Multiple TPR motifs characterize the Fanconi anemia FANCG protein.

PubMed

Blom, Eric; van de Vrugt, Henri J; de Vries, Yne; de Winter, Johan P; Arwert, Fré; Joenje, Hans

2004-01-05

The genome protection pathway that is defective in patients with Fanconi anemia (FA) is controlled by at least eight genes, including BRCA2. A key step in the pathway involves the monoubiquitylation of FANCD2, which critically depends on a multi-subunit nuclear 'core complex' of at least six FANC proteins (FANCA, -C, -E, -F, -G, and -L). Except for FANCL, which has WD40 repeats and a RING finger domain, no significant domain structure has so far been recognized in any of the core complex proteins. By using a homology search strategy comparing the human FANCG protein sequence with its ortholog sequences in Oryzias latipes (Japanese rice fish) and Danio rerio (zebrafish) we identified at least seven tetratricopeptide repeat motifs (TPRs) covering a major part of this protein. TPRs are degenerate 34-amino acid repeat motifs which function as scaffolds mediating protein-protein interactions, often found in multiprotein complexes. In four out of five TPR motifs tested (TPR1, -2, -5, and -6), targeted missense mutagenesis disrupting the motifs at the critical position 8 of each TPR caused complete or partial loss of FANCG function. Loss of function was evident from failure of the mutant proteins to complement the cellular FA phenotype in FA-G lymphoblasts, which was correlated with loss of binding to FANCA. Although the TPR4 mutant fully complemented the cells, it showed a reduced interaction with FANCA, suggesting that this TPR may also be of functional importance. The recognition of FANCG as a typical TPR protein predicts this protein to play a key role in the assembly and/or stabilization of the nuclear FA protein core complex.

Modulation of the multistate folding of designed TPR proteins through intrinsic and extrinsic factors

PubMed Central

Phillips, J J; Javadi, Y; Millership, C; Main, E R G

2012-01-01

Tetratricopeptide repeats (TPRs) are a class of all alpha-helical repeat proteins that are comprised of 34-aa helix-turn-helix motifs. These stack together to form nonglobular structures that are stabilized by short-range interactions from residues close in primary sequence. Unlike globular proteins, they have few, if any, long-range nonlocal stabilizing interactions. Several studies on designed TPR proteins have shown that this modular structure is reflected in their folding, that is, modular multistate folding is observed as opposed to two-state folding. Here we show that TPR multistate folding can be suppressed to approximate two-state folding through modulation of intrinsic stability or extrinsic environmental variables. This modulation was investigated by comparing the thermodynamic unfolding under differing buffer regimes of two distinct series of consensus-designed TPR proteins, which possess different intrinsic stabilities. A total of nine proteins of differing sizes and differing consensus TPR motifs were each thermally and chemically denatured and their unfolding monitored using differential scanning calorimetry (DSC) and CD/fluorescence, respectively. Analyses of both the DSC and chemical denaturation data show that reducing the total stability of each protein and repeat units leads to observable two-state unfolding. These data highlight the intimate link between global and intrinsic repeat stability that governs whether folding proceeds by an observably two-state mechanism, or whether partial unfolding yields stable intermediate structures which retain sufficient stability to be populated at equilibrium. PMID:22170589

The active site of O-GlcNAc transferase imposes constraints on substrate sequence

PubMed Central

Rafie, Karim; Blair, David E.; Borodkin, Vladimir S.; Albarbarawi, Osama; van Aalten, Daan M. F.

2016-01-01

O-GlcNAc transferase (OGT) glycosylates a diverse range of intracellular proteins with O-linked N-acetylglucosamine (O-GlcNAc), an essential and dynamic post-translational modification in metazoa. Although this enzyme modifies hundreds of proteins with O-GlcNAc, it is not understood how OGT achieves substrate specificity. In this study, we describe the application of a high-throughput OGT assay on a library of peptides. The sites of O-GlcNAc modification were mapped by ETD-mass spectrometry, and found to correlate with previously detected O-GlcNAc sites. Crystal structures of four acceptor peptides in complex with human OGT suggest that a combination of size and conformational restriction defines sequence specificity in the −3 to +2 subsites. This work reveals that while the N-terminal TPR repeats of hOGT may play a role in substrate recognition, the sequence restriction imposed by the peptide-binding site makes a significant contribution to O-GlcNAc site specificity. PMID:26237509

Specific Binding of Tetratricopeptide Repeat Proteins to Heat Shock Protein 70 (Hsp70) and Heat Shock Protein 90 (Hsp90) Is Regulated by Affinity and Phosphorylation.

PubMed

Assimon, Victoria A; Southworth, Daniel R; Gestwicki, Jason E

2015-12-08

Heat shock protein 70 (Hsp70) and heat shock protein 90 (Hsp90) require the help of tetratricopeptide repeat (TPR) domain-containing cochaperones for many of their functions. Each monomer of Hsp70 or Hsp90 can interact with only a single TPR cochaperone at a time, and each member of the TPR cochaperone family brings distinct functions to the complex. Thus, competition for TPR binding sites on Hsp70 and Hsp90 appears to shape chaperone activity. Recent structural and biophysical efforts have improved our understanding of chaperone-TPR contacts, focusing on the C-terminal EEVD motif that is present in both chaperones. To better understand these important protein-protein interactions on a wider scale, we measured the affinity of five TPR cochaperones, CHIP, Hop, DnaJC7, FKBP51, and FKBP52, for the C-termini of four members of the chaperone family, Hsc70, Hsp72, Hsp90α, and Hsp90β, in vitro. These studies identified some surprising selectivity among the chaperone-TPR pairs, including the selective binding of FKBP51/52 to Hsp90α/β. These results also revealed that other TPR cochaperones are only able to weakly discriminate between the chaperones or between their paralogs. We also explored whether mimicking phosphorylation of serine and threonine residues near the EEVD motif might impact affinity and found that pseudophosphorylation had selective effects on binding to CHIP but not other cochaperones. Together, these findings suggest that both intrinsic affinity and post-translational modifications tune the interactions between the Hsp70 and Hsp90 proteins and the TPR cochaperones.

Molecular basis for TPR domain-mediated regulation of protein phosphatase 5.

PubMed

Yang, Jing; Roe, S Mark; Cliff, Matthew J; Williams, Mark A; Ladbury, John E; Cohen, Patricia T W; Barford, David

2005-01-12

Protein phosphatase 5 (Ppp5) is a serine/threonine protein phosphatase comprising a regulatory tetratricopeptide repeat (TPR) domain N-terminal to its phosphatase domain. Ppp5 functions in signalling pathways that control cellular responses to stress, glucocorticoids and DNA damage. Its phosphatase activity is suppressed by an autoinhibited conformation maintained by the TPR domain and a C-terminal subdomain. By interacting with the TPR domain, heat shock protein 90 (Hsp90) and fatty acids including arachidonic acid stimulate phosphatase activity. Here, we describe the structure of the autoinhibited state of Ppp5, revealing mechanisms of TPR-mediated phosphatase inhibition and Hsp90- and arachidonic acid-induced stimulation of phosphatase activity. The TPR domain engages with the catalytic channel of the phosphatase domain, restricting access to the catalytic site. This autoinhibited conformation of Ppp5 is stabilised by the C-terminal alphaJ helix that contacts a region of the Hsp90-binding groove on the TPR domain. Hsp90 activates Ppp5 by disrupting TPR-phosphatase domain interactions, permitting substrate access to the constitutively active phosphatase domain, whereas arachidonic acid prompts an alternate conformation of the TPR domain, destabilising the TPR-phosphatase domain interface.

Characterization of Spindle Checkpoint Kinase Mps1 Reveals Domain with Functional and Structural Similarities to Tetratricopeptide Repeat Motifs of Bub1 and BubR1 Checkpoint Kinases*

PubMed Central

Lee, Semin; Thebault, Philippe; Freschi, Luca; Beaufils, Sylvie; Blundell, Tom L.; Landry, Christian R.; Bolanos-Garcia, Victor M.; Elowe, Sabine

2012-01-01

Kinetochore targeting of the mitotic kinases Bub1, BubR1, and Mps1 has been implicated in efficient execution of their functions in the spindle checkpoint, the self-monitoring system of the eukaryotic cell cycle that ensures chromosome segregation occurs with high fidelity. In all three kinases, kinetochore docking is mediated by the N-terminal region of the protein. Deletions within this region result in checkpoint failure and chromosome segregation defects. Here, we use an interdisciplinary approach that includes biophysical, biochemical, cell biological, and bioinformatics methods to study the N-terminal region of human Mps1. We report the identification of a tandem repeat of the tetratricopeptide repeat (TPR) motif in the N-terminal kinetochore binding region of Mps1, with close homology to the tandem TPR motif of Bub1 and BubR1. Phylogenetic analysis indicates that TPR Mps1 was acquired after the split between deutorostomes and protostomes, as it is distinguishable in chordates and echinoderms. Overexpression of TPR Mps1 resulted in decreased efficiency of both chromosome alignment and mitotic arrest, likely through displacement of endogenous Mps1 from the kinetochore and decreased Mps1 catalytic activity. Taken together, our multidisciplinary strategy provides new insights into the evolution, structural organization, and function of Mps1 N-terminal region. PMID:22187426

Characterization of spindle checkpoint kinase Mps1 reveals domain with functional and structural similarities to tetratricopeptide repeat motifs of Bub1 and BubR1 checkpoint kinases.

PubMed

Lee, Semin; Thebault, Philippe; Freschi, Luca; Beaufils, Sylvie; Blundell, Tom L; Landry, Christian R; Bolanos-Garcia, Victor M; Elowe, Sabine

2012-02-17

Kinetochore targeting of the mitotic kinases Bub1, BubR1, and Mps1 has been implicated in efficient execution of their functions in the spindle checkpoint, the self-monitoring system of the eukaryotic cell cycle that ensures chromosome segregation occurs with high fidelity. In all three kinases, kinetochore docking is mediated by the N-terminal region of the protein. Deletions within this region result in checkpoint failure and chromosome segregation defects. Here, we use an interdisciplinary approach that includes biophysical, biochemical, cell biological, and bioinformatics methods to study the N-terminal region of human Mps1. We report the identification of a tandem repeat of the tetratricopeptide repeat (TPR) motif in the N-terminal kinetochore binding region of Mps1, with close homology to the tandem TPR motif of Bub1 and BubR1. Phylogenetic analysis indicates that TPR Mps1 was acquired after the split between deutorostomes and protostomes, as it is distinguishable in chordates and echinoderms. Overexpression of TPR Mps1 resulted in decreased efficiency of both chromosome alignment and mitotic arrest, likely through displacement of endogenous Mps1 from the kinetochore and decreased Mps1 catalytic activity. Taken together, our multidisciplinary strategy provides new insights into the evolution, structural organization, and function of Mps1 N-terminal region.

Endoplasmic Reticulum Protein Quality Control Is Determined by Cooperative Interactions between Hsp/c70 Protein and the CHIP E3 Ligase*

PubMed Central

Matsumura, Yoshihiro; Sakai, Juro; Skach, William R.

2013-01-01

The C terminus of Hsp70 interacting protein (CHIP) E3 ligase functions as a key regulator of protein quality control by binding the C-terminal (M/I)EEVD peptide motif of Hsp/c70(90) with its N-terminal tetratricopeptide repeat (TPR) domain and facilitating polyubiquitination of misfolded client proteins via its C-terminal catalytic U-box. Using CFTR as a model client, we recently showed that the duration of the Hsc70-client binding cycle is a primary determinant of stability. However, molecular features that control CHIP recruitment to Hsp/c70, and hence the fate of the Hsp/c70 client, remain unknown. To understand how CHIP recognizes Hsp/c70, we utilized a dominant negative mutant in which loss of a conserved proline in the U-box domain (P269A) eliminates E3 ligase activity. In a cell-free reconstituted ER-associated degradation system, P269A CHIP inhibited Hsc70-dependent CFTR ubiquitination and degradation in a dose-dependent manner. Optimal inhibition required both the TPR and the U-box, indicating cooperativity between the two domains. Neither the wild type nor the P269A mutant changed the extent of Hsc70 association with CFTR nor the dissociation rate of the Hsc70-CFTR complex. However, the U-box mutation stimulated CHIP binding to Hsc70 while promoting CHIP oligomerization. CHIP binding to Hsc70 binding was also stimulated by the presence of an Hsc70 client with a preference for the ADP-bound state. Thus, the Hsp/c70 (M/I)EEVD motif is not a simple anchor for the TPR domain. Rather CHIP recruitment involves reciprocal allosteric interactions between its TPR and U-box domains and the substrate-binding and C-terminal domains of Hsp/c70. PMID:23990462

TprC/D (Tp0117/131), a trimeric, pore-forming rare outer membrane protein of Treponema pallidum, has a bipartite domain structure.

PubMed

Anand, Arvind; Luthra, Amit; Dunham-Ems, Star; Caimano, Melissa J; Karanian, Carson; LeDoyt, Morgan; Cruz, Adriana R; Salazar, Juan C; Radolf, Justin D

2012-05-01

Identification of Treponema pallidum rare outer membrane proteins (OMPs) has been a longstanding objective of syphilis researchers. We recently developed a consensus computational framework that employs a battery of cellular localization and topological prediction tools to generate ranked clusters of candidate rare OMPs (D. L. Cox et al., Infect. Immun. 78:5178-5194, 2010). TP0117/TP0131 (TprC/D), a member of the T. pallidum repeat (Tpr) family, was a highly ranked candidate. Circular dichroism, heat modifiability by SDS-PAGE, Triton X-114 phase partitioning, and liposome incorporation confirmed that full-length, recombinant TprC (TprC(Fl)) forms a β-barrel capable of integrating into lipid bilayers. Moreover, TprC(Fl) increased efflux of terbium-dipicolinic acid complex from large unilamellar vesicles and migrated as a trimer by blue-native PAGE. We found that in T. pallidum, TprC is heat modifiable, trimeric, expressed in low abundance, and, based on proteinase K accessibility and opsonophagocytosis assays, surface exposed. From these collective data, we conclude that TprC is a bona fide rare OMP as well as a functional ortholog of Escherichia coli OmpF. We also discovered that TprC has a bipartite architecture consisting of a soluble N-terminal portion (TprC(N)), presumably periplasmic and bound directly or indirectly to peptidoglycan, and a C-terminal β-barrel (TprC(C)). Syphilitic rabbits generate antibodies exclusively against TprC(C), while secondary syphilis patients fail to mount a detectable antibody response against either domain. The syphilis spirochete appears to have resolved a fundamental dilemma arising from its extracellular lifestyle, namely, how to enhance OM permeability without increasing its vulnerability to the antibody-mediated defenses of its natural human host.

Localization of Nucleoporin Tpr to the Nuclear Pore Complex Is Essential for Tpr Mediated Regulation of the Export of Unspliced RNA

PubMed Central

Rajanala, Kalpana; Nandicoori, Vinay Kumar

2012-01-01

Nucleoporin Tpr is a component of the nuclear pore complex (NPC) that localizes exclusively to intranuclear filaments. Tpr functions as a scaffolding element in the nuclear phase of the NPC and plays a role in mitotic spindle checkpoint signalling. Export of intron-containing mRNA in Mason Pfizer Monkey Virus is regulated by direct interaction of cellular proteins with the cis-acting Constitutive Transport Element (CTE). In mammalian cells, the transport of Gag/Pol-CTE reporter construct is not very efficient, suggesting a regulatory mechanism to retain this unspliced RNA. Here we report that the knockdown of Tpr in mammalian cells leads to a drastic enhancement in the levels of Gag proteins (p24) in the cytoplasm, which is rescued by siRNA resistant Tpr. Tpr's role in the retention of unspliced RNA is independent of the functions of Sam68 and Tap/Nxf1 proteins, which are reported to promote CTE dependent export. Further, we investigated the possible role for nucleoporins that are known to function in nucleocytoplasmic transport in modulating unspliced RNA export. Results show that depletion of Nup153, a nucleoporin required for NPC anchoring of Tpr, plays a role in regulating the export, while depletion of other FG repeat-containing nucleoporins did not alter the unspliced RNA export. Results suggest that Tpr and Nup153 both regulate the export of unspliced RNA and they are most likely functioning through the same pathway. Importantly, we find that localization of Tpr to the NPC is necessary for Tpr mediated regulation of unspliced RNA export. Collectively, the data indicates that perinuclear localization of Tpr at the nucleopore complex is crucial for regulating intron containing mRNA export by directly or indirectly participating in the processing and degradation of aberrant mRNA transcripts. PMID:22253824

Molecular basis for the interaction between stress-inducible phosphoprotein 1 (STIP1) and S100A1.

PubMed

Maciejewski, Andrzej; Prado, Vania F; Prado, Marco A M; Choy, Wing-Yiu

2017-05-16

Stress-inducible phosphoprotein 1 (STIP1) is a cellular co-chaperone, which regulates heat-shock protein 70 (Hsp70) and Hsp90 activity during client protein folding. Members of the S100 family of dimeric calcium-binding proteins have been found to inhibit Hsp association with STIP1 through binding of STIP1 tetratricopeptide repeat (TPR) domains, possibly regulating the chaperone cycle. Here, we investigated the molecular basis of S100A1 binding to STIP1. We show that three S100A1 dimers associate with one molecule of STIP1 in a calcium-dependent manner. Isothermal titration calorimetry revealed that individual STIP1 TPR domains, TPR1, TPR2A and TPR2B, bind a single S100A1 dimer with significantly different affinities and that the TPR2B domain possesses the highest affinity for S100A1. S100A1 bound each TPR domain through a common binding interface composed of α-helices III and IV of each S100A1 subunit, which is only accessible following a large conformational change in S100A1 upon calcium binding. The TPR2B-binding site for S100A1 was predominately mapped to the C-terminal α-helix of TPR2B, where it is inserted into the hydrophobic cleft of an S100A1 dimer, suggesting a novel binding mechanism. Our data present the structural basis behind STIP1 and S100A1 complex formation, and provide novel insights into TPR module-containing proteins and S100 family member complexes. © 2017 The Author(s); published by Portland Press Limited on behalf of the Biochemical Society.

Amino Acid Substitutions of Coiled-Coil Protein Tpr Abrogate Anchorage to the Nuclear Pore Complex but Not Parallel, In-Register Homodimerization

PubMed Central

Hase, Manuela E.; Kuznetsov, Nikolai V.; Cordes, Volker C.

2001-01-01

Tpr is a protein component of nuclear pore complex (NPC)-attached intranuclear filaments. Secondary structure predictions suggest a bipartite structure, with a large N-terminal domain dominated by heptad repeats (HRs) typical for coiled-coil–forming proteins. Proposed functions for Tpr have included roles as a homo- or heteropolymeric architectural element of the nuclear interior. To gain insight into Tpr's ultrastructural properties, we have studied recombinant Tpr segments by circular dichroism spectroscopy, chemical cross-linking, and rotary shadowing electron microscopy. We show that polypeptides of the N-terminal domain homodimerize in vitro and represent α-helical molecules of extended rod-like shape. With the use of a yeast two-hybrid approach, arrangement of the coiled-coil is found to be in parallel and in register. To clarify whether Tpr can self-assemble further into homopolymeric filaments, the full-length protein and deletion mutants were overexpressed in human cells and then analyzed by confocal immunofluorescence microscopy, cell fractionation, and immuno-electron microscopy. Surplus Tpr, which does not bind to the NPC, remains in a soluble state of ∼7.5 S and occasionally forms aggregates of entangled molecules but neither self-assembles into extended linear filaments nor stably binds to other intranuclear structures. Binding to the NPC is shown to depend on the integrity of individual HRs; amino acid substitutions within these HRs abrogate NPC binding and render the protein soluble but do not abolish Tpr's general ability to homodimerize. Possible contributions of Tpr to the structural organization of the nuclear periphery in somatic cells are discussed. PMID:11514627

Modular elements of the TPR domain in the Mps1 N terminus differentially target Mps1 to the centrosome and kinetochore

PubMed Central

Marquardt, Joseph R.; Perkins, Jennifer L.; Beuoy, Kyle J.; Fisk, Harold A.

2016-01-01

Faithful segregation of chromosomes to two daughter cells is regulated by the formation of a bipolar mitotic spindle and the spindle assembly checkpoint, ensuring proper spindle function. Here we show that the proper localization of the kinase Mps1 (monopolar spindle 1) is critical to both these processes. Separate elements in the Mps1 N-terminal extension (NTE) and tetratricopeptide repeat (TPR) domains govern localization to either the kinetochore or the centrosome. The third TPR (TPR3) and the TPR-capping helix (C-helix) are each sufficient to target Mps1 to the centrosome. TPR3 binds to voltage-dependent anion channel 3, but although this is sufficient for centrosome targeting of Mps1, it is not necessary because of the presence of the C-helix. A version of Mps1 lacking both elements cannot localize to or function at the centrosome, but maintains kinetochore localization and spindle assembly checkpoint function, indicating that TPR3 and the C-helix define a bipartite localization determinant that is both necessary and sufficient to target Mps1 to the centrosome but dispensable for kinetochore targeting. In contrast, elements required for kinetochore targeting (the NTE and first two TPRs) are dispensable for centrosomal localization and function. These data are consistent with a separation of Mps1 function based on localization determinants within the N terminus. PMID:27339139

Modular elements of the TPR domain in the Mps1 N terminus differentially target Mps1 to the centrosome and kinetochore.

PubMed

Marquardt, Joseph R; Perkins, Jennifer L; Beuoy, Kyle J; Fisk, Harold A

2016-07-12

Faithful segregation of chromosomes to two daughter cells is regulated by the formation of a bipolar mitotic spindle and the spindle assembly checkpoint, ensuring proper spindle function. Here we show that the proper localization of the kinase Mps1 (monopolar spindle 1) is critical to both these processes. Separate elements in the Mps1 N-terminal extension (NTE) and tetratricopeptide repeat (TPR) domains govern localization to either the kinetochore or the centrosome. The third TPR (TPR3) and the TPR-capping helix (C-helix) are each sufficient to target Mps1 to the centrosome. TPR3 binds to voltage-dependent anion channel 3, but although this is sufficient for centrosome targeting of Mps1, it is not necessary because of the presence of the C-helix. A version of Mps1 lacking both elements cannot localize to or function at the centrosome, but maintains kinetochore localization and spindle assembly checkpoint function, indicating that TPR3 and the C-helix define a bipartite localization determinant that is both necessary and sufficient to target Mps1 to the centrosome but dispensable for kinetochore targeting. In contrast, elements required for kinetochore targeting (the NTE and first two TPRs) are dispensable for centrosomal localization and function. These data are consistent with a separation of Mps1 function based on localization determinants within the N terminus.

A Bipartite Interaction between Hsp70 and CHIP Regulates Ubiquitination of Chaperoned Client Proteins

DOE PAGES

Zhang, Huaqun; Amick, Joseph; Chakravarti, Ritu; ...

2015-02-12

The ubiquitin ligase CHIP plays an important role in cytosolic protein quality control by ubiquitinating proteins chaperoned by Hsp70/Hsc70 and Hsp90, thereby targeting such substrate proteins for degradation. We present a 2.91 Å resolution structure of the tetratricopeptide repeat (TPR) domain of CHIP in complex with the α-helical lid subdomain and unstructured tail of Hsc70. Surprisingly, the CHIP-TPR interacts with determinants within both the Hsc70-lid subdomain and the C-terminal PTIEEVD motif of the tail, exhibiting an atypical mode of interaction between chaperones and TPR domains. Here, we demonstrate that the interaction between CHIP and the Hsc70-lid subdomain is required formore » proper ubiquitination of Hsp70/Hsc70 or Hsp70/Hsc70-bound substrate proteins. Posttranslational modifications of the Hsc70 lid and tail disrupt key contacts with the CHIP-TPR and may regulate CHIP-mediated ubiquitination. Our study shows how CHIP docks onto Hsp70/Hsc70 and defines a bipartite mode of interaction between TPR domains and their binding partners.« less

Sequence analyses reveal that a TPR–DP module, surrounded by recombinable flanking introns, could be at the origin of eukaryotic Hop and Hip TPR–DP domains and prokaryotic GerD proteins

PubMed Central

Papandreou, Nikolaos; Chomilier, Jacques

2008-01-01

The co-chaperone Hop [heat shock protein (HSP) organising protein] is known to bind both Hsp70 and Hsp90. Hop comprises three repeats of a tetratricopeptide repeat (TPR) domain, each consisting of three TPR motifs. The first and last TPR domains are followed by a domain containing several dipeptide (DP) repeats called the DP domain. These analyses suggest that the hop genes result from successive recombination events of an ancestral TPR–DP module. From a hydrophobic cluster analysis of homologous Hop protein sequences derived from gene families, we can postulate that shifts in the open reading frames are at the origin of the present sequences. Moreover, these shifts can be related to the presence or absence of biological function. We propose to extend the family of Hop co-chaperons into the kingdom of bacteria, as several structurally related genes have been identified by hydrophobic cluster analysis. We also provide evidence of common structural characteristics between hop and hip genes, suggesting a shared precursor of ancestral TPR–DP domains. Electronic supplementary material The online version of this article (doi:10.1007/s12192-008-0083-8) contains supplementary material, which is available to authorized users. PMID:18987995

Protein–Protein Interactions Modulate the Docking-Dependent E3-Ubiquitin Ligase Activity of Carboxy-Terminus of Hsc70-Interacting Protein (CHIP)*

PubMed Central

Narayan, Vikram; Landré, Vivien; Ning, Jia; Hernychova, Lenka; Muller, Petr; Verma, Chandra; Walkinshaw, Malcolm D.; Blackburn, Elizabeth A.; Ball, Kathryn L.

2015-01-01

CHIP is a tetratricopeptide repeat (TPR) domain protein that functions as an E3-ubiquitin ligase. As well as linking the molecular chaperones to the ubiquitin proteasome system, CHIP also has a docking-dependent mode where it ubiquitinates native substrates, thereby regulating their steady state levels and/or function. Here we explore the effect of Hsp70 on the docking-dependent E3-ligase activity of CHIP. The TPR-domain is revealed as a binding site for allosteric modulators involved in determining CHIP's dynamic conformation and activity. Biochemical, biophysical and modeling evidence demonstrate that Hsp70-binding to the TPR, or Hsp70-mimetic mutations, regulate CHIP-mediated ubiquitination of p53 and IRF-1 through effects on U-box activity and substrate binding. HDX-MS was used to establish that conformational-inhibition-signals extended from the TPR-domain to the U-box. This underscores inter-domain allosteric regulation of CHIP by the core molecular chaperones. Defining the chaperone-associated TPR-domain of CHIP as a manager of inter-domain communication highlights the potential for scaffolding modules to regulate, as well as assemble, complexes that are fundamental to protein homeostatic control. PMID:26330542

Novel Entropically Driven Conformation-specific Interactions with Tomm34 Protein Modulate Hsp70 Protein Folding and ATPase Activities*

PubMed Central

Durech, Michal; Trcka, Filip; Man, Petr; Blackburn, Elizabeth A.; Hernychova, Lenka; Dvorakova, Petra; Coufalova, Dominika; Kavan, Daniel; Vojtesek, Borivoj; Muller, Petr

2016-01-01

Co-chaperones containing tetratricopeptide repeat (TPR) domains enable cooperation between Hsp70 and Hsp90 to maintain cellular proteostasis. Although the details of the molecular interactions between some TPR domains and heat shock proteins are known, we describe a novel mechanism by which Tomm34 interacts with and coordinates Hsp70 activities. In contrast to the previously defined Hsp70/Hsp90-organizing protein (Hop), Tomm34 interaction is dependent on the Hsp70 chaperone cycle. Tomm34 binds Hsp70 in a complex process; anchorage of the Hsp70 C terminus by the TPR1 domain is accompanied by additional contacts formed exclusively in the ATP-bound state of Hsp70 resulting in a high affinity entropically driven interaction. Tomm34 induces structural changes in determinants within the Hsp70-lid subdomain and modulates Hsp70/Hsp40-mediated refolding and Hsp40-stimulated Hsp70 ATPase activity. Because Tomm34 recruits Hsp90 through its TPR2 domain, we propose a model in which Tomm34 enables Hsp70/Hsp90 scaffolding and influences the Hsp70 chaperone cycle, providing an additional role for co-chaperones that contain multiple TPR domains in regulating protein homeostasis. PMID:26944342

Defining the molecular basis of BubR1 kinetochore interactions and APC/C-CDC20 inhibition.

PubMed

D'Arcy, Sheena; Davies, Owen R; Blundell, Tom L; Bolanos-Garcia, Victor M

2010-05-07

BubR1 is essential for the mitotic checkpoint that prevents aneuploidy in cellular progeny by triggering anaphase delay in response to kinetochores incorrectly/not attached to the mitotic spindle. Here, we define the molecular architecture of the functionally significant N-terminal region of human BubR1 and present the 1.8 A crystal structure of its tetratricopeptide repeat (TPR) domain. The structure reveals divergence from the classical TPR fold and is highly similar to the TPR domain of budding yeast Bub1. Shared distinctive features include a disordered loop insertion, a 3(10)-helix, a tight turn involving glycine positive Phi angles, and noncanonical packing of and between the TPR motifs. We also define the molecular determinants of the interaction between BubR1 and kinetochore protein Blinkin. We identify a shallow groove on the concave surface of the BubR1 TPR domain that forms multiple discrete and potentially cooperative interactions with Blinkin. Finally, we present evidence for a direct interaction between BubR1 and Bub1 mediated by regions C-terminal to their TPR domains. This interaction provides a mechanism for Bub1-dependent kinetochore recruitment of BubR1. We thus present novel molecular insights into the structure of BubR1 and its interactions at the kinetochore-microtubule interface. Our studies pave the way for future structure-directed engineering aimed at dissecting the roles of kinetochore-bound and other pools of BubR1 in vivo.

Stability of the cancer target DDIAS is regulated by the CHIP/HSP70 pathway in lung cancer cells

PubMed Central

Won, Kyoung-Jae; Im, Joo-Young; Kim, Bo-Kyung; Ban, Hyun Seung; Jung, Young-Jin; Jung, Kyeong Eun; Won, Misun

2017-01-01

DNA damage-induced apoptosis suppressor (DDIAS) rescues lung cancer cells from apoptosis in response to DNA damage. DDIAS is transcriptionally activated by NFATc1 and EGF-mediated ERK5/MEF2B, leading to cisplatin resistance and cell invasion. Therefore, DDIAS is suggested as a therapeutic target for lung cancer. Here, we report that DDIAS stability is regulated by E3 U-box ubiquitin ligase carboxyl terminus of HSP70-interacting protein (CHIP)-mediated proteasomal degradation. We first isolated CHIP as an interacting partner of DDIAS by yeast two-hybrid screening. CHIP physically associated with both the N- and C-terminal regions of DDIAS, targeting it for proteasomal degradation and reducing the DDIAS half-life. CHIP overexpression analyses indicated that the tetratrico peptide repeat (TPR) domain and the U-box are required for DDIAS ubiquitination. It is likely that HSP70-bound DDIAS is recruited to the CHIP E3 ligase via the TPR domain, suggesting DDIAS as a client protein of HSP70. In addition, CHIP overexpression in lung cancer cells expressing high DDIAS levels induced significant growth inhibition by enhancing DDIAS degradation. Furthermore, simultaneous CHIP overexpression and DNA damage agent treatment caused a substantial increase in the apoptosis of lung cancer cells. Taken together, these findings indicate that the stability of the DDIAS protein is regulated by CHIP/HSP70-mediated proteasomal degradation and that CHIP overexpression stimulates the apoptosis of lung cancer cells in response to DNA-damaging agents. PMID:28079882

Stability of the cancer target DDIAS is regulated by the CHIP/HSP70 pathway in lung cancer cells.

PubMed

Won, Kyoung-Jae; Im, Joo-Young; Kim, Bo-Kyung; Ban, Hyun Seung; Jung, Young-Jin; Jung, Kyeong Eun; Won, Misun

2017-01-12

DNA damage-induced apoptosis suppressor (DDIAS) rescues lung cancer cells from apoptosis in response to DNA damage. DDIAS is transcriptionally activated by NFATc1 and EGF-mediated ERK5/MEF2B, leading to cisplatin resistance and cell invasion. Therefore, DDIAS is suggested as a therapeutic target for lung cancer. Here, we report that DDIAS stability is regulated by E3 U-box ubiquitin ligase carboxyl terminus of HSP70-interacting protein (CHIP)-mediated proteasomal degradation. We first isolated CHIP as an interacting partner of DDIAS by yeast two-hybrid screening. CHIP physically associated with both the N- and C-terminal regions of DDIAS, targeting it for proteasomal degradation and reducing the DDIAS half-life. CHIP overexpression analyses indicated that the tetratrico peptide repeat (TPR) domain and the U-box are required for DDIAS ubiquitination. It is likely that HSP70-bound DDIAS is recruited to the CHIP E3 ligase via the TPR domain, suggesting DDIAS as a client protein of HSP70. In addition, CHIP overexpression in lung cancer cells expressing high DDIAS levels induced significant growth inhibition by enhancing DDIAS degradation. Furthermore, simultaneous CHIP overexpression and DNA damage agent treatment caused a substantial increase in the apoptosis of lung cancer cells. Taken together, these findings indicate that the stability of the DDIAS protein is regulated by CHIP/HSP70-mediated proteasomal degradation and that CHIP overexpression stimulates the apoptosis of lung cancer cells in response to DNA-damaging agents.

