1996-01-01

    Nuclear proteins are targeted through the nuclear pore complex (NPC) in an energy-dependent reaction. The import reaction is mediated by nuclear localization sequences (NLS) in the substrate which are recognized by heterodimeric cytoplasmic receptors. hSRP1 alpha is an NLS-binding subunit of the human NLS receptor complex and is complexed in vivo with a second subunit of 97 kDa (p97). We show here that a short amino-terminal domain in hSRP1 alpha is necessary and sufficient for its interaction with p97. This domain is conserved in other SRP1-like proteins and its fusion to a cytoplasmic reporter protein is sufficient to promote complete nuclear import, circumventing the usual requirement for an NLS receptor interaction. The same amino-terminal domain inhibits import of NLS-containing proteins when added to an in vitro nuclear transport assay. While full-length hSRP alpha is able to leave the nucleus, the amino-terminal domain alone is not sufficient to promote exit. We conclude that hSRP1 alpha functions as an adaptor to tether NLS-containing substrates to the protein import machinery. Images PMID:8617227

  10. Insights into the Conformation of the Membrane Proximal Regions Critical to the Trimerization of the HIV-1 gp41 Ectodomain Bound to Dodecyl Phosphocholine Micelles

    PubMed Central

    Louis, John M.; Baber, James L.; Ghirlando, Rodolfo; Aniana, Annie; Bax, Ad; Roche, Julien

    2016-01-01

    The transitioning of the ectodomain of gp41 from a pre-hairpin to a six-helix bundle conformation is a crucial aspect of virus-cell fusion. To gain insight into the intermediary steps of the fusion process we have studied the pH and dodecyl phosphocholine (DPC) micelle dependent trimer association of gp41 by systematic deletion analysis of an optimized construct termed 17–172 (residues 528 to 683 of Env) that spans the fusion peptide proximal region (FPPR) to the membrane proximal external region (MPER) of gp41, by sedimentation velocity and double electron-electron resonance (DEER) EPR spectroscopy. Trimerization at pH 7 requires the presence of both the FPPR and MPER regions. However, at pH 4, the protein completely dissociates to monomers. DEER measurements reveal a partial fraying of the C-terminal MPER residues in the 17–172 trimer while the other regions, including the FPPR, remain compact. In accordance, truncating nine C-terminal MPER residues (675–683) in the 17–172 construct does not shift the trimer-monomer equilibrium significantly. Thus, in the context of the gp41 ectodomain spanning residues 17–172, trimerization is clearly dependent on FPPR and MPER regions even when the terminal residues of MPER unravel. The antibody Z13e1, which spans both the 2F5 and 4E10 epitopes in MPER, binds to 17–172 with a Kd of 1 ± 0.12 μM. Accordingly, individual antibodies 2F5 and 4E10 also recognize the 17–172 trimer/DPC complex. We propose that binding of the C-terminal residues of MPER to the surface of the DPC micelles models a correct positioning of the trimeric transmembrane domain anchored in the viral membrane. PMID:27513582

  11. An enhanced recombinant amino-terminal acetylation system and novel in vivo high-throughput screen for molecules affecting α-synuclein oligomerisation.

    PubMed

    Eastwood, Tara A; Baker, Karen; Brooker, Holly R; Frank, Stefanie; Mulvihill, Daniel P

    2017-03-01

    Amino-terminal acetylation is a ubiquitous protein modification affecting the majority of eukaryote proteins to regulate stability and function. We describe an optimised recombinant expression system for rapid production of amino terminal-acetylated proteins within bacteria. We go on to describe the system's use in a fluorescence based in vivo assay for use in the high-throughput screen to identify drugs that impact amino-terminal acetylation-dependent oligomerisation. These new tools and protocols will allow researchers to enhance routine recombinant protein production and identify new molecules for use in research and clinical applications.

  12. Analysis of high-affinity assembly for AMPA receptor amino-terminal domains

    PubMed Central

    Zhao, Huaying; Berger, Anthony J.; Brown, Patrick H.; Kumar, Janesh; Balbo, Andrea; May, Carrie A.; Casillas, Ernesto; Laue, Thomas M.; Patterson, George H.

    2012-01-01

    Analytical ultracentrifugation (AUC) and steady-state fluorescence anisotropy were used to measure the equilibrium dissociation constant (Kd) for formation of dimers by the amino-terminal domains (ATDs) of the GluA2 and GluA3 subtypes of AMPA receptor. Previous reports on GluA2 dimerization differed in their estimate of the monomer–dimer Kd by a 2,400-fold range, with no consensus on whether the ATD forms tetramers in solution. We find by sedimentation velocity (SV) analysis performed using absorbance detection a narrow range of monomer–dimer Kd values for GluA2, from 5 to 11 nM for six independent experiments, with no detectable formation of tetramers and no effect of glycosylation or the polypeptide linker connecting the ATD and ligand-binding domains; for GluA3, the monomer–dimer Kd was 5.6 µM, again with no detectable tetramer formation. For sedimentation equilibrium (SE) experiments, a wide range of Kd values was obtained for GluA2, from 13 to 284 nM, whereas for GluA3, the Kd of 3.1 µM was less than twofold different from the SV value. Analysis of cell contents after the ∼1-week centrifuge run by silver-stained gels revealed low molecular weight GluA2 breakdown products. Simulated data for SE runs demonstrate that the apparent Kd for GluA2 varies with the extent of proteolysis, leading to artificially high Kd values. SV experiments with fluorescence detection for GluA2 labeled with 5,6-carboxyfluorescein, and fluorescence anisotropy measurements for GluA2 labeled with DyLight405, yielded Kd values of 5 and 11 nM, consistent with those from SV with absorbance detection. However, the sedimentation coefficients measured by AUC using absorbance and fluorescence systems were strikingly different, and for the latter are not consistent with hydrodynamic protein models. Thus, for unknown reasons, the concentration dependence of sedimentation coefficients obtained with fluorescence detection SV may be unreliable, limiting the usefulness of this technique for

  13. The membrane topology of the amino-terminal domain of the red cell calcium pump.

    PubMed Central

    Castello, P. R.; González Flecha, F. L.; Caride, A. J.; Fernández, H. N.; Delfino, J. M.; Rossi, J. P.

    1997-01-01

    A systematic study of the membrane-associated regions in the plasma membrane Ca2+ pump of erythrocytes has been performed by hydrophobic photolabeling. Purified Ca2+ pump was labeled with 3-(trifluoromethyl)-3-(m-[125I]iodophenyl)-diazirine ([125I]TID), a generic photoactivatable hydrophobic probe. These results were compared with the enzyme labeled with a strictly membrane-bound probe, [3H]bis-phosphatidylethanolamine (trifluoromethyl) phenyldiazirine. A significant light-dependent labeling of an M(r) 135,000-140,000 peptide, corresponding to the full Ca2+ pump, was observed with both probes. After proteolysis of the pump labeled with each probe and isolation of fragments by SDS-PAGE, a common pattern of labeled peptides was observed. Similarly, labeling of the Ca2+ pump with [125I]TID, either in isolated red blood cell membranes or after the enzyme was purified, yields a similar pattern of labeled peptides. Taken together, these results validate the use of either probe to study the lipid interface of the membrane-embedded region of this protein, and sustain the notion that the conformation of the pump is maintained throughout the procedures of solubilization, affinity purification, and reconstitution into proteoliposomes. In this work, we put special emphasis on a detailed analysis of the N-terminal domain of the Ca2+ pump. A labeled peptide of M(r) 40,000 belonging to this region was purified and further digested with V8 protease. The specific incorporation of [125I]TID to proteolytic fragments pertaining to the amino-terminal region indicates the existence of two transmembrane stretches in this domain. A theoretical analysis based on the amino acid sequence 1-322 predicts two segments with high probability of membrane insertion, in agreement with the experimental data. Each segment shows a periodicity pattern of hydrophobicity and variability compatible with alpha-helical structure. These results strongly suggest the existence of a transmembrane helical hairpin

  14. Dissection of Amino-Terminal Functional Domains of Murine Coronavirus Nonstructural Protein 3

    PubMed Central

    Hurst-Hess, Kelley R.; Kuo, Lili

    2015-01-01

    ABSTRACT Coronaviruses, the largest RNA viruses, have a complex program of RNA synthesis that entails genome replication and transcription of subgenomic mRNAs. RNA synthesis by the prototype coronavirus mouse hepatitis virus (MHV) is carried out by a replicase-transcriptase composed of 16 nonstructural protein (nsp) subunits. Among these, nsp3 is the largest and the first to be inserted into the endoplasmic reticulum. nsp3 comprises multiple structural domains, including two papain-like proteases (PLPs) and a highly conserved ADP-ribose-1″-phosphatase (ADRP) macrodomain. We have previously shown that the ubiquitin-like domain at the amino terminus of nsp3 is essential and participates in a critical interaction with the viral nucleocapsid protein early in infection. In the current study, we exploited atypical expression schemes to uncouple PLP1 from the processing of nsp1 and nsp2 in order to investigate the requirements of nsp3 domains for viral RNA synthesis. In the first strategy, a mutant was created in which replicase polyprotein translation initiated with nsp3, thereby establishing that complete elimination of nsp1 and nsp2 does not abolish MHV viability. In the second strategy, a picornavirus autoprocessing element was used to separate a truncated nsp1 from nsp3. This provided a platform for further dissection of amino-terminal domains of nsp3. From this, we found that catalytic mutation of PLP1 or complete deletion of PLP1 and the adjacent ADRP domain was tolerated by the virus. These results showed that neither the PLP1 domain nor the ADRP domain of nsp3 provides integral activities essential for coronavirus genomic or subgenomic RNA synthesis. IMPORTANCE The largest component of the coronavirus replicase-transcriptase complex, nsp3, contains multiple modules, many of which do not have clearly defined functions in genome replication or transcription. These domains may play direct roles in RNA synthesis, or they may have evolved for other purposes, such as

  15. Amino-terminal pro-brain natriuretic peptide in children with latent rheumatic heart disease

    PubMed Central

    Zachariah, Justin P; Aliku, Twalib; Scheel, Amy; Hasan, Babar S; Lwabi, Peter; Sable, Craig; Beaton, Andrea Z

    2016-01-01

    Background: Rheumatic heart disease (RHD) is a global cause of early heart failure. Early RHD is characterized by valvar regurgitation, leading to ventricular distention and possible elaboration of amino-terminal pro-brain natriuretic peptide (NT-proBNP). We investigated the ability of NT-proBNP to distinguish cases of latent RHD detected by echocardiographic screening from the controls. Materials and Methods: Ugandan children (N = 44, 36% males, mean age: 12 ± 2 years) with latent RHD (cases) and siblings (controls) by echocardiography were enrolled. Cases and controls were matched for age and sex, and they had normal hemoglobin (mean: 12.8 mg/dL). Children with congenital heart disease, pregnancy, left ventricular dilation or ejection fraction (EF) below 55%, or other acute or known chronic health conditions were excluded. RHD cases were defined by the World Heart Federation (WHF) 2012 consensus guideline criteria as definite. Controls had no echocardiography (echo) evidence for RHD. At the time of echo, venous blood samples were drawn and stored as serum. NT-proBNP levels were measured using sandwich immunoassay. Paired t-tests were used to compare NT-proBNP concentrations including sex-specific analyses. Results: The mean NT-proBNP concentration in the cases was 105.74 ± 67.21 pg/mL while in the controls, it was 86.63 ± 55.77 pg/mL. The cases did not differ from the controls (P = 0.3). In sex-specific analyses, male cases differed significantly from the controls (158.78 ± 68.82 versus 76 ± 42.43, P = 0.008). Female cases did not differ from the controls (75.44 ± 45.03 versus 92.30 ± 62.35 respectively, P = 0.4). Conclusion: Serum NT-proBNP did not distinguish between latent RHD cases and the controls. Sex and within-family exposures may confound this result. More investigation into biomarker-based RHD detection is warranted. PMID:27212845

  16. Structural Characterization of HIV gp41 with the Membrane-proximal External Region

    SciTech Connect

    Shi, W.; Bohon, J; Han, D; Habte, H; Qin, Y; Cho, M; Chance, M

    2010-01-01

    Human immunodeficiency virus, type 1 (HIV-1) envelope glycoprotein (gp120/gp41) plays a critical role in virus infection and pathogenesis. Three of the six monoclonal antibodies considered to have broadly neutralizing activities (2F5, 4E10, and Z13e1) bind to the membrane-proximal external region (MPER) of gp41. This makes the MPER a desirable template for developing immunogens that can elicit antibodies with properties similar to these monoclonal antibodies, with a long term goal of developing antigens that could serve as novel HIV vaccines. In order to provide a structural basis for rational antigen design, an MPER construct, HR1-54Q, was generated for x-ray crystallographic and x-ray footprinting studies to provide both high resolution atomic coordinates and verification of the solution state of the antigen, respectively. The crystal structure of HR1-54Q reveals a trimeric, coiled-coil six-helical bundle, which probably represents a postfusion form of gp41. The MPER portion extends from HR2 in continuation of a slightly bent long helix and is relatively flexible. The structures observed for the 2F5 and 4E10 epitopes agree well with existing structural data, and enzyme-linked immunosorbent assays indicate that the antigen binds well to antibodies that recognize the above epitopes. Hydroxyl radical-mediated protein footprinting of the antigen in solution reveals specifically protected and accessible regions consistent with the predictions based on the trimeric structure from the crystallographic data. Overall, the HR1-54Q antigen, as characterized by crystallography and footprinting, represents a postfusion, trimeric form of HIV gp41, and its structure provides a rational basis for gp41 antigen design suitable for HIV vaccine development.

  17. Amino-terminal sequence of p36 and associated p10: identification of the site of tyrosine phosphorylation and homology with S-100.

    PubMed Central

    Glenney, J R; Tack, B F

    1985-01-01

    p36 is a major substrate of both viral and growth factor-receptor-associated tyrosine protein kinases. p36 can be isolated as a complex consisting of a subunit of Mr 36,000 (p36) and a subunit of Mr 10,000 (p10), and it represents an abundant cellular protein. We have isolated the p36-p10 complex from bovine intestinal epithelium and analyzed the amino terminus of both subunits. Sequence analysis of the first 56 amino acids of p10 demonstrates a striking sequence homology (48% identically placed residues) with the Mr 10,000 calcium-binding proteins from bovine brain, termed S-100. Intestinal p36 could be effectively labeled on a single tyrosine in vitro with immunoprecipitated pp60v-src and [gamma-32P]ATP. Mild proteolysis of p36 with chymotrypsin resulted in the cleavage into large (Mr, 33,000) and small domains (Mr, 3000), with the latter representing the phosphorylated amino terminus. Although the amino terminus is apparently blocked, sequence analysis of a secondary tryptic peptide of the Mr 3000 fragment as well as the amino-terminal sequence of the Mr 33,000 domain and overlapping peptides clearly established the site of tyrosine phosphorylation. Images PMID:2415974

  18. Osteoarthritis-associated basic calcium phosphate crystals activate membrane proximal kinases in human innate immune cells.

    PubMed

    Corr, Emma M; Cunningham, Clare C; Helbert, Laura; McCarthy, Geraldine M; Dunne, Aisling

    2017-02-07

    Osteoarthritis (OA) is a chronic debilitating joint disorder of particularly high prevalence in the elderly population. Intra-articular basic calcium phosphate (BCP) crystals are present in the majority of OA joints and are associated with severe degeneration. They are known to activate macrophages, synovial fibroblasts, and articular chondrocytes, resulting in increased cell proliferation and the production of pro-inflammatory cytokines and matrix metalloproteases (MMPs). This suggests a pathogenic role in OA by causing extracellular matrix degradation and subchondral bone remodelling. There are currently no disease-modifying drugs available for crystal-associated OA; hence, the aim of this study was to explore the inflammatory pathways activated by BCP crystals in order to identify potential therapeutic targets to limit crystal-induced inflammation. Primary human macrophages and dendritic cells were stimulated with BCP crystals, and activation of spleen tyrosine kinase (Syk), phosphoinositide-3 kinase (PI3K), and mitogen-activated protein kinases (MAPKs) was detected by immunoblotting. Lipopolysaccharide (LPS)-primed macrophages were pre-treated with inhibitors of Syk, PI3K, and MAPKs prior to BCP stimulation, and cytokine production was quantified by enzyme-linked immunosorbent assay (ELISA). Aa an alternative, cells were treated with synovial fluid derived from osteoarthritic knees in the presence or absence of BCP crystals, and gene induction was assessed by real-time polymerase chain reaction (PCR). We demonstrate that exposure of primary human macrophages and dendritic cells to BCP crystals leads to activation of the membrane-proximal tyrosine kinases Syk and PI3K. Furthermore, we show that production of the pro-inflammatory cytokines interleukin (IL)-1α and IL-1β and phosphorylation of downstream MEK and ERK MAPKs is suppressed following treatment with inhibitors of Syk or PI3K. Finally, we demonstrate that treatment of macrophages with BCP crystals

  19. The membrane proximal external regions of gp41 from HIV-1 strains HXB2 and JRFL have different sensitivities to alanine mutation.

    PubMed

    Yi, Hyun Ah; Diaz-Rohrer, Barbara; Saminathan, Priyanka; Jacobs, Amy

    2015-03-03

    The transmembrane subunit (gp41) of the HIV envelope protein complex (Env) mediates the viral fusion step of HIV entry. The membrane proximal external region (MPER), one of the functional domains of gp41, has been the focus of a great deal of research because it is a target for neutralizing antibodies. In this study, we examined 23 amino acid residues in the MPER (660-683) in both a CXCR4 coreceptor-utilizing strain (HXB2) and a CCR5-utilizing strain (JRFL) by alanine scanning mutagenesis. Despite the high degree of gp41 sequence conservation, the effects of alanine mutation in the MPER were different between the two strains. Most mutations in HXB2 had fusogenicity and protein expression levels not less than 50% of that of the wild type in the case of cell-cell fusion. However, ∼30% of the mutants in HXB2 showed a severe defect in fusogenicity in viral entry. Mutations in the MPER of strain JRFL had more dramatic effects than that in HXB2 in cell-cell fusion and viral entry. The fact that there are large differences in the effects of mutation between two strains suggests the potential for the interaction of the MPER with nonconserved sequences such as the fusion peptide and/or other NHR domains as well as potential long-range structural effects on the conformational changes that occur with the Env complex during membrane fusion.

  20. Characterization of P0, a ribosomal phosphoprotein of Plasmodium falciparum. Antibody against amino-terminal domain inhibits parasite growth.

    PubMed

    Goswami, A; Singh, S; Redkar, V D; Sharma, S

    1997-05-02

    A cDNA expression clone of the human malarial parasite Plasmodium falciparum, lambdaPf4, which was reactive only to the immune sera and not to the patient sera, has recently been found to be the P. falciparum homologue of the P0 ribosomal phosphoprotein gene. A Northern analysis of the P0 gene revealed the presence of two transcripts, both present in all the different intraerythrocytic stages of the parasite life cycle. A 138-base pair amino-terminal domain of this gene was expressed as a fusion protein with glutathione S-transferase in Escherichia coli. Polyclonal antibodies raised against this domain immunoprecipitated the expected 38-kDa P0 protein from the 35S-labeled as well as 32P-labeled P. falciparum cultures. Monospecific human immune sera affinity-purified using the expression clone lambdaPf4 also immunoprecipitated the same size protein from [35S]methionine-labeled P. falciparum protein extract. Purified IgG from polyclonal antibodies raised against the amino-terminal domain of P0 protein completely inhibited the growth of P. falciparum in vitro. This inhibition appears to be mainly at the step of erythrocyte invasion by the parasites.

  1. Identification of platelet membrane proteins that interact with amino-terminal peptides of pp60c-src.

    PubMed

    Feder, D; Bishop, J M

    1991-10-05

    Platelets contain exceptionally high levels of pp60c-src and, thus, provide a convenient system for investigating the physiological function of this protein-tyrosine kinase. We have employed chemical cross-linking of myristylated amino-terminal peptides of pp60c-src to platelet membranes in order to identify platelet membrane components that interact with pp60c-src to regulate or mediate its activity. We detected specific binding of radioiodinated peptides to platelet membrane proteins of 32, 50, 92, and 105 kDa. The 32-kDa protein may be related to the putative src receptor component recently identified in fibroblast membranes. The most reactive platelet protein, however, is the 50-kDa protein, which is either absent or nonreactive in fibroblast membranes. Binding of src peptides to this protein was saturable, and we estimate the presence of approximately 1 x 10(6) of the 50-kDa binding sites per platelet. The specificity of the peptide binding to the 50- and 32-kDa platelet proteins was analyzed by competition with different peptides. The binding sites displayed an absolute requirement for an N-myristoyl moiety and a strong preference for pp60c-src amino-terminal sequences. The identification of these src-interacting proteins may help to decipher the biochemical pathways in which platelet pp60c-src is involved.

  2. Membrane Association via an Amino-terminal Amphipathic Helix Is Required for the Cellular Organization and Function of RNase II*

    PubMed Central

    Lu, Feng; Taghbalout, Aziz

    2013-01-01

    The subcellular localization of the exoribonuclease RNase II is not known despite the advanced biochemical characterization of the enzyme. Here we report that RNase II is organized into cellular structures that appear to coil around the Escherichia coli cell periphery and that RNase II is associated with the cytoplasmic membrane by its amino-terminal amphipathic helix. The helix also acts as an autonomous transplantable membrane binding domain capable of directing normally cytoplasmic proteins to the membrane. Assembly of the organized cellular structures of RNase II required the RNase II amphipathic membrane binding domain. Co-immunoprecipitation of the protein from cell extracts indicated that RNase II interacts with itself. The RNase II self-interaction and the ability of the protein to assemble into organized cellular structures required the membrane binding domain. The ability of RNase II to maintain cell viability in the absence of the exoribonuclease polynucleotide phosphorylase was markedly diminished when the RNase II cellular structures were lost due to changes in the amphipathicity of the amino-terminal helix, suggesting that membrane association and assembly of RNase II into organized cellular structures play an important role in the normal function of the protein within the bacterial cell. PMID:23344958

  3. Membrane association via an amino-terminal amphipathic helix is required for the cellular organization and function of RNase II.

    PubMed

    Lu, Feng; Taghbalout, Aziz

    2013-03-08

    The subcellular localization of the exoribonuclease RNase II is not known despite the advanced biochemical characterization of the enzyme. Here we report that RNase II is organized into cellular structures that appear to coil around the Escherichia coli cell periphery and that RNase II is associated with the cytoplasmic membrane by its amino-terminal amphipathic helix. The helix also acts as an autonomous transplantable membrane binding domain capable of directing normally cytoplasmic proteins to the membrane. Assembly of the organized cellular structures of RNase II required the RNase II amphipathic membrane binding domain. Co-immunoprecipitation of the protein from cell extracts indicated that RNase II interacts with itself. The RNase II self-interaction and the ability of the protein to assemble into organized cellular structures required the membrane binding domain. The ability of RNase II to maintain cell viability in the absence of the exoribonuclease polynucleotide phosphorylase was markedly diminished when the RNase II cellular structures were lost due to changes in the amphipathicity of the amino-terminal helix, suggesting that membrane association and assembly of RNase II into organized cellular structures play an important role in the normal function of the protein within the bacterial cell.

  4. Species-specific epitopes exist on the cytoplasmic amino-terminal domain of erythrocyte band 3 protein.

    PubMed

    Kimura, A; Uda, T; Nakashima, S; Ikeda, H; Yasuda, S; Osawa, M; Tsuji, T

    1992-08-01

    Monoclonal antibodies (P3-9H, P3-1F, P3-2H, P3-4A, and P3-4C) to human erythrocyte band 3 were produced using human erythrocyte membranes as the immunogen. All epitopes defined by these antibodies were found on the amino-terminal cytoplasmic domain of erythrocyte band 3. The antibodies crossreacted variously with erythrocyte band 3 of primates (chimpanzee, orangutan, Rhesus monkey, Japanese monkey, spider monkey, and capuchin monkey) in enzyme-linked immunosorbent assay. P3-9H did not crossreact with erythrocyte band 3 of any primate examined; P3-1F crossreacted only with that of chimpanzee; P3-2H crossreacted with erythrocyte band 3 of chimpanzee, spider monkey, and capuchin monkey; and P3-4A and P3-4C crossreacted with erythrocyte band 3 of all primates examined. These results suggest that evolutional changes in primates are accumulated in the amino-terminal cytoplasmic domain of band 3 and that species-specific epitopes exist on this domain.

  5. The Arabidopsis Chloroplast Stromal N-Terminome: Complexities of Amino-Terminal Protein Maturation and Stability.

    PubMed

    Rowland, Elden; Kim, Jitae; Bhuiyan, Nazmul H; van Wijk, Klaas J

    2015-11-01

    Protein amino (N) termini are prone to modifications and are major determinants of protein stability in bacteria, eukaryotes, and perhaps also in chloroplasts. Most chloroplast proteins undergo N-terminal maturation, but this is poorly understood due to insufficient experimental information. Consequently, N termini of mature chloroplast proteins cannot be accurately predicted. This motivated an extensive characterization of chloroplast protein N termini in Arabidopsis (Arabidopsis thaliana) using terminal amine isotopic labeling of substrates and mass spectrometry, generating nearly 14,000 tandem mass spectrometry spectra matching to protein N termini. Many nucleus-encoded plastid proteins accumulated with two or three different N termini; we evaluated the significance of these different proteoforms. Alanine, valine, threonine (often in N-α-acetylated form), and serine were by far the most observed N-terminal residues, even after normalization for their frequency in the plastid proteome, while other residues were absent or highly underrepresented. Plastid-encoded proteins showed a comparable distribution of N-terminal residues, but with a higher frequency of methionine. Infrequent residues (e.g. isoleucine, arginine, cysteine, proline, aspartate, and glutamate) were observed for several abundant proteins (e.g. heat shock proteins 70 and 90, Rubisco large subunit, and ferredoxin-glutamate synthase), likely reflecting functional regulation through their N termini. In contrast, the thylakoid lumenal proteome showed a wide diversity of N-terminal residues, including those typically associated with instability (aspartate, glutamate, leucine, and phenylalanine). We propose that, after cleavage of the chloroplast transit peptide by stromal processing peptidase, additional processing by unidentified peptidases occurs to avoid unstable or otherwise unfavorable N-terminal residues. The possibility of a chloroplast N-end rule is discussed.

  6. A Simple Procedure for Constructing 5'-Amino-Terminated Oligodeoxynucleotides in Aqueous Solution

    NASA Technical Reports Server (NTRS)

    Bruick, Richard K.; Koppitz, Marcus; Joyce, Gerald F.; Orgel, Leslie E.

    1997-01-01

    A rapid method for the synthesis of oligodeoxynucleotides (ODNs) terminated by 5'-amino-5'-deoxythymidine is described. A 3'-phosphorylated ODN (the donor) is incubated in aqueous solution with 5'-amino- 5'-deoxythymidine in the presence of N-(3-dimethylaminopropyl)-)N'-ethylcarbodiimide hydrochloride (EDC), extending the donor by one residue via a phosphoramidate bond. Template- directed ligation of the extended donor and an acceptor ODN, followed by acid hydrolysis, yields the acceptor ODN extended by a single 5'-amino-5'-deoxythymidine residue at its 5'terminus.

  7. The Stem of Vesicular Stomatitis Virus G Can Be Replaced With the HIV-1 Env Membrane-Proximal External Region Without Loss of G Function or Membrane-Proximal External Region Antigenic Properties

    PubMed Central

    Lorenz, Ivo C.; Nguyen, Hanh T.; Kemelman, Marina; Lindsay, Ross W.; Yuan, Maoli; Wright, Kevin J.; Arendt, Heather; Back, Jaap Willem; DeStefano, Joanne; Hoffenberg, Simon; Morrow, Gavin; Jurgens, Christy K.; Phogat, Sanjay K.; Zamb, Timothy J.

    2014-01-01

    Abstract The structure of the HIV-1 envelope membrane-proximal external region (MPER) is influenced by its association with the lipid bilayer on the surface of virus particles and infected cells. To develop a replicating vaccine vector displaying MPER sequences in association with membrane, Env epitopes recognized by the broadly neutralizing antibodies 2F5, 4E10, or both were grafted into the membrane-proximal stem region of the vesicular stomatitis virus (VSV) glycoprotein (G). VSV encoding functional G-MPER chimeras based on G from the Indiana or New Jersey serotype propagated efficiently, although grafting of both epitopes (G-2F5-4E10) modestly reduced replication and resulted in the acquisition of one to two adaptive mutations in the grafted MPER sequence. Monoclonal antibodies 2F5 and 4E10 efficiently neutralized VSV G-MPER vectors and bound to virus particles in solution, indicating that the epitopes were accessible in the preattachment form of the G-MPER chimeras. Overall, our results showed that the HIV Env MPER could functionally substitute for the VSV G-stem region implying that both perform similar functions even though they are from unrelated viruses. Furthermore, we found that the MPER sequence grafts induced low but detectable MPER-specific antibody responses in rabbits vaccinated with live VSV, although additional vector and immunogen modifications or use of a heterologous prime-boost vaccination regimen will be required to increase the magnitude of the immune response. PMID:24597516

  8. Fusogenic activity of amino-terminal region of HIV type 1 Nef protein.

    PubMed

    Curtain, C C; Separovic, F; Rivett, D; Kirkpatrick, A; Waring, A J; Gordon, L M; Azad, A A

    1994-10-01

    We have studied two isoforms of Nef, Nef-27 and Nef-25, which were produced in E. coli. Nef-25 lacked the first 18 N-terminal residues of Nef-27 and both were nonmyristylated. Nef-27 fuses small unilamellar dipalmitoyl phosphatidylcholine vesicles (SUVs), as indicated by enhanced light scattering of SUVs and lipid mixing using concentration-dependent fluorescence dequenching. Nef-27 also causes the appearance of a shifted isotropic peak in the 31P NMR spectra of these vesicles, suggesting that protein interactions induce nonlamellar lipid structures. Recombinant Nef-25, which lacks only the 18 N-terminal residues of Nef-27, does not fuse vesicles and has little effect on the 31P NMR spectra. On the other hand, synthetic peptides consisting of 18 or 21 of the N-terminal residues of Nef-27 are strongly membrane perturbing, causing vesicle fusion and inducing isotropic peaks in the 31P NMR spectrum. Endogenous fluorescence spectra of the N-terminal peptide (21 residues) with SUVs show that the N-terminal sequence of Nef may achieve these perturbing effects by inserting its hydrophobic side into the lipid bilayer. Theoretical calculations using hydrophobic moment plot analysis indicate that short-length stretches (i.e., six amino acid residues) of the N-terminal sequence may insert into the lipid bilayer as multimeric alpha helices or beta sheets. The above-described membrane activities of Nef-27, which principally reside in its N-terminal domain, may play critical role(s) in certain functional properties of the full-length protein. For example, the fusogenic activity of the N-terminal sequence may be involved in the extracellular release of Nef-27, much of which appears to be associated with small membrane vesicles. The fusion activity may also be relevant to the ability of Nef-27 to downregulate CD4 and IL-2 receptors when this protein is electroporated into cultured lymphocytes, an activity not possessed by Nef-25.

  9. Amino-terminal sequence of glycoprotein D of herpes simplex virus types 1 and 2

    SciTech Connect

    Eisenberg, R.J.; Long, D.; Hogue-Angeletti, R.; Cohen, G.H.

    1984-01-01

    Glycoprotein D (gD) of herpes simplex virus is a structural component of the virion envelope which stimulates production of high titers of herpes simplex virus type-common neutralizing antibody. The authors caried out automated N-terminal amino acid sequencing studies on radiolabeled preparations of gD-1 (gD of herpes simplex virus type 1) and gD-2 (gD of herpes simplex virus type 2). Although some differences were noted, particularly in the methionine and alanine profiles for gD-1 and gD-2, the amino acid sequence of a number of the first 30 residues of the amino terminus of gD-1 and gD-2 appears to be quite similar. For both proteins, the first residue is a lysine. When we compared out sequence data for gD-1 with those predicted by nucleic acid sequencing, the two sequences could be aligned (with one exception) starting at residue 26 (lysine) of the predicted sequence. Thus, the first 25 amino acids of the predicted sequence are absent from the polypeptides isolated from infected cells.

  10. Terminal Interface Conformations Modulate Dimer Stability Prior to Amino Terminal Autoprocessing of HIV-1 Protease

    SciTech Connect

    Agniswamy, Johnson; Sayer, Jane M.; Weber, Irene T.; Louis, John M.

    2012-04-17

    The HIV-1 protease (PR) mediates its own release (autoprocessing) from the polyprotein precursor, Gag-Pol, flanked by the transframe region (TFR) and reverse transcriptase at its N- and C-termini, respectively. Autoprocessing at the N-terminus of PR mediates stable dimer formation essential for catalytic activity, leading to the formation of infectious virus. An antiparallel {beta}-sheet interface formed by the four N- and C-terminal residues of each subunit is important for dimer stability. Here, we present the first high-resolution crystal structures of model protease precursor-clinical inhibitor (PI darunavir or saquinavir) complexes, revealing varying conformations of the N-terminal flanking (S{sup -4}FNF{sup -1}) and interface residues (P{sup 1}QIT{sup 4}). A 180{sup o} rotation of the T{sup 4}-L{sup 5} peptide bond is accompanied by a new Q{sup 2}-L{sup 5} hydrogen bond and complete disengagement of PQIT from the {beta}-sheet dimer interface, which may be a feature for intramolecular autoprocessing. This result is consistent with drastically lower thermal stability by 14-20 C of PI complexes of precursors and the mature PR lacking its PQIT residues (by 18.3 C). Similar to the TFR-PR precursor, this deletion also results in a darunavir dissociation constant (2 x 10{sup 4})-fold higher and a markedly increased dimer dissociation constant relative to the mature PR. The terminal {beta}-sheet perturbations of the dimeric structure likely account for the drastically poorer inhibition of autoprocessing of TFR-PR relative to the mature PR, even though significant differences in active site-PI interactions in these structures were not observed. The novel conformations of the dimer interface may be exploited to target selectively the protease precursor prior to its N-terminal cleavage.

  11. The amino-terminal domain of yeast U1-70K is necessary and sufficient for function.

    PubMed Central

    Hilleren, P J; Kao, H Y; Siliciano, P G

    1995-01-01

    The Saccharomyces cerevisiae SNP1 gene encodes a protein that shares 30% amino acid identity with the mammalian U1 small nuclear ribonucleoprotein particle protein 70K (U1-70K). We have demonstrated that yeast strains in which the SNP1 gene was disrupted are viable but exhibit greatly increased doubling times and severe temperature sensitivity. Furthermore, snp1-null strains are defective in pre-mRNA splicing. We have tested deletion alleles of SNP1 for their ability to complement these phenotypes. We found that the highly conserved RNA recognition motif consensus domain of Snp1 is not required for complementation of the snp1-null growth or splicing defects nor for the in vivo association with the U1 small nuclear ribonucleoprotein particle. However, the amino-terminal domain of Snp1, less strongly conserved, is necessary and sufficient for complementation. PMID:7565787

  12. An evolutionary tree relating eight alphaviruses, based on amino-terminal sequences of their glycoproteins.

    PubMed Central

    Bell, J R; Kinney, R M; Trent, D W; Strauss, E G; Strauss, J H

    1984-01-01

    The NH2-terminal amino acid sequences of both structural glycoproteins of each of eight alphaviruses have been obtained. These sequences demonstrate that the alphaviruses are all closely related and have in all probability descended from a common ancestor. Cysteines are conserved as well as several other residues important for secondary structure, suggesting that the three-dimensional conformations of the alphavirus glycoproteins are conserved while considerable variation in the primary sequence has evolved. Secondary structure predictions based upon the amino acid sequences are consistent with this hypothesis. An evolutionary tree for these eight alphaviruses has been constructed from the amino acid sequence data and, at many positions in the sequence, the amino acids present in the ancestral glycoproteins have been deduced. Images PMID:6087344

  13. Structure of the flexible amino terminal domain of prion protein bound to a sulfated glycan

    PubMed Central

    Taubner, Lara M.; Bienkiewicz, Ewa A.; Copié, Valérie; Caughey, Byron

    2010-01-01

    The intrinsically disordered amino-proximal domain of hamster prion protein (PrP) contains four copies of a highly conserved octapeptide sequence PHGGGWGQ that is flanked by two polycationic residue clusters. This N-terminal domain mediates the binding of sulfated glycans, which can profoundly influence the conversion of PrP to pathological forms and the progression of prion disease. To investigate the structural consequences of sulfated glycan binding, we performed multidimensional heteronuclear (1H, 13C, 15N) nuclear magnetic resonance (NMR), circular dichroism (CD), and fluorescence studies on hamster PrP residues 23–106 (PrP 23–106) and fragments thereof when bound to pentosan polysulfate (PPS). While the majority of PrP 23–106 remains disordered upon PPS binding, the octarepeat region adopts a repeating loop-turn structure that we have determined by NMR. The β-like turns within the repeats are corroborated by CD data, which demonstrate that these turns are also present, although less pronounced, without PPS. Binding to PPS exposes a hydrophobic surface composed of aligned tryptophan sidechains, the spacing and orientation of which are consistent with a self-association or ligand binding site. The unique tryptophan motif was probed by intrinsic tryptophan fluorescence, which displayed enhanced fluorescence of PrP 23–106 when bound to PPS, consistent with the alignment of tryptophan sidechains. Chemical shift mapping identified binding sites on PrP 23–106 for PPS, which include the octarepeat histidine and an N-terminal basic cluster previously linked to sulfated glycan binding. These data may in part explain how sulfated glycans modulate PrP conformational conversions and oligomerizations. PMID:19913031

  14. Structure of the flexible amino-terminal domain of prion protein bound to a sulfated glycan.

    PubMed

    Taubner, Lara M; Bienkiewicz, Ewa A; Copié, Valérie; Caughey, Byron

    2010-01-22

    The intrinsically disordered amino-proximal domain of hamster prion protein (PrP) contains four copies of a highly conserved octapeptide sequence, PHGGGWGQ, that is flanked by two polycationic residue clusters. This N-terminal domain mediates the binding of sulfated glycans, which can profoundly influence the conversion of PrP to pathological forms and the progression of prion disease. To investigate the structural consequences of sulfated glycan binding, we performed multidimensional heteronuclear ((1)H, (13)C, (15)N) NMR (nuclear magnetic resonance), circular dichroism (CD), and fluorescence studies on hamster PrP residues 23-106 (PrP 23-106) and fragments thereof when bound to pentosan polysulfate (PPS). While the majority of PrP 23-106 remain disordered upon PPS binding, the octarepeat region adopts a repeating loop-turn structure that we have determined by NMR. The beta-like turns within the repeats are corroborated by CD data demonstrating that these turns are also present, although less pronounced, without PPS. Binding to PPS exposes a hydrophobic surface composed of aligned tryptophan side chains, the spacing and orientation of which are consistent with a self-association or ligand binding site. The unique tryptophan motif was probed by intrinsic tryptophan fluorescence, which displayed enhanced fluorescence of PrP 23-106 when bound to PPS, consistent with the alignment of tryptophan side chains. Chemical-shift mapping identified binding sites on PrP 23-106 for PPS, which include the octarepeat histidine and an N-terminal basic cluster previously linked to sulfated glycan binding. These data may in part explain how sulfated glycans modulate PrP conformational conversions and oligomerizations. Published by Elsevier Ltd.

  15. Homochiral preference in peptide synthesis in ribosome: role of amino terminal, peptidyl terminal, and U2620.

    PubMed

    Thirumoorthy, Krishnan; Nandi, Nilashis

    2007-08-23

    Experimental studies have shown that peptide synthesis in ribosome exhibits a homochiral preference. We present, for the first time, an analysis of the origin of the phenomenon using hybrid quantum chemical studies based on a model of peptidyl transferase center from the crystal structure of the ribosomal part of Haloarcula marismortui. The study quantitatively shows that the observed homochiral preference is due to the difference in the nonbonded interaction between amino acids at the A- and P-terminals as well as due to the difference in interaction with the U2620 residue. A major part of the discrimination comes from the variation of nonbonded interaction of rotating A-terminal during the approach of the former toward the P-terminal. The difference indicates that, during the rotatory motion between A- and P-terminals for the proximal positioning of the reactant for reaction to occur, the interaction for a L-L pair is far less repulsive compared to the same process for a D-L pair. The activation barriers for L-L and D-L pairs of the neutral state of phenylalanine leading to corresponding dipeptides are also compared. The corresponding difference in rate constants is 40-fold. The study provides an understanding of how preferred addition of L-L pairs of amino acids rather than D-L pairs leads to retention of homochirality in peptides.

  16. Transcytosis-blocking abs elicited by an oligomeric immunogen based on the membrane proximal region of HIV-1 gp41 target non-neutralizing epitopes.

    PubMed

    Matoba, Nobuyuki; Griffin, Tagan A; Mittman, Michele; Doran, Jeffrey D; Alfsen, Annette; Montefiori, David C; Hanson, Carl V; Bomsel, Morgane; Mor, Tsafrir S

    2008-05-01

    CTB-MPR(649-684), a translational fusion protein consisting of cholera toxin B subunit (CTB) and residues 649 684 of gp41 membrane proximal region (MPR), is a candidate vaccine aimed at blocking early steps of HIV-1 mucosal transmission. Bacterially produced CTB MPR(649-684) was purified to homogeneity by two affinity chromatography steps. Similar to gp41 and derivatives thereof, the MPR domain can specifically and reversibly self-associate. The affinities of the broadly-neutralizing monoclonal Abs 4E10 and 2F5 to CTB MPR(649-684) were equivalent to their nanomolar affinities toward an MPR peptide. The fusion protein's affinity to GM1 ganglioside was comparable to that of native CTB. Rabbits immunized with CTB-MPR(649-684) raised only a modest level of anti-MPR(649-684) Abs. However, a prime-boost immunization with CTB-MPR(649-684) and a second MPR(649-684)-based immunogen elicited a more productive anti-MPR(649-684) antibody response. These Abs strongly blocked the epithelial transcytosis of a primary subtype B HIV-1 isolate in a human tight epithelial model, expanding our previously reported results using a clade D virus. The Abs recognized epitopes at the N-terminal portion of the MPR peptide, away from the 2F5 and 4E10 epitopes and were not effective in neutralizing infection of CD4+ cells. These results indicate distinct vulnerabilities of two separate interactions of HIV-1 with human cells - Abs against the C-terminal portion of the MPR can neutralize CD4+-dependent infection, while Abs targeting the MPR's N-terminal portion can effectively block galactosyl ceramide dependent transcytosis. We propose that Abs induced by MPR(649-684)-based immunogens may provide broad protective value independent of infection neutralization.

  17. Natural Variation of the Amino-Terminal Glutamine-Rich Domain in Drosophila Argonaute2 Is Not Associated with Developmental Defects

    PubMed Central

    Hain, Daniel; Bettencourt, Brian R.; Okamura, Katsutomo; Csorba, Tibor; Meyer, Wibke; Jin, Zhigang; Biggerstaff, Jason; Siomi, Haruhiko; Hutvagner, Gyorgy; Lai, Eric C.

    2010-01-01

    The Drosophila argonaute2 (ago2) gene plays a major role in siRNA mediated RNA silencing pathways. Unlike mammalian Argonaute proteins, the Drosophila protein has an unusual amino-terminal domain made up largely of multiple copies of glutamine-rich repeats (GRRs). We report here that the ago2 locus produces an alternative transcript that encodes a putative short isoform without this amino-terminal domain. Several ago2 mutations previously reported to be null alleles only abolish expression of the long, GRR-containing isoform. Analysis of drop out (dop) mutations had previously suggested that variations in GRR copy number result in defects in RNAi and embryonic development. However, we find that dop mutations genetically complement transcript-null alleles of ago2 and that ago2 alleles with variant GRR copy numbers support normal development. In addition, we show that the assembly of the central RNAi machinery, the RISC (RNA induced silencing complex), is unimpaired in embryos when GRR copy number is altered. In fact, we find that GRR copy number is highly variable in natural D. melanogaster populations as well as in laboratory strains. Finally, while many other insects share an extensive, glutamine-rich Ago2 amino-terminal domain, its primary sequence varies drastically between species. Our data indicate that GRR variation does not modulate an essential function of Ago2 and that the amino-terminal domain of Ago2 is subject to rapid evolution. PMID:21253006

  18. Improved Bioactivity of Antimicrobial Peptides by Addition of Amino-Terminal Copper and Nickel (ATCUN) Binding Motifs

    PubMed Central

    Libardo, M. Daben; Cervantes, Jorge L.; Salazar, Juan C.; Angeles-Boza, Alfredo M.

    2015-01-01

    Antimicrobial peptides (AMPs) are promising candidates to help circumvent antibiotic resistance, which is an increasing clinical problem. Amino-terminal copper and nickel (ATCUN) binding motifs are known to actively form reactive oxygen species (ROS) upon metal binding. The combination of these two peptidic constructs could lead to a novel class of dual-acting antimicrobial agents. To test this hypothesis, a set of ATCUN binding motifs were screened for their ability to induce ROS formation, and the most potent were then used to modify AMPs with different modes of action. ATCUN binding motif-containing derivatives of anoplin (GLLKRIKTLL-NH2), pro-apoptotic peptide (PAP; KLAKLAKKLAKLAK-NH2), and sh-buforin (RAGLQFPVGRVHRLLRK-NH2) were synthesized and found to be more active than the parent AMPs against a panel of clinically relevant bacteria. The lower minimum inhibitory concentration (MIC) values for the ATCUN-anoplin peptides are attributed to the higher pore-forming activity along with their ability to cause ROS-induced membrane damage. The addition of the ATCUN motifs to PAP also increases its ability to disrupt membranes. DNA damage is the major contributor to the activity of the ATCUN-sh-buforin peptides. Our findings indicate that the addition of ATCUN motifs to AMPs is a simple strategy that leads to AMPs with higher antibacterial activity and possibly to more potent, usable antibacterial agents. PMID:24803240

  19. Exogenous expression of the amino-terminal half of the tight junction protein ZO-3 perturbs junctional complex assembly.

    PubMed

    Wittchen, E S; Haskins, J; Stevenson, B R

    2000-11-13

    The functional characteristics of the tight junction protein ZO-3 were explored through exogenous expression of mutant protein constructs in MDCK cells. Expression of the amino-terminal, PSD95/dlg/ZO-1 domain-containing half of the molecule (NZO-3) delayed the assembly of both tight and adherens junctions induced by calcium switch treatment or brief exposure to the actin-disrupting drug cytochalasin D. Junction formation was monitored by transepithelial resistance measurements and localization of junction-specific proteins by immunofluorescence. The tight junction components ZO-1, ZO-2, endogenous ZO-3, and occludin were mislocalized during the early stages of tight junction assembly. Similarly, the adherens junction proteins E-cadherin and beta-catenin were also delayed in their recruitment to the cell membrane, and NZO-3 expression had striking effects on actin cytoskeleton dynamics. NZO-3 expression did not alter expression levels of ZO-1, ZO-2, endogenous ZO-3, occludin, or E-cadherin; however, the amount of Triton X-100-soluble, signaling-active beta-catenin was increased in NZO-3-expressing cells during junction assembly. In vitro binding experiments showed that ZO-1 and actin preferentially bind to NZO-3, whereas both NZO-3 and the carboxy-terminal half of the molecule (CZO-3) contain binding sites for occludin and cingulin. We hypothesize that NZO-3 exerts its dominant-negative effects via a mechanism involving the actin cytoskeleton, ZO-1, and/or beta-catenin.

  20. Exogenous Expression of the Amino-Terminal Half of the Tight Junction Protein Zo-3 Perturbs Junctional Complex Assembly

    PubMed Central

    Wittchen, Erika S.; Haskins, Julie; Stevenson, Bruce R.

    2000-01-01

    The functional characteristics of the tight junction protein ZO-3 were explored through exogenous expression of mutant protein constructs in MDCK cells. Expression of the amino-terminal, PSD95/dlg/ZO-1 domain-containing half of the molecule (NZO-3) delayed the assembly of both tight and adherens junctions induced by calcium switch treatment or brief exposure to the actin-disrupting drug cytochalasin D. Junction formation was monitored by transepithelial resistance measurements and localization of junction-specific proteins by immunofluorescence. The tight junction components ZO-1, ZO-2, endogenous ZO-3, and occludin were mislocalized during the early stages of tight junction assembly. Similarly, the adherens junction proteins E-cadherin and β-catenin were also delayed in their recruitment to the cell membrane, and NZO-3 expression had striking effects on actin cytoskeleton dynamics. NZO-3 expression did not alter expression levels of ZO-1, ZO-2, endogenous ZO-3, occludin, or E-cadherin; however, the amount of Triton X-100–soluble, signaling-active β-catenin was increased in NZO-3–expressing cells during junction assembly. In vitro binding experiments showed that ZO-1 and actin preferentially bind to NZO-3, whereas both NZO-3 and the carboxy-terminal half of the molecule (CZO-3) contain binding sites for occludin and cingulin. We hypothesize that NZO-3 exerts its dominant-negative effects via a mechanism involving the actin cytoskeleton, ZO-1, and/or β-catenin. PMID:11076967

  1. Amino-terminal domains of kainate receptors determine the differential dependence on Neto auxiliary subunits for trafficking

    PubMed Central

    Sheng, Nengyin; Shi, Yun Stone; Nicoll, Roger A.

    2017-01-01

    The kainate receptor (KAR), a subtype of glutamate receptor, mediates excitatory synaptic responses at a subset of glutamatergic synapses. However, the molecular mechanisms underlying the trafficking of its different subunits are poorly understood. Here we use the CA1 hippocampal pyramidal cell, which lacks KAR-mediated synaptic currents, as a null background to determine the minimal requirements for the extrasynaptic and synaptic expression of the GluK2 subunit. We find that the GluK2 receptor itself, in contrast to GluK1, traffics to the neuronal surface and synapse efficiently and the auxiliary subunits Neto1 and Neto2 caused no further enhancement of these two trafficking processes. However, the regulation of GluK2 biophysical properties by Neto proteins is the same as that of GluK1. We further determine that it is the amino-terminal domains (ATDs) of GluK1 and GluK2 that control the strikingly different trafficking properties between these two receptors. Moreover, the ATDs are critical for synaptic expression of heteromeric receptors at mossy fiber–CA3 synapses and also mediate the differential dependence on Neto proteins for surface and synaptic trafficking of GluK1 and GluK2. These results highlight the fundamental differences between the two major KAR subunits and their interplay with Neto auxiliary proteins. PMID:28100490

  2. Zinc phthalocyanine conjugated with the amino-terminal fragment of urokinase for tumor-targeting photodynamic therapy.

    PubMed

    Chen, Zhuo; Xu, Peng; Chen, Jincan; Chen, Hongwei; Hu, Ping; Chen, Xueyuan; Lin, Lin; Huang, Yunmei; Zheng, Ke; Zhou, Shanyong; Li, Rui; Chen, Song; Liu, Jianyong; Xue, Jinping; Huang, Mingdong

    2014-10-01

    Photodynamic therapy (PDT) has attracted much interest for the treatment of cancer due to the increased incidence of multidrug resistance and systemic toxicity in conventional chemotherapy. Phthalocyanine (Pc) is one of main classes of photosensitizers for PDT and possesses optimal photophysical and photochemical properties. A higher specificity can ideally be achieved when Pcs are targeted towards tumor-specific receptors, which may also facilitate specific drug delivery. Herein, we develop a simple and unique strategy to prepare a hydrophilic tumor-targeting photosensitizer ATF-ZnPc by covalently coupling zinc phthalocyanine (ZnPc) to the amino-terminal fragment (ATF) of urokinase-type plasminogen activator (uPA), a fragment responsible for uPA receptor (uPAR, a biomarker overexpressed in cancer cells), through the carboxyl groups of ATF. We demonstrate the high efficacy of this tumor-targeting PDT agent for the inhibition of tumor growth both in vitro and in vivo. Our in vivo optical imaging results using H22 tumor-bearing mice show clearly the selective accumulation of ATF-ZnPc in tumor region, thereby revealing the great potential of ATF-ZnPc for clinical applications such as cancer detection and guidance of tumor resection in addition to photodynamic treatment. Copyright © 2014 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.

  3. Improved bioactivity of antimicrobial peptides by addition of amino-terminal copper and nickel (ATCUN) binding motifs.

    PubMed

    Libardo, M Daben; Cervantes, Jorge L; Salazar, Juan C; Angeles-Boza, Alfredo M

    2014-08-01

    Antimicrobial peptides (AMPs) are promising candidates to help circumvent antibiotic resistance, which is an increasing clinical problem. Amino-terminal copper and nickel (ATCUN) binding motifs are known to actively form reactive oxygen species (ROS) upon metal binding. The combination of these two peptidic constructs could lead to a novel class of dual-acting antimicrobial agents. To test this hypothesis, a set of ATCUN binding motifs were screened for their ability to induce ROS formation, and the most potent were then used to modify AMPs with different modes of action. ATCUN binding motif-containing derivatives of anoplin (GLLKRIKTLL-NH2), pro-apoptotic peptide (PAP; KLAKLAKKLAKLAK-NH2), and sh-buforin (RAGLQFPVGRVHRLLRK-NH2) were synthesized and found to be more active than the parent AMPs against a panel of clinically relevant bacteria. The lower minimum inhibitory concentration (MIC) values for the ATCUN-anoplin peptides are attributed to the higher pore-forming activity along with their ability to cause ROS-induced membrane damage. The addition of the ATCUN motifs to PAP also increases its ability to disrupt membranes. DNA damage is the major contributor to the activity of the ATCUN-sh-buforin peptides. Our findings indicate that the addition of ATCUN motifs to AMPs is a simple strategy that leads to AMPs with higher antibacterial activity and possibly to more potent, usable antibacterial agents.

  4. The photomorphogenesis regulator DET1 binds the amino-terminal tail of histone H2B in a nucleosome context.

    PubMed

    Benvenuto, Giovanna; Formiggini, Fabio; Laflamme, Pierre; Malakhov, Mikhail; Bowler, Chris

    2002-09-03

    Light provides a major source of information from the environment during plant growth and development. Recent results suggest that the key events controlling light-regulated gene expression in plants are translocation of the phytochrome photoreceptors into the nucleus, followed by their binding to transcription factors such as PIF3. Coupled with this, the degradation of positively acting intermediates such as the transcription factor HY5 by COP1 and the COP9 signalosome appears to be an important process whereby photomorphogenesis is repressed in darkness (e.g., ). Genetic analyses in Arabidopsis and tomato have revealed that the nuclear protein DET1 also plays a key role in the repression of photomorphogenesis. However, the function of this protein has remained a mystery. In a series of in vitro experiments, we provide persuasive evidence that DET1 binds to nonacetylated amino-terminal tails of the core histone H2B in the context of the nucleosome. Furthermore, we have utilized FRET (fluorescence resonance energy transfer) imaging with GFP variants to demonstrate this interaction within the nucleus of living plant cells. Given the dramatic photomorphogenic phenotypes of det1 mutants, we propose that chromatin remodeling plays a heretofore unsuspected role in regulating gene expression during photomorphogenesis.

  5. Increase in plasma concentrations of cardiodilatin (amino terminal pro-atrial natriuretic peptide) in cardiac failure and during recumbency.

    PubMed Central

    Meleagros, L; Gibbs, J S; Ghatei, M A; Bloom, S R

    1988-01-01

    Plasma concentrations of cardiodilatin, the peptide sequence at the amino terminal of the pro-atrial natriuretic peptide, in 17 normal subjects ranged from 59 to 202 (mean 118 (SEM) (9] pmol/l. Recumbency increased the mean (SEM) concentration to 160 (13) pmol/l. The plasma concentration of cardiodilatin in 24 patients with congestive cardiac failure was much higher (964 (175) pmol/l) than in the normal subjects. It was highest in those with heart failure in New York Heart Association functional classes III and IV and the concentration correlated both with atrial natriuretic peptide concentrations and left ventricular ejection fraction. Concentrations rose during induced tachycardia in three patients tested. Chromatography showed a single clean peak of plasma cardiodilatin immunoreactivity. It seems that cardiodilatin is a second circulating cardiac peptide that is jointly released with atrial natriuretic peptide by common stimuli. Other workers have reported that, like atrial natriuretic peptide, three partial cardiodilatin sequences can stimulate renal particulate guanylate cyclase and increase cyclic guanosine monophosphate. The simultaneous release of cardiodilatin in higher circulating concentrations than atrial natriuretic peptide may be relevant to the finding that appropriate concentrations of exogenous atrial natiuretic peptide alone do not produce the full renal effects associated with endogenous peptide release. PMID:2970269

  6. Amino-terminal domains of kainate receptors determine the differential dependence on Neto auxiliary subunits for trafficking.

    PubMed

    Sheng, Nengyin; Shi, Yun Stone; Nicoll, Roger A

    2017-01-31

    The kainate receptor (KAR), a subtype of glutamate receptor, mediates excitatory synaptic responses at a subset of glutamatergic synapses. However, the molecular mechanisms underlying the trafficking of its different subunits are poorly understood. Here we use the CA1 hippocampal pyramidal cell, which lacks KAR-mediated synaptic currents, as a null background to determine the minimal requirements for the extrasynaptic and synaptic expression of the GluK2 subunit. We find that the GluK2 receptor itself, in contrast to GluK1, traffics to the neuronal surface and synapse efficiently and the auxiliary subunits Neto1 and Neto2 caused no further enhancement of these two trafficking processes. However, the regulation of GluK2 biophysical properties by Neto proteins is the same as that of GluK1. We further determine that it is the amino-terminal domains (ATDs) of GluK1 and GluK2 that control the strikingly different trafficking properties between these two receptors. Moreover, the ATDs are critical for synaptic expression of heteromeric receptors at mossy fiber-CA3 synapses and also mediate the differential dependence on Neto proteins for surface and synaptic trafficking of GluK1 and GluK2. These results highlight the fundamental differences between the two major KAR subunits and their interplay with Neto auxiliary proteins.

  7. Amino-terminal propeptide of C-type natriuretic peptide (NTproCNP) predicts height velocity in healthy children

    PubMed Central

    Olney, Robert C.; Permuy, Joseph W.; Prickett, Timothy C.R.; Han, Joan C.; Espiner, Eric A.

    2012-01-01

    Objective C-type natriuretic peptide (CNP) is a paracrine regulatory factor of the growth plate and plays a key role in endochondral growth. Its amino-terminal propeptide (NTproCNP) is an equimolar product of CNP biosynthesis and is easily measured in plasma. Preliminary studies suggest that NTproCNP levels correlate with height velocity in sheep and children. The objectives of the study were to correlate NTproCNP levels with height velocity and to define the reference range for plasma CNP and NTproCNP across childhood. Design This was a prospective, cross-sectional, observational study of healthy children. Patients Participants were 258 healthy children between 2 months and 20 years of age. Measurements Anthropometrics were obtained and CNP and NTproCNP levels were determined by radioimmunoassay. Results For both sexes, CNP and NTproCNP levels were high in infancy, lower in early childhood, rising during puberty, then falling to low adult levels. Levels of NTproCNP peaked at 14.1 years in boys and 11.9 years in girls, coincident with the age of peak height velocity. Levels of NTproCNP varied with pubertal status, peaking at genital Tanner stage IV in boys and III in girls. There was a highly significant correlation between NTproCNP and height velocity. Conclusions C-type natriuretic peptide plays an integral role in endochondral growth. We show here that CNP synthesis (as measured by NTproCNP levels in plasma) is closely related to linear growth in healthy children at all ages. We propose NTproCNP as a biomarker of linear growth. PMID:22435455

  8. Amino-terminal propeptide of C-type natriuretic peptide (NTproCNP) predicts height velocity in healthy children.

    PubMed

    Olney, Robert C; Permuy, Joseph W; Prickett, Timothy C R; Han, Joan C; Espiner, Eric A

    2012-09-01

    C-type natriuretic peptide (CNP) is a paracrine regulatory factor of the growth plate and plays a key role in endochondral growth. Its amino-terminal propeptide (NTproCNP) is an equimolar product of CNP biosynthesis and is easily measured in plasma. Preliminary studies suggest that NTproCNP levels correlate with height velocity in sheep and children. The objectives of the study were to correlate NTproCNP levels with height velocity and to define the reference range for plasma CNP and NTproCNP across childhood. This was a prospective, cross-sectional, observational study of healthy children. Participants were 258 healthy children between 2 months and 20 years of age. Anthropometrics were obtained and CNP and NTproCNP levels were determined by radioimmunoassay. For both sexes, CNP and NTproCNP levels were high in infancy, lower in early childhood, rising during puberty, then falling to low adult levels. Levels of NTproCNP peaked at 14·1 years in boys and 11·9 years in girls, coincident with the age of peak height velocity. Levels of NTproCNP varied with pubertal status, peaking at genital Tanner stage IV in boys and III in girls. There was a highly significant correlation between NTproCNP and height velocity. C-type natriuretic peptide plays an integral role in endochondral growth. We show here that CNP synthesis (as measured by NTproCNP levels in plasma) is closely related to linear growth in healthy children at all ages. We propose NTproCNP as a biomarker of linear growth. © 2012 Blackwell Publishing Ltd.

  9. Procollagen type III amino terminal peptide and myocardial fibrosis: A study in hypertensive patients with and without left ventricular hypertrophy.

    PubMed

    dos Santos Moreira, Carlos; Serejo, Fátima; Alcântara, Paula; Ramalhinho, Vítor; Braz Nogueira, J

    2015-05-01

    An exaggerated accumulation of type I and type III fibrillar collagens occurs throughout the free wall and interventricular septum of patients with primary hypertension and left ventricular hypertrophy (LVH). In the present study the serum concentration of procollagen type III amino terminal peptide (PIIIP) was measured to determine the value of this peptide as a potential marker of ventricular fibrosis in hypertensive patients, particularly those with LVH. The study population consisted of patients with never-treated mild to moderate essential hypertension and 30 normotensive control subjects. Clinical, echocardiographic, electrocardiographic and biochemical parameters were assessed in all patients. Heart rate, body mass index and levels of blood pressure were increased in hypertensives, particularly those with LVH, compared to normotensive controls. Posterior wall thickness, left ventricular (LV) mass and LV mass index, and serum PIIIP concentration were also increased in hypertensives, with significant differences between the two hypertensive groups. The ratio between maximal early and late transmitral flow velocity measured during diastole was lower in hypertensives, particularly those with LVH, than in normotensive controls. The increase in PIIIP indicates that type III collagen synthesis increases in hypertensives, particularly those with LVH, implying that alterations in the heart in hypertension are the result not solely of hypertrophied LV muscle, but also of increased collagen deposition within the ventricular wall and around the coronary vessels. Thus, measurement of serum PIIIP could be a practical and useful tool in the non-invasive assessment of myocardial remodeling in hypertension. Copyright © 2014 Sociedade Portuguesa de Cardiologia. Published by Elsevier España. All rights reserved.

  10. Alteration of the mode of antibacterial action of a defensin by the amino-terminal loop substitution

    SciTech Connect

    Gao, Bin; Zhu, Shunyi

    2012-10-05

    Highlights: Black-Right-Pointing-Pointer Al-M is an engineered fungal defensin with the n-loop of an insect defensin. Black-Right-Pointing-Pointer Al-M adopts a native defensin-like structure with high antibacterial potency. Black-Right-Pointing-Pointer Al-M kills bacteria through a membrane disruptive mechanism. Black-Right-Pointing-Pointer This work sheds light on the functional evolution of CS{alpha}{beta}-type defensins. -- Abstract: Ancient invertebrate-type and classical insect-type defensins (AITDs and CITDs) are two groups of evolutionarily related antimicrobial peptides (AMPs) that adopt a conserved cysteine-stabilized {alpha}-helical and {beta}-sheet (CS{alpha}{beta}) fold with a different amino-terminal loop (n-loop) size and diverse modes of antibacterial action. Although they both are identified as inhibitors of cell wall biosynthesis, only CITDs evolved membrane disruptive ability by peptide oligomerization to form pores. To understand how this occurred, we modified micasin, a fungus-derived AITDs with a non-membrane disruptive mechanism, by substituting its n-loop with that of an insect-derived CITDs. After air oxidization, the synthetic hybrid defensin (termed Al-M) was structurally identified by circular dichroism (CD) and functionally evaluated by antibacterial and membrane permeability assays and electronic microscopic observation. Results showed that Al-M folded into a native-like defensin structure, as determined by its CD spectrum that is similar to that of micasin. Al-M was highly efficacious against the Gram-positive bacterium Bacillus megaterium with a lethal concentration of 1.76 {mu}M. As expected, in contrast to micasin, Al-M killed the bacteria through a membrane disruptive mechanism of action. The alteration in modes of action supports a key role of the n-loop extension in assembling functional surface of CITDs for membrane disruption. Our work provides mechanical evidence for evolutionary relationship between AITDs and CITDs.

  11. Severing of F-actin by the amino-terminal half of gelsolin suggests internal cooperativity in gelsolin.

    PubMed Central

    Selden, L A; Kinosian, H J; Newman, J; Lincoln, B; Hurwitz, C; Gershman, L C; Estes, J E

    1998-01-01

    Gelsolin is a Ca2+-regulated actin-binding protein that can sever, cap, and nucleate growth from the pointed ends of actin filaments. In this study we have measured the binding of the amino-terminal half of gelsolin, G1-3, to pyrene-labeled F-actin as a function of Ca2+ concentration. The rate of binding is shown to be dependent on micromolar concentrations of Ca2+. Independent experiments demonstrate that conformational changes in G1-3 are induced by micromolar concentrations of Ca2+. Titrations of pyrene-F-actin with G1-3 and gelsolin show that the quenching of pyrene fluorescence is identical in extent and stoichiometry for both G1-3 and gelsolin. In contrast, severing of F-actin by G1-3 is found to be much less efficient than is severing by gelsolin. In experiments in which F-actin severing is quantitatively measured, the filament number is found to be proportional to the 1.35 power of the G1-3 concentration. This deviation from linearity may be explained by cooperativity; the binding of two G1-3 molecules in close proximity may lead to cooperative severing of the polymer, thus increasing the severing efficiency. This model is supported by experiments that show that the efficiency of G1-3 severing of F-actin increases with increasing G1-3:F-actin ratios. Extrapolating from these results, we conclude that G4-6, the carboxyl-terminal half of gelsolin, has an active role in the severing of F-actin by intact gelsolin. Whereas F-actin severing by G1-3 is enhanced by cooperative binding of two separate G1-3 molecules, cooperativity is inherent to intact gelsolin because the cooperative partners are covalently linked. PMID:9826629

  12. Control of N-methyl-D-aspartate Receptor Function by the NR2 Subunit Amino-Terminal Domain

    PubMed Central

    Yuan, Hongjie; Hansen, Kasper B.; Vance, Katie M.; Ogden, Kevin K.; Traynelis, Stephen F.

    2009-01-01

    NMDA receptors comprised of different NR2 subunits exhibit strikingly unique biophysical and pharmacological properties. Here we report that the extracellular amino-terminal domain (ATD) of the NR2 subunit controls pharmacological and kinetic properties of recombinant NMDA receptors, such as agonist potency, deactivation time course, open probability (POPEN), and mean open/shut duration. Using ATD deletion mutants of NR2A, NR2B, NR2C, NR2D and chimeras of NR2A and NR2D with interchanged ATD (NR2A-(2D-ATD) and NR2D-(2A-ATD)), we show that the ATD contributes to the low glutamate potency of NR2A-containing NMDA receptors and the high glutamate potency of NR2D-containing receptors. The ATD influences the deactivation time courses of NMDA receptors, as removal of the ATD from NR2A slows the deactivation rate, while removal of the ATD from NR2B, NR2C and NR2D accelerates the deactivation rate. Open probability also is influenced by the ATD. Removal of the ATD from NR2A or replacement of the NR2A-ATD with that of NR2D decreases POPEN in single channel recordings from outside-out patches of HEK 293 cells. By contrast, deletion of the ATD from NR2D or replacement of the NR2D ATD with that of NR2A increases POPEN and mean open duration. These data demonstrate the modular nature of NMDA receptors and show that the ATD of the different NR2 subunits plays an important role in fine-tuning the functional properties of the individual NMDA receptor subtypes. PMID:19793963

  13. The amino terminal domain of HIV-1 Rev is required for discrimination of the RRE from nonspecific RNA.

    PubMed

    Daly, T J; Doten, R C; Rusche, J R; Auer, M

    1995-10-20

    The ability of HIV-1 Rev to successfully discriminate between specific Rev-responsive elements (RRE) and nonspecific binding sites in the presence of excess nonspecific RNA was examined using filter binding, gel shift, and gel filtration techniques, using purified M4 Rev mutant protein and endoproteinase Lys-C cleaved wild-type Rev. The M4 Rev displayed a slightly reduced binding affinity to the RRE, as well as a tenfold decrease in its ability to discriminate the RRE from non-specific RNA compared to the wild-type Rev. Gel shift and gel filtration chromotography data also showed decreased ability of the mutant to multimerize in the absence or presence of the RRE. The Lys-C cleaved Rev, which lacks the amino-terminal 20 amino acids of the protein, displayed less ability to discriminate the RRE from nonspecific RNA compared to either the wild-type or the M4 mutant Rev and appeared unable to form protein-protein interactions, yet still bound sense and antisense RNA species with high affinity (Kd was in the nanomolar concentration range). A 40 amino acid peptide containing the arginine-rich RRE binding domain of Rev was also observed to interact with both the RRE and antisense RNA fragments with a binding constant of about 1 x 10(-9) M. However, the peptide displayed almost no ability to discriminate between the RRE and a comparably sized antisense RRE. The loss in ability to discriminate correct from incorrect binding sites correlates with overall decreases in the alpha-helical character of the protein and perturbations within the amino terminus. The amino terminus of Rev is likely to maintain the conformational integrity of the arginine rich RRE binding domain which is required for specific RNA binding site discrimination or stabilization of specific Rev-RRE interactions.

  14. Modulating immunogenic properties of HIV-1 gp41 membrane-proximal external region by destabilizing six-helix bundle structure

    PubMed Central

    Banerjee, Saikat; Shi, Heliang; Habte, Habtom H.; Qin, Yali; Cho, Michael W.

    2016-01-01

    The C-terminal alpha-helix of gp41 membrane-proximal external region (MPER; 671NWFDITNWLWYIK683) encompassing 4E10/10E8 epitopes is an attractive target for HIV-1 vaccine development. We previously reported that gp41-HR1-54Q, a trimeric protein comprised of the MPER in the context of a stable six-helix bundle (6HB), induced strong immune responses against the helix, but antibodies were directed primarily against the non-neutralizing face of the helix. To better target 4E10/10E8 epitopes, we generated four putative fusion intermediates by introducing double point mutations or deletions in the heptad repeat region 1 (HR1) that destabilize 6HB in varying degrees. One variant, HR1-Δ10-54K, elicited antibodies in rabbits that targeted W672, I675 and L679, which are critical for 4E10/10E8 recognition. Overall, the results demonstrated that altering structural parameters of 6HB can influence immunogenic properties of the MPER and antibody targeting. Further exploration of this strategy could allow development of immunogens that could lead to induction of 4E10/10E8-like antibodies. PMID:26803471

  15. Modulating immunogenic properties of HIV-1 gp41 membrane-proximal external region by destabilizing six-helix bundle structure.

    PubMed

    Banerjee, Saikat; Shi, Heliang; Habte, Habtom H; Qin, Yali; Cho, Michael W

    2016-03-01

    The C-terminal alpha-helix of gp41 membrane-proximal external region (MPER; (671)NWFDITNWLWYIK(683)) encompassing 4E10/10E8 epitopes is an attractive target for HIV-1 vaccine development. We previously reported that gp41-HR1-54Q, a trimeric protein comprised of the MPER in the context of a stable six-helix bundle (6HB), induced strong immune responses against the helix, but antibodies were directed primarily against the non-neutralizing face of the helix. To better target 4E10/10E8 epitopes, we generated four putative fusion intermediates by introducing double point mutations or deletions in the heptad repeat region 1 (HR1) that destabilize 6HB in varying degrees. One variant, HR1-∆10-54K, elicited antibodies in rabbits that targeted W672, I675 and L679, which are critical for 4E10/10E8 recognition. Overall, the results demonstrated that altering structural parameters of 6HB can influence immunogenic properties of the MPER and antibody targeting. Further exploration of this strategy could allow development of immunogens that could lead to induction of 4E10/10E8-like antibodies.

  16. Structural basis for KIT receptor tyrosine kinase inhibition by antibodies targeting the D4 membrane-proximal region.

    PubMed

    Reshetnyak, Andrey V; Nelson, Bryce; Shi, Xiarong; Boggon, Titus J; Pavlenco, Alevtina; Mandel-Bausch, Elizabeth M; Tome, Francisco; Suzuki, Yoshihisa; Sidhu, Sachdev S; Lax, Irit; Schlessinger, Joseph

    2013-10-29

    Somatic oncogenic mutations in the receptor tyrosine kinase KIT function as major drivers of gastrointestinal stromal tumors and a subset of acute myeloid leukemia, melanoma, and other cancers. Although treatment of these cancers with tyrosine kinase inhibitors shows dramatic responses and durable disease control, drug resistance followed by clinical progression of disease eventually occurs in virtually all patients. In this report, we describe inhibitory KIT antibodies that bind to the membrane-proximal Ig-like D4 of KIT with significant overlap with an epitope in D4 that mediates homotypic interactions essential for KIT activation. Crystal structures of the anti-KIT antibody in complex with KIT D4 and D5 allowed design of affinity-matured libraries that were used to isolate variants with increased affinity and efficacy. Isolated antibodies showed KIT inhibition together with suppression of cell proliferation driven by ligand-stimulated WT or constitutively activated oncogenic KIT mutant. These antibodies represent a unique therapeutic approach and a step toward the development of "naked" or toxin-conjugated KIT antibodies for the treatment of KIT-driven cancers.

  17. Complexin induces a conformational change at the membrane-proximal C-terminal end of the SNARE complex

    PubMed Central

    Choi, Ucheor B; Zhao, Minglei; Zhang, Yunxiang; Lai, Ying; Brunger, Axel T

    2016-01-01

    Complexin regulates spontaneous and activates Ca2+-triggered neurotransmitter release, yet the molecular mechanisms are still unclear. Here we performed single molecule fluorescence resonance energy transfer experiments and uncovered two conformations of complexin-1 bound to the ternary SNARE complex. In the cis conformation, complexin-1 induces a conformational change at the membrane-proximal C-terminal end of the ternary SNARE complex that specifically depends on the N-terminal, accessory, and central domains of complexin-1. The complexin-1 induced conformation of the ternary SNARE complex may be related to a conformation that is juxtaposing the synaptic vesicle and plasma membranes. In the trans conformation, complexin-1 can simultaneously interact with a ternary SNARE complex via the central domain and a binary SNARE complex consisting of syntaxin-1A and SNAP-25A via the accessory domain. The cis conformation may be involved in activation of synchronous neurotransmitter release, whereas both conformations may be involved in regulating spontaneous release. DOI: http://dx.doi.org/10.7554/eLife.16886.001 PMID:27253060

  18. Inhibition of the Androgen Receptor Amino-Terminal Domain by a Small Molecule as Treatment for Castrate-Resistant Prostate Cancer

    DTIC Science & Technology

    2013-10-01

    Small Molecule as Treatment for Castrate-Resistant Prostate Cancer PRINCIPAL INVESTIGATOR: Stephen R. Plymate CONTRACTING ORGANIZATION...SUBTITLE “Inhibition of the Androgen Receptor Amino-Terminal Domain by a Small Molecule as Treatment for Castrate-Resistant Prostate Cancer” 5a...Sadar MD 2013 EPI small molecules covalently bind intrinsically disordered N-terminal domain of the androgen receptor to inhibit the growth of

  19. Analysis of the Human Immunodeficiency Virus Type 1 gp41 Membrane Proximal External Region Arrayed on Hepatitis B Surface Antigen Particles

    PubMed Central

    Phogat, S; K, Svehla; M, Tang; A, Spadaccini; J, Muller; J, Mascola; Berkower; R, Wyatt

    2009-01-01

    Vaccine immunogens derived from the envelope glycoproteins of the human immunodeficiency virus type 1 (HIV-1) that elicit broad neutralizing antibodies remains an elusive goal. The highly conserved 30 amino acid membrane proximal external region (MPER) of HIV gp41 contains the hydrophobic epitopes for two rare HIV-1 broad cross-reactive neutralizing antibodies, 2F5 and 4E10. Both these antibodies possess relatively hydrophobic HCDR3 loops and demonstrate enhanced binding to their epitopes in the context of the native gp160 precursor envelope glycoprotein by the intimate juxtaposition of a lipid membrane. The Hepatitis B surface antigen (HBsAg) S1 protein forms nanoparticles that can be utilized both as an immunogenic array of the MPER and to provide the lipid environment needed for enhanced 2F5 and 4E10 binding. We show that recombinant HBsAg particles with MPER (HBsAg-MPER) appended at the C-terminus of the S1 protein are recognized by 2F5 and 4E10 with high affinity compared to positioning the MPER at the N-terminus or the extracellular loop (ECL) of S1. Addition of C-terminal hydrophobic residues derived from the HIV-1 Env transmembrane region further enhances recognition of the MPER by both 2F5 and 4E10. Delipidation of the HBsAg-MPER particles decreases 2F5 and 4E10 binding and subsequent reconstitution with synthetic lipids restores optimal binding. Inoculation of the particles into small animals raised cross-reactive antibodies that recognize both the MPER and HIV-1 gp160 envelope glycoproteins expressed on the cell surface; however, no neutralizing activity could be detected. Prime:boost immunization of the HBsAg-MPER particles in sequence with HIV envelope glycoprotein proteoliposomes (Env-PLs) did not raise neutralizing antibodies that could be mapped to the MPER region. However, the Env-PLs did raise anti-Env antibodies that had the ability to neutralize selected HIV-1 isolates. The first generation HBsAg-MPER particles represent a unique means to

  20. Amino-terminal p53 mutations lead to expression of apoptosis proficient p47 and prognosticate better survival, but predispose to tumorigenesis.

    PubMed

    Phang, Beng Hooi; Othman, Rashidah; Bougeard, Gaelle; Chia, Ren Hui; Frebourg, Thierry; Tang, Choong Leong; Cheah, Peh Yean; Sabapathy, Kanaga

    2015-11-17

    Whereas most mutations in p53 occur in the DNA-binding domain and lead to its functional inactivation, their relevance in the amino-terminal transactivation domain is unclear. We show here that amino-terminal p53 (ATp53) mutations often result in the abrogation of full-length p53 expression, but concomitantly lead to the expression of the amino-terminally truncated p47 isoform. Using genetically modified cancer cells that only express p47, we demonstrate it to be up-regulated in response to various stimuli, and to contribute to cell death, through its ability to selectively activate a group of apoptotic target genes. Target gene selectivity is influenced by K382 acetylation, which depends on the amino terminus, and is required for recruitment of selective cofactors. Consistently, cancers capable of expressing p47 had a better overall survival. Nonetheless, retention of the apoptotic function appears insufficient for tumor suppression, because these mutations are also found in the germ line and lead to Li-Fraumeni syndrome. These data from ATp53 mutations collectively demonstrate that p53's apoptosis proficiency is dispensable for tumor suppression, but could prognosticate better survival.

  1. Combined Measurement of Soluble ST2 and Amino-Terminal Pro-B-Type Natriuretic Peptide Provides Early Assessment of Severity in Cardiogenic Shock Complicating Acute Coronary Syndrome.

    PubMed

    Tolppanen, Heli; Rivas-Lasarte, Mercedes; Lassus, Johan; Sadoune, Malha; Gayat, Etienne; Pulkki, Kari; Arrigo, Mattia; Krastinova, Evguenia; Sionis, Alessandro; Parissis, John; Spinar, Jindrich; Januzzi, James; Harjola, Veli-Pekka; Mebazaa, Alexandre

    2017-07-01

    Mortality in cardiogenic shock complicating acute coronary syndrome is high, and objective risk stratification is needed for rational use of advanced therapies such as mechanical circulatory support. Traditionally, clinical variables have been used to judge risk in cardiogenic shock. The aim of this study was to assess the added value of serial measurement of soluble ST2 and amino-terminal pro-B-type natriuretic peptide to clinical parameters for risk stratification in cardiogenic shock. CardShock (www.clinicaltrials.gov NCT01374867) is a prospective European multinational study of cardiogenic shock. The main study introduced CardShock risk score, which is calculated from seven clinical variables at baseline, and was associated with short-term mortality. Nine tertiary care university hospitals. Patients with cardiogenic shock caused by acute coronary syndrome (n=145). In this substudy, plasma samples from the study patients were analyzed at eight time points during the ICU or cardiac care unit stay. Additional prognostic value of the biomarkers was assessed with incremental discrimination improvement. The combination of soluble ST2 and amino-terminal pro-B-type natriuretic peptide showed excellent discrimination for 30-day mortality (area under the curve, 0.77 at 12 hr up to 0.93 at 5-10 d after cardiogenic shock onset). At 12 hours, patients with both biomarkers elevated (soluble ST2, ≥ 500 ng/mL and amino-terminal pro-B-type natriuretic peptide, ≥ 4,500 ng/L) had higher 30-day mortality (79%) compared to those with one or neither biomarkers elevated (31% or 10%, respectively; p < 0.001). Combined measurement of soluble ST2 and amino-terminal pro-B-type natriuretic peptide at 12 hours added value to CardShock risk score, correctly reclassifying 11% of patients. The combination of results for soluble ST2 and amino-terminal pro-B-type natriuretic peptide provides early risk assessment beyond clinical variables in patients with acute coronary syndrome

  2. Recombinant expression, purification, and biophysical characterization of the transmembrane and membrane proximal domains of HIV-1 gp41.

    PubMed

    Gong, Zhen; Kessans, Sarah A; Song, Lusheng; Dörner, Katerina; Lee, Ho-Hsien; Meador, Lydia R; LaBaer, Joshua; Hogue, Brenda G; Mor, Tsafrir S; Fromme, Petra

    2014-11-01

    The transmembrane subunit (gp41) of the envelope glycoprotein of HIV-1 associates noncovalently with the surface subunit (gp120) and together they play essential roles in viral mucosal transmission and infection of target cells. The membrane proximal region (MPR) of gp41 is highly conserved and contains epitopes of broadly neutralizing antibodies. The transmembrane (TM) domain of gp41 not only anchors the envelope glycoprotein complex in the viral membrane but also dynamically affects the interactions of the MPR with the membrane. While high-resolution X-ray structures of some segments of the MPR were solved in the past, they represent the post-fusion forms. Structural information on the TM domain of gp41 is scant and at low resolution. Here we describe the design, expression and purification of a protein construct that includes MPR and the transmembrane domain of gp41 (MPR-TMTEV-6His), which reacts with the broadly neutralizing antibodies 2F5 and 4E10 and thereby may represent an immunologically relevant conformation mimicking a prehairpin intermediate of gp41. The expression level of MPR-TMTEV-6His was improved by fusion to the C-terminus of Mistic protein, yielding ∼ 1 mg of pure protein per liter. The isolated MPR-TMTEV-6His protein was biophysically characterized and is a monodisperse candidate for crystallization. This work will enable further investigation into the structure of MPR-TMTEV-6His, which will be important for the structure-based design of a mucosal vaccine against HIV-1.

  3. Acid-labile formylation of amino terminal proline of human immunodeficiency virus type 1 p24(gag) was found by proteomics using two-dimensional gel electrophoresis and matrix-assisted laser desorption/ionization-time-of-flight mass spectrometry.

    PubMed

    Fuchigami, Takashi; Misumi, Shogo; Takamune, Nobutoki; Takahashi, Ichiro; Takama, Michiho; Shoji, Shozo

    2002-05-10

    HIV-1(LAV-1) particles were collected by ultracentrifugation, treated with subtilisin, and then purified by Sepharose CL-4B column chromatography to remove microvesicles. The lysate of the purified human immunodeficiency virus type 1 (HIV-1) particles was subjected to two-dimensional (2D) gel electrophoresis and stained, and the stained spots were excised and digested with trypsin. The resulting peptide fragments were characterized by matrix-assisted laser desorption/ionization-time-of-flight mass spectrometry (MALDI-TOF MS). Twenty-five proteins were identified as the proteins inside the virion and the acid-labile formyl group of an amino terminal proline residue of HIV-1(LAV-1) p24(gag) was determined by MALDI-TOF MS before and after weak-acid treatments (0.6 N hydrochloric acid) and confirmed by post-source decay (PSD) of the N-formylated N-terminal tryptic peptide (N-formylated Pro(1)-Arg(18)). The role of formylation has been unclear so far, but it is surmised that the acid-labile formylation of HIV-1(LAV-1) p24(gag) may play a critical role in the formation of the HIV-1 core for conferring HIV-1 infectivity.

  4. Association of the amino-terminal half of c-Src with focal adhesions alters their properties and is regulated by phosphorylation of tyrosine 527.

    PubMed Central

    Kaplan, K B; Bibbins, K B; Swedlow, J R; Arnaud, M; Morgan, D O; Varmus, H E

    1994-01-01

    We have characterized the mechanism by which the subcellular distribution of c-Src is controlled by the phosphorylation of tyrosine 527. Mutation of this tyrosine dramatically redistributes c-Src from endosomal membranes to focal adhesions. Redistribution to focal adhesions occurs independently of kinase activity and cellular transformation. In cells lacking the regulatory kinase (CSK) that phosphorylates tyrosine 527, c-Src is also found predominantly in focal adhesions, confirming that phosphorylation of tyrosine 527 affects the location of c-Src inside the cell. The first 251 amino acids of c-Src are sufficient to allow association with focal adhesions, indicating that at least one signal for positioning c-Src in focal adhesions resides in the amino-terminal half. Point mutations and deletions in the first 251 amino acids of c-Src reveal that association with focal adhesions requires the myristylation site needed for membrane attachment, as well as the SH3 domain. Expression of the amino-terminal region alters both the structural and biochemical properties of focal adhesions. Focal adhesions containing this non-catalytic portion of c-Src are larger and exhibit increased levels of phosphotyrosine staining. Our results suggest that c-Src may regulate focal adhesions and cellular adhesion by a kinase-independent mechanism. Images PMID:7525268

  5. Beta-succinyl-coenzyme A synthetase from Trichomonas vaginalis is a soluble hydrogenosomal protein with an amino-terminal sequence that resembles mitochondrial presequences.

    PubMed Central

    Lahti, C J; d'Oliveira, C E; Johnson, P J

    1992-01-01

    We describe studies directed toward understanding the biogenesis and origin of the hydrogenosome, an unusual organelle found exclusively in certain anaerobic eukaryotes that lack mitochondria. Hydrogenosomes are involved in fermentative carbohydrate metabolism and are proposed to have arisen through conversion of mitochondria or via endosymbiosis with an anaerobic bacterium. We cloned a gene encoding the beta subunit of the hydrogenosomal protein succinyl-coenzyme A synthetase (beta-SCS) and isolated the protein from Trichomonas vaginalis. The T. vaginalis beta-SCS gene encodes a protein with a calculated molecular mass of 43,980 Da that has 43% amino acid identity (65% similarity) with beta-SCS from Escherichia coli. The trichomonad protein partitions into the soluble fraction of hydrogenosomes treated with sodium carbonate at high pH, consistent with a matrix localization within the organelle. The protein is encoded by a multigene family composed of at least three members. Amino-terminal sequencing of beta-SCS purified from T. vaginalis hydrogenosomes shows that the mature protein lacks the first nine amino acids encoded in the gene. This apparent amino-terminal leader sequence is strikingly similar to that of another hydrogenosomal protein and to mitochondrial presequences. Images PMID:1400232

  6. Three Contact Modes of Amino Terminal Groups on Gold in Single Molecular Junction of Au/1,4-Diaminobutane/Au

    NASA Astrophysics Data System (ADS)

    Omori, Yasuhiro; Tobita, Junichi; Kato, Yasuyuki; Akiba, Uichi; Fujihira, Masamichi

    2007-12-01

    The effect of the binding modes of two amino terminal groups present on two gold protrusions on currents passing through a single molecular junction of Au/1,4-diaminobutane/Au was studied via the repeated formation of a break junction using a scanning tunneling microscope (STM) under nitrogen at room temperature. In addition to the two reported (high and low) values of the single molecular conductance of diaminobutane, another lower conductance was found through a careful analysis of histograms in a low-current range (0-0.3 nA). The techniques used here to improve the determination of the values of single molecular conductance were the selection of valid i-s curves with current steps and the use of the robust statistical method to correct for the contribution of the background tunneling currents to the current histograms. These three single molecular conductance values are attributed to three different binding modes of the two terminal amino groups giving the three different contact resistances at room temperature. That is, each amino terminal group is likely to be bound to one or the other gold protrusion through a more or a less conductive contact mode. Thus, a single molecular junction between two gold electrodes has three different contact modes, i.e., i) more-more, ii) more-less (or less-more), and iii) less-less conductive contact modes at the two ends.

  7. Recombinant α-chains of type IV collagen demonstrate that the amino terminal of the Goodpasture autoantigen is crucial for antibody recognition

    PubMed Central

    Ryan, J J; Mason, P J; Pusey, C D; Turner, N

    1998-01-01

    Goodpasture's disease, an autoimmune disorder causing severe glomerulonephritis and pulmonary haemorrhage, is characterized by antibodies to the glomerular basement membrane (GBM). The principal target antigen has been identified as the carboxyl terminal non-collagenous (NC1) domain of the α3-chain of type IV collagen. Anti-GBM antibodies appear to recognize one major epitope that is common to all patients, and is largely conformational. We have analysed antibody binding to recombinant α(IV)NC1 domains using a construct and expression system shown to produce correctly folded antigen that is strongly recognized by autoantibodies. In this system, as with the native antigen, α3(IV)NC1 was bound strongly by antibodies from all patients, whereas the closely related α1(IV) and α5(IV)NC1 domains, similarly expressed, showed no such binding. A series of chimeric NC1 domains, between human α3(IV) and α1(IV), and between human and rat α3(IV), were expressed as recombinant molecules, and were recognized by autoantibodies to varying degrees. Strong binding required the presence of human α3(IV) sequence in the amino terminal region of both sets of chimeric molecules. This work strongly suggests that the amino terminal of α3(IV)NC1 is critical for antibody recognition, whereas the carboxyl terminal end of α3(IV)NC1 has a less important role. PMID:9697978

  8. The Arabidopsis Chloroplast Stromal N-Terminome: Complexities of Amino-Terminal Protein Maturation and Stability1[OPEN

    PubMed Central

    Rowland, Elden; Kim, Jitae; Bhuiyan, Nazmul H.; van Wijk, Klaas J.

    2015-01-01

    Protein amino (N) termini are prone to modifications and are major determinants of protein stability in bacteria, eukaryotes, and perhaps also in chloroplasts. Most chloroplast proteins undergo N-terminal maturation, but this is poorly understood due to insufficient experimental information. Consequently, N termini of mature chloroplast proteins cannot be accurately predicted. This motivated an extensive characterization of chloroplast protein N termini in Arabidopsis (Arabidopsis thaliana) using terminal amine isotopic labeling of substrates and mass spectrometry, generating nearly 14,000 tandem mass spectrometry spectra matching to protein N termini. Many nucleus-encoded plastid proteins accumulated with two or three different N termini; we evaluated the significance of these different proteoforms. Alanine, valine, threonine (often in N-α-acetylated form), and serine were by far the most observed N-terminal residues, even after normalization for their frequency in the plastid proteome, while other residues were absent or highly underrepresented. Plastid-encoded proteins showed a comparable distribution of N-terminal residues, but with a higher frequency of methionine. Infrequent residues (e.g. isoleucine, arginine, cysteine, proline, aspartate, and glutamate) were observed for several abundant proteins (e.g. heat shock proteins 70 and 90, Rubisco large subunit, and ferredoxin-glutamate synthase), likely reflecting functional regulation through their N termini. In contrast, the thylakoid lumenal proteome showed a wide diversity of N-terminal residues, including those typically associated with instability (aspartate, glutamate, leucine, and phenylalanine). We propose that, after cleavage of the chloroplast transit peptide by stromal processing peptidase, additional processing by unidentified peptidases occurs to avoid unstable or otherwise unfavorable N-terminal residues. The possibility of a chloroplast N-end rule is discussed. PMID:26371235

  9. Interactions of amino terminal domains of Shaker K channels with a pore blocking site studied with synthetic peptides

    PubMed Central

    1993-01-01

    Synthetic peptides of the five alternative NH2-terminal sequences of Shaker when applied to the cytoplasmic side of ShB channels that have an NH2-terminal deletion (ShB delta 6-46) block the channel with potencies correlated with the rate of inactivation in the corresponding variant. These peptides share no sequence similarity and yet three out of the five have apparent dissociation constants between 2 and 15 microM, suggesting that the specificity requirements for binding are low. To identify the primary structural determinants required for effective block of ShB delta 6-46, we examined the effects of substitutions made to the 20 residue ShB peptide on association and dissociation rates. Nonpolar residues within the peptide appear to be important in stabilizing the binding through hydrophobic interactions. Substitutions to leucine-7 showed there was a clear correlation between hydrophobicity and the dissociation rate constant (koff) with little effect on the association rate constant (kon). Substituting charged residues for hydrophobic residues within the region 4-8 disrupted binding. Within the COOH-terminal half of the peptide, substitutions that increased the net positive charge increased kon with relatively small changes in koff, suggesting the involvement of long-range electrostatic interactions in increasing the effective concentration of the peptide. Neutralizing charged residues produced small changes in koff. Charges within the region 12-20 act equivalently; alterations which conserved net charge produced little effect on either kon or koff. The results are consistent with this region of the peptide having an extended conformation and suggest that when bound this region makes few contacts with the channel protein and remains relatively unconstrained. Analogous mutations within the NH2-terminal domain of the intact ShB channel produced qualitatively similar effects on blocking and unblocking rates. PMID:8133245

  10. The periplasmic membrane proximal domain of MacA acts as a switch in stimulation of ATP hydrolysis by MacB transporter.

    PubMed

    Modali, Sita D; Zgurskaya, Helen I

    2011-08-01

    Escherichia coli MacAB-TolC is a tripartite macrolide efflux transporter driven by hydrolysis of ATP. In this complex, MacA is the periplasmic membrane fusion protein that stimulates the activity of MacB transporter and establishes the link with the outer membrane channel TolC. The molecular mechanism by which MacA stimulates MacB remains unknown. Here, we report that the periplasmic membrane proximal domain of MacA plays a critical role in functional MacA-MacB interactions and stimulation of MacB ATPase activity. Binding of MacA to MacB stabilizes the ATP-bound conformation of MacB, whereas interactions with both MacB and TolC affect the conformation of MacA. A single G353A substitution in the C-terminus of MacA inactivates MacAB-TolC function by changing the conformation of the membrane proximal domain of MacA and disrupting the proper assembly of the MacA-MacB complex. We propose that MacA acts in transport by promoting MacB transition into the closed ATP-bound conformation and in this respect, is similar to the periplasmic solute-binding proteins.

  11. Immunoreactive prohormone atrial natriuretic peptides 1-30 and 31-67 - Existence of a single circulating amino-terminal peptide

    NASA Technical Reports Server (NTRS)

    Chen, Yu-Ming; Whitson, Peggy A.; Cintron, Nitza M.

    1990-01-01

    Sep-Pak C18 extraction of human plasma and radioimmunoassay using antibodies which recognize atrial natriuretic peptide (99-128) and the prohormone sequences 1-30 and 31-67 resulted in mean values from 20 normal subjects of 26.2 (+/- 9.2), 362 (+/- 173) and 368 (+/- 160) pg/ml, respectively. A high correlation coefficient between values obtained using antibodies recognizing prohormone sequences 1-30 and 31-67 was observed (R = 0.84). Extracted plasma immunoreactivity of 1-30 and 31-67 both eluted at 46 percent acetonitrile. In contrast, chromatographic elution of synthetic peptides 1-30 and 31-67 was observed at 48 and 39 percent acetonitrile, respectively. Data suggest that the radioimmunoassay of plasma using antibodies recognizing prohormone sequences 1-30 and 31-67 may represent the measurement of a unique larger amino-terminal peptide fragment containing antigenic sites recognized by both antisera.

  12. Removal of amino-terminal extracellular domains of desmoglein 1 by staphylococcal exfoliative toxin is sufficient to initiate epidermal blister formation.

    PubMed

    Nishifuji, Koji; Shimizu, Atsushi; Ishiko, Akira; Iwasaki, Toshiroh; Amagai, Masayuki

    2010-09-01

    In both bullous impetigo and staphylococcal scalded-skin syndrome (SSSS), exfoliative toxins (ETs) produced by Staphylococcus aureus cause superficial intraepidermal blisters. ETs are known to cleave specifically a single peptide bond in the extracellular domains 3 and 4 of desmoglein (Dsg) 1. However, the precise mechanisms underlying ET-induced epidermal blister formation remain poorly understood. To determine whether cleavage of Dsg1 by an ET is sufficient to induce blister formation in vivo or if the subsequent internalization of cleaved Dsg1 or other desmosomal components is required. Skin samples obtained from neonatal mice injected with ETA were analyzed by time-lapse immunofluorescence and transmission electron microscopy for desmosomal components. Epidermal blister formation was observed as early as 60 min after ETA treatment. At this time, the amino-terminal extracellular domains of Dsg1 disappeared from the surface of keratinocytes, while the cleaved carboxy-terminal domain of Dsg1 (Dsg1-C) as well as the extracellular domains of desmocollin 1 (Dsc1-N) remained on the cell surface. Half-split desmosomes with intracytoplasmic dense plaques and attached tonofilaments were recognized ultrastructurally on the split surface of keratinocytes at 60 min. Subsequent to this, Dsg1-C and Dsc1-N gradually disappeared from the surface layer of keratinocytes. Our findings suggest that the removal of amino-terminal extracellular domains of Dsg1 by ETs is sufficient to initiate epidermal blister formation in bullous impetigo and SSSS. Copyright 2010 Japanese Society for Investigative Dermatology. Published by Elsevier Ireland Ltd. All rights reserved.

  13. A critical role for amino-terminal glutamine/asparagine repeats in the formation and propagation of a yeast prion.

    PubMed

    DePace, A H; Santoso, A; Hillner, P; Weissman, J S

    1998-06-26

    The yeast [PSI+] factor propagates by a prion-like mechanism involving self-replicating Sup35p amyloids. We identified multiple Sup35p mutants that either are poorly recruited into, or cause curing of, wildtype amyloids in vivo. In vitro, these mutants showed markedly decreased rates of amyloid formation, strongly supporting the protein-only prion hypothesis. Kinetic analysis suggests that the prion state replicates by accelerating slow conformational changes rather than by providing stable nuclei. Strikingly, our mutations map exclusively within a short glutamine/asparagine-rich region of Sup35p, and all but one occur at polar residues. Even after replacement of this region with polyglutamine, Sup35p retains its ability to form amyloids. These and other considerations suggest similarities between the prion-like propagation of [PSI+] and polyglutamine-mediated pathogenesis of several neurodegenerative diseases.

  14. Substitutions in the Amino-Terminal Tail of Neurospora Histone H3 Have Varied Effects on DNA Methylation

    PubMed Central

    Adhvaryu, Keyur K.; Berge, Emanuela; Tamaru, Hisashi; Freitag, Michael; Selker, Eric U.

    2011-01-01

    Eukaryotic genomes are partitioned into active and inactive domains called euchromatin and heterochromatin, respectively. In Neurospora crassa, heterochromatin formation requires methylation of histone H3 at lysine 9 (H3K9) by the SET domain protein DIM-5. Heterochromatin protein 1 (HP1) reads this mark and directly recruits the DNA methyltransferase, DIM-2. An ectopic H3 gene carrying a substitution at K9 (hH3K9L or hH3K9R) causes global loss of DNA methylation in the presence of wild-type hH3 (hH3WT). We investigated whether other residues in the N-terminal tail of H3 are important for methylation of DNA and of H3K9. Mutations in the N-terminal tail of H3 were generated and tested for effects in vitro and in vivo, in the presence or absence of the wild-type allele. Substitutions at K4, K9, T11, G12, G13, K14, K27, S28, and K36 were lethal in the absence of a wild-type allele. In contrast, mutants bearing substitutions of R2, A7, R8, S10, A15, P16, R17, K18, and K23 were viable. The effect of substitutions on DNA methylation were variable; some were recessive and others caused a semi-dominant loss of DNA methylation. Substitutions of R2, A7, R8, S10, T11, G12, G13, K14, and P16 caused partial or complete loss of DNA methylation in vivo. Only residues R8-G12 were required for DIM-5 activity in vitro. DIM-5 activity was inhibited by dimethylation of H3K4 and by phosphorylation of H3S10, but not by acetylation of H3K14. We conclude that the H3 tail acts as an integrating platform for signals that influence DNA methylation, in part through methylation of H3K9. PMID:22242002

  15. The membrane-proximal external region of the human immunodeficiency virus type 1 envelope: dominant site of antibody neutralization and target for vaccine design.

    PubMed

    Montero, Marinieve; van Houten, Nienke E; Wang, Xin; Scott, Jamie K

    2008-03-01

    Enormous efforts have been made to produce a protective vaccine against human immunodeficiency virus type 1; there has been little success. However, the identification of broadly neutralizing antibodies against epitopes on the highly conserved membrane-proximal external region (MPER) of the gp41 envelope protein has delineated this region as an attractive vaccine target. Furthermore, emerging structural information on the MPER has provided vaccine designers with new insights for building relevant immunogens. This review describes the current state of the field regarding (i) the structure and function of the gp41 MPER; (ii) the structure and binding mechanisms of the broadly neutralizing antibodies 2F5, 4E10, and Z13; and (iii) the development of an MPER-targeting vaccine. In addition, emerging approaches to vaccine design are presented.

  16. Close membrane-membrane proximity induced by Ca(2+)-dependent multivalent binding of synaptotagmin-1 to phospholipids.

    PubMed

    Araç, Demet; Chen, Xiaocheng; Khant, Htet A; Ubach, Josep; Ludtke, Steven J; Kikkawa, Masahide; Johnson, Arthur E; Chiu, Wah; Südhof, Thomas C; Rizo, Josep

    2006-03-01

    Synaptotagmin acts as a Ca(2+) sensor in neurotransmitter release through its two C(2) domains. Ca(2+)-dependent phospholipid binding is key for synaptotagmin function, but it is unclear how this activity cooperates with the SNARE complex involved in release or why Ca(2+) binding to the C(2)B domain is more crucial for release than Ca(2+) binding to the C(2)A domain. Here we show that Ca(2+) induces high-affinity simultaneous binding of synaptotagmin to two membranes, bringing them into close proximity. The synaptotagmin C(2)B domain is sufficient for this ability, which arises from the abundance of basic residues around its surface. We propose a model wherein synaptotagmin cooperates with the SNAREs in bringing the synaptic vesicle and plasma membranes together and accelerates membrane fusion through the highly positive electrostatic potential of its C(2)B domain.

  17. The plasmid RK2 replication initiator protein (TrfA) binds to the sliding clamp beta subunit of DNA polymerase III: implication for the toxicity of a peptide derived from the amino-terminal portion of 33-kilodalton TrfA.

    PubMed

    Kongsuwan, Kritaya; Josh, Peter; Picault, Marc J; Wijffels, Gene; Dalrymple, Brian

    2006-08-01

    The broad-host-range plasmid RK2 is capable of replication and stable maintenance within a wide range of gram-negative bacterial hosts. It encodes the essential replication initiation protein TrfA, which binds to the host initiation protein, DnaA, at the plasmid origin of replication (oriV). There are two versions of the TrfA protein, 44 and 33 kDa, resulting from alternate in-frame translational starts. We have shown that the smaller protein, TrfA-33, and its 64-residue amino-terminal peptide (designated T1) physically interact with the Escherichia coli beta sliding clamp (beta(2)). This interaction appears to be mediated through a QLSLF peptide motif located near the amino-terminal end of TrfA-33 and T1, which is identical to the previously described eubacterial clamp-binding consensus motif. T1 forms a stable complex with beta(2) and was found to inhibit plasmid RK2 replication in vitro. This specific interaction between T1 and beta(2) and the ability of T1 to block DNA replication have implications for the previously reported cell lethality caused by overproduction of T1. The toxicity of T1 was suppressed when wild-type T1 was replaced with mutant T1, carrying an LF deletion in the beta-binding motif. Previously, T1 toxicity has been shown to be suppressed by Hda, an intermediate regulatory protein which helps prevent over-initiation in E. coli through its interaction with the initiator protein, DnaA, and beta(2). Our results support a model in which T1 toxicity is caused by T1 binding to beta(2), especially when T1 is overexpressed, preventing beta(2) from interacting with host replication proteins such as Hda during the early events of chromosome replication.

  18. The Plasmid RK2 Replication Initiator Protein (TrfA) Binds to the Sliding Clamp β Subunit of DNA Polymerase III: Implication for the Toxicity of a Peptide Derived from the Amino-Terminal Portion of 33-Kilodalton TrfA

    PubMed Central

    Kongsuwan, Kritaya; Josh, Peter; Picault, Marc J.; Wijffels, Gene; Dalrymple, Brian

    2006-01-01

    The broad-host-range plasmid RK2 is capable of replication and stable maintenance within a wide range of gram-negative bacterial hosts. It encodes the essential replication initiation protein TrfA, which binds to the host initiation protein, DnaA, at the plasmid origin of replication (oriV). There are two versions of the TrfA protein, 44 and 33 kDa, resulting from alternate in-frame translational starts. We have shown that the smaller protein, TrfA-33, and its 64-residue amino-terminal peptide (designated T1) physically interact with the Escherichia coli β sliding clamp (β2). This interaction appears to be mediated through a QLSLF peptide motif located near the amino-terminal end of TrfA-33 and T1, which is identical to the previously described eubacterial clamp-binding consensus motif. T1 forms a stable complex with β2 and was found to inhibit plasmid RK2 replication in vitro. This specific interaction between T1 and β2 and the ability of T1 to block DNA replication have implications for the previously reported cell lethality caused by overproduction of T1 (P. D. Kim, T. M. Rosche, and W. Firshein, Plasmid 43:214-222, 2000). The toxicity of T1 was suppressed when wild-type T1 was replaced with mutant T1, carrying an LF deletion in the β-binding motif. Previously, T1 toxicity has been shown to be suppressed by Hda, an intermediate regulatory protein which helps prevent overinitiation in E. coli through its interaction with the initiator protein, DnaA, and β2. Our results support a model in which T1 toxicity is caused by T1 binding to β2, especially when T1 is overexpressed, preventing β2 from interacting with host replication proteins such as Hda during the early events of chromosome replication. PMID:16855240

  19. Molecular Basis for Subtype Specificity and High-Affinity Zinc Inhibition in the GluN1-GluN2A NMDA Receptor Amino-Terminal Domain.

    PubMed

    Romero-Hernandez, Annabel; Simorowski, Noriko; Karakas, Erkan; Furukawa, Hiro

    2016-12-21

    Zinc is vastly present in the mammalian brain and controls functions of various cell surface receptors to regulate neurotransmission. A distinctive characteristic of N-methyl-D-aspartate (NMDA) receptors containing a GluN2A subunit is that their ion channel activity is allosterically inhibited by a nano-molar concentration of zinc that binds to an extracellular domain called an amino-terminal domain (ATD). Despite physiological importance, the molecular mechanism underlying the high-affinity zinc inhibition has been incomplete because of the lack of a GluN2A ATD structure. Here we show the first crystal structures of the heterodimeric GluN1-GluN2A ATD, which provide the complete map of the high-affinity zinc-binding site and reveal distinctive features from the ATD of the GluN1-GluN2B subtype. Perturbation of hydrogen bond networks at the hinge of the GluN2A bi-lobe structure affects both zinc inhibition and open probability, supporting the general model in which the bi-lobe motion in ATD regulates the channel activity in NMDA receptors. Copyright © 2016 Elsevier Inc. All rights reserved.

  20. The amino-terminal half of rotavirus SA114fM VP4 protein contains a hemagglutination domain and primes for neutralizing antibodies to the virus.

    PubMed Central

    Lizano, M; López, S; Arias, C F

    1991-01-01

    We have previously reported the synthesis in Escherichia coli of polypeptide MS2-VP8', which contains the amino-terminal half of the SA114fM VP4 protein fused to MS2 bacteriophage polymerase sequences (C. F. Arias, M. Lizano, and S. López, J. Gen. Virol. 68:633-642, 1987). In this work we have synthesized the carboxy-terminal half of the VP4 protein also fused to the MS2 polymerase. This protein, designated MS2-VP5', was recognized by sera to the complete virion and was able to induce antibodies to the virus when administered to mice; however, these antibodies had no neutralizing activity. The two chimeric polypeptides were tested for their ability to agglutinate erythrocytes and to prime the immune system of mice. Bacterial lysates enriched for the MS2-VP8' hybrid polypeptide, but not those enriched for the MS2-VP5' protein or those containing proteins from the host E. coli strain, had hemagglutinating activity. This hemagglutination was inhibited by sera to SA114fM rotavirus. In addition, a single dose of the MS2-VP8' polypeptide was able to prime the immune system of mice for an augmented neutralizing antibody response when the animals were subsequently immunized with purified SA114fM virus. Images PMID:1847459

  1. High CO2 Leads to Na,K-ATPase Endocytosis via c-Jun Amino-Terminal Kinase-Induced LMO7b Phosphorylation

    PubMed Central

    Trejo Bittar, Humberto E.; Welch, Lynn C.; Vagin, Olga; Deiss-Yehiely, Nimrod; Kelly, Aileen M.; Baker, Mairead R.; Capri, Joseph; Cohn, Whitaker; Whitelegge, Julian P.; Vadász, István; Gruenbaum, Yosef; Sznajder, Jacob I.

    2015-01-01

    The c-Jun amino-terminal kinase (JNK) plays a role in inflammation, proliferation, apoptosis, and cell adhesion and cell migration by phosphorylating paxillin and β-catenin. JNK phosphorylation downstream of AMP-activated protein kinase (AMPK) activation is required for high CO2 (hypercapnia)-induced Na,K-ATPase endocytosis in alveolar epithelial cells. Here, we provide evidence that during hypercapnia, JNK promotes the phosphorylation of LMO7b, a scaffolding protein, in vitro and in intact cells. LMO7b phosphorylation was blocked by exposing the cells to the JNK inhibitor SP600125 and by infecting cells with dominant-negative JNK or AMPK adenovirus. The knockdown of the endogenous LMO7b or overexpression of mutated LMO7b with alanine substitutions of five potential JNK phosphorylation sites (LMO7b-5SA) or only Ser-1295 rescued both LMO7b phosphorylation and the hypercapnia-induced Na,K-ATPase endocytosis. Moreover, high CO2 promoted the colocalization and interaction of LMO7b and the Na,K-ATPase α1 subunit at the plasma membrane, which were prevented by SP600125 or by transfecting cells with LMO7b-5SA. Collectively, our data suggest that hypercapnia leads to JNK-induced LMO7b phosphorylation at Ser-1295, which facilitates the interaction of LMO7b with Na,K-ATPase at the plasma membrane promoting the endocytosis of Na,K-ATPase in alveolar epithelial cells. PMID:26370512

  2. High CO2 Leads to Na,K-ATPase Endocytosis via c-Jun Amino-Terminal Kinase-Induced LMO7b Phosphorylation.

    PubMed

    Dada, Laura A; Trejo Bittar, Humberto E; Welch, Lynn C; Vagin, Olga; Deiss-Yehiely, Nimrod; Kelly, Aileen M; Baker, Mairead R; Capri, Joseph; Cohn, Whitaker; Whitelegge, Julian P; Vadász, István; Gruenbaum, Yosef; Sznajder, Jacob I

    2015-12-01

    The c-Jun amino-terminal kinase (JNK) plays a role in inflammation, proliferation, apoptosis, and cell adhesion and cell migration by phosphorylating paxillin and β-catenin. JNK phosphorylation downstream of AMP-activated protein kinase (AMPK) activation is required for high CO2 (hypercapnia)-induced Na,K-ATPase endocytosis in alveolar epithelial cells. Here, we provide evidence that during hypercapnia, JNK promotes the phosphorylation of LMO7b, a scaffolding protein, in vitro and in intact cells. LMO7b phosphorylation was blocked by exposing the cells to the JNK inhibitor SP600125 and by infecting cells with dominant-negative JNK or AMPK adenovirus. The knockdown of the endogenous LMO7b or overexpression of mutated LMO7b with alanine substitutions of five potential JNK phosphorylation sites (LMO7b-5SA) or only Ser-1295 rescued both LMO7b phosphorylation and the hypercapnia-induced Na,K-ATPase endocytosis. Moreover, high CO2 promoted the colocalization and interaction of LMO7b and the Na,K-ATPase α1 subunit at the plasma membrane, which were prevented by SP600125 or by transfecting cells with LMO7b-5SA. Collectively, our data suggest that hypercapnia leads to JNK-induced LMO7b phosphorylation at Ser-1295, which facilitates the interaction of LMO7b with Na,K-ATPase at the plasma membrane promoting the endocytosis of Na,K-ATPase in alveolar epithelial cells.

  3. Cytoplasmic expression of mature glycylglycine endopeptidase lysostaphin with an amino terminal hexa-histidine in a soluble and catalytically active form in Escherichia coli.

    PubMed

    Sharma, Rahul; Sharma, Poonam R; Choudhary, Manohar L; Pande, Amit; Khatri, Ghan Shyam

    2006-01-01

    Methicillin-resistant Staphylococcus aureus is a major problem in the world, causing hospital acquired infections and the infections/pathogenesis in community. Lysostaphin is a novel therapeutic molecule to kill the multidrug-resistant S. aureus. Mature lysostaphin is a single polypeptide (approximately 27 kDa) chain metalloprotease glycylglycine endopeptidase, capable of specifically hydrolyzing penta-glycine crosslinks present in the peptidoglycan of the S. aureus cell wall. The mature lysostaphin gene of Staphylococcus simulans has been cloned and overexpressed in the cytoplasm of E. coli with amino terminal hexa-histidine as a fusion partner under the transcriptional control of bacteriophage T7 phi 10 promoter/lac operator and ribosome binding site. The transformed E. coli BL21 (lambdaDE3) cells produced catalytically active soluble (His)6-lysostaphin fusion protein in the cytoplasm representing approximately 20% of the total cellular proteins. The fusion protein was purified to homogeneity using a single chromatographic step of IMAC on Ni-NTA agarose. The present cloning, expression, and purification procedure of recombinant lysostaphin from a non-pathogenic organism E. coli enables preparation of large quantity of r-lysostaphin for structure function studies and evaluation of its clinical potential in therapy and prophylaxis of staphylococcal infections.