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

PubMed Central

Nijenhuis, Wilco; von Castelmur, Eleonore; Littler, Dene; De Marco, Valeria; Tromer, Eelco; Vleugel, Mathijs; van Osch, Maria H.J.; Snel, Berend

2013-01-01

The mitotic checkpoint ensures correct chromosome segregation by delaying cell cycle progression until all kinetochores have attached to the mitotic spindle. In this paper, we show that the mitotic checkpoint kinase MPS1 contains an N-terminal localization module, organized in an N-terminal extension (NTE) and a tetratricopeptide repeat (TPR) domain, for which we have determined the crystal structure. Although the module was necessary for kinetochore localization of MPS1 and essential for the mitotic checkpoint, the predominant kinetochore binding activity resided within the NTE. MPS1 localization further required HEC1 and Aurora B activity. We show that MPS1 localization to kinetochores depended on the calponin homology domain of HEC1 but not on Aurora B–dependent phosphorylation of the HEC1 tail. Rather, the TPR domain was the critical mediator of Aurora B control over MPS1 localization, as its deletion rendered MPS1 localization insensitive to Aurora B inhibition. These data are consistent with a model in which Aurora B activity relieves a TPR-dependent inhibitory constraint on MPS1 localization. PMID:23569217

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

PubMed

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

2013-04-15

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

Crystal structure of P58(IPK) TPR fragment reveals the mechanism for its molecular chaperone activity in UPR

DOE Office of Scientific and Technical Information (OSTI.GOV)

Tao, Jiahui; Petrova, Kseniya; Ron, David

2010-05-25

P58(IPK) might function as an endoplasmic reticulum molecular chaperone to maintain protein folding homeostasis during unfolded protein responses. P58(IPK) contains nine tetratricopeptide repeat (TPR) motifs and a C-terminal J-domain within its primary sequence. To investigate the mechanism by which P58(IPK) functions to promote protein folding within the endoplasmic reticulum, we have determined the crystal structure of P58(IPK) TPR fragment to 2.5 {angstrom} resolution by the SAD method. The crystal structure of P58(IPK) revealed three domains (I-III) with similar folds and each domain contains three TPR motifs. An ELISA assay indicated that P58(IPK) acts as a molecular chaperone by interacting withmore » misfolded proteins such as luciferase and rhodanese. The P58(IPK) structure reveals a conserved hydrophobic patch located in domain I that might be involved in binding the misfolded polypeptides. Structure-based mutagenesis for the conserved hydrophobic residues located in domain I significantly reduced the molecular chaperone activity of P58(IPK).« less

Identification of amino acids in the tetratricopeptide repeat and C-terminal domains of protein phosphatase 5 involved in autoinhibition and lipid activation.

PubMed

Kang, H; Sayner, S L; Gross, K L; Russell, L C; Chinkers, M

2001-09-04

Protein phosphatase 5 (PP5) exhibits low basal activity due to the autoinhibitory properties of its N-terminal and C-terminal domains but can be activated approximately 40-fold in vitro by polyunsaturated fatty acids. To identify residues involved in regulating PP5 activity, we performed scanning mutagenesis of its N-terminal tetratricopeptide repeat (TPR) domain and deletion mutagenesis of its C-terminal domain. Mutating residues in a groove of the TPR domain that binds to heat shock protein 90 had no effect on basal phosphatase activity. Mutation of Glu-76, however, whose side chain projects away from this groove, resulted in a 10-fold elevation of basal activity without affecting arachidonic acid-stimulated activity. Thus, the interface of the TPR domain involved in PP5 autoinhibition appears to be different from that involved in heat shock protein 90 binding. We also observed a 10-fold elevation of basal phosphatase activity upon removing the C-terminal 13 amino acids of PP5, with a concomitant 50% decrease in arachidonic acid-stimulated activity. These two effects were accounted for by two distinct amino acid deletions: deleting the four C-terminal residues (496-499) of PP5 had no effect on its activity, but removing Gln-495 elevated basal activity 10-fold. Removal of a further three amino acids had no additional effect, but deleting Asn-491 resulted in a 50% reduction in arachidonic acid-stimulated activity. Thus, Glu-76 in the TPR domain and Gln-495 at the C-terminus were implicated in maintaining the low basal activity of PP5. While the TPR domain alone has been thought to mediate fatty acid activation of PP5, our data suggest that Asn-491, near its C-terminus, may also be involved in this process.

Molecular cloning and characterization of an Hsp90/70 organizing protein gene from Frankliniella occidentalis (Insecta: Thysanoptera, Thripidae).

PubMed

Li, Hong-Bo; Du, Yu-Zhou

2013-05-15

The heat shock 90/70 organizing protein (Hop), also known as Sti-1 (stress-induced protein-1), is a co-chaperone that usually mediates the interaction of Hsp90 and Hsp70 and has been extensively characterized in mammals and plants. However, its role in insects remains unknown. In the present study, we isolated and characterized a Hop homologue gene from Frankliniella occidentalis (Fohop). The Fohop contains a 1659bp ORF encoding a protein of 552 amino acids with a caculated molecular mass of approximately 62.25kDa, which displays a reasonable degree of identity with the known Hops and shares several canonical motifs, including three tetratricopeptide repeated motif domains (TPR1, TPR2A and TPR2B) and two aspartic acid-proline (DP) repeat motifs (DP1 and DP2). As in other hops, Fohop contains introns, but the number and the position are quite variable. The mRNA expression patterns indicated that Fohop was constitutively expressed throughout the developmental stages, but was obviously upregulated by heat stress both in larvae and adults. Our studies imply that Hop, as in other Hsps, may play an important role in heat shock response of F. occidentalis. Copyright © 2013 Elsevier B.V. All rights reserved.

Dissection of Swa2p/Auxilin Domain Requirements for Cochaperoning Hsp70 Clathrin-uncoating Activity In Vivo

PubMed Central

Xiao, Jing; Kim, Leslie S.

2006-01-01

The auxilin family of J-domain proteins load Hsp70 onto clathrin-coated vesicles (CCVs) to drive uncoating. In vitro, auxilin function requires its ability to bind clathrin and stimulate Hsp70 ATPase activity via its J-domain. To test these requirements in vivo, we performed a mutational analysis of Swa2p, the yeast auxilin ortholog. Swa2p is a modular protein with three N-terminal clathrin-binding (CB) motifs, a ubiquitin association (UBA) domain, a tetratricopeptide repeat (TPR) domain, and a C-terminal J-domain. In vitro, clathrin binding is mediated by multiple weak interactions, but a Swa2p truncation lacking two CB motifs and the UBA domain retains nearly full function in vivo. Deletion of all CB motifs strongly abrogates clathrin disassembly but does not eliminate Swa2p function in vivo. Surprisingly, mutation of the invariant HPD motif within the J-domain to AAA only partially affects Swa2p function. Similarly, a TPR point mutation (G388R) causes a modest phenotype. However, Swa2p function is abolished when these TPR and J mutations are combined. The TPR and J-domains are not functionally redundant because deletion of either domain renders Swa2p nonfunctional. These data suggest that the TPR and J-domains collaborate in a bipartite interaction with Hsp70 to regulate its activity in clathrin disassembly. PMID:16687570

Implication of Proteins Containing Tetratricopeptide Repeats in Conditional Virulence Phenotypes of Legionella pneumophila

PubMed Central

Bandyopadhyay, Purnima; Sumer, Eren U.; Jayakumar, Deepak; Liu, Shuqing; Xiao, Huifang

2012-01-01

Legionella pneumophila, the causative agent of Legionnaires' disease, is a ubiquitous freshwater bacterium whose virulence phenotypes require a type IV secretion system (T4SS). L. pneumophila strain JR32 contains two virulence-associated T4SSs, the Dot/Icm and Lvh T4SSs. Defective entry and phagosome acidification phenotypes of dot/icm mutants are conditional and reversed by incubating broth-grown stationary-phase cultures in water (WS treatment) prior to infection, as a mimic of the aquatic environment of Legionella. Reversal of dot/icm virulence defects requires the Lvh T4SS and is associated with a >10-fold induction of LpnE, a tetratricopeptide repeat (TPR)-containing protein. In the current study, we demonstrated that defective entry and phagosome acidification phenotypes of mutants with changes in LpnE and EnhC, another TPR-containing protein, were similarly reversed by WS treatment. In contrast to dot/icm mutants for which the Lvh T4SS was required, reversal for the ΔlpnE or the ΔenhC mutant required that the other TPR-containing protein be present. The single and double ΔlpnE and ΔenhC mutants showed a hypersensitivity to sodium ion, a phenotype associated with dysfunction of the Dot/Icm T4SS. The ΔlpnE single and the ΔlpnE ΔenhC double mutant showed 3- to 9-fold increases in translocation of Dot/Icm T4SS substrates, LegS2/SplY and LepB. Taken together, these data identify TPR-containing proteins in a second mechanism by which the WS mimic of a Legionella environmental niche can reverse virulence defects of broth-grown cultures and implicate LpnE and EnhC directly or indirectly in translocation of Dot/Icm T4SS protein substrates. PMID:22563053

Identification of novel host factors via conserved domain search: Cns1 cochaperone is a novel restriction factor of tombusvirus replication in yeast.

PubMed

Lin, Jing-Yi; Nagy, Peter D

2013-12-01

A large number of host-encoded proteins affect the replication of plus-stranded RNA viruses by acting as susceptibility factors. Many other cellular proteins are known to function as restriction factors of viral infections. Previous studies with tomato bushy stunt tombusvirus (TBSV) in a yeast model host have revealed the inhibitory function of TPR (tetratricopeptide repeat) domain-containing cyclophilins, which are members of the large family of host prolyl isomerases, in TBSV replication. In this paper, we tested additional TPR-containing yeast proteins in a cell-free TBSV replication assay and identified the Cns1p cochaperone for heat shock protein 70 (Hsp70) and Hsp90 chaperones as a strong inhibitor of TBSV replication. Cns1p interacted with the viral replication proteins and inhibited the assembly of the viral replicase complex and viral RNA synthesis in vitro. Overexpression of Cns1p inhibited TBSV replication in yeast. The use of a temperature-sensitive (TS) mutant of Cns1p in yeast revealed that at a semipermissive temperature, TS Cns1p could not inhibit TBSV replication. Interestingly, Cns1p and the TPR-containing Cpr7p cyclophilin have similar inhibitory functions during TBSV replication, although some of the details of their viral restriction mechanisms are different. Our observations indicate that TPR-containing cellular proteins could act as virus restriction factors.

Determination of diphenylether herbicides in water samples by solid-phase microextraction coupled to liquid chromatography.

PubMed

Sheu, Hong-Li; Sung, Yu-Hsiang; Melwanki, Mahaveer B; Huang, Shang-Da

2006-11-01

Solid-phase microextraction (SPME) coupled to LC for the analysis of five diphenylether herbicides (aclonifen, bifenox, fluoroglycofen-ethyl, oxyfluorfen, and lactofen) is described. Various parameters of extraction of analytes onto the fiber (such as type of fiber, extraction time and temperature, pH, impact of salt and organic solute) and desorption from the fiber in the desorption chamber prior to separation (such as type and composition of desorption solvent, desorption mode, soaking time, and flush-out time) were studied and optimized. Four commercially available SPME fibers were studied. PDMS/divinylbenzene (PDMS/DVB, 60 microm) and carbowax/ templated resin (CW/TPR, 50 microm) fibers were selected due to better extraction efficiencies. Repeatability (RSD, < 7%), correlation coefficient (> 0.994), and detection limit (0.33-1.74 and 0.22-1.94 ng/mL, respectively, for PDMS/DVB and CW/TPR) were investigated. Relative recovery (81-104% for PDMS/DVB and 83-100% for CW/TPR fiber) values have also been calculated. The developed method was successfully applied to the analysis of river water and water collected from a vegetable garden.

OsBRI1 Activates BR Signaling by Preventing Binding between the TPR and Kinase Domains of OsBSK3 via Phosphorylation.

PubMed

Zhang, Baowen; Wang, Xiaolong; Zhao, Zhiying; Wang, Ruiju; Huang, Xiahe; Zhu, Yali; Yuan, Li; Wang, Yingchun; Xu, Xiaodong; Burlingame, Alma L; Gao, Yingjie; Sun, Yu; Tang, Wenqiang

2016-02-01

Many plant receptor kinases transduce signals through receptor-like cytoplasmic kinases (RLCKs); however, the molecular mechanisms that create an effective on-off switch are unknown. The receptor kinase BR INSENSITIVE1 (BRI1) transduces brassinosteroid (BR) signal by phosphorylating members of the BR-signaling kinase (BSK) family of RLCKs, which contain a kinase domain and a C-terminal tetratricopeptide repeat (TPR) domain. Here, we show that the BR signaling function of BSKs is conserved in Arabidopsis (Arabidopsis thaliana) and rice (Oryza sativa) and that the TPR domain of BSKs functions as a "phospho-switchable" autoregulatory domain to control BSKs' activity. Genetic studies revealed that OsBSK3 is a positive regulator of BR signaling in rice, while in vivo and in vitro assays demonstrated that OsBRI1 interacts directly with and phosphorylates OsBSK3. The TPR domain of OsBSK3, which interacts directly with the protein's kinase domain, serves as an autoinhibitory domain to prevent OsBSK3 from interacting with bri1-SUPPRESSOR1 (BSU1). Phosphorylation of OsBSK3 by OsBRI1 disrupts the interaction between its TPR and kinase domains, thereby increasing the binding between OsBSK3's kinase domain and BSU1. Our results not only demonstrate that OsBSK3 plays a conserved role in regulating BR signaling in rice, but also provide insight into the molecular mechanism by which BSK family proteins are inhibited under basal conditions but switched on by the upstream receptor kinase BRI1. © 2016 American Society of Plant Biologists. All Rights Reserved.

Structural studies on the co-chaperone Hop and its complexes with Hsp90.

PubMed

Onuoha, S C; Coulstock, E T; Grossmann, J G; Jackson, S E

2008-06-13

The tetratricopeptide repeat domain (TPR)-containing co-chaperone Hsp-organising protein (Hop) plays a critical role in mediating interactions between Heat Shock Protein (Hsp)70 and Hsp90 as part of the cellular assembly machine. It also modulates the ATPase activity of both Hsp70 and Hsp90, thus facilitating client protein transfer between the two. Despite structural work on the individual domains of Hop, no structure for the full-length protein exists, nor is it clear exactly how Hop interacts with Hsp90, although it is known that its primary binding site is the C-terminal MEEVD motif. Here, we have undertaken a biophysical analysis of the structure and binding of Hop to Hsp90 using a variety of truncation mutants of both Hop and Hsp90, in addition to mutants of Hsp90 that are thought to modulate the conformation, in particular the N-terminal dimerisation of the chaperone. The results establish that whilst the primary binding site of Hop is the C-terminal MEEVD peptide of Hsp90, binding also occurs at additional sites in the C-terminal and middle domain. In contrast, we show that another TPR-containing co-chaperone, CyP40, binds solely to the C-terminus of Hsp90. Truncation mutants of Hop were generated and used to investigate the dimerisation interface of the protein. In good agreement with recently published data, we find that the TPR2a domain that contains the Hsp90-binding site is also the primary site for dimerisation. However, our results suggest that residues within the TPR2b may play a role. Together, these data along with shape reconstruction analysis from small-angle X-ray scattering measurements are used to generate a solution structure for full-length Hop, which we show has an overall butterfly-like quaternary structure. Studies on the nucleotide dependence of Hop binding to Hsp90 establish that Hop binds to the nucleotide-free, 'open' state of Hsp90. However, the Hsp90-Hop complex is weakened by the conformational changes that occur in Hsp90 upon ATP binding. Together, the data are used to propose a detailed model of how Hop may help present the client protein to Hsp90 by aligning the bound client on Hsp70 with the middle domain of Hsp90. It is likely that Hop binds to both monomers of Hsp90 in the form of a clamp, interacting with residues in the middle domain of Hsp90, thus preventing ATP hydrolysis, possibly by the prevention of association of N-terminal and middle domains in individual Hsp90 monomers.

Identification and Expression Analysis of the Interferon-Induced Protein with Tetratricopeptide Repeats 5 (IFIT5) Gene in Duck (Anas platyrhynchos domesticus)

PubMed Central

Mu, Chunyu; Su, Yanhui; Liu, Ran; Huang, Zhengyang; Li, Yang; Yu, Qingming; Chang, Guobin; Xu, Qi; Chen, Guohong

2015-01-01

The interferon-induced proteins with tetratricopeptide repeats (IFITs) protein family mediates antiviral effects by inhibiting translation initiation, cell proliferation, and migration in the interferon (IFN) dependent innate immune system. Several members of this family, including IFIT1, IFIT2, IFIT3 and IFIT5, have been heavily studied in mammals. Avian species contain only one family member, IFIT5, and little is known about the role of this protein in birds. In this study, duck IFIT5 (duIFIT5) full-length mRNA was cloned by reverse transcription polymerase chain reaction (RT-PCR) and rapid amplification of the cDNA ends (RACE). Based on the sequence obtained, we performed a series of bioinformatics analyses, and found that duIFIT5 was most similar to homologs in other avian species. Also, duIFIT5 contained eight conserved TPR motifs and two conserved multi-domains (TPR_11 and TPR_12). Finally, we used duck hepatitis virus type 1 (DHV-1) and polyriboinosinicpolyribocytidylic acid (poly (I:C)) as a pathogen or a pathogen-associated molecular pattern induction to infect three-day-old domestic ducklings. The liver and spleen were collected to detect the change in duIFIT5 transcript level upon infection by quantitative real-time PCR (qRT-PCR). DuIFIT5 expression rapidly increased after DHV-1 infection and maintained a high level, while the transcripts of duIFIT5 peaked at 8h after poly (I:C) infection and then returned to normal. Taken together, these results provide a greater understanding of avian IFIT5. PMID:25816333

O-GlcNAc site-mapping of liver X receptor-α and O-GlcNAc transferase.

PubMed

Fan, Qiong; Moen, Anders; Anonsen, Jan Haug; Bindesbøll, Christian; Sæther, Thomas; Carlson, Cathrine Rein; Grønning-Wang, Line M

2018-05-05

The Liver X Receptor α (LXRα) belongs to the nuclear receptor superfamily and plays an essential role in regulating cholesterol, lipid and glucose metabolism and inflammatory responses. We have previously shown that LXRα is post-translationally modified by O-linked β-N-acetyl-glucosamine (O-GlcNAc) with increased transcriptional activity. Moreover, we showed that LXRα associates with O-GlcNAc transferase (OGT) in vitro and in vivo in mouse liver. In this study, we report that human LXRα is O-GlcNAc modified in its N-terminal domain (NTD) by identifying a specific O-GlcNAc site S49 and a novel O-GlcNAc modified peptide 20 LWKPGAQDASSQAQGGSSCILRE 42 . However, O-GlcNAc site-mutations did not modulate LXRα transactivation of selected target gene promoters in vitro. Peptide array and co-immunoprecipitation assays demonstrate that LXRα interacts with OGT in its NTD and ligand-binding domain (LBD) in a ligand-independent fashion. Moreover, we map two new O-GlcNAc sites in the longest OGT isoform (ncOGT): S437 in the tetratricopeptide repeat (TPR) 13 domain and T1043 in the far C-terminus, and a new O-GlcNAc modified peptide (amino acids 826-832) in the intervening region (Int-D) within the catalytic domain. We also map four new O-GlcNAc sites in the short isoform sOGT: S391, T393, S399 and S437 in the TPRs 11-13 domain. Future studies will reveal the biological role of identified O-GlcNAc sites in LXRα and OGT. Copyright © 2018 Elsevier Inc. All rights reserved.

Prediction of Nucleotide Binding Peptides Using Star Graph Topological Indices.

PubMed

Liu, Yong; Munteanu, Cristian R; Fernández Blanco, Enrique; Tan, Zhiliang; Santos Del Riego, Antonino; Pazos, Alejandro

2015-11-01

The nucleotide binding proteins are involved in many important cellular processes, such as transmission of genetic information or energy transfer and storage. Therefore, the screening of new peptides for this biological function is an important research topic. The current study proposes a mixed methodology to obtain the first classification model that is able to predict new nucleotide binding peptides, using only the amino acid sequence. Thus, the methodology uses a Star graph molecular descriptor of the peptide sequences and the Machine Learning technique for the best classifier. The best model represents a Random Forest classifier based on two features of the embedded and non-embedded graphs. The performance of the model is excellent, considering similar models in the field, with an Area Under the Receiver Operating Characteristic Curve (AUROC) value of 0.938 and true positive rate (TPR) of 0.886 (test subset). The prediction of new nucleotide binding peptides with this model could be useful for drug target studies in drug development. © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Ca2+/S100 Proteins Act as Upstream Regulators of the Chaperone-associated Ubiquitin Ligase CHIP (C Terminus of Hsc70-interacting Protein)*

PubMed Central

Shimamoto, Seiko; Kubota, Yasuo; Yamaguchi, Fuminori; Tokumitsu, Hiroshi; Kobayashi, Ryoji

2013-01-01

The U-box E3 ubiquitin ligase CHIP (C terminus of Hsc70-interacting protein) binds Hsp90 and/or Hsp70 via its tetratricopeptide repeat (TPR), facilitating ubiquitination of the chaperone-bound client proteins. Mechanisms that regulate the activity of CHIP are, at present, poorly understood. We previously reported that Ca2+/S100 proteins directly associate with the TPR proteins, such as Hsp70/Hsp90-organizing protein (Hop), kinesin light chain, Tom70, FKBP52, CyP40, and protein phosphatase 5 (PP5), leading to the dissociation of the interactions of the TPR proteins with their target proteins. Therefore, we have hypothesized that Ca2+/S100 proteins can interact with CHIP and regulate its function. GST pulldown assays indicated that Ca2+/S100A2 and S100P bind to the TPR domain and lead to interference with the interactions of CHIP with Hsp70, Hsp90, HSF1, and Smad1. In vitro ubiquitination assays indicated that Ca2+/S100A2 and S100P are efficient and specific inhibitors of CHIP-mediated ubiquitination of Hsp70, Hsp90, HSF1, and Smad1. Overexpression of S100A2 and S100P suppressed CHIP-chaperone complex-dependent mutant p53 ubiquitination and degradation in Hep3B cells. The association of the S100 proteins with CHIP provides a Ca2+-dependent regulatory mechanism for the ubiquitination and degradation of intracellular proteins by the CHIP-proteasome pathway. PMID:23344957

Structural and functional insights into the role of the N-terminal Mps1 TPR domain in the SAC (spindle assembly checkpoint).

PubMed

Thebault, Philippe; Chirgadze, Dimitri Y; Dou, Zhen; Blundell, Tom L; Elowe, Sabine; Bolanos-Garcia, Victor M

2012-12-15

The SAC (spindle assembly checkpoint) is a surveillance system that ensures the timely and accurate transmission of the genetic material to offspring. The process implies kinetochore targeting of the mitotic kinases Bub1 (budding uninhibited by benzamidine 1), BubR1 (Bub1 related) and Mps1 (monopolar spindle 1), which is mediated by the N-terminus of each kinase. In the present study we report the 1.8 Å (1 Å=0.1 nm) crystal structure of the TPR (tetratricopeptide repeat) domain in the N-terminal region of human Mps1. The structure reveals an overall high similarity to the TPR motif of the mitotic checkpoint kinases Bub1 and BubR1, and a number of unique features that include the absence of the binding site for the kinetochore structural component KNL1 (kinetochore-null 1; blinkin), and determinants of dimerization. Moreover, we show that a stretch of amino acids at the very N-terminus of Mps1 is required for dimer formation, and that interfering with dimerization results in mislocalization and misregulation of kinase activity. The results of the present study provide an important insight into the molecular details of the mitotic functions of Mps1 including features that dictate substrate selectivity and kinetochore docking.

C-terminal domain of SMYD3 serves as a unique HSP90-regulated motif in oncogenesis

PubMed Central

Harriss, June; Das, Chhaya; Zhu, Li; Edwards, Melissa; Shaaban, Salam; Tucker, Haley

2015-01-01

The SMYD3 histone methyl transferase (HMTase) and the nuclear chaperone, HSP90, have been independently implicated as proto-oncogenes in several human malignancies. We show that a degenerate tetratricopeptide repeat (TPR)-like domain encoded in the SMYD3 C-terminal domain (CTD) mediates physical interaction with HSP90. We further demonstrate that the CTD of SMYD3 is essential for its basal HMTase activity and that the TPR-like structure is required for HSP90-enhanced enzyme activity. Loss of SMYD3-HSP90 interaction leads to SMYD3 mislocalization within the nucleus, thereby losing its chromatin association. This results in reduction of SMYD3-mediated cell proliferation and, potentially, impairment of SMYD3′s oncogenic activity. These results suggest a novel approach for blocking HSP90-driven malignancy in SMYD3-overexpressing cells with a reduced toxicity profile over current HSP90 inhibitors. PMID:25738358

Structure and function of Hip, an attenuator of the Hsp70 chaperone cycle.

PubMed

Li, Zhuo; Hartl, F Ulrich; Bracher, Andreas

2013-08-01

The Hsp70-interacting protein, Hip, cooperates with the chaperone Hsp70 in protein folding and prevention of aggregation. Hsp70 interacts with non-native protein substrates in an ATP-dependent reaction cycle regulated by J-domain proteins and nucleotide exchange factors (NEFs). Hip is thought to delay substrate release by slowing ADP dissociation from Hsp70. Here we present crystal structures of the dimerization domain and the tetratricopeptide repeat (TPR) domain of rat Hip. As shown in a cocrystal structure, the TPR core of Hip interacts with the Hsp70 ATPase domain through an extensive interface, to form a bracket that locks ADP in the binding cleft. Hip and NEF binding to Hsp70 are mutually exclusive, and thus Hip attenuates active cycling of Hsp70-substrate complexes. This mechanism explains how Hip enhances aggregation prevention by Hsp70 and facilitates transfer of specific proteins to downstream chaperones or the proteasome.

Repeatability and Reproducibility in Proteomic Identifications by Liquid Chromatography—Tandem Mass Spectrometry

PubMed Central

Tabb, David L.; Vega-Montoto, Lorenzo; Rudnick, Paul A.; Variyath, Asokan Mulayath; Ham, Amy-Joan L.; Bunk, David M.; Kilpatrick, Lisa E.; Billheimer, Dean D.; Blackman, Ronald K.; Cardasis, Helene L.; Carr, Steven A.; Clauser, Karl R.; Jaffe, Jacob D.; Kowalski, Kevin A.; Neubert, Thomas A.; Regnier, Fred E.; Schilling, Birgit; Tegeler, Tony J.; Wang, Mu; Wang, Pei; Whiteaker, Jeffrey R.; Zimmerman, Lisa J.; Fisher, Susan J.; Gibson, Bradford W.; Kinsinger, Christopher R.; Mesri, Mehdi; Rodriguez, Henry; Stein, Steven E.; Tempst, Paul; Paulovich, Amanda G.; Liebler, Daniel C.; Spiegelman, Cliff

2009-01-01

The complexity of proteomic instrumentation for LC-MS/MS introduces many possible sources of variability. Data-dependent sampling of peptides constitutes a stochastic element at the heart of discovery proteomics. Although this variation impacts the identification of peptides, proteomic identifications are far from completely random. In this study, we analyzed interlaboratory data sets from the NCI Clinical Proteomic Technology Assessment for Cancer to examine repeatability and reproducibility in peptide and protein identifications. Included data spanned 144 LC-MS/MS experiments on four Thermo LTQ and four Orbitrap instruments. Samples included yeast lysate, the NCI-20 defined dynamic range protein mix, and the Sigma UPS 1 defined equimolar protein mix. Some of our findings reinforced conventional wisdom, such as repeatability and reproducibility being higher for proteins than for peptides. Most lessons from the data, however, were more subtle. Orbitraps proved capable of higher repeatability and reproducibility, but aberrant performance occasionally erased these gains. Even the simplest protein digestions yielded more peptide ions than LC-MS/MS could identify during a single experiment. We observed that peptide lists from pairs of technical replicates overlapped by 35–60%, giving a range for peptide-level repeatability in these experiments. Sample complexity did not appear to affect peptide identification repeatability, even as numbers of identified spectra changed by an order of magnitude. Statistical analysis of protein spectral counts revealed greater stability across technical replicates for Orbitraps, making them superior to LTQ instruments for biomarker candidate discovery. The most repeatable peptides were those corresponding to conventional tryptic cleavage sites, those that produced intense MS signals, and those that resulted from proteins generating many distinct peptides. Reproducibility among different instruments of the same type lagged behind repeatability of technical replicates on a single instrument by several percent. These findings reinforce the importance of evaluating repeatability as a fundamental characteristic of analytical technologies. PMID:19921851

Macrocycles that inhibit the binding between heat shock protein 90 and TPR-containing proteins

PubMed Central

Ardi, Veronica C.; Alexander, Leslie D.; Johnson, Victoria; McAlpine, Shelli R.

2011-01-01

Heat shock protein 90 (Hsp90) accounts for 1–2% of the total proteins in normal cells and functions as a molecular chaperone that folds, assembles, and stabilizes client proteins. Hsp90 is over-expressed (3–6-fold increase) in stressed cells, including cancer cells, and regulates over 200 client and co-chaperone proteins. Hsp90 client proteins are involved in a plethora of cellular signaling events including numerous growth and apoptotic pathways. Since pathway-specific inhibitors can be problematic in drug-resistant cancers, shutting down multiple pathways at once is a promising approach when developing new therapeutics. Hsp90’s ability to modulate many growth and signaling pathways simultaneously makes this protein an attractive target in the field of cancer therapeutics. Herein we present evidence that a small molecule modulates Hsp90 via binding between the N and middle domain and allosterically inhibiting the binding interaction between Hsp90 and four C-terminal binding client proteins: IP6K2, FKBP38, FKBP52, and HOP. These last three clients contain a tetratricopeptide-repeat (TPR) region, which is known to interact with the MEEVD sequence on the C-terminus of Hsp90. Thus, this small molecule modulates the activity between co-chaperones that contain TPR motifs and Hsp90’s MEEVD region. This mechanism of action is unique from that of all Hsp90 inhibitors currently in clinical trials where these molecules have no effect on proteins that bind to the C-terminus of Hsp90. Further, our small molecule induces a Caspase-3 dependent apoptotic event. Thus, we describe the mechanism of a novel scaffold that is a useful tool for studying cell-signaling events that result when blocking the MEEVD-TPR interaction between Hsp90 and co-chaperone proteins. PMID:21950602

Structural, Bioinformatic, and In Vivo Analyses of Two Treponema pallidum Lipoproteins Reveal a Unique TRAP Transporter

DOE Office of Scientific and Technical Information (OSTI.GOV)

Deka, Ranjit K.; Brautigam, Chad A.; Goldberg, Martin

2012-05-25

Treponema pallidum, the bacterial agent of syphilis, is predicted to encode one tripartite ATP-independent periplasmic transporter (TRAP-T). TRAP-Ts typically employ a periplasmic substrate-binding protein (SBP) to deliver the cognate ligand to the transmembrane symporter. Herein, we demonstrate that the genes encoding the putative TRAP-T components from T. pallidum, tp0957 (the SBP), and tp0958 (the symporter), are in an operon with an uncharacterized third gene, tp0956. We determined the crystal structure of recombinant Tp0956; the protein is trimeric and perforated by a pore. Part of Tp0956 forms an assembly similar to those of 'tetratricopeptide repeat' (TPR) motifs. The crystal structure ofmore » recombinant Tp0957 was also determined; like the SBPs of other TRAP-Ts, there are two lobes separated by a cleft. In these other SBPs, the cleft binds a negatively charged ligand. However, the cleft of Tp0957 has a strikingly hydrophobic chemical composition, indicating that its ligand may be substantially different and likely hydrophobic. Analytical ultracentrifugation of the recombinant versions of Tp0956 and Tp0957 established that these proteins associate avidly. This unprecedented interaction was confirmed for the native molecules using in vivo cross-linking experiments. Finally, bioinformatic analyses suggested that this transporter exemplifies a new subfamily of TPATs (TPR-protein-associated TRAP-Ts) that require the action of a TPR-containing accessory protein for the periplasmic transport of a potentially hydrophobic ligand(s).« less

On the role, ecology, phylogeny, and structure of dual-family immunophilins.

PubMed

Barik, Sailen

2017-11-01

The novel class of dual-family immunophilins (henceforth abbreviated as DFI) represents naturally occurring chimera of classical FK506-binding protein (FKBP) and cyclophilin (CYN), connected by a flexible linker that may include a three-unit tetratricopeptide (TPR) repeat. Here, I report a comprehensive analysis of all current DFI sequences and their host organisms. DFIs are of two kinds: CFBP (cyclosporin- and FK506-binding protein) and FCBP (FK506- and cyclosporin-binding protein), found in eukaryotes. The CFBP type occurs in select bacteria that are mostly extremophiles, such as psychrophilic, thermophilic, halophilic, and sulfur-reducing. Essentially all DFI organisms are unicellular. I suggest that DFIs are specialized bifunctional chaperones that use their flexible interdomain linker to associate with large polypeptides or multisubunit megacomplexes to promote simultaneous folding or renaturation of two clients in proximity, essential in stressful and denaturing environments. Analysis of sequence homology and predicted 3D structures of the FKBP and CYN domains as well as the TPR linkers upheld the modular nature of the DFIs and revealed the uniqueness of their TPR domain. The CFBP and FCBP genes appear to have evolved in parallel pathways with no obvious single common ancestor. The occurrence of both types of DFI in multiple unrelated phylogenetic clades supported their selection in metabolic and environmental niche roles rather than a traditional taxonomic relationship. Nonetheless, organisms with these rare immunophilins may define an operational taxonomic unit (OTU) bound by the commonality of chaperone function.