  4. Amino-terminal fragment of pro-brain natriuretic hormone identifies functional impairment and right ventricular overload in operated tetralogy of Fallot patients.

    PubMed

    Festa, P; Ait-Ali, L; Prontera, C; De Marchi, D; Fontana, M; Emdin, M; Passino, C

    2007-01-01

    To evaluate the relationship between plasma concentration of amino-terminal fragment of pro-brain natriuretic peptide (NT-proBNP), functional capacity, and right ventricular overload in survivors of tetralogy of Fallot (TOF) repair, we prospectively studied 70 operated TOF patients (44 males, 21 +/- 1 years old; mean +/- SEM) who underwent, during the same day, echocardiography, cardiac magnetic resonance imaging, neurohormonal characterization (plasma NT-proBNP, catecholamines, plasma renin activity, and aldosterone assay), and cardiopulmonary exercise testing. Forty-eight age- and sex-matched healthy volunteers served as the control group. Compared to controls, maximal workload and peak oxygen consumption (VO2/kg) were lower in operated TOF patients (p < 0.001), whereas NT-proBNP concentration was elevated (p < 0.001). No difference was found among the other neurohormones. In operated TOF patients, NT-proBNP showed a significant positive correlation with right ventricular (RV) end systolic and end diastolic volumes and RV systolic pressure, and it showed a negative correlation with peak VO2/kg and RV ejection fraction. From multivariable analysis, NT-proBNP concentration was found to be an independent predictor of peak VO2/kg, RV end systolic volume, and RV systolic pressure. These results show an association among RV overload, decrease in functional capacity, and cardiac natriuretic peptide expression in operated TOF patients. NT-proBNP plasma assay may be a useful tool for diagnostic purposes and for decision making in this setting.

  5. Improving solubility of NR2B amino-terminal domain of N-methyl-D-aspartate receptor expressed in Escherichia coli

    SciTech Connect

    Ng, F.-M.; Soh Wanqin; Geballe, Matthew T.; Low, C.-M.

    2007-10-12

    The amino-terminal domains (ATDs) of N-methyl-D-aspartate (NMDA) receptors contain binding sites for modulators and may serve as potential drug targets in neurological diseases. Here, three fusion tags (6xHis-, GST-, and MBP-) were fused to the ATD of NMDA receptor NR2B subunit (ATD2B) and expressed in Escherichia coli. Each tag's ability to confer enhanced solubility to ATD2B was assessed. Soluble ATD2B was successfully obtained as a MBP fusion protein. Dynamic light scattering revealed the protein (1 mg/ml) exists as monodispersed species at 25 {sup o}C. Functional studies using circular dichroism showed that the soluble MBP-ATD2B bound ifenprodil in a dose-dependent manner. The dissociation constants obtained for ifenprodil were similar in the absence (64 nM) and presence (116 nM) of saturating concentration of maltose. Moreover, the yield of soluble MBP-ATD2B is 18 times higher than the refolded 6xHis-ATD2B. We have reported a systematic comparison of three different affinity tagging strategies and identified a rapid and efficient method to obtain large amount of ATD2B recombinant protein for biochemical and structural studies.

  6. The Atomic Structure of the HIV-1 gp41 Transmembrane Domain and Its Connection to the Immunogenic Membrane-proximal External Region.

    PubMed

    Apellániz, Beatriz; Rujas, Edurne; Serrano, Soraya; Morante, Koldo; Tsumoto, Kouhei; Caaveiro, Jose M M; Jiménez, M Ángeles; Nieva, José L

    2015-05-22

    The membrane-proximal external region (MPER) C-terminal segment and the transmembrane domain (TMD) of gp41 are involved in HIV-1 envelope glycoprotein-mediated fusion and modulation of immune responses during viral infection. However, the atomic structure of this functional region remains unsolved. Here, based on the high resolution NMR data obtained for peptides spanning the C-terminal segment of MPER and the TMD, we report two main findings: (i) the conformational variability of the TMD helix at a membrane-buried position; and (ii) the existence of an uninterrupted α-helix spanning MPER and the N-terminal region of the TMD. Thus, our structural data provide evidence for the bipartite organization of TMD predicted by previous molecular dynamics simulations and functional studies, but they do not support the breaking of the helix at Lys-683, as was suggested by some models to mark the initiation of the TMD anchor. Antibody binding energetics examined with isothermal titration calorimetry and humoral responses elicited in rabbits by peptide-based vaccines further support the relevance of a continuous MPER-TMD helix for immune recognition. We conclude that the transmembrane anchor of HIV-1 envelope is composed of two distinct subdomains: 1) an immunogenic helix at the N terminus also involved in promoting membrane fusion; and 2) an immunosuppressive helix at the C terminus, which might also contribute to the late stages of the fusion process. The unprecedented high resolution structural data reported here may guide future vaccine and inhibitor developments.

  7. Identification of a new epitope for HIV-neutralizing antibodies in the gp41 membrane proximal external region by an Env-tailored phage display library.

    PubMed

    Zhou, Mingkui; Meyer, Torsten; Koch, Stefanie; Koch, Joachim; von Briesen, Hagen; Benito, José M; Soriano, Vincent; Haberl, Annette; Bickel, Markus; Dübel, Stefan; Hust, Michael; Dietrich, Ursula

    2013-02-01

    HIV controllers are a valuable source for the identification of HIV-neutralizing antibodies, as chronic infection over decades allows extensive affinity maturation of antibodies for improved Ag recognition. We analyzed a small cohort of elite controllers (ECs) for HIV-neutralizing antibodies using a panel of standardized HIV-1 pseudovirions on TZM-bl cells. An HIV-1 Env-tailored phage display library was generated to select epitopes targeted by neutralizing antibodies in the EC26 plasma sample showing the broadest neutralizing activity. Selected Env fragments were mostly allocated to the membrane proximal external region of gp41. After preabsorbing the EC26 plasma with the selected phage EC26-2A4, we achieved 50% depletion of its neutralizing activity. Furthermore, antibodies affinity-purified with the EC26-2A4 epitope from EC26 plasma showed neutralizing activity, proving that the selected phage indeed contains an epitope targeted by neutralizing plasma antibodies. Epitope fine mapping of the purified plasma antibodies on peptide arrays identified a new epitope overlapping, but clearly distinct, from the prominent 2F5 epitope. Of note, the purified antibodies did not show autoreactivity with cardiolipin, whereas low reactivity with phosphatidylserine comparable to mAb 2F5 was observed. Thus, this new epitope represents a promising candidate for further analysis in view of HIV vaccine development. © 2012 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  8. Membrane-proximal TRAIL species are incapable of inducing short circuit apoptosis signaling: Implications for drug development and basic cytokine biology

    PubMed Central

    Tatzel, Katharina; Kuroki, Lindsay; Dmitriev, Igor; Kashentseva, Elena; Curiel, David T.; Goedegebuure, S. Peter; Powell, Matthew A.; Mutch, David G.; Hawkins, William G.; Spitzer, Dirk

    2016-01-01

    TRAIL continues to garner substantial interest as a recombinant cancer therapeutic while the native cytokine itself serves important tumor surveillance functions when expressed in membrane-anchored form on activated immune effector cells. We have recently developed the genetically stabilized TRAIL platform TR3 in efforts to improve the limitations associated with currently available drug variants. While in the process of characterizing mesothelin-targeted TR3 variants using a single chain antibody (scFv) delivery format (SS-TR3), we discovered that the membrane-tethered cytokine had a substantially increased activity profile compared to non-targeted TR3. However, cell death proceeded exclusively via a bystander mechanism and protected the mesothelin-positive targets from apoptosis rather than leading to their elimination. Incorporation of a spacer-into the mesothelin surface antigen or the cancer drug itself-converted SS-TR3 into a cis-acting phenotype. Further experiments with membrane-anchored TR3 variants and the native cytokine confirmed our hypothesis that membrane-proximal TRAIL species lack the capacity to physically engage their cognate receptors coexpressed on the same cell membrane. Our findings not only provide an explanation for the “peaceful” coexistence of ligand and receptor of a representative member of the TNF superfamily but give us vital clues for the design of activity-enhanced TR3-based cancer therapeutics. PMID:26935795

  9. Eliciting neutralizing antibodies against the membrane proximal external region of HIV-1 Env by chimeric live attenuated influenza A virus vaccines.

    PubMed

    Zang, Yang; Du, Dongchuan; Li, Na; Su, Weiheng; Liu, Xintao; Zhang, Yan; Nie, Jianhui; Wang, Youchun; Kong, Wei; Jiang, Chunlai

    2015-07-31

    Despite significant efforts directed toward research on HIV-1 vaccines, a truly effective immunogen has not been achieved. However, the broadly neutralizing antibodies (BnAbs) 2F5 and 4E10, targeting the highly conserved membrane proximal external region (MPER) of HIV-1, are two promising tools for vaccine development. Here we engrafted the MPER into the linker domain between the trimeric core structure and the transmembrane domain of influenza A virus HA2 to investigate the potential of such chimeric viruses to elicit HIV-1 neutralizing antibodies. In the context of proliferating attenuated influenza A viruses, these HIV-1 neutralizing antibody epitopes could be continuously expressed and mimicked their native conformation to induce humoral immune responses. While MPER-specific antibodies could be detected in serum of guinea pigs vaccinated with the chimeric viruses, they exhibited only weakly neutralizing activities. These antisera from vaccinated animals neutralized viruses of clades B and BC (tier 1), but not of clades AE (tier 1) and C (tier 2). These results suggest that influenza A virus can be used as a vehicle for displaying MPER and inducing BnAbs, but it provides limited protection against HIV-1 infection. In the future development of HIV-1 vaccines by rational design, a more effective live virus vector or multiple antigens should be chosen to facilitate the process of neutralizing antibody maturation. Copyright © 2015 Elsevier Ltd. All rights reserved.

  10. The effects of the β-agonist isoproterenol on the down-regulation, functional responsiveness, and trafficking of β2-adrenergic receptors with amino-terminal polymorphisms

    PubMed Central

    Koryakina, Yulia; Jones, Stacie M.; Cornett, Lawrence E.; Seely, Kathryn; Brents, Lisa; Prather, Paul L.; Kofman, Alexander; Kurten, Richard C.

    2014-01-01

    The β2-adrenergic receptor (β2AR) is an important target for respiratory and cardiovascular disease medications. Clinical studies suggest that amino-terminal polymorphisms of the β2AR may act as disease modifiers. We hypothesized that polymorphisms at amino acids 16 and 27 result in differential trafficking and down-regulation of β2AR variants following β-agonist exposure. The functional consequences of the four possible combinations of these polymorphisms in the human β2AR (designated β2AR-RE, -GE, -RQ and -GQ) were studied using site-directed mutagenesis and recombinant expression in HEK 293 cells. Ligand binding assays demonstrated that after 24 h exposure to 1 μM isoproterenol, isoforms with Arg16 (β2AR-RE and β2AR-RQ) underwent increased down-regulation compared to isoforms with Gly16 (β2AR-GE and β2AR-GQ). Consistent with these differences in down-regulation between isoforms, prolonged isoproterenol treatment resulted in diminished cyclic AMP response to subsequent isoproterenol challenge in β2AR-RE relative to β2AR-GE. Confocal microscopy revealed that the receptor isoforms had similar co-localization with the early endosomal marker EEA1 following isoproterenol treatment, suggesting that they had similar patterns of internalization. None of the isoforms exhibited significant co-localization with the recycling endosome marker Rab11 in response to isoproterenol treatment. Furthermore, we found that prolonged isoproterenol treatment led to a higher degree of co-localization of β2AR-RE with the lysosomal marker Lamp1 compared to that of β2AR-GE. Taken together, these results indicate that a mechanism responsible for differential responses of these receptor isoforms to β-agonist involves differences in the efficiency with which agonist-activated receptors are trafficked to lysosomes for degradation, or differences in degradation in the lysosomes. PMID:22938397

  11. An efficient approach to identify ilvA mutations reveals an amino-terminal catalytic domain in biosynthetic threonine deaminase from Escherichia coli.

    PubMed Central

    Fisher, K E; Eisenstein, E

    1993-01-01

    High-level expression of the regulatory enzyme threonine deaminase in Escherichia coli strains grown on minimal medium that are deficient in the activities of enzymes needed for branched-chain amino acid biosynthesis result in growth inhibition, possibly because of the accumulation of toxic levels of alpha-ketobutyrate, the product of the committed step in isoleucine biosynthesis. This condition affords a means for selecting genetic variants of threonine deaminase that are deficient in catalysis by suppression of growth inhibition. Strains harboring mutations in ilvA that decreased the catalytic activity of threonine deaminase were found to grow more rapidly than isogenic strains containing wild-type ilvA. Modification of the ilvA gene to introduce additional unique, evenly spaced restriction enzyme sites facilitated the identification of suppressor mutations by enabling small DNA fragments to be subcloned for sequencing. The 10 mutations identified in ilvA code for enzymes with significantly reduced activity relative to that of wild-type threonine deaminase. Values for their specific activities range from 40% of that displayed by wild-type enzyme to complete inactivation as evidenced by failure to complement an ilvA deletion strain to isoleucine prototrophy. Moreover, some mutant enzymes showed altered allosteric properties with respect to valine activation and isoleucine inhibition. The location of the 10 mutations in the 5' two-thirds of the ilvA gene is consistent with suggestions that threonine deaminase is organized functionally with an amino-terminal domain that is involved in catalysis and a carboxy-terminal domain that is important for regulation. Images PMID:8407838

  12. Procollagen type 1 amino-terminal propeptide (P1NP) and risk of hip fractures in elderly Norwegian men and women. A NOREPOS study.

    PubMed

    Finnes, T E; Lofthus, C M; Meyer, H E; Eriksen, E F; Apalset, E M; Tell, G S; Torjesen, P; Samuelsen, S O; Holvik, K

    2014-07-01

    The current study aimed to assess a possible association between the bone turnover marker procollagen type 1 amino-terminal propeptide (P1NP) and future hip fractures in elderly Norwegian men and women and to elucidate the relation between P1NP, bone mineral density and 25-hydroxyvitamin D (25(OH)D). Men and women aged 71 to 77 from two population based health studies in Norway (1999-2001) were followed for a median period of 7.3 years with respect to hip fractures. The study was designed as a case-cohort study. P1NP and 25(OH)D were analysed in frozen serum samples obtained at baseline in hip fracture patients (n=340) and in randomly selected sex stratified sub-cohorts. Bone mineral density was measured by dual-energy X-ray absorptiometry (DXA) in a subset of participants. Cox proportional hazards regression with inverse probability weighting and robust variance was performed. No significant correlation between 25(OH)D and P1NP was found. A negative correlation between P1NP and BMD was observed in women (Rho=-0.36, p=0.001). A similar trend was observed in men. No association between quartiles of P1NP and rate of subsequent hip fractures was found. Spline analyses suggested a higher rate of hip fracture at P1NP levels above 60 μg/L in both men and women. A higher hip fracture rate, which was independent of BMD, was also indicated in women with very low levels of P1NP.

  13. Identification of a phosphorylation site in the hinge region of the human progesterone receptor and additional amino-terminal phosphorylation sites.

    PubMed

    Knotts, T A; Orkiszewski, R S; Cook, R G; Edwards, D P; Weigel, N L

    2001-03-16

    We have previously reported the identification of seven in vivo phosphorylation sites in the amino-terminal region of the human progesterone receptor (PR). From our previous in vivo studies, it was evident that several phosphopeptides remained unidentified. In particular, we wished to determine whether human PR contains a phosphorylation site in the hinge region, as do other steroid receptors including chicken PR, human androgen receptor, and mouse estrogen receptor. Previously, problematic trypsin cleavage sites hampered our ability to detect phosphorylation sites in large incomplete tryptic peptides. Using a combination of mass spectrometry and in vitro phosphorylation, we have identified six previously unidentified phosphorylation sites in human PR. Using nanoelectrospray ionization mass spectrometry, we have identified two new in vivo phosphorylation sites, Ser(20) and Ser(676), in baculovirus-expressed human PR. Ser(676) is analogous to the hinge site identified in other steroid receptors. Additionally, precursor ion scans identified another phosphopeptide that contains Ser(130)-Pro(131), a likely candidate for phosphorylation. In vitro phosphorylation of PR with Cdk2 has revealed five additional in vitro Cdk2 phosphorylation sites: Ser(25), Ser(213), Thr(430), Ser(554), and Ser(676). At least two of these, Ser(213) and Ser(676), are authentic in vivo sites. We confirmed the presence of the Cdk2-phosphorylated peptide containing Ser(213) in PR from in vivo labeled T47D cells, indicating that this is an in vivo site. Our combined studies indicate that most, if not all, of the Ser-Pro motifs in human PR are sites for phosphorylation. Taken together, these data indicate that the phosphorylation of PR is highly complex, with at least 14 phosphorylation sites.

  14. Early intraplatelet signaling enhances the release of human platelet PAR-1 and -4 amino-terminal peptides in response to thrombin.

    PubMed

    Ofosu, Frederick A; Dewar, Lori; Song, Yingqi; Cedrone, Aisha C; Hortelano, Gonzalo; Craven, Sharon J

    2009-02-24

    Activation of washed human platelets initiated with alpha-thrombin, SFLLRN, or AYPGKF invariably results in the generation of PAR-1-(1-41) and PAR-4-(1-47). PAR-1-(1-41) and PAR-4-(1-47) are amino-terminal peptides generated when PAR-1 and -4 are cleaved in their first extracellular domains after R(41) and R(47), respectively, to expose the tethered ligand domains of PAR-1 and -4. Since soybean trypsin inhibitor decreases generation of PAR-1-(1-41) and PAR-4-(1-47) and other platelet aggregation-related responses to these three agonists, but does not inactivate alpha-thrombin, a platelet trypsin-like proteinase apparently activates PAR-1 and -4 to propagate PAR-dependent platelet responses. This study identified the signaling pathways implicated in the generation of the platelet proteinase that in turn produces PAR-1-(1-41) and PAR-4-(1-47), to thereby drive the subsequent PAR-dependent platelet aggregation-related responses to alpha-thrombin, SFLLRN, or AYPGKF. Only inhibitors of signaling enzymes that prevented ATP release (forskolin, PGE(1), or BIMI-1) prevented or delayed the generation of PAR-1-(1-41) and PAR-4-(1-47) in response to all three agonists. SBTI prevented platelet aggregation initiated by alpha-thrombin, SFLLRN, or AYPGKF but did so less effectively when it was added 10 s after each agonist. Thus, the platelet-derived proteinase acts within 10 s of each agonist addition to generate PAR-1-(1-41) and PAR-4-(1-47). Furthermore, alpha-thrombin may not effectively catalyze PAR-1-(1-41) and PAR-4-(1-47) generation. We propose that unidentified ATP-dependent phosphorylation reactions catalyzed by PKC help to generate the platelet-derived proteinase that propagates human platelet PAR-1 and -4 activation by the three agonists.

  15. Amino-Terminal proB-Type Natriuretic Peptide Levels in the Umbilical Cord Blood of Neonates Differ According to the Type of Prenatally Diagnosed Congenital Heart Disease.

    PubMed

    Bae, Jin Young; Cha, Hyun-Hwa; Seong, Won Joon

    2015-12-01

    The aim of this study was to investigate differences in amino-terminal proB-type natriuretic peptide (NT-proBNP) levels in the cord blood of neonates according to the type of congenital heart disease (CHD) and to evaluate the usefulness of NT-proBNP as a prognostic marker. We included 76 neonates with prenatally diagnosed CHD and 45 controls without CHD. Neonates were classified into five groups based on echocardiographic findings. The levels of NT-proBNP in the cord blood were examined and analyzed according to the neonatal outcomes. The levels of NT-proBNP were significantly elevated in the cord blood of neonates with CHD compared with that in the cord blood of controls. The levels of NT-proBNP in the group with right ventricular outflow tract obstruction without a ventricular septal defect were significantly increased compared to that in the other groups. The neonates that required acute surgical correction had higher levels of NT-proBNP in the cord blood, though they were not statistically significant. Meanwhile, NT-proBNP levels in the cord blood of neonates with functional single ventricle were significantly higher than that in the cord blood of those with functional biventricles. Significant differences in the levels of NT-proBNP between survivors and nonsurvivors were observed within 1 year of birth. In this study, we found that the levels of NT-proBNP in the cord blood of neonates with CHD were higher than the levels in controls. This finding was striking in the group with right ventricular outflow tract obstruction, and it was associated with surgery for functional single ventricle and 1-year survival.

  16. Amino-Terminal Oriented Mass Spectrometry of Substrates (ATOMS) N-terminal sequencing of proteins and proteolytic cleavage sites by quantitative mass spectrometry.

    PubMed

    Doucet, Alain; Overall, Christopher M

    2011-01-01

    Edman degradation is a long-established technique for N-terminal sequencing of proteins and cleavage fragments. However, for accurate data analysis and amino acid assignments, Edman sequencing proceeds on samples of single proteins only and so lacks high-throughput capabilities. We describe a new method for the high-throughput determination of N-terminal sequences of multiple protein fragments in solution. Proteolytic processing can change the activity of bioactive proteins and also reveal cryptic binding sites and generate proteins with new functions (neoproteins) not found in the parent molecule. For example, extracellular matrix (ECM) protein processing often produces multiple proteolytic fragments with the generation of cryptic binding sites and neoproteins by ECM protein processing being well documented. The exact proteolytic cleavage sites need to be identified to fully understand the functions of the cleavage fragments and biological roles of proteases in vivo. However, the identification of cleavage sites in complex high molecular proteins such as those composing the ECM is not trivial. N-terminal microsequencing of proteolytic fragments is the usual method employed, but it suffers from poor resolution of sodium dodecylsulfate-polyacrylamide gel electrophoresis gels and is inefficient at identifying multiple cleavages, requiring preparation of numerous gels or membrane slices for analysis. We recently developed Amino-Terminal Oriented Mass spectrometry of Substrates (ATOMS) to overcome these limitations as a complement for N-terminal sequencing. ATOMS employs isotopic labeling and quantitative tandem mass spectrometry to identify cleavage sites in a fast and accurate manner. We successfully used ATOMS to identify nearly 100 cleavage sites in the ECM proteins laminin and fibronectin. Presented herein is the detailed step-by-step protocol for ATOMS. Copyright © 2011 Elsevier Inc. All rights reserved.

  17. Interaction of the 70,000-mol-wt amino-terminal fragment of fibronectin with the matrix-assembly receptor of fibroblasts

    PubMed Central

    1985-01-01

    Plasma fibronectin binds saturably and reversibly to substrate-attached fibroblasts and is subsequently incorporated into the extracellular matrix (McKeown-Longo, P.J., and D. F. Mosher, 1983, J. Cell Biol., 97:466-472). We examined whether fragments of fibronectin are processed in a similar way. The amino-terminal 70,000-mol-wt catheptic D fragment of fibronectin bound reversibly to cell surfaces with the same affinity as intact fibronectin but did not become incorporated into extracellular matrix. The 70,000-mol-wt fragment blocked binding of intact fibronectin to cell surfaces and incorporation of intact fibronectin into extracellular matrix. Binding of the 70,000-mol-wt fragment to cells was partially abolished by cleavage into 27,000-mol- wt heparin-binding and 40,000-mol-wt gelatin-binding fragments and more completely abolished by reduction and alkylation of disulfide bonds. Binding of the 70,000-mol-wt fragment to cells was not blocked by gelatin or heparin. When coated onto plastic, the 70,000-mol-wt fragment did not mediate attachment and spreading of suspended fibroblasts. Conversely, fibronectin fragments that had attachment and spreading activity did not block binding of exogenous fibronectin to substrate-attached cells. These results indicate that there is a cell binding site in the 70,000-mol-wt fragment that is distinct from the previously described cell attachment site and is required for assembly of exogenous fibronectin into extracellular matrix. PMID:3155749

  18. Focal adhesion kinase and mitogen-activated protein kinases are involved in chondrocyte activation by the 29-kDa amino-terminal fibronectin fragment.

    PubMed

    Gemba, Takefumi; Valbracht, Jean; Alsalameh, Saifeddin; Lotz, Martin

    2002-01-11

    The 29-kDa amino-terminal fibronectin fragment (FN-f) has a potent chondrolytic effect and is thought to be involved in cartilage degradation in arthritis. However, little is known about signal transduction pathways that are activated by FN-f. Here we demonstrated that FN-f induced nitric oxide (NO) production from human articular chondrocytes. Expression of inducible nitric-oxide synthase (iNOS) mRNA and NO production were observed at 6 and 48 h after FN-f treatment, respectively. Interleukin-1beta (IL-1beta) mRNA up-regulation was stimulated by FN-f in human chondrocytes. To address the possibility that FN-f-induced NO release is mediated by IL-1beta production, the effect of IL-1 receptor antagonist (IL-1ra) was determined. IL-1ra partially inhibited FN-f-induced NO release although it almost completely inhibited IL-1beta-induced NO release. Tyrosine phosphorylation of focal adhesion kinase was induced transiently by FN-f treatment. Blocking antibodies to alpha(5) or beta(1) integrin and Arg-Gly-Asp-containing peptides did not inhibit FN-f-induced NO production. PP2, a Src family kinase inhibitor, or cytochalasin D, which selectively disrupts the network of actin filaments, inhibited both FAK phosphorylation and NO production induced by FN-f, but the phosphatidylinositol 3-kinase inhibitor wortmannin had no effect. Analysis of mitogen-activated protein kinases (MAPK) showed activation of extracellular signal-regulated kinase (ERK), c-Jun NH(2)-terminal kinase, and p38 MAPK. High concentrations of SB203580, which inhibit both JNK and p38 MAPK, and PD98059 a selective inhibitor of MEK1/2 that blocks ERK activation, inhibited FN-f induced NO production. These data suggest that focal adhesion kinase and MAPK mediate FN-f induced activation of human articular chondrocytes.

  19. Partial agonist activity of the progesterone receptor antagonist RU486 mediated by an amino-terminal domain coactivator and phosphorylation of serine400.

    PubMed

    Wardell, Suzanne E; Narayanan, Ramesh; Weigel, Nancy L; Edwards, Dean P

    2010-02-01

    Jun dimerization protein-2 (JDP-2) is a progesterone receptor (PR) coregulatory protein that acts by inducing structure and transcriptional activity in the disordered amino-terminal domain (NTD) of PR. JDP-2 can also potentiate the partial agonist activity of the PR antagonist RU486 by mechanisms that have not been defined. Functional mutagenesis experiments revealed that a subregion of the NTD (amino acids 323-427) was required for the partial agonist activity of RU486 induced by PR interaction with JDP-2. However, this subregion was not required for JDP-2 enhancement of the activity of progestin agonists. Mutation of phosphorylation sites within this region of the NTD showed that phosphorylation of serine 400 was required for the partial agonist activity of RU486 stimulated by JDP-2, but was not required for activity of hormone agonist, either in the presence or absence of JDP-2. Cyclin-dependent kinase 2 (Cdk2)/cyclin A is a novel PR coregulator that binds the NTD and acts by phosphorylating steroid receptor coactivator-1 and modulating steroid receptor coactivator-1 interaction with PR. Cdk2/cyclin A also potentiated the partial agonist activity of RU486; however, phosphorylation of serine 400 was not required, indicating that JDP-2 and Cdk2/cyclin A act by distinct mechanisms. We conclude that PR bound to RU486 and associated with JDP-2 adopts an active conformation in a subregion of the NTD requiring phosphorylation of serine 400 that is distinct from that promoted by progestin agonists. These data underscore the structural flexibility of the NTD of PR, and the ability of steroid ligands together with interacting proteins to affect the conformation and activity of the NTD.

  20. Amino-terminal extension generated from an upstream AUG codon increases the efficiency of mitochondrial import of yeast N2,N2-dimethylguanosine-specific tRNA methyltransferases.

    PubMed Central

    Ellis, S R; Hopper, A K; Martin, N C

    1989-01-01

    Fusions between the TRM1 gene of Saccharomyces cerevisiae and COXIV or DHFR were made to examine the mitochondrial targeting signals of N2,N2-dimethylguanosine-specific tRNA methyltransferase [tRNA (m2(2)G)dimethyltransferase]. This enzyme is responsible for the modification of both mitochondrial and cytoplasmic tRNAs. We have previously shown that two forms of the enzyme are translated from two in-frame ATGs in this gene, that they differ by a 16-amino-acid amino-terminal extension, and that both the long and short forms are imported into mitochondria. Results of studies to test the ability of various TRM1 sequences to serve as surrogate mitochondrial targeting signals for passenger protein import in vitro and in vivo showed that the most efficient signal derived from tRNA (m2(2)G)dimethyltransferase included a combination of sequences from both the amino-terminal extension and the amino terminus of the shorter form of the enzyme. The amino-terminal extension itself did not serve as an independent mitochondrial targeting signal, whereas the amino terminus of the shorter form of tRNA (m2(2)G)dimethyltransferase did function in this regard, albeit inefficiently. We analyzed the first 48 amino acids of tRNA (m2(2)G)dimethyltransferase for elements of primary and secondary structure shared with other known mitochondrial targeting signals. The results lead us to propose that the most efficient signal spans the area around the second ATG of TRM1 and is consistent with the idea that there is a mitochondrial targeting signal present at the amino terminus of the shorter form of the enzyme and that the amino-terminal extension augments this signal by extending it to form a larger, more efficient mitochondrial targeting signal. Images PMID:2657400

  1. Immunogens Modeling a Fusion-Intermediate Conformation of gp41 Elicit Antibodies to the Membrane Proximal External Region of the HIV Envelope Glycoprotein.

    PubMed

    Vassell, Russell; He, Yong; Vennakalanti, Prasad; Dey, Antu K; Zhuang, Min; Wang, Wei; Sun, Yide; Biron-Sorek, Zohar; Srivastava, Indresh K; LaBranche, Celia C; Montefiori, David C; Barnett, Susan W; Weiss, Carol D

    2015-01-01

    The membrane proximal external region (MPER) of the gp41 subunit of the HIV-1 envelope glycoprotein (Env) contains determinants for broadly neutralizing antibodies and has remained an important focus of vaccine design. However, creating an immunogen that elicits broadly neutralizing antibodies to this region has proven difficult in part due to the relative inaccessibility of the MPER in the native conformation of Env. Here, we describe the antigenicity and immunogenicity of a panel of oligomeric gp41 immunogens designed to model a fusion-intermediate conformation of Env in order to enhance MPER exposure in a relevant conformation. The immunogens contain segments of the gp41 N- and C-heptad repeats to mimic a trapped intermediate, followed by the MPER, with variations that include different N-heptad lengths, insertion of extra epitopes, and varying C-termini. These well-characterized immunogens were evaluated in two different immunization protocols involving gp41 and gp140 proteins, gp41 and gp160 DNA primes, and different immunization schedules and adjuvants. We found that the immunogens designed to reduce extension of helical structure into the MPER elicited the highest MPER antibody binding titers, but these antibodies lacked neutralizing activity. The gp41 protein immunogens also elicited higher MPER titers than the gp140 protein immunogen. In prime-boost studies, the best MPER responses were seen in the groups that received DNA priming with gp41 vectors followed by gp41 protein boosts. Finally, although titers to the entire protein immunogen were similar in the two immunization protocols, MPER-specific titers differed, suggesting that the immunization route, schedule, dose, or adjuvant may differentially influence MPER immunogenicity. These findings inform the design of future MPER immunogens and immunization protocols.

  2. Receptor-Targeted Nipah Virus Glycoproteins Improve Cell-Type Selective Gene Delivery and Reveal a Preference for Membrane-Proximal Cell Attachment

    PubMed Central

    Bender, Ruben R.; Muth, Anke; Schneider, Irene C.; Friedel, Thorsten; Hartmann, Jessica; Plückthun, Andreas; Maisner, Andrea; Buchholz, Christian J.

    2016-01-01

    Receptor-targeted lentiviral vectors (LVs) can be an effective tool for selective transfer of genes into distinct cell types of choice. Moreover, they can be used to determine the molecular properties that cell surface proteins must fulfill to act as receptors for viral glycoproteins. Here we show that LVs pseudotyped with receptor-targeted Nipah virus (NiV) glycoproteins effectively enter into cells when they use cell surface proteins as receptors that bring them closely enough to the cell membrane (less than 100 Å distance). Then, they were flexible in receptor usage as demonstrated by successful targeting of EpCAM, CD20, and CD8, and as selective as LVs pseudotyped with receptor-targeted measles virus (MV) glycoproteins, the current standard for cell-type specific gene delivery. Remarkably, NiV-LVs could be produced at up to two orders of magnitude higher titers compared to their MV-based counterparts and were at least 10,000-fold less effectively neutralized than MV glycoprotein pseudotyped LVs by pooled human intravenous immunoglobulin. An important finding for NiV-LVs targeted to Her2/neu was an about 100-fold higher gene transfer activity when particles were targeted to membrane-proximal regions as compared to particles binding to a more membrane-distal epitope. Likewise, the low gene transfer activity mediated by NiV-LV particles bound to the membrane distal domains of CD117 or the glutamate receptor subunit 4 (GluA4) was substantially enhanced by reducing receptor size to below 100 Å. Overall, the data suggest that the NiV glycoproteins are optimally suited for cell-type specific gene delivery with LVs and, in addition, for the first time define which parts of a cell surface protein should be targeted to achieve optimal gene transfer rates with receptor-targeted LVs. PMID:27281338

  3. Role of lipid structure in the humoral immune response in mice to covalent lipid-peptides from the membrane proximal region of HIV-1 gp41

    PubMed Central

    Watson, Douglas S.; Szoka, Francis C.

    2009-01-01

    The membrane proximal region (MPR) of HIV-1 gp41 is a desirable target for development of a vaccine that elicits neutralizing antibodies since the patient-derived monoclonal antibodies, 2F5 and 4E10, bind to the MPR and neutralize primary HIV isolates. The 2F5 and 4E10 antibodies cross-react with lipids and structural studies suggest that MPR immunogens may be presented in a membrane environment. We hypothesized that covalent attachment of lipid anchors would enhance the humoral immune response to MPR-derived peptides presented in liposomal bilayers. In a comparison of eight lipids conjugated to an extended 2F5 epitope peptide, a sterol, cholesterol hemisuccinate (CHEMS), was found to promote the strongest anti-peptide IgG titers (6.4 × 104) in sera of BALB/C mice. Two lipid anchors, palmitic acid and phosphatidylcholine, failed to elicit a detectable serum anti-peptide IgG response. Association with the liposomal vehicle contributed to the ability of a lipopeptide to elicit anti-peptide antibodies, but no other single factor, such as position of the lipid anchor, peptide helical content, lipopeptide partition coefficient, or presence of phosphate on the anchor clearly determined lipopeptide potency. Conjugation to CHEMS also rendered a 4E10 epitope peptide immunogenic (5.6 × 102 IgG titer in serum). Finally, attachment of CHEMS to a peptide spanning both the 2F5 and 4E10 epitopes elicited serum IgG antibodies that bound to each of the individual epitopes as well as to recombinant gp140. Further research into the mechanism of how structure influences the immune response to the MPR may lead to immunogens that could be useful in prime-boost regimens for focusing the immune response in an HIV vaccine. PMID:19520200

  4. Serum concentration of amino-terminal propeptide of type III procollagen (PIIINP) as a prognostic marker for skin fibrosis after scar correction in burned patients.

    PubMed

    Ulrich, Dietmar; Noah, Ernst-Magnus; Burchardt, Elmar Reinhardt; Atkins, Derek; Pallua, Norbert

    2002-12-01

    The amino-terminal propeptide of procollagen type III (PIIINP) has been proposed as a marker for fibrogenesis in patients with different fibroproliferative disorders, e.g. liver and lung fibrosis. In this study, serum concentrations of PIIINP were measured by ELISA as a marker for excessive cicatrization in burned patients before and after scar correction. All patients were followed 6 months to determine a new fibrotic reaction during the wound healing process using the Burn Scar Index and to correlate pre- and post-operative concentrations of PIIINP in their sera with the risk to develop a new severe tissue fibrosis leading to pathological scar formation. Furthermore, PIIINP was determined in the excised scar tissue by immunohistochemistry. The study included 38 patients. Nineteen patients (8 female, 11 male, average age 48.3+/-18.9 years) had hypertrophic scars after major burn injury (TBSA, 21+/-12%; Burn Scar Index, 10.4+/-3.7 points) and underwent scar correction. Nineteen patients (12 female, 7 male, average age 42.3+/-25.5 years) who underwent elective plastic-surgical operations served as the control group. Blood samples were drawn immediately before operation, at the 1st, 3rd, 7th, and 14th post-operative days, as well as 1st, 3rd, and 6th months after operation.Pre-operatively, PIIINP was significantly elevated (P<0.05) in burned patients who underwent scar correction. There was a significant increase (P<0.05) of PIIINP in burned patients from 9.8+/-3.7ng/ml pre-operatively to 13.9+/-4.2ng/ml at the 7th post-operation day. Up to 3 months after operation, the 6 months after scar correction concentration remained at a significantly elevated level compared to pre-operative values. The burned patients had a Burn Scar Index of 7.8+/-3.6 points. Pre-operative PIIINP serum concentrations correlated with the Burn Scar Index (r(2)=0.7 and 0.68; P<0.05). Scar tissue stained intensively positive for PIIINP. There was a significant correlation between pre

  5. B-type natriuretic peptide and amino-terminal pro-B-type natriuretic peptide in pediatric patients with pulmonary arterial hypertension

    PubMed Central

    Takatsuki, Shinichi; Wagner, Brandie D; Ivy, David Dunbar

    2011-01-01

    Objectives B-type natriuretic peptide (BNP) and the amino-terminal fragment (NTproBNP) correlate with clinical variables, but have not been simultaneously studied in a large number of pediatric patients with pulmonary arterial hypertension (PAH). The purpose of our investigation was to compare BNP and NTproBNP with clinical indicators of disease in a pediatric PAH population for which biomarkers are much needed. Design We retrospectively compared BNP and NTproBNP levels with exercise capacity, echocardiographic data, and hemodynamics in PAH patients under 21 years-old. Two hundred sixty three blood samples from 88 pediatric PAH patients were obtained, with BNP and NTproBNP drawn at the same time. Results There was a correlation between BNP and NTproBNP with mean pulmonary arterial pressure/mean arterial pressure (mPAP/mSAP) ratio (r=0.40 p<0.01, r=0.45 p<0.01, respectively), mean right atrial pressure (mRAP) (r=0.48 p<0.01, r=0.48 p<0.01), and tricuspid regurgitant (TR) velocity (r=0.36 p<0.01, r=0.41 p<0.01). BNP and NTproBNP are associated with 6 minute walking distance, mPAP, mPAP/mSAP ratio, mRAP, pulmonary vascular resistance index (PVRI), and TR velocity when investigated longitudinally. On the average, a 1 unit increase in log BNP or NTproBNP was associated with 4.5 unitsxm2 or 3.4 unitsxm2 increase in PVRI, respectively. There was a strong correlation between log BNP and log NTproBNP measurements (r= 0.87, p<0.01). Conclusion In pediatric PAH, BNP and NTProBNP are strongly correlated and predict changes in clinical variables and hemodynamics. In a cross-sectional analysis, NTproBNP correlated with echocardiographic and exercise data better than BNP; NTproBNP showed less within patient variability over time, therefore NTproBNP can add additional information towards predicting these clinical measurements. PMID:22325151

  6. Crystal Structures of the Glutamate Receptor Ion Channel GluK3 and GluK5 Amino-Terminal Domains

    SciTech Connect

    Kumar, Janesh; Mayer, Mark L.

    2010-11-30

    Ionotropic glutamate receptors (iGluRs) mediate the majority of fast excitatory synaptic neurotransmission in the central nervous system. The selective assembly of iGluRs into AMPA, kainate, and N-methyl-d-aspartic acid (NMDA) receptor subtypes is regulated by their extracellular amino-terminal domains (ATDs). Kainate receptors are further classified into low-affinity receptor families (GluK1-GluK3) and high-affinity receptor families (GluK4-GluK5) based on their affinity for the neurotoxin kainic acid. These two families share a 42% sequence identity for the intact receptor but only a 27% sequence identity at the level of ATD. We have determined for the first time the high-resolution crystal structures of GluK3 and GluK5 ATDs, both of which crystallize as dimers but with a strikingly different dimer assembly at the R1 interface. By contrast, for both GluK3 and GluK5, the R2 domain dimer assembly is similar to those reported previously for other non-NMDA iGluRs. This observation is consistent with the reports that GluK4-GluK5 cannot form functional homomeric ion channels and require obligate coassembly with GluK1-GluK3. Our analysis also reveals that the relative orientation of domains R1 and R2 in individual non-NMDA receptor ATDs varies by up to 10{sup o}, in contrast to the 50{sup o} difference reported for the NMDA receptor GluN2B subunit. This restricted domain movement in non-NMDA receptor ATDs seems to result both from extensive intramolecular contacts between domain R1 and domain R2 and from their assembly as dimers, which interact at both R1 and R2 domains. Our results provide the first insights into the structure and function of GluK4-GluK5, the least understood family of iGluRs.

  7. Generation of Long-Lived Bone Marrow Plasma Cells Secreting Antibodies Specific for the HIV-1 gp41 Membrane-Proximal External Region in the Absence of Polyreactivity

    PubMed Central

    Donius, Luke R.; Cheng, Yuxing; Choi, Jaewon; Sun, Zhen-Yu J.; Hanson, Melissa; Zhang, Michael; Gierahn, Todd M.; Marquez, Susanna; Uduman, Mohammed; Kleinstein, Steven H.; Irvine, Darrell; Love, J. Christopher; Reinherz, Ellis L.

    2016-01-01

    ABSTRACT An effective preventive vaccine is highly sought after in order to stem the current HIV-1 pandemic. Both conservation of contiguous gp41 membrane-proximal external region (MPER) amino acid sequences across HIV-1 clades and the ability of anti-MPER broadly neutralizing antibodies (BNAbs) to block viral hemifusion/fusion establish the MPER as a prime vaccination target. In earlier studies, we described the development of an MPER vaccine formulation that takes advantage of liposomes to array the MPER on a lipid bilayer surface, paralleling its native configuration on the virus membrane while also incorporating molecular adjuvant and CD4 T cell epitope cargo. Here we demonstrate that several immunizations with MPER/liposomes induce high levels of bone marrow long-lived plasma cell (LLPC) antibody production. Single-cell immunoglobulin gene retrieval analysis shows that these plasma cells are derived from a germ line repertoire of B cells with a diverse representation of immunoglobulin genes, exhibiting antigen-driven positive selection. Characterization of LLPC recombinant monoclonal antibodies (rMAbs) indicates that antigen recognition is achieved through convergence on a common epitopic focus by utilizing various complementarity-determining region H3 (CDRH3) lengths. Importantly, the vast majority of rMAbs produced from these cells lack polyreactivity yet manifest antigen specificity in the context of lipids, shaping MPER-specific paratopes through selective pressure. Taken together, these findings demonstrate that the MPER is a vaccine target with minimal risk of generating off-target autoimmunity. IMPORTANCE A useful vaccine must generate desired long-term, antigen-specific antibody responses devoid of polyreactivity or autoreactivity. The common polyreactive features of some HIV-1 BNAbs have raised concern about elicitation of anti-MPER antibodies. Utilizing single-LLPC repertoire analysis and biophysical characterization of anti-MPER rMAbs, we show that

  8. Substitution of conserved cysteine residues in Wheat streak mosaic virus HC-Pro abolishes virus transmission by the wheat curl mite

    USDA-ARS?s Scientific Manuscript database

    Substitutions in the amino-terminal region of Wheat streak mosaic virus (WSMV) HC-Pro were evaluated for effects on transmission by the wheat curl mite (Aceria tosichella Keifer). Alanine substitution at cysteine residues 16, 46 and 49 abolished vector transmission. Although alanine substitution a...

  9. L718P mutation in the membrane-proximal cytoplasmic tail of β3 promotes abnormal αIIbβ3 clustering and lipid microdomain coalescence, and associates with a thrombasthenia-like phenotype

    PubMed Central

    Jayo, Asier; Conde, Isabel; Lastres, Pedro; Martínez, Constantino; Rivera, José; Vicente, Vicente; González-Manchón, Consuelo

    2010-01-01

    Background Support for the role of transmembrane and membrane-proximal domains of αIIbβ3 integrin in the maintenance of receptor low affinity comes from mutational studies showing that activating mutations can induce constitutive bi-directional transmembrane signaling. Design and Methods We report the functional characterization of a mutant αIIbβ3 integrin carrying the Leu718Pro mutation in the membrane-proximal region of the β3 cytoplasmic domain, identified in heterozygosis in a patient with a severe bleeding phenotype and defective platelet aggregation and adhesion. Results Transiently transfected cells expressed similar levels of normal and mutant αIIbβ3, but surface expression of mutant αvβ3 was reduced due to its retention in intracellular compartments. Cells stably expressing mutant αIIbβ3 showed constitutive binding to soluble multivalent ligands as well as spontaneous fibrinogen-dependent aggregation, but their response to DTT was markedly reduced. Fibrinogen-adherent cells exhibited a peculiar spreading phenotype with long protrusions. Immunofluorescence analysis revealed the formation of αIIbβ3 clusters underneath the entire cell body and the presence of atypical high-density patches of clustered αIIbβ3 containing encircled areas devoid of integrin that showed decreased affinity for the fluorescent lipid analog DiIC16 and were disrupted in cholesterol-depleted cells. Conclusions These findings are consistent with an important role of the membrane-proximal region of β3 in modulating αIIbβ3 clustering and lateral redistribution of membrane lipids. Since the β3 mutant was associated with a thrombasthenic phenotype in a patient carrying one normal β3 allele, these results support a dominant role of clustering in regulating integrin αIIbβ3 functions in vivo. PMID:20081061

  10. The Amino-Terminal Region of Drosophila MSL1 Contains Basic, Glycine-Rich, and Leucine Zipper-Like Motifs That Promote X Chromosome Binding, Self-Association, and MSL2 Binding, Respectively

    PubMed Central

    Li, Fang; Parry, David A. D.; Scott, Maxwell J.

    2005-01-01

    In Drosophila melanogaster, X chromosome dosage compensation is achieved by doubling the transcription of most X-linked genes. The male-specific lethal (MSL) complex is required for this process and binds to hundreds of sites on the male X chromosome. The MSL1 protein is essential for X chromosome binding and serves as a central scaffold for MSL complex assembly. We find that the amino-terminal region of MSL1 binds to hundreds of sites on the X chromosome in normal males but only to approximately 30 high-affinity sites in the absence of endogenous MSL1. Binding to the high-affinity sites requires a basic motif at the amino terminus that is conserved among Drosophila species. X chromosome binding also requires a conserved leucine zipper-like motif that binds to MSL2. A glycine-rich motif between the basic and leucine-zipper-like motifs mediates MSL1 self-association in vitro and binding of the amino-terminal region of MSL1 to the MSL complex assembled on the male X chromosome. We propose that the basic region may mediate DNA binding and that the glycine-rich region may promote the association of MSL complexes to closely adjacent sites on the X chromosome. PMID:16199870

  11. Potentiation of Neuronal Nicotinic Receptors by 17β-Estradiol: Roles of the Carboxy-Terminal and the Amino-Terminal Extracellular Domains

    PubMed Central

    Jin, Xiaochun; Steinbach, Joe Henry

    2015-01-01

    The endogenous steroid 17β-estradiol (βEST) potentiates activation of neuronal nicotinic receptors containing α4 subunits. Previous work has shown that the final 4 residues of the α4 subunit are required for potentiation. However, receptors containing the α2 subunit are not potentiated although it has these 4 residues, and only one amino acid difference in the C-terminal tail (FLAGMI vs. WLAGMI). Previous work had indicated that the tryptophan residue was involved in binding an analog of βEST, but not in potentiation by βEST. To determine the structural basis for the loss of potentiation we analyzed data from chimeric subunits, which indicated that the major factor underlying the difference between α2 and α4 is the tryptophan/phenylalanine difference, while the N-terminal extracellular domain is a less significant factor. When the tryptophan in α4 was mutated, both phenylalanine and tyrosine conferred lower potentiation while lysine and leucine did not. The reduction reflected a reduced maximal magnitude of potentiation, indicating that the tryptophan is involved in transduction of steroid effects. The regions of the α4 N-terminal extracellular domain involved in potentiation lie near the agonist-binding pocket, rather than close to the membrane or the C-terminal tail, and appear to be involved in transduction rather than binding. These observations indicate that the C-terminal region is involved in both steroid binding (AGMI residues) and transduction (W). The role of the N-terminus appears to be independent of the C-terminal tryptophan and likely reflects an influence on conformational changes caused during channel activation by agonist and potentiation by estradiol. PMID:26684647

  12. A 43-kilodalton pneumococcal surface protein, PspA: isolation, protective abilities, and structural analysis of the amino-terminal sequence.

    PubMed Central

    Talkington, D F; Crimmins, D L; Voellinger, D C; Yother, J; Briles, D E

    1991-01-01

    PspA is an antigenically variable surface protein of Streptococcus pneumoniae that appears to be essential for full pneumococcal virulence. In addition, monoclonal antibodies to PspA protect mice against infection with specific strains of pneumococci virulent for mice. In this study, we have isolated the 43-kDa N-terminal half of the native 84-kDa PspA and determined the sequence of the first 45 amino acids. This sequence, the first obtained for a pneumococcal surface protein, is consistent with that of an amphiphatic coiled-coil alpha helix with a 7-residue periodicity common to fibrous proteins such as tropomyosin and streptococcal M protein. The 7-residue periodicity begins with residue 8 and extends throughout the remaining sequence for nearly 11 turns of the helix. Mice immunized with this purified PspA segment were protected from fatal pneumococcal challenge, thus demonstrating that those PspA epitopes eliciting protection were present in the N-terminal half of the molecule. Images PMID:2004810

  13. The amino-terminal region of the neuraminidase protein from avian H5N1 influenza virus is important for its biosynthetic transport to the host cell surface.

    PubMed

    Qian, Guomin; Wang, Song; Chi, Xiaojuan; Li, Hua; Wei, Haitao; Zhu, Xiaomei; Chen, Yuhai; Chen, Ji-Long

    2014-12-01

    Influenza virus neuraminidase (NA) is a major viral envelope glycoprotein, which plays a critical role in viral infection. Although NA functional domains have been determined previously, the precise role of the amino acids located at the N-terminus of avian H5N1 NA for protein expression and intracellular transport to the host plasma membrane is not fully understood. In the present study, a series of N-terminal truncation or deletion mutants of H5N1 NA were generated and their expression and intracellular trafficking were investigated. Protein expression from mutants NAΔ20, NAΔ35, NAΔ40, NAΔ7-20 and NAΔ7-35 was undetectable by immunoblotting and by performing NA activity assays. Mutants NAΔ6, NAΔ11 and NAΔ15-20 showed a marked decreased in protein expression, whereas mutants NAΔ7-15 and NAΔ15 displayed a slight increase in protein expression, compared with that of the native NA protein. These data suggest that amino acid residues 16-20 are vital for NA protein expression, while amino acids 7-15 might suppress NA protein expression. In deletion mutants NAΔ7-15 and NAΔ15 there was an accumulation of NA protein at the juxta-nuclear region, with reduced expression of NA at the cell surface. Although active Cdc42 could promote transport of wild-type NA to the host cell surface, this member of the Rho family of GTPases failed to regulate transport of mutants NAΔ7-15 and NAΔ15. The results of the study reveal that amino acid residues 7-15 of H5N1 NA are critical for its biosynthetic transport to the host cell surface.

  14. Relief of autoinhibition of the electrogenic Na-HCO(3) [corrected] cotransporter NBCe1-B: role of IRBIT vs.amino-terminal truncation.

    PubMed

    Lee, Seong-Ki; Boron, Walter F; Parker, Mark D

    2012-02-01

    Two maneuvers known to stimulate electrogenic sodium bicarbonate cotransporter 1 (NBCe1) activity are 1) deletion from the cytosolic amino-terminus (Nt) of NBCe1-C of an 87-amino acid sequence that contains an autoinhibitory domain (AID); and 2) binding of the protein IRBIT to elements within the same 87-amino acid module in a different variant, NBCe1-B. Helpful to understanding the relationship between these two phenomena would be an appreciation of the relative magnitude of stimulation caused by each maneuver for the same NBCe1 variant. In the present study, we performed two-electrode voltage-clamp on Xenopus oocytes expressing human NBCe1-B constructs, with and without human IRBIT constructs. We find that removal of the AID stimulates NBCe1-B to the same extent as coexpression of wild-type IRBIT. The potency of wild-type IRBIT apparently is reduced by the action of endogenous oocyte protein phosphatases: a mutant IRBIT that cannot be influenced by the action of protein phosphatase-1 stimulates NBCe1-B to an extent 50% greater than can be achieved by removal of the NBCe1-B AID. Thus the stimulatory effect of IRBIT cannot be explained solely by masking of autoinhibitory determinants within the AID. Finally, we find that an NBCe1-B construct that lacks amino acid residues 2-16 of the Nt is fully autoinhibited, but cannot be stimulated by IRBIT, indicating that autoinhibitory and IRBIT-binding determinants within the cytosolic Nt are not identical.

  15. The amino-terminal conserved domain of 4-hydroxy-3-methylbut-2-enyl diphosphate reductase is critical for its function in oxygen-evolving photosynthetic organisms

    PubMed Central

    Hsieh, Wei-Yu; Hsieh, Ming-Hsiun

    2015-01-01

    4-hydroxy-3-methylbut-2-enyl diphosphate reductase (HDR), also known as isoprenoid synthesis H (IspH) or lysis-tolerant B (LytB), catalyzes the last step of the methylerythritol phosphate pathway to synthesize isopentenyl diphosphate and dimethylallyl diphosphate. The structure and reaction mechanism of IspH have been actively investigated in Escherichia coli but little is known in plants. Compared with the bacterial IspH, cyanobacterial and plant HDRs all contain an extra N-terminal conserved domain (NCD) that is essential for their function. Tyr72 in the NCD and several plant-specific residues around the central active site are critical for Arabidopsis HDR function. These results suggest that the structure and reaction mechanism of HDR/IspH may be different between plants and bacteria. The E. coli IspH is an iron-sulfur protein that is sensitive to oxygen. It is possible that the cyanobacterial HDR may independently evolve from the common ancestor of prokaryotes to obtain the NCD, which may protect the enzyme from high concentration of oxygen during photosynthesis. PMID:25723575

  16. Crystal structure of type I ryanodine receptor amino-terminal [beta]-trefoil domain reveals a disease-associated mutation 'hot spot' loop

    SciTech Connect

    Amador, Fernando J.; Liu, Shuang; Ishiyama, Noboru; Plevin, Michael J.; Wilson, Aaron; MacLennan, David H.; Ikura, Mitsuhiko

    2009-12-01

    Muscle contraction and relaxation is regulated by transient elevations of myoplasmic Ca{sup 2+}. Ca{sup 2+} is released from stores in the lumen of the sarco(endo)plasmic reticulum (SER) to initiate formation of the Ca{sup 2+} transient by activation of a class of Ca{sup 2+} release channels referred to as ryanodine receptors (RyRs) and is pumped back into the SER lumen by Ca{sup 2+}-ATPases (SERCAs) to terminate the Ca{sup 2+} transient. Mutations in the type 1 ryanodine receptor gene, RYR1, are associated with 2 skeletal muscle disorders, malignant hyperthermia (MH), and central core disease (CCD). The evaluation of proposed mechanisms by which RyR1 mutations cause MH and CCD is hindered by the lack of high-resolution structural information. Here, we report the crystal structure of the N-terminal 210 residues of RyR1 (RyR{sub NTD}) at 2.5 {angstrom}. The RyR{sub NTD} structure is similar to that of the suppressor domain of type 1 inositol 1,4,5-trisphosphate receptor (IP3Rsup), but lacks most of the long helix-turn-helix segment of the 'arm' domain in IP3Rsup. The N-terminal {beta}-trefoil fold, found in both RyR and IP{sub 3}R, is likely to play a critical role in regulatory mechanisms in this channel family. A disease-associated mutation 'hot spot' loop was identified between strands 8 and 9 in a highly basic region of RyR1. Biophysical studies showed that 3 MH-associated mutations (C36R, R164C, and R178C) do not adversely affect the global stability or fold of RyRNTD, supporting previously described mechanisms whereby mutations perturb protein-protein interactions.

  17. Amino-terminal alteration of the HLA-A*0201-restricted human immunodeficiency virus pol peptide increases complex stability and in vitro immunogenicity.