Low-Resolution Structure of the Full-Length Barley (Hordeum vulgare) SGT1 Protein in Solution, Obtained Using Small-Angle X-Ray Scattering

PubMed Central

Taube, Michał; Pieńkowska, Joanna R.; Jarmołowski, Artur; Kozak, Maciej

2014-01-01

SGT1 is an evolutionarily conserved eukaryotic protein involved in many important cellular processes. In plants, SGT1 is involved in resistance to disease. In a low ionic strength environment, the SGT1 protein tends to form dimers. The protein consists of three structurally independent domains (the tetratricopeptide repeats domain (TPR), the CHORD- and SGT1-containing domain (CS), and the SGT1-specific domain (SGS)), and two less conserved variable regions (VR1 and VR2). In the present study, we provide the low-resolution structure of the barley (Hordeum vulgare) SGT1 protein in solution and its dimer/monomer equilibrium using small-angle scattering of synchrotron radiation, ab-initio modeling and circular dichroism spectroscopy. The multivariate curve resolution least-square method (MCR-ALS) was applied to separate the scattering data of the monomeric and dimeric species from a complex mixture. The models of the barley SGT1 dimer and monomer were formulated using rigid body modeling with ab-initio structure prediction. Both oligomeric forms of barley SGT1 have elongated shapes with unfolded inter-domain regions. Circular dichroism spectroscopy confirmed that the barley SGT1 protein had a modular architecture, with an α-helical TPR domain, a β-sheet sandwich CS domain, and a disordered SGS domain separated by VR1 and VR2 regions. Using molecular docking and ab-initio protein structure prediction, a model of dimerization of the TPR domains was proposed. PMID:24714665

Cardiovascular responses to military antishock trouser inflation during standing arm exercise.

PubMed

Ng, A V; Hanson, P; Aaron, E A; Demment, R B; Conviser, J M; Nagle, F J

1987-09-01

Military antishock trousers (MAST) inflated to 50 mmHg were used with 12 healthy males (mean age 28 +/- 1 yr) to determine the effects of lower-body positive pressure on cardiac output (Q), stroke volume (SV), heart rate (HR), systolic blood pressure (SBP), diastolic blood pressure (DBP), mean arterial blood pressure (MABP), total peripheral resistance (TPR), and O2 uptake (VO2) during graded arm-cranking exercise. Subjects were studied while standing at rest and at 25, 50, and 75% of maximal arm-cranking VO2. At each level, rest or work was continued for 6 min with MAST inflated and for 6 min with MAST deflated. Order of inflation and deflation was alternated at each experimental rest or exercise level. Measurements were obtained during the last 2 min at each level. Repeated-measures analysis of variance revealed significant increases (P less than 0.001) in Q, SV, and MABP and a consistent decrease in HR with MAST inflation. There was no apparent change in Q/VO2 between inflated and control conditions. There was no effect of MAST inflation on VO2 or TPR. MAST inflation counteracts the gravitational effect of venous return in upright exercise, restoring central blood volume and thereby increasing Q and MABP from control. HR is decreased consequent to increased MABP through arterial baroreflexes. The associated decrease in TPR is not observed, being offset by the mechanical compression of leg vasculature with MAST inflation.

A comparison of small-field tissue phantom ratio data generation methods for an Elekta Agility 6 MV photon beam

DOE Office of Scientific and Technical Information (OSTI.GOV)

Richmond, Neil, E-mail: neil.richmond@stees.nhs.uk; Brackenridge, Robert

2014-04-01

Tissue-phantom ratios (TPRs) are a common dosimetric quantity used to describe the change in dose with depth in tissue. These can be challenging and time consuming to measure. The conversion of percentage depth dose (PDD) data using standard formulae is widely employed as an alternative method in generating TPR. However, the applicability of these formulae for small fields has been questioned in the literature. Functional representation has also been proposed for small-field TPR production. This article compares measured TPR data for small 6 MV photon fields against that generated by conversion of PDD using standard formulae to assess the efficacymore » of the conversion data. By functionally fitting the measured TPR data for square fields greater than 4 cm in length, the TPR curves for smaller fields are generated and compared with measurements. TPRs and PDDs were measured in a water tank for a range of square field sizes. The PDDs were converted to TPRs using standard formulae. TPRs for fields of 4 × 4 cm{sup 2} and larger were used to create functional fits. The parameterization coefficients were used to construct extrapolated TPR curves for 1 × 1 cm{sup 2}, 2 × 2-cm{sup 2}, and 3 × 3-cm{sup 2} fields. The TPR data generated using standard formulae were in excellent agreement with direct TPR measurements. The TPR data for 1 × 1-cm{sup 2}, 2 × 2-cm{sup 2}, and 3 × 3-cm{sup 2} fields created by extrapolation of the larger field functional fits gave inaccurate initial results. The corresponding mean differences for the 3 fields were 4.0%, 2.0%, and 0.9%. Generation of TPR data using a standard PDD-conversion methodology has been shown to give good agreement with our directly measured data for small fields. However, extrapolation of TPR data using the functional fit to fields of 4 × 4 cm{sup 2} or larger resulted in generation of TPR curves that did not compare well with the measured data.« less

Method for predicting peptide detection in mass spectrometry

DOEpatents

Kangas, Lars [West Richland, WA; Smith, Richard D [Richland, WA; Petritis, Konstantinos [Richland, WA

2010-07-13

A method of predicting whether a peptide present in a biological sample will be detected by analysis with a mass spectrometer. The method uses at least one mass spectrometer to perform repeated analysis of a sample containing peptides from proteins with known amino acids. The method then generates a data set of peptides identified as contained within the sample by the repeated analysis. The method then calculates the probability that a specific peptide in the data set was detected in the repeated analysis. The method then creates a plurality of vectors, where each vector has a plurality of dimensions, and each dimension represents a property of one or more of the amino acids present in each peptide and adjacent peptides in the data set. Using these vectors, the method then generates an algorithm from the plurality of vectors and the calculated probabilities that specific peptides in the data set were detected in the repeated analysis. The algorithm is thus capable of calculating the probability that a hypothetical peptide represented as a vector will be detected by a mass spectrometry based proteomic platform, given that the peptide is present in a sample introduced into a mass spectrometer.

Simulation and fitting of complex reaction network TPR: The key is the objective function

DOE PAGES

Savara, Aditya Ashi

2016-07-07

In this research, a method has been developed for finding improved fits during simulation and fitting of data from complex reaction network temperature programmed reactions (CRN-TPR). It was found that simulation and fitting of CRN-TPR presents additional challenges relative to simulation and fitting of simpler TPR systems. The method used here can enable checking the plausibility of proposed chemical mechanisms and kinetic models. The most important finding was that when choosing an objective function, use of an objective function that is based on integrated production provides more utility in finding improved fits when compared to an objective function based onmore » the rate of production. The response surface produced by using the integrated production is monotonic, suppresses effects from experimental noise, requires fewer points to capture the response behavior, and can be simulated numerically with smaller errors. For CRN-TPR, there is increased importance (relative to simple reaction network TPR) in resolving of peaks prior to fitting, as well as from weighting of experimental data points. Using an implicit ordinary differential equation solver was found to be inadequate for simulating CRN-TPR. Lastly, the method employed here was capable of attaining improved fits in simulation and fitting of CRN-TPR when starting with a postulated mechanism and physically realistic initial guesses for the kinetic parameters.« less

Design and Analysis of Temperature Preference Behavior and its Circadian Rhythm in Drosophila

PubMed Central

Goda, Tadahiro; Leslie, Jennifer R.; Hamada, Fumika N.

2014-01-01

The circadian clock regulates many aspects of life, including sleep, locomotor activity, and body temperature (BTR) rhythms1,2. We recently identified a novel Drosophila circadian output, called the temperature preference rhythm (TPR), in which the preferred temperature in flies rises during the day and falls during the night 3. Surprisingly, the TPR and locomotor activity are controlled through distinct circadian neurons3. Drosophila locomotor activity is a well known circadian behavioral output and has provided strong contributions to the discovery of many conserved mammalian circadian clock genes and mechanisms4. Therefore, understanding TPR will lead to the identification of hitherto unknown molecular and cellular circadian mechanisms. Here, we describe how to perform and analyze the TPR assay. This technique not only allows for dissecting the molecular and neural mechanisms of TPR, but also provides new insights into the fundamental mechanisms of the brain functions that integrate different environmental signals and regulate animal behaviors. Furthermore, our recently published data suggest that the fly TPR shares features with the mammalian BTR3. Drosophila are ectotherms, in which the body temperature is typically behaviorally regulated. Therefore, TPR is a strategy used to generate a rhythmic body temperature in these flies5-8. We believe that further exploration of Drosophila TPR will facilitate the characterization of the mechanisms underlying body temperature control in animals. PMID:24457268

Preliminary application of a novel algorithm to monitor changes in pre-flight total peripheral resistance for prediction of post-flight orthostatic intolerance in astronauts

NASA Astrophysics Data System (ADS)

Arai, Tatsuya; Lee, Kichang; Stenger, Michael B.; Platts, Steven H.; Meck, Janice V.; Cohen, Richard J.

2011-04-01

Orthostatic intolerance (OI) is a significant challenge for astronauts after long-duration spaceflight. Depending on flight duration, 20-80% of astronauts suffer from post-flight OI, which is associated with reduced vascular resistance. This paper introduces a novel algorithm for continuously monitoring changes in total peripheral resistance (TPR) by processing the peripheral arterial blood pressure (ABP). To validate, we applied our novel mathematical algorithm to the pre-flight ABP data previously recorded from twelve astronauts ten days before launch. The TPR changes were calculated by our algorithm and compared with the TPR value estimated using cardiac output/heart rate before and after phenylephrine administration. The astronauts in the post-flight presyncopal group had lower pre-flight TPR changes (1.66 times) than those in the non-presyncopal group (2.15 times). The trend in TPR changes calculated with our algorithm agreed with the TPR trend calculated using measured cardiac output in the previous study. Further data collection and algorithm refinement are needed for pre-flight detection of OI and monitoring of continuous TPR by analysis of peripheral arterial blood pressure.

Nucleoporins as components of the nuclear pore complex core structure and Tpr as the architectural element of the nuclear basket.

PubMed

Krull, Sandra; Thyberg, Johan; Björkroth, Birgitta; Rackwitz, Hans-Richard; Cordes, Volker C

2004-09-01

The vertebrate nuclear pore complex (NPC) is a macromolecular assembly of protein subcomplexes forming a structure of eightfold radial symmetry. The NPC core consists of globular subunits sandwiched between two coaxial ring-like structures of which the ring facing the nuclear interior is capped by a fibrous structure called the nuclear basket. By postembedding immunoelectron microscopy, we have mapped the positions of several human NPC proteins relative to the NPC core and its associated basket, including Nup93, Nup96, Nup98, Nup107, Nup153, Nup205, and the coiled coil-dominated 267-kDa protein Tpr. To further assess their contributions to NPC and basket architecture, the genes encoding Nup93, Nup96, Nup107, and Nup205 were posttranscriptionally silenced by RNA interference (RNAi) in HeLa cells, complementing recent RNAi experiments on Nup153 and Tpr. We show that Nup96 and Nup107 are core elements of the NPC proper that are essential for NPC assembly and docking of Nup153 and Tpr to the NPC. Nup93 and Nup205 are other NPC core elements that are important for long-term maintenance of NPCs but initially dispensable for the anchoring of Nup153 and Tpr. Immunogold-labeling for Nup98 also results in preferential labeling of NPC core regions, whereas Nup153 is shown to bind via its amino-terminal domain to the nuclear coaxial ring linking the NPC core structures and Tpr. The position of Tpr in turn is shown to coincide with that of the nuclear basket, with different Tpr protein domains corresponding to distinct basket segments. We propose a model in which Tpr constitutes the central architectural element that forms the scaffold of the nuclear basket.

Cost utility of telaprevir-PR (peginterferon-ribavirin) versus boceprevir-PR and versus PR alone in chronic hepatitis C in The Netherlands.

PubMed

Vellopoulou, Aikaterini; van Agthoven, Michel; van der Kolk, Annemarie; de Knegt, Robert J; Berdeaux, Gilles; Cure, Sandrine; Bianic, Florence; Lamotte, Mark

2014-12-01

The hepatitis C virus may lead to cirrhosis, liver cancer, liver transplant, and increased mortality. With standard treatment peginterferon-alpha and ribavirin (PR), sustained viral response (SVR) was less than 50 %. SVR rates improve greatly when PR is combined with telaprevir or boceprevir. The aim of this study was to assess the cost utility of telaprevir-peginterferon-ribavirin (TPR) versus PR and boceprevir-peginterferon-ribavirin (BPR) in treatment-naïve (TN) and treatment-experienced (TE) adults with chronic hepatitis C in the Netherlands. A Markov model with a lifelong time horizon and annual cycles was developed. Clinical data stemmed from phase III trials (TPR vs PR, BPR vs PR). A mixed treatment comparison (MTC) was developed to compare TPR and BPR indirectly. Unit costs and utilities based on EQ-5D were established in a Dutch cross-sectional study. Cost per quality-adjusted life-years (QALYs) was calculated according to the societal perspective. Treating TN patients with TPR generates 1.12 additional QALYs with €333 additional cost compared with PR, resulting in an incremental cost-utility ratio of €299/QALY. In TE patients, TPR dominates PR with cost savings (-€7,819) and 1.63 additional QALYs. TPR dominates BPR yielding additional QALYs (0.26 in TN; 0.71 in TE) and cost savings (-€7,296, -€18,144, respectively). TPR seems a cost-effective alternative to PR in TN patients and dominant in TE patients. TPR was a dominant, more effective and less costly alternative to BPR in both patient types. The cost effectiveness of both TPR and BPR is well below generally accepted willingness-to-pay thresholds and may be considered cost effective.

Learning Language through Total Physical Response.

ERIC Educational Resources Information Center

Marlatt, Edward A.

1995-01-01

The Total Physical Response (TPR) method of language instruction is introduced, and guidelines for designing and implementing TPR lessons for students with hearing impairments are provided. In TPR instruction, students develop understanding before speech or signing, understanding is demonstrated through actions, and new vocabulary is developed…

Using TPR Communicatively.

ERIC Educational Resources Information Center

Tomscha, Terry

The Total Physical Response (TPR) method of teaching languages, while found to be effective and theoretically interesting, appears to have limitations in some circumstances. In a British program of English as a second language, TPR was found to be useful only for teaching vocabulary and only at low levels; the language it produced (imperatives and…

Diversity of indigenous endophytic bacteria associated with the roots of Chinese cabbage (Brassica campestris L.) cultivars and their antagonism towards pathogens.

PubMed

Haque, Md Azizul; Yun, Han Dae; Cho, Kye Man

2016-05-01

The study aimed to reveal the diversity of endophytic bacteria in the roots of Chinese cabbage (CC) cultivated in two areas in Korea, namely, Seosang-gun (SS) and Haenam-gun (HN), and also in a transgenic plant (TP) from the laboratory. A total of 653 colonies were isolated from the interior of CC roots, comprising 118, 302, and 233 isolates from SS, HN, and TP samples, respectively. Based on 16S rRNA gene sequence analysis, the isolates belonged to four major phylogenetic groups: high-G+C Gram-positive bacteria (HGC-GPB), low-G+C Gram-positive bacteria (LGC-GPB), Proteobacteria, and Bacteriodetes. The most dominant groups in the roots of the SS, HN, and TP cultivars were LGC-GPB (48.3%), Proteobacteria (50.2%), and HGC-GPB (38.2%), respectively. Importantly, most of the isolates that produced cell-walldegrading enzymes belonged to the genus Bacillus. Bacillus sp. (HNR03, TPR06), Bacillus pumilus (SSR07, HNR11, TPR07), and Bacillus subtilis (TPR03) showed high antagonism against the tested food-borne pathogenic bacteria. In addition, Bacillus sp. (HNR03, TPR06), Bacillus pumilus (SSR07, HNR11, HNR17, TPR11), Microbacterium oxidans (SSR09, TPR04), Bacillus cereus HNR10, Pseudomonas sp. HNR13, and Bacillus subtilis (TPR02, TPR03) showed strong antagonistic activity against the fungi Phythium ultimum, Phytophthora capsici, Fusarium oxysporum, and Rhizoctonia solani. The endophytes isolated from the TP cultivar showed the strongest antagonistic reactions against pathogens. This study is the first report on endophytic bacteria from Chinese cabbage roots.

Fine Analysis of Genetic Diversity of the tpr Gene Family among Treponemal Species, Subspecies and Strains

PubMed Central

Centurion-Lara, Arturo; Giacani, Lorenzo; Godornes, Charmie; Molini, Barbara J.; Brinck Reid, Tara; Lukehart, Sheila A.

2013-01-01

Background The pathogenic non-cultivable treponemes include three subspecies of Treponema pallidum (pallidum, pertenue, endemicum), T. carateum, T. paraluiscuniculi, and the unclassified Fribourg-Blanc treponeme (Simian isolate). These treponemes are morphologically indistinguishable and antigenically and genetically highly similar, yet cross-immunity is variable or non-existent. Although all of these organisms cause chronic, multistage skin and systemic disease, they have historically been classified by mode of transmission, clinical presentations and host ranges. Whole genome studies underscore the high degree of sequence identity among species, subspecies and strains, pinpointing a limited number of genomic regions for variation. Many of these “hot spots” include members of the tpr gene family, composed of 12 paralogs encoding candidate virulence factors. We hypothesize that the distinct clinical presentations, host specificity, and variable cross-immunity might reside on virulence factors such as the tpr genes. Methodology/Principal Findings Sequence analysis of 11 tpr loci (excluding tprK) from 12 strains demonstrated an impressive heterogeneity, including SNPs, indels, chimeric genes, truncated gene products and large deletions. Comparative analyses of sequences and 3D models of predicted proteins in Subfamily I highlight the striking co-localization of discrete variable regions with predicted surface-exposed loops. A hallmark of Subfamily II is the presence of chimeric genes in the tprG and J loci. Diversity in Subfamily III is limited to tprA and tprL. Conclusions/Significance An impressive sequence variability was found in tpr sequences among the Treponema isolates examined in this study, with most of the variation being consistent within subspecies or species, or between syphilis vs. non-syphilis strains. Variability was seen in the pallidum subspecies, which can be divided into 5 genogroups. These findings support a genetic basis for the classification of these organisms into their respective subspecies and species. Future functional studies will determine whether the identified genetic differences relate to cross-immunity, clinical differences, or host ranges. PMID:23696912

Three-phase Bone Scintigraphy Can Predict the Analgesic Efficacy of Ketamine Therapy in CRPS.

PubMed

Sorel, Marc; Beatrix, Jacques-Christian; Locko, Blanche; Armessen, Catherine; Domec, Anne-Marie; Lecompte, Otilia; Boucheneb, Sofiane; Harache, Benoit; Robert, Jacques; Lefaucheur, Jean-Pascal

2018-03-13

The efficacy of ketamine in relieving complex regional pain syndrome (CRPS) lacks predictive factors. The value of three-phase bone scintigraphy (TPBS) was assessed or this purpose. TPBS was performed in 105 patients with unilateral, focal CRPS of type 1 before 5 days of ketamine infusions. Tracer uptake was measured in the region of interest concerned by CRPS and the contralateral homologous region. For the three scintigraphic phases (vascular, tissular, and bone phases), an asymmetry ratio of fixation was calculated between the affected and the unaffected sides (VPr, TPr, and BPr). Ketamine efficacy was assessed on pain intensity scores. Ketamine-induced pain relief did not correlate with VPr, TPr, and BPr, but with the ratios of these ratios: BPr/TPr (r=0.32, P=0.009), BPr/VPr (r=0.34, P=0.005), and TPr/VPr (r=0.23, P=0.02). The optimum cut-off value for predicting the response to ketamine therapy was >1.125 for BPr/TPr, >1.075 for BPr/VPr, and >0.935 for TPr/VPr. The combination of increased values of BPr/TPr, BPr/VPr, and TPr/VPr was extremely significantly associated with ketamine therapy outcome. The relative hyperfixation of the radioactive tracer in the limb region concerned by CRPS in phases 2 and 3 versus phase 1 of TPBS correlated positively to the analgesic efficacy of ketamine. This study shows for the first time the potential predictive value of TPBS regarding ketamine therapy outcome. In addition, these results suggest that the analgesic action of ketamine is not restricted to "central" mechanisms, but may also involve "peripheral" mechanisms related to tissue inflammation and bone remodeling.

Cyclophilin 40 facilitates HSP90-mediated RISC assembly in plants.

PubMed

Iki, Taichiro; Yoshikawa, Manabu; Meshi, Tetsuo; Ishikawa, Masayuki

2012-01-18

Posttranscriptional gene silencing is mediated by RNA-induced silencing complexes (RISCs) that contain AGO proteins and single-stranded small RNAs. The assembly of plant AGO1-containing RISCs depends on the molecular chaperone HSP90. Here, we demonstrate that cyclophilin 40 (CYP40), protein phosphatase 5 (PP5), and several other proteins with the tetratricopeptide repeat (TPR) domain associates with AGO1 in an HSP90-dependent manner in extracts of evacuolated tobacco protoplasts (BYL). Intriguingly, CYP40, but not the other TPR proteins, could form a complex with small RNA duplex-bound AGO1. Moreover, CYP40 that was synthesized by in-vitro translation using BYL uniquely facilitated binding of small RNA duplexes to AGO1, and as a result, increased the amount of mature RISCs that could cleave target RNAs. CYP40 was not contained in mature RISCs, indicating that the association is transient. Addition of PP5 or cyclophilin-binding drug cyclosporine A prevented the association of endogenous CYP40 with HSP90-AGO1 complex and inhibited RISC assembly. These results suggest that a complex of AGO1, HSP90, CYP40, and a small RNA duplex is a key intermediate of RISC assembly in plants.

Triangulated Proxy Reporting: a technique for improving how communication partners come to know people with severe cognitive impairment.

PubMed

Lyons, Gordon; De Bortoli, Tania; Arthur-Kelly, Michael

2017-09-01

This paper explains and demonstrates the pilot application of Triangulated Proxy Reporting (TPR); a practical technique for enhancing communication around people who have severe cognitive impairment (SCI). An introduction explains SCI and how this impacts on communication; and consequently on quality of care and quality of life. This is followed by an explanation of TPR and its origins in triangulation research techniques. An illustrative vignette explicates its utility and value in a group home for a resident with profound multiple disabilities. The Discussion and Conclusion sections propose the wider application of TPR for different cohorts of people with SCIs, their communication partners and service providers. TPR presents as a practical technique for enhancing communication interactions with people who have SCI. The paper demonstrates the potential of the technique for improving engagement amongst those with profound multiple disabilities, severe acquired brain injury and advanced dementia and their partners in and across different care settings. Implications for Rehabilitation Triangulated Proxy Reporting (TPR) shows potential to improve communications between people with severe cognitive impairments and their communication partners. TPR can lead to improved quality of care and quality of life for people with profound multiple disabilities, very advanced dementia and severe acquired brain injury, who otherwise are very difficult to support. TPR is a relatively simple and inexpensive technique that service providers can incorporate into practice to improving communications between clients with severe cognitive impairments, their carers and other support professionals.

Characterization of LaRhO3 perovskites for dry (CO2) reforming of methane (DRM)

DOE Office of Scientific and Technical Information (OSTI.GOV)

Johansson, Ted; Pakhare, Devendra; Haynes, Daniel

2014-01-01

Abstract This work reports on the characterization of LaRhO3 perovskite as a catalyst for dry reforming of methane. The catalyst was studied using CH4-temperature programmed reduction (TPR), H2-TPR, and temperature programmed surface reaction (TPSR), and the changes in the crystal structure of the catalyst due to these treatments were studied by X-ray diffraction (XRD). XRD pattern of the freshly calcined perovskites showed the formation of highly crystalline LaRhO3 and La2O3 phases. H2-TPR of the fresh calcined catalyst showed a shoulder at 342°C and a broad peak at 448°C, suggesting that the reduction of Rh in perovskite occurs in multiple steps.more » XRD pattern of the reduced catalyst suggests complete reduction of the LaRhO3 phase and the formation of metallic Rh and minor amounts of La(OH)3. The CH4-TPR data show qualitatively similar results as H2-TPR, with a shoulder and a broad peak in the same temperature range. Following the H2-TPR up to 950°C, the same batch of catalyst was oxidized by flowing 5 vol. % O2/He up to 500°C and a second H2-TPR (also up to 950°C) was conducted. This second H2-TPR differed significantly from that of the fresh calcined catalyst. The single sharp peak at 163°C in the second H2-TPR suggests a significant change in the catalyst, probably causedby the transformation of about 90 % of the perovskite into Rh/La2O3. This was confirmed by the XRD studies of the catalyst reduced after the oxidation at 500°C. TPSR of the dry reforming reaction on the fresh calcined catalyst showed CO and H2 formation starting at 400°C, with complete consumption of the reactants at 650°C. The uneven consumption of reactants between 400°C and 650°C suggests that reactions other than DRM occur, including reverse water gas shift (RWGS) and the Boudouard reaction (BR), probably as a result of in-situ changes in the catalyst, consistent with the H2-TPR results. TPSR, after a H2-TPR up to 950°C, showed that the dry reforming reaction did not light off until 570°C, which is much higher temperature than the one observed using fresh calcined catalyst. This shows that the uniform sites produced during the 950°C H2-TPR are catalytically less active than those of the fresh calcined catalyst, and that no significant side reactions such as RWGS or the Boudouard reaction occur. This suggests that reduction leads to the formation of a single type of sites which do not catalyze simultaneous side reactions.« less

The O-GlcNAc Transferase Intellectual Disability Mutation L254F Distorts the TPR Helix.

PubMed

Gundogdu, Mehmet; Llabrés, Salomé; Gorelik, Andrii; Ferenbach, Andrew T; Zachariae, Ulrich; van Aalten, Daan M F

2018-05-17

O-linked β-N-acetyl- D -glucosamine (O-GlcNAc) transferase (OGT) regulates protein O-GlcNAcylation, an essential post-translational modification that is abundant in the brain. Recently, OGT mutations have been associated with intellectual disability, although it is not understood how they affect OGT structure and function. Using a multi-disciplinary approach we show that the L254F OGT mutation leads to conformational changes of the tetratricopeptide repeats and reduced activity, revealing the molecular mechanisms contributing to pathogenesis. Copyright © 2018 The Authors. Published by Elsevier Ltd.. All rights reserved.

WISE-2005: Adrenergic Responses Before and After 60 Days of 6 Degree Head-Down Bed-Rest in Women

NASA Technical Reports Server (NTRS)

Edgell, H.; Dyson, K.; Shoemaker, J. K.; Custaud, M. A.; Arbeille, Ph.; Greaves, D.; Hughson, R. L.; Hughson, R. L.

2006-01-01

Sixteen women who participated in the WISE-2005 headdown bed rest (HDBR) were studied before and on day 56 of bed rest to test the hypothesis that chronic changes in circulating norepinephrine (NOR) would change the response to adrenergic receptor agonists. Five minute infusions of 2 doses of isoproterenol (ISO), and 2 doses of NOR were administered while heart rate (HR), mean arterial pressure (MAP) and total peripheral resistance (TPR) were measured. Before HDBR, the higher dose of ISO increased HR by 13 beats/min (P

Functional characterization, localization, and inhibitor sensitivity of the TPR-FGFR1 fusion in 8p11 myeloproliferative syndrome.

PubMed

Malli, Theodora; Buxhofer-Ausch, Veronika; Rammer, Melanie; Erdel, Martin; Kranewitter, Wolfgang; Rumpold, Holger; Marschon, Renate; Deutschbauer, Sabine; Simonitsch-Klupp, Ingrid; Valent, Peter; Muellner-Ammer, Kirsten; Sebesta, Christian; Birkner, Thomas; Webersinke, Gerald

2016-01-01

Myeloid and lymphoid neoplasms with fibroblast growth factor receptor 1 (FGFR1) abnormalities, also known as 8p11 myeloproliferative syndrome (EMS), represent rare and aggressive disorders, associated with chromosomal aberrations that lead to the fusion of FGFR1 to different partner genes. We report on a third patient with a fusion of the translocated promoter region (TPR) gene, a component of the nuclear pore complex, to FGFR1 due to a novel ins(1;8)(q25;p11p23). The fact that this fusion is a rare but recurrent event in EMS prompted us to examine the localization and transforming potential of the chimeric protein. TPR-FGFR1 localizes in the cytoplasm, although the nuclear pore localization signal of TPR is retained in the fusion protein. Furthermore, TPR-FGFR1 enables cytokine-independent survival, proliferation, and granulocytic differentiation of the interleukin-3 dependent myeloid progenitor cell line 32Dcl3, reflecting the chronic phase of EMS characterized by myeloid hyperplasia. 32Dcl3 cells transformed with the TPR-FGFR1 fusion and treated with increasing concentrations of the tyrosine kinase inhibitors ponatinib (AP24534) and infigratinib (NVP-BGJ398) displayed reduced survival and proliferation with IC50 values of 49.8 and 7.7 nM, respectively. Ponatinib, a multitargeted tyrosine kinase inhibitor, is already shown to be effective against several FGFR1-fusion kinases. Infigratinib, tested only against FGFR1OP2-FGFR1 to date, is also efficient against TPR-FGFR1. Taking its high specificity for FGFRs into account, infigratinib could be beneficial for EMS patients and should be further investigated for the treatment of myeloproliferative neoplasms with FGFR1 abnormalities. © 2015 Wiley Periodicals, Inc.

Activation of the JNK pathway is essential for transformation by the Met oncogene.

PubMed

Rodrigues, G A; Park, M; Schlessinger, J

1997-05-15

The Met/Hepatocyte Growth Factor (HGF) receptor tyrosine kinase is oncogenically activated through a rearrangement that creates a hybrid gene Tpr-Met. The resultant chimeric p65(Tpr-Met) protein is constitutively phosphorylated on tyrosine residues in vivo and associates with a number of SH2-containing signaling molecules including the p85 subunit of PI-3 kinase and the Grb2 adaptor protein, which couples receptor tyrosine kinases to the Ras signaling pathway. Mutation of the binding site for Grb2 impairs the ability of Tpr-Met oncoprotein to transform fibroblasts, suggesting that the activation of the Ras/MAP kinase signaling pathway through Grb2 may be essential for cellular transformation. To test this hypothesis dominant-negative mutants of Grb2 with deletions of the SH3 domains were introduced into Tpr-Met transformed fibroblasts. Cells overexpressing the mutants were found to be morphologically reverted and exhibited reduced growth in soft agar. Surprisingly, the Grb2 mutants blocked activation of the JNK/SAPK but not MAP kinase activity induced by the Tpr-Met oncoprotein. Additionally, cells expressing dominant-negative Grb2 mutants had reduced PI-3-kinase activity and dominant-negative mutants of Rac1 blocked both Tpr-Met-induced transformation and activation of JNK. These experiments reveal a novel link between Met and the JNK pathway, which is essential for transformation by this oncogene.

Large enhancement of oscillating chemiluminescence with [Ru(bpy)3 ](2+) -catalyzed Belousov-Zhabotinsky reaction in the presence of tri-n-propylamine.

PubMed

Lan, Xiaolan; Zheng, Baozhan; Zhao, Yan; Yuan, Hongyan; Du, Juan; Xiao, Dan

2013-01-01

Oscillating chemiluminescence enhanced by the addition of tri-n-propylamine (TPrA) to the typical Belousov-Zhabotinsky (BZ) reaction system catalyzed by ruthenium(II)tris(2.2'-bipyridine)(Ru(bpy)3 (2+) ) was investigated using a luminometry method. The [Ru(bpy)3 ](2+) /TPrA system was first used as the catalyst for a BZ oscillator in a closed system, which exhibited a shorter induction period, higher amplitude and much more stable chemiluminescence (CL) oscillation. The effects of various concentrations of TPrA, oxygen and nitrogen flow rate on the oscillating behavior of this system were examined. In addition, the CL intensity of the [Ru(bpy)3 ](2+) /TPrA-BZ system was found to be inhibited by phenol, thus providing a way for use of the BZ system in the determination of phenolic compounds. Moreover, the possible mechanism of the oscillating CL reaction catalyzed by [Ru(bpy)3 ](2+) /TPrA and the inhibition effects of oxygen and phenol on this oscillating CL system were considered. Copyright © 2012 John Wiley & Sons, Ltd.