    PubMed Central

    Pogue, R R; Eron, J; Frelinger, J A; Matsui, M

    1995-01-01

    Initial studies suggested that major histocompatibility complex class I-restricted viral epitopes could be predicted by the presence of particular residues termed anchors. However, recent studies showed that nonanchor positions of the epitopes are also significant for class I binding and recognition by cytotoxic T lymphocytes (CTLs). We investigated if changing nonanchor amino acids could increase class I affinity, complex stability, and T-cell recognition of a natural viral epitope. This concept was tested by using the HLA-A 0201-restricted human immunodeficiency virus type 1 epitope from reverse transcriptase (pol). Position 1 (P1) amino acid substitutions were emphasized because P1 alterations may not alter the T-cell receptor interaction. The peptide with the P1 substitution of tyrosine for isoleucine (I1Y) showed a binding affinity for HLA-A 0201 similar to that of the wild-type pol peptide in a cell lysate assembly assay. Surprisingly, I1Y significantly increased the HLA-A 0201-peptide complex stability at the cell surface. I1Y sensitized HLA-A 0201-expressing target cells for wild-type pol-specific CTL lysis as well as wild-type pol. Peripheral blood lymphocytes from three HLA-A2 HIV-seropositive individuals were stimulated in vitro with I1Y and wild-type pol. I1Y stimulated a higher wild-type pol-specific CTL response than wild-type pol in all three donors. Thus, I1Y may be an "improved" epitope for use as a CTL-based human immunodeficiency virus vaccine component. The design of improved epitopes has important ramifications for prophylaxis and therapeutic vaccine development. PMID:7545295

  18. XA27 Depends on an Amino-Terminal Signal-Anchor-Like Sequence to Localize to the Apoplast for Resistance to Xanthomonas oryzae pv oryzae1[W

    PubMed Central

    Wu, Lifang; Goh, Mei Ling; Sreekala, Chellamma; Yin, Zhongchao

    2008-01-01

    The rice (Oryza sativa) gene Xa27 confers resistance to Xanthomonas oryzae pv oryzae, the causal agent of bacterial blight disease in rice. Sequence analysis of the deduced XA27 protein provides little or no clue to its mode of action, except that a signal-anchor-like sequence is predicted at the amino (N)-terminal region of XA27. As part of an effort to characterize the biochemical function of XA27, we decided to determine its subcellular localization. Initial studies showed that a functional XA27-green fluorescent protein fusion protein accumulated in vascular elements, the host sites where the bacterial blight pathogens multiply. The localization of XA27-green fluorescent protein to the apoplast was verified by detection of the protein on cell walls of leaf sheath and root cells after plasmolysis. Similarly, XA27-FLAG localizes to xylem vessels and cell walls of xylem parenchyma cells, revealed by immunogold electron microscopy. XA27-FLAG could be secreted from electron-dense vesicles in cytoplasm to the apoplast via exocytosis. The signal-anchor-like sequence has an N-terminal positively charged region including a triple arginine motif followed by a hydrophobic region. Deletion of the hydrophobic region or substitution of the triple arginine motif with glycine or lysine residues abolished the localization of the mutated proteins to the cell wall and impaired the plant's resistance to X. oryzae pv oryzae. These results indicate that XA27 depends on the N-terminal signal-anchor-like sequence to localize to the apoplast and that this localization is important for resistance to X. oryzae pv oryzae. PMID:18784285

  19. Uniform {sup 15}N- and {sup 15}N/{sup 13}C-labeling of proteins in mammalian cells and solution structure of the amino terminal fragment of u-PA

    SciTech Connect

    Hansen, A.P.; Petros, A.M.; Meadows, R.P.; Mazar, A.P.; Nettesheim, D.G.; Pederson, T.M.; Fesik, S.W.

    1994-12-01

    Urokinase-type plasminogen activator (u-PA) is a 54-kDa glycoprotein that catalyzes the conversion of plasminogen to plasmin, a broad-specificity protease responsible for the degradation of fibrin clots and extracellular matrix components. The u-PA protein consists of three individual modules: a growth factor domain (GFD), a kringle, and a serine protease domain. The amino terminal fragment (ATF) includes the GFD-responsible for u-PA binding to its receptor-and the kringle domains. This protein was expressed and uniformly {sup 15}N-and {sup 15}N/{sup 13}C-labeled in mammalian cells by methods that will be described. In addition, we present the three-dimensional structure of ATF that was derived from 1299 NOE-derived distance restraints along with the {phi} angle and hydrogen bonding restraints. Although the individual domains in the structures were highly converged, the two domains are structurally independent. The overall structures of the individual domains are very similar to the structures of homologous proteins. However, important structural differences between the growth factor domain of u-PA and other homologous proteins were observed in the region that has been implicated in binding the urokinase receptor. These results may explain, in part, why other growth factors show no appreciable affinity for the urokinase receptor.

  20. Evaluation of a novel multi-immunogen vaccine strategy for targeting 4E10/10E8 neutralizing epitopes on HIV-1 gp41 membrane proximal external region.

    PubMed

    Banerjee, Saikat; Shi, Heliang; Banasik, Marisa; Moon, Hojin; Lees, William; Qin, Yali; Harley, Andrew; Shepherd, Adrian; Cho, Michael W

    2017-02-23

    The membrane proximal external region (MPER) of HIV-1 gp41 is targeted by broadly neutralizing antibodies (bnAbs) 4E10 and 10E8. In this proof-of-concept study, we evaluated a novel multi-immunogen vaccine strategy referred to as Incremental, Phased Antigenic Stimulation for Rapid Antibody Maturation (IPAS-RAM) to induce 4E10/10E8-like bnAbs. Rabbits were immunized sequentially, but in a phased manner, with three immunogens that are progressively more native (gp41-28×3, gp41-54CT, and rVV-gp160DH12). Although nAbs were not induced, epitope-mapping analyses indicated that IPAS-RAM vaccination was better able to target antibodies towards the 4E10/10E8 epitopes than homologous prime-boost immunization using gp41-28×3 alone. MPER-specific rabbit monoclonal antibodies were generated, including 9F6. Although it lacked neutralizing activity, the target epitope profile of 9F6 closely resembled those of 4E10 and 10E8 ((671)NWFDITNWLWYIK(683)). B-cell repertoire analyses suggested the importance of co-immunizations for maturation of 9F6, which warrants further evaluation of our IPAS-RAM vaccine strategy using an improved priming immunogen.

  1. Cry1A toxins of Bacillus thuringiensis bind specifically to a region adjacent to the membrane-proximal extracellular domain of BT-R(1) in Manduca sexta: involvement of a cadherin in the entomopathogenicity of Bacillus thuringiensis.

    PubMed

    Dorsch, J A; Candas, M; Griko, N B; Maaty, W S A; Midboe, E G; Vadlamudi, R K; Bulla, L A

    2002-09-01

    Many subspecies of the soil bacterium Bacillus thuringiensis produce various parasporal crystal proteins, also known as Cry toxins, that exhibit insecticidal activity upon binding to specific receptors in the midgut of susceptible insects. One such receptor, BT-R(1) (210 kDa), is a cadherin located in the midgut epithelium of the tobacco hornworm, Manduca sexta. It has a high binding affinity (K(d) approximately 1nM) for the Cry1A toxins of B. thuringiensis. Truncation analysis of BT-R(1) revealed that the only fragment capable of binding the Cry1A toxins of B. thuringiensis was a contiguous 169-amino acid sequence adjacent to the membrane-proximal extracellular domain. The purified toxin-binding fragment acted as an antagonist to Cry1Ab toxin by blocking the binding of toxin to the tobacco hornworm midgut and inhibiting insecticidal action. Exogenous Cry1Ab toxin bound to intact COS-7 cells expressing BT-R(1) cDNA, subsequently killing the cells. Recruitment of BT-R(1) by B. thuringiensis indicates that the bacterium interacts with a specific cell adhesion molecule during its pathogenesis. Apparently, Cry toxins, like other bacterial toxins, attack epithelial barriers by targeting cell adhesion molecules within susceptible insect hosts.

  2. [Impact of plasma pro-B-type natriuretic peptide amino-terminal and galectin-3 levels on the predictive capacity of the LIPID Clinical Risk Scale in stable coronary disease].

    PubMed

    Higueras, Javier; Martín-Ventura, José Luis; Blanco-Colio, Luis; Cristóbal, Carmen; Tarín, Nieves; Huelmos, Ana; Alonso, Joaquín; Pello, Ana; Aceña, Álvaro; Carda, Rocío; Lorenzo, Óscar; Mahíllo-Fernández, Ignacio; Asensio, Dolores; Almeida, Pedro; Rodríguez-Artalejo, Fernando; Farré, Jerónimo; López Bescós, Lorenzo; Egido, Jesús; Tuñón, José

    2015-01-01

    At present, there is no tool validated by scientific societies for risk stratification of patients with stable coronary artery disease (SCAD). It has been shown that plasma levels of monocyte chemoattractant protein-1 (MCP-1), galectin-3 and pro-B-type natriuretic peptide amino-terminal (NT-proBNP) have prognostic value in this population. To analyze the prognostic value of a clinical risk scale published in Long-term Intervention with Pravastatin in Ischemic Disease (LIPID) study and determining its predictive capacity when combined with plasma levels of MCP-1, galectin-3 and NT-proBNP in patients with SCAD. A total of 706 patients with SCAD and a history of acute coronary syndrome (ACS) were analyzed over a follow up period of 2.2 ± 0.99 years. The primary endpoint was the occurrence of an ischemic event (any SCA, stroke or transient ischemic attack), heart failure, or death. A clinical risk scale derived from the LIPID study significantly predicted the development of the primary endpoint, with an area under the ROC curve (Receiver Operating Characteristic) of 0.642 (0.579 to 0.705); P<0.001. A composite score was developed by adding the scores of the LIPID and scale decile levels of MCP -1, galectin -3 and NT-proBNP. The predictive value improved with an area under the curve of 0.744 (0.684 to 0.805); P<0.001 (P=0.022 for comparison). A score greater than 21.5 had a sensitivity of 74% and a specificity of 61% for the development of the primary endpoint (P<0.001, log -rank test). Plasma levels of MCP-1, galectin -3 and NT-proBNP improve the ability of the LIPID clinical scale to predict the prognosis of patients with SCAD. Copyright © 2014 Sociedad Española de Arteriosclerosis. Published by Elsevier España. All rights reserved.

  3. The development of decision limits for the GH-2000 detection methodology using additional insulin-like growth factor-I and amino-terminal pro-peptide of type III collagen assays.

    PubMed

    Holt, Richard I G; Böhning, Walailuck; Guha, Nishan; Bartlett, Christiaan; Cowan, David A; Giraud, Sylvain; Bassett, E Eryl; Sönksen, Peter H; Böhning, Dankmar

    2015-09-01

    The GH-2000 and GH-2004 projects have developed a method for detecting GH misuse based on measuring insulin-like growth factor-I (IGF-I) and the amino-terminal pro-peptide of type III collagen (P-III-NP). The objectives were to analyze more samples from elite athletes to improve the reliability of the decision limit estimates, to evaluate whether the existing decision limits needed revision, and to validate further non-radioisotopic assays for these markers. The study included 998 male and 931 female elite athletes. Blood samples were collected according to World Anti-Doping Agency (WADA) guidelines at various sporting events including the 2011 International Association of Athletics Federations (IAAF) World Athletics Championships in Daegu, South Korea. IGF-I was measured by the Immunotech A15729 IGF-I IRMA, the Immunodiagnostic Systems iSYS IGF-I assay and a recently developed mass spectrometry (LC-MS/MS) method. P-III-NP was measured by the Cisbio RIA-gnost P-III-P, Orion UniQ™ PIIINP RIA and Siemens ADVIA Centaur P-III-NP assays. The GH-2000 score decision limits were developed using existing statistical techniques. Decision limits were determined using a specificity of 99.99% and an allowance for uncertainty because of the finite sample size. The revised Immunotech IGF-I - Orion P-III-NP assay combination decision limit did not change significantly following the addition of the new samples. The new decision limits are applied to currently available non-radioisotopic assays to measure IGF-I and P-III-NP in elite athletes, which should allow wider flexibility to implement the GH-2000 marker test for GH misuse while providing some resilience against manufacturer withdrawal or change of assays. Copyright © 2015 John Wiley & Sons, Ltd.

  4. Amino-terminal domain of the v-fms oncogene product includes a functional signal peptide that directs synthesis of a transforming glycoprotein in the absence of feline leukemia virus gag sequences

    SciTech Connect

    Wheeler, E.F.; Roussel, M.F.; Hampe, A.; Walker, M.H.; Fried, V.A.; Look, A.T.; Rettenmier, C.W.; Sherr, C.J.

    1986-08-01

    The nucleotide sequence of a 5' segment of the human genomic c-fms proto-oncogene suggested that recombination between feline leukemia virus and feline c-fms sequences might have occurred in a region encoding the 5' untranslated portion of c-fms mRNA. The polyprotein precursor gP180/sup gag-fms/ encoded by the McDonough strain of feline sarcoma virus was therefore predicted to contain 34 v-fms-coded amino acids derived from sequences of the c-fms gene that are not ordinarily translated from the proto-oncogene mRNA. The (gP180/sup gag-fms/) polyprotein was cotranslationally cleaved near the gag-fms junction to remove its gag gene-coded portion. Determination of the amino-terminal sequence of the resulting v-fms-coded glycoprotein, gp120/sup v-fms/, showed that the site of proteolysis corresponded to a predicted signal peptidase cleavage site within the c-fms gene product. Together, these analyses suggested that the linked gag sequences may not be necessary for expression of a biologically active v-fms gene product. The gag-fms sequences of feline sarcoma virus strain McDonough and the v-fms sequences alone were inserted into a murine retroviral vector containing a neomycin resistance gene. The authors conclude that a cryptic hydrophobic signal peptide sequence in v-fms was unmasked by gag deletion, thereby allowing the correct orientation and transport of the v-fms was unmasked by gag deletion, thereby allowing the correct orientation and transport of the v-fms gene product within membranous organelles. It seems likely that the proteolytic cleavage of gP180/gag-fms/ is mediated by signal peptidase and that the amino termini of gp140/sup v-fms/ and the c-fms gene product are identical.

  5. The development of decision limits for the implementation of the GH-2000 detection methodology using current commercial insulin-like growth factor-I and amino-terminal pro-peptide of type III collagen assays.

    PubMed

    Erotokritou-Mulligan, Ioulietta; Guha, Nishan; Stow, Michael; Bassett, E Eryl; Bartlett, Christiaan; Cowan, David A; Sönksen, Peter H; Holt, Richard I G

    2012-04-01

    The GH-2000 project developed a method for detecting GH misuse based on the measurement of insulin-like growth factor-I (IGF-I) and the amino-terminal pro-peptide of type III collagen (P-III-NP). The objective of this study was to develop decision limits for the GH-2000 score to detect GH misuse in elite athletes using two currently available commercial assays for each analyte. 404 male (mean age 23.9 yrs, range 12-37 yrs) and 94 female elite athletes (mean age 24.5 yrs, range 18-34 yrs) participated. Blood samples were collected according to World Anti-Doping Agency (WADA) guidelines at various sporting events including 238 samples collected as part of the UK Anti-Doping Testing Programme. Laboratory analysis: IGF-I was measured by Siemens Immulite IGF-I assay and Immunotech A15729 IGF-I IRMA. P-III-NP was measured by RIA-gnost P-III-P and the UniQ™ PIIINP RIA. The GH-2000 score decision limits were developed through the analysis of the elite athlete samples. For males and females separately, the distributions of GH-2000 scores were consistent with Normal distributions. Using a specificity of 99.99% new decision limits were determined which included an allowance for uncertainty associated with calculations based on a finite sample size. One outlier was identified with results incompatible with normal physiology and tested positive with the current isoform GH test. We have developed decision limits using currently available commercial assays to measure IGF-I and P-III-NP in elite athletes. This should allow the introduction of a test for GH misuse based on the measurement of these GH sensitive biomarkers. Copyright © 2012 Elsevier Ltd. All rights reserved.

  6. The Amino-terminal Domain of the Androgen Receptor Co-opts Extracellular Signal-regulated Kinase (ERK) Docking Sites in ELK1 Protein to Induce Sustained Gene Activation That Supports Prostate Cancer Cell Growth*

    PubMed Central

    Rosati, Rayna; Patki, Mugdha; Chari, Venkatesh; Dakshnamurthy, Selvakumar; McFall, Thomas; Saxton, Janice; Kidder, Benjamin L.; Shaw, Peter E.; Ratnam, Manohar

    2016-01-01

    The ETS domain transcription factor ELK1 is in a repressive association with growth genes and is transiently activated through phosphorylation by ERK1/2. In prostate cancer (PCa) cells the androgen receptor (AR) is recruited by ELK1, via its amino-terminal domain (A/B), as a transcriptional co-activator, without ELK1 hyper-phosphorylation. Here we elucidate the structural basis of the interaction of AR with ELK1. The ELK1 polypeptide motifs required for co-activation by AR versus those required for activation of ELK1 by ERK were systematically mapped using a mammalian two-hybrid system and confirmed using a co-immunoprecipitation assay. The mapping precisely identified the two ERK-docking sites in ELK1, the D-box and the DEF (docking site for ERK, FXFP) motif, as the essential motifs for its cooperation with AR(A/B) or WTAR. In contrast, the transactivation domain in ELK1 was only required for activation by ERK. ELK1-mediated transcriptional activity of AR(A/B) was optimal in the absence of ELK1 binding partners, ERK1/2 and serum-response factor. Purified ELK1 and AR bound with a dissociation constant of 1.9 × 10−8 m. A purified mutant ELK1 in which the D-box and DEF motifs were disrupted did not bind AR. An ELK1 mutant with deletion of the D-box region had a dominant-negative effect on androgen-dependent growth of PCa cells that were insensitive to MEK inhibition. This novel mechanism in which a nuclear receptor impinges on a signaling pathway by co-opting protein kinase docking sites to constitutively activate growth genes could enable rational design of a new class of targeted drug interventions. PMID:27793987

  7. Folded monomers and hexamers of the ectodomain of the HIV gp41 membrane fusion protein: potential roles in fusion and synergy between the fusion peptide, hairpin, and membrane-proximal external region.

    PubMed

    Banerjee, Koyeli; Weliky, David P

    2014-11-25

    HIV is an enveloped virus and fusion between the HIV and host cell membranes is catalyzed by the ectodomain of the HIV gp41 membrane protein. Both the N-terminal fusion peptide (FP) and C-terminal membrane-proximal external region (MPER) are critical for fusion and are postulated to bind to the host cell and HIV membranes, respectively. Prior to fusion, the gp41 on the virion is a trimer in noncovalent complex with larger gp120 subunits. The gp120 bind host cell receptors and move away or dissociate from gp41 which subsequently catalyzes fusion. In the present work, large gp41 ectodomain constructs were produced and biophysically and structurally characterized. One significant finding is observation of synergy between the FP, hairpin, and MPER in vesicle fusion. The ectodomain-induced fusion can be very efficient with only ∼15 gp41 per vesicle, which is comparable to the number of gp41 on a virion. Conditions are found with predominant monomer or hexamer but not trimer and these may be oligomeric states during fusion. Monomer gp41 ectodomain is hyperthermostable and has helical hairpin structure. A new HIV fusion model is presented where (1) hemifusion is catalyzed by folding of gp41 ectodomain monomers into hairpins and (2) subsequent fusion steps are catalyzed by assembly into a hexamer with FPs in an antiparallel β sheet. There is also significant interest in the gp41 MPER because it is the epitope of several broadly neutralizing antibodies. Two of these antibodies bind our gp41 ectodomain constructs and support investigation of the gp41 ectodomain as an immunogen in HIV vaccine development.

  8. Activation of JAK3, but not JAK1, is critical to interleukin-4 (IL4) stimulated proliferation and requires a membrane-proximal region of IL4 receptor alpha.

    PubMed

    Malabarba, M G; Kirken, R A; Rui, H; Koettnitz, K; Kawamura, M; O'Shea, J J; Kalthoff, F S; Farrar, W L

    1995-04-21

    The tyrosine kinases JAK1 and JAK3 have been shown to undergo tyrosine phosphorylation in response to interleukin-2 (IL), IL4, IL7, and IL9, cytokines which share the common IL2 receptor gamma-chain (IL2R gamma), and evidence has been found for a preferential coupling of JAK3 to IL2R gamma and JAK1 to IL2R beta. Here we show, using human premyeloid TF-1 cells, that IL4 stimulates JAK3 to a larger extent than JAK1, based upon three different evaluation criteria. These include a more vigorous tyrosine phosphorylation of JAK3 as measured by anti-phosphotyrosine immunoblotting, a more marked activation of JAK3 as determined by in vitro tyrosine kinase assays and a more manifest presence of JAK3 in activated IL4-receptor complexes. These observations suggest that IL4 receptor signal transduction does not depend on equimolar heterodimerization of JAK1 and JAK3 following IL4-induced heterodimerization of IL4R alpha and IL2R gamma. Indeed, when human IL4R alpha was stably expressed in mouse BA/F3 cells, robust IL4-induced proliferation and JAK3 activation occurred without detectable involvement of JAK1, JAK2, or TYK2. The present study suggests that JAK1 plays a subordinate role in IL4 receptor signaling, and that in certain cells exclusive JAK3 activation may mediate IL4-induced cell growth. Moreover, mutational analysis of human IL4R alpha showed that a membrane-proximal cytoplasmic region was critical for JAK3 activation, while the I4R motif was not, which is compatible with a role of JAK3 upstream of the recruitment of the insulin receptor substrate-1/4PS signaling proteins by IL4 receptors.

  9. Neutralization Efficiency Is Greatly Enhanced by Bivalent Binding of an Antibody to Epitopes in the V4 Region and the Membrane-Proximal External Region within One Trimer of Human Immunodeficiency Virus Type 1 Glycoproteins▿

    PubMed Central

    Wang, Pengcheng; Yang, Xinzhen

    2010-01-01

    Most antibodies are multivalent, with the potential to bind with high avidity. However, neutralizing antibodies commonly bind to virions monovalently. Bivalent binding of a monoclonal antibody (MAb) to a virion has been documented only in a single case. Thus, the role of high avidity in antibody-mediated neutralization of viruses has not been defined clearly. In this study, we demonstrated that when an artificial 2F5 epitope was inserted in the gp120 V4 region so that an HIV-1 envelope glycoprotein (Env) trimer contains a natural 2F5 epitope in the gp41 membrane-proximal envelope region (MPER) and an artificially engineered 2F5 epitope in the gp120 V4 region, bivalent 2F5 IgG achieved greatly enhanced neutralization efficiency, with a 50% inhibitory concentration (IC50) decrease over a 2-log scale. In contrast, the monovalent 2F5 Fab fragment did not exhibit any appreciable change in neutralization efficiency in the same context. These results demonstrate that bivalent binding of 2F5 IgG to a single HIV-1 Env trimer results in dramatic enhancement of neutralization, probably through an increase in binding avidity. Furthermore, we demonstrated that bivalent binding of MAb 2F5 to the V4 region and MPER of an HIV-1 Env trimer can be achieved only in a specific configuration, providing an important insight into the structure of a native/infectious HIV-1 Env trimer. This specific binding configuration also establishes a useful standard that can be applied to evaluate the biological relevance of structural information on the HIV-1 Env trimer. PMID:20463081

  10. Extracellular membrane-proximal domain of HAb18G/CD147 binds to metal ion-dependent adhesion site (MIDAS) motif of integrin β1 to modulate malignant properties of hepatoma cells.

    PubMed

    Li, Yong; Wu, Jiao; Song, Fei; Tang, Juan; Wang, Shi-Jie; Yu, Xiao-Ling; Chen, Zhi-Nan; Jiang, Jian-Li

    2012-02-10

    Several lines of evidence suggest that HAb18G/CD147 interacts with the integrin variants α3β1 and α6β1. However, the mechanism of the interaction remains largely unknown. In this study, mammalian protein-protein interaction trap (MAPPIT), a mammalian two-hybrid method, was used to study the CD147-integrin β1 subunit interaction. CD147 in human hepatocellular carcinoma (HCC) cells was interfered with by small hairpin RNA. Nude mouse xenograft model and metastatic model of HCC were used to detect the role of CD147 in carcinogenesis and metastasis. We found that the extracellular membrane-proximal domain of HAb18G/CD147 (I-type domain) binds at the metal ion-dependent adhesion site in the βA domain of the integrin β1 subunit, and Asp(179) in the I-type domain of HAb18G/CD147 plays an important role in the interaction. The levels of the proteins that act downstream of integrin, including focal adhesion kinase (FAK) and phospho-FAK, were decreased, and the cytoskeletal structures of HCC cells were rearranged bearing the HAb18G/CD147 deletion. Simultaneously, the migration and invasion capacities, secretion of matrix metalloproteinases, colony formation rate in vitro, and tumor growth and metastatic potential in vivo were decreased. These results indicate that the interaction of HAb18G/CD147 extracellular I-type domain with the integrin β1 metal ion-dependent adhesion site motif activates the downstream FAK signaling pathway, subsequently enhancing the malignant properties of HCC cells.

  11. In vivo suppression of vein graft disease by nonviral, electroporation-mediated, gene transfer of tissue inhibitor of metalloproteinase-1 linked to the amino terminal fragment of urokinase (TIMP-1.ATF), a cell-surface directed matrix metalloproteinase inhibitor.

    PubMed

    Eefting, Daniel; de Vries, Margreet R; Grimbergen, Jos M; Karper, Jacco C; van Bockel, J Hajo; Quax, Paul H A

    2010-02-01

    Smooth muscle cell (SMC) migration and proliferation are important in the development of intimal hyperplasia, the major cause of vein graft failure. Proteases of the plasminogen activator (PA) system and of the matrix metalloproteinase (MMP) system are pivotal in extracellular matrix degradation and, by that, SMC migration. Previously, we demonstrated that inhibition of both protease systems simultaneously with viral gene delivery of the hybrid protein TIMP-1.ATF, consisting of the tissue inhibitor of metalloproteinase-1 (TIMP-1) and the receptor-binding amino terminal fragment (ATF) of urokinase, reduces SMC migration and neointima formation in an in vitro restenosis model using human saphenous vein cultures more efficiently than both protease systems separately. Because use of viral gene delivery is difficult in clinical application, this study used nonviral delivery of TIMP-1.ATF plasmid to reduce vein graft disease in a murine bypass model. Nonviral gene transfer by electroporation was used to avert major disadvantages of viral gene delivery, such as immune responses and short-term expression. Plasmids encoding ATF, TIMP-1, TIMP-1.ATF, or luciferase, as a control, were injected and electroporated in both calf muscles of hypercholesterolemic apolipoprotein E3-Leiden (APOE*3Leiden) mice (n = 8). One day after electroporation, a venous interposition of a donor mouse was placed into the carotid artery of a recipient mouse. In this model, vein graft thickening develops with features of accelerated atherosclerosis. Vein grafts were harvested 4 weeks after electroporation and surgery, and histologic analysis of the vessel wall was performed. Electroporation-mediated overexpression of the plasmid vectors resulted in a prolonged expression of the transgenes and resulted in a significant reduction of vein graft thickening (ATF: 36% +/- 9%, TIMP-1: 49% +/- 5%, TIMP-1.ATF: 58% +/- 5%; P < .025). Although all constructs reduced vein graft thickening compared with the

  12. The prognostic value of estimated glomerular filtration rate, amino-terminal portion of the pro-hormone B-type natriuretic peptide and parameters of cardiopulmonary exercise testing in patients with chronic heart failure.

    PubMed

    Verberne, Hein J; van der Spank, Aukje; Bresser, Paul; Somsen, G Aernout

    2012-06-05

    The aim of this study was to evaluate the prognostic value of renal function in relation to amino-terminal portion of the pro-hormone B-type natriuretic peptide (NT-proBNP) and parameters of cardiopulmonary exercise testing in predicting mortality and morbidity in patients with moderate chronic heart failure (CHF). Sixty-one CHF patients were included in the study. Patients' characteristics were: age 64.3±11.6 years; New York Heart Association class I/II/III: 14/37/10; left ventricular ejection fraction: 0.30±0.13 (%); NT-proBNP: 252.2±348.0 (ng/L); estimated creatinine clearance (e-CC): 73.6±31.4 (mL/min); estimated glomerular filtration rate (e-GFR): 66.1±24.6 (mL/min/1.73 m(2)); the highest O2 uptake during exercise (VO(2-peak)): 1.24±0.12 mL/kg/min; VO(2)/workload: 8.52±1.81 (mL/min/W)]. During follow up (59.5±4.0 months) there were 15 cardiac deaths and 16 patients were hospitalized due to progression of heart failure. NT-proBNP and VO(2)/workload were independently associated with cardiac death (P=0.007 and P=0.006, respectively). Hospitalization for progressive CHF was only associated with NT-proBNP (P=0.002). The combined cardiac events (cardiac death and hospitalization) were associated with NT-proBNP and VO(2)/ workload (P=0.007 and P=0.005, respectively). The addition of estimates of renal function (neither serum creatinine nor e-GFR) did not improve the prognostic value for any of the models.In conclusion, in patients with moderate CHF, increased NT-proBNP and reduced VO(2)/ work-load identify those with increased mortality and morbidity, irrespective of estimates of renal function.

  13. Alanine scanning mutagenesis of the second extracellular loop of type 1 corticotropin-releasing factor receptor revealed residues critical for peptide binding.

    PubMed

    Gkountelias, Kostas; Tselios, Theodoros; Venihaki, Maria; Deraos, George; Lazaridis, Iakovos; Rassouli, Olga; Gravanis, Achille; Liapakis, George

    2009-04-01

    Upon binding of the corticotropin-releasing factor (CRF) analog sauvagine to the type 1 CRF receptor (CRF(1)), the amino-terminal portion of the peptide has been shown to lie near Lys257 in the receptor's second extracellular loop (EL2). To test the hypothesis that EL2 residues play a role in the binding of sauvagine to CRF(1) we carried out an alanine-scanning mutagenesis study to determine the functional role of EL2 residues (Leu251 to Val266). Only the W259A, F260A, and W259A/F260A mutations reduced the binding affinity and potency of sauvagine. In contrast, these mutations did not seem to significantly alter the overall receptor conformation, in that they left unchanged the affinities of the ligands astressin and antalarmin that have been suggested to bind to different regions of CRF(1). The W259A, F260A, and W259A/F260A mutations also decreased the affinity of the endogenous ligand, CRF, implying that these residues may play a common important role in the binding of different peptides belonging to CRF family. Parallel amino acid deletions of the two peptides produced ligands with various affinities for wild-type CRF(1) compared with the W259A, F260A, and W259A/F260A mutants, supporting the interaction between the amino-terminal residues 8 to 10 of sauvagine and the corresponding region in CRF with EL2 of CRF(1). This is the first time that a specific region of CRF(1) has been implicated in detailed interactions between the receptor and the amino-terminal portion of peptides belonging to the CRF family.

  14. The amino-terminal portion of CD1 of the adenovirus E1A proteins is required to induce susceptibility to tumor necrosis factor cytolysis in adenovirus-infected mouse cells.

    PubMed Central

    Duerksen-Hughes, P J; Hermiston, T W; Wold, W S; Gooding, L R

    1991-01-01

    Previous work by our laboratory and others has shown that mouse cells normally resistant to tumor necrosis factor can be made sensitive to the cytokine by the expression of adenovirus E1A. The E1A gene can be introduced by either infection or transfection, and either of the two major E1A proteins, 289R or 243R, can induce this sensitivity. The E1A proteins are multifunctional and modular, with specific domains associated with specific functions. Here, we report that the CD1 domain of E1A is required to induce susceptibility to tumor necrosis factor cytolysis in adenovirus-infected mouse C3HA fibroblasts. Amino acids C terminal to residue 60 and N terminal to residue 36 are not necessary for this function. This conclusion is based on 51Cr-release assays for cytolysis in cells infected with adenovirus mutants with deletions in various portions of E1A. These E1A mutants are all in an H5dl309 background and therefore they lack the tumor necrosis factor protection function provided by the 14.7-kilodalton (14.7K) protein encoded by region E3. Western blot (immunoblot) analysis indicated that most of the mutant E1A proteins were stable in infected C3HA cells, although with certain large deletions the E1A proteins were unstable. The region between residues 36 and 60 is included within but does not precisely correlate with domains in E1A that have been implicated in nuclear localization, enhancer repression, cellular immortalization, cell transformation in cooperation with ras, induction of cellular DNA synthesis and proliferation, induction of DNA degradation, and binding to the 300K protein and the 105K retinoblastoma protein. Images PMID:1825340

  15. Single mutation in Shine-Dalgarno-like sequence present in the amino terminal of lactate dehydrogenase of Plasmodium effects the production of an eukaryotic protein expressed in a prokaryotic system.

    PubMed

    Cicek, Mustafa; Mutlu, Ozal; Erdemir, Aysegul; Ozkan, Ebru; Saricay, Yunus; Turgut-Balik, Dilek

    2013-06-01

    One of the most important step in structure-based drug design studies is obtaining the protein in active form after cloning the target gene. In one of our previous study, it was determined that an internal Shine-Dalgarno-like sequence present just before the third methionine at N-terminus of wild type lactate dehydrogenase enzyme of Plasmodium falciparum prevent the translation of full length protein. Inspection of the same region in P. vivax LDH, which was overproduced as an active enzyme, indicated that the codon preference in the same region was slightly different than the codon preference of wild type PfLDH. In this study, 5'-GGAGGC-3' sequence of P. vivax that codes for two glycine residues just before the third methionine was exchanged to 5'-GGAGGA-3', by mimicking P. falciparum LDH, to prove the possible effects of having an internal SD-like sequence when expressing an eukaryotic protein in a prokaryotic system. Exchange was made by site-directed mutagenesis. Results indicated that having two glycine residues with an internal SD-like sequence (GGAGGA) just before the third methionine abolishes the enzyme activity due to the preference of the prokaryotic system used for the expression. This study emphasizes the awareness of use of a prokaryotic system to overproduce an eukaryotic protein.

  16. The amino-terminal fusion domain peptide of human immunodeficiency virus type 1 gp41 inserts into the sodium dodecyl sulfate micelle primarily as a helix with a conserved glycine at the micelle-water interface.

    PubMed Central

    Chang, D K; Cheng, S F; Chien, W J

    1997-01-01

    A peptide based on the N-terminal fusion domain of gp41 of human immunodeficiency virus type 1 (HIV-1) and its tryptophan analog were synthesized to examine the secondary structure in the micellar environment. Nuclear magnetic resonance (NMR), circular dichroism and electron paramagnetic resonance experiments indicated that the gp41 fusion peptide inserted into the micelle primarily as a helix (59%), with substantial beta-structure (26.7%). Deep penetration of the peptide into the apolar hydrocarbon core was supported by the results of fluorescence experiments in which the tryptophan analog exhibited a blue shift of about 30 nm in the presence of a sodium dodecyl sulfate micelle, in 1,2-dimyristoyl-rac-glycero-3-phosphocholine, and in 1,2-dipalmitoyl-sn-glycero-3-phospho-L-serine vesicular solutions. The results of spin label-attenuated 1H resonance experiments show that the region C-terminal to G16, which contains a turn structure, exhibited substantial interaction with the micelle, suggesting that it lies on the surface of micelle. Molecular simulation based on data from NMR experiments revealed a flexible hinge at residues 15 and 16 (alanine and glycine, respectively) from the N terminus of the peptide located at the micelle-solution interface. The highly conserved A15-G16 dipeptide may play a role in the function of fusion domain of HIV-1 envelope glycoprotein. PMID:9261381

  17. Residual Cap

    NASA Image and Video Library

    2006-05-10

    This MOC image shows a summertime view of the south polar residual cap of Mars. In this image, mesas composed largely of solid carbon dioxide are separated from one another by irregularly-shaped depressions

  18. RESIDUAL RISK ASSESSMENTS - RESIDUAL RISK ...

    EPA Pesticide Factsheets

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

  19. Model peptides provide new insights into the role of histidine residues as potential ligands in human cellular copper acquisition via Ctr1.

    PubMed

    Haas, Kathryn L; Putterman, Allison B; White, Daniel R; Thiele, Dennis J; Franz, Katherine J

    2011-03-30

    Cellular acquisition of copper in eukaryotes is primarily accomplished through the Ctr family of copper transport proteins. In both humans and yeast, methionine-rich "Mets" motifs in the amino-terminal extracellular domain of Ctr1 are thought to be responsible for recruitment of copper at the cell surface. Unlike yeast, mammalian Ctr1 also contains extracellular histidine-rich motifs, although a role for these regions in copper uptake has not been explored in detail. Herein, synthetic model peptides containing the first 14 residues of the extracellular domain of human Ctr1 (MDHSHHMGMSYMDS) have been prepared and evaluated for their apparent binding affinity to both Cu(I) and Cu(II). These studies reveal a high affinity Cu(II) binding site (log K = 11.0 ± 0.3 at pH 7.4) at the amino-terminus of the peptide as well as a high affinity Cu(I) site (log K = 10.2 ± 0.2 at pH 7.4) that utilizes adjacent HH residues along with an additional His or Met ligand. These model studies suggest that the histidine domains may play a direct role in copper acquisition from serum copper-binding proteins and in facilitating the reduction of Cu(II) to the active Ctr1 substrate, Cu(I). We tested this hypothesis by expressing a Ctr1 mutant lacking only extracellular histidine residues in Ctr1-knockout mouse embryonic fibroblasts. Results from live cell studies support the hypothesis that extracellular amino-terminal His residues directly participate in the copper transport function of Ctr1.

  20. Model Peptides Provide New Insights into the Role of Histidine Residues as Potential Ligands in Human Cellular Copper Acquisition via Ctr1

    PubMed Central

    Haas, Kathryn L.; Putterman, Allison B.; White, Daniel R.; Thiele, Dennis J.; Franz, Katherine J.

    2011-01-01

    Cellular acquisition of copper in eukaryotes is primarily accomplished through the Ctr family of copper transport proteins. In both humans and yeast, methionine-rich “Mets” motifs in the amino-terminal extracellular domain of Ctr1 are thought to be responsible for recruitment of copper at the cell surface. Unlike yeast, mammalian Ctr1 also contains extracellular histidine-rich motifs, although a role for these regions in copper uptake has not been explored in detail. Herein, synthetic model peptides containing the first 14 residues of the extracellular domain of human Ctr1 (MDHSHHMGMSYMDS) have been prepared and evaluated for their apparent binding affinity to both Cu(I) and Cu(II). These studies reveal a high affinity Cu(II) binding site (logK = 11.0 ± 0.3 at pH 7.4) at the amino-terminus of the peptide as well as a high affinity Cu(I) site (logK = 10.2 ± 0.2 at pH 7.4) that utilizes adjacent HH residues along with an additional His or Met ligand. These model studies suggest that the histidine domains may play a direct role in copper acquisition from serum copper-binding proteins and in facilitating the reduction of Cu(II) to the active Ctr1 substrate, Cu(I). We tested this hypothesis by expressing a Ctr1 mutant lacking only extracellular histidine residues in Ctr1-knockout mouse embryonic fibroblasts. Results from live cell studies support the hypothesis that extracellular amino-terminal His residues directly participate in the copper transport function of Ctr1. PMID:21375246

  1. Residual Cap

    NASA Technical Reports Server (NTRS)

    2006-01-01

    10 May 2006 This Mars Global Surveyor (MGS) Mars Orbiter Camera (MOC) image shows a summertime view of the south polar residual cap of Mars. In this image, mesas composed largely of solid carbon dioxide are separated from one another by irregularly-shaped depressions. The variation in brightness across this scene is a function of several factors including, but not limited to, varying proportions of dust and solid carbon dioxide, undulating topography, and differences in the roughness of the slopes versus the flat surfaces.

    Location near: 86.7oS, 343.3oW Image width: 3 km (1.9 mi) Illumination from: upper left Season: Southern Summer

  2. Distinct tyrosine residues within the interleukin-2 receptor beta chain drive signal transduction specificity, redundancy, and diversity.

    PubMed

    Gaffen, S L; Lai, S Y; Ha, M; Liu, X; Hennighausen, L; Greene, W C; Goldsmith, M A

    1996-08-30

    To explore the basis for interleukin (IL)-2 receptor (IL-2R) signaling specificity, the roles of tyrosine-based sequences located within the cytoplasmic tails of the beta and gammac chains were examined in the murine helper T cell line HT-2. Activation of the Janus kinase/signal transducer and activator of transcription (JAK/STAT) pathway, cellular proliferation, and the induction of various genes were monitored. All four of the cytoplasmic tyrosine residues as well as the distal portion of the gammac proved dispensable for the entire spectrum of IL-2R signaling responses studied. Conversely, select tyrosine residues within the beta chain were essential and differentially required for various signaling events. Specifically, activation of c-fos gene expression was found to occur exclusively through the most membrane proximal tyrosine, Tyr-338, whereas proliferation and the activation of STAT-5 were induced either through Tyr-338 or through the two C-terminal tyrosine residues, Tyr-392 and Tyr-510. These tyrosine residues mediated the induction of two different STAT-5 isoforms, which were found to form heterodimers upon receptor activation. In contrast to the tyrosine dependence of c-fos and STAT-5 induction, bcl-2 gene induction proceeded independently of all IL-2Rbeta tyrosine residues. Thus, the tyrosine-based modules present within the IL-2Rbeta cytoplasmic tail play a critical role in IL-2R signaling, mediating specificity, redundancy, and multifunctionality.

  3. Early region 1B of adenovirus 2 encodes two coterminal proteins of 495 and 155 amino acid residues.

    PubMed Central

    Anderson, C W; Schmitt, R C; Smart, J E; Lewis, J B

    1984-01-01

    Partial sequence analysis of tryptic peptides has identified the E1B-495R (E1b-57K) (early transcription region 1B of 495 amino acid residues, with an approximate molecular weight of 57,000) protein of adenovirus 2 as encoded by the 495 amino acid open reading frame located in the adenovirus 2 DNA sequence between nucleotides 2016 and 3500. Additional proteins of 16,000 Mr and 18,000 Mr that are related to the E1B-495R protein were identified by cell-free translation of hybridization-selected mRNA. Analysis of [35S]methionine-containing amino terminal tryptic peptides by thin-layer chromatography showed that the E1B-495R, E1B-18K, and E1B-16K proteins all begin at the same initiation codon. The E1B-495R protein from 293 cells also has the same initial tryptic peptide, acetyl-methionyl-glutamyl-arginine. Sequence analysis of E1B-18K tryptic peptides indicated that this protein also has the same carboxy terminus as the E1B-495R protein and that it is derived from an mRNA that is spliced to remove sequences between nucleotides 2250 and 3269, resulting in a protein product of 155 amino acid residues. Analysis of E1B-16K tryptic peptides has not yet revealed the carboxy terminal structure of this protein. Both the E1B-495R and the E1B-155R (E1B-18K) proteins, as well as the E1B-16K protein, were precipitated from cell-free translations and from extracts of infected cells by antiserum against an amino terminal nonapeptide common to these proteins. Images PMID:6323739

  4. Identification of Two Tyrosine Residues Required for the Intramolecular Mechanism Implicated in GIT1 Activation

    PubMed Central

    Totaro, Antonio; Astro, Veronica; Tonoli, Diletta; de Curtis, Ivan

    2014-01-01

    GIT1 is an ArfGAP and scaffolding protein regulating cell adhesion and migration. The multidomain structure of GIT1 allows the interaction with several partners. Binding of GIT1 to some of its partners requires activation of the GIT1 polypeptide. Our previous studies indicated that binding of paxillin to GIT1 is enhanced by release of an intramolecular interaction between the amino-terminal and carboxy-terminal portions that keeps the protein in a binding-incompetent state. Here we have addressed the mechanism mediating this intramolecular inhibitory mechanism by testing the effects of the mutation of several formerly identified GIT1 phosphorylation sites on the binding to paxillin. We have identified two tyrosines at positions 246 and 293 of the human GIT1 polypeptide that are needed to keep the protein in the inactive conformation. Interestingly, mutation of these residues to phenylalanine did not affect binding to paxillin, while mutation to either alanine or glutamic acid enhanced binding to paxillin, without affecting the constitutive binding to the Rac/Cdc42 exchange factor βPIX. The involvement of the two tyrosine residues in the intramolecular interaction was supported by reconstitution experiments showing that these residues are important for the binding between the amino-terminal fragment and carboxy-terminal portions of GIT1. Either GIT1 or GIT1-N tyrosine phosphorylation by Src and pervanadate treatment to inhibit protein tyrosine phosphatases did not affect the intramolecular binding between the amino- and carboxy-terminal fragments, nor the binding of GIT1 to paxillin. Mutations increasing the binding of GIT1 to paxillin positively affected cell motility, measured both by transwell migration and wound healing assays. Altogether these results show that tyrosines 246 and 293 of GIT1 are required for the intramolecular inhibitory mechanism that prevents the binding of GIT1 to paxillin. The data also suggest that tyrosine phosphorylation may not be

  5. Acidic Residues Control the Dimerization of the N-terminal Domain of Black Widow Spiders’ Major Ampullate Spidroin 1

    PubMed Central

    Bauer, Joschka; Schaal, Daniel; Eisoldt, Lukas; Schweimer, Kristian; Schwarzinger, Stephan; Scheibel, Thomas

    2016-01-01

    Dragline silk is the most prominent amongst spider silks and comprises two types of major ampullate spidroins (MaSp) differing in their proline content. In the natural spinning process, the conversion of soluble MaSp into a tough fiber is, amongst other factors, triggered by dimerization and conformational switching of their helical amino-terminal domains (NRN). Both processes are induced by protonation of acidic residues upon acidification along the spinning duct. Here, the structure and monomer-dimer-equilibrium of the domain NRN1 of Latrodectus hesperus MaSp1 and variants thereof have been investigated, and the key residues for both could be identified. Changes in ionic composition and strength within the spinning duct enable electrostatic interactions between the acidic and basic pole of two monomers which prearrange into an antiparallel dimer. Upon naturally occurring acidification this dimer is stabilized by protonation of residue E114. A conformational change is independently triggered by protonation of clustered acidic residues (D39, E76, E81). Such step-by-step mechanism allows a controlled spidroin assembly in a pH- and salt sensitive manner, preventing premature aggregation of spider silk proteins in the gland and at the same time ensuring fast and efficient dimer formation and stabilization on demand in the spinning duct. PMID:27681031

  6. Acidic Residues Control the Dimerization of the N-terminal Domain of Black Widow Spiders’ Major Ampullate Spidroin 1

    NASA Astrophysics Data System (ADS)

    Bauer, Joschka; Schaal, Daniel; Eisoldt, Lukas; Schweimer, Kristian; Schwarzinger, Stephan; Scheibel, Thomas

    2016-09-01

    Dragline silk is the most prominent amongst spider silks and comprises two types of major ampullate spidroins (MaSp) differing in their proline content. In the natural spinning process, the conversion of soluble MaSp into a tough fiber is, amongst other factors, triggered by dimerization and conformational switching of their helical amino-terminal domains (NRN). Both processes are induced by protonation of acidic residues upon acidification along the spinning duct. Here, the structure and monomer-dimer-equilibrium of the domain NRN1 of Latrodectus hesperus MaSp1 and variants thereof have been investigated, and the key residues for both could be identified. Changes in ionic composition and strength within the spinning duct enable electrostatic interactions between the acidic and basic pole of two monomers which prearrange into an antiparallel dimer. Upon naturally occurring acidification this dimer is stabilized by protonation of residue E114. A conformational change is independently triggered by protonation of clustered acidic residues (D39, E76, E81). Such step-by-step mechanism allows a controlled spidroin assembly in a pH- and salt sensitive manner, preventing premature aggregation of spider silk proteins in the gland and at the same time ensuring fast and efficient dimer formation and stabilization on demand in the spinning duct.

  7. The primary structure of skeletal muscle myosin heavy chain: IV. Sequence of the rod, and the complete 1,938-residue sequence of the heavy chain.

    PubMed

    Maita, T; Yajima, E; Nagata, S; Miyanishi, T; Nakayama, S; Matsuda, G

    1991-07-01

    In the preceding paper [Maita, T., Miyanishi, T., Matsuzono, K., Tanioka, Y., & Matsuda, G. (1991) J. Biochem. 110, 68-74], we reported the amino-terminal 837-residue sequence of the heavy chain of adult chicken pectoralis muscle myosin. This paper describes the carboxyl terminal 1,097-residue sequence and the linkage of the two sequences. Rod obtained by digesting myosin filaments with alpha-chymotrypsin was redigested with the protease at high KCl concentration, and two fragments, subfragment-2 and light meromyosin, were isolated and sequenced by conventional methods. The linkage of the two fragments was deduced from the sequence of an overlapping peptide obtained by cleaving the rod with cyanogen bromide. The rod contained 1,039 amino acid residues, but lacked the carboxyl-terminal 58 residues of the heavy chain. A carboxyl-terminal 63-residue peptide obtained by cleaving the whole heavy chain with cyanogen bromide was sequenced. Thus, the carboxyl terminal 1,097-residue sequence of the heavy chain was completed. The linkage of subfragment-1 and the rod was deduced from the sequence of an overlapping peptide between the two which was obtained by cleaving heavy meromyosin with cyanogen bromide. Comparing the sequence of the adult myosin thus determined with that of chicken embryonic myosin reported by Molina et al. [Molina, M.I., Kropp, K.E., Gulick, J., & Robbins, J. (1987) J. Biol. Chem. 262, 6478-6488], we found that the sequence homology is 94%.

  8. Identification of critical amino acid residues and functional conservation of the Neurospora crassa and Rattus norvegicus orthologues of neuronal calcium sensor-1.

    PubMed

    Gohain, Dibakar; Deka, Rekha; Tamuli, Ranjan

    2016-12-01

    Neuronal calcium sensor-1 (NCS-1) is a member of neuronal calcium sensor family of proteins consisting of an amino terminal myristoylation domain and four conserved calcium (Ca(2+)) binding EF-hand domains. We performed site-directed mutational analysis of three key amino acid residues that are glycine in the conserved site for the N-terminal myristoylation, a conserved glutamic acid residue responsible for Ca(2+) binding in the third EF-hand (EF3), and an unusual non-conserved amino acid arginine at position 175 in the Neurospora crassa NCS-1. The N. crassa strains possessing the ncs-1 mutant allele of these three amino acid residues showed impairment in functions ranging from growth, Ca(2+) stress tolerance, and ultraviolet survival. In addition, heterologous expression of the NCS-1 from Rattus norvegicus in N. crassa confirmed its interspecies functional conservation. Moreover, functions of glutamic acid at position 120, the first Ca(2+) binding residue among all the EF-hands of the R. norvegicus NCS-1 was found conserved. Thus, we identified three critical amino acid residues of N. crassa NCS-1, and demonstrated its functional conservation across species using the orthologue from R. norvegicus.

  9. Regulation of catabolism of microinjected ribonuclease A. Identification of residues 7-11 as the essential pentapeptide

    SciTech Connect

    Dice, J.F.; Chiang, H.L.; Spencer, E.P.; Backer, J.M.

    1986-05-25

    We have identified a pentapeptide region of microinjected ribonuclease A that is required for enhanced degradation of this protein during serum withdrawal. We introduced reductively methylated (/sup 3/H)ribonuclease A, (/sup 3/H)ribonuclease S-protein (residues 21-124), and (/sup 3/H)ribonuclease S-peptide (residues 1-20) into the cytosol of human fibroblasts by red cell-mediated microinjection and osmotic lysis of pinosomes. The degradative rates of ribonuclease A and ribonuclease S-peptide are increased 2-fold upon withdrawal of serum, while catabolism of ribonuclease S-protein is not regulated in this manner. Certain fragments of ribonuclease S-peptide are also degraded in a serum-dependent fashion (residues 1-14 and 4-13), while other fragments are not (residues 1-10 and 2-8). (/sup 3/H)Ribonuclease S-peptide is cleaved into two smaller radioactive peptides during loading into red cell ghosts. We tentatively identified the larger fragment as residues 7-11 based on its molecular weight determined by Sephadex chromatography in the presence of 8 M urea combined with sequential Edman degradation to identify the position of radioactive lysines. The smaller peptide fragment appears to be the amino-terminal dipeptide, Lys-Glu, and/or residues 7-8, Lys-Phe. After microinjection into fibroblasts, the pentapeptide is degraded at an enhanced rate in the absence of serum, while degradation of the dipeptide is not affected. We confirmed that residues 7-11 constitute the larger hydrolysis product of S-peptide by synthesizing this pentapeptide and radiolabeling it by reductive methylation. It migrated at the expected position after Sephadex chromatography in 8 M urea and was further hydrolyzed only slightly during loading into red cells.

  10. A natural, single-residue substitution yields a less active peptaibiotic: the structure of bergofungin A at atomic resolution.

    PubMed

    Gessmann, Renate; Axford, Danny; Brückner, Hans; Berg, Albrecht; Petratos, Kyriacos

    2017-02-01

    Bergofungin is a peptide antibiotic that is produced by the ascomycetous fungus Emericellopsis donezkii HKI 0059 and belongs to peptaibol subfamily 2. The crystal structure of bergofungin A has been determined and refined to 0.84 Å resolution. This is the second crystal structure of a natural 15-residue peptaibol, after that of samarosporin I. The amino-terminal phenylalanine residue in samarosporin I is exchanged to a valine residue in bergofungin A. According to agar diffusion tests, this results in a nearly inactive antibiotic peptide compared with the moderately active samarosporin I. Crystals were obtained from methanol solutions of purified bergofungin mixed with water. Although there are differences in the intramolecular hydrogen-bonding scheme of samarosporin I, the overall folding is very similar for both peptaibols, namely 310-helical at the termini and α-helical in the middle of the molecules. Bergofungin A and samarosporin I molecules are arranged in a similar way in both lattices. However, the packing of bergofungin A exhibits a second solvent channel along the twofold axis. This latter channel occurs in the vicinity of the N-terminus, where the natural substitution resides.

  11. Utilization of oak residues

    Treesearch

    Richard C. Allison

    1971-01-01

    Residues should be thought of as a raw material for specific uses rather than as a waste. Fines and solid wood residues are usually kept separated in waste-collection system, but species are rarely kept separated. The properties of each species dictate what uses can be made of them. Quantity, location, cost, moisture content, physical size, and presence of foreign...

  12. Nuclear respiratory factors 1 and 2 utilize similar glutamine-containing clusters of hydrophobic residues to activate transcription.

    PubMed Central

    Gugneja, S; Virbasius, C M; Scarpulla, R C

    1996-01-01

    Nuclear respiratory factors 1 and 2 (NRF-1 and NRF-2) are ubiquitous transcription factors that have been implicated in the control of nuclear genes required for respiration, heme biosynthesis, and mitochondrial DNA transcription and replication. Recently, both factors have been found to be major transcriptional determinants for a subset of these genes that define a class of simple promoters involved in respiratory chain expression. Here, functional domains required for transactivation by NRF-1 have been defined. An atypical nuclear localization signal resides in a conserved amino-terminal region adjacent to the DNA binding domain and consists of functionally redundant clusters of basic residues. A second domain in the carboxy-terminal half of the molecule is necessary for transcriptional activation. The activation domains of both NRF-1 and NRF-2 were extensively characterized by both deletion and alanine substitution mutagenesis. The results show that these domains do not fall into known classes defined by a preponderance of amino acid residues, including glutamines, prolines, or isoleucines, as found in other eukaryotic activators. Rather, in both factors, a series of tandemly arranged clusters of hydrophobic amino acids were required for activation. Although all of the functional clusters contain glutamines, the glutamines differ from the hydrophobic residues in that they are inconsequential for activation. Unlike the NRF-2 domain, which contains its essential hydrophobic motifs within 40 residues, the NRF-1 domain spans about 40% of the molecule and appears to have a bipartite structure. The findings indicate that NRF-1 and NRF-2 utilize similar hydrophobic structural motifs for activating transcription. PMID:8816484

  13. Identification of Key Residues Determining Isomerohydrolase Activity of Human RPE65*

    PubMed Central

    Takahashi, Yusuke; Moiseyev, Gennadiy; Ma, Jian-xing

    2014-01-01

    RPE65 is the retinoid isomerohydrolase that converts all-trans-retinyl ester to 11-cis-retinol, a key reaction in the retinoid visual cycle. We have previously reported that cone-dominant chicken RPE65 (cRPE65) shares 90% sequence identity with human RPE65 (hRPE65) but exhibits substantially higher isomerohydrolase activity than that of bovine RPE65 or hRPE65. In this study, we sought to identify key residues responsible for the higher enzymatic activity of cRPE65. Based on the amino acid sequence comparison of mammalian and other lower vertebrates' RPE65, including cone-dominant chicken, 8 residues of hRPE65 were separately replaced by their counterparts of cRPE65 using site-directed mutagenesis. The enzymatic activities of cRPE65, hRPE65, and its mutants were measured by in vitro isomerohydrolase activity assay, and the retinoid products were analyzed by HPLC. Among the mutants analyzed, two single point mutants, N170K and K297G, and a double mutant, N170K/K297G, of hRPE65 exhibited significantly higher catalytic activity than WT hRPE65. Further, when an amino-terminal fragment (Met1–Arg33) of the N170K/K297G double mutant of hRPE65 was replaced with the corresponding cRPE65 fragment, the isomerohydrolase activity was further increased to a level similar to that of cRPE65. This finding contributes to the understanding of the structural basis for isomerohydrolase activity. This highly efficient human isomerohydrolase mutant can be used to improve the efficacy of RPE65 gene therapy for retinal degeneration caused by RPE65 mutations. PMID:25112876

  14. Positive and negative regulation of type II TGF-beta receptor signal transduction by autophosphorylation on multiple serine residues.

    PubMed Central

    Luo, K; Lodish, H F

    1997-01-01

    The type II transforming growth factor-beta (TGF-beta) receptor Ser/Thr kinase (TbetaRII) is responsible for the initiation of multiple TGF-beta signaling pathways, and loss of its function is associated with many types of human cancer. Here we show that TbetaRII kinase is regulated intricately by autophosphorylation on at least three serine residues. Ser213, in the membrane-proximal segment outside the kinase domain, undergoes intra-molecular autophosphorylation which is essential for the activation of TbetaRII kinase activity, activation of TbetaRI and TGF-beta-induced growth inhibition. In contrast, phosphorylation of Ser409 and Ser416, located in a segment corresponding to the substrate recognition T-loop region in a three-dimensional structural model of protein kinases, is enhanced by receptor dimerization and can occur via an intermolecular mechanism. Phosphorylation of Ser409 is essential for TbetaRII kinase signaling, while phosphorylation of Ser416 inhibits receptor function. Mutation of Ser416 to alanine results in a hyperactive receptor that is better able than wild-type to induce TbetaRI activation and subsequent cell cycle arrest. Since on a single receptor either Ser409 or Ser416, but not both simultaneously, can become autophosphorylated, our results show that TbetaRII phosphorylation is regulated intricately and affects TGF-beta receptor signal transduction both positively and negatively. PMID:9155023

  15. TENORM: Coal Combustion Residuals

    EPA Pesticide Factsheets

    Burning coal in boilers to create steam for power generation and industrial applications produces a number of combustion residuals. Naturally radioactive materials that were in the coal mostly end up in fly ash, bottom ash and boiler slag.

  16. Identifying SARS-CoV membrane protein amino acid residues linked to virus-like particle assembly.

    PubMed

    Tseng, Ying-Tzu; Chang, Chia-Hui; Wang, Shiu-Mei; Huang, Kuo-Jung; Wang, Chin-Tien

    2013-01-01

    Severe acute respiratory syndrome coronavirus (SARS-CoV) membrane (M) proteins are capable of self-assembly and release in the form of membrane-enveloped vesicles, and of forming virus-like particles (VLPs) when coexpressed with SARS-CoV nucleocapsid (N) protein. According to previous deletion analyses, M self-assembly involves multiple M sequence regions. To identify important M amino acid residues for VLP assembly, we coexpressed N with multiple M mutants containing substitution mutations at the amino-terminal ectodomain, carboxyl-terminal endodomain, or transmembrane segments. Our results indicate that a dileucine motif in the endodomain tail (218LL219) is required for efficient N packaging into VLPs. Results from cross-linking VLP analyses suggest that the cysteine residues 63, 85 and 158 are not in close proximity to the M dimer interface. We noted a significant reduction in M secretion due to serine replacement for C158, but not for C63 or C85. Further analysis suggests that C158 is involved in M-N interaction. In addition to mutations of the highly conserved 107-SWWSFNPE-114 motif, substitutions at codons W19, W57, P58, W91, Y94 or F95 all resulted in significantly reduced VLP yields, largely due to defective M secretion. VLP production was not significantly affected by a tryptophan replacement of Y94 or F95 or a phenylalanine replacement of W19, W57 or W91. Combined, these results indicate the involvement of specific M amino acids during SARS-CoV virus assembly, and suggest that aromatic residue retention at specific positions is critical for M function in terms of directing virus assembly.

  17. Forest Residues Bundling Project

    Treesearch

    U.S. Forest Service

    2007-01-01

    During the summer of 2003, the U.S. Forest Service conducted an evaluation of biomass bundling for forest residue extraction. This CD provides a report of the project results, a video documentary project record, and a collection of images from the project. Additional information is available at:

  18. [China's crop residues resources evaluation].

    PubMed

    Xie, Guanghui; Wang, Xiaoyu; Ren, Lantian

    2010-07-01

    The availability of crop residues in China is reviewed in this article. The definition of crop residues is clarified as the total byproducts of field production and processing industry thereafter, and methodology for evaluating crop residues is discussed. Based on literature, the progress on the crop residue assessment is addressed. The annual field crops residues in China from 1991 to 1999 were estimated between 6.0-6.8 hundred million tons, while the data for the process residues were not available. From 2000 to 2007, the annual crop residues were estimated between 5.9-7.3 hundred million tons, while the processing residues at the range of 0.9-1.1 hundred million tons. The reasons for the significant variations are due to the disagreement on crop residue definition, different, even inaccurate residue to grain ratio data used in the estimations, and the lacking of clear understanding on the statistical analysis and grain outputs related to the crop residue evaluation. With the complete statistic analysis method, the author's group evaluated the residues in 2006 and 2007 to be 7.4 hundred million tones in total, including 6.5 hundred million tons for field crop residues and 0.9 hundred million tons for process residues. Moreover, the geographic distribution of the field crop residues was analyzed based on the harvest indices (HI) tested within the near five years.

  19. The bovine mannose 6-phosphate/insulin-like growth factor II receptor. The role of arginine residues in mannose 6-phosphate binding.

    PubMed

    Dahms, N M; Rose, P A; Molkentin, J D; Zhang, Y; Brzycki, M A

    1993-03-15

    The extracytoplasmic region of the bovine cation-independent mannose 6-phosphate/insulin-like growth factor II receptor (M6P/IGF-II receptor) consists of 15 homologous repeating domains, each of which is approximately 147 residues in length. The receptor contains two high affinity mannose 6-phosphate (Man-6-P) binding sites and our recent studies (Westlund, B., Dahms, N. M., and Kornfeld, S. (1991) J. Biol. Chem. 266, 23233-23239) have localized these two binding sites to domains 1-3 and 7-11. To further define the location of the Man-6-P binding sites and to determine the role of specific arginine residues in Man-6-P binding, site-directed mutagenesis was utilized to create truncated soluble forms of the M6P/IGF-II receptor in conjunction with either conservative (Lys) or nonconservative (Ala) replacement of arginine residues. These mutants were expressed transiently in COS-1 cells and assayed for their ability to bind phosphomannosyl residues by affinity chromatography. Analysis of the ligand binding activity of carboxyl-terminal truncated forms of the receptor's extracytoplasmic region demonstrated that the second Man-6-P binding site is contained within domains 7-9. Substitution of Arg435 in domain 3 of the amino-terminal binding site and Arg1334 in domain 9 of the second binding site results in a dramatic loss of ligand binding activity. However, substitutions at positions 435 and/or 1334 did not affect the secretion, glycosylation, or immunoreactivity of these truncated proteins. Taken together, these results indicate that Arg435 and Arg1334 are essential components of the M6P/IGF-II receptor's high affinity Man-6-P binding sites.

  20. The ASCE Residuals Transport Manual

    SciTech Connect

    Albertson, O.E.; Bizier, P.A.; Brown, J.; Koch, C.; Sadick, T.

    1999-07-01

    This presentation will highlight the ASCE Residuals Transport Manual, which has been published by ASCE this year. This document, which represents the state of the art in information on residuals transport, is designed to be used by both the active practitioner, as well as for instructional purposes. The authors will present the various chapters which cover the following topics: Conveyance of Water and Wastewater Residuals, Rheology, Sludge Characteristics, Quality and Quantity, Overview of Residuals Conveyance Devices, Pumping of Viscous Sludges and Slurries, Transport of Thickened Residuals, Conveyance of Dewatered Residuals, Transport of Granular and Compactable Residuals, and Case Studies. The Objective of the Transport Monograph is to summarize in one concise volume the general state of knowledge regarding residuals transport from both water and wastewater residuals. The presentation will cover each chapter and will review the pertinent information contained in the manual.

  1. Residual stresses in material processing

    SciTech Connect

    Kozaczek, K.J.; Watkins, T.R.; Hubbard, C.R.; Wang, Xun-Li; Spooner, S.

    1994-09-01

    Material manufacturing processes often introduce residual stresses into the product. The residual stresses affect the properties of the material and often are detrimental. Therefore, the distribution and magnitude of residual stresses in the final product are usually an important factor in manufacturing process optimization or component life prediction. The present paper briefly discusses the causes of residual stresses. It then adresses the direct, nondestructive methods of residual stress measurement by X-ray and neutron diffraction. Examples are presented to demonstrate the importance of residual stress measurement in machining and joining operations.

  2. Residual Neuromuscular Blockade.

    PubMed

    Plummer-Roberts, Anna L; Trost, Christina; Collins, Shawn; Hewer, Ian

    2016-02-01

    This article provides an update on residual neuromuscular blockade for nurse anesthetists. The neuromuscular junction, pharmacology for producing and reversing neuromuscular blockade, monitoring sites and methods, and patient implications relating to incomplete reversal of neuromuscular blockade are reviewed. Overall recommendations include using multiple settings when employing a peripheral nerve stimulator for monitoring return of neuromuscular function and administering pharmacologic reversal when the train-of-four ratio is below 0.9.

  3. Examination of Residuals

    DTIC Science & Technology

    1961-01-01

    observations have been made to test a prediction of the theory. Another example would be an experiment on extrasensory perception ; most people believe that...to have the same n and v and yet differ perceptibly in the properties of their residuals. This will be illustrated by two examples with very small n...logarithmically. In recording sensory perceptions or value judgments arbitrary numerical scores are sometimes used, and on the face of it these might as well be

  4. SRC Residual fuel oils

    DOEpatents

    Tewari, Krishna C.; Foster, Edward P.

    1985-01-01

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

  5. Wood Residue Distribution Simulator (WORDS)

    Treesearch

    Douglas A. Eza; James W. McMinn; Peter E. Dress

    1984-01-01

    Successful development of woody biomass for energy will depend on the distribution of local supply and demand within subregions, rather than on the total inventory of residues. The Wood Residue Distribution Simulator (WORDS) attempts to find a least-cost allocation of residues from local sources of supply to local sources of demand, given the cost of the materials,...

  6. An immunogen containing four tandem 10E8 epitope repeats with exposed key residues induces antibodies that neutralize HIV-1 and activates an ADCC reporter gene.

    PubMed

    Sun, Zhiwu; Zhu, Yun; Wang, Qian; Ye, Ling; Dai, Yanyan; Su, Shan; Yu, Fei; Ying, Tianlei; Yang, Chinglai; Jiang, Shibo; Lu, Lu

    2016-06-22

    After three decades of intensive research efforts, an effective vaccine against HIV-1 remains to be developed. Several broadly neutralizing antibodies to HIV-1, such as 10E8, recognize the membrane proximal external region (MPER) of the HIV-1 gp41 protein. Thus, the MPER is considered to be a very important target for vaccine design. However, the MPER segment has very weak immunogenicity and tends to insert its epitope residues into the cell membrane, thereby avoiding antibody binding. To address this complication in vaccine development, we herein designed a peptide, designated 10E8-4P, containing four copies of the 10E8 epitope as an immunogen. As predicted by structural simulation, 10E8-4P exhibits a well-arranged tandem helical conformation, with the key residues in the 10E8 epitope oriented at different angles, thus suggesting that some of these key residues may be exposed outside of the lipid membrane. Compared with a peptide containing a single 10E8 epitope (10E8-1P), 10E8-4P not only exhibited better antigenicity but also elicited neutralizing antibody response against HIV-1 pseudoviruses, whereas 10E8-1P could not induce detectable neutralizing antibody response. Importantly, antibodies elicited by 10E8-4P also possessed a strong ability to activate an antibody-dependent cell-mediated cytotoxicity (ADCC) reporter gene, thus suggesting that they may have ADCC activity. Therefore, this strategy shows promise for further optimization and application in future HIV-1 vaccine design.

  7. Lack of association of a variable number of aspartic acid residues in the asporin gene with osteoarthritis susceptibility: case-control studies in Spanish Caucasians

    PubMed Central

    Rodriguez-Lopez, Julio; Pombo-Suarez, Manuel; Liz, Myriam; Gomez-Reino, Juan J; Gonzalez, Antonio

    2006-01-01

    A recent genetic association study has identified a microsatellite in the coding sequence of the asporin gene as a susceptibility factor for osteoarthritis (OA). Alleles of this microsatellite determine the variable number of aspartic acid residues in the amino-terminal end of the asporin protein. Asporin binds directly to the growth factor transforming growth factor beta and inhibits its anabolic effects in cartilage, which include stimulation of collagen and aggrecan synthesis. The OA-associated allele, with 14 aspartic acid residues, inhibits the anabolic effects of transforming growth factor beta more strongly than other asporin alleles, leading to increased OA liability. We have explored whether the association found in several cohorts of Japanese hip OA and knee OA patients was also present in Spanish Caucasians. We studied patients that had undergone total joint replacement for primary OA in the hip (n = 303) or the knee (n = 188) and patients with hand OA (n = 233), and we compared their results with controls (n = 294) lacking overt OA clinical symptoms. No significant differences were observed in any of the multiple comparisons performed, which included global tests of allele frequency distributions and specific comparisons as well as stratification by affected joint and by sex. Our results, together with reports from the United Kingdom and Greece, indicate that the stretch of aspartic acid residues in asporin is not an important factor in OA susceptibility among European Caucasians. It remains possible that lifestyle, environmental or genetic differences allow for an important effect of asporin variants in other ethnic groups as has been reported in the Japanese, but this should be supported by additional studies. PMID:16542493

  8. Effects of amino-terminal extensions and specific mutations on the activity of restrictocin.

    PubMed

    Yang, R; Kenealy, W R

    1992-08-25

    The cytotoxic activities of restrictocin with aminoterminal extensions and specific mutations were investigated using in vivo and in vitro systems. Genes were constructed from the cDNA clone of restrictocin which encode: the native form of restrictocin (including the leader sequence); Met-prorestrictocin, in which a codon for methionine was placed before a putative pro region; Met-mature restrictocin, with a methionine codon prior to the mature form of restrictocin; and three mutated forms of Met-mature restrictocin, E95G, E115G/H136L, and H136L. These constructions were placed under the control of the GAL1 promoter and were transformed into Saccharomyces cerevisiae. Transformants were killed, and a new RNA band formed when any of these genes except those containing the H136L mutation were expressed. Restrictocin protein was detected by immunoblot only in cells expressing the native form of restrictocin and the forms containing the H136L mutation. Native restrictocin, Met-prorestrictocin, and Met-mature restrictocin mRNA were translated in an in vitro system resulting in proteins of the expected molecular weight and inactivation of the translation system. Restrictocin was not inactivated by the presence of the leader sequence and the putative prosequence. Amino acid His136 is putatively in the active site of restrictocin by analogy to ribonuclease U2 and the elimination of toxic effects in the S. cerevisiae expression and in vitro translation systems.

  9. Free-Energy Landscape of the Amino-Terminal Fragment of Huntingtin in Aqueous Solution

    PubMed Central

    Binette, Vincent; Côté, Sébastien; Mousseau, Normand

    2016-01-01

    The first exon of Huntingtin—a protein with multiple biological functions whose misfolding is related to Huntington’s disease—modulates its localization, aggregation, and function within the cell. It is composed of a 17-amino-acid amphipathic segment (Htt17), an amyloidogenic segment of consecutive glutamines (QN), and a proline-rich segment. Htt17 is of fundamental importance: it serves as a membrane anchor to control the localization of huntingtin, it modulates huntingtin’s function through posttranslational modifications, and it controls the self-assembly of the amyloidogenic QN segment into oligomers and fibrils. Experimentally, the conformational ensemble of the Htt17 monomer, as well as the impact of the polyglutamine and proline-rich segments, remains, however, mostly uncharacterized at the atomic level due to its intrinsic flexibility. Here, we unveil the free-energy landscape of Htt17, Htt17Q17, and Htt17Q17P11 using Hamiltonian replica exchange combined with well-tempered metadynamics. We characterize the free-energy landscape of these three fragments in terms of a few selected collective variables. Extensive simulations reveal that the free energy of Htt17 is dominated by a broad ensemble of configurations that agree with solution NMR chemical shifts. Addition of Q17 at its carboxy-terminus reduces the extent of the main basin to more extended configurations of Htt17 with lower helix propensity. Also, the aliphatic carbons of Q17 partially sequester the nonpolar amino acids of Htt17. For its part, addition of Q17P11 shifts the overall landscape to a more extended and helical Htt17 stabilized by interactions with Q17 and P11, which almost exclusively form a PPII-helix, as well as by intramolecular H-bonds and salt bridges. Our characterization of Huntingtin’s amino-terminus provides insights into the structural origin of its ability to oligomerize and interact with phospholipid bilayers, processes closely linked to the biological functions of this protein. PMID:26958885

  10. Amino Terminal Region of Dengue Virus NS4A Cytosolic Domain Binds to Highly Curved Liposomes.

    PubMed

    Hung, Yu-Fu; Schwarten, Melanie; Hoffmann, Silke; Willbold, Dieter; Sklan, Ella H; Koenig, BerndW

    2015-07-21

    Dengue virus (DENV) is an important human pathogen causing millions of disease cases and thousands of deaths worldwide. Non-structural protein 4A (NS4A) is a vital component of the viral replication complex (RC) and plays a major role in the formation of host cell membrane-derived structures that provide a scaffold for replication. The N-terminal cytoplasmic region of NS4A(1-48) is known to preferentially interact with highly curved membranes. Here, we provide experimental evidence for the stable binding of NS4A(1-48) to small liposomes using a liposome floatation assay and identify the lipid binding sequence by NMR spectroscopy. Mutations L6E;M10E were previously shown to inhibit DENV replication and to interfere with the binding of NS4A(1-48) to small liposomes. Our results provide new details on the interaction of the N-terminal region of NS4A with membranes and will prompt studies of the functional relevance of the curvature sensitive membrane anchor at the N-terminus of NS4A.

  11. Changes in the conformation of the Vsr endonuclease amino-terminal domain accompany DNA cleavage.

    PubMed

    Polosina, Yaroslava Y; Cupples, Claire G

    2009-10-01

    In Escherichia coli, T/G mismatches arising from deamination of 5-methylcytosine to thymine are converted to CG base pairs by the very short patch (VSP) repair pathway. DNA Polymerase I removes and resynthesizes the mismatched T starting from a 5'-nick created by the Vsr endonuclease. We used limited trypsinolysis to probe conformational changes in the N-terminal domain of Vsr in response to DNA binding, DNA cleavage and interaction with the polymerase. Our data show that the domain becomes trypsin resistant only under conditions that allow DNA cleavage, while interaction with the polymerase restores trypsin sensitivity. We suggest that the domain changes its conformation as a result of DNA nicking, and that DNA Pol I releases Vsr from the nick by reversing that conformational change.

  12. Amino terminal region of Phytophthora sojae cel12 endoglucanase confers tissue collapse function in Nicotiana

    USDA-ARS?s Scientific Manuscript database

    Phytophthora encodes an unusually large number of glycosyl hydrolases (GH), with many large gene families resulting from duplication events. There are ten copies of GH 12 (cel12) present in Phytophthora sojae. This is the only pathogen endoglucanase family to which plants produce an inhibitory pr...

  13. The amino-terminal domain of glutamate receptor {delta}2 triggers presynaptic differentiation

    SciTech Connect

    Uemura, Takeshi; Mishina, Masayoshi

    2008-12-26

    Glutamate receptor (GluR) {delta}2 selectively expressed in cerebellar Purkinje cells plays key roles in synapse formation, long-term depression and motor learning. We propose that GluR{delta}2 regulates synapse formation by making a physical linkage between the active zone and postsynaptic density. To examine the issue, GluR{delta}2-transfected 293T cells were cultured with cerebellar neurons. We found numerous punctate signals for presynaptic markers on the surface of 293T cells expressing GluR{delta}2. The presynaptic specializations induced by GluR{delta}2 were capable of exo- and endocytosis as indicated by FM1-43 dye labeling. Replacement of the extracellular N-terminal domain (NTD) of GluR{delta}2 with that of the AMPA receptor GluR{alpha}1 abolished the inducing activity. The NTD of GluR{delta}2 fused to the immunoglobulin constant region successfully induced the accumulation of presynaptic specializations on the surface of beads bearing the fusion protein. These results suggest that GluR{delta}2 triggers presynaptic differentiation by direct interaction with presynaptic components through the NTD.

  14. The biological functions of Naa10 — From amino-terminal acetylation to human disease

    PubMed Central

    Dörfel, Max J.; Lyon, Gholson J.

    2015-01-01

    N-terminal acetylation (NTA) is one of the most abundant protein modifications known, and the N-terminal acetyltransferase (NAT) machinery is conserved throughout all Eukarya. Over the past 50 years, the function of NTA has begun to be slowly elucidated, and this includes the modulation of protein–protein interaction, protein-stability, protein function, and protein targeting to specific cellular compartments. Many of these functions have been studied in the context of Naa10/NatA; however, we are only starting to really understand the full complexity of this picture. Roughly, about 40% of all human proteins are substrates of Naa10 and the impact of this modification has only been studied for a few of them. Besides acting as a NAT in the NatA complex, recently other functions have been linked to Naa10, including post-translational NTA, lysine acetylation, and NAT/KAT-independent functions. Also, recent publications have linked mutations in Naa10 to various diseases, emphasizing the importance of Naa10 research in humans. The recent design and synthesis of the first bisubstrate inhibitors that potently and selectively inhibit the NatA/Naa10 complex, monomeric Naa10, and hNaa50 further increases the toolset to analyze Naa10 function. PMID:25987439

  15. residue and shunting pinholes

    NASA Astrophysics Data System (ADS)

    Gorji, Nima E.

    2014-09-01

    The present work considers two observable phenomena through the experimental fabrication and electrical characterization of the rf-sputtered CdS/CdTe thin film solar cells that extremely reduce the overall conversion efficiency of the device: CdCl2 residue on the surface of the semiconductor and shunting pinholes. The former happens through nonuniform treatment of the As-deposited solar cells before annealing at high temperature and the latter occurs by shunting pinholes when the cell surface is shunted by defects, wire-like pathways or scratches on the metallic back contact caused from the external contacts. Such physical problems may be quite common in the experimental activities and reduce the performance down to 4-5 % which leads to dismantle the device despite its precise fabrication. We present our electrical characterization on the samples that received wet CdCl2 surface treatment (uniform or nonuniform) and are damaged by the pinholes.

  16. Residual gas analyzer calibration

    NASA Technical Reports Server (NTRS)

    Lilienkamp, R. H.

    1972-01-01

    A technique which employs known gas mixtures to calibrate the residual gas analyzer (RGA) is described. The mass spectra from the RGA are recorded for each gas mixture. This mass spectra data and the mixture composition data each form a matrix. From the two matrices the calibration matrix may be computed. The matrix mathematics requires the number of calibration gas mixtures be equal to or greater than the number of gases included in the calibration. This technique was evaluated using a mathematical model of an RGA to generate the mass spectra. This model included shot noise errors in the mass spectra. Errors in the gas concentrations were also included in the valuation. The effects of these errors was studied by varying their magnitudes and comparing the resulting calibrations. Several methods of evaluating an actual calibration are presented. The effects of the number of gases in then, the composition of the calibration mixture, and the number of mixtures used are discussed.

  17. Experimental determination of residual stress

    NASA Technical Reports Server (NTRS)

    Ferguson, Milton W.

    1991-01-01

    Residual stresses in finished parts have often been regarded as factors contributing to premature part failure and geometric distortions. Currently, residual stresses in welded structures and railroad components are being investigated. High residual stresses formed in welded structures due primarily to the differential contractions of the weld material as it cools and solidifies can have a profound effect on the surface performance of the structure. In railroad wheels, repeated use of the brakes causes high residual stresses in the rims which may lead to wheel failure and possible derailment. The goals of the study were: (1) to develop strategies for using x-ray diffraction to measure residual stress; (2) to subject samples of Inconel 718 to various mechanical and heat treatments and to measure the resulting stress using x-ray diffraction; and (3) to measure residual stresses in ferromagnetic alloys using magnetoacoustics.

  18. Materials recovery from shredder residues

    SciTech Connect

    Daniels, E. J.; Jody, B. J.; Pomykala, J., Jr.

    2000-07-24

    Each year, about five (5) million ton of shredder residues are landfilled in the US. Similar quantities are landfilled in Europe and the Pacific Rim. Landfilling of these residues results in a cost to the existing recycling industry and also represents a loss of material resources that are otherwise recyclable. In this paper, the authors outline the resources recoverable from typical shredder residues and describe technology that they have developed to recover these resources.

  19. Microwave emission and crop residues

    NASA Technical Reports Server (NTRS)

    Jackson, Thomas J.; O'Neill, Peggy E.

    1991-01-01

    A series of controlled experiments were conducted to determine the significance of crop residues or stubble in estimating the emission of the underlying soil. Observations using truck-mounted L and C band passive microwave radiometers showed that for dry wheat and soybeans the dry residue caused negligible attenuation of the background emission. Green residues, with water contents typical of standing crops, did have a significant effect on the background emission. Results for these green residues also indicated that extremes in plant structure, as created using parallel and perpendicular stalk orientations, can cause very large differences in the degree of attenuation.

  20. Microwave emission and crop residues

    NASA Technical Reports Server (NTRS)

    Jackson, Thomas J.; O'Neill, Peggy E.

    1991-01-01

    A series of controlled experiments were conducted to determine the significance of crop residues or stubble in estimating the emission of the underlying soil. Observations using truck-mounted L and C band passive microwave radiometers showed that for dry wheat and soybeans the dry residue caused negligible attenuation of the background emission. Green residues, with water contents typical of standing crops, did have a significant effect on the background emission. Results for these green residues also indicated that extremes in plant structure, as created using parallel and perpendicular stalk orientations, can cause very large differences in the degree of attenuation.

  1. Detailed analysis of the IL-5-IL-5R alpha interaction: characterization of crucial residues on the ligand and the receptor.

    PubMed Central

    Cornelis, S; Plaetinck, G; Devos, R; Van der Heyden, J; Tavernier, J; Sanderson, C J; Guisez, Y; Fiers, W

    1995-01-01

    The receptor for interleukin-5 (IL-5) is composed of two different subunits. The IL-5 receptor alpha (IL-5R alpha) is required for ligand-specific binding while association with the beta-chain results in increased binding affinity. Murine IL-5 (mIL-5) has similar activity on human and murine cells, whereas human IL-5 (hIL-5) has marginal activity on murine cells. We found that the combined substitution of K84 and N108 on hIL-5 by their respective murine counterpart yields a molecule which is as potent as mIL-5 for growth stimulation of a murine cell line. Since the unidirectional species specificity is due only to the interaction with the IL-5R alpha subunit, we have used chimeric IL-5R alpha molecules to define regions of hIL-5R alpha involved in species-specific hIL-5 ligand binding. We found that this property is largely determined by the NH2-terminal module of hIL-5R alpha, and detailed analysis defined D56 and to a lesser extent E58 as important for binding. Moreover, two additional residues, D55 and Y57, were identified by alanine scanning mutagenesis within the same region. Based on the observed homology between the NH2-terminal module and the membrane proximal (WSXWS-containing) module of hIL-5R alpha we located this stretch of four amino acid residues (D55, D56, Y57 and E58) in the loop region that connects the C and D beta-strands on the proposed tertiary structure of the NH2-terminal module.(ABSTRACT TRUNCATED AT 250 WORDS) Images PMID:7628440

  2. On tide-induced lagrangian residual current and residual transport: 1. Lagrangian residual current

    USGS Publications Warehouse

    Feng, Shizuo; Cheng, Ralph T.; Pangen, Xi

    1986-01-01

    Residual currents in tidal estuaries and coastal embayments have been recognized as fundamental factors which affect the long-term transport processes. It has been pointed out by previous studies that it is more relevant to use a Lagrangian mean velocity than an Eulerian mean velocity to determine the movements of water masses. Under weakly nonlinear approximation, the parameter k, which is the ratio of the net displacement of a labeled water mass in one tidal cycle to the tidal excursion, is assumed to be small. Solutions for tides, tidal current, and residual current have been considered for two-dimensional, barotropic estuaries and coastal seas. Particular attention has been paid to the distinction between the Lagrangian and Eulerian residual currents. When k is small, the first-order Lagrangian residual is shown to be the sum of the Eulerian residual current and the Stokes drift. The Lagrangian residual drift velocity or the second-order Lagrangian residual current has been shown to be dependent on the phase of tidal current. The Lagrangian drift velocity is induced by nonlinear interactions between tides, tidal currents, and the first-order residual currents, and it takes the form of an ellipse on a hodograph plane. Several examples are given to further demonstrate the unique properties of the Lagrangian residual current.

  3. Taking inventory of woody residuals

    Treesearch

    David McKeever

    2003-01-01

    USDA Forest Service analysis finds 104 million tons of woody residuals available for recovery in the U.S., with wood in MSW and C&D debris streams comprising 28 million tons. The U.S. Department of Agriculture Forest Service conducts a variety of analyses to estimate the quantity of woody residuals in the United States. Its Forest Products Laboratory in Madison,...

  4. Residue-based scattering factors.

    PubMed

    Xu, Hongliang

    2016-11-01

    A glob is defined as a group of atoms in the crystal which can be chosen in various ways. Globs themselves can be used as scattering elements in the theory of structure determination, just as atoms are used at present. In this paper, amino-acid residues are chosen to form globs and empirical formulas for residue-based scattering factors have been developed.

  5. Residual strains in conduit arteries.

    PubMed

    Rachev, A; Greenwald, S E

    2003-05-01

    Residual strains and stresses are those that exist in a body when all external loads are removed. Residual strains in arteries can be characterized by the opening angle of the sector-like cross-section which arises when an unloaded ring segment is radially cut. A review of experimental methods for measuring residual strains and the main results about the variation of the opening angle with arterial localization, age, smooth muscle activity, mechanical environment and certain vascular pathologies are presented and discussed. It is shown that, in addition to their well-established ability to homogenize the stress field in the arterial wall, residual strains make arteries more compliant and thereby improve their performance as elastic reservoirs and ensure more effective local control of the arterial lumen by smooth muscle cells. Finally, evidence that, in some cases, residual strains remain in arteries even after they have been cut radially is discussed.

  6. Proteolysis of SNAP-25 isoforms by botulinum neurotoxin types A, C, and E: domains and amino acid residues controlling the formation of enzyme-substrate complexes and cleavage.

    PubMed

    Vaidyanathan, V V; Yoshino, K; Jahnz, M; Dörries, C; Bade, S; Nauenburg, S; Niemann, H; Binz, T

    1999-01-01

    Tetanus toxin and the seven serologically distinct botulinal neurotoxins (BoNT/A to BoNT/G) abrogate synaptic transmission at nerve endings through the action of their light chains (L chains), which proteolytically cleave VAMP (vesicle-associated membrane protein)/synaptobrevin, SNAP-25 (synaptosome-associated protein of 25 kDa), or syntaxin. BoNT/C was reported to proteolyze both syntaxin and SNAP-25. Here, we demonstrate that cleavage of SNAP-25 occurs between Arg198 and Ala199, depends on the presence of regions Asn93 to Glu145 and Ile156 to Met202, and requires about 1,000-fold higher L chain concentrations in comparison with BoNT/A and BoNT/E. Analyses of the BoNT/A and BoNT/E cleavage sites revealed that changes in the carboxyl-terminal residues, in contrast with changes in the amino-terminal residues, drastically impair proteolysis. A proteolytically inactive BoNT/A L chain mutant failed to bind to VAMP/synaptobrevin and syntaxin, but formed a stable complex (KD = 1.9 x 10(-7) M) with SNAP-25. The minimal essential domain of SNAP-25 required for cleavage by BoNT/A involves the segment Met146-Gln197, and binding was optimal only with full-length SNAP-25. Proteolysis by BoNT/E required the presence of the domain Ile156-Asp186. Murine SNAP-23 was cleaved by BoNT/E and, to a reduced extent, by BoNT/A, whereas human SNAP-23 was resistant to all clostridial L chains. Lys185Asp or Pro182Arg mutations of human SNAP-23 induced susceptibility toward BoNT/E or toward both BoNT/A and BoNT/E, respectively.

  7. Importance of the residue Asp 290 on chain length selectivity and catalytic efficiency of recombinant Staphylococcus simulans lipase expressed in E. coli.

    PubMed

    Sayari, Adel; Mosbah, Habib; Gargouri, Youssef

    2007-05-01

    In addition to their physiological importance, microbial lipases, like staphylococcal ones, are of considerable commercial interest for biotechnological applications such as detergents, food production, and pharmaceuticals and industrial synthesis of fine chemicals. The gene encoding the extracellular lipase of Staphylococcus simulans (SSL) was subcloned in the pET-14b expression vector and expressed in Esherichia coli BL21 (DE3). The wild-type SSL was expressed as amino terminal His6-tagged recombinant protein. One-step purification of the recombinant lipase was achieved with nickel metal affinity column. The purified His-tagged SSL (His6-SSL) is able to hydrolyse triacylglycerols without chain length selectivity. The major differences among lipases are reflected in their chemical specificity in the hydrolysis of peculiar ester bonds, and their respective capacity to hydrolyse substrates having different physico-chemical properties. It has been proposed, using homology alignment, that the region around the residue 290 of Staphylococcus hyicus lipase could be involved in the selection of the substrate. To evaluate the importance of this environment, the residue Asp290 of Staphylococcus simulans lipase was mutated to Ala using site-directed mutagenesis. The mutant expression plasmid was also overexpressed in Esherichia coli and purified with a nickel metal affinity column. The substitution of Asp290 by Ala was accompanied by a significant shift of the acyl-chain length specificity of the mutant towards short chain fatty acid esters. Kinetic studies of wild-type SSL and its mutant D290A were carried out, and show essentially that the catalytic efficiency (k cat /K M ) of the mutant was affected. Our results confirmed that Asp290 is important for the chain length selectivity and catalytic efficiency of Staphylococcus simulans lipase.

  8. RESIDUAL RISK ASSESSMENT: MAGNETIC TAPE ...

    EPA Pesticide Factsheets

    This document describes the residual risk assessment for the Magnetic Tape Manufacturing source category. For stationary sources, section 112 (f) of the Clean Air Act requires EPA to assess risks to human health and the environment following implementation of technology-based control standards. If these technology-based control standards do not provide an ample margin of safety, then EPA is required to promulgate addtional standards. This document describes the methodology and results of the residual risk assessment performed for the Magnetic Tape Manufacturing source category. The results of this analyiss will assist EPA in determining whether a residual risk rule for this source category is appropriate.

  9. Electromagnetic zonal flow residual responses

    NASA Astrophysics Data System (ADS)

    Catto, Peter J.; Parra, Felix I.; Pusztai, István

    2017-08-01

    The collisionless axisymmetric zonal flow residual calculation for a tokamak plasma is generalized to include electromagnetic perturbations. We formulate and solve the complete initial value zonal flow problem by retaining the fully self-consistent axisymmetric spatial perturbations in the electric and magnetic fields. Simple expressions for the electrostatic, shear and compressional magnetic residual responses are derived that provide a fully electromagnetic test of the zonal flow residual in gyrokinetic codes. Unlike the electrostatic potential, the parallel vector potential and the parallel magnetic field perturbations need not relax to flux functions for all possible initial conditions.

  10. RESIDUAL RISK ASSESSMENT: INDUSTRIAL PROCESS ...

    EPA Pesticide Factsheets

    This document describes the residual risk assessment for the Industrial Process Cooling Towers source category. For stationary sources, section 112 (f) of the Clean Air Act requires EPA to assess risks to human health and the environment following implementation of technology-based control standards. If these technology-based control standards do not provide an ample margin of safety, then EPA is required to promulgate addtional standards. This document describes the methodology and results of the residual risk assessment performed for the Industrial Process Cooling Towers source category. The results of this analyiss will assist EPA in determining whether a residual risk rule for this source category is appropriate.

  11. RESIDUAL RISK ASSESSMENT: ETHYLENE OXIDE ...

    EPA Pesticide Factsheets

    This document describes the residual risk assessment for the Ethylene Oxide Commercial Sterilization source category. For stationary sources, section 112 (f) of the Clean Air Act requires EPA to assess risks to human health and the environment following implementation of technology-based control standards. If these technology-based control standards do not provide an ample margin of safety, then EPA is required to promulgate addtional standards. This document describes the methodology and results of the residual risk assessment performed for the Ethylene Oxide Commercial Sterilization source category. The results of this analyiss will assist EPA in determining whether a residual risk rule for this source category is appropriate.

  12. RESIDUAL RISK ASSESSMENT: ETHYLENE OXIDE ...

    EPA Pesticide Factsheets

    This document describes the residual risk assessment for the Ethylene Oxide Commercial Sterilization source category. For stationary sources, section 112 (f) of the Clean Air Act requires EPA to assess risks to human health and the environment following implementation of technology-based control standards. If these technology-based control standards do not provide an ample margin of safety, then EPA is required to promulgate addtional standards. This document describes the methodology and results of the residual risk assessment performed for the Ethylene Oxide Commercial Sterilization source category. The results of this analyiss will assist EPA in determining whether a residual risk rule for this source category is appropriate.

  13. OECD Maximum Residue Limit Calculator

    EPA Pesticide Factsheets

    With the goal of harmonizing the calculation of maximum residue limits (MRLs) across the Organisation for Economic Cooperation and Development, the OECD has developed an MRL Calculator. View the calculator.

  14. Americium recovery from reduction residues

    DOEpatents

    Conner, W.V.; Proctor, S.G.

    1973-12-25

    A process for separation and recovery of americium values from container or bomb'' reduction residues comprising dissolving the residues in a suitable acid, adjusting the hydrogen ion concentration to a desired level by adding a base, precipitating the americium as americium oxalate by adding oxalic acid, digesting the solution, separating the precipitate, and thereafter calcining the americium oxalate precipitate to form americium oxide. (Official Gazette)

  15. Evaluation of residue-residue contact prediction in CASP10

    PubMed Central

    Monastyrskyy, Bohdan; D’Andrea, Daniel; Fidelis, Krzysztof; Tramontano, Anna; Kryshtafovych, Andriy

    2013-01-01

    We present the results of the assessment of the intra-molecular residue-residue contact predictions from 26 prediction groups participating in the 10th round of the CASP experiment. The most recently developed direct coupling analysis methods did not take part in the experiment likely because they require a very deep sequence alignment not available for any of the 114 CASP10 targets. The performance of contact prediction methods was evaluated with the measures used in previous CASPs (i.e., prediction accuracy and the difference between the distribution of the predicted contacts and that of all pairs of residues in the target protein), as well as new measures, such as the Matthews correlation coefficient, the area under the precision-recall curve and the ranks of the first correctly and incorrectly predicted contact. We also evaluated the ability to detect inter-domain contacts and tested whether the difficulty of predicting contacts depends upon the protein length and the depth of the family sequence alignment. The analyses were carried out on the target domains for which structural homologs did not exist or were difficult to identify. The evaluation was performed for all types of contacts (short, medium, and long-range), with emphasis placed on long-range contacts, i.e. those involving residues separated by at least 24 residues along the sequence. The assessment suggests that the best CASP10 contact prediction methods perform at approximately the same level, and comparably to those participating in CASP9. PMID:23760879

  16. DISSOLUTION OF NEPTUNIUM OXIDE RESIDUES

    SciTech Connect

    Kyser, E

    2009-01-12

    This report describes the development of a dissolution flowsheet for neptunium (Np) oxide (NpO{sub 2}) residues (i.e., various NpO{sub 2} sources, HB-Line glovebox sweepings, and Savannah River National Laboratory (SRNL) thermogravimetric analysis samples). Samples of each type of materials proposed for processing were dissolved in a closed laboratory apparatus and the rate and total quantity of off-gas were measured. Samples of the off-gas were also analyzed. The quantity and type of solids remaining (when visible) were determined after post-dissolution filtration of the solution. Recommended conditions for dissolution of the NpO{sub 2} residues are: Solution Matrix and Loading: {approx}50 g Np/L (750 g Np in 15 L of dissolver solution), using 8 M nitric acid (HNO{sub 3}), 0.025 M potassium fluoride (KF) at greater than 100 C for at least 3 hours. Off-gas: Analysis of the off-gas indicated nitric oxide (NO), nitrogen dioxide (NO{sub 2}) and nitrous oxide (N{sub 2}O) as the only identified components. No hydrogen (H{sub 2}) was detected. The molar ratio of off-gas produced per mole of Np dissolved ranged from 0.25 to 0.4 moles of gas per mole of Np dissolved. A peak off-gas rate of {approx}0.1 scfm/kg bulk oxide was observed. Residual Solids: Pure NpO{sub 2} dissolved with little or no residue with the proposed flowsheet but the NpCo and both sweepings samples left visible solid residue after dissolution. For the NpCo and Part II Sweepings samples the residue amounted to {approx}1% of the initial material, but for the Part I Sweepings sample, the residue amounted to {approx}8 % of the initial material. These residues contained primarily aluminum (Al) and silicon (Si) compounds that did not completely dissolve under the flowsheet conditions. The residues from both sweepings samples contained minor amounts of plutonium (Pu) particles. Overall, the undissolved Np and Pu particles in the residues were a very small fraction of the total solids.

  17. Residual stresses in welded plates

    NASA Technical Reports Server (NTRS)

    Bernstein, Edward L.

    1994-01-01

    The purpose of this project was to develop a simple model which could be used to study residual stress. The mechanism that results in residual stresses in the welding process starts with the deposition of molten weld metal which heats the immediately adjacent material. After solidification of weld material, normal thermal shrinkage is resisted by the adjacent, cooler material. When the thermal strain exceeds the elastic strain corresponding to the yield point stress, the stress level is limited by this value, which decreases with increasing temperature. Cooling then causes elastic unloading which is restrained by the adjoining material. Permanent plastic strain occurs, and tension is caused in the region immediately adjacent to the weld material. Compression arises in the metal farther from the weld in order to maintain overall static equilibrium. Subsequent repair welds may add to the level of residual stresses. The level of residual stress is related to the onset of fracture during welding. Thus, it is of great importance to be able to predict the level of residual stresses remaining after a weld procedure, and to determine the factors, such as weld speed, temperature, direction, and number of passes, which may affect the magnitude of remaining residual stress. It was hoped to use traditional analytical modeling techniques so that it would be easier to comprehend the effect of these variables on the resulting stress. This approach was chosen in place of finite element methods so as to facilitate the understanding of the physical processes. The accuracy of the results was checked with some existing experimental studies giving residual stress levels found from x-ray diffraction measurements.

  18. Hydrothermal carbonization of agricultural residues.

    PubMed

    Oliveira, Ivo; Blöhse, Dennis; Ramke, Hans-Günter

    2013-08-01

    The work presented in this article addresses the application of hydrothermal carbonization (HTC) to produce a solid fuel named HTC-Biochar, whose characteristics are comparable to brown coal. Several batch HTC experiments were performed using agricultural residues (AR) as substrates, commonly treated in farm-based biogas plants in Germany. Different AR were used in different combinations with other biomass residues. The biogas potential from the resulting process water was also determined. The combination of different AR lead to the production of different qualities of HTC-Biochars as well as different mass and energy yields. Using more lignocellulosic residues lead to higher mass and energy yields for the HTC-Biochar produced. Whilst residues rich in carbohydrates of lower molecular weight such as corn silage and dough residues lead to the production of a HTC-Biochar of better quality and more similar to brown coal. Process water achieved a maximum of 16.3 L CH4/kg FM (fresh matter).

  19. Dry fermentation of agricultural residues

    NASA Astrophysics Data System (ADS)

    Jewell, W. J.; Chandler, J. A.; Dellorto, S.; Fanfoni, K. J.; Fast, S.; Jackson, D.; Kabrick, R. M.

    1981-09-01

    A dry fermentation process is discussed which converts agricultural residues to methane, using the residues in their as produced state. The process appears to simplify and enhance the possibilities for using crop residues as an energy source. The major process variables investigated include temperature, the amount and type of inoculum, buffer requirements, compaction, and pretreatment to control the initial available organic components that create pH problems. A pilot-scale reactor operation on corn stover at a temperature of 550 C, with 25 percent initial total solids, a seed-to-feed ratio of 2.5 percent, and a buffer-to-feed ratio of 8 percent achieved 33 percent total volatile solids destruction in 60 days. Volumetric biogas yields from this unit were greater than 1 vol/vol day for 12 days, and greater than 0.5 vol/vol day for 32 days, at a substrate density of 169 kg/m (3).

  20. RESIDUAL RISK ASSESSMENT: SYNTHETIC ORGANIC ...

    EPA Pesticide Factsheets

    This document describes the residual risk assessment for the Synthetic Organic Chemical Manufacturing Industry source category. For stationary sources, section 112 (f) of the Clean Air Act requires EPA to assess risks to human health and the environment following implementation of technology-based control standards. If these technology-based control standards do not provide an ample margin of safety, then EPA is required to promulgate addtional standards. This document describes the methodology and results of the residual risk assessment performed for the Synthetic Organic Chemical Manufacturing Industry source category. The results of this analyiss will assist EPA in determining whether a residual risk rule for this source category is appropriate. Update 9/19/2006: Proposed Rule June 14, 2006 - Risk Assessment complete September 2005 and only available in the Docket.

  1. Chemistry of combined residual chlorination

    SciTech Connect

    Leao, S.F.; Selleck, R.E.

    1982-01-01

    The decay of the combined chlorine residual was investigated in this work. Recent concerns about the formation of undesirable compounds such as chloroform with free residual chlorination have focused attention on the alternative use of combined residual chlorination. This work investigates the applicability of reactions proposed to describe the transformations and decay of the combined residual with time. Sodium hypochlorite was added to buffered solutions of ammonia with the chlorine residual being monitored over periods extending up to 10 days. The reaction was studied at four initial concentrations of hypochlorite of 100, 50, 25 and 10 mg/L as Cl/sub 2/ with molar application ratios of chlorine to ammonia, defined herein as M ratios, of 0.90, 0.50, 0.25 and 0.05 at each hypochlorite dose. Sixty-eight experiments were conducted at the pH of 6.6 and 7.2. The conclusions are: (1) in the absence of free chlorine, the concentration of NH/sub 3/ does not seem to affect the rate of disappearance of the residual other than through the formation of NHCl/sub 2/ by NH/sub 2/Cl hydrolysis; (2) the reaction between NHCl/sub 2/ and NH/sub 4//sup +/ to form NH/sub 2/Cl is either much slower than reported by Gray et. al. or the mechanism is different with a rate limiting step not involving NH/sub 3/ or NH/sub 4//sup +/; (3) a redox reaction in addition to the first-order decomposition of NHCl/sub 2/ appears necessary. Model simulation results indicated that a reaction of the type NH/sub 2/Cl + NHCl/sub 2/ ..-->.. P added to the first-order NHCl/sub 2/ decomposition can explain the results observed except at the higher chlorine doses.

  2. Residual contact restraints in cryogenics

    NASA Astrophysics Data System (ADS)

    Cretegny, J. F.; Demonicault, J. M.

    The use of residual stress measurements to evaluate the state of cryogenic turbomachines, whose surfaces are worn by the working conductions in dry contact, is addressed. Their contribution to the understanding of the reasons of possible ruptures is considered. It is stated that residual stress measurements should be used as a complementary tool rather than as input data for models. It is shown, thanks to two examples concerning the ball bearings and splines of the liquid hydrogen turbopump of the Vulcain engine, what can be expected from such techniques. Total exploitation of the results has still to be done, but preliminary results are quite encouraging.

  3. Pesticide residues in olive oil.

    PubMed

    Lentza-Rizos, C; Avramides, E J

    1995-01-01

    The attacks of pests and diseases and the presence of weeds make it necessary to apply pesticides to olive trees to ensure crop protection. Residues of these compounds may remain and contaminate the oil produced. For the analysis of pesticide residues in olive oil, the most common methods are multiresidue methods for fatty substrates, based on partitioning between hexane or light petroleum and acetonitrile. Recently, other methods have been applied, such as ready-to-use, disposable minicolumns or direct injection of oil into a capillary gas chromatograph equipped with a precolumn with an oil recovery tank. Although several pesticides are registered in oil-producing countries for use on olive trees, available literature on the level and fate of residues is very limited. However, it is clear that fat-soluble pesticides tend to concentrate in the oil, both after full coverage and bait spraying, and their use close to harvest should therefore be avoided. Because it is sometimes necessary to use such pesticides late in autumn because of their effectiveness in cases of severe attack, residue trials should be carried out to determine the residue concentration in oil and to set a reasonable preharvest safety interval. Data produced by such trials would permit the establishment of MRLs (tolerances) in olive oil to cover cases where the residues, although relatively high, are not of toxicological significance for consumers (risk assessment). Such is the case with corn oil and the fat-soluble insecticide methyl pirimiphos, registered in the U.S. for use on corn. The U.S. EPA tolerance for methyl pirimiphos in corn is 8 mg/kg, whereas it is 11 times higher (88 mg/kg) for corn oil because it is known to concentrate in the oil. Similar provisions for olive oil, based on data from residue trials according to Good Agricultural Practice, the long-term toxicity of each pesticide as expressed by its ADI for man, and olive oil consumption patterns, would facilitate international trade

  4. Collection of sugarcane crop residue for energy

    SciTech Connect

    Eiland, B.R.; Clayton, J.E.

    1982-12-01

    Crop residue left after sugarcane harvesting was recovered using a forage harvester and a large round baler. The quantity, bulk density and moisture content of the crop residue was determined in four fields. Crop residue from 7 ha was burned in boilers at a sugar mill. Samples of this residue were tested by a laboratory and compared to sugarcane bagasse.

  5. Residual Structures in Latent Growth Curve Modeling

    ERIC Educational Resources Information Center

    Grimm, Kevin J.; Widaman, Keith F.

    2010-01-01

    Several alternatives are available for specifying the residual structure in latent growth curve modeling. Two specifications involve uncorrelated residuals and represent the most commonly used residual structures. The first, building on repeated measures analysis of variance and common specifications in multilevel models, forces residual variances…

  6. Choosing forest residues management alternatives.

    Treesearch

    John M. Pierovich; Richard C. Smith

    1973-01-01

    Forest residues management involves disposal, modification, or utilization of wood products. The costs and benefits of the several alternatives available to forest managers must be evaluated in relation to land management goals and constraints in four areas: (1) unused wood fiber, (2) conflagrations, (3) impairment of forest resources, and (4) opposition to treatment...

  7. Residual Stresses in Ground Steels.

    DTIC Science & Technology

    1979-06-13

    stress near the surface can be lower. The level of residual stress is also strongly affected by carbon,’3 which influences the microplastic behaviour of...1966, Vol. 14, 99-104. 14. C. 3. )4cMahon: “ Microplastic Behaviour in Iron” in Mv. in Mater . S d . Res., Vol. 2, 121-140, Interscience, New York

  8. Managing woodwaste: Yield from residue

    SciTech Connect

    Nielson, E.; Rayner, S.

    1993-12-31

    Historically, the majority of sawmill waste has been burned or buried for the sole purpose of disposal. In most jurisdictions, environmental legislation will prohibit, or render uneconomic, these practices. Many reports have been prepared to describe the forest industry`s residue and its environmental effect; although these help those looking for industry-wide or regional solutions, such as electricity generation, they have limited value for the mill manager, who has the on-hands responsibility for generation and disposal of the waste. If the mill manager can evaluate waste streams and break them down into their usable components, he can find niche market solutions for portions of the plant residue and redirect waste to poor/no-return, rather than disposal-cost, end uses. In the modern mill, residue is collected at the individual machine centre by waste conveyors that combine and mix sawdust, shavings, bark, etc. and send the result to the hog-fuel pile. The mill waste system should be analyzed to determine the measures that can improve the quality of residues and determine the volumes of any particular category before the mixing, mentioned above, occurs. After this analysis, the mill may find a niche market for a portion of its woodwaste.

  9. Potential hazards of fumigant residues.

    PubMed Central

    Fishbein, L

    1976-01-01

    A spectrum of fumigants (primarily ethylene dibromide, 1,2-dibromo-3-chloropropane, ethylene oxide, symdibromotetetrachloroethane, 1,3-dichloropropene, dichlorovos, carbon tetrachloride, methyl bromide) as well as their degradation products in foodstuffs and soil have been examined mainly in regard to the potential mutagenicity of their residues. PMID:789068

  10. Catalytic combustion of residual fuels

    NASA Technical Reports Server (NTRS)

    Bulzan, D. L.; Tacina, R. R.

    1981-01-01

    A noble metal catalytic reactor was tested using two grades of petroleum derived residual fuels at specified inlet air temperatures, pressures, and reference velocities. Combustion efficiencies greater than 99.5 percent were obtained. Steady state operation of the catalytic reactor required inlet air temperatures of at least 800 K. At lower inlet air temperatures, upstream burning in the premixing zone occurred which was probably caused by fuel deposition and accumulation on the premixing zone walls. Increasing the inlet air temperature prevented this occurrence. Both residual fuels contained about 0.5 percent nitrogen by weight. NO sub x emissions ranged from 50 to 110 ppm by volume at 15 percent excess O2. Conversion of fuel-bound nitrogen to NO sub x ranged from 25 to 50 percent.

  11. Limits of adaptation, residual interferences

    NASA Technical Reports Server (NTRS)

    Mokry, Miroslav (Editor); Erickson, J. C., Jr.; Goodyer, Michael J.; Mignosi, Andre; Russo, Giuseppe P.; Smith, J.; Wedemeyer, Erich H.; Newman, Perry A.

    1990-01-01

    Methods of determining linear residual wall interference appear to be well established theoretically; however they need to be validated, for example by comparative studies of test data on the same model in different adaptive-wall wind tunnels as well as in passive, ventilated-wall tunnels. The GARTEur CAST 7 and the CAST 10/DOA 2 investigations are excellent examples of such comparative studies. Results to date in both one-variable and two-variable methods for nonlinear wall interference indicate that a great deal more research and validation are required. The status in 2D flow is advanced over that in 3D flow as is the case generally with adaptive-wall development. Nevertheless, it is now well established that for transonic testing with extensive supercritical flow present, significant wall interference is likely to exist in conventional ventilated test sections. Consequently, residual correction procedures require further development hand-in-hand with further adaptive-wall development.

  12. 40 CFR 180.185 - DCPA; tolerances for residues.

    Code of Federal Regulations, 2011 CFR

    2011-07-01

    ...; tolerances for residues. (a) General. Tolerances for the combined residues of the herbicide dimethyl... residues of the herbicide dimethyl tetrachloroterephthalate (DCPA) and its metabolites monomethyl... residues. Tolerances are established for the combined indirect or inadvertent residues of the...

  13. 40 CFR 180.185 - DCPA; tolerances for residues.

    Code of Federal Regulations, 2014 CFR

    2014-07-01

    ...; tolerances for residues. (a) General. Tolerances for the combined residues of the herbicide dimethyl... residues of the herbicide dimethyl tetrachloroterephthalate (DCPA) and its metabolites monomethyl... residues. Tolerances are established for the combined indirect or inadvertent residues of the...

  14. 40 CFR 180.185 - DCPA; tolerances for residues.

    Code of Federal Regulations, 2013 CFR

    2013-07-01

    ...; tolerances for residues. (a) General. Tolerances for the combined residues of the herbicide dimethyl... residues of the herbicide dimethyl tetrachloroterephthalate (DCPA) and its metabolites monomethyl... residues. Tolerances are established for the combined indirect or inadvertent residues of the...

  15. 40 CFR 180.185 - DCPA; tolerances for residues.

    Code of Federal Regulations, 2010 CFR

    2010-07-01

    ...; tolerances for residues. (a) General. Tolerances for the combined residues of the herbicide dimethyl... residues of the herbicide dimethyl tetrachloroterephthalate (DCPA) and its metabolites monomethyl... residues. Tolerances are established for the combined indirect or inadvertent residues of the...

  16. 40 CFR 180.185 - DCPA; tolerances for residues.

    Code of Federal Regulations, 2012 CFR

    2012-07-01

    ...; tolerances for residues. (a) General. Tolerances for the combined residues of the herbicide dimethyl... residues of the herbicide dimethyl tetrachloroterephthalate (DCPA) and its metabolites monomethyl... residues. Tolerances are established for the combined indirect or inadvertent residues of the...

  17. Calcination/dissolution residue treatment

    SciTech Connect

    Knight, R.C.; Creed, R.F.; Patello, G.K.; Hollenberg, G.W.; Buehler, M.F.; O`Rourke, S.M.; Visnapuu, A.; McLaughlin, D.F.

    1994-09-01

    Currently, high-level wastes are stored underground in steel-lined tanks at the Hanford site. Current plans call for the chemical pretreatment of these wastes before their immobilization in stable glass waste forms. One candidate pretreatment approach, calcination/dissolution, performs an alkaline fusion of the waste and creates a high-level/low-level partition based on the aqueous solubilities of the components of the product calcine. Literature and laboratory studies were conducted with the goal of finding a residue treatment technology that would decrease the quantity of high-level waste glass required following calcination/dissolution waste processing. Four elements, Fe, Ni, Bi, and U, postulated to be present in the high-level residue fraction were identified as being key to the quantity of high-level glass formed. Laboratory tests of the candidate technologies with simulant high-level residues showed reductive roasting followed by carbonyl volatilization to be successful in removing Fe, Ni, and Bi. Subsequent bench-scale tests on residues from calcination/dissolution processing of genuine Hanford Site tank waste showed Fe was separated with radioelement decontamination factors of 70 to 1,000 times with respect to total alpha activity. Thermodynamic analyses of the calcination of five typical Hanford Site tank waste compositions also were performed. The analyses showed sodium hydroxide to be the sole molten component in the waste calcine and emphasized the requirement for waste blending if fluid calcines are to be achieved. Other calcine phases identified in the thermodynamic analysis indicate the significant thermal reconstitution accomplished in calcination.

  18. Electromechanical Apparatus Measures Residual Stress

    NASA Technical Reports Server (NTRS)

    Chern, Engmin J.; Flom, Yury

    1993-01-01

    Nondestructive test exploits relationship between stress and eddy-current-probe resistance. Yields data on residual stress or strain in metal tension/compression specimen (stress or strain remaining in specimen when no stress applied from without). Apparatus is assembly of commercial equipment: tension-or-compression testing machine, eddy-current probe, impedance gain-and-phase analyzer measuring impedance of probe coil, and desktop computer, which controls other equipment and processes data received from impedance gain-and-phase analyzer.