Mechanical Modulation of Tunneling Current in Transition Metal Dichalcogenides Heterostructures: A First Principles Study

NASA Astrophysics Data System (ADS)

Kuroda, Marcelo

Recent experiments in MoS2 heterostructures reported that out-of-plane tunneling piezoresistivity (TPR) - mechanical modulation of the tunneling current - achieves sensitivities of one decade per Ådisplacement. Owing to their nanometer scale, a quantitative theoretical framework providing the TPR structure-property relationship is necessary to further improve sensitivities. To this end, first principles calculations within density functional theory are used to characterize the phenomenon in MoX2 (with X = S, Se). The TPR is quantified in relation to electrode composition and film thickness showing remarkable agreement with experiments. The origin of the TPR is attributed to the heterostructure compliance rather than band alignment changes with strain, and differs from mechanisms in other nanometer-thick bulk films. Large work function metals (Pt, Au) are singled out as best candidates for enhanced TPR gauges due to weak bonding and negligible thermionic emission; compliant bilayers show larger stress-sensitivity than monolayers. By accounting for the atomistic details and material composition of 2D material-based heterostructures, this work has the potential to advance sensor and nano-electro-mechanical system technologies.

Selective activators of protein phosphatase 5 target the auto-inhibitory mechanism.

PubMed

Haslbeck, Veronika; Drazic, Adrian; Eckl, Julia M; Alte, Ferdinand; Helmuth, Martin; Popowicz, Grzegorz; Schmidt, Werner; Braun, Frank; Weiwad, Matthias; Fischer, Gunter; Gemmecker, Gerd; Sattler, Michael; Striggow, Frank; Groll, Michael; Richter, Klaus

2015-04-20

Protein phosphatase 5 (PP5) is an evolutionary conserved serine/threonine phosphatase. Its dephosphorylation activity modulates a diverse set of cellular factors including protein kinases and the microtubule-associated tau protein involved in neurodegenerative disorders. It is auto-regulated by its heat-shock protein (Hsp90)-interacting tetratricopeptide repeat (TPR) domain and its C-terminal α-helix. In the present study, we report the identification of five specific PP5 activators [PP5 small-molecule activators (P5SAs)] that enhance the phosphatase activity up to 8-fold. The compounds are allosteric modulators accelerating efficiently the turnover rate of PP5, but do barely affect substrate binding or the interaction between PP5 and the chaperone Hsp90. Enzymatic studies imply that the compounds bind to the phosphatase domain of PP5. For the most promising compound crystallographic comparisons of the apo PP5 and the PP5-P5SA-2 complex indicate a relaxation of the auto-inhibited state of PP5. Residual electron density and mutation analyses in PP5 suggest activator binding to a pocket in the phosphatase/TPR domain interface, which may exert regulatory functions. These compounds thus may expose regulatory mechanisms in the PP5 enzyme and serve to develop optimized activators based on these scaffolds. © 2015 Authors.

Proteolysis of HCF-1 by Ser/Thr glycosylation-incompetent O-GlcNAc transferase:UDP-GlcNAc complexes

PubMed Central

Kapuria, Vaibhav; Röhrig, Ute F.; Bhuiyan, Tanja; Borodkin, Vladimir S.; van Aalten, Daan M.F.; Zoete, Vincent; Herr, Winship

2016-01-01

In complex with the cosubstrate UDP-N-acetylglucosamine (UDP-GlcNAc), O-linked-GlcNAc transferase (OGT) catalyzes Ser/Thr O-GlcNAcylation of many cellular proteins and proteolysis of the transcriptional coregulator HCF-1. Such a dual glycosyltransferase–protease activity, which occurs in the same active site, is unprecedented and integrates both reversible and irreversible forms of protein post-translational modification within one enzyme. Although occurring within the same active site, we show here that glycosylation and proteolysis occur through separable mechanisms. OGT consists of tetratricopeptide repeat (TPR) and catalytic domains, which, together with UDP-GlcNAc, are required for both glycosylation and proteolysis. Nevertheless, a specific TPR domain contact with the HCF-1 substrate is critical for proteolysis but not Ser/Thr glycosylation. In contrast, key catalytic domain residues and even a UDP-GlcNAc oxygen important for Ser/Thr glycosylation are irrelevant for proteolysis. Thus, from a dual glycosyltransferase–protease, essentially single-activity enzymes can be engineered both in vitro and in vivo. Curiously, whereas OGT-mediated HCF-1 proteolysis is limited to vertebrate species, invertebrate OGTs can cleave human HCF-1. We present a model for the evolution of HCF-1 proteolysis by OGT. PMID:27056667

The E3 Ligase CHIP: Insights into Its Structure and Regulation

PubMed Central

Paul, Indranil; Ghosh, Mrinal K.

2014-01-01

The carboxy-terminus of Hsc70 interacting protein (CHIP) is a cochaperone E3 ligase containing three tandem repeats of tetratricopeptide (TPR) motifs and a C-terminal U-box domain separated by a charged coiled-coil region. CHIP is known to function as a central quality control E3 ligase and regulates several proteins involved in a myriad of physiological and pathological processes. Recent studies have highlighted varied regulatory mechanisms operating on the activity of CHIP which is crucial for cellular homeostasis. In this review article, we give a concise account of our current knowledge on the biochemistry and regulation of CHIP. PMID:24868554

Theory and Practice in Participatory Research: Lessons from the Native Elder Care Study

PubMed Central

Goins, R. Turner; Garroutte, Eva Marie; Fox, Susan Leading; Dee Geiger, Sarah; Manson, Spero M.

2011-01-01

Models for community-based participatory research (CBPR) urge academic investigators to collaborate with communities to identify and pursue research questions, processes, and outcomes valuable to both partners. The tribal participatory research (TPR) conceptual model suggests modifications to CBPR to fit the special needs of American Indian communities. This paper draws upon authors’ collaboration with one American Indian tribe to recommend theoretical revision and practical strategies for conducting gerontological research in tribal communities. We rated the TPR model as a strong, specialized adaptation of participatory research principles. Although the need for some TPR mechanisms may vary, our experience recommends incorporating dissemination as a central TPR mechanism. Researchers and communities can expect well-crafted collaborative projects to generate particular types of positive project outcomes for both partners, but should prepare for both predictable and unique challenges. PMID:21292753

Structure of an APC3–APC16 Complex: Insights into Assembly of the Anaphase-Promoting Complex/Cyclosome

DOE PAGES

Yamaguchi, Masaya; Yu, Shanshan; Qiao, Renping; ...

2014-12-06

The anaphase-promoting complex/cyclosome (APC/C) is a massive E3 ligase that controls mitosis by catalyzing ubiquitination of key cell cycle regulatory proteins. The APC/C assembly contains two subcomplexes: the “Platform” centers around a cullin-RING-like E3 ligase catalytic core; the “Arc Lamp” is a hub that mediates transient association with regulators and ubiquitination substrates. The Arc Lamp contains the small subunits APC16, CDC26, and APC13, and tetratricopeptide repeat (TPR) proteins (APC7, APC3, APC6, and APC8) that homodimerize and stack with quasi-2-fold symmetry. Within the APC/C complex, APC3 serves as center for regulation. APC3's TPR motifs recruit substrate-binding coactivators, CDC20 and CDH1, viamore » their C-terminal conserved Ile-Arg (IR) tail sequences. Human APC3 also binds APC16 and APC7 and contains a > 200-residue loop that is heavily phosphorylated during mitosis, although the basis for APC3 interactions and whether loop phosphorylation is required for ubiquitination are unclear. Here, we map the basis for human APC3 assembly with APC16 and APC7, report crystal structures of APC3Δloop alone and in complex with the C-terminal domain of APC16, and test roles of APC3's loop and IR tail binding surfaces in APC/C-catalyzed ubiquitination. The structures show how one APC16 binds asymmetrically to the symmetric APC3 dimer and, together with biochemistry and prior data, explain how APC16 recruits APC7 to APC3, show how APC3's C-terminal domain is rearranged in the full APC/C assembly, and visualize residues in the IR tail binding cleft important for coactivator-dependent ubiquitination. Overall, the results provide insights into assembly, regulation, and interactions of TPR proteins and the APC/C.« less

Tethering of SCFDia2 to the Replisome Promotes Efficient Ubiquitylation and Disassembly of the CMG Helicase

PubMed Central

Maculins, Timurs; Nkosi, Pedro Junior; Nishikawa, Hiroko; Labib, Karim

2015-01-01

Summary Disassembly of the Cdc45-MCM-GINS (CMG) DNA helicase, which unwinds the parental DNA duplex at eukaryotic replication forks, is the key regulated step during replication termination but is poorly understood [1, 2]. In budding yeast, the F-box protein Dia2 drives ubiquitylation of the CMG helicase at the end of replication, leading to a disassembly pathway that requires the Cdc48 segregase [3]. The substrate-binding domain of Dia2 comprises leucine-rich repeats, but Dia2 also has a TPR domain at its amino terminus that interacts with the Ctf4 and Mrc1 subunits of the replisome progression complex [4, 5], which assembles around the CMG helicase at replication forks [6]. Previous studies suggested two disparate roles for the TPR domain of Dia2, either mediating replisome-specific degradation of Mrc1 and Ctf4 [4] or else tethering SCFDia2 (SCF [Skp1/cullin/F-box protein]) to the replisome to increase its local concentration at replication forks [5]. Here, we show that SCFDia2 does not mediate replisome-specific degradation of Mrc1 and Ctf4, either during normal S phase or in response to replication stress. Instead, the tethering of SCFDia2 to the replisome progression complex increases the efficiency of ubiquitylation of the Mcm7 subunit of CMG, both in vitro and in vivo. Correspondingly, loss of tethering reduces the efficiency of CMG disassembly in vivo and is synthetic lethal in combination with a disassembly-defective allele of CDC48. Residual ubiquitylation of Mcm7 in dia2-ΔTPR cells is still CMG specific, highlighting the complex regulation of the final stages of chromosome replication, about which much still remains to be learned. PMID:26255844

T-piece resuscitators: how do they compare?

PubMed

Hinder, Murray; McEwan, Alistair; Drevhammer, Thomas; Donaldson, Snorri; Tracy, Mark Brian

2018-05-04

The T-piece resuscitator (TPR) has seen increased use as a primary resuscitation device with newborns. Traditional TPR design uses a high resistance expiratory valve to produce positive end expiratory pressure (PEEP) or continuous positive airway pressure (CPAP) at resuscitation. A new TPR device that uses a dual flow ratio valve (fluidic flip) to produce PEEP/CPAP is now available (rPAP). We aimed to compare the measured ventilation performance of different TPR devices in a controlled bench test study. Single operator provided positive pressure ventilation to an incremental testlung compliance (Crs) model (0.5-5 mL/cmH 2 O) with five different brands of TPR device (Atom, Neopuff, rPAP, GE Panda warmer and Draeger Resuscitaire). At recommended peak inflation pressure (PIP) 20 cmH 2 O, PEEP of 5 cmH 2 O and rate of 60 inflations per minute. 1864 inflations were analysed. Four of the five devices tested demonstrated inadvertent elevations in mean PEEP (5.5-10.3 cmH 2 O, p<0.001) from set value as Crs was increased, while one device (rPAP) remained at the set value. Measured PIP exceeded the set value in two infant warmer devices (GE and Draeger) with inbuilt TPR at Crs of 0.5 (24.5 and 23.5 cmH 2 O, p<0.001). Significant differences were seen in tidal volumes across devices particularly at higher Crs (p<0.001). Results show important variation in delivered ventilation from set values due to inherent TPR device design characteristics with a range of lung compliances expected at birth. Device-generated inadvertent PEEP and overdelivery of PIP may be clinically deleterious for term and preterm newborns or infants with larger Crs during resuscitation. © Article author(s) (or their employer(s) unless otherwise stated in the text of the article) 2018. All rights reserved. No commercial use is permitted unless otherwise expressly granted.

Clinical implementation of photon beam flatness measurements to verify beam quality.

PubMed

Goodall, Simon; Harding, Nicholas; Simpson, Jake; Alexander, Louise; Morgan, Steve

2015-11-08

This work describes the replacement of Tissue Phantom Ratio (TPR) measurements with beam profile flatness measurements to determine photon beam quality during routine quality assurance (QA) measurements. To achieve this, a relationship was derived between the existing TPR15/5 energy metric and beam flatness, to provide baseline values and clinically relevant tolerances. The beam quality was varied around two nominal beam energy values for four matched Elekta linear accelerators (linacs) by varying the bending magnet currents and reoptimizing the beam. For each adjusted beam quality the TPR15/5 was measured using an ionization chamber and Solid Water phantom. Two metrics of beam flatness were evaluated using two identical commercial ionization chamber arrays. A linear relationship was found between TPR15/5 and both metrics of flatness, for both nominal energies and on all linacs. Baseline diagonal flatness (FDN) values were measured to be 103.0% (ranging from 102.5% to 103.8%) for 6 MV and 102.7% (ranging from 102.6% to 102.8%) for 10 MV across all four linacs. Clinically acceptable tolerances of ± 2% for 6 MV, and ± 3% for 10 MV, were derived to equate to the current TPR15/5 clinical tolerance of ± 0.5%. Small variations in the baseline diagonal flatness values were observed between ionization chamber arrays; however, the rate of change of TPR15/5 with diagonal flatness was found to remain within experimental uncertainty. Measurements of beam flatness were shown to display an increased sensitivity to variations in the beam quality when compared to TPR measurements. This effect is amplified for higher nominal energy photons. The derivation of clinical baselines and associated tolerances has allowed this method to be incorporated into routine QA, streamlining the process whilst also increasing versatility. In addition, the effect of beam adjustment can be observed in real time, allowing increased practicality during corrective and preventive maintenance interventions.

The inclusion of disability as a condition for termination of parental rights.

PubMed

Lightfoot, Elizabeth; Hill, Katharine; LaLiberte, Traci

2010-12-01

All 50 states and the District of Columbia have statutes outlining the grounds for terminating parental rights (TPR) in relation to child abuse and neglect. Although recent research has found that parents with disabilities are not more likely to maltreat their children than parents without disabilities (Glaun & Brown, 1999; Oyserman, Mowbray, Meares, & Firminger, 2000), studies have found very high rates of TPR of parents with disabilities (Accardo & Whitman, 1989). The objective of this study is to examine how states are including disability in their TPR statutes. This study used legal document analysis, consisting of a comprehensive Boolean search of the state codes of the 50 states and District of Columbia (DC) relating to TPR, using the most recent state code available on Lexis-Nexis in August 2005. TPR and related statutes were searched for contemporary and historical disability related terms and their common cognates, such as: "mental," "disability," "handicap," and "incapacity." Two researchers independently conducted the searches, and the searches were reconciled. A code list was then developed to measure for inclusion of disability, preciseness, scope, use of language, and references to accessibility or fairness. Statutes were then reanalyzed, and groupings developed. Thirty-seven states included disability-related grounds for termination of parental rights, while 14 states did not include disability language as grounds for termination. Many of these state codes used outdated terminology, imprecise definitions, and emphasized disability status rather than behavior. All of the 14 states that do not include disability in TPR grounds allowed for termination based on neglectful parental behavior that may be influenced by a disability. The use of disability language in TPR statutes can put an undue focus on the condition of having a disability, rather than parenting behavior. This paper recommends that states consider removing disability language from their statutes, as such language risks taking the emphasis away from the assessment based on parenting behavior. Copyright © 2010 Elsevier Ltd. All rights reserved.

The association of oxidative stress with arterial compliance and vascular resistance in a bi-ethnic population: the SABPA study.

PubMed

Mokhaneli, Maserame Cleopatra; Fourie, Carla Maria T; Botha, Shani; Mels, Catharina Martha C

2016-08-01

A loss of arterial elasticity increases the risk for cardiovascular events. Oxidative injury to the vessel wall may be one of the underlying mechanisms influencing arterial elasticity. We compared markers of oxidative stress, antioxidant capacity, inflammation, windkessel compliance (Cwk), and total peripheral resistance (TPR) in black and white South Africans. Associations of arterial compliance and vascular resistance (as indicated by TPR) with oxidative stress, antioxidant capacity and inflammatory markers were also investigated. We included 146 black and 181 white men and women. Measurements from the Finometer device were used to calculate Cwk and TPR while thiobarbituric acids reactive substances (TBARS), glutathione peroxidase (GPx), C-reactive protein (CRP), and interleukin-6 (IL-6) were analyzed in serum or urine samples. Black participants had higher TPR, TBARS, GPx, CRP, and IL-6 levels (all p ≤ 0.018) and lower Cwk (both p ≤ 0.013) compared to white participants. Multiple regression analyses revealed independent associations of Cwk (β = -0.27, p = 0.015) and TPR (β = 0.18, p = 0.018) with TBARS in black participants, while Cwk (β = -0.10; p = 0.019) and TPR (β = 0.13, p = 0.047) were independently associated with GPx in white participants. Decreased arterial compliance and increased vascular resistance associated with increased oxidative damage independent of hypertensive status in black participants. These results suggest that oxidative stress plays a role in early vascular changes in a black population prone to the development of cardiovascular disease.

Total plankton respiration in the Chesapeake Bay plume

NASA Technical Reports Server (NTRS)

Robertson, C. N.; Thomas, J. P.

1981-01-01

Total plankton respiration (TPR) was measured at 17 stations within the Chesapeake Bay plume off the Virginia coast during March, June, and October 1980. Elevated rates of TPR, as well as higher concentrations of chlorophyll a and phaeopigment a, were found to be associated with the Bay plume during each survey. The TPR rates within the Bay plume were close to those found associated with the Hudson River plume for comparable times of the year. The data examined indicate that the Chesapeake Bay plume stimulates biological activity and is a source of organic loading to the contiguous shelf ecosystem.

Role of molecular chaperones and TPR-domain proteins in the cytoplasmic transport of steroid receptors and their passage through the nuclear pore.

PubMed

Galigniana, Mario D; Echeverría, Pablo C; Erlejman, Alejandra G; Piwien-Pilipuk, Graciela

2010-01-01

In the absence of hormone, corticosteroid receptors such as GR (glucocorticoid receptor) and (mineralocorticoid receptor) are primarily located in the cytoplasm. Upon steroid-binding, they rapidly accumulate in the nucleus. Regardless of their primary location, these receptors and many other nuclear factors undergo a constant and dynamic nucleocytoplasmic shuttling. All members of the steroid receptor family are known to form large oligomeric structures with the heat-shock proteins of 90-kDa (hsp90) and 70-kDa (hsp70), the small acidic protein p23, and a tetratricopeptide repeat (TPR) -domain protein such as FK506-binding proteins (FKBPs), cyclophilins (CyPs) or the serine/threonine protein phosphatase 5 (PP5). It has always been stated that the dissociation of the chaperone heterocomplex (a process normally referred to as receptor "transformation") is the first step that permits the nuclear import of steroid receptors. However the experimental evidence is consistent with a model where the chaperone machinery is required for the retrotransport of the receptor through the cytoplasm and also facilitates the passage through the nuclear pore. Recent evidence indicates that the hsp90-based chaperone system also interacts with structures of the nuclear pore such as importin β and the integral nuclear pore glycoprotein Nup62 facilitating the passage of the untransformed receptor through the nuclear pore.

Prion-specific Hsp40 function: The role of the auxilin homolog Swa2

PubMed Central

Oliver, Emily E.; Troisi, Elizabeth M.; Hines, Justin K.

2017-01-01

ABSTRACT Yeast prions are protein-based genetic elements that propagate through cell populations via cytosolic transfer from mother to daughter cell. Molecular chaperone proteins including Hsp70, the Hsp40/J-protein Sis1, and Hsp104 are required for continued prion propagation, however the specific requirements of chaperone proteins differ for various prions. We recently reported that Swa2, the yeast homolog of the mammalian protein auxilin, is specifically required for the propagation of the prion [URE3].1 [URE3] propagation requires both a functional J-domain and the tetratricopeptide repeat (TPR) domain of Swa2, but does not require Swa2 clathrin binding. We concluded that the TPR domain determines the specificity of the genetic interaction between Swa2 and [URE3], and that this domain likely interacts with one or more proteins with a C-terminal EEVD motif. Here we extend that analysis to incorporate additional data that supports this hypothesis. We also present new data eliminating Hsp104 as the relevant Swa2 binding partner and discuss our findings in the context of other recent work involving Hsp90. Based on these findings, we propose a new model for Swa2's involvement in [URE3] propagation in which Swa2 and Hsp90 mediate the formation of a multi-protein complex that increases the number of sites available for Hsp104 disaggregation. PMID:28574745

Gender differences in autonomic cardiovascular regulation: spectral, hormonal, and hemodynamic indexes

NASA Technical Reports Server (NTRS)

Evans, J. M.; Ziegler, M. G.; Patwardhan, A. R.; Ott, J. B.; Kim, C. S.; Leonelli, F. M.; Knapp, C. F.

2001-01-01

The autonomic nervous system drives variability in heart rate, vascular tone, cardiac ejection, and arterial pressure, but gender differences in autonomic regulation of the latter three parameters are not well documented. In addition to mean values, we used spectral analysis to calculate variability in arterial pressure, heart rate (R-R interval, RRI), stroke volume, and total peripheral resistance (TPR) and measured circulating levels of catecholamines and pancreatic polypeptide in two groups of 25 +/- 1.2-yr-old, healthy men and healthy follicular-phase women (40 total subjects, 10 men and 10 women per group). Group 1 subjects were studied supine, before and after beta- and muscarinic autonomic blockades, administered singly and together on separate days of study. Group 2 subjects were studied supine and drug free with the additional measurement of skin perfusion. In the unblocked state, we found that circulating levels of epinephrine and total spectral power of stroke volume, TPR, and skin perfusion ranged from two to six times greater in men than in women. The difference (men > women) in spectral power of TPR was maintained after beta- and muscarinic blockades, suggesting that the greater oscillations of vascular resistance in men may be alpha-adrenergically mediated. Men exhibited muscarinic buffering of mean TPR whereas women exhibited beta-adrenergic buffering of mean TPR as well as TPR and heart rate oscillations. Women had a greater distribution of RRI power in the breathing frequency range and a less negative slope of ln RRI power vs. ln frequency, both indicators that parasympathetic stimuli were the dominant influence on women's heart rate variability. The results of our study suggest a predominance of sympathetic vascular regulation in men compared with a dominant parasympathetic influence on heart rate regulation in women.

The UNC-45 Myosin Chaperone: From Worms to Flies to Vertebrates

PubMed Central

Lee, Chi F.; Melkani, Girish C.; Bernstein, Sanford I.

2014-01-01

UNC-45 is a UCS domain protein that is critical for myosin stability and function. It likely aides in folding myosin during cellular differentiation and maintenance and protects myosin from denaturation during stress. Invertebrates have a single unc-45 gene that is expressed in both muscle and non-muscle tissues. Vertebrates possess one gene expressed in striated muscle (unc-45b) and one that is more generally expressed (unc-45a). Structurally, UNC-45 is composed of a series of alpha-helices connected by loops. It has an N-terminal TPR domain that binds to Hsp90 and a central domain composed of armadillo repeats. Its C-terminal UCS domain, which is also comprised of helical armadillo repeats, interacts with myosin. In this review, we present biochemical, structural and genetic analyses of UNC-45 in Caenorhabditis elegans, Drosophila melanogaster and various vertebrates. Further, we provide insights into UNC-45 functions, its potential mechanism of action and its roles in human disease. PMID:25376491

Subglottic injury, gastric juice, corticosteroids, and peptide growth factors in a porcine model.

PubMed

Yellon, R F; Szeremeta, W; Grandis, J R; Diguisseppe, P; Dickman, P S

1998-06-01

To study the effects of mucosal injury, gastric juice, and corticosteroids and to determine the presence of peptide growth factors in the subglottic mucosa in a porcine model. Prospective cohort animal study. In this model of subglottic injury, five groups (n = 5 each) of piglets were used. Injury was induced by electrocautery (acute), electrocautery plus repeated saline application (chronic), electrocautery plus repeated gastric juice application (chronic plus gastric juice), or repeated gastric juice application (gastric). Control piglets had normal saline applied repeatedly. Histopathologic findings for the gastric juice group included basal cell hyperplasia (80%), squamous metaplasia (80%), and mucosal ulceration (40%). Control piglets showed squamous metaplasia (80%) but no basilar hyperplasia or ulceration. Immunohistochemistry detected peptide growth factors and epidermal growth factor receptor (EGFR) in all groups. Decreased staining was most frequent in the acute injury group. Quantitative reverse transcriptase polymerase chain reaction (RT-PCR) documented lower expression of EGFR in the gastric juice group (P = .01). These findings suggest that peptide growth factors and EGFR are part of normal subglottic mucosal turnover. Noxious stimuli decrease production of these factors. Gastric juice had adverse effects documented by histopathology and molecular techniques.

The Test Validation Summary

ERIC Educational Resources Information Center

Frederick, Richard I.; Bowden, Stephen C.

2009-01-01

Common rates employed in classificatory testing are the true positive rate (TPR), false positive rate (FPR), positive predictive power (PPP), and negative predictive power (NPP). FPR and TPR are estimated from research samples representing populations to be distinguished by classificatory testing. PPP and NPP are used by clinicians to classify…

Total Physical Response: A Technique for Teaching All Skills in Spanish.

ERIC Educational Resources Information Center

Glisan, Eileen W.

1986-01-01

Presents a strategy for using an expanded version of Total Physical Response (TPR) as one tool for teaching listening, speaking, reading, and writing in Spanish. Variations of TPR are suggested for the purpose of implementing the technique within the foreign language curriculum. (Author/CB)

Effects of the choice of reference on the selectivity of a multi-contact nerve cuff electrode.

PubMed

Koh, Ryan G L; Zariffa, Jose

2016-08-01

Tripolar referencing is typically used in nerve cuff electrode recordings due to its ability to maximize the signal-to-noise ratio of contacts at the centre, but this may not be the optimal choice for a multi-contact nerve cuff consisting of contacts in off-centre rings. We conducted a simulation study to compare the effects of 3 different reference types on the recording selectivity of a multi-contact nerve cuff: the tripolar reference (TPR), common average reference (CAR), and multiple tripolar references based on consecutive groups of 3 rings (cTPR). For this purpose, we introduce a novel measure called the contact information metric (CIM). Selectivity was tested in 2 noise settings, one in which white Gaussian noise was added inside the nerve cuff electrode and the other in which electromyogram (EMG) noise was added outside the nerve cuff electrode. The mean CIMs values calculated for the best 8 contacts were 3.42±6.25, 2.70±3.37, and 3.65±1.90 for the TPR, the CAR and the cTPR, respectively, in the case of EMG noise added outside the nerve cuff electrode. This study shows that the use of cTPR reference is the optimal choice for selectivity when using a multi-contact nerve cuff electrode which contains off-centre rings.

The genome of the amoeba symbiont "Candidatus Amoebophilus asiaticus" encodes an afp-like prophage possibly used for protein secretion.

PubMed

Penz, Thomas; Horn, Matthias; Schmitz-Esser, Stephan

2010-01-01

The recently sequenced genome of the obligate intracellular amoeba symbiont 'Candidatus Amoebophilus asiaticus' is unique among prokaryotic genomes due to its extremely large fraction of genes encoding proteins harboring eukaryotic domains such as ankyrin-repeats, TPR/SEL1 repeats, leucine-rich repeats, as well as F- and U-box domains, most of which likely serve in the interaction with the amoeba host. Here we provide evidence for the presence of additional proteins which are presumably presented extracellularly and should thus also be important for host cell interaction. Surprisingly, we did not find homologues of any of the well-known protein secretion systems required to translocate effector proteins into the host cell in the A. asiaticus genome, and the type six secretion systems seems to be incomplete. Here we describe the presence of a putative prophage in the A. asiaticus genome, which shows similarity to the antifeeding prophage from the insect pathogen Serratia entomophila. In S. entomophila this system is used to deliver toxins into insect hosts. This putative antifeeding-like prophage might thus represent the missing protein secretion apparatus in A. asiaticus.

The Energy Landscapes of Repeat-Containing Proteins: Topology, Cooperativity, and the Folding Funnels of One-Dimensional Architectures

PubMed Central

Komives, Elizabeth A.; Wolynes, Peter G.

2008-01-01

Repeat-proteins are made up of near repetitions of 20– to 40–amino acid stretches. These polypeptides usually fold up into non-globular, elongated architectures that are stabilized by the interactions within each repeat and those between adjacent repeats, but that lack contacts between residues distant in sequence. The inherent symmetries both in primary sequence and three-dimensional structure are reflected in a folding landscape that may be analyzed as a quasi–one-dimensional problem. We present a general description of repeat-protein energy landscapes based on a formal Ising-like treatment of the elementary interaction energetics in and between foldons, whose collective ensemble are treated as spin variables. The overall folding properties of a complete “domain” (the stability and cooperativity of the repeating array) can be derived from this microscopic description. The one-dimensional nature of the model implies there are simple relations for the experimental observables: folding free-energy (ΔGwater) and the cooperativity of denaturation (m-value), which do not ordinarily apply for globular proteins. We show how the parameters for the “coarse-grained” description in terms of foldon spin variables can be extracted from more detailed folding simulations on perfectly funneled landscapes. To illustrate the ideas, we present a case-study of a family of tetratricopeptide (TPR) repeat proteins and quantitatively relate the results to the experimentally observed folding transitions. Based on the dramatic effect that single point mutations exert on the experimentally observed folding behavior, we speculate that natural repeat proteins are “poised” at particular ratios of inter- and intra-element interaction energetics that allow them to readily undergo structural transitions in physiologically relevant conditions, which may be intrinsically related to their biological functions. PMID:18483553

A Novel Peptide Derived from the Fusion Protein Heptad Repeat Inhibits Replication of Subacute Sclerosing Panencephalitis Virus In Vitro and In Vivo.

PubMed

Watanabe, Masahiro; Hashimoto, Koichi; Abe, Yusaku; Kodama, Eiichi N; Nabika, Ryota; Oishi, Shinya; Ohara, Shinichiro; Sato, Masatoki; Kawasaki, Yukihiko; Fujii, Nobutaka; Hosoya, Mitsuaki

2016-01-01

Subacute sclerosing panencephalitis (SSPE) is a persistent, progressive, and fatal degenerative disease resulting from persistent measles virus (MV) infection of the central nervous system. Most drugs used to treat SSPE have been reported to have limited effects. Therefore, novel therapeutic strategies are urgently required. The SSPE virus, a variant MV strain, differs virologically from wild-type MV strain. One characteristic of the SSPE virus is its defective production of cell-free virus, which leaves cell-to-cell infection as the major mechanism of viral dissemination. The fusion protein plays an essential role in this cell-to-cell spread. It contains two critical heptad repeat regions that form a six-helix bundle in the trimer similar to most viral fusion proteins. In the case of human immunodeficiency virus type-1 (HIV-1), a synthetic peptide derived from the heptad repeat region of the fusion protein enfuvirtide inhibits viral replication and is clinically approved as an anti-HIV-1 agent. The heptad repeat regions of HIV-1 are structurally and functionally similar to those of the MV fusion protein. We therefore designed novel peptides derived from the fusion protein heptad repeat region of the MV and examined their effects on the measles and SSPE virus replication in vitro and in vivo. Some of these synthetic novel peptides demonstrated high antiviral activity against both the measles (Edmonston strain) and SSPE (Yamagata-1 strain) viruses at nanomolar concentrations with no cytotoxicity in vitro. In particular, intracranial administration of one of the synthetic peptides increased the survival rate from 0% to 67% in an SSPE virus-infected nude mouse model.

A Novel Peptide Derived from the Fusion Protein Heptad Repeat Inhibits Replication of Subacute Sclerosing Panencephalitis Virus In Vitro and In Vivo

PubMed Central

Watanabe, Masahiro; Hashimoto, Koichi; Abe, Yusaku; Kodama, Eiichi N.; Nabika, Ryota; Oishi, Shinya; Ohara, Shinichiro; Sato, Masatoki; Kawasaki, Yukihiko; Fujii, Nobutaka; Hosoya, Mitsuaki

2016-01-01

Subacute sclerosing panencephalitis (SSPE) is a persistent, progressive, and fatal degenerative disease resulting from persistent measles virus (MV) infection of the central nervous system. Most drugs used to treat SSPE have been reported to have limited effects. Therefore, novel therapeutic strategies are urgently required. The SSPE virus, a variant MV strain, differs virologically from wild-type MV strain. One characteristic of the SSPE virus is its defective production of cell-free virus, which leaves cell-to-cell infection as the major mechanism of viral dissemination. The fusion protein plays an essential role in this cell-to-cell spread. It contains two critical heptad repeat regions that form a six-helix bundle in the trimer similar to most viral fusion proteins. In the case of human immunodeficiency virus type-1 (HIV-1), a synthetic peptide derived from the heptad repeat region of the fusion protein enfuvirtide inhibits viral replication and is clinically approved as an anti-HIV-1 agent. The heptad repeat regions of HIV-1 are structurally and functionally similar to those of the MV fusion protein. We therefore designed novel peptides derived from the fusion protein heptad repeat region of the MV and examined their effects on the measles and SSPE virus replication in vitro and in vivo. Some of these synthetic novel peptides demonstrated high antiviral activity against both the measles (Edmonston strain) and SSPE (Yamagata-1 strain) viruses at nanomolar concentrations with no cytotoxicity in vitro. In particular, intracranial administration of one of the synthetic peptides increased the survival rate from 0% to 67% in an SSPE virus-infected nude mouse model. PMID:27612283

Computationally assisted screening and design of cell-interactive peptides by a cell-based assay using peptide arrays and a fuzzy neural network algorithm.