  19. Pesticidal residues in animal tissues

    USGS Publications Warehouse

    DeWitt, J.B.; Menzie, C.M.; Adomaitis, V.A.; Reichel, W.L.

    1960-01-01

    Tests with penned starlings, rats, pheasants, and ducks indicated that each species differs in sensitivity to the various pesticides. Residues in tissues are proportional to the degree of exposure during area treatment and they are also found in animals shot six or more months after treatment. The presence of more than 20-30 ppm of DDT, 20 ppm of chlordan, and 6-20 ppm of heptachlor epoxide in quail tissues indicated that the birds had ingested lethal dosages of the pesticides.

  20. Electromechanical Apparatus Measures Residual Stress

    NASA Technical Reports Server (NTRS)

    Chern, Engmin J.; Flom, Yury

    1993-01-01

    Nondestructive test exploits relationship between stress and eddy-current-probe resistance. Yields data on residual stress or strain in metal tension/compression specimen (stress or strain remaining in specimen when no stress applied from without). Apparatus is assembly of commercial equipment: tension-or-compression testing machine, eddy-current probe, impedance gain-and-phase analyzer measuring impedance of probe coil, and desktop computer, which controls other equipment and processes data received from impedance gain-and-phase analyzer.

  1. Characterization of Human and Murine T-Cell Immunoglobulin Mucin Domain 4 (TIM-4) IgV Domain Residues Critical for Ebola Virus Entry

    PubMed Central

    Rhein, Bethany A.; Brouillette, Rachel B.; Schaack, Grace A.; Chiorini, John A.

    2016-01-01

    ABSTRACT Phosphatidylserine (PtdSer) receptors that are responsible for the clearance of dying cells have recently been found to mediate enveloped virus entry. Ebola virus (EBOV), a member of the Filoviridae family of viruses, utilizes PtdSer receptors for entry into target cells. The PtdSer receptors human and murine T-cell immunoglobulin mucin (TIM) domain proteins TIM-1 and TIM-4 mediate filovirus entry by binding to PtdSer on the virion surface via a conserved PtdSer binding pocket within the amino-terminal IgV domain. While the residues within the TIM-1 IgV domain that are important for EBOV entry are characterized, the molecular details of virion–TIM-4 interactions have yet to be investigated. As sequences and structural alignments of the TIM proteins suggest distinct differences in the TIM-1 and TIM-4 IgV domain structures, we sought to characterize TIM-4 IgV domain residues required for EBOV entry. Using vesicular stomatitis virus pseudovirions bearing EBOV glycoprotein (EBOV GP/VSVΔG), we evaluated virus binding and entry into cells expressing TIM-4 molecules mutated within the IgV domain, allowing us to identify residues important for entry. Similar to TIM-1, residues in the PtdSer binding pocket of murine and human TIM-4 (mTIM-4 and hTIM-4) were found to be important for EBOV entry. However, additional TIM-4-specific residues were also found to impact EBOV entry, with a total of 8 mTIM-4 and 14 hTIM-4 IgV domain residues being critical for virion binding and internalization. Together, these findings provide a greater understanding of the interaction of TIM-4 with EBOV virions. IMPORTANCE With more than 28,000 cases and over 11,000 deaths during the largest and most recent Ebola virus (EBOV) outbreak, there has been increased emphasis on the development of therapeutics against filoviruses. Many therapies under investigation target EBOV cell entry. T-cell immunoglobulin mucin (TIM) domain proteins are cell surface factors important for the entry of many

  2. Characterization of Human and Murine T-Cell Immunoglobulin Mucin Domain 4 (TIM-4) IgV Domain Residues Critical for Ebola Virus Entry.

    PubMed

    Rhein, Bethany A; Brouillette, Rachel B; Schaack, Grace A; Chiorini, John A; Maury, Wendy

    2016-07-01

    Phosphatidylserine (PtdSer) receptors that are responsible for the clearance of dying cells have recently been found to mediate enveloped virus entry. Ebola virus (EBOV), a member of the Filoviridae family of viruses, utilizes PtdSer receptors for entry into target cells. The PtdSer receptors human and murine T-cell immunoglobulin mucin (TIM) domain proteins TIM-1 and TIM-4 mediate filovirus entry by binding to PtdSer on the virion surface via a conserved PtdSer binding pocket within the amino-terminal IgV domain. While the residues within the TIM-1 IgV domain that are important for EBOV entry are characterized, the molecular details of virion-TIM-4 interactions have yet to be investigated. As sequences and structural alignments of the TIM proteins suggest distinct differences in the TIM-1 and TIM-4 IgV domain structures, we sought to characterize TIM-4 IgV domain residues required for EBOV entry. Using vesicular stomatitis virus pseudovirions bearing EBOV glycoprotein (EBOV GP/VSVΔG), we evaluated virus binding and entry into cells expressing TIM-4 molecules mutated within the IgV domain, allowing us to identify residues important for entry. Similar to TIM-1, residues in the PtdSer binding pocket of murine and human TIM-4 (mTIM-4 and hTIM-4) were found to be important for EBOV entry. However, additional TIM-4-specific residues were also found to impact EBOV entry, with a total of 8 mTIM-4 and 14 hTIM-4 IgV domain residues being critical for virion binding and internalization. Together, these findings provide a greater understanding of the interaction of TIM-4 with EBOV virions. With more than 28,000 cases and over 11,000 deaths during the largest and most recent Ebola virus (EBOV) outbreak, there has been increased emphasis on the development of therapeutics against filoviruses. Many therapies under investigation target EBOV cell entry. T-cell immunoglobulin mucin (TIM) domain proteins are cell surface factors important for the entry of many enveloped viruses

  3. Geotechnical characteristics of residual soils

    SciTech Connect

    Townsend, F.C.

    1985-01-01

    Residual soils are products of chemical weathering and thus their characteristics are dependent upon environmental factors of climate, parent material, topography and drainage, and age. These conditions are optimized in the tropics where well-drained regions produce reddish lateritic soils rich in iron and aluminum sesquioxides and kaolinitic clays. Conversely, poorly drained areas tend towards montmorillonitic expansive black clays. Andosols develop over volcanic ash and rock regions and are rich in allophane (amorphous silica) and metastable halloysite. The geological origins greatly affect the resulting engineering characteristics. Both lateritic soils and andosols are susceptible to property changes upon drying, and exhibit compaction and strength properties not indicative of their classification limits. Both soils have been used successfully in earth dam construction, but attention must be given to seepage control through the weathered rock. Conversely, black soils are unpopular for embankments. Lateritic soils respond to cement stabilization and, in some cases, lime stabilization. Andosols should also respond to lime treatment and cement treatments if proper mixing can be achieved. Black expansive residual soils respond to lime treatment by demonstrating strength gains and decreased expansiveness. Rainfall induced landslides are typical of residual soil deposits.

  4. Actinide recovery from pyrochemical residues

    SciTech Connect

    Avens, L.R.; Clifton, D.G.; Vigil, A.R.

    1985-05-01

    We demonstrated a new process for recovering plutonium and americium from pyrochemical waste. The method is based on chloride solution anion exchange at low acidity, or acidity that eliminates corrosive HCl fumes. Developmental experiments of the process flow chart concentrated on molten salt extraction (MSE) residues and gave >95% plutonium and >90% americium recovery. The recovered plutonium contained <500 ppM americium and <2500 ppM magnesium. The process operates by sorbing PuCl/sub 6//sup 2 -/ from high-chloride low-acid solution. Americium and other metals are washed from the ion exchange column with lN HNO/sub 3/-4.8M NaCl. After elution, plutonium is recovered by hydroxide precipitation, and americium is recovered by NaHCO/sub 3/ precipitation. All filtrates from the process can be discardable as low-level contaminated waste. Production-scale experiments are in progress for MSE residues. Flow charts for actinide recovery from electro-refining and direct oxide reduction residues are presented and discussed.

  5. Evaluation of residue drum storage safety risks

    SciTech Connect

    Conner, W.V.

    1994-06-17

    A study was conducted to determine if any potential safety problems exist in the residue drum backlog at the Rocky Flats Plant. Plutonium residues stored in 55-gallon drums were packaged for short-term storage until the residues could be processed for plutonium recovery. These residues have now been determined by the Department of Energy to be waste materials, and the residues will remain in storage until plans for disposal of the material can be developed. The packaging configurations which were safe for short-term storage may not be safe for long-term storage. Interviews with Rocky Flats personnel involved with packaging the residues reveal that more than one packaging configuration was used for some of the residues. A tabulation of packaging configurations was developed based on the information obtained from the interviews. A number of potential safety problems were identified during this study, including hydrogen generation from some residues and residue packaging materials, contamination containment loss, metal residue packaging container corrosion, and pyrophoric plutonium compound formation. Risk factors were developed for evaluating the risk potential of the various residue categories, and the residues in storage at Rocky Flats were ranked by risk potential. Preliminary drum head space gas sampling studies have demonstrated the potential for formation of flammable hydrogen-oxygen mixtures in some residue drums.

  6. PESTICIDE RESIDUE RECOVERIES FROM SURFACE WIPES

    EPA Science Inventory

    Human exposure is a consequence of pesticide use indoors with a primary source resulting from residue deposition on household surfaces. Accurate measurements of surface residues is essential for estimating exposure from different routes. Various procedures have been developed ...

  7. 48 CFR 970.5001 - Residual powers.

    Code of Federal Regulations, 2010 CFR

    2010-10-01

    ... 48 Federal Acquisition Regulations System 5 2010-10-01 2010-10-01 false Residual powers. 970.5001 Section 970.5001 Federal Acquisition Regulations System DEPARTMENT OF ENERGY AGENCY SUPPLEMENTARY....5001 Residual powers....

  8. 48 CFR 250.104 - Residual powers.

    Code of Federal Regulations, 2010 CFR

    2010-10-01

    ... 48 Federal Acquisition Regulations System 3 2010-10-01 2010-10-01 false Residual powers. 250.104 Section 250.104 Federal Acquisition Regulations System DEFENSE ACQUISITION REGULATIONS SYSTEM, DEPARTMENT... Contractual Actions 250.104 Residual powers....

  9. 48 CFR 250.104 - Residual powers.

    Code of Federal Regulations, 2011 CFR

    2011-10-01

    ... 48 Federal Acquisition Regulations System 3 2011-10-01 2011-10-01 false Residual powers. 250.104 Section 250.104 Federal Acquisition Regulations System DEFENSE ACQUISITION REGULATIONS SYSTEM, DEPARTMENT... Contractual Actions 250.104 Residual powers....

  10. 48 CFR 970.5001 - Residual powers.

    Code of Federal Regulations, 2014 CFR

    2014-10-01

    ... 48 Federal Acquisition Regulations System 5 2014-10-01 2014-10-01 false Residual powers. 970.5001 Section 970.5001 Federal Acquisition Regulations System DEPARTMENT OF ENERGY AGENCY SUPPLEMENTARY....5001 Residual powers....

  11. 48 CFR 1850.104 - Residual powers.

    Code of Federal Regulations, 2012 CFR

    2012-10-01

    ... 48 Federal Acquisition Regulations System 6 2012-10-01 2012-10-01 false Residual powers. 1850.104 Section 1850.104 Federal Acquisition Regulations System NATIONAL AERONAUTICS AND SPACE ADMINISTRATION... 1850.104 Residual powers....

  12. 48 CFR 250.104 - Residual powers.

    Code of Federal Regulations, 2014 CFR

    2014-10-01

    ... 48 Federal Acquisition Regulations System 3 2014-10-01 2014-10-01 false Residual powers. 250.104 Section 250.104 Federal Acquisition Regulations System DEFENSE ACQUISITION REGULATIONS SYSTEM, DEPARTMENT... Contractual Actions 250.104 Residual powers....

  13. 48 CFR 250.104 - Residual powers.

    Code of Federal Regulations, 2013 CFR

    2013-10-01

    ... 48 Federal Acquisition Regulations System 3 2013-10-01 2013-10-01 false Residual powers. 250.104 Section 250.104 Federal Acquisition Regulations System DEFENSE ACQUISITION REGULATIONS SYSTEM, DEPARTMENT... Contractual Actions 250.104 Residual powers....

  14. 48 CFR 1850.104 - Residual powers.

    Code of Federal Regulations, 2013 CFR

    2013-10-01

    ... 48 Federal Acquisition Regulations System 6 2013-10-01 2013-10-01 false Residual powers. 1850.104 Section 1850.104 Federal Acquisition Regulations System NATIONAL AERONAUTICS AND SPACE ADMINISTRATION... 1850.104 Residual powers....

  15. 48 CFR 1850.104 - Residual powers.

    Code of Federal Regulations, 2014 CFR

    2014-10-01

    ... 48 Federal Acquisition Regulations System 6 2014-10-01 2014-10-01 false Residual powers. 1850.104 Section 1850.104 Federal Acquisition Regulations System NATIONAL AERONAUTICS AND SPACE ADMINISTRATION... 1850.104 Residual powers....

  16. 48 CFR 970.5001 - Residual powers.

    Code of Federal Regulations, 2012 CFR

    2012-10-01

    ... 48 Federal Acquisition Regulations System 5 2012-10-01 2012-10-01 false Residual powers. 970.5001 Section 970.5001 Federal Acquisition Regulations System DEPARTMENT OF ENERGY AGENCY SUPPLEMENTARY....5001 Residual powers....

  17. 48 CFR 970.5001 - Residual powers.

    Code of Federal Regulations, 2011 CFR

    2011-10-01

    ... 48 Federal Acquisition Regulations System 5 2011-10-01 2011-10-01 false Residual powers. 970.5001 Section 970.5001 Federal Acquisition Regulations System DEPARTMENT OF ENERGY AGENCY SUPPLEMENTARY....5001 Residual powers....

  18. 48 CFR 970.5001 - Residual powers.

    Code of Federal Regulations, 2013 CFR

    2013-10-01

    ... 48 Federal Acquisition Regulations System 5 2013-10-01 2013-10-01 false Residual powers. 970.5001 Section 970.5001 Federal Acquisition Regulations System DEPARTMENT OF ENERGY AGENCY SUPPLEMENTARY....5001 Residual powers....

  19. 48 CFR 250.104 - Residual powers.

    Code of Federal Regulations, 2012 CFR

    2012-10-01

    ... 48 Federal Acquisition Regulations System 3 2012-10-01 2012-10-01 false Residual powers. 250.104 Section 250.104 Federal Acquisition Regulations System DEFENSE ACQUISITION REGULATIONS SYSTEM, DEPARTMENT... Contractual Actions 250.104 Residual powers....

  20. Interpretation on Recycling Plastics from Shredder Residue

    EPA Pesticide Factsheets

    EPA is considering an interpretation of its regulations that would generally allow for recycling of plastic separated from shredder residue under the conditions described in the Voluntary Procedures for Recycling Plastics from Shredder Residue.

  1. PESTICIDE RESIDUE RECOVERIES FROM SURFACE WIPES

    EPA Science Inventory

    Human exposure is a consequence of pesticide use indoors with a primary source resulting from residue deposition on household surfaces. Accurate measurements of surface residues is essential for estimating exposure from different routes. Various procedures have been developed ...

  2. 9 CFR 311.39 - Biological residues.

    Code of Federal Regulations, 2011 CFR

    2011-01-01

    ... 9 Animals and Animal Products 2 2011-01-01 2011-01-01 false Biological residues. 311.39 Section... Biological residues. Carcasses, organs, or other parts of carcasses of livestock shall be condemned if it is determined that they are adulterated because of the presence of any biological residues. ...

  3. 9 CFR 311.39 - Biological residues.

    Code of Federal Regulations, 2010 CFR

    2010-01-01

    ... 9 Animals and Animal Products 2 2010-01-01 2010-01-01 false Biological residues. 311.39 Section... Biological residues. Carcasses, organs, or other parts of carcasses of livestock shall be condemned if it is determined that they are adulterated because of the presence of any biological residues. ...

  4. Polychlorinated Biphenyls (PCB) Residue Effects Database

    EPA Pesticide Factsheets

    The PCB Residue Effects (PCBRes) Database was developed to assist scientists and risk assessors in correlating PCB and dioxin-like compound residues with toxic effects. The purpose is to develop PCB critical residue values for fish, mammals and birds, especially as these relate to aquatic and aquatic-dependent species.

  5. 48 CFR 50.104 - Residual powers.

    Code of Federal Regulations, 2010 CFR

    2010-10-01

    ... 48 Federal Acquisition Regulations System 1 2010-10-01 2010-10-01 false Residual powers. 50.104... EXTRAORDINARY CONTRACTUAL ACTIONS AND THE SAFETY ACT Extraordinary Contractual Actions 50.104 Residual powers. This section prescribes standards and procedures for exercising residual powers under Pub. L....

  6. 48 CFR 50.104 - Residual powers.

    Code of Federal Regulations, 2011 CFR

    2011-10-01

    ... 48 Federal Acquisition Regulations System 1 2011-10-01 2011-10-01 false Residual powers. 50.104... EXTRAORDINARY CONTRACTUAL ACTIONS AND THE SAFETY ACT Extraordinary Contractual Actions 50.104 Residual powers. This section prescribes standards and procedures for exercising residual powers under Pub. L....

  7. 48 CFR 50.104 - Residual powers.

    Code of Federal Regulations, 2014 CFR

    2014-10-01

    ... 48 Federal Acquisition Regulations System 1 2014-10-01 2014-10-01 false Residual powers. 50.104... EXTRAORDINARY CONTRACTUAL ACTIONS AND THE SAFETY ACT Extraordinary Contractual Actions 50.104 Residual powers. This section prescribes standards and procedures for exercising residual powers under Pub. L....

  8. 48 CFR 50.104 - Residual powers.

    Code of Federal Regulations, 2012 CFR

    2012-10-01

    ... 48 Federal Acquisition Regulations System 1 2012-10-01 2012-10-01 false Residual powers. 50.104... EXTRAORDINARY CONTRACTUAL ACTIONS AND THE SAFETY ACT Extraordinary Contractual Actions 50.104 Residual powers. This section prescribes standards and procedures for exercising residual powers under Pub. L....

  9. 48 CFR 50.104 - Residual powers.

    Code of Federal Regulations, 2013 CFR

    2013-10-01

    ... 48 Federal Acquisition Regulations System 1 2013-10-01 2013-10-01 false Residual powers. 50.104... EXTRAORDINARY CONTRACTUAL ACTIONS AND THE SAFETY ACT Extraordinary Contractual Actions 50.104 Residual powers. This section prescribes standards and procedures for exercising residual powers under Pub. L....

  10. Process to recycle shredder residue

    DOEpatents

    Jody, Bassam J.; Daniels, Edward J.; Bonsignore, Patrick V.

    2001-01-01

    A system and process for recycling shredder residue, in which separating any polyurethane foam materials are first separated. Then separate a fines fraction of less than about 1/4 inch leaving a plastics-rich fraction. Thereafter, the plastics rich fraction is sequentially contacted with a series of solvents beginning with one or more of hexane or an alcohol to remove automotive fluids; acetone to remove ABS; one or more of EDC, THF or a ketone having a boiling point of not greater than about 125.degree. C. to remove PVC; and one or more of xylene or toluene to remove polypropylene and polyethylene. The solvents are recovered and recycled.

  11. Managing residual limb hyperhidrosis in wounded warriors.

    PubMed

    Pace, Sarah; Kentosh, Joshua

    2016-06-01

    Residual limb dermatologic problems are a common concern among young active traumatic amputee patients who strive to maintain an active lifestyle. Hyperhidrosis of residual limbs is a recognized inciting factor that often contributes to residual limb dermatoses and is driven by the design of the prosthetic liner covering the residual limb. Treatment of hyperhidrosis in this population presents a unique challenge. Several accepted treatments of hyperhidrosis can offer some relief but have been limited by lack of results or side-effect profiles. Microwave thermal ablation has presented an enticing potential for residual limb hyperhidrosis.

  12. South Polar Residual Ice Cap

    NASA Technical Reports Server (NTRS)

    1990-01-01

    This mosaic is composed of 18 Viking Orbiter images (6 each in red, green, and violet filters), acquired on September 28, 1977, during revolution 407 of Viking Orbiter 2. The south pole is located just off the lower left edge of the polar cap, and the 0 degree longitude meridian extends toward the top of the mosaic. The large crater near the right edge (named 'South') is about 100 km in diameter. These images were acquired during southern summer on Mars (Ls = 341 degrees); the sub-solar declination was 8 degrees S., and the south polar cap was nearing its final stage of retreat just prior to vernal equinox. The south residual cap is approximately 400 km across, and the exposed surface is thought to consist dominantly of carbon-dioxide frost. This is in contrast to the water-ice surface of the north polar residual cap. It is likely that water ice is present in layers that underlie the south polar cap and that comprise the surrounding layered terrains. Near the top of this image, irregular pits with sharp-rimmed cliffs appear 'etched', presumably by wind. A series of rugged mountains (extending toward the upper right corner of the image) are of unknown origin.

  13. Intercompatibility of residual fuel blends

    SciTech Connect

    Ben-Asher, J.; Krenis, G.; Luria, D.

    1995-04-01

    It is a well known fact that two fuel oils, thermally stable by themselves, may produce sludge of asphaltenic nature when blended together. Settling out of asphaltenes from the fuel medium will result in strainer and burner plugging, causing serious operational difficulties in industrial and marine fuel systems. It was the aim of the present study to establish criteria for the phenomenon of incompatibility. The parameters which influence thermal stability of the blend were assessed, and an attempt was made to predict possible separation of asphaltenes from fuel oil mixtures. Fuel oils originating from Brazil, France, Hong-Kong, Greece, U.S.A., Japan as well as locally (Israel) produced residual fuels were mixed. Thermal stability of the blend was determined by ASTM D-4740 method. In some cases, total sediment was measured by ASTM D-4870. Blends of fuels were stored at 50{degrees}C to assess the effect of elevated temperatures on thermal stability. It was found that most of the blends were compatible though composed of fuels originating from different refineries. Density, Pour Point, Sulfur content and Xylene Number served as useful tools for predicting the thermal stability of residual fuel blends.

  14. RESIDUAL STRESSES IN 3013 CONTAINERS

    SciTech Connect

    Mickalonis, J.; Dunn, K.

    2009-11-10

    The DOE Complex is packaging plutonium-bearing materials for storage and eventual disposition or disposal. The materials are handled according to the DOE-STD-3013 which outlines general requirements for stabilization, packaging and long-term storage. The storage vessels for the plutonium-bearing materials are termed 3013 containers. Stress corrosion cracking has been identified as a potential container degradation mode and this work determined that the residual stresses in the containers are sufficient to support such cracking. Sections of the 3013 outer, inner, and convenience containers, in both the as-fabricated condition and the closure welded condition, were evaluated per ASTM standard G-36. The standard requires exposure to a boiling magnesium chloride solution, which is an aggressive testing solution. Tests in a less aggressive 40% calcium chloride solution were also conducted. These tests were used to reveal the relative stress corrosion cracking susceptibility of the as fabricated 3013 containers. Significant cracking was observed in all containers in areas near welds and transitions in the container diameter. Stress corrosion cracks developed in both the lid and the body of gas tungsten arc welded and laser closure welded containers. The development of stress corrosion cracks in the as-fabricated and in the closure welded container samples demonstrates that the residual stresses in the 3013 containers are sufficient to support stress corrosion cracking if the environmental conditions inside the containers do not preclude the cracking process.

  15. South Polar Residual Ice Cap

    NASA Technical Reports Server (NTRS)

    1990-01-01

    This mosaic is composed of 18 Viking Orbiter images (6 each in red, green, and violet filters), acquired on September 28, 1977, during revolution 407 of Viking Orbiter 2. The south pole is located just off the lower left edge of the polar cap, and the 0 degree longitude meridian extends toward the top of the mosaic. The large crater near the right edge (named 'South') is about 100 km in diameter. These images were acquired during southern summer on Mars (Ls = 341 degrees); the sub-solar declination was 8 degrees S., and the south polar cap was nearing its final stage of retreat just prior to vernal equinox. The south residual cap is approximately 400 km across, and the exposed surface is thought to consist dominantly of carbon-dioxide frost. This is in contrast to the water-ice surface of the north polar residual cap. It is likely that water ice is present in layers that underlie the south polar cap and that comprise the surrounding layered terrains. Near the top of this image, irregular pits with sharp-rimmed cliffs appear 'etched', presumably by wind. A series of rugged mountains (extending toward the upper right corner of the image) are of unknown origin.

  16. Residual number processing in dyscalculia.

    PubMed

    Cappelletti, Marinella; Price, Cathy J

    2014-01-01

    Developmental dyscalculia - a congenital learning disability in understanding numerical concepts - is typically associated with parietal lobe abnormality. However, people with dyscalculia often retain some residual numerical abilities, reported in studies that otherwise focused on abnormalities in the dyscalculic brain. Here we took a different perspective by focusing on brain regions that support residual number processing in dyscalculia. All participants accurately performed semantic and categorical colour-decision tasks with numerical and non-numerical stimuli, with adults with dyscalculia performing slower than controls in the number semantic tasks only. Structural imaging showed less grey-matter volume in the right parietal cortex in people with dyscalculia relative to controls. Functional MRI showed that accurate number semantic judgements were maintained by parietal and inferior frontal activations that were common to adults with dyscalculia and controls, with higher activation for participants with dyscalculia than controls in the right superior frontal cortex and the left inferior frontal sulcus. Enhanced activation in these frontal areas was driven by people with dyscalculia who made faster rather than slower numerical decisions; however, activation could not be accounted for by response times per se, because it was greater for fast relative to slow dyscalculics but not greater for fast controls relative to slow dyscalculics. In conclusion, our results reveal two frontal brain regions that support efficient number processing in dyscalculia.

  17. Residual number processing in dyscalculia☆

    PubMed Central

    Cappelletti, Marinella; Price, Cathy J.

    2013-01-01

    Developmental dyscalculia – a congenital learning disability in understanding numerical concepts – is typically associated with parietal lobe abnormality. However, people with dyscalculia often retain some residual numerical abilities, reported in studies that otherwise focused on abnormalities in the dyscalculic brain. Here we took a different perspective by focusing on brain regions that support residual number processing in dyscalculia. All participants accurately performed semantic and categorical colour-decision tasks with numerical and non-numerical stimuli, with adults with dyscalculia performing slower than controls in the number semantic tasks only. Structural imaging showed less grey-matter volume in the right parietal cortex in people with dyscalculia relative to controls. Functional MRI showed that accurate number semantic judgements were maintained by parietal and inferior frontal activations that were common to adults with dyscalculia and controls, with higher activation for participants with dyscalculia than controls in the right superior frontal cortex and the left inferior frontal sulcus. Enhanced activation in these frontal areas was driven by people with dyscalculia who made faster rather than slower numerical decisions; however, activation could not be accounted for by response times per se, because it was greater for fast relative to slow dyscalculics but not greater for fast controls relative to slow dyscalculics. In conclusion, our results reveal two frontal brain regions that support efficient number processing in dyscalculia. PMID:24266008

  18. Residual symptoms in elderly major depression remitters.

    PubMed

    Gastó, C; Navarro, V; Catalán, R; Portella, M J; Marcos, T

    2003-07-01

    To assess residual symptoms in severe geriatric major depression in remission, and to determine baseline clinical and sociodemographic predictors of residual symptoms in remitters. A total of 108 elderly patients with unipolar major depression were evaluated and treated naturalistically for 9 months so as to record the predictors of residual symptoms in remitters. In order to reduce the likelihood of confusing residual symptoms with normal effects of age, 30 control subjects were also monitored. Seventy-nine patients (73.1%) were considered remitters and 82.3% of remitters showed residual symptoms. Medical burden, chronic stress and subjective social support were the only variables which predicted the severity of residual symptoms in remitters. Residual symptoms in elderly patients with major depression in remission should not only be attributed exclusively to intrinsic factors of the illness or the age of the individual patient, but also to external factors.

  19. An essential tyrosine residue of Aspergillus polygalacturonase.

    PubMed

    Stratilová, E; Dzúrová, M; Markovic, O; Jörnvall, H

    1996-03-11

    Based on strict conservation of a tyrosine residue in 24 polygalacturonases, tyrosine modification was assessed in two different forms of the Aspergillus enzyme. The second subform was unknown in structure but submitted to sequence analysis and was found also to have the conserved tyrosine residue. Results of chemical modifications are consistent in showing inactivation of the proteins with all tyrosine-reactive agents tested, acetic anhydride, N-acetyl imidazole, and tetranitromethane. Furthermore, after acetylation, regeneration of enzyme activity was possible with hydroxylamine. Spectrophotometric pH titration showed that one accessible tyrosine residue is ionized at pH 9.3-9.5, whereas the remaining, masked residues are all ionized at pH 10.5. It is concluded that one tyrosine residue is catalytically important, in agreement with the inactivation and reactivation data, that this residue is accessible, and that it is likely to correspond to the strictly conserved residue observed in all forms.

  20. Determination of pymetrozine residues in cucumber.

    PubMed

    Talebi, K; Ghazizadeh Ahsaii, H

    2006-01-01

    An experiment was carried out to investigate the residues of pymetrozin in cucumber under field conditions. Plots of cucumber (cv. Daminus) were sprayed with pymetrozine (Chess, WG50) at two different rates; 0.5 and 1.0 kg/ha. Samples were collected 2 hours and 1, 2, 3, and 4 days after spraying and pymetrozin residues were determined using high performance liquid chromatography with UV detector. Two hours after spraying, the residues was 0.326 mg/kg for 0.5 kg/ha treatment. The residues decreased to 0.013 mg/kg on the third day and no residue was detected four days after spraying. In 1.0 kg/ha treatment, the residue was 0.627 mg/kg, two hours after spraying, which decreased to 0.028 mg/kg, on the third day, but no residues was found in samples which collected four days after spraying. In farm plots which received 0.5 kg/ha pymetrozine, the residues in peels and peeled cucumber were 0.359 and 0.308 mg/kg respectively, two hours after spraying, but in plots which sprayed at 1.0 kg/ha, the residues were 0.648 and 0.609 mg/kg respectively. Furthermore the residues in peels showed a significant difference with those of peeled and unpeeled cucumber under field conditions.

  1. Self-assembled monolayer of light-harvesting core complexes of photosynthetic bacteria on an amino-terminated ITO electrode

    PubMed Central

    Suemori, Yoshiharu; Nagata, Morio; Nakamura, Yukari; Nakagawa, Katsunori; Okuda, Ayumi; Inagaki, Jun-ichi; Shinohara, Kiyoshi; Ogawa, Makiko; Iida, Kouji; Dewa, Takehisa; Yamashita, Keiji; Gardiner, Alastair; Cogdell, Richard J.

    2006-01-01

    Light-harvesting antenna core (LH1-RC) complexes isolated from Rhodospirillum rubrum and Rhodopseudomonas palustris were successfully self-assembled on an ITO electrode modified with 3-aminopropyltriethoxysilane. Near infra-red (NIR) absorption, fluorescence, and IR spectra of these LH1-RC complexes indicated that these LH1-RC complexes on the electrode were stable on the electrode. An efficient energy transfer and photocurrent responses of these LH1-RC complexes on the electrode were observed upon illumination of the LH1 complex at 880 nm. PMID:17111238

  2. Inhibition of the Androgen Receptor Amino Terminal Domain by a Small Molecule as Treatment for Castrate Resistant Prostate Cancer

    DTIC Science & Technology

    2016-01-01

    androgen deprivation or blockade. Scope : Aim 1 will determine the impact of EPI-001 on castration sensitive tumor regression and re-growth in LuCaP...final report on this award. The SOW has been completed and the EPI compound had transitioned to Phase 1 clinical trial . Findings: We have clearly shown...suppression of AR activity. Significance: Based on these studies to this point EPI has entered Phase 1 clinical trials . 15. SUBJECT TERMS 16

  3. [The determination of serum amino-terminal procollagen type-III propeptide (PIIINP) in occupational exposure to rock wool fiber].

    PubMed

    Cavalleri, A; Gobba, F; Ferrari, D; Bacchella, L; Robotti, M; Mineo, F; Pedroni, C

    1992-01-01

    Fifty-six males workers exposed to rock wool during production, and 20 referents were examined. Exposure, evaluated by personal sampling, ranged from 0.05 to 0.74 fibres/ml (median 0.15). The subjects underwent a medical examination, chest X-ray according to ILO recommendations and pulmonary function tests. In all subjects the serum levels of type III procollagen N-terminal propeptide (PIIINPs) were determined. No evidence of pulmonary fibrosis, nor work-related lung diseases were observed. PIIINPs mean values in the exposed (9.8 ng/ml; 2.8 S.D.) were slightly higher, but not significantly different when compared to referents (8.5 ng/ml; 2.5 S.D.). No significant correlation between PIIINPs and rock wool exposure (both airborne levels and exposure duration) was observed. Furthermore, peptide levels were not related to pulmonary function test results. Our results suggest that occupational exposure to rock wool fibres lower than 0.75 fibres/ml for less than 20 years does not induce definite cases of pulmonary fibrosis nor an increase of type III collagen synthesis in the lung.

  4. Amino-terminal domains of c-myc and N-myc proteins mediate binding to the retinoblastoma gene product

    NASA Astrophysics Data System (ADS)

    Rustgi, Anil K.; Dyson, Nicholas; Bernards, Rene

    1991-08-01

    THE proteins encoded by the myc gene family are involved in the control of cell proliferation and differentiation, and aberrant expression of myc proteins has been implicated in the genesis of a variety of neoplasms1. In the carboxyl terminus, myc proteins have two domains that encode a basic domain/helix-loop-helix and a leucine zipper motif, respectively. These motifs are involved both in DNA binding and in protein dimerization2-5. In addition, myc protein family members share several regions of highly conserved amino acids in their amino termini that are essential for transformation6,7. We report here that an N-terminal domain present in both the c-myc and N-myc proteins mediates binding to the retinoblastoma gene product, pRb. We show that the human papilloma virus E7 protein competes with c-myc for binding to pRb, indicating that these proteins share overlapping binding sites on pRb. Furthermore, a mutant Rb protein from a human tumour cell line that carried a 35-amino-acid deletion in its C terminus failed to bind to c-myc. Our results suggest that c-myc and pRb cooperate through direct binding to control cell proliferation.

  5. The amino-terminal structure of human fragile X mental retardation protein obtained using precipitant-immobilized imprinted polymers.

    PubMed

    Hu, Yufeng; Chen, Zhenhang; Fu, Yanjun; He, Qingzhong; Jiang, Lun; Zheng, Jiangge; Gao, Yina; Mei, Pinchao; Chen, Zhongzhou; Ren, Xueqin

    2015-03-23

    Flexibility is an intrinsic property of proteins and essential for their biological functions. However, because of structural flexibility, obtaining high-quality crystals of proteins with heterogeneous conformations remain challenging. Here, we show a novel approach to immobilize traditional precipitants onto molecularly imprinted polymers (MIPs) to facilitate protein crystallization, especially for flexible proteins. By applying this method, high-quality crystals of the flexible N-terminus of human fragile X mental retardation protein are obtained, whose absence causes the most common inherited mental retardation. A novel KH domain and an intermolecular disulfide bond are discovered, and several types of dimers are found in solution, thus providing insights into the function of this protein. Furthermore, the precipitant-immobilized MIPs (piMIPs) successfully facilitate flexible protein crystal formation for five model proteins with increased diffraction resolution. This highlights the potential of piMIPs for the crystallization of flexible proteins.

  6. The amino-terminal structure of human fragile X mental retardation protein obtained using precipitant-immobilized imprinted polymers

    NASA Astrophysics Data System (ADS)

    Hu, Yufeng; Chen, Zhenhang; Fu, Yanjun; He, Qingzhong; Jiang, Lun; Zheng, Jiangge; Gao, Yina; Mei, Pinchao; Chen, Zhongzhou; Ren, Xueqin

    2015-03-01

    Flexibility is an intrinsic property of proteins and essential for their biological functions. However, because of structural flexibility, obtaining high-quality crystals of proteins with heterogeneous conformations remain challenging. Here, we show a novel approach to immobilize traditional precipitants onto molecularly imprinted polymers (MIPs) to facilitate protein crystallization, especially for flexible proteins. By applying this method, high-quality crystals of the flexible N-terminus of human fragile X mental retardation protein are obtained, whose absence causes the most common inherited mental retardation. A novel KH domain and an intermolecular disulfide bond are discovered, and several types of dimers are found in solution, thus providing insights into the function of this protein. Furthermore, the precipitant-immobilized MIPs (piMIPs) successfully facilitate flexible protein crystal formation for five model proteins with increased diffraction resolution. This highlights the potential of piMIPs for the crystallization of flexible proteins.

  7. Normal amino-terminal pro-brain natriuretic peptide (NT-proBNP) values in amniotic fluid.

    PubMed

    Carvajal, Jorge Andrés; Ferrer, Fernando Andrés; Araya, Francisca Isabel; Delpiano, Ana María

    2017-01-01

    Brain natriuretic peptide (BNP) is synthesized by human fetal membranes, both the amnion and chorion. This locally produced BNP inhibits the contraction of the human myometrium, contributing to the maintenance of myometrial quiescence during pregnancy. Reference values for NT-proBNP concentrations in amniotic fluid at different gestational ages have not been completely defined. We aimed to investigate the range of fetal NT-proBNP values in amniotic fluid in normal pregnancy between 17 and 41weeks of gestation. Samples of amniotic fluid were obtained from women meeting the following inclusion criteria: gestational age defined by early ultrasound, singleton gestation and not in labor. The exclusion criteria were as follows: multiple gestation, clinically evident chorioamnionitis, laboratory signs of infection in the amniotic fluid sample and fetal conditions that may alter NT Pro-BNP levels (anemia, hydrops, etc.). NT-proBNP concentrations in amniotic fluid were measured using the automated Elecsys® proBNP assay. We analyzed 218 samples of amniotic fluid at various gestational ages. Half of the samples were obtained by amniocentesis (118 samples), and the other half (100 samples) were obtained by direct puncture at the time of cesarean section. We found a significant decline in NT-proBNP concentrations with advancing gestational age. Gestational age has to be taken into consideration in the assessment of NT-proBNP values. Our data may be used as reference values in fetal medicine, as a possible predictor of preterm delivery risk using the inferior limit (0.5 multiples of the median (MoM)) of our normal curve. Copyright © 2016 The Canadian Society of Clinical Chemists. Published by Elsevier Inc. All rights reserved.

  8. Conserved cell cycle regulatory properties within the amino terminal domain of the Epstein-Barr virus nuclear antigen 3C

    SciTech Connect

    Sharma, Nikhil; Knight, Jason S.; Robertson, Erle S. . E-mail: erle@mail.med.upenn.edu

    2006-03-15

    The gammaherpesviruses Rhesus lymphocryptovirus (LCV) and Epstein-Barr virus (EBV) are closely related phylogenetically. Rhesus LCV efficiently immortalizes Rhesus B cells in vitro. However, despite a high degree of conservation between the Rhesus LCV and EBV genomes, Rhesus LCV fails to immortalize human B cells in vitro. This species restriction may, at least in part, be linked to the EBV nuclear antigens (EBNAs) and latent membrane proteins (LMPs), known to be essential for B cell transformation. We compared specific properties of EBNA3C, a well-characterized and essential EBV protein, with its Rhesus counterpart to determine whether EBNA3C phenotypes which contribute to cell cycle regulation are conserved in the Rhesus LCV. We show that both EBNA3C and Rhesus EBNA3C bind to a conserved region of mammalian cyclins, regulate pRb stability, and modulate SCF{sup Skp2}-dependent ubiquitination. These results suggest that Rhesus LCV restriction from human B cell immortalization is independent of the conserved cell cycle regulatory functions of the EBNA3C protein.

  9. Amino-terminal arginylation targets endoplasmic reticulum chaperone BiP for autophagy through p62 binding.

    PubMed

    Cha-Molstad, Hyunjoo; Sung, Ki Sa; Hwang, Joonsung; Kim, Kyoung A; Yu, Ji Eun; Yoo, Young Dong; Jang, Jun Min; Han, Dong Hoon; Molstad, Michael; Kim, Jung Gi; Lee, Yoon Jee; Zakrzewska, Adriana; Kim, Su-Hyeon; Kim, Sung Tae; Kim, Sun Yong; Lee, Hee Gu; Soung, Nak Kyun; Ahn, Jong Seog; Ciechanover, Aaron; Kim, Bo Yeon; Kwon, Yong Tae

    2015-07-01

    We show that ATE1-encoded Arg-transfer RNA transferase (R-transferase) of the N-end rule pathway mediates N-terminal arginylation of multiple endoplasmic reticulum (ER)-residing chaperones, leading to their cytosolic relocalization and turnover. N-terminal arginylation of BiP (also known as GRP78), protein disulphide isomerase and calreticulin is co-induced with autophagy during innate immune responses to cytosolic foreign DNA or proteasomal inhibition, associated with increased ubiquitylation. Arginylated BiP (R-BiP) is induced by and associated with cytosolic misfolded proteins destined for p62 (also known as sequestosome 1, SQSTM1) bodies. R-BiP binds the autophagic adaptor p62 through the interaction of its N-terminal arginine with the p62 ZZ domain. This allosterically induces self-oligomerization and aggregation of p62 and increases p62 interaction with LC3, leading to p62 targeting to autophagosomes and selective lysosomal co-degradation of R-BiP and p62 together with associated cargoes. In this autophagic mechanism, Nt-arginine functions as a delivery determinant, a degron and an activating ligand. Bioinformatics analysis predicts that many ER residents use arginylation to regulate non-ER processes.

  10. Prognostic value of circulating amino-terminal pro-C-type natriuretic peptide in critically ill patients

    PubMed Central

    2011-01-01

    Introduction C-type natriuretic peptide (CNP) is a paracrine molecule which is mainly synthesized in the vasculature. High levels have been reported in sepsis, and CNP has been proposed as a biomarker predicting sepsis in traumatized patients. We aimed at evaluating the diagnostic and prognostic value of N-terminal pro-CNP (NT-proCNP) for predicting sepsis, disease severity and mortality in critically ill medical patients. Methods 273 critically ill patients (197 patients with sepsis or septic shock, 76 without evidence of sepsis) and 43 healthy controls were consecutively included in a prospective clinical single-center non-interventional study at the Medical Intensive Care Unit, RWTH-University Aachen, Germany. Patients' outcome was followed for about 1 year. NT-proCNP serum concentrations were determined upon ICU admission, as well as in the mornings of day 3 and day 7 after admission. Intensive care treatment measures as well as routine and experimental laboratory parameters were recorded and analyzed. Results NT-proCNP serum concentrations upon admission to the ICU were elevated in critically ill patients as compared with healthy controls. Patients with sepsis had significantly higher NT-proCNP levels than non-sepsis patients. NT-proCNP was strongly associated with inflammatory parameters (i.e. C-reactive protein, procalcitonin and TNF-α), biomarkers of organ dysfunction and clinical composite scores (APACHE-II, SOFA, SAPS2). NT-proCNP levels at admission and day 3 were found to be a strong predictive marker for ICU- and overall survival. Moreover, a decline of serum NT-proCNP after admission to the ICU was associated with reduced mortality. The predictive power of serum NT-proCNP was similar to 'conventional' prognostic tools such as clinical scores. Conclusions NT-proCNP is significantly elevated in critically ill patients, with highest levels in sepsis. Inflammation as well as organ function are strongly associated with NT-proCNP serum concentrations. Low initial NT-proCNP levels and a decline during initial treatment indicate a favourable ICU- and long-term outcome. PMID:21281508

  11. Separation of polyethylene glycols and amino-terminated polyethylene glycols by high-performance liquid chromatography under near critical conditions.

    PubMed

    Wei, Y-Z; Zhuo, R-X; Jiang, X-L

    2016-05-20

    The separation and characterization of polyethylene glycols (PEGs) and amino-substituted derivatives on common silica-based reversed-phase packing columns using isocratic elution is described. This separation is achieved by liquid chromatography under the near critical conditions (LCCC), based on the number of amino functional end groups without obvious effect of molar mass for PEGs. The mobile phase is acetonitrile in water with an optimal ammonium acetate buffer. The separation mechanism of PEG and amino-substituted PEG under the near LCCC on silica-based packing columns is confirmed to be ion-exchange interaction. Under the LCCC of PEG backbone, with fine tune of buffer concentration, the retention factor ratios for benzylamine and phenol in buffered mobile phases, α(benzylamine/phenol)-values, were used to assess the ion-exchange capacity on silica-based reversed-phase packing columns. To the best of our knowledge, this is the first report on separation of amino-functional PEGs independent of the molar mass by isocratic elution using common C18 or phenyl reversed-phase packing columns.

  12. An Internally Translated MAVS Variant Exposes Its Amino-terminal TRAF-Binding Motifs to Deregulate Interferon Induction.

    PubMed

    Minassian, Arlet; Zhang, Junjie; He, Shanping; Zhao, Jun; Zandi, Ebrahim; Saito, Takeshi; Liang, Chengyu; Feng, Pinghui

    2015-07-01

    Activation of pattern recognition receptors and proper regulation of downstream signaling are crucial for host innate immune response. Upon infection, the NF-κB and interferon regulatory factors (IRF) are often simultaneously activated to defeat invading pathogens. Mechanisms concerning differential activation of NF-κB and IRF are not well understood. Here we report that a MAVS variant inhibits interferon (IFN) induction, while enabling NF-κB activation. Employing herpesviral proteins that selectively activate NF-κB signaling, we discovered that a MAVS variant of ~50 kDa, thus designated MAVS50, was produced from internal translation initiation. MAVS50 preferentially interacts with TRAF2 and TRAF6, and activates NF-κB. By contrast, MAVS50 inhibits the IRF activation and suppresses IFN induction. Biochemical analysis showed that MAVS50, exposing a degenerate TRAF-binding motif within its N-terminus, effectively competed with full-length MAVS for recruiting TRAF2 and TRAF6. Ablation of the TRAF-binding motif of MAVS50 impaired its inhibitory effect on IRF activation and IFN induction. These results collectively identify a new means by which signaling events is differentially regulated via exposing key internally embedded interaction motifs, implying a more ubiquitous regulatory role of truncated proteins arose from internal translation and other related mechanisms.

  13. Amino-terminal enhancer of split gene AES encodes a tumor and metastasis suppressor of prostate cancer.

    PubMed

    Okada, Yoshiyuki; Sonoshita, Masahiro; Kakizaki, Fumihiko; Aoyama, Naoki; Itatani, Yoshiro; Uegaki, Masayuki; Sakamoto, Hiromasa; Kobayashi, Takashi; Inoue, Takahiro; Kamba, Tomomi; Suzuki, Akira; Ogawa, Osamu; Taketo, M Mark

    2017-04-01

    A major cause of cancer death is its metastasis to the vital organs. Few effective therapies are available for metastatic castration-resistant prostate cancer (PCa), and progressive metastatic lesions such as lymph nodes and bones cause mortality. We recently identified AES as a metastasis suppressor for colon cancer. Here, we have studied the roles of AES in PCa progression. We analyzed the relationship between AES expression and PCa stages of progression by immunohistochemistry of human needle biopsy samples. We then performed overexpression and knockdown of AES in human PCa cell lines LNCaP, DU145 and PC3, and determined the effects on proliferation, invasion and metastasis in culture and in a xenograft model. We also compared the PCa phenotypes of Aes/Pten compound knockout mice with those of Pten simple knockout mice. Expression levels of AES were inversely correlated with clinical stages of human PCa. Exogenous expression of AES suppressed the growth of LNCaP cells, whereas the AES knockdown promoted it. We also found that AES suppressed transcriptional activities of androgen receptor and Notch signaling. Notably, AES overexpression in AR-defective DU145 and PC3 cells reduced invasion and metastasis to lymph nodes and bones without affecting proliferation in culture. Consistently, prostate epithelium-specific inactivation of Aes in Pten(flox/flox) mice increased expression of Snail and MMP9, and accelerated growth, invasion and lymph node metastasis of the mouse prostate tumor. These results suggest that AES plays an important role in controlling tumor growth and metastasis of PCa by regulating both AR and Notch signaling pathways. © 2017 The Authors. Cancer Science published by John Wiley & Sons Australia, Ltd on behalf of Japanese Cancer Association.

  14. Detection of antibiotic residues in poultry meat.

    PubMed

    Sajid, Abdul; Kashif, Natasha; Kifayat, Nasira; Ahmad, Shabeer

    2016-09-01

    The antibiotic residues in poultry meat can pose certain hazards to human health among them are sensitivity to antibiotics, allergic reactions, mutation in cells, imbalance of intestinal micro biota and bacterial resistance to antibiotics. The purpose of the present paper was to detect antibiotic residue in poultry meat. During the present study a total of 80 poultry kidney and liver samples were collected and tested for detection of different antibiotic residues at different pH levels Eschericha coli at pH 6, 7 and Staphyloccocus aureus at pH 8 & 9. Out of 80 samples only 4 samples were positive for antibiotic residues. The highest concentrations of antibiotic residue found in these tissues were tetracycline (8%) followed by ampicilin (4%), streptomycine (2%) and aminoglycosides (1%) as compared to other antibiotics like sulfonamides, neomycine and gentamycine. It was concluded that these microorganism at these pH levels could be effectively used for detection of antibiotic residues in poultry meat.

  15. Optical systolic array processor using residue arithmetic

    NASA Technical Reports Server (NTRS)

    Jackson, J.; Casasent, D.

    1983-01-01

    The use of residue arithmetic to increase the accuracy and reduce the dynamic range requirements of optical matrix-vector processors is evaluated. It is determined that matrix-vector operations and iterative algorithms can be performed totally in residue notation. A new parallel residue quantizer circuit is developed which significantly improves the performance of the systolic array feedback processor. Results are presented of a computer simulation of this system used to solve a set of three simultaneous equations.

  16. Studies Of Residual Flexibility And Vibration Testing

    NASA Technical Reports Server (NTRS)

    Admire, John R.; Tinker, Michael L.; Bookout, Paul S.; Ivey, Edward W.

    1995-01-01

    Collection of reports presents theoretical and experimental studies in which concept of residual flexibility applied to modal vibration testing and verification of mathematical models of vibrations of flexible structure constrained by another structure. "Residual flexibility" denotes that part of interface flexibility due to mode shapes out of frequency range of test. Studies directed toward assessing residual-flexibility approach as substitute for fixed-base vibrational testing of payloads installed in spacecraft.

  17. Residual stresses in polymer matrix composite laminates

    NASA Technical Reports Server (NTRS)

    Hahn, H. T.

    1976-01-01

    Residual stresses in composites are induced during fabrication and by environmental exposure. The theory formulated can describe the shrinkage commonly observed after a thermal expansion test. Comparison between the analysis and experimental data for laminates of various material systems indicates that the residual stress-free temperature can be lower than the curing temperature, depending on the curing process. Effects of residual stresses on ply failure including the acoustic emission characteristics are discussed.

  18. Particulate residue separators for harvesting devices

    DOEpatents

    Hoskinson, Reed L.; Kenney, Kevin L.; Wright, Christopher T.; Hess, John R.

    2010-06-29

    A particulate residue separator and a method for separating a particulate residue stream may include a plenum borne by a harvesting device, and have a first, intake end and a second, exhaust end; first and second particulate residue air streams which are formed by the harvesting device and which travel, at least in part, along the plenum and in a direction of the second, exhaust end; and a baffle assembly which is located in partially occluding relation relative to the plenum, and which substantially separates the first and second particulate residue air streams.

  19. Methods of separating particulate residue streams

    DOEpatents

    Hoskinson, Reed L [Rigby, ID; Kenney, Kevin L [Idaho Falls, ID; Wright, Christopher T [Idaho Falls, ID; Hess, J Richard [Idaho Falls, ID

    2011-04-05

    A particulate residue separator and a method for separating a particulate residue stream may include an air plenum borne by a harvesting device, and have a first, intake end and a second, exhaust end; first and second particulate residue air streams that are formed by the harvesting device and that travel, at least in part, along the air plenum and in a direction of the second, exhaust end; and a baffle assembly that is located in partially occluding relation relative to the air plenum and that substantially separates the first and second particulate residue air streams.

  20. Identification of kinetically hot residues in proteins.

    PubMed Central

    Demirel, M. C.; Atilgan, A. R.; Jernigan, R. L.; Erman, B.; Bahar, I.

    1998-01-01

    A number of recent studies called attention to the presence of kinetically important residues underlying the formation and stabilization of folding nuclei in proteins, and to the possible existence of a correlation between conserved residues and those participating in the folding nuclei. Here, we use the Gaussian network model (GNM), which recently proved useful in describing the dynamic characteristics of proteins for identifying the kinetically hot residues in folded structures. These are the residues involved in the highest frequency fluctuations near the native state coordinates. Their high frequency is a manifestation of the steepness of the energy landscape near their native state positions. The theory is applied to a series of proteins whose kinetically important residues have been extensively explored: chymotrypsin inhibitor 2, cytochrome c, and related C2 proteins. Most of the residues previously pointed out to underlie the folding process of these proteins, and to be critically important for the stabilization of the tertiary fold, are correctly identified, indicating a correlation between the kinetic hot spots and the early forming structural elements in proteins. Additionally, a strong correlation between kinetically hot residues and loci of conserved residues is observed. Finally, residues that may be important for the stability of the tertiary structure of CheY are proposed. PMID:9865946

  1. Partitioning Residue-derived and Residue-induced Emissions of N2O Using 15N-labelled Crop Residues

    NASA Astrophysics Data System (ADS)

    Farrell, R. E.; Carverhill, J.; Lemke, R.; Knight, J. D.

    2014-12-01

    Estimates of N2O emissions in Canada indicate that 17% of all agriculture-based emissions are associated with the decomposition of crop residues. However, research specific to the western Canadian prairies (including Saskatchewan) has shown that the N2O emission factor for N sources in this region typically ranges between 0.2 and 0.6%, which is well below the current IPCC default emission factor of 1.0%. Thus, it stands to reason that emissions from crop residues should also be lower than those calculated using the current IPCC emission factor. Current data indicates that residue decomposition, N mineralization and N2O production are affected by a number of factors such as C:N ratio and chemical composition of the residue, soil type, and soil water content; thus, a bench-scale incubation study was conducted to examine the effects of soil type and water content on N2O emissions associated with the decomposition of different crop residues. The study was carried out using soils from the Black, Dark Brown, Brown, and Gray soil zones and was conducted at both 50% and 70% water-filled pore space (WFPS); the soils were amended with 15N-labeled residues of wheat, pea, canola, and flax, or with an equivalent amount of 15N-labeled urea; 15N2O production was monitored using a Picarro G5101-i isotopic N2O analyzer. Crop residue additions to the soils resulted in both direct and indirect emissions of N2O, with residue derived emissions (RDE; measured as 15N2O) generally exceeding residue-induced emissions (RIE) at 50% WFPS—with RDEs ranging from 42% to 88% (mean = 58%) of the total N2O. Conversely, at 70% WFPS, RDEs were generally lower than RIEs—ranging from 21% to 83% (mean = 48%). Whereas both water content and soil type had an impact on N2O production, there was a clear and consistent trend in the emission factors for the residues; i.e., emissions were always greatest for the canola residue and lowest for the wheat residue and urea fertilizer; and intermediate for pea

  2. Thermal Insulation from Hardwood Residues

    NASA Astrophysics Data System (ADS)

    Sable, I.; Grinfelds, U.; Vikele, L.; Rozenberga, L.; Zeps, M.; Luguza, S.

    2015-11-01

    Adequate heat is one of the prerequisites for human wellbeing; therefore, building insulation is required in places where the outside temperature is not suitable for living. The climate change, with its rising temperatures and longer dry periods, promotes enlargement of the regions with conditions more convenient for hardwood species than for softwood species. Birch (Betula pendula) is the most common hardwood species in Latvia. The aim of this work was to obtain birch fibres from wood residues of plywood production and to form low-density thermal insulation boards. Board formation and production was done in the presence of water; natural binder, fire retardant and fungicide were added in different concentrations. Board properties such as density, transportability or resistance to particulate loss, thermal conductivity and reaction to fire were investigated. This study included thermal insulation boards with the density of 102-120 kg/m3; a strong correlation between density and the binder amount was found. Transportability also improved with the addition of a binder, and 0.1-0.5% of the binder was the most appropriate amount for this purpose. The measured thermal conductivity was in the range of 0.040-0.043 W/(m·K). Fire resistance increased with adding the fire retardant. We concluded that birch fibres are applicable for thermal insulation board production, and it is possible to diversify board properties, changing the amount of different additives.

  3. Catalyst deactivation in residue hydrocracking

    SciTech Connect

    Oballa, M.C.; Wong, C.; Krzywicki, A.

    1994-12-31

    The existence of a computer-controlled bench scale hydrocracking units at the authors site has made cheaper the non-stop running of experiments for long periods of time. It was, therefore possible to show, at minimal costs, when three hydrocracking catalysts in service reach their maximum lifetime. Different parameters which are helpful for catalyst life and activity predictions were calculated, e.g., relative catalyst age and the effectiveness factor. Experimental results compared well with model, giving them the minimum and maximum catalyst lifetime, as well as the deactivation profile with regard to sulfur and metals removal. Reaction rate constants for demetallization and desulfurization were also determined. Six commercial catalysts were evaluated at short term runs and the three most active were used for long term runs. Out of three catalysts tested for deactivation at long term runs, it was possible to choose one whose useful life was higher than the others. All runs were carried out in a Robinson-Mahoney continuous flow stirred tank reactor, using 50/50 volumetric mixture of Cold Lake/Lloydminster atmospheric residue and NiMo/Al{sub 2}O{sub 3} catalyst.

  4. Gunshot residue preservation in seawater.

    PubMed

    Lindström, Anne-Christine; Hoogewerff, Jurian; Athens, Josie; Obertova, Zuzana; Duncan, Warwick; Waddell, Neil; Kieser, Jules

    2015-08-01

    Little is known about the persistence of gunshot residue (GSR) in soft tissue and bones during decomposition in marine environments. For a better understanding, qualitative and quantitative data were obtained on GSR retention on soft tissue and bony gunshot wounds (GSWs). A quantity of 36 fleshed and 36 defleshed bovine ribs were shot at contact range with 0.22 calibre hollow point ammunition using a Stirling 0.22 calibre long rifle. Bone specimens in triplicate were placed in three environments: submerged, intertidal and in supralittoral zone. Sets of triplicates were recovered on day 3, 10, 24 and 38, and analysed with scanning electron microscopy with energy dispersive X-ray spectrometry (SEM-EDX), and inductive coupled plasma mass spectrometry (ICP-MS). The SEM-EDX recorded GSR-indicative particles surrounding the bullet entrance on all bone types (fleshed and defleshed) in all environments throughout the study. GSR-unique particles were only detected on the supralittoral bones. The ICP-MS analysis showed faster GSR loss on submerged than intertidal and supralittoral defleshed specimens. Fleshed specimens showed a faster GSR loss on intertidal than submerged and supralittoral specimens. In conclusion, the GSR disappeared faster from submerged and intertidal than non-submerged specimens. The difference of detection of GSR between analysed specimens (defleshed versus fleshed) disappeared upon defleshing. This study highlights the potential of finding evidence of GSR in a submerged body and the potential of microscopic and analytical methods for examining suspected GSW in highly decomposed bodies in marine habitats.

  5. Fgd residues: a commercial opportunity

    SciTech Connect

    Juzwiak, J.H.; Smith, C.L. )

    1992-01-01

    This paper focuses on actual operating experience in the handling and processing of ash and FGD by-products by Conversion Systems at the Orlando Utility Commission Stanton Energy Center at Orlando, Florida. The discussion discloses how the Poz-O-Tec stabilization technology, the most widely used disposal technology for wet FGD sludge, has been modified and improved to maximize the by-product recycling and produce the most cost effective disposal option. Recently, CSI has directed 100% of the Stanton Facility FGD stabilization production into the generation of the commercial aggregate called Poz-O-Lite Aggregate. This aggregate product is used in the manufacture of concrete block, replacing ordinary crushed stone. CSI has been able to develop this reuse application using the low permeability and superior strength of the stabilized material. The utility also benefits from the stabilized material which is not recycled as it offers opportunity for co-disposal of other combustion residues from some of the utility's oil fired plants in cells that are lined and then capped with the stabilized material. In summary, virtually all production of the by-products, from combustion or flue gas desulfurization at the Stanton Energy Center are now being used for commercial application, except that specifically required as liners for the disposal of other materials on site.

  6. Assessment of secondary crop residues. Final report

    SciTech Connect

    Ashare, E.; Leuschner, A.P.; West, C.E.; Langton, B.

    1981-03-01

    This report is the first of three reports assessing the feasibility of converting secondary agricultural residues to energy in the form of either methane gas or ethyl alcohol. Secondary agricultural residues are defined in this study as those residues resulting from biomass processing to produce primary products; e.g., whey from cheese processing, vegetable processing wastes, residues from paper pulping, etc. This report summarizes the first two phases of this study, data compilation, and evaluation. Subsequent reports will analyze the technical and economic feasibility of converting these residues to energy and the implementability of this technology. The industries for which data has been compiled in this report include vegetable, fruit, seafood, meat, poultry, and dairy processing and the pulp, paper, and paperboard industry. The data collected include raw product input, final processed product output, residue types, and quantity, residue concentration, biodegradability, seasonality of production, and geographic distribution of processing facilities. In general, these industries produce a relatively solid residue ranging in total solids concentration from 10 to 50% and a dilute liquid residue with an organic content (measured as COD or BOD) ranging from a few hundred to a few thousand mg/l. Due to the significant quantities of residues generated in each of the industries, it appears that the potential exists for generating a substantial quantity of energy. For a particular industry this quantity of energy can range from only one percent upwards to nearly thirty-five percent of the total processing energy required. The total processing energy required for the industries included in this study is approximately 2.5 quads per year. The potential energy which can be generated from these industrial residues will be 0.05 to 0.10 quads per year or approximately 2 to 4 percent of the total demand.

  7. Use of Combined Uncertainty of Pesticide Residue Results for Testing Compliance with Maximum Residue Limits (MRLs).

    PubMed

    Farkas, Zsuzsa; Slate, Andrew; Whitaker, Thomas B; Suszter, Gabriella; Ambrus, Árpád

    2015-05-13

    The uncertainty of pesticide residue levels in crops due to sampling, estimated for 106 individual crops and 24 crop groups from residue data obtained from supervised trials, was adjusted with a factor of 1.3 to accommodate the larger variability of residues under normal field conditions. Further adjustment may be necessary in the case of mixed lots. The combined uncertainty of residue data including the contribution of sampling is used for calculation of an action limit, which should not be exceeded when compliance with maximum residue limits is certified as part of premarketing self-control programs. On the contrary, for testing compliance of marketed commodities the residues measured in composite samples should be greater than or equal to the decision limit calculated only from the combined uncertainty of the laboratory phase of the residue determination. The options of minimizing the combined uncertainty of measured residues are discussed. The principles described are also applicable to other chemical contaminants.

  8. Characterization Report on Sand, Slag, and Crucible Residues and on Fluoride Residues

    SciTech Connect

    Murray, A.M.

    1999-02-10

    This paper reports on the chemical characterization of the sand, slag, and crucible (SS and C) residues and the fluoride residues that may be shipped from the Rocky Flats Environmental Technology Site (RFETS) to Savannah River Site (SRS).

  9. Soil water evaporation and crop residues

    USDA-ARS?s Scientific Manuscript database

    Crop residues have value when left in the field and also when removed from the field and sold as a commodity. Reducing soil water evaporation (E) is one of the benefits of leaving crop residues in place. E was measured beneath a corn canopy at the soil suface with nearly full coverage by corn stover...

  10. Measurment Of Residual Stress In Ferromagnetic Materials

    NASA Technical Reports Server (NTRS)

    Namkung, Min; Yost, William T.; Kushnick, Peter W.; Grainger, John L.

    1992-01-01

    Magnetoacoustic (MAC) and magnetoacoustic emission (MAE) techniques combined to provide complete characterization of residual stresses in ferromagnetic structural materials. Combination of MAC and MAE techniques makes it possible to characterize residual tension and compression without being limited by surface conditions and unavailability of calibration standards. Significant in field of characterization of materials as well as detection of fatigue failure.

  11. Residuals Management and Water Pollution Control Planning.

    ERIC Educational Resources Information Center

    Environmental Protection Agency, Washington, DC. Office of Public Affairs.

    This pamphlet addresses the problems associated with residuals and water quality especially as it relates to the National Water Pollution Control Program. The types of residuals and appropriate management systems are discussed. Additionally, one section is devoted to the role of citizen participation in developing management programs. (CS)

  12. 40 CFR 158.2290 - Residue chemistry.

    Code of Federal Regulations, 2013 CFR

    2013-07-01

    ... pesticide may be used in a food-handling establishment unless data including, but not limited to... show that residues will not occur in food or feed. 4. If an antimicrobial pesticide may be applied to a... residues of concern, as part of programs to monitor pesticides in the U.S. food supply. 12. Data are...

  13. 40 CFR 158.2290 - Residue chemistry.

    Code of Federal Regulations, 2014 CFR

    2014-07-01

    ... pesticide may be used in a food-handling establishment unless data including, but not limited to... show that residues will not occur in food or feed. 4. If an antimicrobial pesticide may be applied to a... residues of concern, as part of programs to monitor pesticides in the U.S. food supply. 12. Data are...

  14. 40 CFR 240.208 - Residue.

    Code of Federal Regulations, 2012 CFR

    2012-07-01

    ... 40 Protection of Environment 26 2012-07-01 2011-07-01 true Residue. 240.208 Section 240.208 Protection of Environment ENVIRONMENTAL PROTECTION AGENCY (CONTINUED) SOLID WASTES GUIDELINES FOR THE THERMAL PROCESSING OF SOLID WASTES Requirements and Recommended Procedures § 240.208 Residue. ...

  15. 40 CFR 240.208 - Residue.

    Code of Federal Regulations, 2013 CFR

    2013-07-01

    ... 40 Protection of Environment 26 2013-07-01 2013-07-01 false Residue. 240.208 Section 240.208 Protection of Environment ENVIRONMENTAL PROTECTION AGENCY (CONTINUED) SOLID WASTES GUIDELINES FOR THE THERMAL PROCESSING OF SOLID WASTES Requirements and Recommended Procedures § 240.208 Residue. ...

  16. 40 CFR 240.208 - Residue.

    Code of Federal Regulations, 2011 CFR

    2011-07-01

    ... 40 Protection of Environment 25 2011-07-01 2011-07-01 false Residue. 240.208 Section 240.208 Protection of Environment ENVIRONMENTAL PROTECTION AGENCY (CONTINUED) SOLID WASTES GUIDELINES FOR THE THERMAL PROCESSING OF SOLID WASTES Requirements and Recommended Procedures § 240.208 Residue. ...

  17. Crop Residues: The Rest of the Story

    USDA-ARS?s Scientific Manuscript database

    A recent scientific publication stated that to remove CO2 from the atmosphere, the most permanent and rapid solution would be to sink crop residues to the ocean floor where they would be buried in deep ocean sediments. However, mitigating rising atmospheric CO2 concentrations by removing crop residu...

  18. Unicystic ameloblastoma arising from a residual cyst

    PubMed Central

    Mahajan, Amit D; Manjunatha, Bhari Sharanesha; Khurana, Neha M; Shah, Navin

    2014-01-01

    Intraoral swellings involving alveolar ridges in edentulous patients are clinically diagnosed as residual cysts, traumatic bone cysts, Stafne's jaw bone cavity, ameloblastoma and metastatic tumours of the jaw. This case report describes a residual cyst in a 68-year-old edentulous male patient which was enucleated and histopathologically confirmed as a unicystic ameloblastoma. PMID:25199192

  19. Soil water evaporation and crop residues

    USDA-ARS?s Scientific Manuscript database

    Crop residues have value when left in the field and also when removed from the field and sold as a commodity. Reducing soil water evaporation (E) is one of the benefits of leaving crop residues in place. E was measured beneath a corn canopy at the soil suface with nearly full coverage by corn stover...

  20. Does Bt Corn Really Produce Tougher Residues

    USDA-ARS?s Scientific Manuscript database

    Bt corn hybrids produce insecticidal proteins that are derived from a bacterium, Bacillus thuringiensis. There have been concerns that Bt corn hybrids produce residues that are relatively resistant to decomposition. We conducted four experiments that examined the decomposition of corn residues und...

  1. Residuals Management and Water Pollution Control Planning.

    ERIC Educational Resources Information Center

    Environmental Protection Agency, Washington, DC. Office of Public Affairs.

    This pamphlet addresses the problems associated with residuals and water quality especially as it relates to the National Water Pollution Control Program. The types of residuals and appropriate management systems are discussed. Additionally, one section is devoted to the role of citizen participation in developing management programs. (CS)

  2. Residue decomposition of submodel of WEPS

    USDA-ARS?s Scientific Manuscript database

    The Residue Decomposition submodel of the Wind Erosion Prediction System (WEPS) simulates the decrease in crop residue biomass due to microbial activity. The decomposition process is modeled as a first-order reaction with temperature and moisture as driving variables. Decomposition is a function of ...

  3. RECOVERY OF URANIUM VALUES FROM RESIDUES

    DOEpatents

    Schaap, W.B.

    1959-08-18

    A process is described for the recovery of uranium from insoluble oxide residues resistant to repeated leaching with mineral acids. The residue is treated with gaseous hydrogen fluoride, then with hydrogen and again with hydrogen fluoride, preferably at 500 to 700 deg C, prior to the mineral acid leaching.

  4. Processing hardwood bark residues by screening

    Treesearch

    David M. Emanuel

    1978-01-01

    Most of the hardwood bark residues removed by floating-cutterhead or rosserhead debarkers can be processed into acceptable bark products by screening alone. And by prescreening bark residues, operators of bark processing plants can use smaller hammermills than otherwise are required, thus lowering investment and energy costs.

  5. 48 CFR 1450.104 - Residual powers.

    Code of Federal Regulations, 2010 CFR

    2010-10-01

    ... 48 Federal Acquisition Regulations System 5 2010-10-01 2010-10-01 false Residual powers. 1450.104 Section 1450.104 Federal Acquisition Regulations System DEPARTMENT OF THE INTERIOR CONTRACT MANAGEMENT EXTRAORDINARY CONTRACTUAL ACTIONS AND THE SAFETY ACT Extraordinary Contractual Actions 1450.104 Residual powers....

  6. BOOSTER CHLORINATION FOR MANAGING DISINFECTANT RESIDUALS

    EPA Science Inventory

    Booster chlorination is an approach to residual maintenance in which chlorine is applied at strategic locations within the distribution system. Situations in which booster chlorination may be most effective for maintaining a residual are explained informally in the context of a ...

  7. BOOSTER CHLORINATION FOR MANAGING DISINFECTANT RESIDUALS

    EPA Science Inventory

    Booster chlorination is an approach to residual maintenance in which chlorine is applied at strategic locations within the distribution system. Situations in which booster chlorination may be most effective for maintaining a residual are explained informally in the context of a ...

  8. Distribution of veterinary drug residues among muscles

    USDA-ARS?s Scientific Manuscript database

    The U.S. Food and Drug Administration sets tolerances for veterinary drug residues in muscle, but does not specify which muscle should be sampled for analysis. The goal of this research was to determine if antibiotic residue levels are dependent on muscle type. In this study, penicillin G (Pen G) d...

  9. 48 CFR 1450.104 - Residual powers.

    Code of Federal Regulations, 2014 CFR

    2014-10-01

    ... 48 Federal Acquisition Regulations System 5 2014-10-01 2014-10-01 false Residual powers. 1450.104 Section 1450.104 Federal Acquisition Regulations System DEPARTMENT OF THE INTERIOR CONTRACT MANAGEMENT EXTRAORDINARY CONTRACTUAL ACTIONS AND THE SAFETY ACT Extraordinary Contractual Actions 1450.104 Residual powers....

  10. 48 CFR 1450.104 - Residual powers.

    Code of Federal Regulations, 2013 CFR

    2013-10-01

    ... 48 Federal Acquisition Regulations System 5 2013-10-01 2013-10-01 false Residual powers. 1450.104 Section 1450.104 Federal Acquisition Regulations System DEPARTMENT OF THE INTERIOR CONTRACT MANAGEMENT EXTRAORDINARY CONTRACTUAL ACTIONS AND THE SAFETY ACT Extraordinary Contractual Actions 1450.104 Residual powers....

  11. 48 CFR 1450.104 - Residual powers.

    Code of Federal Regulations, 2012 CFR

    2012-10-01

    ... 48 Federal Acquisition Regulations System 5 2012-10-01 2012-10-01 false Residual powers. 1450.104 Section 1450.104 Federal Acquisition Regulations System DEPARTMENT OF THE INTERIOR CONTRACT MANAGEMENT EXTRAORDINARY CONTRACTUAL ACTIONS AND THE SAFETY ACT Extraordinary Contractual Actions 1450.104 Residual powers....

  12. 48 CFR 1450.104 - Residual powers.

    Code of Federal Regulations, 2011 CFR

    2011-10-01

    ... 48 Federal Acquisition Regulations System 5 2011-10-01 2011-10-01 false Residual powers. 1450.104 Section 1450.104 Federal Acquisition Regulations System DEPARTMENT OF THE INTERIOR CONTRACT MANAGEMENT EXTRAORDINARY CONTRACTUAL ACTIONS AND THE SAFETY ACT Extraordinary Contractual Actions 1450.104 Residual powers....

  13. Residue management: Back to the roots

    USDA-ARS?s Scientific Manuscript database

    Addressing the issues of climate change and sustainable biomass feedstocks have soil as a common theme. Managing crop residues is directly related to soil management. Understanding how soil and crop residue management interact provides insight on how to assure agricultural soil can serve as a carbon...

  14. Multi-state complex angular momentum residues

    NASA Astrophysics Data System (ADS)

    Thylwe, Karl-Erik

    2006-09-01

    A relation between a multi-state complex angular momentum (CAM) pole residue and the corresponding CAM-state wavefunction is derived for a real symmetric potential matrix. The result generalizes a residue formula available for single-channel atomical collision systems and it is based on a diagonalization of the S matrix together with the use of exact Wronskian relations.

  15. Tank 12H residuals sample analysis report

    SciTech Connect

    Oji, L. N.; Shine, E. P.; Diprete, D. P.; Coleman, C. J.; Hay, M. S.

    2015-06-11

    The Savannah River National Laboratory (SRNL) was requested by Savannah River Remediation (SRR) to provide sample preparation and analysis of the Tank 12H final characterization samples to determine the residual tank inventory prior to grouting. Eleven Tank 12H floor and mound residual material samples and three cooling coil scrape samples were collected and delivered to SRNL between May and August of 2014.

  16. 40 CFR 240.208 - Residue.

    Code of Federal Regulations, 2010 CFR

    2010-07-01

    ... 40 Protection of Environment 24 2010-07-01 2010-07-01 false Residue. 240.208 Section 240.208 Protection of Environment ENVIRONMENTAL PROTECTION AGENCY (CONTINUED) SOLID WASTES GUIDELINES FOR THE THERMAL PROCESSING OF SOLID WASTES Requirements and Recommended Procedures § 240.208 Residue. ...

  17. Residue contact-count potentials are as effective as residue-residue contact-type potentials for ranking protein decoys

    PubMed Central

    Bolser, Dan M; Filippis, Ioannis; Stehr, Henning; Duarte, Jose; Lappe, Michael

    2008-01-01

    Background For over 30 years potentials of mean force have been used to evaluate the relative energy of protein structures. The most commonly used potentials define the energy of residue-residue interactions and are derived from the empirical analysis of the known protein structures. However, single-body residue 'environment' potentials, although widely used in protein structure analysis, have not been rigorously compared to these classical two-body residue-residue interaction potentials. Here we do not try to combine the two different types of residue interaction potential, but rather to assess their independent contribution to scoring protein structures. Results A data set of nearly three thousand monomers was used to compare pairwise residue-residue 'contact-type' propensities to single-body residue 'contact-count' propensities. Using a large and standard set of protein decoys we performed an in-depth comparison of these two types of residue interaction propensities. The scores derived from the contact-type and contact-count propensities were assessed using two different performance metrics and were compared using 90 different definitions of residue-residue contact. Our findings show that both types of score perform equally well on the task of discriminating between near-native protein decoys. However, in a statistical sense, the contact-count based scores were found to carry more information than the contact-type based scores. Conclusion Our analysis has shown that the performance of either type of score is very similar on a range of different decoys. This similarity suggests a common underlying biophysical principle for both types of residue interaction propensity. However, several features of the contact-count based propensity suggests that it should be used in preference to the contact-type based propensity. Specifically, it has been shown that contact-counts can be predicted from sequence information alone. In addition, the use of a single-body term allows

  18. 40 CFR 180.519 - Bromide ion and residual bromine; tolerances for residues.

    Code of Federal Regulations, 2014 CFR

    2014-07-01

    ... polybrominated ion-exchange resin (as a source of bromine) under the supervision of trained personnel. (2... 40 Protection of Environment 24 2014-07-01 2014-07-01 false Bromide ion and residual bromine... Tolerances § 180.519 Bromide ion and residual bromine; tolerances for residues. (a) General. The food...

  19. Residuals and the Residual-Based Statistic for Testing Goodness of Fit of Structural Equation Models

    ERIC Educational Resources Information Center

    Foldnes, Njal; Foss, Tron; Olsson, Ulf Henning

    2012-01-01

    The residuals obtained from fitting a structural equation model are crucial ingredients in obtaining chi-square goodness-of-fit statistics for the model. The authors present a didactic discussion of the residuals, obtaining a geometrical interpretation by recognizing the residuals as the result of oblique projections. This sheds light on the…

  20. Residuals and the Residual-Based Statistic for Testing Goodness of Fit of Structural Equation Models

    ERIC Educational Resources Information Center

    Foldnes, Njal; Foss, Tron; Olsson, Ulf Henning

    2012-01-01

    The residuals obtained from fitting a structural equation model are crucial ingredients in obtaining chi-square goodness-of-fit statistics for the model. The authors present a didactic discussion of the residuals, obtaining a geometrical interpretation by recognizing the residuals as the result of oblique projections. This sheds light on the…

  1. Computational Prediction of Hot Spot Residues

    PubMed Central

    Morrow, John Kenneth; Zhang, Shuxing

    2013-01-01

    Most biological processes involve multiple proteins interacting with each other. It has been recently discovered that certain residues in these protein-protein interactions, which are called hot spots, contribute more significantly to binding affinity than others. Hot spot residues have unique and diverse energetic properties that make them challenging yet important targets in the modulation of protein-protein complexes. Design of therapeutic agents that interact with hot spot residues has proven to be a valid methodology in disrupting unwanted protein-protein interactions. Using biological methods to determine which residues are hot spots can be costly and time consuming. Recent advances in computational approaches to predict hot spots have incorporated a myriad of features, and have shown increasing predictive successes. Here we review the state of knowledge around protein-protein interactions, hot spots, and give an overview of multiple in silico prediction techniques of hot spot residues. PMID:22316154

  2. Process for treatment of residual gas

    SciTech Connect

    Nolden, K.

    1980-01-01

    A process is disclosed for the treatment of the residual gases which are produced when hydrogen sulfide is reduced, by combustion, to elementary sulfur by the Claus process. The residual gases are fed through a heated conduit and gas scrubber, wherein the temperature of those residual gases are maintained above the melting point of sulfur. A portion of the raw coke oven gas condensate is admitted to the gas scrubber to be returned to the coke oven battery main from the flushing liquid separator as flushing liquor. The residual gases are then conducted through the coke oven gas purification process equipment along with the raw coke oven gas where the residual gases are intermixed with the raw coke oven gas prior to tar separation.

  3. Recycling of auto shredder residue.

    PubMed

    Nourreddine, Menad

    2007-01-31

    Currently, about 75% of end-of-life vehicle's (ELV) total weight is recycled in EU countries. The remaining 25%, which is called auto shredder residues (ASR) or auto fluff, is disposed of as landfill because of its complexity. It is a major challenge to reduce this percentage of obsolete cars. The European draft directive states that by the year 2006, only 15% of the vehicle's weight can be disposed of at landfill sites and by 2015, this will be reduced to 5%. The draft directive states that a further 10% can be incinerated. The quantities of shredder fluff are likely to increase in the coming years. This is because of the growing number of cars being scrapped, coupled with the increase in the amount of plastics used in cars. In Sweden, some current projects are focusing on recycling of ASR material. In this paper some different alternatives for using this material are reported. The hypothetical injection of ASR into a blast furnace concentrating on ASR's effect to some blast furnace (BF) parameters has been completed using a blast furnace mass balance model. As a result, in principle, ASR can be used as reducing agent in the BF process if certain conditions are met. The particle size of ASR material must be controlled to ensure optimal gasification of the material in the raceway. Regarding the chemical composition of ASR, the non-ferrous content can affect the pig iron quality, which is difficult to rectify at a later point. The most attractive recycling alternative is to use the products obtained from pyrolysis of ASR in appropriate metallurgical processes.

  4. Residues of veterinary drugs at injection sites.

    PubMed

    Reeves, P T

    2007-02-01

    Residues of veterinary drugs have potential implications for human food safety and international trade in animal-derived food commodities. A particular concern is the slow depletion of residues of some injectable formulations from the site of administration. Licensing authorities have adopted different approaches to the human food safety assessment of injection site residues. European agencies apply the maximum residue limit (MRL) for muscle to muscle at the injection site and specify a withdrawal period sufficient to ensure the ingestion of a 300 g portion of muscle, if comprised entirely of injection site tissue, does not exceed the acceptable daily intake. The agencies in Australia, Canada and the USA also exclude injection site residues from the MRL-setting process. These agencies evaluate the risk to consumers posed by potential acute manifestations resulting from the infrequent ingestion of injection site residues based on acute dietary exposure considerations. While all of these approaches protect the safety of consumers, the adoption of different approaches has potential implications for residue surveillance programs in the international trade in meat. In particular, when an exporting country establishes standards for residues at injection sites based on acute dietary exposure considerations and the importing country assesses these residues against the MRL for muscle, the unnecessary condemnation of meat and disruption to market access may result. The latter may represent a potential economical impost to the exporting country. An internationally harmonized approach to the risk analysis of residues of veterinary drugs at injection sites, which protects the safety of consumers and facilitates the international trade in meat, is needed.

  5. The Sec7 N-terminal regulatory domains facilitate membrane-proximal activation of the Arf1 GTPase

    PubMed Central

    Richardson, Brian C; Halaby, Steve L; Gustafson, Margaret A; Fromme, J Christopher

    2016-01-01

    The Golgi complex is the central sorting compartment of eukaryotic cells. Arf guanine nucleotide exchange factors (Arf-GEFs) regulate virtually all traffic through the Golgi by activating Arf GTPase trafficking pathways. The Golgi Arf-GEFs contain multiple autoregulatory domains, but the precise mechanisms underlying their function remain largely undefined. We report a crystal structure revealing that the N-terminal DCB and HUS regulatory domains of the Arf-GEF Sec7 form a single structural unit. We demonstrate that the established role of the N-terminal region in dimerization is not conserved; instead, a C-terminal autoinhibitory domain is responsible for dimerization of Sec7. We find that the DCB/HUS domain amplifies the ability of Sec7 to activate Arf1 on the membrane surface by facilitating membrane insertion of the Arf1 amphipathic helix. This enhancing function of the Sec7 N-terminal domains is consistent with the high rate of Arf1-dependent trafficking to the plasma membrane necessary for maximal cell growth. DOI: http://dx.doi.org/10.7554/eLife.12411.001 PMID:26765562

  6. Structure of the Membrane Proximal Oxioreductase Domain of Human Steap3, the Dominant Ferrireductase of the Erythroid Transferrin Cycle

    SciTech Connect

    Sendamarai, A.K.; Ohgami, R.S.; Fleming, M.D.; Lawrence, C.M.

    2009-05-27

    The daily production of 200 billion erythrocytes requires 20 mg of iron, accounting for nearly 80% of the iron demand in humans. Thus, erythroid precursor cells possess an efficient mechanism for iron uptake in which iron loaded transferrin (Tf) binds to the transferrin receptor (TfR) at the cell surface. The Tf:TfR complex then enters the endosome via receptor-mediated endocytosis. Upon endosomal acidification, iron is released from Tf, reduced to Fe{sup 2+} by Steap3, and transported across the endosomal membrane by divalent metal iron transporter 1. Steap3, the major ferrireductase in erythrocyte endosomes, is a member of a unique family of reductases. Steap3 is comprised of an N-terminal cytosolic oxidoreductase domain and a C-terminal heme-containing transmembrane domain. Cytosolic NADPH and a flavin are predicted cofactors, but the NADPH/flavin binding domain differs significantly from those in other eukaryotic reductases. Instead, Steap3 shows remarkable, although limited homology to FNO, an archaeal oxidoreductase. We have determined the crystal structure of the human Steap3 oxidoreductase domain in the absence and presence of NADPH. The structure reveals an FNO-like domain with an unexpected dimer interface and substrate binding sites that are well positioned to direct electron transfer from the cytosol to a heme moiety predicted to be fixed within the transmembrane domain. Here, we discuss possible gating mechanisms for electron transfer across the endosomal membrane.

  7. Non-local residue-residue contacts in proteins are more conserved than local ones.

    PubMed

    Noivirt-Brik, Orly; Hazan, Gershon; Unger, Ron; Ofran, Yanay

    2013-02-01

    Non-covalent residue-residue contacts drive the folding of proteins and stabilize them. They may be local-i.e. involve residues that are close in sequence, or non-local. It has been suggested that, in most proteins, local contacts drive protein folding by providing crucial constraints of the conformational space, thus allowing proteins to fold. We compared residues that are involved in local contacts to residues that are involved in non-local contacts and found that, in most proteins, residues in non-local contacts are significantly more conserved evolutionarily than residues in local contacts. Moreover, non-local contacts are more structurally conserved: a contact between positions that are distant in sequence is more likely to exist in many structural homologues compared with a contact between positions that are close in sequence. These results provide new insights into the mechanisms of protein folding and may allow for better prediction of critical intra-chain contacts.

  8. Antioxidant properties of roasted coffee residues.

    PubMed

    Yen, Wen-Jye; Wang, Bor-Sen; Chang, Lee-Wen; Duh, Pin-Der

    2005-04-06

    The antioxidant activity of roasted coffee residues was evaluated. Extraction with four solvents (water, methanol, ethanol, and n-hexane) showed that water extracts of roasted coffee residues (WERCR) produced higher yields and gave better protection for lipid peroxidation. WERCR showed a remarkable protective effect on oxidative damage of protein. In addition, WERCR showed scavenging of free radicals as well as the reducing ability and to bind ferrous ions, indicating that WERCR acts as both primary and secondary antioxidants. The HPLC analyses showed that phenolic acids (chlorogenic acid and caffeic acid) and nonphenolic compounds [caffeine, trigonelline, nicotinic acid, and 5-(hydroxymethyl)furfuraldehyde] remained in roasted coffee residues. These compounds showed a protective effect on a liposome model system. The concentrations of flavonoids and polyphenolic compounds in roasted coffee residues were 8,400 and 20,400 ppm, respectively. In addition, the Maillard reaction products (MRPs) remaining in roasted coffee residues were believed to show antioxidant activity. These data indicate that roasted coffee residues have excellent potential for use as a natural antioxidant source because the antioxidant compounds remained in roasted coffee residues.

  9. Prediction of machining induced residual stresses

    NASA Astrophysics Data System (ADS)

    Pramod, Monangi; Reddy, Yarkareddy Gopi; Prakash Marimuthu, K.

    2017-07-01

    Whenever a component is machined, residual stresses are induced in it. These residual stresses induced in the component reduce its fatigue life, corrosion resistance and wear resistance. Thus it is important to predict and control the machining-induced residual stress. A lot of research is being carried out in this area in the past decade. This paper aims at prediction of residual stresses during machining of Ti-6Al-4V. A model was developed and under various combinations of cutting conditions such as, speed, feed and depth of cut, the behavior of residual stresses were simulated using Finite Element Model. The present work deals with the development of thermo-mechanical model to predict the machining induced residual stresses in Titanium alloy. The simulation results are compared with the published results. The results are in good agreement with the published results. Future work involves optimization or the cutting parameters that effect the machining induced residual stresses. The results obtained were validated with previous work.

  10. Simple approach for ranking structure determining residues.

    PubMed

    Luna-Martínez, Oscar D; Vidal-Limón, Abraham; Villalba-Velázquez, Miryam I; Sánchez-Alcalá, Rosalba; Garduño-Juárez, Ramón; Uversky, Vladimir N; Becerril, Baltazar

    2016-01-01

    Mutating residues has been a common task in order to study structural properties of the protein of interest. Here, we propose and validate a simple method that allows the identification of structural determinants; i.e., residues essential for preservation of the stability of global structure, regardless of the protein topology. This method evaluates all of the residues in a 3D structure of a given globular protein by ranking them according to their connectivity and movement restrictions without topology constraints. Our results matched up with sequence-based predictors that look up for intrinsically disordered segments, suggesting that protein disorder can also be described with the proposed methodology.

  11. Simple approach for ranking structure determining residues

    PubMed Central

    Luna-Martínez, Oscar D.; Vidal-Limón, Abraham; Villalba-Velázquez, Miryam I.; Sánchez-Alcalá, Rosalba; Garduño-Juárez, Ramón; Uversky, Vladimir N.

    2016-01-01

    Mutating residues has been a common task in order to study structural properties of the protein of interest. Here, we propose and validate a simple method that allows the identification of structural determinants; i.e., residues essential for preservation of the stability of global structure, regardless of the protein topology. This method evaluates all of the residues in a 3D structure of a given globular protein by ranking them according to their connectivity and movement restrictions without topology constraints. Our results matched up with sequence-based predictors that look up for intrinsically disordered segments, suggesting that protein disorder can also be described with the proposed methodology. PMID:27366642

  12. Diazinon residues in insects from sprayed tobacco

    USGS Publications Warehouse

    Stromborg, K.L.; Beyer, W.N.; Kolbe, E.

    1982-01-01

    Pooled samples of tobacco hornworms collected from a field sprayed with 0.84 kg/ha of diazinon were analyzed for residues at various intervals after application. No residues of the toxic metabolite diazoxon were detected (sensitivity 0.5 ppm) in any sample. Only one sample exceeded 1.0 ppm of the parent compound and was collected 4 hours after spraying. Residues declined over time (P<0.01) and none were detected (sensitivity 0.1 ppm) 18 days after spraying. the potential hazard to birds eating these insects appeared to be minimal.

  13. Residual entanglement of accelerated fermions is useful

    NASA Astrophysics Data System (ADS)

    Farahmand, Mehrnoosh; Mohammadzadeh, Hosein; Rahimi, Robabeh; Mehri-Dehnavi, Hossein

    2017-08-01

    The non-vanishing residual entanglement, between the fermionic modes in the infinite acceleration limit, does not violate CHSH inequality, therefore it is not non-local. In this paper, we study the usefulness of the residual fermionic entanglement in single mode approximation and beyond single mode approximation. It is shown that there are some cases where the CHSH inequality is not violated by the residual entanglement, but the state is useful for quantum teleportation. Conditions for the violation of the CHSH inequality in terms of the ;presence probability; of the particle in different Rindler regions are given for the state to be useful for teleportation and superdense coding.

  14. RESIDUAL RISK ASSESSMENT: PCE DRY CLEANERS ...

    EPA Pesticide Factsheets

    This document describes the residual risk assessement for the Perchloroethylene Dry Cleaners source category. For stationary sources, section 112(f) of the Clean Air Act requires EPA to assess risks to human health and the environment following implementation of technology-based control standards. If these technology-based control standards do not provide an ample margin of safety, then EPA is required to promulgate additional standards. The purpose of this document is to describe the methodology and results of teh residual risk assessment performed for the Perchloroethylene Dry Cleaners source category. The results of this analysis will assist EPA in determining whether a residual risk rule for this source category is appropriate.

  15. Biomass energy from crop and forest residues.

    PubMed

    Pimentel, D; Moran, M A; Fast, S; Weber, G; Bukantis, R; Balliett, L; Boveng, P; Cleveland, C; Hindman, S; Young, M

    1981-06-05

    Residues remaining after the harvest of crop and forestry products are being proposed as a substantial energy source for the nation. An estimated 22 percent of the residues might be utilized, providing a renewable source of high-grade energy with the potential of supplying 1 percent of the current U.S. gasoline consumption as ethanol or 4 percent of the total electrical energy used. These net energy benefits are limited by high energy costs to collect, transport, and process the residues. Environmental threats include soil erosion, water runoff, and nutrient loss.

  16. Image-based Measurement of Post-Swallow Residue: The Normalized Residue Ratio Scale

    PubMed Central

    Pearson, William G.; Smith, Zachary M.; Steele, Catriona M.

    2012-01-01

    Post-swallow residue is considered a sign of swallowing impairment. Existing methods for capturing post-swallow residue (perceptual and quantitative) have inherent limitations. We employed several different perceptual and quantitative (ratio) methods for measuring post-swallow residue on the same 40 swallows and addressed the following questions: (1) Do perceptual and quantitative methods demonstrate good agreement? (2) What differences in precision are apparent by measurement method (one-dimensional, two-dimensional, and circumscribed area ratios)? (3) Do residue ratios agree strongly with residue area measures that are anatomically normalized? Based on the findings of this series of questions, a new method for capturing residue is proposed: the Normalized Residue Ratio Scale (NRRS). The NRRS is a continuous measurement that incorporates both the ratio of residue relative to the available pharyngeal space and the residue proportionate to the size of the individual. A demonstration of this method is presented to illustrate the added precision of the NRRS measurement in comparison to other approaches for measuring residue severity. PMID:23089830

  17. Recovery of transuranics from process residues

    SciTech Connect

    Gray, J.H.; Gray, L.W.

    1987-01-01

    Process residues are generated at both the Rocky Flats Plant (RFP) and the Savannah River Plant (SRP) during aqueous chemical and pyrochemical operations. Frequently, process operations will result in either impure products or produce residues sufficiently contaminated with transuranics to be nondiscardable as waste. Purification and recovery flowsheets for process residues have been developed to generate solutions compatible with subsequent Purex operations and either solid or liquid waste suitable for disposal. The ''scrub alloy'' and the ''anode heel alloy'' are examples of materials generated at RFP which have been processed at SRP using the developed recovery flowsheets. Examples of process residues being generated at SRP for which flowsheets are under development include LECO crucibles and alpha-contaminated hydraulic oil.

  18. Quantifying logging residue - before the fact

    SciTech Connect

    Bones, J.T.

    1982-06-01

    Tree biomass estimation, which is being integrated into the U.S. Forest Service Renewable Resources Evaluation Program, will give foresters the ability to estimate the amount of logging residues they might expect from harvested treetops and branches and residual rough, rotten, and small trees before the actual harvest. With planning, and increased demand for such timber products as pulpwood and fuelwood, product recovery could be increased by up to 43 percent in softwood stands and 99% in hardwoods. Recovery levels affect gross product receipts and site preparation costs. An example of product recovery and residue generation is presented for three harvesting options in Pennsylvania hardwood stands. Under the whole-tree harvesting option, 46% more product was recovered than in single product harvesting, and logging residue levels were reduced by 58%.

  19. Properties of Rasch residual fit statistics.

    PubMed

    Wu, Margaret; Adams, Richard J

    2013-01-01

    This paper examines the residual-based fit statistics commonly used in Rasch measurement. In particular, the paper analytically examines some of the theoretical properties of the residual-based fit statistics with a view to establishing the inferences that can be made using these fit statistics. More specifically, the relationships between the distributional properties of the fit statistics and sample size are discussed; some research that erroneously concludes that residual-based fit statistics are unstable is reviewed; and finally, it is analytically illustrated that, for dichotomous items, residual-based fit statistics provide a measure of the relative slope of empirical item characteristic curves. With a clear understanding of the theoretical properties of the fit statistics, the use and limitations of these statistics can be placed in the right light.

  20. Residual strength of damaged marine structures

    NASA Astrophysics Data System (ADS)

    Ghose, Dhruba J.; Nappi, Natale S.; Wiernicki, Christopher J.

    1994-09-01

    Traditionally assessment of ship's longitudinal strength has been made by comparing the elastic stresses at the deck or bottom shell to fractions of the material yield strength. This results in high reserve capacity due to inherent redundancies in ship structures. Residual strength, which is defined as the strength of the structure after damage, has rarely been considered either during design or at the time of repair. In this report, key elements required to undertake an engineering analysis to evaluate the residual strength have been identified. Emphasis has been placed on assessing the residual strength of marine structures damaged due to normal operating loads. Methods available to industry for evaluation of damage such as, fracture and ultimate strength have been summarized. An example problem, illustrating the application of an integrated approach to residual strength assessment on a particular ship type, is presented.

  1. SAR impulse response with residual chirps.

    SciTech Connect

    Doerry, Armin Walter

    2009-06-01

    A Linear Frequency-Modulated (LFM) chirp is a function with unit amplitude and quadratic phase characteristic. In a focused Synthetic Aperture Radar (SAR) image, a residual chirp is undesired for targets of interest, as it coarsens the manifested resolution. However, for undesired spurious signals, a residual chirp is often advantageous because it spreads the energy and thereby diminishes its peak value. In either case, a good understanding of the effects of a residual LFM chirp on a SAR Impulse Response (IPR) is required to facilitate system analysis and design. This report presents an analysis of the effects of a residual chirp on the IPR. As reference, there is a rich body of publications on various aspects of LFM chirps. A quick search reveals a plethora of articles, going back to the early 1950s. We mention here purely as trivia one of the earlier analysis papers on this waveform by Klauder, et al.

  2. Ultrasonics used to measure residual stress

    NASA Technical Reports Server (NTRS)

    1967-01-01

    Ultrasonic method is used to measure residual stress in metal structures. By using this method, various forms of wave propagation in metals are possible, and more thorough analysis of complex geometric structures may be had.

  3. Study of Selected Petroleum Refining Residuals

    EPA Pesticide Factsheets

    The document describes EPA's approach to conducting the industry study required by the Environmental Defense Fund (EDF)/EPA consent decree and characterizes the study residuals and how they are managed.

  4. RESIDUAL STRESS IN HARDENED STEEL CYLINDERS

    DTIC Science & Technology

    ultimate strength of the steel and in some instances caused cracking, and (4) stress patterns of interrupted quench specimens were not consistent enough to warrant a conclusion. (Author)...A study was conducted to (1) measure residual stress in hardened steel solid cylinders, (2) correlate the stress values with heat treatments, and (3...develop a dissolution technique. Residual stress patterns for 12 solid cylinders of 4160 steel, heat treated by various methods, were determined

  5. GLC determination of quinaldine residue in fish

    USGS Publications Warehouse

    Allen, J.L.; Sills, J.B.

    1970-01-01

    A procedure for the determination of quinaldine residue in various fish tissues is described. Homogenized tissues are extracted wi th hexane-ethyl ether, the extracts are concentrated by partitioning through O.IN sulfuric acid, and the residues are measured by alkali Harne ionization gas chromatography. Muscle tissues containing from 0.01 to 10.0 ppm quinaldine were successfully analyzed with recoveries from 75 to 100%.

  6. Detecting organic gunpowder residues from handgun use

    NASA Astrophysics Data System (ADS)

    MacCrehan, William A.; Ricketts, K. Michelle; Baltzersen, Richard A.; Rowe, Walter F.

    1999-02-01

    The gunpowder residues that remain after the use of handguns or improvised explosive devices pose a challenge for the forensic investigator. Can these residues be reliably linked to a specific gunpowder or ammunition? We investigated the possibility by recovering and measuring the composition of organic additives in smokeless powder and its post-firing residues. By determining gunpowder additives such as nitroglycerin, dinitrotoluene, ethyl- and methylcentralite, and diphenylamine, we hope to identify the type of gunpowder in the residues and perhaps to provide evidence of a match to a sample of unfired powder. The gunpowder additives were extracted using an automated technique, pressurized fluid extraction (PFE). The conditions for the quantitative extraction of the additives using neat and solvent-modified supercritical carbon dioxide were investigated. All of the major gunpowder additives can be determined with baseline resolution using capillary electrophoresis (CE) with a micellar agent and UV absorbance detection. A study of candidate internal standards for use in the CE method is also presented. The PFE/CE technique is used to evaluate a new residue sampling protocol--asking shooters to blow their noses. In addition, an initial investigation of the compositional differences among unfired and post-fired .22 handgun residues is presented.

  7. Axial residual stresses in boron fibers

    NASA Technical Reports Server (NTRS)

    Behrendt, D. R.

    1978-01-01

    The axial residual stress distribution as a function of radius was determined from the fiber surface to the core including the average residual stress in the core. Such measurements on boron on tungsten (B/W) fibers show that the residual stresses for 102, 142, 203, and 366 micron diameter fibers were similar, being compressive at the surface and changing monotonically to a region of tensile within the boron. At approximately 25 percent of the original radius, the stress reaches a maximum tensile stress of about 860 mn/sq.m and then decreases to a compressive stress near the tungsten boride core. Data were presented for 203 micron diameter B/W fibers that show annealing above 900 C reduces the residual stresses. A comparison between 102 micron diameter B/W and boron on carbon (b/C) shows that the residual stresses were similar in the outer regions of the fibers, but that large differences near and in the core were observed. The effects of these residual stresses on the fracture of boron fibers were discussed.

  8. Method for residual household waste composition studies.

    PubMed

    Sahimaa, Olli; Hupponen, Mari; Horttanainen, Mika; Sorvari, Jaana

    2015-12-01

    The rising awareness of decreasing natural resources has brought forward the idea of a circular economy and resource efficiency in Europe. As a part of this movement, European countries have identified the need to monitor residual waste flows in order to make recycling more efficient. In Finland, studies on the composition of residual household waste have mostly been conducted using different methods, which makes the comparison of the results difficult. The aim of this study was to develop a reliable method for residual household waste composition studies. First, a literature review on European study methods was performed. Also, 19 Finnish waste composition studies were compared in order to identify the shortcomings of the current Finnish residual household waste composition data. Moreover, the information needs of different waste management authorities concerning residual household waste were studied through a survey and personal interviews. Stratification, sampling, the classification of fractions and statistical analysis were identified as the key factors in a residual household waste composition study. The area studied should be divided into non-overlapping strata in order to decrease the heterogeneity of waste and enable comparisons between different waste producers. A minimum of six subsamples, each 100 kg, from each stratum should be sorted. Confidence intervals for each waste category should be determined in order to evaluate the applicability of the results. A new three-level classification system was created based on Finnish stakeholders' information needs and compared to four other European waste composition study classifications.

  9. Residues of oxytetracycline in cultured rainbow trout.

    PubMed

    Sharafati-Chaleshtori, R; Mardani, G; Rafieian-Kopaei, M; Sharafati-Chaleshtori, A; Drees, F

    2013-11-01

    Nowadays, antibiotics are widely used in aquatic animals to control and treatment of infections or as food supplement for growth increase and animal output. With increasing use of veterinary drugs in food production, there is global consideration about the consumption of antimicrobial residues in aquatic foods and their effects on human health. This study was aimed to evaluate the Oxytetracycline (OTC) residues in Rainbow trout meat in Shahre-kord (Iran) markets before and after frying. After randomized collection of 50 samples of fish in Shahre-kord markets in a six months period were examined. The prepared samples were examined for OTC residues using HPLC analytical method before and after frying. Results showed that 3 (6%) of the samples before frying and 12 (24%) after frying were having lower than Maximum residual limits (MRLs) in Codex alimentarius. However, mean OTC residues before and after frying samples were above MRLs. The mean amounts of OTC were 2260 +/- 1090 and 1110 +/- 930 ng g(-1) before and after frying, respectively. These findings show that the frying of fish reduces OTC residual. Nevertheless, the usage of OTC should be reduced to an acceptable level in fishery industry.

  10. Pyrethroid insecticide residues on vegetable crops.

    PubMed

    Ripley, B D; Ritcey, G M; Harris, C R; Denommé, M A; Brown, P D

    2001-08-01

    Pyrethroid insecticides were applied on various vegetable crops as foliar treatments to determine dissipation rates. On Chinese broccoli (Guy Lon), Chinese mustard (Pak Choi) and Chinese cabbage (Kasumi, napa), fenvalerate was persistent with residues of 0.10, 0.14 and 0.11 mg kg-1, respectively, by day 21. Cypermethrin residues on head lettuce were below 0.1 mg kg-1 by day 10 but on the leafier romaine and endive varieties it was more persistent and required 14-19 days to dissipate below this concentration. After three applications, residues of cypermethrin in harvested carrots and of permethrin in eggplant were not detected on the day of application. On asparagus, deltamethrin and cypermethrin residues declined to less than 0.1 mg kg-1 by days 1 and 2, respectively; permethrin was more persistent, requiring more than 2 days to decline to less than 0.1 mg kg-1. Deltamethrin on dry (cooking) and Spanish onions was not detected on the day of application. On tomatoes, the concentration of permethrin was 0.093 mg kg-1 on the day of application and declined to about 0.05 mg kg-1 after 2-4 days. In general, permethrin, cypermethrin and deltamethrin residues declined to acceptable concentrations within an acceptable pre-harvest interval. Fenvalerate may be too persistent on these speciality crops unless a maximum residue limit > 0.1 mg kg-1 is permitted.

  11. RESIDUAL ENERGY SPECTRUM OF SOLAR WIND TURBULENCE

    SciTech Connect

    Chen, C. H. K.; Bale, S. D.; Salem, C. S.; Maruca, B. A.

    2013-06-20

    It has long been known that the energy in velocity and magnetic field fluctuations in the solar wind is not in equipartition. In this paper, we present an analysis of 5 yr of Wind data at 1 AU to investigate the reason for this. The residual energy (difference between energy in velocity and magnetic field fluctuations) was calculated using both the standard magnetohydrodynamic (MHD) normalization for the magnetic field and a kinetic version, which includes temperature anisotropies and drifts between particle species. It was found that with the kinetic normalization, the fluctuations are closer to equipartition, with a mean normalized residual energy of {sigma}{sub r} = -0.19 and mean Alfven ratio of r{sub A} = 0.71. The spectrum of residual energy, in the kinetic normalization, was found to be steeper than both the velocity and magnetic field spectra, consistent with some recent MHD turbulence predictions and numerical simulations, having a spectral index close to -1.9. The local properties of residual energy and cross helicity were also investigated, showing that globally balanced intervals with small residual energy contain local patches of larger imbalance and larger residual energy at all scales, as expected for nonlinear turbulent interactions.

  12. Total residual chlorine as a regulatory tool

    SciTech Connect

    Mattice, J.S.; Tsai, S.C.

    1981-01-01

    Inherent in the choice of total residual chlorine (TRC) as a basis for establishing water quality criteria or effluent guidelines for chlorine are assumptions that components of TRC are roughly equivalent in toxicity to aquatic species and that the toxicities of these components are additive. Most of the studies of this assumption involved comparisons of mixtures of residual chlorine components and thus led to conflicting conclusions. In addition, studies designed to avoid the mixture problem omitted one or more of the most likely products of chlorination from the comparison(s). On the other hand, the assumption of additive toxicity of residual chlorine species has largely been ignored. In view of the continuing controversy that focused on the levels of residual chlorine which can be discharged without deleterious impact on receiving waters, it is important that these assumptions be evaluated comprehensively. New data on the toxicities to the mosquitofish, Gambusia affinis, of inorganic monochloramine, inorganic dichloramine, a mixture of these two compounds, and two mixtures with free chlorine are reported. These and other data on the toxicity of residual chlorine species to mosquitofish are then used to examine the questions of toxicity equivalence and additivity of species of residual chlorine. (ERB)

  13. Fluorescence imaging to quantify crop residue cover

    NASA Technical Reports Server (NTRS)

    Daughtry, C. S. T.; Mcmurtrey, J. E., III; Chappelle, E. W.

    1994-01-01

    Crop residues, the portion of the crop left in the field after harvest, can be an important management factor in controlling soil erosion. Methods to quantify residue cover are needed that are rapid, accurate, and objective. Scenes with known amounts of crop residue were illuminated with long wave ultraviolet (UV) radiation and fluorescence images were recorded with an intensified video camera fitted with a 453 to 488 nm band pass filter. A light colored soil and a dark colored soil were used as background for the weathered soybean stems. Residue cover was determined by counting the proportion of the pixels in the image with fluorescence values greater than a threshold. Soil pixels had the lowest gray levels in the images. The values of the soybean residue pixels spanned nearly the full range of the 8-bit video data. Classification accuracies typically were within 3(absolute units) of measured cover values. Video imaging can provide an intuitive understanding of the fraction of the soil covered by residue.

  14. 40 CFR 180.417 - Triclopyr; tolerances for residues.

    Code of Federal Regulations, 2014 CFR

    2014-07-01

    ... § 180.417 Triclopyr; tolerances for residues. (a) General. (1) Tolerances for residues of the herbicide...) Tolerances for the combined residues of the herbicide triclopyr ((3,5,6-trichloro-2-pyridinyl)oxy)...

  15. 40 CFR 180.417 - Triclopyr; tolerances for residues.

    Code of Federal Regulations, 2012 CFR

    2012-07-01

    ... § 180.417 Triclopyr; tolerances for residues. (a) General. (1) Tolerances for residues of the herbicide...) Tolerances for the combined residues of the herbicide triclopyr ((3,5,6-trichloro-2-pyridinyl)oxy)...

  16. 40 CFR 180.417 - Triclopyr; tolerances for residues.

    Code of Federal Regulations, 2013 CFR

    2013-07-01

    ... § 180.417 Triclopyr; tolerances for residues. (a) General. (1) Tolerances for residues of the herbicide...) Tolerances for the combined residues of the herbicide triclopyr ((3,5,6-trichloro-2-pyridinyl)oxy)...

  17. 40 CFR 180.417 - Triclopyr; tolerances for residues.

    Code of Federal Regulations, 2011 CFR

    2011-07-01

    ... § 180.417 Triclopyr; tolerances for residues. (a) General. (1) Tolerances for residues of the herbicide...) Tolerances for the combined residues of the herbicide triclopyr ((3,5,6-trichloro-2-pyridinyl)oxy)...

  18. 40 CFR 180.432 - Lactofen; tolerances for residues.

    Code of Federal Regulations, 2012 CFR

    2012-07-01

    ... Lactofen; tolerances for residues. (a) General. Tolerances are established for residues of the herbicide... for residues of the herbicide lactofen, including its metabolites and degradates, in or on the...

  19. 40 CFR 180.339 - MCPA; tolerances for residues.

    Code of Federal Regulations, 2011 CFR

    2011-07-01

    ...; tolerances for residues. (a) General. (1) Tolerances are established for residues of the herbicide MCPA ((4... for residues of the herbicide MCPA ((4-chloro-2-methylphenoxy)acetic acid) resulting from the direct...

  20. 40 CFR 180.339 - MCPA; tolerances for residues.

    Code of Federal Regulations, 2013 CFR

    2013-07-01

    ...; tolerances for residues. (a) General. (1) Tolerances are established for residues of the herbicide MCPA ((4... for residues of the herbicide MCPA ((4-chloro-2-methylphenoxy)acetic acid) resulting from the direct...

  1. 40 CFR 180.432 - Lactofen; tolerances for residues.

    Code of Federal Regulations, 2013 CFR

    2013-07-01

    ... Lactofen; tolerances for residues. (a) General. Tolerances are established for residues of the herbicide... for residues of the herbicide lactofen, including its metabolites and degradates, in or on the...

  2. 40 CFR 180.339 - MCPA; tolerances for residues.

    Code of Federal Regulations, 2014 CFR

    2014-07-01

    ...; tolerances for residues. (a) General. (1) Tolerances are established for residues of the herbicide MCPA ((4... for residues of the herbicide MCPA ((4-chloro-2-methylphenoxy)acetic acid) resulting from the direct...

  3. 40 CFR 180.432 - Lactofen; tolerances for residues.

    Code of Federal Regulations, 2011 CFR

    2011-07-01

    ... Lactofen; tolerances for residues. (a) General. Tolerances are established for residues of the herbicide... for residues of the herbicide lactofen, including its metabolites and degradates, in or on the...

  4. 40 CFR 180.432 - Lactofen; tolerances for residues.

    Code of Federal Regulations, 2014 CFR

    2014-07-01

    ... Lactofen; tolerances for residues. (a) General. Tolerances are established for residues of the herbicide... for residues of the herbicide lactofen, including its metabolites and degradates, in or on the...

  5. 40 CFR 180.339 - MCPA; tolerances for residues.

    Code of Federal Regulations, 2012 CFR

    2012-07-01

    ...; tolerances for residues. (a) General. (1) Tolerances are established for residues of the herbicide MCPA ((4... for residues of the herbicide MCPA ((4-chloro-2-methylphenoxy)acetic acid) resulting from the direct...

  6. 40 CFR 180.432 - Lactofen; tolerances for residues.

    Code of Federal Regulations, 2010 CFR

    2010-07-01

    ... Lactofen; tolerances for residues. (a) Tolerances are established for residues of the herbicide lactofen, 1... residues of the herbicide, lactofen, 1-(carboethoxy)ethyl 5- -2- nitrobenzoate, in or on the following food...

  7. A survey of residual analysis and a new test of residual trend.

    PubMed

    McDowell, J J; Calvin, Olivia L; Klapes, Bryan

    2016-05-01

    A survey of residual analysis in behavior-analytic research reveals that existing methods are problematic in one way or another. A new test for residual trends is proposed that avoids the problematic features of the existing methods. It entails fitting cubic polynomials to sets of residuals and comparing their effect sizes to those that would be expected if the sets of residuals were random. To this end, sampling distributions of effect sizes for fits of a cubic polynomial to random data were obtained by generating sets of random standardized residuals of various sizes, n. A cubic polynomial was then fitted to each set of residuals and its effect size was calculated. This yielded a sampling distribution of effect sizes for each n. To test for a residual trend in experimental data, the median effect size of cubic-polynomial fits to sets of experimental residuals can be compared to the median of the corresponding sampling distribution of effect sizes for random residuals using a sign test. An example from the literature, which entailed comparing mathematical and computational models of continuous choice, is used to illustrate the utility of the test.

  8. Flexibility of active-site gorge aromatic residues and non-gorge aromatic residues in acetylcholinesterase

    SciTech Connect

    Ghattyvenkatakrishna, Pavan K; Uberbacher, Edward C

    2013-01-01

    The presence of an unusually large number of aromatic residues in the active site gorge of acetylcholinesterase has been a topic of great interest. Flexibility of these residues has been suspected to be a key player in controlling ligand traversal in the gorge. This raises the question of whether the over representation of aromatic residues in the gorge implies higher than normal flexibility of those residues. The current study suggests that it does not. Large changes in the hydrophobic cross sectional area due to dihedral oscillations are probably the reason behind their presence in the gorge.