PubMed

Kaga, Chiaki; Okochi, Mina; Tomita, Yasuyuki; Kato, Ryuji; Honda, Hiroyuki

2008-03-01

We developed a method of effective peptide screening that combines experiments and computational analysis. The method is based on the concept that screening efficiency can be enhanced from even limited data by use of a model derived from computational analysis that serves as a guide to screening and combining the model with subsequent repeated experiments. Here we focus on cell-adhesion peptides as a model application of this peptide-screening strategy. Cell-adhesion peptides were screened by use of a cell-based assay of a peptide array. Starting with the screening data obtained from a limited, random 5-mer library (643 sequences), a rule regarding structural characteristics of cell-adhesion peptides was extracted by fuzzy neural network (FNN) analysis. According to this rule, peptides with unfavored residues in certain positions that led to inefficient binding were eliminated from the random sequences. In the restricted, second random library (273 sequences), the yield of cell-adhesion peptides having an adhesion rate more than 1.5-fold to that of the basal array support was significantly high (31%) compared with the unrestricted random library (20%). In the restricted third library (50 sequences), the yield of cell-adhesion peptides increased to 84%. We conclude that a repeated cycle of experiments screening limited numbers of peptides can be assisted by the rule-extracting feature of FNN.

Improvement of an enzyme-linked immunosorbent assay for equine herpesvirus type 4 by using a synthetic-peptide 24-mer repeat sequence of glycoprotein G as an antigen

PubMed Central

BANNAI, Hiroshi; NEMOTO, Manabu; TSUJIMURA, Koji; YAMANAKA, Takashi; MAEDA, Ken; KONDO, Takashi

2015-01-01

To increase the sensitivity of an enzyme-linked immunosorbent assay (ELISA) for equine herpesvirus type 4 (EHV-4) that uses a 12-mer peptide of glycoprotein G (gG4-12-mer: MKNNPIYSEGSL) [4], we used a longer peptide consisting of a 24-mer repeat sequence (gG4-24-mer: MKNNPIYSEGSLMLNVQHDDSIHT) as an antigen. Sera of horses experimentally infected with EHV-4 reacted much more strongly to the gG4-24-mer peptide than to the gG4-12-mer peptide. We used peptide ELISAs to test paired sera from horses naturally infected with EHV-4 (n=40). gG4-24-mer ELISA detected 37 positive samples (92.5%), whereas gG4-12-mer ELISA detected only 28 (70.0%). gG4-24-mer ELISA was much more sensitive than gG4-12-mer ELISA. PMID:26424485

Improvement of an enzyme-linked immunosorbent assay for equine herpesvirus type 4 by using a synthetic-peptide 24-mer repeat sequence of glycoprotein G as an antigen.

PubMed

Bannai, Hiroshi; Nemoto, Manabu; Tsujimura, Koji; Yamanaka, Takashi; Maeda, Ken; Kondo, Takashi

2016-02-01

To increase the sensitivity of an enzyme-linked immunosorbent assay (ELISA) for equine herpesvirus type 4 (EHV-4) that uses a 12-mer peptide of glycoprotein G (gG4-12-mer: MKNNPIYSEGSL) [4], we used a longer peptide consisting of a 24-mer repeat sequence (gG4-24-mer: MKNNPIYSEGSLMLNVQHDDSIHT) as an antigen. Sera of horses experimentally infected with EHV-4 reacted much more strongly to the gG4-24-mer peptide than to the gG4-12-mer peptide. We used peptide ELISAs to test paired sera from horses naturally infected with EHV-4 (n=40). gG4-24-mer ELISA detected 37 positive samples (92.5%), whereas gG4-12-mer ELISA detected only 28 (70.0%). gG4-24-mer ELISA was much more sensitive than gG4-12-mer ELISA.

Characterisation and evaluation of antiviral recombinant peptides based on the heptad repeat regions of NDV and IBV fusion glycoproteins

DOE Office of Scientific and Technical Information (OSTI.GOV)

Wang Xiaojia, E-mail: wangxj@cau.edu.cn; Li Chuangen; Chi Xiaojing

Mixed virus infections can cause livestock losses that are more devastating than those caused by single virus infections. Newcastle disease virus (NDV) and infectious bronchitis virus (IBV), serious threats to the poultry industry, can give rise to complex mixed infections that hinder diagnosis and prevention. In this study, we show that newly designed peptides, which are based on the heptad repeat (HR) region of the fusion glycoproteins from NDV and IBV, have more potent antiviral activity than the mother HR peptides. Plaque formation and chicken embryo infectivity assays confirmed these results. The novel peptides completely inhibited single virus infections andmore » mixed infections caused by NDV and IBV. Furthermore, we assessed cell toxicity and possible targets for the peptides, thereby strengthening the notion that HR2 is an attractive site for therapeutic intervention. These results suggest the possibility of designing a relatively broad-spectrum class of antiviral peptides that can reduce the effects of mixed-infections.« less

How to Incorporate Total Physical Response into the English Programme.

ERIC Educational Resources Information Center

Sano, Masayuki

1986-01-01

An experimental project attempted to incorporate Total Physical Response (TPR--a method which requires learners to respond physically to imperatives given by the teacher to teach certain aspects of the language) into an English course in a Japanese junior high school. Results indicated that TPR shows potential for usefulness in second language…

Traditional, Natural, and TPR Approaches to ESL: A Study of Japanese Students.

ERIC Educational Resources Information Center

Furuhata, Hamako

1999-01-01

Reports a study of Japanese students' perception of traditional methods versus the natural approach and total physical response (TPR) methods for learning English, and their preferred styles of learning. Subjects were Japanese students attending intensive language schools in the U.S. Students generally preferred innovative methods, such as…

Nanoengineered analytical immobilized metal affinity chromatography stationary phase by atom transfer radical polymerization: Separation of synthetic prion peptides

PubMed Central

McCarthy, P.; Chattopadhyay, M.; Millhauser, G.L.; Tsarevsky, N.V.; Bombalski, L.; Matyjaszewski, K.; Shimmin, D.; Avdalovic, N.; Pohl, C.

2010-01-01

Atom transfer radical polymerization (ATRP) was employed to create isolated, metal-containing nanoparticles on the surface of non-porous polymeric beads with the goal of developing a new immobilized metal affnity chromatography (IMAC) stationary phase for separating prion peptides and proteins. Transmission electron microscopy was used to visualize nanoparticles on the substrate surface. Individual ferritin molecules were also visualized as ferritin–nanoparticle complexes. The column's resolving power was tested by synthesizing peptide analogs to the copper binding region of prion protein and injecting mixtures of these analogs onto the column. As expected, the column was capable of separating prion-related peptides differing in number of octapeptide repeat units (PHGGGWGQ), (PHGGGWGQ)2, and (PHGGGWGQ)4. Unexpectedly, the column could also resolve peptides containing the same number of repeats but differing only in the presence of a hydrophilic tail, Q → A substitution, or amide nitrogen methylation. PMID:17481564

Optimizing and Interpreting Insular Functional Connectivity Maps Obtained During Acute Experimental Pain: The Effects of Global Signal and Task Paradigm Regression.

PubMed

Ibinson, James W; Vogt, Keith M; Taylor, Kevin B; Dua, Shiv B; Becker, Christopher J; Loggia, Marco; Wasan, Ajay D

2015-12-01

The insula is uniquely located between the temporal and parietal cortices, making it anatomically well-positioned to act as an integrating center between the sensory and affective domains for the processing of painful stimulation. This can be studied through resting-state functional connectivity (fcMRI) imaging; however, the lack of a clear methodology for the analysis of fcMRI complicates the interpretation of these data during acute pain. Detected connectivity changes may reflect actual alterations in low-frequency synchronous neuronal activity related to pain, may be due to changes in global cerebral blood flow or the superimposed task-induced neuronal activity. The primary goal of this study was to investigate the effects of global signal regression (GSR) and task paradigm regression (TPR) on the changes in functional connectivity of the left (contralateral) insula in healthy subjects at rest and during acute painful electric nerve stimulation of the right hand. The use of GSR reduced the size and statistical significance of connectivity clusters and created negative correlation coefficients for some connectivity clusters. TPR with cyclic stimulation gave task versus rest connectivity differences similar to those with a constant task, suggesting that analysis which includes TPR is more accurately reflective of low-frequency neuronal activity. Both GSR and TPR have been inconsistently applied to fcMRI analysis. Based on these results, investigators need to consider the impact GSR and TPR have on connectivity during task performance when attempting to synthesize the literature.

Mechanism study on inorganic oxidants induced inhibition of Ru(bpy)₃²+ electrochemiluminescence and its application for sensitive determination of some inorganic oxidants.

PubMed

Qiu, Bin; Xue, Lingling; Wu, Yanping; Lin, Zhenyu; Guo, Longhua; Chen, Guonan

2011-07-15

Inhibited Ru(bpy)(3)(2+) electrochemiluminescence by inorganic oxidants is investigated. Results showed that a number of inorganic oxidants can quench the ECL of Ru(bpy)(3)(2+)/tri-n-propylamine (TPrA) system, and the logarithm of the decrease in ECL intensity (ΔI) was proportional to the logarithm of analyte concentrations. Based on which, a sensitive approach for detection of these inorganic oxidants was established, e.g. the log-log plots of ΔI versus the concentration of MnO(4)(-), Cr(2)O(7)(2-) and Fe(CN)(6)(3-) are linear in the range of 1×10(-7) to 3×10(-4)M for MnO(4)(-) and Cr(2)O(7)(2-), and 1×10(-7) to 1×10(-4)M for Fe(CN)(6)(3-), with the limit of detection (LOD) of 8.0×10(-8)M, 2×10(-8)M, and 1×10(-8)M, respectively. A series of experiments such as a comparison of the inhibitory effect of different compounds on Ru(bpy)(3)(2+)/TPrA ECL, ECL emission spectra, UV-Vis absorption spectra etc. were investigated in order to discover how these inorganic analytes quench the ECL of Ru(bpy)(3)(2+)/TPrA system. A mechanism based on consumption of TPrA intermediate (TPrA(·)) by inorganic oxidants was proposed. Copyright © 2011 Elsevier B.V. All rights reserved.

Structure of the TPR Domain of AIP: Lack of Client Protein Interaction with the C-Terminal α-7 Helix of the TPR Domain of AIP Is Sufficient for Pituitary Adenoma Predisposition

PubMed Central

Morgan, Rhodri M. L.; Hernández-Ramírez, Laura C.; Trivellin, Giampaolo; Zhou, Lihong; Roe, S. Mark; Korbonits, Márta; Prodromou, Chrisostomos

2012-01-01

Mutations of the aryl hydrocarbon receptor interacting protein (AIP) have been associated with familial isolated pituitary adenomas predisposing to young-onset acromegaly and gigantism. The precise tumorigenic mechanism is not well understood as AIP interacts with a large number of independent proteins as well as three chaperone systems, HSP90, HSP70 and TOMM20. We have determined the structure of the TPR domain of AIP at high resolution, which has allowed a detailed analysis of how disease-associated mutations impact on the structural integrity of the TPR domain. A subset of C-terminal α-7 helix (Cα-7h) mutations, R304* (nonsense mutation), R304Q, Q307* and R325Q, a known site for AhR and PDE4A5 client-protein interaction, occur beyond those that interact with the conserved MEEVD and EDDVE sequences of HSP90 and TOMM20. These C-terminal AIP mutations appear to only disrupt client-protein binding to the Cα-7h, while chaperone binding remains unaffected, suggesting that failure of client-protein interaction with the Cα-7h is sufficient to predispose to pituitary adenoma. We have also identified a molecular switch in the AIP TPR-domain that allows recognition of both the conserved HSP90 motif, MEEVD, and the equivalent sequence (EDDVE) of TOMM20. PMID:23300914

DOE Office of Scientific and Technical Information (OSTI.GOV)

Hinshaw, Stephen M.; Makrantoni, Vasso; Kerr, Alastair

The cohesin ring holds newly replicated sister chromatids together until their separation at anaphase. Initiation of sister chromatid cohesion depends on a separate complex, Scc2NIPBL/Scc4Mau2 (Scc2/4), which loads cohesin onto DNA and determines its localization across the genome. Proper cohesin loading is essential for cell division, and partial defects cause chromosome missegregation and aberrant transcriptional regulation, leading to severe developmental defects in multicellular organisms. We present here a crystal structure showing the interaction between Scc2 and Scc4. Scc4 is a TPR array that envelops an extended Scc2 peptide. Using budding yeast, we demonstrate that a conserved patch on the surfacemore » of Scc4 is required to recruit Scc2/4 to centromeres and to build pericentromeric cohesion. These findings reveal the role of Scc4 in determining the localization of cohesin loading and establish a molecular basis for Scc2/4 recruitment to centromeres.« less

Neopuff T-piece resuscitator mask ventilation: Does mask leak vary with different peak inspiratory pressures in a manikin model?

PubMed

Maheshwari, Rajesh; Tracy, Mark; Hinder, Murray; Wright, Audrey

2017-08-01

The aim of this study was to compare mask leak with three different peak inspiratory pressure (PIP) settings during T-piece resuscitator (TPR; Neopuff) mask ventilation on a neonatal manikin model. Participants were neonatal unit staff members. They were instructed to provide mask ventilation with a TPR with three PIP settings (20, 30, 40 cm H 2 O) chosen in a random order. Each episode was for 2 min with 2-min rest period. Flow rate and positive end-expiratory pressure (PEEP) were kept constant. Airway pressure, inspiratory and expiratory tidal volumes, mask leak, respiratory rate and inspiratory time were recorded. Repeated measures analysis of variance was used for statistical analysis. A total of 12 749 inflations delivered by 40 participants were analysed. There were no statistically significant differences (P > 0.05) in the mask leak with the three PIP settings. No statistically significant differences were seen in respiratory rate and inspiratory time with the three PIP settings. There was a significant rise in PEEP as the PIP increased. Failure to achieve the desired PIP was observed especially at the higher settings. In a neonatal manikin model, the mask leak does not vary as a function of the PIP when the flow rate is constant. With a fixed rate and inspiratory time, there seems to be a rise in PEEP with increasing PIP. © 2017 Paediatrics and Child Health Division (The Royal Australasian College of Physicians).

Swa2, the yeast homolog of mammalian auxilin, is specifically required for the propagation of the prion variant [URE 3‐1

PubMed Central

Troisi, Elizabeth M.; Rockman, Michael E.; Nguyen, Phil P.; Oliver, Emily E.

2015-01-01

Summary Yeast prions require a core set of chaperone proteins including Sis1, Hsp70 and Hsp104 to generate new amyloid templates for stable propagation, yet emerging studies indicate that propagation of some prions requires additional chaperone activities, demonstrating chaperone specificity beyond the common amyloid requirements. To comprehensively assess such prion‐specific requirements for the propagation of the [URE 3] prion variant [URE 3‐1], we screened 12 yeast cytosolic J‐proteins, and here we report a novel role for the J‐protein Swa2/Aux1. Swa2 is the sole yeast homolog of the mammalian protein auxilin, which, like Swa2, functions in vesicle‐mediated endocytosis by disassembling the structural lattice formed by the protein clathrin. We found that, in addition to Sis1, [URE 3‐1] is specifically dependent upon Swa2, but not on any of the 11 other J‐proteins. Further, we show that [URE 3‐1] propagation requires both a functional J‐domain and the tetratricopeptide repeat (TPR) domain, but surprisingly does not require Swa2‐clathrin binding. Because the J‐domain of Swa2 can be replaced with the J‐domains of other proteins, our data strongly suggest that prion‐chaperone specificity arises from the Swa2 TPR domain and supports a model where Swa2 acts through Hsp70, most likely to provide additional access points for Hsp104 to promote prion template generation. PMID:26031938

Social Studies in Motion: Learning with the Whole Person

ERIC Educational Resources Information Center

Schulte, Paige L.

2005-01-01

Total Physical Response (TPR), developed by James Asher, is defined as a teaching technique whereby a learner responds to language input with body motions. Performing a chant or the game "Robot" is an example of a TPR activity, where the teacher commands her robots to do some task in the classroom. Acting out stories and giving imperative commands…

Ten tandem repeats of {beta}-hCG 109-118 enhance immunogenicity and anti-tumor effects of {beta}-hCG C-terminal peptide carried by mycobacterial heat-shock protein HSP65

DOE Office of Scientific and Technical Information (OSTI.GOV)

Zhang Yankai; Yan Rong; He Yi

2006-07-14

The {beta}-subunit of human chorionic gonadotropin ({beta}-hCG) is secreted by many kinds of tumors and it has been used as an ideal target antigen to develop vaccines against tumors. In view of the low immunogenicity of this self-peptide,we designed a method based on isocaudamer technique to repeat tandemly the 10-residue sequence X of {beta}-hCG (109-118), then 10 tandemly repeated copies of the 10-residue sequence combined with {beta}-hCG C-terminal 37 peptides were fused to mycobacterial heat-shock protein 65 to construct a fusion protein HSP65-X10-{beta}hCGCTP37 as an immunogen. In this study, we examined the effect of the tandem repeats of this 10-residuemore » sequence in eliciting an immune by comparing the immunogenicity and anti-tumor effects of the two immunogens, HSP65-X10-{beta}hCGCTP37 and HSP65-{beta}hCGCTP37 (without the 10 tandem repeats). Immunization of mice with the fusion protein HSP65-X10-{beta}hCGCTP37 elicited much higher levels of specific anti-{beta}-hCG antibodies and more effectively inhibited the growth of Lewis lung carcinoma (LLC) in vivo than with HSP65-{beta}hCGCTP37, which should suggest that HSP65-X10-{beta}hCGCTP37 may be an effective protein vaccine for the treatment of {beta}-hCG-dependent tumors and multiple tandem repeats of a certain epitope are an efficient method to overcome the low immunogenicity of self-peptide antigens.« less

Serological profiling of the EBV immune response in Chronic Fatigue Syndrome using a peptide microarray.

PubMed

Loebel, Madlen; Eckey, Maren; Sotzny, Franziska; Hahn, Elisabeth; Bauer, Sandra; Grabowski, Patricia; Zerweck, Johannes; Holenya, Pavlo; Hanitsch, Leif G; Wittke, Kirsten; Borchmann, Peter; Rüffer, Jens-Ulrich; Hiepe, Falk; Ruprecht, Klemens; Behrends, Uta; Meindl, Carola; Volk, Hans-Dieter; Reimer, Ulf; Scheibenbogen, Carmen

2017-01-01

Epstein-Barr-Virus (EBV) plays an important role as trigger or cofactor for various autoimmune diseases. In a subset of patients with Chronic Fatigue Syndrome (CFS) disease starts with infectious mononucleosis as late primary EBV-infection, whereby altered levels of EBV-specific antibodies can be observed in another subset of patients. We performed a comprehensive mapping of the IgG response against EBV comparing 50 healthy controls with 92 CFS patients using a microarray platform. Patients with multiple sclerosis (MS), systemic lupus erythematosus (SLE) and cancer-related fatigue served as controls. 3054 overlapping peptides were synthesised as 15-mers from 14 different EBV proteins. Array data was validated by ELISA for selected peptides. Prevalence of EBV serotypes was determined by qPCR from throat washing samples. EBV type 1 infections were found in patients and controls. EBV seroarray profiles between healthy controls and CFS were less divergent than that observed for MS or SLE. We found significantly enhanced IgG responses to several EBNA-6 peptides containing a repeat sequence in CFS patients compared to controls. EBNA-6 peptide IgG responses correlated well with EBNA-6 protein responses. The EBNA-6 repeat region showed sequence homologies to various human proteins. Patients with CFS had a quite similar EBV IgG antibody response pattern as healthy controls. Enhanced IgG reactivity against an EBNA-6 repeat sequence and against EBNA-6 protein is found in CFS patients. Homologous sequences of various human proteins with this EBNA-6 repeat sequence might be potential targets for antigenic mimicry.

Prognostic value of noninvasive hemodynamic evaluation of the acute effect of levosimendan in advanced heart failure.

PubMed

Malfatto, Gabriella; Della Rosa, Francesco; Rella, Valeria; Villani, Alessandra; Branzi, Giovanna; Blengino, Simonetta; Giglio, Alessia; Facchini, Mario; Parati, Gianfranco

2014-04-01

Optimization of inotropic treatment in worsening heart failure sometimes requires invasive hemodynamic assessment in selected patients. Impedance cardiography (ICG) may be useful for a noninvasive hemodynamic evaluation. ICG was performed in 40 patients (69 ± 8 years; left ventricular ejection fraction 27.5 ± 5.6%; New York Heart Association 3.18 ± 0.34; Interagency Registry for Mechanically Assisted Circulatory Support 5.48 ± 0.96, before and after infusion of Levosimendan (0.1–0.2 µg/kg per min for up to 24 h). Echocardiogram, ICG [measuring cardiac index (CI), total peripheral resistances (TPRs) and thoracic fluid content (TFC)] and plasma levels of brain natriuretic peptide (BNP) were obtained; in nine patients, right heart catheterization was also carried out. When right catheterization and ICG were performed simultaneously, a significant relationship was observed between values of CI and TPR, and between TFC and pulmonary wedge pressure. ICG detected the Levosimendan-induced recovery of the hemodynamic status, associated with improved systolic and diastolic function and reduction in BNP levels. One-year mortality was 4.4%. At multivariate analysis, independent predictors of mortality were: no improvement in the severity of mitral regurgitation, a persistent restrictive filling pattern (E/E’ > 15), a reduction of BNP levels below 30% and a change below 10% in CI, TPR and TFC. When combined, absence of hemodynamic improvement at ICG could predict 1-year mortality with better sensitivity (86%) and specificity (85%) than the combination of echocardiographic and BNP criteria only (sensitivity 80% and specificity 36%). Noninvasive hemodynamic evaluation of heart failure patients during infusion of inodilator drugs is reliable and may help in their prognostic stratification.

Automated discrimination of dicentric and monocentric chromosomes by machine learning-based image processing.

PubMed

Li, Yanxin; Knoll, Joan H; Wilkins, Ruth C; Flegal, Farrah N; Rogan, Peter K

2016-05-01

Dose from radiation exposure can be estimated from dicentric chromosome (DC) frequencies in metaphase cells of peripheral blood lymphocytes. We automated DC detection by extracting features in Giemsa-stained metaphase chromosome images and classifying objects by machine learning (ML). DC detection involves (i) intensity thresholded segmentation of metaphase objects, (ii) chromosome separation by watershed transformation and elimination of inseparable chromosome clusters, fragments and staining debris using a morphological decision tree filter, (iii) determination of chromosome width and centreline, (iv) derivation of centromere candidates, and (v) distinction of DCs from monocentric chromosomes (MC) by ML. Centromere candidates are inferred from 14 image features input to a Support Vector Machine (SVM). Sixteen features derived from these candidates are then supplied to a Boosting classifier and a second SVM which determines whether a chromosome is either a DC or MC. The SVM was trained with 292 DCs and 3135 MCs, and then tested with cells exposed to either low (1 Gy) or high (2-4 Gy) radiation dose. Results were then compared with those of 3 experts. True positive rates (TPR) and positive predictive values (PPV) were determined for the tuning parameter, σ. At larger σ, PPV decreases and TPR increases. At high dose, for σ = 1.3, TPR = 0.52 and PPV = 0.83, while at σ = 1.6, the TPR = 0.65 and PPV = 0.72. At low dose and σ = 1.3, TPR = 0.67 and PPV = 0.26. The algorithm differentiates DCs from MCs, overlapped chromosomes and other objects with acceptable accuracy over a wide range of radiation exposures. © 2016 Wiley Periodicals, Inc.

Phosphorus release from ash and remaining tissues of two wetland species after a prescribed fire.

PubMed

Liu, G D; Gu, B; Miao, S L; Li, Y C; Migliaccio, K W; Qian, Y

2010-01-01

Dead plant tissues and ash from a prescribed fire play an important role in nutrient balance and cycling in the Florida Everglades ecosystem. The objective of this study was to assess the dynamic changes in total phosphorus release (TPr) from ash or tissues of either cattail (Typha domingensis Pers.) or sawgrass (Cladium jamaicense Crantz) to water. Natural-dead (senesced-dead) and burning-dead (standing-dead due to a prescribed fire) cattail and sawgrass were collected from highly (H) and moderately (M) impacted zones in the Florida Everglades. This experiment was conducted by incubation and water-extraction of the materials in plastic bottles for 65 d at room temperature (24 +/- 1 degrees C). Results showed that 63 to 88%, 17 to 48%, 9 to 20%, and 13 to 28% of total P (TPp) were released as TPr from cattail and sawgrass ash, cattail tissues from the H zone, cattail tissues, and sawgrass tissues from the M zone, respectively. TPp means total P of plant tissues, whereas TPr is total P release from the tissues or ash. Most of the TPr was released within 24 h after burning. The quick release of TPr observed in this experiment may help explain the P surge in the surface water immediately following a fire in the marsh. These findings suggest that prescribed burning accelerates P release from cattail and sawgrass. They also imply that it is very important to keep the water stagnant in the first 24 h to maximize the benefits of a prescribed fire in the Everglades.

Integrating Total Physical Response Strategy in a Level I Spanish Class.

ERIC Educational Resources Information Center

Wolfe, David E.; Jones, Gwendolyn

1982-01-01

Reports on an experiment in which one group of high school students was taught using Total Physical Response (TPR) for 20 minutes a day and a control group was taught by the usual manner. Results showed students in the TPR group scored better on standard unit tests and expressed greater satisfaction with their teacher and course. (EKN)

A Comparison of Foreign Language Teaching Methods: Total Physical Response versus Song/Chants with Kindergartners.

ERIC Educational Resources Information Center

Omari, Deena Rae

Several teaching methods aid young children in learning foreign languages, all of which include continuous repetition and review of learned information. The two methods used in this study were Total Physical Response (TPR) and songs/chants. The TPR method used a gesture for each vocabulary card, and the songs/chants method incorporated Spanish…

A novel TPR-BEN domain interaction mediates PICH-BEND3 association.

PubMed

Pitchai, Ganesha P; Kaulich, Manuel; Bizard, Anna H; Mesa, Pablo; Yao, Qi; Sarlos, Kata; Streicher, Werner W; Nigg, Erich A; Montoya, Guillermo; Hickson, Ian D

2017-11-02

PICH is a DNA translocase required for the maintenance of chromosome stability in human cells. Recent data indicate that PICH co-operates with topoisomerase IIα to suppress pathological chromosome missegregation through promoting the resolution of ultra-fine anaphase bridges (UFBs). Here, we identify the BEN domain-containing protein 3 (BEND3) as an interaction partner of PICH in human cells in mitosis. We have purified full length PICH and BEND3 and shown that they exhibit a functional biochemical interaction in vitro. We demonstrate that the PICH-BEND3 interaction occurs via a novel interface between a TPR domain in PICH and a BEN domain in BEND3, and have determined the crystal structure of this TPR-BEN complex at 2.2 Å resolution. Based on the structure, we identified amino acids important for the TPR-BEN domain interaction, and for the functional interaction of the full-length proteins. Our data reveal a proposed new function for BEND3 in association with PICH, and the first example of a specific protein-protein interaction mediated by a BEN domain. © The Author(s) 2017. Published by Oxford University Press on behalf of Nucleic Acids Research.

Optimisation of solid-phase microextraction coupled to HPLC-UV for the determination of organochlorine pesticides and their metabolites in environmental liquid samples.

PubMed

Torres Padrón, M E; Sosa Ferrera, Z; Santana Rodríguez, J J

2006-09-01

A solid-phase microextraction (SPME) procedure using two commercial fibers coupled with high-performance liquid chromatography (HPLC) is presented for the extraction and determination of organochlorine pesticides in water samples. We have evaluated the extraction efficiency of this kind of compound using two different fibers: 60-mum polydimethylsiloxane-divinylbenzene (PDMS-DVB) and Carbowax/TPR-100 (CW/TPR). Parameters involved in the extraction and desorption procedures (e.g. extraction time, ionic strength, extraction temperature, desorption and soaking time) were studied and optimized to achieve the maximum efficiency. Results indicate that both PDMS-DVB and CW/TPR fibers are suitable for the extraction of this type of compound, and a simple calibration curve method based on simple aqueous standards can be used. All the correlation coefficients were better than 0.9950, and the RSDs ranged from 7% to 13% for 60-mum PDMS-DVB fiber and from 3% to 10% for CW/TPR fiber. Optimized procedures were applied to the determination of a mixture of six organochlorine pesticides in environmental liquid samples (sea, sewage and ground waters), employing HPLC with UV-diode array detector.

The central repeat domain 1 of Kaposi's sarcoma-associated herpesvirus (KSHV) latency associated-nuclear antigen 1 (LANA1) prevents cis MHC class I peptide presentation

DOE Office of Scientific and Technical Information (OSTI.GOV)

Kwun, Hyun Jin; Ramos da Silva, Suzane; Department of Pathology, Botucatu School of Medicine at Sao Paulo State University, Sao Paulo

KSHV LANA1, a latent protein expressed during chronic infection to maintain a viral genome, inhibits major histocompatibility complex class I (MHC I) peptide presentation in cis as a means of immune evasion. Through deletional cloning, we localized this function to the LANA1 central repeat 1 (CR1) subregion. Other CR subregions retard LANA1 translation and proteasomal processing but do not markedly inhibit LANA1 peptide processing by MHC I. Inhibition of proteasomal processing ablates LANA1 peptide presentation. Direct expression of LANA1 within the endoplasmic reticulum (ER) overcomes CR1 inhibition suggesting that CR1 acts prior to translocation of cytoplasmic peptides into the ER.more » By physically separating CR1 from other subdomains, we show that LANA1 evades MHC I peptide processing by a mechanism distinct from other herpesviruses including Epstein-Barr virus (EBV). Although LANA1 and EBV EBNA1 are functionally similar, they appear to use different mechanisms to evade host cytotoxic T lymphocyte surveillance.« less

Distinct C9orf72-Associated Dipeptide Repeat Structures Correlate with Neuronal Toxicity

PubMed Central

Krans, Amy; Sawaya, Michael R.; Paulson, Henry L.; Todd, Peter K.; Barmada, Sami J.; Ivanova, Magdalena I.

2016-01-01

Hexanucleotide repeat expansions in C9orf72 are the most common inherited cause of amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). The expansions elicit toxicity in part through repeat-associated non-AUG (RAN) translation of the intronic (GGGGCC)n sequence into dipeptide repeat-containing proteins (DPRs). Little is known, however, about the structural characteristics and aggregation propensities of the dipeptide units comprising DPRs. To address this question, we synthesized dipeptide units corresponding to the three sense-strand RAN translation products, analyzed their structures by circular dichroism, electron microscopy and dye binding assays, and assessed their relative toxicity when applied to primary cortical neurons. Short, glycine-arginine (GR)3 dipeptides formed spherical aggregates and selectively reduced neuronal survival compared to glycine-alanine (GA)3 and glycine-proline (GP)3 dipeptides. Doubling peptide length had little effect on the structure of GR or GP peptides, but (GA)6 peptides formed β-sheet rich aggregates that bound thioflavin T and Congo red yet lacked the typical fibrillar morphology of amyloids. Aging of (GA)6 dipeptides increased their β-sheet content and enhanced their toxicity when applied to neurons. We also observed that the relative toxicity of each tested dipeptide was proportional to peptide internalization. Our results demonstrate that different C9orf72-related dipeptides exhibit distinct structural properties that correlate with their relative toxicity. PMID:27776165

Cyclic Peptidic Mimetics of Apollo Peptides Targeting Telomeric Repeat Binding Factor 2 (TRF2) and Apollo Interaction.

PubMed

Chen, Xia; Liu, Liu; Chen, Yong; Yang, Yuting; Yang, Chao-Yie; Guo, Tianyue; Lei, Ming; Sun, Haiying; Wang, Shaomeng

2018-05-10

Telomeric repeat binding factor 2 (TRF2) is a telomere-associated protein that plays an important role in the formation of the 3' single strand DNA overhang and the "T loop", two structures critical for the stability of the telomeres. Apollo is a 5'-exonuclease recruited by TRF2 to the telomere and contributes to the formation of the 3' single strand DNA overhang. Knocking down of Apollo can induce DNA damage response similar to that caused by the knocking down of TRF2. In this Letter, we report the design and synthesis of a class of cyclic peptidic mimetics of the TRFH binding motif of Apollo (Apollo TBM ). We found conformational control of the C terminal residues of Apollo TBM can effectively improve the binding affinity. We have obtained a crystal structure of a cyclic peptidic Apollo peptide mimetic ( 34 ) complexed with TRF2, which provides valuable guidance to the future design of TRF2 inhibitors.

Drosophila DH31 Neuropeptide and PDF Receptor Regulate Night-Onset Temperature Preference

PubMed Central

Goda, Tadahiro; Tang, Xin; Umezaki, Yujiro; Chu, Michelle L.