  9. 40 CFR 180.439 - Thifensulfuron methyl; tolerances for residues.

    Code of Federal Regulations, 2010 CFR

    2010-07-01

    ... residues of thifensulfuron methyl, including its metabolites and degradates, in or on the commodities... established for residues of thifensulfuron methyl, including its metabolites and degradates, in or on...

  10. 40 CFR 180.659 - Pyroxasulfone; tolerances for residues.

    Code of Federal Regulations, 2012 CFR

    2012-07-01

    ... residues of the herbicide pyroxasulfone, including its metabolites and degradates, in or on the commodities....015 (2) Tolerances are established for residues of the herbicide pyroxasulfone, including its...

  11. Sustainable System for Residual Hazards Management

    SciTech Connect

    Kevin M. Kostelnik; James H. Clarke; Jerry L. Harbour

    2004-06-01

    Hazardous, radioactive and other toxic substances have routinely been generated and subsequently disposed of in the shallow subsurface throughout the world. Many of today’s waste management techniques do not eliminate the problem, but rather only concentrate or contain the hazardous contaminants. Residual hazards result from the presence of hazardous and/or contaminated material that remains on-site following active operations or the completion of remedial actions. Residual hazards pose continued risk to humans and the environment and represent a significant and chronic problem that require continuous longterm management (i.e. >1000 years). To protect human health and safeguard the natural environment, a sustainable system is required for the proper management of residual hazards. A sustainable system for the management of residual hazards will require the integration of engineered, institutional and land-use controls to isolate residual contaminants and thus minimize the associated hazards. Engineered controls are physical modifications to the natural setting and ecosystem, including the site, facility, and/or the residual materials themselves, in order to reduce or eliminate the potential for exposure to contaminants of concern (COCs). Institutional controls are processes, instruments, and mechanisms designed to influence human behavior and activity. System failure can involve hazardous material escaping from the confinement because of system degradation (i.e., chronic or acute degradation) or by externalintrusion of the biosphere into the contaminated material because of the loss of institutional control. An ongoing analysis of contemporary and historic sites suggests that the significance of the loss of institutional controls is a critical pathway because decisions made during the operations/remedial action phase, as well as decisions made throughout the residual hazards management period, are key to the longterm success of the prescribed system. In fact

  12. Crop Residue Coverage Estimation Using ASTER Imagery

    NASA Astrophysics Data System (ADS)

    Lewis, D.; Yao, H.; Kincaid, R.

    2006-12-01

    Soil erosion and its related runoff is a serious problem in U.S. agriculture. USDA has classified 33 percent of U.S. agricultural land as being highly erodible. It is well recognized that residue coverage on the soil surface can reduce soil erosion. The National Food Security Act of 1985 requires that agricultural producers protect all highly erodible cropland from excessive erosion. The 2002 Farm Bill gave U.S. Department of Agriculture's (USDA) Natural Resource Conservation Service (NRCS) the authority to make a determination of compliance. NRCS is currently running several programs to implement conservation practices and to monitor compliance. To be in compliance, growers must keep crop residue cover more than 30 percent of the field. This requires field-level assessment. The NRCS does not have the resources to regularly survey every field. One potential approach for compliance decision making is using data from the Advanced Spaceborne Thermal Emission and Reflection Radiometer (ASTER) sensor onboard NASA's Terra satellite. ASTER data provides 15 bands of 15 meter visible/NIR (VNIR) and 30 meter SWIR resolution data. Both the spatial resolution and spectral wavelength range and resolution are suitable for field level residue cover estimation. The objective of this study was to explore the potential of using ASTER data for crop residue cover estimation. The results indicate that ASTER imagery has good capability to identify residue within the corn fields and moderate capability in soybean residue estimation. SWIR bands have the most promise in separating crop residue when compared to the VNIR bands. Satellite based remote sensing imagery could be a potential rapid decision making tool for NRCS's compliance programs.

  13. Pesticide residues in birds and mammals

    USGS Publications Warehouse

    Stickel, L.F.; Edwards, C.A.

    1973-01-01

    SUMMARY: Residues of organochlorine pesticides and their breakdown products are present in the tissues of essentially all wild birds throughout the world. These chemicals accumulate in fat from a relatively small environmental exposure. DDE and dieldrin are most prevalent. Others, such as heptachlor epoxide, chlordane, endrin, and benzene hexachloride also occur, the quantities and kinds generally reflecting local or regional use. Accumulation may be sufficient to kill animals following applications for pest control. This has occurred in several large-scale programmes in the United States. Mortality has also resulted from unintentional leakage of chemical from commercial establishments. Residues may persist in the environment for many years, exposing successive generations of animals. In general, birds that eat other birds, or fish, have higher residues than those that eat seeds and vegetation. The kinetic processes of absorption, metabolism, storage, and output differ according to both kind of chemical and species of animal. When exposure is low and continuous, a balance between intake and excretion may be achieved. Residues reach a balance at an approximate animal body equilibrium or plateau; the storage is generally proportional to dose. Experiments with chickens show that dieldrin and heptachlor epoxide have the greatest propensity for storage, endrin next, then DDT, then lindane. The storage of DDT was complicated by its metabolism to DDE and DDD, but other studies show that DDE has a much greater propensity for storage than either DDD or DDT. Methoxychlor has little cumulative capacity in birds. Residues in eggs reflect and parallel those in the parent bird during accumulation, equilibrium, and decline when dosage is discontinued. Residues with the greatest propensity for storage are also lost most slowly. Rate of loss of residues can be modified by dietary components and is speeded by weight loss of the animal. Under sublethal conditions of continuous

  14. Residual gravimetric method to measure nebulizer output.

    PubMed

    Vecellio None, Laurent; Grimbert, Daniel; Bordenave, Joelle; Benoit, Guy; Furet, Yves; Fauroux, Brigitte; Boissinot, Eric; De Monte, Michele; Lemarié, Etienne; Diot, Patrice

    2004-01-01

    The aim of this study was to assess a residual gravimetric method based on weighing dry filters to measure the aerosol output of nebulizers. This residual gravimetric method was compared to assay methods based on spectrophotometric measurement of terbutaline (Bricanyl, Astra Zeneca, France), high-performance liquid chromatography (HPLC) measurement of tobramycin (Tobi, Chiron, U.S.A.), and electrochemical measurements of NaF (as defined by the European standard). Two breath-enhanced jet nebulizers, one standard jet nebulizer, and one ultrasonic nebulizer were tested. Output produced by the residual gravimetric method was calculated by weighing the filters both before and after aerosol collection and by filter drying corrected by the proportion of drug contained in total solute mass. Output produced by the electrochemical, spectrophotometric, and HPLC methods was determined after assaying the drug extraction filter. The results demonstrated a strong correlation between the residual gravimetric method (x axis) and assay methods (y axis) in terms of drug mass output (y = 1.00 x -0.02, r(2) = 0.99, n = 27). We conclude that a residual gravimetric method based on dry filters, when validated for a particular agent, is an accurate way of measuring aerosol output.

  15. Analytical electron microscopy of LDEF impactor residues

    SciTech Connect

    Bernhard, R.P.; Barrett, R.A.; Zolensky, M.E.

    1995-02-01

    The LDEF contained 57 individual experiment trays or tray portions specifically designed to characterize critical aspects of meteoroid and debris environment in low-Earth orbit (LEO). However, it was realized from the beginning that the most efficient use of the satellite would be to characterize impact features from the entire surface of the LDEF. With this in mind particular interest has focused on common materials facing in all 26 LDEF facing directions; among the most important of these materials has been the tray clamps. Therefore, in an effort to better understand the nature and flux of particulates in LEO, and their effects on spacecraft hardware, the authors are analyzing residues found in impact features on LDEF tray clamp surfaces. This paper summarizes all data from 79 clamps located on Bay A & B of the LDEF. The authors also describe current efforts to characterize impactor residues recovered from the impact craters, and they have found that a low, but significant, fraction of these residues have survived in a largely unmelted state. These residues can be characterized sufficiently to permit resolution of the impactor origin. The authors have concentrated on the residue from chondritic interplanetary dust particles (micrometeoroids), as these represent the harshest test of their analytical capabilities.

  16. Analytical electron microscopy of LDEF impactor residues

    NASA Technical Reports Server (NTRS)

    Bernhard, Ronald P.; Barrett, Ruth A.; Zolensky, Michael E.

    1995-01-01

    The LDEF contained 57 individual experiment trays or tray portions specifically designed to characterize critical aspects of meteoroid and debris environment in low-Earth orbit (LEO). However, it was realized from the beginning that the most efficient use of the satellite would be to characterize impact features from the entire surface of the LDEF. With this in mind particular interest has focused on common materials facing in all 26 LDEF facing directions; among the most important of these materials has been the tray clamps. Therefore, in an effort to better understand the nature and flux of particulates in LEO, and their effects on spacecraft hardware, we are analyzing residues found in impact features on LDEF tray clamp surfaces. This paper summarizes all data from 79 clamps located on Bay A & B of the LDEF. We also describe current efforts to characterize impactor residues recovered from the impact craters, and we have found that a low, but significant, fraction of these residues have survived in a largely unmelted state. These residues can be characterized sufficiently to permit resolution of the impactor origin. We have concentrated on the residue from chondritic interplanetary dust particles (micrometeoroids), as these represent the harshest test of our analytical capabilities.

  17. Emerging pesticide residue issues and analytical approaches.

    PubMed

    Fintschenko, Yolanda; Krynitsky, Alexander J; Wong, Jon W

    2010-05-26

    The 46th Annual Florida Pesticide Residue Workshop of 2009 (FPRW 2009) held in St. Pete Beach, FL, is the latest in an annual tradition drawing scientists from U.S. federal and state government laboratories, industry, and other laboratories worldwide. In 2009, selected FPRW presenters were invited to contribute to this special issue of the Journal of Agricultural and Food Chemistry with a section devoted to emerging pesticide residue issues and analytical approaches. What follows is the written record of what should become a scientific conversation launched at FPRW 2009. There are two distinct approaches to organic residue analysis: instrumental methods and assays. In much of the world, scientists primarily rely on laboratories equipped with instrumentation for analysis, usually gas chromatography and liquid chromatography with some type of selective detector. In the discussion of instrumental approaches, the focus is on chromatography with mass spectrometry as a detection method. Approaches such as biomonitoring and assays fall outside the traditional instrumental method approach to residue analysis. Assays that do not require laboratory equipment are of greater interest for screening and are well-suited to field use. Regardless of the analytical method, the success of multiresidue analysis relies on the appropriate choice of sample preparation and cleanup methodologies. Many new sample preparation and cleanup approaches used for pesticide and other small molecule contaminant residue analyses in a variety of complex sample matrices are discussed in this special issue. The goal of these approaches is to reduce overall analysis time and solvent consumption without compromising the analytical results.

  18. Treatment of vacuum residues in hydroconversion conditions

    NASA Astrophysics Data System (ADS)

    León, A. Y.; Mendoza, D. L.; Espinosa, J. O.; Laverde, D.

    2016-02-01

    In this paper the use of a liquid homogeneous catalyst has been studied in reactivity vacuum residues by hydroconversion under different conditions. To cover a wide range of compositions, six (6) vacuum residues were selected from crude mixtures. Hydroconversion test were performed in batch reactor with hydrogen atmosphere at about 2000psi in a temperature range between 430 and 480°C. The results allowed to establish that the reactivity hydroconversion conditions about coke formation is higher in vacuum residues with higher content of resins and asphaltenes. The reaction conditions promote distillate formation, however, with increasing stringency conditions, the distillate yield decreases due to distillate transformation into temperature range 430 and 460°C compared to the tests performed without catalyst demonstrating that the use of homogeneous catalyst is an alternative to treating vacuum residues and results are satisfactory in the conversion processes. Finally, predictive expressions have been developed in the formation of products depending on the conditions of temperature and physicochemical properties of processed vacuum residue.

  19. Residual stress characterization for laminated composites

    NASA Astrophysics Data System (ADS)

    Liu, Shao-Chun

    With increasing applications of advanced laminated composites, process-induced residual stress has drawn more and more attention in recent years. Efforts have been devoted to understanding residual stress both quantitatively and qualitatively. In the current study, a novel technique called the Cure Referencing Method was developed which has the capability for measuring the residual stress on the symmetric laminated composite plates. It can also differentiate residual stress into two components: one is due to the mismatch of the coefficient of thermal expansion, the other is caused by the matrix chemical curing shrinkage. The chemical curing shrinkage of the polymer matrix was investigated in further detail. A technique was developed to measure the post-gel chemical curing shrinkage which is the portion of curing shrinkage that really induces the residual stress in the polymer matrix composites. Time-dependent material property is another issue associated with polymer matrix composite materials. The data of several short-term tensile creep tests run at different temperature were used to construct a linear viscoelastic: model for describing the behavior of the composites over a long period of time. It was found that physical aging of the polymer matrix needs to be taken into account in order to have a more accurate representation of the long-term behavior. A fair agreement was obtained between the result of the long-term creep test and the master curve constructed from several momentary creep tests.

  20. 40 CFR 721.4500 - Isopropylamine distillation residues and ethylamine distillation residues.

    Code of Federal Regulations, 2012 CFR

    2012-07-01

    ... 40 Protection of Environment 32 2012-07-01 2012-07-01 false Isopropylamine distillation residues and ethylamine distillation residues. 721.4500 Section 721.4500 Protection of Environment... SUBSTANCES Significant New Uses for Specific Chemical Substances § 721.4500 Isopropylamine...

  1. 40 CFR 721.4500 - Isopropylamine distillation residues and ethylamine distillation residues.

    Code of Federal Regulations, 2014 CFR

    2014-07-01

    ... 40 Protection of Environment 31 2014-07-01 2014-07-01 false Isopropylamine distillation residues and ethylamine distillation residues. 721.4500 Section 721.4500 Protection of Environment... SUBSTANCES Significant New Uses for Specific Chemical Substances § 721.4500 Isopropylamine...

  2. 40 CFR 721.4500 - Isopropylamine distillation residues and ethylamine distillation residues.

    Code of Federal Regulations, 2010 CFR

    2010-07-01

    ... 40 Protection of Environment 30 2010-07-01 2010-07-01 false Isopropylamine distillation residues and ethylamine distillation residues. 721.4500 Section 721.4500 Protection of Environment... SUBSTANCES Significant New Uses for Specific Chemical Substances § 721.4500 Isopropylamine...

  3. 40 CFR 721.4500 - Isopropylamine distillation residues and ethylamine distillation residues.

    Code of Federal Regulations, 2013 CFR

    2013-07-01

    ... 40 Protection of Environment 32 2013-07-01 2013-07-01 false Isopropylamine distillation residues and ethylamine distillation residues. 721.4500 Section 721.4500 Protection of Environment... SUBSTANCES Significant New Uses for Specific Chemical Substances § 721.4500 Isopropylamine...

  4. 40 CFR 721.4500 - Isopropylamine distillation residues and ethylamine distillation residues.

    Code of Federal Regulations, 2011 CFR

    2011-07-01

    ... 40 Protection of Environment 31 2011-07-01 2011-07-01 false Isopropylamine distillation residues and ethylamine distillation residues. 721.4500 Section 721.4500 Protection of Environment... SUBSTANCES Significant New Uses for Specific Chemical Substances § 721.4500 Isopropylamine...

  5. Distribution of Penicillin G Residues in Culled Dairy Cow Muscles: Implications for Residue Monitoring

    USDA-ARS?s Scientific Manuscript database

    The U.S. Food and Drug Administration sets tolerances for veterinary drug residues in muscle, but does not specify which type of muscle should be analyzed. In order to determine if antibiotic residue levels are dependent on muscle type, 7 culled dairy cows were dosed with Penicillin G (Pen G) from ...

  6. Detection of gunshot residues using mass spectrometry.

    PubMed

    Taudte, Regina Verena; Beavis, Alison; Blanes, Lucas; Cole, Nerida; Doble, Philip; Roux, Claude

    2014-01-01

    In recent years, forensic scientists have become increasingly interested in the detection and interpretation of organic gunshot residues (OGSR) due to the increasing use of lead- and heavy metal-free ammunition. This has also been prompted by the identification of gunshot residue- (GSR-) like particles in environmental and occupational samples. Various techniques have been investigated for their ability to detect OGSR. Mass spectrometry (MS) coupled to a chromatographic system is a powerful tool due to its high selectivity and sensitivity. Further, modern MS instruments can detect and identify a number of explosives and additives which may require different ionization techniques. Finally, MS has been applied to the analysis of both OGSR and inorganic gunshot residue (IGSR), although the "gold standard" for analysis is scanning electron microscopy with energy dispersive X-ray microscopy (SEM-EDX). This review presents an overview of the technical attributes of currently available MS and ionization techniques and their reported applications to GSR analysis.

  7. Field Test Kit for Gun Residue Detection

    SciTech Connect

    WALKER, PAMELA K.; RODACY, PHILIP J.

    2002-01-01

    One of the major needs of the law enforcement field is a product that quickly, accurately, and inexpensively identifies whether a person has recently fired a gun--even if the suspect has attempted to wash the traces of gunpowder off. The Field Test Kit for Gunshot Residue Identification based on Sandia National Laboratories technology works with a wide variety of handguns and other weaponry using gunpowder. There are several organic chemicals in small arms propellants such as nitrocellulose, nitroglycerine, dinitrotoluene, and nitrites left behind after the firing of a gun that result from the incomplete combustion of the gunpowder. Sandia has developed a colorimetric shooter identification kit for in situ detection of gunshot residue (GSR) from a suspect. The test kit is the first of its kind and is small, inexpensive, and easily transported by individual law enforcement personnel requiring minimal training for effective use. It will provide immediate information identifying gunshot residue.

  8. Residual Stress Analysis in Thick Uranium Films

    SciTech Connect

    Hodge, A M; Foreman, R J; Gallegos, G F

    2004-12-06

    Residual stress analysis was performed on thick, 1.0 to 25 {micro}m, depleted Uranium (DU) films deposited on an Al substrate by magnetron sputtering. Two distinct characterization techniques were used to measure substrate curvature before and after deposition. Stress evaluation was performed using the Benabdi/Roche equation, which is based on beam theory of a bi-layer material. The residual stress evolution was studied as a function of coating thickness and applied negative bias voltage (0-300V). The stresses developed were always compressive; however, increasing the coating thickness and applying a bias voltage presented a trend towards more tensile stresses and thus an overall reduction of residual stresses.

  9. Residual neuromuscular blockade in critical care.

    PubMed

    Wilson, Jason; Collins, Angela S; Rowan, Brea O

    2012-06-01

    Neuromuscular blockade is a pharmacological adjunct for anesthesia and for surgical interventions. Neuromuscular blockers can facilitate ease of instrumentation and reduce complications associated with intubation. An undesirable sequela of these agents is residual neuromuscular blockade. Residual neuromuscular blockade is linked to aspiration, diminished response to hypoxia, and obstruction of the upper airway that may occur soon after extubation. If an operation is particularly complex or requires a long anesthesia time, residual neuromuscular blockade can contribute to longer stays in the intensive care unit and more hours of mechanical ventilation. Given the risks of this medication class, it is essential to have an understanding of the mechanism of action of, assessment of, and factors affecting blockade and to be able to identify factors that affect pharmacokinetics.

  10. Plasma treatment of air pollution control residues.

    PubMed

    Amutha Rani, D; Gomez, E; Boccaccini, A R; Hao, L; Deegan, D; Cheeseman, C R

    2008-01-01

    Air pollution control (APC) residues from waste incineration have been blended with silica and alumina and the mix melted using DC plasma arc technology. The chemical composition of the fully amorphous homogeneous glass formed has been determined. Waste acceptance criteria compliance leach testing demonstrates that the APC residue derived glass releases only trace levels of heavy metals (Pb (<0.007mg/kg) and Zn (0.02mg/kg)) and Cl(-) (0.2mg/kg). These are significantly below the limit values for disposal to inert landfill. It is concluded that plasma treatment of APC residues can produce an inert glass that may have potential to be used either in bulk civil engineering applications or in the production of higher value glass-ceramic products.

  11. Residual bone growth after lengthening procedures.

    PubMed

    Journeau, Pierre; Lascombes, Pierre; Barbier, Dominique; Popkov, Dmitry

    2016-12-01

    The prognosis of limb length discrepancy is a major subject in paediatric orthopaedic surgery. The strategy depends on the prognosis and must be adapted to each patient. The residual growth of the lengthened segment often remains unknown, but is dependent on age, the percentage of lengthening and other factors. Using a large cohort of 150 children who had undergone bone lengthening procedures, we describe five patterns of post-intervention growth and identify factors that are favourable for normal residual growth. The criteria for bone lengthening which should maintain good residual growth are-bone age at lengthening should be before the pubertal growth spurt; the interval between two lengthening procedures should be over three years; the percentage of lengthening should be <30% of the initial segment; and no more than two lengthening procedures should be carried out during infancy.

  12. Residual Defect Density in Random Disks Deposits

    PubMed Central

    Topic, Nikola; Pöschel, Thorsten; Gallas, Jason A. C.

    2015-01-01

    We investigate the residual distribution of structural defects in very tall packings of disks deposited randomly in large channels. By performing simulations involving the sedimentation of up to 50 × 109 particles we find all deposits to consistently show a non-zero residual density of defects obeying a characteristic power-law as a function of the channel width. This remarkable finding corrects the widespread belief that the density of defects should vanish algebraically with growing height. A non-zero residual density of defects implies a type of long-range spatial order in the packing, as opposed to only local ordering. In addition, we find deposits of particles to involve considerably less randomness than generally presumed. PMID:26235809

  13. System and method for measuring residual stress

    DOEpatents

    Prime, Michael B.

    2002-01-01

    The present invention is a method and system for determining the residual stress within an elastic object. In the method, an elastic object is cut along a path having a known configuration. The cut creates a portion of the object having a new free surface. The free surface then deforms to a contour which is different from the path. Next, the contour is measured to determine how much deformation has occurred across the new free surface. Points defining the contour are collected in an empirical data set. The portion of the object is then modeled in a computer simulator. The points in the empirical data set are entered into the computer simulator. The computer simulator then calculates the residual stress along the path which caused the points within the object to move to the positions measured in the empirical data set. The calculated residual stress is then presented in a useful format to an analyst.

  14. Determination of Pesticide Residues in Cannabis Smoke

    PubMed Central

    Sullivan, Nicholas; Elzinga, Sytze; Raber, Jeffrey C.

    2013-01-01

    The present study was conducted in order to quantify to what extent cannabis consumers may be exposed to pesticide and other chemical residues through inhaled mainstream cannabis smoke. Three different smoking devices were evaluated in order to provide a generalized data set representative of pesticide exposures possible for medical cannabis users. Three different pesticides, bifenthrin, diazinon, and permethrin, along with the plant growth regulator paclobutrazol, which are readily available to cultivators in commercial products, were investigated in the experiment. Smoke generated from the smoking devices was condensed in tandem chilled gas traps and analyzed with gas chromatography-mass spectrometry (GC-MS). Recoveries of residues were as high as 69.5% depending on the device used and the component investigated, suggesting that the potential of pesticide and chemical residue exposures to cannabis users is substantial and may pose a significant toxicological threat in the absence of adequate regulatory frameworks. PMID:23737769

  15. Firearms discharge residue sample collection techniques.

    PubMed

    Goleb, J A; Midkiff, C R

    1975-10-01

    Critical comparisons of Ba and Sb in firearms discharge residue were made on samples collected by three independent collection technqiues. Collection materials studied were transparent adhesive tape, (Scotch Brand), a solution of cellulose acetate in acetone ("Film Lift"), and plastic-shafted cotton swabs wetted with dilute nitric acid. Flameless atomic absorption analyses were performed with a Jarrell-Ash Model 810 instrument equipped with a tantalum strip atomizer. Tape and cotton swabs gave comparable positive indications of residue, with frequencies of 90 and 80%, respectively. The plastic Film Lift gave fewer positives, with a frequency of 50%. With the transparent tape lift, gunshot residue particles are discernible, making nondestructive microscopic identification possible prior to destructive elemental analysis.

  16. Electromagnetic corrections to the zonal flow residual

    NASA Astrophysics Data System (ADS)

    Pusztai, Istvan; Catto, Peter J.; Parra, Felix I.

    2014-10-01

    The axisymmetric zonal flow residual calculation in tokamak plasmas is generalized to include electromagnetic perturbations. Instead of imposing magnetic perturbations externally, we formulate and solve a description retaining the fully self-consistent temporal and spatial perturbations in the electric and magnetic fields. Simple expressions for the electrostatic, shear and compressional magnetic residual responses derived provide a fully electromagnetic test of the zonal flow residual in gyrokinetic codes. We find that at β ~ O (1) the most easily testable quantity is the compressional magnetic perturbation generated by the density perturbation corresponding to the zonal flow potential, while at small values of β, the electrostatic and shear magnetic responses to an initial compressional magnetic perturbation can also be detectable. Without collisions any initial magnetic perturbation remain completely undamped. Supported by US Department of Energy grant at DE-FG02-91ER-54109 at MIT. IP is supported by the International Postdoc grant of Vetenskapsradet.

  17. Genomic Prediction Accounting for Residual Heteroskedasticity.

    PubMed

    Ou, Zhining; Tempelman, Robert J; Steibel, Juan P; Ernst, Catherine W; Bates, Ronald O; Bello, Nora M

    2015-11-12

    Whole-genome prediction (WGP) models that use single-nucleotide polymorphism marker information to predict genetic merit of animals and plants typically assume homogeneous residual variance. However, variability is often heterogeneous across agricultural production systems and may subsequently bias WGP-based inferences. This study extends classical WGP models based on normality, heavy-tailed specifications and variable selection to explicitly account for environmentally-driven residual heteroskedasticity under a hierarchical Bayesian mixed-models framework. WGP models assuming homogeneous or heterogeneous residual variances were fitted to training data generated under simulation scenarios reflecting a gradient of increasing heteroskedasticity. Model fit was based on pseudo-Bayes factors and also on prediction accuracy of genomic breeding values computed on a validation data subset one generation removed from the simulated training dataset. Homogeneous vs. heterogeneous residual variance WGP models were also fitted to two quantitative traits, namely 45-min postmortem carcass temperature and loin muscle pH, recorded in a swine resource population dataset prescreened for high and mild residual heteroskedasticity, respectively. Fit of competing WGP models was compared using pseudo-Bayes factors. Predictive ability, defined as the correlation between predicted and observed phenotypes in validation sets of a five-fold cross-validation was also computed. Heteroskedastic error WGP models showed improved model fit and enhanced prediction accuracy compared to homoskedastic error WGP models although the magnitude of the improvement was small (less than two percentage points net gain in prediction accuracy). Nevertheless, accounting for residual heteroskedasticity did improve accuracy of selection, especially on individuals of extreme genetic merit. Copyright © 2016 Ou et al.

  18. Genomic Prediction Accounting for Residual Heteroskedasticity

    PubMed Central

    Ou, Zhining; Tempelman, Robert J.; Steibel, Juan P.; Ernst, Catherine W.; Bates, Ronald O.; Bello, Nora M.

    2015-01-01

    Whole-genome prediction (WGP) models that use single-nucleotide polymorphism marker information to predict genetic merit of animals and plants typically assume homogeneous residual variance. However, variability is often heterogeneous across agricultural production systems and may subsequently bias WGP-based inferences. This study extends classical WGP models based on normality, heavy-tailed specifications and variable selection to explicitly account for environmentally-driven residual heteroskedasticity under a hierarchical Bayesian mixed-models framework. WGP models assuming homogeneous or heterogeneous residual variances were fitted to training data generated under simulation scenarios reflecting a gradient of increasing heteroskedasticity. Model fit was based on pseudo-Bayes factors and also on prediction accuracy of genomic breeding values computed on a validation data subset one generation removed from the simulated training dataset. Homogeneous vs. heterogeneous residual variance WGP models were also fitted to two quantitative traits, namely 45-min postmortem carcass temperature and loin muscle pH, recorded in a swine resource population dataset prescreened for high and mild residual heteroskedasticity, respectively. Fit of competing WGP models was compared using pseudo-Bayes factors. Predictive ability, defined as the correlation between predicted and observed phenotypes in validation sets of a five-fold cross-validation was also computed. Heteroskedastic error WGP models showed improved model fit and enhanced prediction accuracy compared to homoskedastic error WGP models although the magnitude of the improvement was small (less than two percentage points net gain in prediction accuracy). Nevertheless, accounting for residual heteroskedasticity did improve accuracy of selection, especially on individuals of extreme genetic merit. PMID:26564950

  19. Catalytic combustion with incompletely vaporized residual fuel

    NASA Technical Reports Server (NTRS)

    Rosfjord, T. J.

    1981-01-01

    Catalytic combustion of fuel lean mixtures of incompletely vaporized residual fuel and air was investigated. The 7.6 cm diameter, graded cell reactor was constructed from zirconia spinel substrate and catalyzed with a noble metal catalyst. Streams of luminous particles exited the rector as a result of fuel deposition and carbonization on the substrate. Similar results were obtained with blends of No. 6 and No. 2 oil. Blends of shale residual oil and No. 2 oil resulted in stable operation. In shale oil blends the combustor performance degraded with a reduced degree of fuel vaporization. In tests performed with No. 2 oil a similar effect was observed.

  20. Mechanically induced residual stresses: Modelling and characterisation

    NASA Astrophysics Data System (ADS)

    Stranart, Jean-Claude E.

    Accurate characterisation of residual stress represents a major challenge to the engineering community. This is because it is difficult to validate the measurement and the accuracy is doubtful. It is with this in mind that the current research program concerning the characterisation of mechanically induced residual stresses was undertaken. Specifically, the cold expansion of fastener holes and the shot peening treatment of aerospace alloys, aluminium 7075 and titanium Ti-6Al-4V, are considered. The objective of this study is to characterise residual stresses resulting from cold working using three powerful techniques. These are: (i) theoretical using three dimensional non-linear finite element modelling, (ii) semi-destructive using a modified incremental hole drilling technique and (iii) nondestructive using a newly developed guided wave method supplemented by traditional C-scan measurements. The three dimensional finite element results of both simultaneous and sequential cold expansion of two fastener holes revealed the importance of the separation distance, the expansion level and the loading history upon the development and growth of the plastic zone and unloading residual stresses. It further showed that the commonly adopted two dimensional finite element models are inaccurate and incapable of predicting these residual stresses. Similarly, the dynamic elasto-plastic finite element studies of shot peening showed that the depth of the compressed layer, surface and sub-surface residual stresses are significantly influenced by the shot characteristics. Furthermore, the results reveal that the separation distance between two simultaneously impacting shots governs the plastic zone development and its growth. In the semi-destructive incremental hole drilling technique, the accuracy of the newly developed calibration coefficients and measurement techniques were verified with a known stress field and the method was used to measure peening residual stresses. Unlike

  1. 51st North American Chemical Residue Workshop.

    PubMed

    Yang, Paul; Martos, Perry; Barrett, Brad

    2015-06-03

    Manuscripts collected in this 51st North American Chemical Residue Workshop (NACRW) Symposium issue of the Journal of Agricultural and Food Chemistry (JAFC) were originally presented at the 51st NACRW meeting. The 2014 NACRW JAFC symposium collects 14 publications representing the broad range of topics in chemical analyses presented at the 2014 meeting. These include the analysis of chemical residues and contaminants in food, environment, feed, botanical, and bee samples as well as the application of quality control/quality assurance protocols in routine and method development.

  2. [Total amitraz residues in bee honeys].

    PubMed

    Hemmerling, C; Augustyniak, B; Risto, C

    1991-01-01

    A total of 330 bee honey samples was analysed in 1986-1990, and the results are reported. Analysis was performed according to the method for the amitraz total residue determination by hydrolysis and steam distillation as described. 60% of the honeys were practically not contamined (total residue content amounting to 0.01 mg/kg), 8.5% of the honeys contained more than 0.05 mg/kg. Maximal values of 0.2-0.5 mg/kg were stated. 54% of the rape-honeys contained more than 0.01 mg/kg, 19% more than 0.05 mg/kg.

  3. Residual contact stresses in cryotechnical environments

    NASA Astrophysics Data System (ADS)

    Cretegny, J. F.; Demonicault, J. M.

    Two examples were chosen to show the use of residual stress measurements in the evaluation and comprehension of possible ruptures of parts subjected to the working conditions of cryogenic turbomachines which induce wear of the surfaces in dry contact. The examples concern the ball bearings and spline of the liquid hydrogen pump of the Vulcain engine to be used on Ariane 5. The Ariane program is introduced and tribological problems of the cryogenic technique are discussed. The utility of the residual stress measurements is assessed.

  4. Residual radioactivity of treated green diamonds.

    PubMed

    Cassette, Philippe; Notari, Franck; Lépy, Marie-Christine; Caplan, Candice; Pierre, Sylvie; Hainschwang, Thomas; Fritsch, Emmanuel

    2017-08-01

    Treated green diamonds can show residual radioactivity, generally due to immersion in radium salts. We report various activity measurements on two radioactive diamonds. The activity was characterized by alpha and gamma ray spectrometry, and the radon emanation was measured by alpha counting of a frozen source. Even when no residual radium contamination can be identified, measurable alpha and high-energy beta emissions could be detected. The potential health impact of radioactive diamonds and their status with regard to the regulatory policy for radioactive products are discussed. Copyright © 2017. Published by Elsevier Ltd.

  5. Continuous Removal of Coal-Gasification Residue

    NASA Technical Reports Server (NTRS)

    Collins, Earl R., Jr.; Suitor, J.; Dubis, D.

    1986-01-01

    Continuous-flow hopper processes solid residue from coal gasification, converting it from ashes, cinders, and clinkers to particles size of sand granules. Unit does not require repeated depressurization of lockhopper to admit and release materials. Therefore consumes less energy. Because unit has no airlock valves opened and closed repeatedly on hot, abrasive particles, subjected to lesser wear. Coal-gasification residue flows slowly through pressure-letdown device. Material enters and leaves continuously. Cleanout door on each pressure-letdown chamber allows access for maintenance and emergencies.

  6. Pesticide residue levels in Argentinian pasteurised milk.

    PubMed

    Maitre, M I; de la Sierra, P; Lenardon, A; Enrique, S; Marino, F

    1994-10-14

    Pasteurised milk--120 samples--was analysed (in Santa Fe City, Argentina) to determine organochlorinated pesticides. Almost all samples showed the presence of such residues; the compounds more frequently detected being: heptachlor and its epoxide (98%) and hexachlorocyclohexane .HCH. alpha and gamma isomers. Residues of aldrin and dieldrin; chlordane (alpha and gamma); endosulfan (I and II); DDT (o,p-DDT, p,p'-DDE, o,p-DDD, p,p'-DDD) as well as hexachlorocyclobenzene (HCB) were also found. Despite their presence high concentrations are sporadic, the mean values being less than FAO/OMS tolerance limits. Relations between maxima concentrations and seasons were not found.

  7. Rigid Residue Scan Simulations Systematically Reveal Residue Entropic Roles in Protein Allostery.

    PubMed

    Kalescky, Robert; Zhou, Hongyu; Liu, Jin; Tao, Peng

    2016-04-01

    Intra-protein information is transmitted over distances via allosteric processes. This ubiquitous protein process allows for protein function changes due to ligand binding events. Understanding protein allostery is essential to understanding protein functions. In this study, allostery in the second PDZ domain (PDZ2) in the human PTP1E protein is examined as model system to advance a recently developed rigid residue scan method combining with configurational entropy calculation and principal component analysis. The contributions from individual residues to whole-protein dynamics and allostery were systematically assessed via rigid body simulations of both unbound and ligand-bound states of the protein. The entropic contributions of individual residues to whole-protein dynamics were evaluated based on covariance-based correlation analysis of all simulations. The changes of overall protein entropy when individual residues being held rigid support that the rigidity/flexibility equilibrium in protein structure is governed by the La Châtelier's principle of chemical equilibrium. Key residues of PDZ2 allostery were identified with good agreement with NMR studies of the same protein bound to the same peptide. On the other hand, the change of entropic contribution from each residue upon perturbation revealed intrinsic differences among all the residues. The quasi-harmonic and principal component analyses of simulations without rigid residue perturbation showed a coherent allosteric mode from unbound and bound states, respectively. The projection of simulations with rigid residue perturbation onto coherent allosteric modes demonstrated the intrinsic shifting of ensemble distributions supporting the population-shift theory of protein allostery. Overall, the study presented here provides a robust and systematic approach to estimate the contribution of individual residue internal motion to overall protein dynamics and allostery.

  8. Rigid Residue Scan Simulations Systematically Reveal Residue Entropic Roles in Protein Allostery

    PubMed Central

    Liu, Jin

    2016-01-01

    Intra-protein information is transmitted over distances via allosteric processes. This ubiquitous protein process allows for protein function changes due to ligand binding events. Understanding protein allostery is essential to understanding protein functions. In this study, allostery in the second PDZ domain (PDZ2) in the human PTP1E protein is examined as model system to advance a recently developed rigid residue scan method combining with configurational entropy calculation and principal component analysis. The contributions from individual residues to whole-protein dynamics and allostery were systematically assessed via rigid body simulations of both unbound and ligand-bound states of the protein. The entropic contributions of individual residues to whole-protein dynamics were evaluated based on covariance-based correlation analysis of all simulations. The changes of overall protein entropy when individual residues being held rigid support that the rigidity/flexibility equilibrium in protein structure is governed by the La Châtelier’s principle of chemical equilibrium. Key residues of PDZ2 allostery were identified with good agreement with NMR studies of the same protein bound to the same peptide. On the other hand, the change of entropic contribution from each residue upon perturbation revealed intrinsic differences among all the residues. The quasi-harmonic and principal component analyses of simulations without rigid residue perturbation showed a coherent allosteric mode from unbound and bound states, respectively. The projection of simulations with rigid residue perturbation onto coherent allosteric modes demonstrated the intrinsic shifting of ensemble distributions supporting the population-shift theory of protein allostery. Overall, the study presented here provides a robust and systematic approach to estimate the contribution of individual residue internal motion to overall protein dynamics and allostery. PMID:27115535

  9. Managing logging residue under the timber sale contract.

    Treesearch

    Thomas C. Adams

    1980-01-01

    Management of logging residue is becoming an important part of timber sale planning. This involves controlling the amount of residue remaining on the ground and its distribution by diameter size class. Some residue is beneficial.An interdisciplinary team specified a desired residue level for one clearcutting unit of this trial. For comparison another cutting...

  10. Utilizing hardwood logging residue: a case study in the Appalachians

    Treesearch

    E. Paul Craft

    1976-01-01

    An Appalachian hardwood timber stand that contained 6,700 board feet per acre of sawtimber was harvested by clearcutting. After the merchantable sawlogs were removed, this stand contained 69.3 tons per acre of green wood residue. Thirty-three and one-third tons of residue were from tops of merchantable sawtimber; 36 tons were from residual trees. Treetop residue...

  11. Predicting logging residue volumes in the Pacific Northwest

    Treesearch

    Erik C. Berg; Todd A. Morgan; Eric A. Simmons; Stan Zarnoch; Micah G. Scudder

    2016-01-01

    Pacific Northwest forest managers seek estimates of post-timber-harvest woody residue volumes and biomass that can be related to readily available site- and tree-level attributes. To better predict residue production, researchers investigated variability in residue ratios, growing-stock residue volume per mill-delivered volume, across Idaho, Montana, Oregon, and...

  12. 40 CFR 180.184 - Linuron; tolerances for residues.

    Code of Federal Regulations, 2014 CFR

    2014-07-01

    ... Linuron; tolerances for residues. (a) General. Tolerances are established for residues of the herbicide.... Time-limited tolerances are established for residues of the herbicide linuron , including its... defined in § 180.1(l), are established for residues of the herbicide linuron...

  13. 40 CFR 180.584 - Tolylfluanid; tolerances for residues.

    Code of Federal Regulations, 2010 CFR

    2010-07-01

    ... 40 Protection of Environment 23 2010-07-01 2010-07-01 false Tolylfluanid; tolerances for residues...) PESTICIDE PROGRAMS TOLERANCES AND EXEMPTIONS FOR PESTICIDE CHEMICAL RESIDUES IN FOOD Specific Tolerances § 180.584 Tolylfluanid; tolerances for residues. (a) General. Tolerances are established for residues...

  14. 40 CFR 180.581 - Iprovalicarb; tolerances for residues.

    Code of Federal Regulations, 2010 CFR

    2010-07-01

    ... 40 Protection of Environment 23 2010-07-01 2010-07-01 false Iprovalicarb; tolerances for residues...) PESTICIDE PROGRAMS TOLERANCES AND EXEMPTIONS FOR PESTICIDE CHEMICAL RESIDUES IN FOOD Specific Tolerances § 180.581 Iprovalicarb; tolerances for residues. (a) General. Tolerances are established for residues...

  15. 40 CFR 158.1410 - Residue chemistry data requirements table.

    Code of Federal Regulations, 2012 CFR

    2012-07-01

    ... 40 Protection of Environment 25 2012-07-01 2012-07-01 false Residue chemistry data requirements...) PESTICIDE PROGRAMS DATA REQUIREMENTS FOR PESTICIDES Residue Chemistry § 158.1410 Residue chemistry data... the residue chemistry data requirements for a particular pesticide product. Notes that apply to an...

  16. 40 CFR 158.1410 - Residue chemistry data requirements table.

    Code of Federal Regulations, 2013 CFR

    2013-07-01

    ... 40 Protection of Environment 25 2013-07-01 2013-07-01 false Residue chemistry data requirements...) PESTICIDE PROGRAMS DATA REQUIREMENTS FOR PESTICIDES Residue Chemistry § 158.1410 Residue chemistry data... the residue chemistry data requirements for a particular pesticide product. Notes that apply to an...

  17. 40 CFR 158.1410 - Residue chemistry data requirements table.

    Code of Federal Regulations, 2011 CFR

    2011-07-01

    ... 40 Protection of Environment 24 2011-07-01 2011-07-01 false Residue chemistry data requirements...) PESTICIDE PROGRAMS DATA REQUIREMENTS FOR PESTICIDES Residue Chemistry § 158.1410 Residue chemistry data... the residue chemistry data requirements for a particular pesticide product. Notes that apply to an...

  18. 40 CFR 158.1410 - Residue chemistry data requirements table.

    Code of Federal Regulations, 2014 CFR

    2014-07-01

    ... 40 Protection of Environment 24 2014-07-01 2014-07-01 false Residue chemistry data requirements...) PESTICIDE PROGRAMS DATA REQUIREMENTS FOR PESTICIDES Residue Chemistry § 158.1410 Residue chemistry data... the residue chemistry data requirements for a particular pesticide product. Notes that apply to an...

  19. 40 CFR 180.471 - Furilazole; tolerances for residues.

    Code of Federal Regulations, 2010 CFR

    2010-07-01

    ... 40 Protection of Environment 23 2010-07-01 2010-07-01 false Furilazole; tolerances for residues...) PESTICIDE PROGRAMS TOLERANCES AND EXEMPTIONS FOR PESTICIDE CHEMICAL RESIDUES IN FOOD Specific Tolerances § 180.471 Furilazole; tolerances for residues. (a) General. Tolerances are established for residues...

  20. 40 CFR 180.214 - Fenthion; tolerances for residues.

    Code of Federal Regulations, 2010 CFR

    2010-07-01

    ... 40 Protection of Environment 23 2010-07-01 2010-07-01 false Fenthion; tolerances for residues. 180... PROGRAMS TOLERANCES AND EXEMPTIONS FOR PESTICIDE CHEMICAL RESIDUES IN FOOD Specific Tolerances § 180.214 Fenthion; tolerances for residues. (a) General. Tolerances are established for residues of the...

  1. 40 CFR 180.461 - Cadusafos; tolerances for residues.

    Code of Federal Regulations, 2010 CFR

    2010-07-01

    ... 40 Protection of Environment 23 2010-07-01 2010-07-01 false Cadusafos; tolerances for residues...) PESTICIDE PROGRAMS TOLERANCES AND EXEMPTIONS FOR PESTICIDE CHEMICAL RESIDUES IN FOOD Specific Tolerances § 180.461 Cadusafos; tolerances for residues. A tolerance is established for the residues of...

  2. 40 CFR 180.209 - Terbacil; tolerances for residues.

    Code of Federal Regulations, 2010 CFR

    2010-07-01

    ... 40 Protection of Environment 23 2010-07-01 2010-07-01 false Terbacil; tolerances for residues. 180... PROGRAMS TOLERANCES AND EXEMPTIONS FOR PESTICIDE CHEMICAL RESIDUES IN FOOD Specific Tolerances § 180.209 Terbacil; tolerances for residues. (a) General. Tolerances are established for combined residues of...

  3. 40 CFR 180.455 - Procymidone; tolerances for residues.

    Code of Federal Regulations, 2010 CFR

    2010-07-01

    ... 40 Protection of Environment 23 2010-07-01 2010-07-01 false Procymidone; tolerances for residues...) PESTICIDE PROGRAMS TOLERANCES AND EXEMPTIONS FOR PESTICIDE CHEMICAL RESIDUES IN FOOD Specific Tolerances § 180.455 Procymidone; tolerances for residues. A tolerance is established for the residues of...

  4. 40 CFR 180.468 - Flumetsulam; tolerances for residues.

    Code of Federal Regulations, 2010 CFR

    2010-07-01

    ... 40 Protection of Environment 23 2010-07-01 2010-07-01 false Flumetsulam; tolerances for residues...) PESTICIDE PROGRAMS TOLERANCES AND EXEMPTIONS FOR PESTICIDE CHEMICAL RESIDUES IN FOOD Specific Tolerances § 180.468 Flumetsulam; tolerances for residues. Tolerances are established for residues of the...

  5. 40 CFR 180.543 - Diclosulam; tolerances for residues.

    Code of Federal Regulations, 2010 CFR

    2010-07-01

    ... 40 Protection of Environment 23 2010-07-01 2010-07-01 false Diclosulam; tolerances for residues...) PESTICIDE PROGRAMS TOLERANCES AND EXEMPTIONS FOR PESTICIDE CHEMICAL RESIDUES IN FOOD Specific Tolerances § 180.543 Diclosulam; tolerances for residues. (a) General. Tolerances are established for residues...

  6. 40 CFR 180.460 - Benoxacor; tolerances for residues.

    Code of Federal Regulations, 2010 CFR

    2010-07-01

    ... 40 Protection of Environment 23 2010-07-01 2010-07-01 false Benoxacor; tolerances for residues...) PESTICIDE PROGRAMS TOLERANCES AND EXEMPTIONS FOR PESTICIDE CHEMICAL RESIDUES IN FOOD Specific Tolerances § 180.460 Benoxacor; tolerances for residues. (a) General. Tolerances are established for residues...

  7. 40 CFR 180.132 - Thiram; tolerances for residues.

    Code of Federal Regulations, 2010 CFR

    2010-07-01

    ... 40 Protection of Environment 23 2010-07-01 2010-07-01 false Thiram; tolerances for residues. 180... PROGRAMS TOLERANCES AND EXEMPTIONS FOR PESTICIDE CHEMICAL RESIDUES IN FOOD Specific Tolerances § 180.132 Thiram; tolerances for residues. (a) General. Tolerances are established for residues of the...

  8. 40 CFR 180.496 - Thiazopyr; tolerances for residues.

    Code of Federal Regulations, 2010 CFR

    2010-07-01

    ... 40 Protection of Environment 23 2010-07-01 2010-07-01 false Thiazopyr; tolerances for residues...) PESTICIDE PROGRAMS TOLERANCES AND EXEMPTIONS FOR PESTICIDE CHEMICAL RESIDUES IN FOOD Specific Tolerances § 180.496 Thiazopyr; tolerances for residues. Tolerances are established for combined residues of...

  9. 40 CFR 180.315 - Methamidophos; tolerances for residues.

    Code of Federal Regulations, 2010 CFR

    2010-07-01

    ... 40 Protection of Environment 23 2010-07-01 2010-07-01 false Methamidophos; tolerances for residues...) PESTICIDE PROGRAMS TOLERANCES AND EXEMPTIONS FOR PESTICIDE CHEMICAL RESIDUES IN FOOD Specific Tolerances § 180.315 Methamidophos; tolerances for residues. (a) Tolerances are established for residues of...

  10. 40 CFR 180.614 - Kasugamycin; tolerances for residues.

    Code of Federal Regulations, 2010 CFR

    2010-07-01

    ... 40 Protection of Environment 23 2010-07-01 2010-07-01 false Kasugamycin; tolerances for residues...) PESTICIDE PROGRAMS TOLERANCES AND EXEMPTIONS FOR PESTICIDE CHEMICAL RESIDUES IN FOOD Specific Tolerances § 180.614 Kasugamycin; tolerances for residues. (a) General. Tolerances are established for residues...

  11. 40 CFR 180.243 - Propazine; tolerances for residues.

    Code of Federal Regulations, 2010 CFR

    2010-07-01

    ... 40 Protection of Environment 23 2010-07-01 2010-07-01 false Propazine; tolerances for residues...) PESTICIDE PROGRAMS TOLERANCES AND EXEMPTIONS FOR PESTICIDE CHEMICAL RESIDUES IN FOOD Specific Tolerances § 180.243 Propazine; tolerances for residues. Tolerances are established for negligible residues (N)...

  12. 40 CFR 180.337 - Oxytetracycline; tolerance for residues.

    Code of Federal Regulations, 2010 CFR

    2010-07-01

    ... residues. 180.337 Section 180.337 Protection of Environment ENVIRONMENTAL PROTECTION AGENCY (CONTINUED) PESTICIDE PROGRAMS TOLERANCES AND EXEMPTIONS FOR PESTICIDE CHEMICAL RESIDUES IN FOOD Specific Tolerances § 180.337 Oxytetracycline; tolerance for residues. Tolerances are established for residues of...

  13. Occurrence of insecticide residues in selected crops and natural resources.

    PubMed

    Ratna Kumari, B; Ranga Rao, G V; Sahrawat, K L; Rajasekhar, P

    2012-07-01

    Pesticide residue monitoring was taken up at Kothapally and Enkepally villages of Ranga Reddy district, Andhra Pradesh in food crops (rice, maize, pigeonpea), vegetables (tomato and brinjal), cotton besides soil and water during 2008-2009 seasons. Of the 80 food crop and cotton samples, only two rice grain samples (3 %) showed beta endosulfan residues and two (3 %) out of 80 soil samples of food crops and cotton showed alpha and beta endosulfan residues. Out of 75 tomato samples, 26 (35 %) were contaminated and 4 % had residues above maximum residue limit (MRLs). Out of the 50 soil samples from tomato fields, 13 (26 %) contained residues. Among the 80 brinjal samples, 46 (56 %) contained residues and 4 % of samples had residues above MRLs. Only 13 % of the soil samples from brinjal fields were contaminated. Water samples found free from residues. In general the incidence of residues was below MRL in food crops.

  14. Chemical Stabilization of Hanford Tank Residual Waste

    SciTech Connect

    Cantrell, Kirk J.; Um, Wooyong; Williams, Benjamin D.; Bowden, Mark E.; Gartman, Brandy N.; Lukens, Wayne W.; Buck, Edgar C.; Mausolf, Edward J.

    2014-03-01

    Three different chemical treatment methods were tested for their ability to stabilize residual waste from Hanford tank C-202 for reducing contaminant release (Tc, Cr, and U in particular). The three treatment methods tested were lime addition [Ca(OH)2], an in-situ Ceramicrete waste form based on chemically bonded phosphate ceramics, and a ferrous iron/goethite treatment. These approaches rely on formation of insoluble forms of the contaminants of concern (lime addition and ceramicrete) and chemical reduction followed by co-precipitation (ferrous iron/goethite incorporation treatment). The results have demonstrated that release of the three most significant mobile contaminants of concern from tank residual wastes can be dramatically reduced after treatment compared to contact with simulated grout porewater without treatment. For uranium, all three treatments methods reduced the leachable uranium concentrations by well over three orders of magnitude. In the case of uranium and technetium, released concentrations were well below their respective MCLs for the wastes tested. For tank C-202 residual waste, chromium release concentrations were above the MCL but were considerably reduced relative to untreated tank waste. This innovative approach has the potential to revolutionize Hanford’s tank retrieval process, by allowing larger volumes of residual waste to be left in tanks while providing an acceptably low level of risk with respect to contaminant release that is protective of the environment and human health. Such an approach could enable DOE to realize significant cost savings through streamlined retrieval and closure operations.

  15. Substratum, Adstratum, and Residual Bilingualism in Brussels.

    ERIC Educational Resources Information Center

    Beardsmore, Hugo Baetens

    1983-01-01

    Discusses residual bilingualism as a means of identifying the nature, quantity, and distribution of Dutch-origin elements in the speech of different users of French in Brussels. Observations on code switching in a community of monoglots, bilinguals, and immigrants help provide a frame of reference for similar complex bilingual contexts elsewhere.…

  16. Vitrification for stability of scrap and residue

    SciTech Connect

    Forsberg, C.W.

    1996-05-01

    A conference breakout discussion was held on the subject of vitrification for stabilization of plutonium scrap and residue. This was one of four such sessions held within the vitrification workshop for participants to discuss specific subjects in further detail. The questions and issues were defined by the participants.

  17. Selenium speciation in flue desulfurization residues.

    PubMed

    Zhong, Liping; Cao, Yan; Li, Wenying; Xie, Kechang; Pan, Wei-Ping

    2011-01-01

    Flue gas from coal combustion contains significant amounts of volatile selenium (Se). The capture of Se in the flue gas desulfurization (FGD) scrubber unit has resulted in a generation of metal-laden residues. It is important to determine Se speciation to understand the environmental impact of its disposal. A simple method has been developed for selective inorganic Se(IV), Se(VI) and organic Se determination in the liquid-phase FGD residues by hydride generation atomic fluorescence spectrometry (AFS). It has been determined that Se(IV), Se(VI) and organic Se can be accurately determined with detection limits (DL) of 0.05, 0.06 and 0.06 microg/L, respectively. The accuracy of the proposed method was evaluated by analyzing the certified reference material, NIST CRM 1632c, and also by analyzing spiked tap-water samples. Analysis indicates that the concentration of Se is high in FGD liquid residues and primarily exists in a reduced state as selenite (Se(IV)). The toxicity of Se(IV) is the strongest of all Se species. Flue gas desulfurization residues pose a serious environmental risk.

  18. RESRAD. Site-Specific Residual Radioactivity

    SciTech Connect

    Yu, C.

    1989-06-01

    RESRAD is designed to derive site-specific guidelines for allowable residual concentrations of radionuclides in soil. A guideline is defined as a radionuclide concentration or a level of radiation or radioactivity that is acceptable if a site is to be used without radiological restrictions. Guidelines are expressed as (1) concentrations of residual radionuclides in soil, (2) concentrations of airborne radon decay products, (3) levels of external gamma radiation, (4) levels of radioactivity from surface contamination, and (5) concentrations of residual radionuclides in air and water. Soil is defined as unconsolidated earth material, including rubble and debris that may be present. The controlling principles of all guidelines are (1) the annual radiation dose received by a member of the critical population group from the residual radioactive material - predicted by a realistic but reasonably conservative analysis and averaged over a 50 year period - should not exceed 100 mrem/yr, and (2) doses should be kept as low as reasonably achievable. All significant exposure pathways for the critical population group are considered in deriving soil guidelines. These pathways include direct exposure to external radiation from the contaminated soil material; internal radiation from inhalation of airborne radionuclides; and internal radiation from ingestion of plant foods grown in the contaminated soil, meat and milk from livestock fed with contaminated fodder and water, drinking water from a contaminated well, and fish from a contaminated pond.

  19. Monitoring of pesticide residues in vegetarian diet.

    PubMed

    Kumari, Beena; Kathpal, T S

    2009-04-01

    Samples (28) of complete vegetarian diet consumed from morning till night i.e. tea, milk, breakfast, lunch, snacks, dinner, sweet dish etc. were collected from homes, hostels and hotels periodically from Hisar and analysed for detecting the residues of organochlorine, synthetic pyrethriod, organophosphate and carbamate insecticides. The estimation was carried out by using multi-residue analytical technique employing gas chromatograph (GC)-electron capture detector and GC-nitrogen phosphorous detector systems equipped with capillary columns. The whole diet sample was macerated in a mixer grinder and a representative sample in duplicate was analyzed for residues keeping the average daily diet of an adult to be 1,300 g. On comparing the data, it was found that actual daily intake (microgram/person/day) of lindane in two and endosulfan in four samples exceeded the acceptable daily intake. Residues of other pesticides in all the diet samples were lower than the acceptable daily intake (ADI) of the respective pesticides. The study concluded that although all the diet samples were found contaminated with one or the other pesticide, the actual daily intake of only a few pesticides was higher than their respective ADI. More extensive study covering other localities of Haryana has been suggested to know the overall scenario of contamination of vegetarian diet.

  20. Pesticide Residues in Food: Your Daily Dose.

    ERIC Educational Resources Information Center

    Mott, Lawrie

    1985-01-01

    Extensive use of pesticides during food production has created concerns for certain involuntary risks. Examines these concerns: government role in control and monitoring pesticide use, proposals for reform, and how consumer awareness might be an effective pressure for finding remedies. A table listing produce and pesticide residues is included.…