2016-01-01

Body temperature exhibits rhythmic fluctuations over a 24 h period (Refinetti and Menaker, 1992) and decreases during the night, which is associated with sleep initiation (Gilbert et al., 2004; Kräuchi, 2007a,b). However, the underlying mechanism of this temperature decrease is largely unknown. We have previously shown that Drosophila exhibit a daily temperature preference rhythm (TPR), in which their preferred temperatures increase during the daytime and then decrease at the transition from day to night (night-onset) (Kaneko et al., 2012). Because Drosophila are small ectotherms, their body temperature is very close to that of the ambient temperature (Stevenson, 1985), suggesting that their TPR generates their body temperature rhythm. Here, we demonstrate that the neuropeptide diuretic hormone 31 (DH31) and pigment-dispersing factor receptor (PDFR) contribute to regulate the preferred temperature decrease at night-onset. We show that PDFR and tethered-DH31 expression in dorsal neurons 2 (DN2s) restore the preferred temperature decrease at night-onset, suggesting that DH31 acts on PDFR in DN2s. Notably, we previously showed that the molecular clock in DN2s is important for TPR. Although PDF (another ligand of PDFR) is a critical factor for locomotor activity rhythms, Pdf mutants exhibit normal preferred temperature decreases at night-onset. This suggests that DH31-PDFR signaling specifically regulates a preferred temperature decrease at night-onset. Thus, we propose that night-onset TPR and locomotor activity rhythms are differentially controlled not only by clock neurons but also by neuropeptide signaling in the brain. SIGNIFICANCE STATEMENT Body temperature rhythm (BTR) is fundamental for the maintenance of functions essential for homeostasis, such as generating metabolic energy and sleep. One major unsolved question is how body temperature decreases dramatically during the night. Previously, we demonstrated that a BTR-like mechanism, referred to as temperature preference rhythm (TPR), exists in Drosophila. Here, we demonstrate that the diuretic hormone 31 (DH31) neuropeptide and pigment-dispersing factor receptor (PDFR) regulate preferred temperature decreases at night-onset via dorsal neurons 2. This is the first in vivo evidence that DH31 could function as a ligand of PDFR. Although both DH31 and PDF are ligands of PDFR, we show that DH31 regulates night-onset TPR, but PDF does not, suggesting that night-onset TPR and locomotor activity rhythms are controlled by different neuropeptides via different clock cells. PMID:27852781

Drosophila DH31 Neuropeptide and PDF Receptor Regulate Night-Onset Temperature Preference.

PubMed

Goda, Tadahiro; Tang, Xin; Umezaki, Yujiro; Chu, Michelle L; Kunst, Michael; Nitabach, Michael N; Hamada, Fumika N

2016-11-16

Body temperature exhibits rhythmic fluctuations over a 24 h period (Refinetti and Menaker, 1992) and decreases during the night, which is associated with sleep initiation (Gilbert et al., 2004; Kräuchi, 2007a,b). However, the underlying mechanism of this temperature decrease is largely unknown. We have previously shown that Drosophila exhibit a daily temperature preference rhythm (TPR), in which their preferred temperatures increase during the daytime and then decrease at the transition from day to night (night-onset) (Kaneko et al., 2012). Because Drosophila are small ectotherms, their body temperature is very close to that of the ambient temperature (Stevenson, 1985), suggesting that their TPR generates their body temperature rhythm. Here, we demonstrate that the neuropeptide diuretic hormone 31 (DH31) and pigment-dispersing factor receptor (PDFR) contribute to regulate the preferred temperature decrease at night-onset. We show that PDFR and tethered-DH31 expression in dorsal neurons 2 (DN2s) restore the preferred temperature decrease at night-onset, suggesting that DH31 acts on PDFR in DN2s. Notably, we previously showed that the molecular clock in DN2s is important for TPR. Although PDF (another ligand of PDFR) is a critical factor for locomotor activity rhythms, Pdf mutants exhibit normal preferred temperature decreases at night-onset. This suggests that DH31-PDFR signaling specifically regulates a preferred temperature decrease at night-onset. Thus, we propose that night-onset TPR and locomotor activity rhythms are differentially controlled not only by clock neurons but also by neuropeptide signaling in the brain. Body temperature rhythm (BTR) is fundamental for the maintenance of functions essential for homeostasis, such as generating metabolic energy and sleep. One major unsolved question is how body temperature decreases dramatically during the night. Previously, we demonstrated that a BTR-like mechanism, referred to as temperature preference rhythm (TPR), exists in Drosophila Here, we demonstrate that the diuretic hormone 31 (DH31) neuropeptide and pigment-dispersing factor receptor (PDFR) regulate preferred temperature decreases at night-onset via dorsal neurons 2. This is the first in vivo evidence that DH31 could function as a ligand of PDFR. Although both DH31 and PDF are ligands of PDFR, we show that DH31 regulates night-onset TPR, but PDF does not, suggesting that night-onset TPR and locomotor activity rhythms are controlled by different neuropeptides via different clock cells. Copyright © 2016 the authors 0270-6474/16/3611739-16$15.00/0.

Effects of calcium feeding strategy on true ileal phosphorus digestibility and true phosphorus retention determined with growing broilers.

PubMed

Perryman, K R; Masey O'Neill, H V; Bedford, M R; Dozier, W A

2016-05-01

An experiment utilizing 960 Ross × Ross 708 male broilers was conducted to determine the effects of Ca feeding strategy on true ileal (prececal) P digestibility (TIPD) and true P retention (TPR) of corn. Experimental diets were formulated with 1 of 3 dietary Ca feeding strategies (0.95%, 0.13%, or variable Ca concentrations to maintain a 2.1:1 Ca:P ratio) and contain 0, 25, 50, or 75% corn. A practical corn-soybean meal diet (1.4:1 Ca:P ratio) was fed as a control. After receiving a common starter diet, experimental diets were fed from 19 to 26 d of age. After a 48-h dietary adaptation period, a 48-h retention assay was conducted. At 25 and 26 d of age, ileal digesta were collected from 8 birds per cage. Broilers consuming the control diet had higher (P<0.001) BW gain, feed intake, digesta P, and excreta P than broilers consuming the corn titration diets. Digesta and excreta P increased (linear, P<0.05) with graded increases of corn. True ileal P digestibility and TPR were highest (P<0.05) for diets with 0.13% Ca (57.3 and 69.5%, respectively) compared with diets formulated with a 2.1:1 Ca:P ratio (41.2 and 37.8%, respectively) or 0.95% Ca (25.4 and 39.0%, respectively). Values for TPR were higher (P<0.05) than those for TIPD except when the dietary Ca:P ratio was fixed. Additionally, negative endogenous P losses were predicted by regression equations when TPR was estimated for birds fed titration diets with the fixed Ca:P ratio. Changing the Ca concentration of the diets to maintain a fixed Ca:P ratio influenced (P<0.001) apparent P retention, which affected the estimate for TPR due to the prediction of negative endogenous P losses. These data demonstrated that regression analysis may have limitations when estimating the TIPD or TPR of corn when formulating diets with different Ca feeding strategies. More research is necessary to elucidate the factors that contributed to regression equations predicting negative endogenous P losses. © 2016 Poultry Science Association Inc.

Adherence to recommendations of the Therapeutic Positioning Report about treatment with oral anticoagulants in elderly patients with atrial fibrillation. The ESPARTA study.

PubMed

Suárez Fernández, Carmen; Mostaza, Jose María; Castilla Guerra, Luis; Cantero Hinojosa, Jesus; Suriñach, Josep Maria; Acosta de Bilbao, Fernando; Tamarit, Juan José; Diaz Diaz, José Luis; Hernandez, Jose Luis; Cazorla, Daniel; Ràfols, Carles

2017-10-06

To evaluate the adherence to the recommendations in clinical practice performed by the Therapeutic Positioning Report (TPR) of the Spanish Agency of Medicines and Sanitary Products about the treatment with oral anticoagulants in patients aged≥75 years old with nonvalvular atrial fibrillation (NVAF) treated in Internal Medicine departments in Spain. Observational, cross-sectional and multicenter study in which 837 patients aged≥75 years old with NVAF, with stable treatment with oral anticoagulants at least 3 months before inclusion, and that had started treatment with oral anticoagulants before the inclusion period were included. Mean age was 83.0±5.0 years old, mean CHADS 2 score 3.2±1.2, mean CHA 2 DS 2 -VASc score 5.0±1.4, and mean HAS-BLED score 2.1±0.9. A percentage of 70.8 of patients were treated with vitamin K antagonists (VKA) and the rest of patients with direct oral anticoagulants (DOACs). A percentage of 65.6 of patients treated with VKA did not follow the recommendations made by the TPR compared with 43.0% of patients treated with DOACs (P<.0001). In the case of VKA, the main reason for being considered as not appropriate according to the TPR was having poor control of anticoagulation and not switching to DOACs, whereas in the case of DOACs, it was not receiving the adequate dose according to the TPR. In a high proportion of anticoagulated elderly patients with NVAF in Spain, the recommendations performed by the TPR are not followed, particularly with VKA, since patients are not switched to DOACs despite time in therapeutic range. Copyright © 2017 Elsevier España, S.L.U. All rights reserved.

A Critical Role for the Transient Receptor Potential Channel Type 6 in Human Platelet Activation

PubMed Central

Conlon, Christine; Khasawneh, Fadi T.

2015-01-01

While calcium signaling is known to play vital roles in platelet function, the mechanisms underlying its receptor-operated calcium entry component (ROCE) remain poorly understood. It has been proposed, but never proven in platelets, that the canonical transient receptor potential channel-6 (TRPC6) mediates ROCE. Nonetheless, we have previously shown that the mouse TRPC6 regulates hemostasis, thrombogenesis by regulating platelet aggregation. In the present studies, we used a pharmacological approach to characterize the role of TRPC6 in human platelet biology. Thus, interestingly, we observed that a TRPC6 inhibitor exerted significant inhibitory effects on human platelet aggregation in a thromboxane receptor (TPR)-selective manner; no additional inhibition was observed in the presence of the calcium chelator BAPTA. This inhibitor also significantly inhibited human platelet secretion (dense and alpha granules), integrin IIb-IIIa, Akt and ERK phosphorylation, again, in a TPR-selective manner; no effects were observed in response to ADP receptor stimulation. Furthermore, there was a causal relationship between these inhibitory effects, and the capacity of the TRPC6 inhibitor to abrogate elevation in intracellular calcium, that was again found to be TPR-specific. This effect was not found to be due to antagonism of TPR, as the TRPC6 inhibitor did not displace the radiolabeled antagonist [3H]SQ29,548 from its binding sites. Finally, our studies also revealed that TRPC6 regulates human clot retraction, as well as physiological hemostasis and thrombus formation, in mice. Taken together, our findings demonstrate, for the first time, that TRPC6 directly regulates TPR-dependent ROCE and platelet function. Moreover, these data highlight TRPC6 as a novel promising therapeutic strategy for managing thrombotic disorders. PMID:25928636

Molecular dynamics simulations of site point mutations in the TPR domain of cyclophilin 40 identify conformational states with distinct dynamic and enzymatic properties

NASA Astrophysics Data System (ADS)

Gur, Mert; Blackburn, Elizabeth A.; Ning, Jia; Narayan, Vikram; Ball, Kathryn L.; Walkinshaw, Malcolm D.; Erman, Burak

2018-04-01

Cyclophilin 40 (Cyp40) is a member of the immunophilin family that acts as a peptidyl-prolyl-isomerase enzyme and binds to the heat shock protein 90 (Hsp90). Its structure comprises an N-terminal cyclophilin domain and a C-terminal tetratricopeptide (TPR) domain. Cyp40 is overexpressed in prostate cancer and certain T-cell lymphomas. The groove for Hsp90 binding on the TPR domain includes residues Lys227 and Lys308, referred to as the carboxylate clamp, and is essential for Cyp40-Hsp90 binding. In this study, the effect of two mutations, K227A and K308A, and their combinative mutant was investigated by performing a total of 5.76 μs of all-atom molecular dynamics (MD) simulations in explicit solvent. All simulations, except the K308A mutant, were found to adopt two distinct (extended or compact) conformers defined by different cyclophilin-TPR interdomain distances. The K308A mutant was only observed in the extended form which is observed in the Cyp40 X-ray structure. The wild-type, K227A, and combined mutant also showed bimodal distributions. The experimental melting temperature, Tm, values of the mutants correlate with the degree of compactness with the K308A extended mutant having a marginally lower melting temperature. Another novel measure of compactness determined from the MD data, the "coordination shell volume," also shows a direct correlation with Tm. In addition, the MD simulations show an allosteric effect with the mutations in the remote TPR domain having a pronounced effect on the molecular motions of the enzymatic cyclophilin domain which helps rationalise the experimentally observed increase in enzyme activity measured for all three mutations.

The production and characterization of novel heavy-chain antibodies against the tandem repeat region of MUC1 mucin.

PubMed

Rahbarizadeh, Fatemeh; Rasaee, Mohammad J; Forouzandeh, Mehdi; Allameh, Abdolamir; Sarrami, Ramin; Nasiry, Habib; Sadeghizadeh, Majid

2005-01-01

Camelidae are known to produce immunoglobulins (Igs) devoid of light chains and constant heavy-chain domains (CH1). Antigen-specific fragments of these heavy-chain IgGs (VHH) are of great interest in biotechnology applications. This paper describes the first example of successfully raised heavy-chain antibodies in Camelus dromedarius (single-humped camel) and Camelus bactrianus (two-humped camel) against a MUC1 related peptide that is found to be an important epitope expressed in cancerous tissue. Camels were immunized against a synthetic peptide corresponding to the tandem repeat region of MUC1 mucin and cancerous tissue preparation obtained from patients suffering from breast carcinoma. Three IgG subclasses with different binding properties to protein A and G were purified by affinity chromatography. Both conventional and heavy-chain IgG antibodies were produced in response to MUC1-related peptide. The elicited antibodies could react specifically with the tandem repeat region of MUC1 mucin in an enzyme linked immunosorbant assay (ELISA). Anti-peptide antibodies were purified after passing antiserum over two affinity chromatography columns. Using ELISA, immunocytochemistry and Western blotting, the interaction of purified antibodies with different antigens was evaluated. The antibodies were observed to be selectively bound to antigens namely: MUC1 peptide (tandem repeat region), human milk fat globule membrane (HMFG), deglycosylated human milk fat globule membrane (D-HMFG), homogenized cancerous breast tissue and a native MUC1 purified from ascitic fluid. Ka values of specific polyclonal antipeptide antibodies were estimated in C. dromedarius and C. bactrianus, as 7 x 10(10) M(-1) and 1.4 x 10(10) M(-1) respectively.

Non-equilibrium plasma reactors for organic solvent destruction

DOE Office of Scientific and Technical Information (OSTI.GOV)

Yang, C.L.; Beltran, M.R.; Kravets, Z.

1997-12-31

Two non-equilibrium plasma reactors were evaluated for their ability to destroy three widely used organic solvents, i.e., 2-butanone, toluene and ethyl acetate. The catalyzed plasma reactor (CPR) with 6 mm glass beads destroys 98% of 50 ppm toluene in air at 24 kV/cm and space velocities of 1,400 v/v/hr. Eighty-five percent of ethyl acetate and 2-butanone are destroyed under the same conditions. The tubular plasma reactor (TPR) has an efficiency of 10% to 20% lower than that of a CPR under the same conditions. The 1,400 v/v/hr in a CPR is equal to a residence time of 2.6 seconds inmore » a TPR. The operating temperatures, corona characteristics, as well as the kinetics of VOC destruction in both TPR and CPR were studied.« less

Preliminary X-ray crystallographic studies of mouse UPR responsive protein P58(IPK) TPR fragment

DOE Office of Scientific and Technical Information (OSTI.GOV)

Tao, Jiahui; Wu, Yunkun; Ron, David

2008-02-01

To investigate the mechanism by which P58(IPK) functions to promote protein folding within the ER, a P58(IPK) TPR fragment without the C-terminal J-domain has been crystallized. Endoplasmic reticulum (ER) stress induces the unfolded protein response (UPR), which can promote protein folding and misfolded protein degradation and attenuate protein translation and protein translocation into the ER. P58(IPK) has been proposed to function as a molecular chaperone to maintain protein-folding homeostasis in the ER under normal and stressed conditions. P58(IPK) contains nine TPR motifs and a C-terminal J-domain within its primary sequence. To investigate the mechanism by which P58(IPK) functions to promotemore » protein folding within the ER, a P58(IPK) TPR fragment without the C-terminal J-domain was crystallized. The crystals diffract to 2.5 Å resolution using a synchrotron X-ray source. The crystals belong to space group P2{sub 1}, with unit-cell parameters a = 83.53, b = 92.75, c = 84.32 Å, α = 90.00, β = 119.36, γ = 90.00°. There are two P58(IPK) molecules in the asymmetric unit, which corresponds to a solvent content of approximately 60%. Structure determination by MAD methods is under way.« less

Secretion of CyaA-PrtB and HlyA-PrtB fusion proteins in Escherichia coli: involvement of the glycine-rich repeat domain of Erwinia chrysanthemi protease B.

PubMed Central

Létoffé, S; Wandersman, C

1992-01-01

Protease B from Erwinia chrysanthemi was shown previously to have a C-terminal secretion signal located downstream of a domain that contains six glycine-rich repeats. This domain is conserved in all known bacterial proteins secreted by the signal peptide-independent pathway. The role of these repeats in the secretion process is controversial. We compared the secretion processes of various heterologous polypeptides fused either directly to the signal or separated from it by the glycine-rich domain. Although the repeats are not involved in the secretion of small truncated protease B carboxy-terminal peptides, they are required for the secretion of higher-molecular-weight fusion proteins. Secretion efficiency was also dependent on the size of the passenger polypeptide. Images PMID:1629152

Functional analysis of recombinant human and Yarrowia lipolytica O-GlcNAc transferases expressed in Saccharomyces cerevisiae.

PubMed

Oh, Hye Ji; Moon, Hye Yun; Cheon, Seon Ah; Hahn, Yoonsoo; Kang, Hyun Ah

2016-10-01

O-linked β-N-acetylglucosamine (O-GlcNAc) glycosylation is an important post-translational modification in many cellular processes. It is mediated by O-GlcNAc transferases (OGTs), which catalyze the addition of O-GlcNAc to serine or threonine residues of the target proteins. In this study, we expressed a putative Yarrowia lipolytica OGT (YlOGT), the only homolog identified in the subphylum Saccharomycotina through bioinformatics analysis, and the human OGT (hOGT) as recombinant proteins in Saccharomyces cerevisiae, and performed their functional characterization. Immunoblotting assays using antibody against O-GlcNAc revealed that recombinant hOGT (rhOGT), but not the recombinant YlOGT (rYlOGT), undergoes auto-O-GlcNAcylation in the heterologous host S. cerevisiae. Moreover, the rhOGT expressed in S. cerevisiae showed a catalytic activity during in vitro assays using casein kinase II substrates, whereas no such activity was obtained in rYlOGT. However, the chimeric human-Y. lipolytica OGT, carrying the human tetratricopeptide repeat (TPR) domain along with the Y. lipolytica catalytic domain (CTD), mediated the transfer of O-GlcNAc moiety during the in vitro assays. Although the overexpression of full-length OGTs inhibited the growth of S. cerevisiae, no such inhibition was obtained upon overexpression of only the CTD fragment, indicating the role of TPR domain in growth inhibition. This is the first report on the functional analysis of the fungal OGT, indicating that the Y. lipolytica OGT retains its catalytic activity, although the physiological role and substrates of YlOGT remain to be elucidated.

Ensemble characterization of an intrinsically disordered FG-Nup peptide and its F>A mutant in DMSO-d6.

PubMed

Reid, Korey M; Sunanda, Punnepalli; Raghothama, S; Krishnan, V V

2017-11-01

Intrinsically disordered proteins (IDP) lack a well-defined 3D-structure under physiological conditions, yet, the inherent disorder represented by an ensemble of conformation plays a critical role in many cellular and regulatory processes. Nucleoporins, or Nups, are the proteins found in the nuclear pore complex (NPC). The central pore of the NPC is occupied by Nups, which have phenylalanine-glycine domain repeats and are intrinsically disordered, and therefore are termed FG-Nups. These FG-domain repeats exhibit differing cohesiveness character and differ from least (FG) to most (GLFG) cohesive. The designed FG-Nup is a 25 AA model peptide containing a noncohesive FG-motif flanked by two cohesive GLFG-motifs (WT peptide). Complete NMR-based ensemble characterization of this peptide along with a control peptide with an F>A substitution (MU peptide) are discussed. Ensemble characterization of the NMR-determined models suggests that both the peptides do not have consistent secondary structures and continue to be disordered. Nonetheless, the role of cohesive elements mediated by the GLFG motifs is evident in the WT ensemble of structures that are more compact than the MU peptide. The approach presented here allows an alternate way to investigate the specific roles of distinct amino acid motifs that translate into the long-range organization of the ensemble of structures and in general on the nature of IDPs. © 2017 Wiley Periodicals, Inc.

The expanded amelogenin polyproline region preferentially binds to apatite versus carbonate and promotes apatite crystal elongation

PubMed Central

Gopinathan, Gokul; Jin, Tianquan; Liu, Min; Li, Steve; Atsawasuwan, Phimon; Galang, Maria-Therese; Allen, Michael; Luan, Xianghong; Diekwisch, Thomas G. H.

2014-01-01

The transition from invertebrate calcium carbonate-based calcite and aragonite exo- and endoskeletons to the calcium phosphate-based vertebrate backbones and jaws composed of microscopic hydroxyapatite crystals is one of the great revolutions in the evolution of terrestrial organisms. To identify potential factors that might have played a role in such a transition, three key domains of the vertebrate tooth enamel protein amelogenin were probed for calcium mineral/protein interactions and their ability to promote calcium phosphate and calcium carbonate crystal growth. Under calcium phosphate crystal growth conditions, only the carboxy-terminus augmented polyproline repeat peptide, but not the N-terminal peptide nor the polyproline repeat peptide alone, promoted the formation of thin and parallel crystallites resembling those of bone and initial enamel. In contrast, under calcium carbonate crystal growth conditions, all three amelogenin-derived polypeptides caused calcium carbonate to form fused crystalline conglomerates. When examined for long-term crystal growth, polyproline repeat peptides of increasing length promoted the growth of shorter calcium carbonate crystals with broader basis, contrary to the positive correlation between polyproline repeat element length and apatite mineralization published earlier. To determine whether the positive correlation between polyproline repeat element length and apatite crystal growth versus the inverse correlation between polyproline repeat length and calcium carbonate crystal growth were related to the binding affinity of the polyproline domain to either apatite or carbonate, a parallel series of calcium carbonate and calcium phosphate/apatite protein binding studies was conducted. These studies demonstrated a remarkable binding affinity between the augmented amelogenin polyproline repeat region and calcium phosphates, and almost no binding to calcium carbonates. In contrast, the amelogenin N-terminus bound to both carbonate and apatite, but preferentially to calcium carbonate. Together, these studies highlight the specific binding affinity of the augmented amelogenin polyproline repeat region to calcium phosphates versus calcium carbonate, and its unique role in the growth of thin apatite crystals as they occur in vertebrate biominerals. Our data suggest that the rise of apatite-based biominerals in vertebrates might have been facilitated by a rapid evolution of specialized polyproline repeat proteins flanked by a charged domain, resulting in apatite crystals with reduced width, increased length, and tailored biomechanical properties. PMID:25426079

Fabrication of artificial toroid nanostructures by modified β-sheet peptides.

PubMed

Li, Wen; Li, Jingfang; Lee, Myongsoo

2013-09-25

Facial peptide P1 carrying repeating hydrophobic and hydrophilic residues as well as lysine terminals self-assemble into uniform toroid structures. The sensitive balance between the hydrophobic interactions and electrostatic repulsion dominates the formation of highly curved assemblies.

Structural and functional analysis of RopB: A major virulence regulator in Streptococcus pyogenes

DOE PAGES

Makthal, Nishanth; Gavagan, Maire; Do, Hackwon; ...

2016-02-19

Group A Streptococcus (GAS) is an exclusive human pathogen that causes significant disease burden. Global regulator RopB of GAS controls the expression of several major virulence factors including secreted protease SpeB during high cell density. However, the molecular mechanism for RopB-dependent speB expression remains unclear. To understand the mechanism of transcription activation by RopB, we determined the crystal structure of the C-terminal domain of RopB. RopB-CTD has the TPR motif, a signature motif involved in protein-peptide interactions and shares significant structural homology with the quorum sensing RRNPP family regulators. Characterization of the high cell density-specific cell-free growth medium demonstrated themore » presence of a low molecular weight proteinaceous secreted factor that upregulates RopB-dependent speB expression. Together, these results suggest that RopB and its cognate peptide signals constitute an intercellular signalling machinery that controls the virulence gene expression in concert with population density. Structure-guided mutational analyses of RopB dimer interface demonstrated that single alanine substitutions at this critical interface significantly altered RopB-dependent speB expression and attenuated GAS virulence. Finally, results presented here suggested that a properly aligned RopB dimer interface is important for GAS pathogenesis and highlighted the dimerization interactions as a plausible therapeutic target for the development of novel antimicrobials.« less

Structural and functional analysis of RopB: A major virulence regulator in Streptococcus pyogenes

DOE Office of Scientific and Technical Information (OSTI.GOV)

Makthal, Nishanth; Gavagan, Maire; Do, Hackwon

Group A Streptococcus (GAS) is an exclusive human pathogen that causes significant disease burden. Global regulator RopB of GAS controls the expression of several major virulence factors including secreted protease SpeB during high cell density. However, the molecular mechanism for RopB-dependent speB expression remains unclear. To understand the mechanism of transcription activation by RopB, we determined the crystal structure of the C-terminal domain of RopB. RopB-CTD has the TPR motif, a signature motif involved in protein-peptide interactions and shares significant structural homology with the quorum sensing RRNPP family regulators. Characterization of the high cell density-specific cell-free growth medium demonstrated themore » presence of a low molecular weight proteinaceous secreted factor that upregulates RopB-dependent speB expression. Together, these results suggest that RopB and its cognate peptide signals constitute an intercellular signalling machinery that controls the virulence gene expression in concert with population density. Structure-guided mutational analyses of RopB dimer interface demonstrated that single alanine substitutions at this critical interface significantly altered RopB-dependent speB expression and attenuated GAS virulence. Finally, results presented here suggested that a properly aligned RopB dimer interface is important for GAS pathogenesis and highlighted the dimerization interactions as a plausible therapeutic target for the development of novel antimicrobials.« less

Influence of a heptad repeat stutter on the pH-dependent conformational behavior of the central coiled-coil from influenza hemagglutinin HA2.

PubMed

Higgins, Chelsea D; Malashkevich, Vladimir N; Almo, Steven C; Lai, Jonathan R

2014-09-01

The coiled-coil is one of the most common protein structural motifs. Amino acid sequences of regions that participate in coiled-coils contain a heptad repeat in which every third then forth residue is occupied by a hydrophobic residue. Here we examine the consequences of a "stutter," a deviation of the idealized heptad repeat that is found in the central coiled-coil of influenza hemagluttinin HA2. Characterization of a peptide containing the native stutter-containing HA2 sequence, as well as several variants in which the stutter was engineered out to restore an idealized heptad repeat pattern, revealed that the stutter is important for allowing coiled-coil formation in the WT HA2 at both neutral and low pH (7.1 and 4.5). By contrast, all variants that contained idealized heptad repeats exhibited marked pH-dependent coiled-coil formation with structures forming much more stably at low pH. A crystal structure of one variant containing an idealized heptad repeat, and comparison to the WT HA2 structure, suggest that the stutter distorts the optimal interhelical core packing arrangement, resulting in unwinding of the coiled-coil superhelix. Interactions between acidic side chains, in particular E69 and E74 (present in all peptides studied), are suggested to play a role in mediating these pH-dependent conformational effects. This conclusion is partially supported by studies on HA2 variant peptides in which these positions were altered to aspartic acid. These results provide new insight into the structural role of the heptad repeat stutter in HA2. © 2014 Wiley Periodicals, Inc.

Human lactoferricin derived di-peptides deploying loop structures induce apoptosis specifically in cancer cells through targeting membranous phosphatidylserine.

PubMed

Riedl, Sabrina; Leber, Regina; Rinner, Beate; Schaider, Helmut; Lohner, Karl; Zweytick, Dagmar

2015-11-01

Host defense-derived peptides have emerged as a novel strategy for the development of alternative anticancer therapies. In this study we report on characteristic features of human lactoferricin (hLFcin) derivatives which facilitate specific killing of cancer cells of melanoma, glioblastoma and rhabdomyosarcoma compared with non-specific derivatives and the synthetic peptide RW-AH. Changes in amino acid sequence of hLFcin providing 9-11 amino acids stretched derivatives LF11-316, -318 and -322 only yielded low antitumor activity. However, the addition of the repeat (di-peptide) and the retro-repeat (di-retro-peptide) sequences highly improved cancer cell toxicity up to 100% at 20 μM peptide concentration. Compared to the complete parent sequence hLFcin the derivatives showed toxicity on the melanoma cell line A375 increased by 10-fold and on the glioblastoma cell line U-87mg by 2-3-fold. Reduced killing velocity, apoptotic blebbing, activation of caspase 3/7 and formation of apoptotic DNA fragments proved that the active and cancer selective peptides, e.g. R-DIM-P-LF11-322, trigger apoptosis, whereas highly active, though non-selective peptides, such as DIM-LF11-318 and RW-AH seem to kill rapidly via necrosis inducing membrane lyses. Structural studies revealed specific toxicity on cancer cells by peptide derivatives with loop structures, whereas non-specific peptides comprised α-helical structures without loop. Model studies with the cancer membrane mimic phosphatidylserine (PS) gave strong evidence that PS only exposed by cancer cells is an important target for specific hLFcin derivatives. Other negatively charged membrane exposed molecules as sialic acid, heparan and chondroitin sulfate were shown to have minor impact on peptide activity. Copyright © 2015. Published by Elsevier B.V.

Amphipathic Polyproline Peptides Stimulate Cholesterol Efflux by the ABCA1 Transporter

PubMed Central

Sviridov, D.O.; Drake, S.K.; Freeman, L.A.; Remaley, A.T.

2016-01-01

ApoA-I mimetics are short synthetic peptides that contain an amphipathic αα-helix and stimulate cholesterol efflux by the ABCA1 transporter in a detergent-like extraction mechanism. We investigated the use of amphipathic peptides with a polypro helix for stimulating cholesterol efflux by ABCA1. Polypro peptides were synthesized with modified prolines, containing either a hydrophobic phenol group (Prop) or a polar N-acetylgalactosamine (Prog) attached to the pyrrolidine ring and were designated as either PP-2, 3, 4, or 5, depending on the number of 3 amino acid repeat units (Prop - Prog - Prop). Based on molecular modeling, these peptides were predicted to be relatively rigid and to bind to a phospholipid bilayer. By CD spectroscopy, PP peptides formed a Type-II polypro helix in an aqueous solution. PP-2 was inactive in promoting cholesterol efflux, but peptides with more than 2 repeat units were active. PP-4 showed a similar Vmax as a much longer amphipathic α-αhelical peptide, containing 37 amino acids, but had a Km that was approximately 20-fold lower. PP peptides were specific in that they did not stimulate cholesterol efflux from cells not expressing ABCA1 and were also non-cytotoxic. Addition of PP-3, 4 and 5 to serum promoted the formation of smaller size HDL species (7 nM) and increased its capacity for ABCA1-dependent cholesterol efflux by approximately 20-35% (p<0.05). Because of their relatively small size and increased potency, amphipathic peptides with a polypro helix may represent an alternative structural motif for the development of apoA-I mimetic peptides. PMID:26879139

Combining tissue-phantom ratios to provide a beam-quality specifier for flattening filter free photon beams

DOE Office of Scientific and Technical Information (OSTI.GOV)

Dalaryd, Mårten, E-mail: Marten.Dalaryd@med.lu.se; Knöös, Tommy; Ceberg, Crister

Purpose: There are currently several commercially available radiotherapy treatment units without a flattening filter in the beam line. Unflattened photon beams have an energy and lateral fluence distribution that is different from conventional beams and, thus, their attenuation properties differ. As a consequence, for flattening filter free (FFF) beams, the relationship between the beam-quality specifier TPR{sub 20,10} and the Spencer–Attix restricted water-to-air mass collision stopping-power ratios, (L{sup -}/ρ){sub air}{sup water}, may have to be refined in order to be used with equivalent accuracy as for beams with a flattening filter. The purpose of this work was twofold. First, to studymore » the relationship between TPR{sub 20,10} and (L{sup -}/ρ){sub air}{sup water} for FFF beams, where the flattening filter has been replaced by a metal plate as in most clinical FFF beams. Second, to investigate the potential of increasing the accuracy in determining (L{sup -}/ρ){sub air}{sup water} by adding another beam-quality metric, TPR{sub 10,5}. The relationship between (L{sup -}/ρ){sub air}{sup water} and %dd(10){sub x} for beams with and without a flattening filter was also included in this study. Methods: A total of 24 realistic photon beams (10 with and 14 without a flattening filter) from three different treatment units have been used to calculate (L{sup -}/ρ){sub air}{sup water}, TPR{sub 20,10}, and TPR{sub 10,5} using the EGSnrc Monte Carlo package. The relationship between (L{sup -}/ρ){sub air}{sup water} and the dual beam-quality specifier TPR{sub 20,10} and TPR{sub 10,5} was described by a simple bilinear equation. The relationship between the photon beam-quality specifier %dd(10){sub x} used in the AAPM’s TG-51 dosimetry protocol and (L{sup -}/ρ){sub air}{sup water} was also investigated for the beams used in this study, by calculating the photon component of the percentage depth dose at 10 cm depth with SSD 100 cm. Results: The calculated (L{sup -}/ρ){sub air}{sup water} for beams without a flattening filter was 0.3% lower, on average, than for beams with a flattening filter and comparable TPR{sub 20,10}. Using the relationship in IAEA, TRS-398 resulted in a root mean square deviation (RMSD) of 0.0028 with a maximum deviation of 0.0043 (0.39%) from Monte Carlo calculated values. For all beams in this study, the RMSD between the proposed model and the Monte Carlo calculated values was 0.0006 with a maximum deviation of 0.0013 (0.1%). Using an earlier proposed relationship [Xiong and Rogers, Med. Phys. 35, 2104–2109 (2008)] between %dd(10){sub x} and (L{sup -}/ρ){sub air}{sup water} gave a RMSD of 0.0018 with a maximum deviation of 0.0029 (0.26%) for all beams in this study (compared to RMSD 0.0015 and a maximum deviation of 0.0048 (0.47%) for the relationship used in AAPM TG-51 published by Almond et al. [Med. Phys. 26, 1847–1870 (1999)]). Conclusions: Using TPR{sub 20,10} as a beam-quality specifier, for the flattening filter free beams used in this study, gave a maximum difference of 0.39% between (L{sup -}/ρ){sub air}{sup water} predicted using IAEA TRS-398 and Monte Carlo calculations. An additional parameter for determining (L{sup -}/ρ){sub air}{sup water} has been presented. This parameter is easy to measure; it requires only an additional dose measurement at 5 cm depth with SSD 95 cm, and provides information for accurate determination of the (L{sup -}/ρ){sub air}{sup water} ratio for beams both with and without a flattening filter at the investigated energies.« less

Phenylephrine-induced elevations in arterial blood pressure are attenuated in heat-stressed humans

NASA Technical Reports Server (NTRS)

Cui, Jian; Wilson, Thad E.; Crandall, Craig G.

2002-01-01

To test the hypothesis that phenylephrine-induced elevations in blood pressure are attenuated in heat-stressed humans, blood pressure was elevated via steady-state infusion of three doses of phenylephrine HCl in 10 healthy subjects in both normothermic and heat stress conditions. Whole body heating significantly increased sublingual temperature by 0.5 degrees C, muscle sympathetic nerve activity (MSNA), heart rate, and cardiac output and decreased total peripheral vascular resistance (TPR; all P < 0.005) but did not change mean arterial blood pressure (MAP; P > 0.05). At the highest dose of phenylephrine, the increase in MAP and TPR from predrug baselines was significantly attenuated during the heat stress [DeltaMAP 8.4 +/- 1.2 mmHg; DeltaTPR 0.96 +/- 0.85 peripheral resistance units (PRU)] compared with normothermia (DeltaMAP 15.4 +/- 1.4 mmHg, DeltaTPR 7.13 +/- 1.18 PRU; all P < 0.001). The sensitivity of baroreflex control of MSNA and heart rate, expressed as the slope of the relationship between MSNA and diastolic blood pressure, as well as the slope of the relationship between heart rate and systolic blood pressure, respectively, was similar between thermal conditions (each P > 0.05). These data suggest that phenylephrine-induced elevations in MAP are attenuated in heat-stressed humans without affecting baroreflex control of MSNA or heart rate.

A New Transgenic Mouse Model of Heart Failure and Cardiac Cachexia Raised by Sustained Activation of Met Tyrosine Kinase in the Heart.

PubMed

Sala, Valentina; Gatti, Stefano; Gallo, Simona; Medico, Enzo; Cantarella, Daniela; Cimino, James; Ponzetto, Antonio; Crepaldi, Tiziana

2016-01-01

Among other diseases characterized by the onset of cachexia, congestive heart failure takes a place of relevance, considering the high prevalence of this pathology in most European countries and in the United States, and is undergoing a rapid increase in developing countries. Actually, only few models of cardiac cachexia exist. Difficulties in the recruitment and follow-up of clinical trials implicate that new reproducible and well-characterized animal models are pivotal in developing therapeutic strategies for cachexia. We generated a new model of cardiac cachexia: a transgenic mouse expressing Tpr-Met receptor, the activated form of c-Met receptor of hepatocyte growth factor, specifically in the heart. We showed that the cardiac-specific induction of Tpr-Met raises a cardiac hypertrophic remodelling, which progresses into concentric hypertrophy with concomitant increase in Gdf15 mRNA levels. Hypertrophy progresses to congestive heart failure with preserved ejection fraction, characterized by reduced body weight gain and food intake and skeletal muscle wasting. Prevention trial by suppressing Tpr-Met showed that loss of body weight could be prevented. Skeletal muscle wasting was also associated with altered gene expression profiling. We propose transgenic Tpr-Met mice as a new model of cardiac cachexia, which will constitute a powerful tool to understand such complex pathology and test new drugs/approaches at the preclinical level.

MCMEG: Simulations of both PDD and TPR for 6 MV LINAC photon beam using different MC codes

NASA Astrophysics Data System (ADS)

Fonseca, T. C. F.; Mendes, B. M.; Lacerda, M. A. S.; Silva, L. A. C.; Paixão, L.; Bastos, F. M.; Ramirez, J. V.; Junior, J. P. R.

2017-11-01

The Monte Carlo Modelling Expert Group (MCMEG) is an expert network specializing in Monte Carlo radiation transport and the modelling and simulation applied to the radiation protection and dosimetry research field. For the first inter-comparison task the group launched an exercise to model and simulate a 6 MV LINAC photon beam using the Monte Carlo codes available within their laboratories and validate their simulated results by comparing them with experimental measurements carried out in the National Cancer Institute (INCA) in Rio de Janeiro, Brazil. The experimental measurements were performed using an ionization chamber with calibration traceable to a Secondary Standard Dosimetry Laboratory (SSDL). The detector was immersed in a water phantom at different depths and was irradiated with a radiation field size of 10×10 cm2. This exposure setup was used to determine the dosimetric parameters Percentage Depth Dose (PDD) and Tissue Phantom Ratio (TPR). The validation process compares the MC calculated results to the experimental measured PDD20,10 and TPR20,10. Simulations were performed reproducing the experimental TPR20,10 quality index which provides a satisfactory description of both the PDD curve and the transverse profiles at the two depths measured. This paper reports in detail the modelling process using MCNPx, MCNP6, EGSnrc and Penelope Monte Carlo codes, the source and tally descriptions, the validation processes and the results.

Immunologically active peptides capable of inducing immunization against malaria and genes encoding therefor

DOE Office of Scientific and Technical Information (OSTI.GOV)

Dame, J.B.; Williams, J.L.; McCutchan, T.F.

An antimalarial immunogenic stimulant is described comprising an immunogenic carrier and a peptide sequence of between 2 and 1000 consecutive repeats of a sequence Asn-X-Y-Pro, wherein X is Ala or Val and Y is Asn or Asp.

Evidence that a proposed repeated segment of glutamine residues is expressed in the Huntington disease protein

DOE Office of Scientific and Technical Information (OSTI.GOV)

Jou, Y.S.; Myers, R.M.

1994-09-01

Huntington disease (HD) appears to be caused by a mutation that results in an expanded number of CAG repeats at the 5{prime} end of the gene. The nucleotide sequence of the gene and cDNA clones predicts a 347 kd protein that contains a stretch of polyglutamine, encoded by the CAG repeat, located 17 amino acids downstream from the proposed translation initiation site. Because understanding the mechanisms of the pathology of HD depends on whether the CAG-repeat is expressed in the protein, we used antibodies directed against portions of the predicted HD gene product to probe the structure of the proteinmore » in tissue culture cells. Two peptides, one located amino-terminal to the proposed polyglutamine stretch (hd1 peptide FESLKSFQQ from amino acids 11-19) and one located in the carboxy-terminal half of the predicted protein (hd2 peptide QQPRNKPLK from amino acids 2531-2539), were used to elicit polyclonal antibodies in NZW rabbits. We affinity-purified the antibodies and used them to analyze the HD protein. Both antisera specifically recognize the peptides used to elicit them, as well as the appropriate portions of the HD protein expressed in E. coli. Western blot analysis showed that both antisera recognize a protein with an apparent molecular weight of approximately 350,000 in human, monkey, rat and mouse cell lines, including two neutronal cell lines. These results, in combination with immunoprecipitation experiments, suggest strongly that the proposed polyglutamine stretch is indeed translated in the HD protein and is evolutionarily conserved in various mammalian species.« less

A capillary electrophoresis method to explore the self-assembly of a novel polypeptide ligand with quantum dots.

PubMed

Wang, Jianhao; Zhang, Chencheng; Liu, Li; Kalesh, Karunakaran A; Qiu, Lin; Ding, Shumin; Fu, Minli; Gao, Li-Qian; Jiang, Pengju

2016-08-01

Polyhistidine peptides are effective ligands to coat quantum dots (QDs). It is known that both the number of histidine (His) residues repeats and their structural arrangements in a peptide ligand play important roles in the assembly of the peptide onto CdSe/ZnS QDs. However, due to steric hindrance, a peptide sequence with more than six His residue tandem repeats would hardly coordinate well with Zn(2+) in the QD shell to further enhance the binding affinity. To solve this problem, a His-containing peptide ligand, ATTO 590-E2 G (NH)6 (ATTO-NH), was specifically designed and synthesized for assembly with QDs. With sequential injection of QDs and ATTO-NH into the capillary electrophoresis with fluorescence detection, strong Förster resonance energy transfer phenomenon between the QDs and the ATTO 590 dye was observed, indicating efficient self-assembly of the novel peptide onto the QDs to form ATTO-NH capped QDs inside the capillary. The binding stability of the ligand onto the QD was then systematically investigated by titrating with imidazole, His, and a his-tag containing competitive peptide. It is believed that this new in-capillary assay significantly reduced the sample consumption and the analysis time. By functionalizing QDs with certain metal cation-specific group fused peptide ligand, the QD-based probes could be even extended to the online detection of metal cations for monitoring environment in the future. © 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Amino acid polymorphisms in the fibronectin-binding repeats of fibronectin-binding protein A affect bond strength and fibronectin conformation

PubMed Central

Casillas-Ituarte, Nadia N.; Cruz, Carlos H. B.; Lins, Roberto D.; DiBartola, Alex C.; Howard, Jessica; Liang, Xiaowen; Höök, Magnus; Viana, Isabelle F. T.; Sierra-Hernández, M. Roxana; Lower, Steven K.

2017-01-01

The Staphylococcus aureus cell surface contains cell wall-anchored proteins such as fibronectin-binding protein A (FnBPA) that bind to host ligands (e.g. fibronectin; Fn) present in the extracellular matrix of tissue or coatings on cardiac implants. Recent clinical studies have found a correlation between cardiovascular infections caused by S. aureus and nonsynonymous SNPs in FnBPA. Atomic force microscopy (AFM), surface plasmon resonance (SPR), and molecular simulations were used to investigate interactions between Fn and each of eight 20-mer peptide variants containing amino acids Ala, Asn, Gln, His, Ile, and Lys at positions equivalent to 782 and/or 786 in Fn-binding repeat-9 of FnBPA. Experimentally measured bond lifetimes (1/koff) and dissociation constants (Kd = koff/kon), determined by mechanically dissociating the Fn·peptide complex at loading rates relevant to the cardiovascular system, varied from the lowest-affinity H782A/K786A peptide (0.011 s, 747 μm) to the highest-affinity H782Q/K786N peptide (0.192 s, 15.7 μm). These atomic force microscopy results tracked remarkably well to metadynamics simulations in which peptide detachment was defined solely by the free-energy landscape. Simulations and SPR experiments suggested that an Fn conformational change may enhance the stability of the binding complex for peptides with K786I or H782Q/K786I (Kdapp = 0.2–0.5 μm, as determined by SPR) compared with the lowest-affinity double-alanine peptide (Kdapp = 3.8 μm). Together, these findings demonstrate that amino acid substitutions in Fn-binding repeat-9 can significantly affect bond strength and influence the conformation of Fn upon binding. They provide a mechanistic explanation for the observation of nonsynonymous SNPs in fnbA among clinical isolates of S. aureus that cause endovascular infections. PMID:28400484

Amino acid polymorphisms in the fibronectin-binding repeats of fibronectin-binding protein A affect bond strength and fibronectin conformation.

PubMed

Casillas-Ituarte, Nadia N; Cruz, Carlos H B; Lins, Roberto D; DiBartola, Alex C; Howard, Jessica; Liang, Xiaowen; Höök, Magnus; Viana, Isabelle F T; Sierra-Hernández, M Roxana; Lower, Steven K

2017-05-26

The Staphylococcus aureus cell surface contains cell wall-anchored proteins such as fibronectin-binding protein A (FnBPA) that bind to host ligands ( e.g. fibronectin; Fn) present in the extracellular matrix of tissue or coatings on cardiac implants. Recent clinical studies have found a correlation between cardiovascular infections caused by S. aureus and nonsynonymous SNPs in FnBPA. Atomic force microscopy (AFM), surface plasmon resonance (SPR), and molecular simulations were used to investigate interactions between Fn and each of eight 20-mer peptide variants containing amino acids Ala, Asn, Gln, His, Ile, and Lys at positions equivalent to 782 and/or 786 in Fn-binding repeat-9 of FnBPA. Experimentally measured bond lifetimes (1/ k off ) and dissociation constants ( K d = k off / k on ), determined by mechanically dissociating the Fn·peptide complex at loading rates relevant to the cardiovascular system, varied from the lowest-affinity H782A/K786A peptide (0.011 s, 747 μm) to the highest-affinity H782Q/K786N peptide (0.192 s, 15.7 μm). These atomic force microscopy results tracked remarkably well to metadynamics simulations in which peptide detachment was defined solely by the free-energy landscape. Simulations and SPR experiments suggested that an Fn conformational change may enhance the stability of the binding complex for peptides with K786I or H782Q/K786I ( K d app = 0.2-0.5 μm, as determined by SPR) compared with the lowest-affinity double-alanine peptide ( K d app = 3.8 μm). Together, these findings demonstrate that amino acid substitutions in Fn-binding repeat-9 can significantly affect bond strength and influence the conformation of Fn upon binding. They provide a mechanistic explanation for the observation of nonsynonymous SNPs in fnbA among clinical isolates of S. aureus that cause endovascular infections. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

Micelle-Triggered β-Hairpin to α-Helix Transition in a 14-Residue Peptide from a Choline-Binding Repeat of the Pneumococcal Autolysin LytA

PubMed Central

Zamora-Carreras, Héctor; Maestro, Beatriz; Strandberg, Erik; Ulrich, Anne S; Sanz, Jesús M; Jiménez, M Ángeles

2015-01-01

Choline-binding modules (CBMs) have a ββ-solenoid structure composed of choline-binding repeats (CBR), which consist of a β-hairpin followed by a short linker. To find minimal peptides that are able to maintain the CBR native structure and to evaluate their remaining choline-binding ability, we have analysed the third β-hairpin of the CBM from the pneumococcal LytA autolysin. Circular dichroism and NMR data reveal that this peptide forms a highly stable native-like β-hairpin both in aqueous solution and in the presence of trifluoroethanol, but, strikingly, the peptide structure is a stable amphipathic α-helix in both zwitterionic (dodecylphosphocholine) and anionic (sodium dodecylsulfate) detergent micelles, as well as in small unilamellar vesicles. This β-hairpin to α-helix conversion is reversible. Given that the β-hairpin and α-helix differ greatly in the distribution of hydrophobic and hydrophilic side chains, we propose that the amphipathicity is a requirement for a peptide structure to interact and to be stable in micelles or lipid vesicles. To our knowledge, this “chameleonic” behaviour is the only described case of a micelle-induced structural transition between two ordered peptide structures. PMID:25917218

Effect of liquid swine manure rate, incorporation, and timing of rainfall on phosphorus loss with surface runoff.

PubMed

Allen, Brett L; Mallarino, Antonio P

2008-01-01

Excessive manure phosphorus (P) application increases risk of P loss from fields. This study assessed total runoff P (TPR), bioavailable P (BAP), and dissolved reactive P (DRP) concentrations and loads in surface runoff after liquid swine (Sus scrofa domesticus) manure application with or without incorporation into soil and different timing of rainfall. Four replicated manure P treatments were applied in 2002 and in 2003 to two Iowa soils testing low in P managed with corn (Zea mays L.)-soybean [Glycine max (L.) Merr.] rotations. Total P applied each time was 0 to 80 kg P ha(-1) at one site and 0 to 108 kg P ha(-1) at the other. Simulated rainfall was applied within 24 h of P application or after 10 to 16 d and 5 to 6 mo. Nonincorporated manure P increased DRP, BAP, and TPR concentrations and loads linearly or exponentially for 24-h and 10- to 16-d runoff events. On average for the 24-h events, DRP, BAP, and TPR concentrations were 5.4, 4.7, and 2.2 times higher, respectively, for nonincorporated manure than for incorporated manure; P loads were 3.8, 7.7, and 3.6 times higher; and DRP and BAP concentrations were 54% of TPR for nonincorporated manure and 22 to 25% for incorporated manure. A 10- to 16-d rainfall delay resulted in DRP, BAP, and TPR concentrations that were 3.1, 2.7, and 1.1 times lower, respectively, than for 24-h events across all nonincorporated P rates, sites, and years, whereas runoff P loads were 3.8, 3.6, and 1.6 times lower, respectively. A 5- to 6-mo simulated rainfall delay reduced runoff P to levels similar to control plots. Incorporating swine manure when the probability of immediate rainfall is high reduces the risk of P loss in surface runoff; however, this benefit sharply decreases with time.

Predicting Structure and Function for Novel Proteins of an Extremophilic Iron Oxidizing Bacterium

NASA Astrophysics Data System (ADS)

Wheeler, K.; Zemla, A.; Banfield, J.; Thelen, M.

2007-12-01

Proteins isolated from uncultivated microbial populations represent the functional components of microbial processes and contribute directly to community fitness under natural conditions. Investigations into proteins in the environment are hindered by the lack of genome data, or where available, the high proportion of proteins of unknown function. We have identified thousands of proteins from biofilms in the extremely acidic drainage outflow of an iron mine ecosystem (1). With an extensive genomic and proteomic foundation, we have focused directly on the problem of several hundred proteins of unknown function within this well-defined model system. Here we describe the geobiological insights gained by using a high throughput computational approach for predicting structure and function of 421 novel proteins from the biofilm community. We used a homology based modeling system to compare these proteins to those of known structure (AS2TS) (2). This approach has resulted in the assignment of structures to 360 proteins (85%) and provided functional information for up to 75% of the modeled proteins. Detailed examination of the modeling results enables confident, high-throughput prediction of the roles of many of the novel proteins within the microbial community. For instance, one prediction places a protein in the phosphoenolpyruvate/pyruvate domain superfamily as a carboxylase that fills in a gap in an otherwise complete carbon cycle. Particularly important for a community in such a metal rich environment is the evolution of over 25% of the novel proteins that contain a metal cofactor; of these, one third are likely Fe containing proteins. Two of the most abundant proteins in biofilm samples are unusual c-type cytochromes. Both of these proteins catalyze iron- oxidation, a key metabolic reaction supporting the energy requirements of this community. Structural models of these cytochromes verify our experimental results on heme binding and electron transfer reactivity, and provide details for a working hypothesis of electron flow within the biofilm's major bacterium. Nearly 7% of the novel proteins contain tetratrico peptide repeat (TPR) modules, a protein-protein interaction domain that participates in signal transduction and a wide variety of other cellular functions. Like many biofilms, the various organisms in this community use unknown mechanisms to communicate, relying upon each other for survival. Especially interesting is evidence that most of these novel TPR proteins are located in the extracellular or membrane fractions, suggesting their role in intracellular communication. (1) Ram et al, 2005, Science 308:1915-20, "Community Proteomics of a Natural Microbial Biofilm" (2) Zemla et al, 2005, Nucleic Acids Res 33 (Web Server issue):W111-5, "AS2TS system for protein structure modeling and analysis" This work was funded by the DOE Genomics: GTL Program and was performed under the auspices of the DOE by the University of California, Lawrence Livermore National Laboratory under contract W-7405-Eng-48.

A designed point mutant in Fis1 disrupts dimerization and mitochondrial fission

PubMed Central

Lees, Jonathan P. B.; Manlandro, Cara Marie; Picton, Lora K.; Ebie Tan, Alexandra Z.; Casares, Salvador; Flanagan, John M.; Fleming, Karen G.; Hill, R. Blake

2012-01-01

Mitochondrial and peroxisomal fission are essential processes with defects resulting in cardiomyopathy and neonatal lethality. Central to organelle fission is Fis1, a monomeric tetratricopeptide-like repeat (TPR) protein whose role in assembly of the fission machinery remains obscure. Two non-functional, Saccharomyces cerevisiae Fis1 mutants (L80P or E78D/I85T/Y88H) were previously identified in genetic screens. Here, we find that these two variants in the cytosolic domain of Fis1 (Fis1ΔTM) are unexpectedly dimeric. A truncation variant of Fis1ΔTM that lacks an N-terminal regulatory domain is also found to be dimeric. The ability to dimerize is a property innate to the native Fis1ΔTM amino acid sequence as we find this domain is dimeric after transient exposure to elevated temperature or chemical denaturants and is kinetically trapped at room temperature. This is the first demonstration of a specific self-association in solution for the Fis1 cytoplasmic domain. We propose a three-dimensional domain-swapped model for dimerization that is validated by a designed mutation, A72P, which potently disrupts dimerization of wild type Fis1. A72P also disrupts dimerization of non-functional variants indicating a common structural basis for dimerization. The obligate monomer variant A72P, like the dimer-promoting variants, is non-functional in fission consistent with a model in which Fis1 activity depends on its ability to interconvert between monomer and dimer species. These studies suggest a new functionally important manner in which TPR containing proteins may reversibly self-associate. PMID:22789569

Carboxyl Terminus of HSC70-interacting Protein (CHIP) Down-regulates NF-κB-inducing Kinase (NIK) and Suppresses NIK-induced Liver Injury*

PubMed Central

Jiang, Bijie; Shen, Hong; Chen, Zheng; Yin, Lei; Zan, Linsen; Rui, Liangyou

2015-01-01

Ser/Thr kinase NIK (NF-κB-inducing kinase) mediates the activation of the noncanonical NF-κB2 pathway, and it plays an important role in regulating immune cell development and liver homeostasis. NIK levels are extremely low in quiescent cells due to ubiquitin/proteasome-mediated degradation, and cytokines stimulate NIK activation through increasing NIK stability; however, regulation of NIK stability is not fully understood. Here we identified CHIP (carboxyl terminus of HSC70-interacting protein) as a new negative regulator of NIK. CHIP contains three N-terminal tetratricopeptide repeats (TPRs), a middle dimerization domain, and a C-terminal U-box. The U-box domain contains ubiquitin E3 ligase activity that promotes ubiquitination of CHIP-bound partners. We observed that CHIP bound to NIK via its TPR domain. In both HEK293 and primary hepatocytes, overexpression of CHIP markedly decreased NIK levels at least in part through increasing ubiquitination and degradation of NIK. Accordingly, CHIP suppressed NIK-induced activation of the noncanonical NF-κB2 pathway. CHIP also bound to TRAF3, and CHIP and TRAF3 acted coordinately to efficiently promote NIK degradation. The TPR but not the U-box domain was required for CHIP to promote NIK degradation. In mice, hepatocyte-specific overexpression of NIK resulted in liver inflammation and injury, leading to death, and liver-specific expression of CHIP reversed the detrimental effects of hepatic NIK. Our data suggest that CHIP/TRAF3/NIK interactions recruit NIK to E3 ligase complexes for ubiquitination and degradation, thus maintaining NIK at low levels. Defects in CHIP regulation of NIK may result in aberrant NIK activation in the liver, contributing to live injury, inflammation, and disease. PMID:25792747

Cardiovascular effects of simulated zero-gravity in humans

NASA Astrophysics Data System (ADS)

Bonde-Petersen, F.; Suzuki, Y.; Sadámoto, T.; Juel Christensen, N.

Head-down and heat-up tilted bedrest (5 degrees) and head out water immersion (HOWI) for 6 hr were compared. Parameters: Cardiac output (rebreathing method), blood pressure (arm cuff), forearm blood flow (venous occlusion plethysmography), total peripheral (TPR), and forearm vascular (FVR) resistances, Hct, Hb, relativē plasma volume (PV) changes, and plasma catecholamines (single-isotope assay). During HOWI there was as expected a decrement in TPR, FVR, Mean arterial pressure (MAP, from 100 to 80 mmHg), Hct, and PV, and—as a new finding—catecholamines, which were 30-50% lower compared with both + 5 and - 5 degrees bedrest. During head down tilt, MAP was elevated (to 100-110 mmHg) and catecholamines did not fall, while TPR and FVR slowly decreased over 6 hr. HOWI is a stronger stimulus than - 5 degrees bedrest, probably because HOWI elevates central venous pressure more markedly emptying the peripheral veins, while bedrest permits a distension of veins, which induces an increase in sympathetic nervous activity.

Noninvasive identification of the total peripheral resistance baroreflex

NASA Technical Reports Server (NTRS)

Mukkamala, Ramakrishna; Toska, Karin; Cohen, Richard J.

2003-01-01

We propose two identification algorithms for quantitating the total peripheral resistance (TPR) baroreflex, an important contributor to short-term arterial blood pressure (ABP) regulation. Each algorithm analyzes beat-to-beat fluctuations in ABP and cardiac output, which may both be obtained noninvasively in humans. For a theoretical evaluation, we applied both algorithms to a realistic cardiovascular model. The results contrasted with only one of the algorithms proving to be reliable. This algorithm was able to track changes in the static gains of both the arterial and cardiopulmonary TPR baroreflex. We then applied both algorithms to a preliminary set of human data and obtained contrasting results much like those obtained from the cardiovascular model, thereby making the theoretical evaluation results more meaningful. This study suggests that, with experimental testing, the reliable identification algorithm may provide a powerful, noninvasive means for quantitating the TPR baroreflex. This study also provides an example of the role that models can play in the development and initial evaluation of algorithms aimed at quantitating important physiological mechanisms.

Cardiovascular Differences after 4 Hours of Head-Down Bed Rest (HDBR) in Men and Women

NASA Astrophysics Data System (ADS)

Edgell, Heather; Grinberg, Anna; Beavers, Keith; Gagne, Nathalie; Geaves, Danielle; Hughson, Richard

2008-06-01

In both sexes, orthostatic responses are impaired by spaceflight or head-down bed-rest (HDBR), with a greater impact in women. We measured heart rate (HR), mean arterial blood pressure (MAP), cardiac output (Q), stroke volume (SV), central venous pressure (CVP), and total peripheral resistance (TPR) in response to lower body negative pressure (LBNP) in men (n=6) and women (n=6; day 8-11 after menstruation) before and after 4-hr HDBR or seated control (SEAT). Women exhibit lower MAP and TPR with higher SV and Q than men (normalized for body size). LBNP increased HR, decreased CVP, decreased SV, increased TPR and decreased Q. In women 4-hr SEAT results in lower CVP (P=0.015). After 4 hr HDBR men and women exhibit: 1) higher HR response (P<0.001), 2) lower resting CVP (P=0.001), 3) a tendency for higher resting Q (p=0.061). Our results indicate that inactivity and/or circadian rhythm may be important factors to consider when studying women.

Inducing unconscious stress: Cardiovascular activity in response to subliminal presentation of threatening and neutral words.

PubMed

van der Ploeg, Melanie M; Brosschot, Jos F; Verkuil, Bart; Gillie, Brandon L; Williams, DeWayne P; Koenig, Julian; Vasey, Michael W; Thayer, Julian F

2017-10-01

Stress-related cognitive processes may occur outside of awareness, here referred to as unconscious stress, and affect one's physiological state. Evidence supporting this idea would provide necessary clarification of the relationship between psychological stress and cardiovascular (CV) health problems. We tested the hypothesis that increases in mean arterial pressure (MAP) and total peripheral resistance (TPR) and decreases in heart rate variability (HRV) would be larger when threatening stimuli are presented outside of awareness, or subliminally, compared with neutral stimuli. Additionally, it was expected that trait worry and resting HRV, as common risk factors for CV disease, would moderate the effect. We presented a subliminal semantic priming paradigm to college students that were randomly assigned to the threat (n = 56) or neutral condition (n = 60) and assessed changes from baseline of MAP, TPR, and HRV. Level of trait worry was assessed with the Penn State Worry Questionnaire. The findings indicate that CV activity changed according to the hypothesized pattern: A higher MAP and TPR and a lower HRV in the threat condition compared with the neutral condition were found with practically meaningful effect sizes. However, these findings were only statistically significant for TPR. Furthermore, changes in CV activity were not moderated by trait worry or resting HRV. This is the first study to explicitly address the role of subliminally presented threat words on health-relevant outcome measures and suggests that unconscious stress can influence peripheral vascular resistance. © 2017 Society for Psychophysiological Research.

Effects of hypercapnia and hypoxia on the cardiovascular system: vascular capacitance and aortic chemoreceptors.

PubMed

Rothe, C F; Maass-Moreno, R; Flanagan, A D

1990-09-01

Aortic chemoreceptor influences on vascular capacitance after changes in blood carbon dioxide and oxygen were studied in mongrel dogs anesthetized with methoxyflurane and nitrous oxide. The mean circulatory filling pressure (Pmcf), measured during transient cardiac fibrillation, provided a measure of capacitance vessel tone. Hypercapnia, hypoxia, and hypoxic hypercapnia significantly increased most variables, except that hypercapnia caused the total peripheral resistance (TPR) to decrease. Hypocapnia caused a significant decrease in mean systemic (Psa) and pulmonary (Ppa) arterial blood pressures, cardiac output (CO), and central blood volume and an increase in TPR and heart rate. The changes in Pmcf on changing blood gas tensions could be described by the equation delta Pmcf = -1.60 + 0.036 (arterial PCO2) + 50.8/arterial PO2. Thus a 10 mmHg increase in arterial PCO2 caused a 0.36 mmHg increase in Pmcf with receptors intact. Cold block (2 degrees C) of the cervical vagosympathetic trunks did not significantly influence the measured variables at control. During severe hypercapnia, vagal cooling caused a small but significant decrease in Pmcf, Psa, Ppa, and CO but not TPR. During hypoxia, vagal cooling caused the Pmcf, Psa, and TPR to decrease. We conclude that although hypercapnia or hypoxia acts reflexly to increase the capacitance vessel tone (an increase in Pmcf), the aortic and cardiopulmonary chemoreceptors with afferents in the vagi have only a small influence on the capacitance system, accounting for only approximately 25% of the total body response.

Cardiovascular responses to postural changes: differences with age for women and men

NASA Technical Reports Server (NTRS)

Frey, M. A.; Tomaselli, C. M.; Hoffler, W. G.

1994-01-01

The cardiovascular responses to postural change, and how they are affected by aging, are inadequately described in women. Therefore, the authors examined the influence of age and sex on the responses of blood pressure, cardiac output, heart rate, and other variables to change in posture. Measurements were made after 10 minutes each in the supine, seated, and standing positions in 22 men and 25 women who ranged in age from 21 to 59 years. Several variables differed, both by sex and by age, when subjects were supine. On rising, subjects' diastolic and mean arterial pressures, heart rate, total peripheral resistance (TPR), and thoracic impedance increased; cardiac output, stroke volume, and mean stroke ejection rate decreased; and changes in all variables, except heart rate, were greater from supine to sitting than sitting to standing. The increase in heart rate was greater in the younger subjects, and increases in TPR and thoracic impedance were greater in the older subjects. Stroke volume decreased less, and TPR and thoracic impedance increased more, in the women than in the men. The increase in TPR was particularly pronounced in the older women. These studies show that the cardiovascular responses to standing differ, in some respects, between the sexes and with age. The authors suggest that the sex differences are, in part, related to greater decrease of thoracic blood volume with standing in women than in men, and that the age differences result, in part, from decreased responsiveness of the high-pressure baroreceptor system.

Gab1 Is Required for Cell Cycle Transition, Cell Proliferation, and Transformation Induced by an Oncogenic Met Receptor

PubMed Central

Mood, Kathleen; Saucier, Caroline; Bong, Yong-Sik; Lee, Hyun-Shik; Park, Morag

2006-01-01

We have shown previously that either Grb2- or Shc-mediated signaling from the oncogenic Met receptor Tpr-Met is sufficient to trigger cell cycle progression in Xenopus oocytes. However, direct binding of these adaptors to Tpr-Met is dispensable, implying that another Met binding partner mediates these responses. In this study, we show that overexpression of Grb2-associated binder 1 (Gab1) promotes cell cycle progression when Tpr-Met is expressed at suboptimal levels. This response requires that Gab1 possess an intact Met-binding motif, the pleckstrin homology domain, and the binding sites for phosphatidylinositol 3-kinase and tyrosine phosphatase SHP-2, but not the Grb2 and CrkII/phospholipase Cγ binding sites. Importantly, we establish that Gab1-mediated signals are critical for cell cycle transition promoted by the oncogenic Met and fibroblast growth factor receptors, but not by progesterone, the natural inducer of cell cycle transition in Xenopus oocytes. Moreover, Gab1 is essential for Tpr-Met–mediated morphological transformation and proliferation of fibroblasts. This study provides the first evidence that Gab1 is a key binding partner of the Met receptor for induction of cell cycle progression, proliferation, and oncogenic morphological transformation. This study identifies Gab1 and its associated signaling partners as potential therapeutic targets to impair proliferation or transformation of cancer cells in human malignancies harboring a deregulated Met receptor. PMID:16775003

Limb venous compliance responses to lower body negative pressure in humans with high blood pressure.

PubMed

Goulopoulou, S; Deruisseau, K C; Carhart, R; Kanaley, J A

2012-05-01

This study tested the hypothesis that limb venous responses to baroreceptor unloading are altered in individuals with high blood pressure (HBP) compared with normotensive (NT) controls. Calf venous compliance was assessed in 20 subjects with prehypertension and stage-1 hypertension (mean arterial pressure, MAP: 104±1 mm Hg) and 13 NT controls (MAP: 86±2 mm Hg) at baseline and during lower body negative pressure (LBNP), using venous occlusion plethysmography. Baroreflex sensitivity (BRS) was measured using the sequence technique and total peripheral resistance (TPR) was estimated from finger plethysmography. Baseline venous compliance was not different between groups, but the HBP group had lower baseline lnBRS (2.22±0.14 vs 2.7±0.18 ms mm Hg(-1)) and greater baseline TPR (3828±138 vs 3250±111 dyn sec(-1) cm(-5) m(2), P<0.05). Calf venous compliance was reduced in response to LBNP only in the NT group (P<0.05). The HBP group had a greater increase in TPR (ΔTPR) compared with the NT group (+1649±335 vs +718±196 dyn sec(-1) cm(-5) m(2), P<0.05). In conclusion, the early stages of hypertension are characterized by an attenuated venoconstrictor response to baroreceptor unloading, which may compensate for an exaggerated vasoconstrictor response and protect against further increases in blood pressure.

A pollen-specific calmodulin-binding protein, NPG1, interacts with putative pectate lyases.

PubMed

Shin, Sung-Bong; Golovkin, Maxim; Reddy, Anireddy S N

2014-06-12

Previous genetic studies have revealed that a pollen-specific calmodulin-binding protein, No Pollen Germination 1 (NPG1), is required for pollen germination. However, its mode of action is unknown. Here we report direct interaction of NPG1 with pectate lyase-like proteins (PLLs). A truncated form of AtNPG1 lacking the N-terminal tetratricopeptide repeat 1 (TPR1) failed to interact with PLLs, suggesting that it is essential for NPG1 interaction with PLLs. Localization studies with AtNPG1 fused to a fluorescent reporter driven by its native promoter revealed its presence in the cytosol and cell wall of the pollen grain and the growing pollen tube of plasmolyzed pollen. Together, our data suggest that the function of NPG1 in regulating pollen germination is mediated through its interaction with PLLs, which may modify the pollen cell wall and regulate pollen tube emergence and growth.

The M-T Hook Structure Is Critical for Design of HIV-1 Fusion Inhibitors*

PubMed Central

Chong, Huihui; Yao, Xue; Sun, Jianping; Qiu, Zonglin; Zhang, Meng; Waltersperger, Sandro; Wang, Meitian; Cui, Sheng; He, Yuxian

2012-01-01

CP621-652 is a potent HIV-1 fusion inhibitor peptide derived from the C-terminal heptad repeat of gp41. We recently identified that its N-terminal residues Met-626 and Thr-627 adopt a unique hook-like structure (termed M-T hook) thus stabilizing the interaction of the inhibitor with the deep pocket on the N-terminal heptad repeat. In this study, we further demonstrated that the M-T hook structure is a key determinant of CP621-652 in terms of its thermostability and anti-HIV activity. To directly define the structure and function of the M-T hook, we generated the peptide MT-C34 by incorporating Met-626 and Thr-627 into the N terminus of the C-terminal heptad repeat-derived peptide C34. The high resolution crystal structure (1.9 Å) of MT-C34 complexed by an N-terminal heptad repeat-derived peptide reveals that the M-T hook conformation is well preserved at the N-terminal extreme of the inhibitor. Strikingly, addition of two hook residues could dramatically enhance the binding affinity and thermostability of 6-helix bundle core. Compared with C34, MT-C34 exhibited significantly increased activity to inhibit HIV-1 envelope-mediated cell fusion (6.6-fold), virus entry (4.5-fold), and replication (6-fold). Mechanistically, MT-C34 had a 10.5-fold higher increase than C34 in blocking 6-helix bundle formation. We further showed that MT-C34 possessed higher potency against T20 (Enfuvirtide, Fuzeon)-resistant HIV-1 variants. Therefore, this study provides convincing data for our proposed concept that the M-T hook structure is critical for designing HIV-1 fusion inhibitors. PMID:22879603

Autophagy Is an Innate Mechanism Associated with Leprosy Polarization

PubMed Central

Andrade, Priscila Ribeiro; Ferreira, Helen; Nery, José Augusto da Costa; Côrte-Real, Suzana; da Silva, Gilberto Marcelo Sperandio; Rosa, Patricia Sammarco; Fabri, Mario; Sarno, Euzenir Nunes

2017-01-01

Leprosy is a chronic infectious disease that may present different clinical forms according to the immune response of the host. Levels of IFN-γ are significantly raised in paucibacillary tuberculoid (T-lep) when compared with multibacillary lepromatous (L-lep) patients. IFN-γ primes macrophages for inflammatory activation and induces the autophagy antimicrobial mechanism. The involvement of autophagy in the immune response against Mycobacterium leprae remains unexplored. Here, we demonstrated by different autophagic assays that LC3-positive autophagosomes were predominantly observed in T-lep when compared with L-lep lesions and skin-derived macrophages. Accumulation of the autophagic receptors SQSTM1/p62 and NBR1, expression of lysosomal antimicrobial peptides and colocalization analysis of autolysosomes revealed an impairment of the autophagic flux in L-lep cells, which was restored by IFN-γ or rapamycin treatment. Autophagy PCR array gene-expression analysis revealed a significantly upregulation of autophagy genes (BECN1, GPSM3, ATG14, APOL1, and TPR) in T-lep cells. Furthermore, an upregulation of autophagy genes (TPR, GFI1B and GNAI3) as well as LC3 levels was observed in cells of L-lep patients that developed type 1 reaction (T1R) episodes, an acute inflammatory condition associated with increased IFN-γ levels. Finally, we observed increased BCL2 expression in L-lep cells that could be responsible for the blockage of BECN1-mediated autophagy. In addition, in vitro studies demonstrated that dead, but not live M. leprae can induce autophagy in primary and lineage human monocytes, and that live mycobacteria can reduce the autophagy activation triggered by dead mycobacteria, suggesting that M. leprae may hamper the autophagic machinery as an immune escape mechanism. Together, these results indicate that autophagy is an important innate mechanism associated with the M. leprae control in skin macrophages. PMID:28056107

Development of short and highly potent self-assembling elastin-derived pentapeptide repeats containing aromatic amino acid residues.

PubMed

Taniguchi, Suguru; Watanabe, Noriko; Nose, Takeru; Maeda, Iori

2016-01-01

Tropoelastin is the primary component of elastin, which forms the elastic fibers that make up connective tissues. The hydrophobic domains of tropoelastin are thought to mediate the self-assembly of elastin into fibers, and the temperature-mediated self-assembly (coacervation) of one such repetitive peptide sequence (VPGVG) has been utilized in various bio-applications. To elucidate a mechanism for coacervation activity enhancement and to develop more potent coacervatable elastin-derived peptides, we synthesized two series of peptide analogs containing an aromatic amino acid, Trp or Tyr, in addition to Phe-containing analogs and tested their functional characteristics. Thus, position 1 of the hydrophobic pentapeptide repeat of elastin (X(1)P(2)G(3)V(4)G(5)) was substituted by Trp or Tyr. Eventually, we acquired a novel, short Trp-containing elastin-derived peptide analog (WPGVG)3 with potent coacervation ability. From the results obtained during this process, we determined the importance of aromaticity and hydrophobicity for the coacervation potency of elastin-derived peptide analogs. Generally, however, the production of long-chain synthetic polypeptides in quantities sufficient for commercial use remain cost-prohibitive. Therefore, the identification of (WPGVG)3, which is a 15-mer short peptide consisting simply of five natural amino acids and shows temperature-dependent self-assembly activity, might serve as a foundation for the development of various kinds of biomaterials. Copyright © 2015 European Peptide Society and John Wiley & Sons, Ltd.

SM50 Repeat-Polypeptides Self-Assemble into Discrete Matrix Subunits and Promote Appositional Calcium Carbonate Crystal Growth during Sea Urchin Tooth Biomineralization

PubMed Central

Mao, Yelin; Satchell, Paul G.; Luan, Xianghong; Diekwisch, Thomas G.H.

2015-01-01

The two major proteins involved in vertebrate enamel formation and echinoderm sea urchin tooth biomineralization, amelogenin and SM50, are both characterized by elongated polyproline repeat domains in the center of the macromolecule. To determine the role of polyproline repeat polypeptides in basal deuterostome biomineralization, we have mapped the localization of SM50 as it relates to crystal growth, conducted self-assembly studies of SM50 repeat polypeptides, and examined their effect on calcium carbonate and apatite crystal growth. Electron micrographs of the growth zone of Strongylocentrotus purpuratus sea urchin teeth documented a series of successive events from intravesicular mineral nucleation to mineral deposition at the interface between tooth surface and odontoblast syncytium. Using immunohistochemistry, SM50 was detected within the cytoplasm of cells associated with the developing tooth mineral, at the mineral secreting front, and adjacent to initial mineral deposits, but not in muscles and ligaments. Polypeptides derived from the SM50 polyproline alternating hexa- and hepta-peptide repeat region (SM50P6P7) formed highly discrete, donut-shaped self-assembly patterns. In calcium carbonate crystal growth studies, SM50P6P7 repeat peptides triggered the growth of expansive networks of fused calcium carbonate crystals while in apatite growth studies, SM50P6P7 peptides facilitated the growth of needle-shaped and parallel arranged crystals resembling those found in developing vertebrate enamel. In comparison, SM50P6P7 surpassed the PXX24 polypeptide repeat region derived from the vertebrate enamel protein amelogenin in its ability to promote crystal nucleation and appositional crystal growth. Together, these studies establish the SM50P6P7 polyproline repeat region as a potent regulator in the protein-guided appositional crystal growth that occurs during continuous tooth mineralization and eruption. In addition, our studies highlight the role of species-specific polyproline repeat motifs in the formation of discrete self-assembled matrices and the resulting control of mineral growth. PMID:26194158

SM50 repeat-polypeptides self-assemble into discrete matrix subunits and promote appositional calcium carbonate crystal growth during sea urchin tooth biomineralization.

PubMed

Mao, Yelin; Satchell, Paul G; Luan, Xianghong; Diekwisch, Thomas G H

2016-01-01

The two major proteins involved in vertebrate enamel formation and echinoderm sea urchin tooth biomineralization, amelogenin and SM50, are both characterized by elongated polyproline repeat domains in the center of the macromolecule. To determine the role of polyproline repeat polypeptides in basal deuterostome biomineralization, we have mapped the localization of SM50 as it relates to crystal growth, conducted self-assembly studies of SM50 repeat polypeptides, and examined their effect on calcium carbonate and apatite crystal growth. Electron micrographs of the growth zone of Strongylocentrotus purpuratus sea urchin teeth documented a series of successive events from intravesicular mineral nucleation to mineral deposition at the interface between tooth surface and odontoblast syncytium. Using immunohistochemistry, SM50 was detected within the cytoplasm of cells associated with the developing tooth mineral, at the mineral secreting front, and adjacent to initial mineral deposits, but not in muscles and ligaments. Polypeptides derived from the SM50 polyproline alternating hexa- and hepta-peptide repeat region (SM50P6P7) formed highly discrete, donut-shaped self-assembly patterns. In calcium carbonate crystal growth studies, SM50P6P7 repeat peptides triggered the growth of expansive networks of fused calcium carbonate crystals while in apatite growth studies, SM50P6P7 peptides facilitated the growth of needle-shaped and parallel arranged crystals resembling those found in developing vertebrate enamel. In comparison, SM50P6P7 surpassed the PXX24 polypeptide repeat region derived from the vertebrate enamel protein amelogenin in its ability to promote crystal nucleation and appositional crystal growth. Together, these studies establish the SM50P6P7 polyproline repeat region as a potent regulator in the protein-guided appositional crystal growth that occurs during continuous tooth mineralization and eruption. In addition, our studies highlight the role of species-specific polyproline repeat motifs in the formation of discrete self-assembled matrices and the resulting control of mineral growth. Copyright © 2015 Elsevier GmbH. All rights reserved.

PGLa-H tandem-repeat peptides active against multidrug resistant clinical bacterial isolates.

PubMed

Rončević, Tomislav; Gajski, Goran; Ilić, Nada; Goić-Barišić, Ivana; Tonkić, Marija; Zoranić, Larisa; Simunić, Juraj; Benincasa, Monica; Mijaković, Marijana; Tossi, Alessandro; Juretić, Davor

2017-02-01

Antimicrobial peptides (AMPs) are promising candidates for new antibiotic classes but often display an unacceptably high toxicity towards human cells. A naturally produced C-terminal fragment of PGLa, named PGLa-H, has been reported to have a very low haemolytic activity while maintaining a moderate antibacterial activity. A sequential tandem repeat of this fragment, diPGLa-H, was designed, as well as an analogue with a Val to Gly substitution at a key position. These peptides showed markedly improved in vitro bacteriostatic and bactericidal activity against both reference strains and multidrug resistant clinical isolates of Gram-negative and Gram-positive pathogens, with generally low toxicity for human cells as assessed by haemolysis, cell viability, and DNA damage assays. The glycine substitution analogue, kiadin, had a slightly better antibacterial activity and reduced haemolytic activity, which may correlate with an increased flexibility of its helical structure, as deduced using molecular dynamics simulations. These peptides may serve as useful lead compounds for developing anti-infective agents against resistant Gram-negative and Gram-positive species. Copyright © 2016 Elsevier B.V. All rights reserved.

Hydroxyapatite-binding peptides for bone growth and inhibition

DOEpatents

Bertozzi, Carolyn R [Berkeley, CA; Song, Jie [Shrewsbury, MA; Lee, Seung-Wuk [Walnut Creek, CA

2011-09-20

Hydroxyapatite (HA)-binding peptides are selected using combinatorial phage library display. Pseudo-repetitive consensus amino acid sequences possessing periodic hydroxyl side chains in every two or three amino acid sequences are obtained. These sequences resemble the (Gly-Pro-Hyp).sub.x repeat of human type I collagen, a major component of extracellular matrices of natural bone. A consistent presence of basic amino acid residues is also observed. The peptides are synthesized by the solid-phase synthetic method and then used for template-driven HA-mineralization. Microscopy reveal that the peptides template the growth of polycrystalline HA crystals .about.40 nm in size.

Production of monoclonal antibodies recognising the peptide core of MUC2 intestinal mucin.

PubMed

Durrant, L G; Jacobs, E; Price, M R

1994-01-01

A peptide based on the tandem repeat sequence of MUC2 mucin was used to produce a series of monoclonal antibodies (MAb). The fine specificity of these antibodies and their implications for MUC2 expression are presented. Three of the MAbs, 996/1, 996/7 and 995/25, were specific to the MUC2p and failed to bind to peptides based on the MUC1,3,4 tandem repeat sequences whereas three others, 994/152, 994/91 and 996/36, cross reacted with the MUC2p and the MUC3 tandem repeat peptide but not the MUC1 and MUC4 peptides. An antigen, affinity purified from a colorectal tumour on one of the MUC2p-specific MAbs, 996/1, was shown to be a high molecular weight polydisperse, mucin-like antigen. Two of the MAbs, 996/1 and 994/152, recognised MUC2 in tissue sections, although the fine specificity varied between the two MAbs, with 994/152 strongly staining gastric, ileum and kidney epithelia, and MAb 996/1 intensely staining colon, liver and prostate tissues. These antibodies also stained a colorectal cell line, and MAb 994/152 also stained a gastric and an ovarian cell line. Six of the MAbs were used to stain colorectal tumour and adjacent 'normal' colonic mucosa sections. All six stained normal mucosa, but only two of the MAbs, 996/1 and 994/91, stained tumour tissue. The staining probably reflects exposure of cryptic epitopes due to varying levels of glycosylation in different tissues. These anti-MUC2p MAbs may help in determining the normal role of MUC2 mucin and how it is subverted in malignancy.

Methionine peptide formation under primordial earth conditions.

PubMed

Li, Feng; Fitz, Daniel; Fraser, Donald G; Rode, Bernd M

2008-01-01

According to recent research on the origin of life it seems more and more likely that amino acids and peptides were among the first biomolecules formed on earth and that a peptide/protein world was thus a key starting point in evolution towards life. Salt-induced Peptide Formation (SIPF) has repeatedly been shown to be the most universal and plausible peptide-forming reaction currently known under prebiotic conditions and forms peptides from amino acids with the help of copper ions and sodium chloride. In this paper we present experimental results for salt-induced peptide formation from methionine. This is the first time that a sulphur-containing amino acid was investigated in this reaction. The possible catalytic effects of glycine and L-histidine in this reaction were also investigated and a possible distinction between the L- and D-forms of methionine was studied as well.

Poly-dipeptides encoded by the C9ORF72 repeats block global protein translation.

PubMed

Kanekura, Kohsuke; Yagi, Takuya; Cammack, Alexander J; Mahadevan, Jana; Kuroda, Masahiko; Harms, Matthew B; Miller, Timothy M; Urano, Fumihiko

2016-05-01

The expansion of the GGGGCC hexanucleotide repeat in the non-coding region of the Chromosome 9 open-reading frame 72 (C9orf72) gene is the most common genetic cause of frontotemporal dementia (FTD) and amyotrophic lateral sclerosis (ALS). This genetic alteration leads to the accumulation of five types of poly-dipeptides translated from the GGGGCC hexanucleotide repeat. Among these, poly-proline-arginine (poly-PR) and poly-glycine-arginine (poly-GR) peptides are known to be neurotoxic. However, the mechanisms of neurotoxicity associated with these poly-dipeptides are not clear. A proteomics approach identified a number of interacting proteins with poly-PR peptide, including mRNA-binding proteins, ribosomal proteins, translation initiation factors and translation elongation factors. Immunostaining of brain sections from patients with C9orf72 ALS showed that poly-GR was colocalized with a mRNA-binding protein, hnRNPA1. In vitro translation assays showed that poly-PR and poly-GR peptides made insoluble complexes with mRNA, restrained the access of translation factors to mRNA, and blocked protein translation. Our results demonstrate that impaired protein translation mediated by poly-PR and poly-GR peptides plays a role in neurotoxicity and reveal that the pathways altered by the poly-dipeptides-mRNA complexes are potential therapeutic targets for treatment of C9orf72 FTD/ALS. © The Author 2016. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com.

Evaluation of antimicrobial peptides as novel bactericidal agents for room temperature-stored platelets.

PubMed

Mohan, Ketha V K; Rao, Shilpakala Sainath; Atreya, Chintamani D

2010-01-01

A single cost-effective pathogen inactivation approach would help to improve the safety of our nation's blood supply. Several methods and technologies are currently being studied to help reduce bacterial contamination of blood components. There is clearly need for simple and easy-to-use pathogen inactivation techniques specific to plasma, platelets (PLTs), and red blood cells. In this report, we introduce a novel proof of concept: using known therapeutic antimicrobial peptides (AMPs) as bactericidal agents for room temperature-stored PLT concentrates (PCs). Nine synthetic AMPs, four from PLT microbicidal protein-derived peptides (PD1-4) and five Arg-Trp (RW) repeat peptides containing one to five repeats, were tested for bactericidal activity in plasma and PC samples spiked with Staphylococcus aureus, S. epidermidis, Escherichia coli, Pseudomonas aeruginosa, Klebsiella pneumoniae, and Bacillus cereus. A 3-log reduction of viable bacteria was considered as the bactericidal activity of a given peptide. In both plasma alone and PCs, RW3 peptide demonstrated bactericidal activity against S. aureus, S. epidermidis, E. coli, P. aeruginosa, and K. pneumoniae; PD4 and RW2 against P. aeruginosa; and RW4 against K. pneumoniae. The activity of each of these four peptides against the remaining bacterial species in the test panel resulted in less than a 3-log reduction in the number of viable bacteria and hence considered ineffective. These findings suggest a new approach to improving the safety of blood components, demonstrating the potential usefulness of screening therapeutic AMPs against selected bacteria to identify suitable bactericidal agents for stored plasma, PCs, and other blood products.

Calcitonin receptors are ancient modulators for rhythms of preferential temperature in insects and body temperature in mammals.

PubMed

Goda, Tadahiro; Doi, Masao; Umezaki, Yujiro; Murai, Iori; Shimatani, Hiroyuki; Chu, Michelle L; Nguyen, Victoria H; Okamura, Hitoshi; Hamada, Fumika N

2018-01-15

Daily body temperature rhythm (BTR) is essential for maintaining homeostasis. BTR is regulated separately from locomotor activity rhythms, but its molecular basis is largely unknown. While mammals internally regulate BTR, ectotherms, including Drosophila , exhibit temperature preference rhythm (TPR) behavior to regulate BTR. Here, we demonstrate that the diuretic hormone 31 receptor (DH31R) mediates TPR during the active phase in Drosophila DH31R is expressed in clock cells, and its ligand, DH31, acts on clock cells to regulate TPR during the active phase. Surprisingly, the mouse homolog of DH31R, calcitonin receptor (Calcr), is expressed in the suprachiasmatic nucleus (SCN) and mediates body temperature fluctuations during the active phase in mice. Importantly, DH31R and Calcr are not required for coordinating locomotor activity rhythms. Our results represent the first molecular evidence that BTR is regulated distinctly from locomotor activity rhythms and show that DH31R/Calcr is an ancient specific mediator of BTR during the active phase in organisms ranging from ectotherms to endotherms. © 2018 Goda et al.; Published by Cold Spring Harbor Laboratory Press.

Comparison of partial oxidation and steam-CO{sub 2} mixed reforming of CH{sub 4} to syngas on MgO-supported metals

DOE Office of Scientific and Technical Information (OSTI.GOV)

Qin, D.; Lapszewicz, J.; Jiang, X.

1996-03-01

Partial oxidation (POX) and steam-CO{sub 2} mixed reforming of CH{sub 4} on MgO-supported noble metals were investigated at high space velocity (5.5 x 10{sup 5} h{sup -1}). Temperature-programmed reaction (TPR) and isotope transient techniques were used to study the mechanism of POX and mixed reforming. TPR profiles of POX and mixed reforming showed similar ignition reaction behaviors, which implied that there are similar characteristics in their mechanisms. Steam reforming and CO{sub 2} reforming were found to start at the same time in mixed reforming. TPR and CH{sub 4}-D{sub 2} exchange experiments indicated that CH{sub 4} was activated at low temperaturemore » on Rh/MgO. POX showed much higher activity than mixed reforming although their C, H, and O atomic concentrations were the same at the beginning of each reaction. Mechanisms for POX and mixed reforming are suggested and the effect of oxygen-metal bond strength on activity is discussed. 31 refs., 11 figs., 3 tabs.« less

Pedestrian detection in infrared image using HOG and Autoencoder

NASA Astrophysics Data System (ADS)

Chen, Tianbiao; Zhang, Hao; Shi, Wenjie; Zhang, Yu

2017-11-01

In order to guarantee the safety of driving at night, vehicle-mounted night vision system was used to detect pedestrian in front of cars and send alarm to prevent the potential dangerous. To decrease the false positive rate (FPR) and increase the true positive rate (TPR), a pedestrian detection method based on HOG and Autoencoder (HOG+Autoencoder) was presented. Firstly, the HOG features of input images were computed and encoded by Autoencoder. Then the encoded features were classified by Softmax. In the process of training, Autoencoder was trained unsupervised. Softmax was trained with supervision. Autoencoder and Softmax were stacked into a model and fine-tuned by labeled images. Experiment was conducted to compare the detection performance between HOG and HOG+Autoencoder, using images collected by vehicle-mounted infrared camera. There were 80000 images for training set and 20000 for the testing set, with a rate of 1:3 between positive and negative images. The result shows that when TPR is 95%, FPR of HOG+Autoencoder is 0.4%, while the FPR of HOG is 5% with the same TPR.

Antimicrobial activities of amphiphilic peptides covalently bonded to a water-insoluble resin.

PubMed Central

Haynie, S L; Crum, G A; Doele, B A

1995-01-01

A series of polymer-bound antimicrobial peptides was prepared, and the peptides were tested for their antimicrobial activities. The immobilized peptides were prepared by a strategy that used solid-phase peptide synthesis that linked the carboxy-terminal amino acid with an ethylenediamine-modified polyamide resin (PepsynK). The acid-stable, permanent amide bond between the support and the nascent peptide renders the peptide resistant to cleavage from the support during the final acid-catalyzed deprotection step in the synthesis. Select immobilized peptides containing amino acid sequences that ranged from the naturally occurring magainin to simpler synthetic sequences with idealized secondary structures were excellent antimicrobial agents against several organisms. The immobilized peptides typically reduced the number of viable cells by > or = 5 log units. We show that the reduction in cell numbers cannot be explained by the action of a soluble component. We observed no leached or hydrolyzed peptide from the resin, nor did we observe any antimicrobial activity in soluble extracts from the immobilized peptide. The immobilized peptides were washed and reused for repeated microbial contact and killing. These results suggest that the surface actions by magainins and structurally related antimicrobial peptides are sufficient for their lethal activities. PMID:7726486

Poly-dipeptides encoded by the C9orf72 repeats bind nucleoli, impede RNA biogenesis, and kill cells.

PubMed

Kwon, Ilmin; Xiang, Siheng; Kato, Masato; Wu, Leeju; Theodoropoulos, Pano; Wang, Tao; Kim, Jiwoong; Yun, Jonghyun; Xie, Yang; McKnight, Steven L

2014-09-05

Many RNA regulatory proteins controlling pre-messenger RNA splicing contain serine:arginine (SR) repeats. Here, we found that these SR domains bound hydrogel droplets composed of fibrous polymers of the low-complexity domain of heterogeneous ribonucleoprotein A2 (hnRNPA2). Hydrogel binding was reversed upon phosphorylation of the SR domain by CDC2-like kinases 1 and 2 (CLK1/2). Mutated variants of the SR domains changing serine to glycine (SR-to-GR variants) also bound to hnRNPA2 hydrogels but were not affected by CLK1/2. When expressed in mammalian cells, these variants bound nucleoli. The translation products of the sense and antisense transcripts of the expansion repeats associated with the C9orf72 gene altered in neurodegenerative disease encode GRn and PRn repeat polypeptides. Both peptides bound to hnRNPA2 hydrogels independent of CLK1/2 activity. When applied to cultured cells, both peptides entered cells, migrated to the nucleus, bound nucleoli, and poisoned RNA biogenesis, which caused cell death. Copyright © 2014, American Association for the Advancement of Science.

PepPat, a pattern-based oligopeptide homology search method and the identification of a novel tachykinin-like peptide.

PubMed

Jiang, Ying; Gao, Ge; Fang, Gang; Gustafson, Eric L; Laverty, Maureen; Yin, Yanbin; Zhang, Yong; Luo, Jingchu; Greene, Jonathan R; Bayne, Marvin L; Hedrick, Joseph A; Murgolo, Nicholas J

2003-05-01

PepPat, a hybrid method that combines pattern matching with similarity scoring, is described. We also report PepPat's application in the identification of a novel tachykinin-like peptide. PepPat takes as input a query peptide and a user-specified regular expression pattern within the peptide. It first performs a database pattern match and then ranks candidates on the basis of their similarity to the query peptide. PepPat calculates similarity over the pattern spanning region, enhancing PepPat's sensitivity for short query peptides. PepPat can also search for a user-specified number of occurrences of a repeated pattern within the target sequence. We illustrate PepPat's application in short peptide ligand mining. As a validation example, we report the identification of a novel tachykinin-like peptide, C14TKL-1, and show it is an NK1 (neuokinin receptor 1) agonist whose message is widely expressed in human periphery. PepPat is offered online at: http://peppat.cbi.pku.edu.cn.

Hydroconversion of methyl laurate on bifunctional Ni2P/AlMCM-41 catalyst prepared via in situ phosphorization using triphenylphosphine

NASA Astrophysics Data System (ADS)

Zhao, Sha; Zhang, Zhena; Zhu, Kongying; Chen, Jixiang

2017-05-01

A series of Ni2P/AlMCM-41-x bifunctional catalysts with different Si/Al ratios (x) were synthesized by in situ phosphorization of Ni/AlMCM-41-x with triphenylphosphine (nominal Ni/P ratio of 0.75) at 300 °C on a fixed-bed reactor. For comparison, NiP/AlMCM-41-5-TPR was also prepared by the TPR method from the supported nickel phosphate with the Ni/P ratio of 1.0, during which metallic Ni rather than Ni2P formed. TEM images show that Ni and Ni2P particles uniformly distributed in Ni2P/AlMCM-41-x and NiP/AlMCM-41-5-TPR. The Ni2P/AlMCM-41-x acidity increased with decreasing the Si/Al ratio. In the hydroconversion of methyl laurate, the conversions were close to 100% on all catalysts at 360 °C, 3.0 MPa, methyl laurate WHSV of 2 h-1 and H2/methyl laurate ratio of 25. As to Ni2P/AlMCM-41-x, with decreasing the Si/Al ratio, the total selectivity to C11 and C12 hydrocarbons decreased, while the total selectivity to isoundecane and isododecane (Si-C11+i-C12) firstly increased and then decreased. Ni2P/AlMCM-41-5 gave the largest Si-C11+i-C12 of 43.2%. While NiP/AlMCM-41-5-TPR gave higher Si-C11+i-C12 than Ni2P/AlMCM-41-5, it was more active for the undesired Csbnd C bond cleavage and methanation. We propose that the in-situ phosphorization adopted here is a promising approach to preparing Ni2P-based bifunctional catalysts.

Spontaneous mutations in CYC8 and MIG1 suppress the short chronological lifespan of budding yeast lacking SNF1/AMPK

PubMed Central

Maqani, Nazif; Fine, Ryan D.; Shahid, Mehreen; Li, Mingguang; Enriquez-Hesles, Elisa; Smith, Jeffrey S.

2018-01-01

Chronologically aging yeast cells are prone to adaptive regrowth, whereby mutants with a survival advantage spontaneously appear and re-enter the cell cycle in stationary phase cultures. Adaptive regrowth is especially noticeable with short-lived strains, including those defective for SNF1, the homolog of mammalian AMP-activated protein kinase (AMPK). SNF1 becomes active in response to multiple environmental stresses that occur in chronologically aging cells, including glucose depletion and oxidative stress. SNF1 is also required for the extension of chronological lifespan (CLS) by caloric restriction (CR) as defined as limiting glucose at the time of culture inoculation. To identify specific downstream SNF1 targets responsible for CLS extension during CR, we screened for adaptive regrowth mutants that restore chronological longevity to a short-lived snf1∆ parental strain. Whole genome sequencing of the adapted mutants revealed missense mutations in TPR motifs 9 and 10 of the transcriptional co-repressor Cyc8 that specifically mediate repression through the transcriptional repressor Mig1. Another mutation occurred in MIG1 itself, thus implicating the activation of Mig1-repressed genes as a key function of SNF1 in maintaining CLS. Consistent with this conclusion, the cyc8 TPR mutations partially restored growth on alternative carbon sources and significantly extended CLS compared to the snf1∆ parent. Furthermore, cyc8 TPR mutations reactivated multiple Mig1-repressed genes, including the transcription factor gene CAT8, which is responsible for activating genes of the glyoxylate and gluconeogenesis pathways. Deleting CAT8 completely blocked CLS extension by the cyc8 TPR mutations on CLS, identifying these pathways as key Snf1-regulated CLS determinants.

A novel peptide from the ACEI/BPP-CNP precursor in the venom of Crotalus durissus collilineatus.

PubMed

Higuchi, Shigesada; Murayama, Nobuhiro; Saguchi, Ken-ichi; Ohi, Hiroaki; Fujita, Yoshiaki; da Silva, Nelson Jorge; de Siqueira, Rodrigo José Bezerra; Lahlou, Saad; Aird, Steven D

2006-10-01

In crotaline venoms, angiotensin-converting enzyme inhibitors [ACEIs, also known as bradykinin potentiating peptides (BPPs)], are products of a gene coding for an ACEI/BPP-C-type natriuretic peptide (CNP) precursor. In the genes from Bothrops jararaca and Gloydius blomhoffii, ACEI/BPP sequences are repeated. Sequencing of a cDNA clone from venom glands of Crotalus durissus collilineatus showed that two ACEIs/BPPs are located together at the N-terminus, but without repeats. An additional sequence for CNP was unexpectedly found at the C-terminus. Homologous genes for the ACEI/BPP-CNP precursor suggest that most crotaline venoms contain both ACEIs/BPPs and CNP. The sequence of ACEIs/BPPs is separated from the CNP sequence by a long spacer sequence. Previously, there was no evidence that this spacer actually coded any expressed peptides. Aird and Kaiser (1986, unpublished) previously isolated and sequenced a peptide of 11 residues (TPPAGPDVGPR) from Crotalus viridis viridis venom. In the present study, analysis of the cDNA clone from C. d. collilineatus revealed a nearly identical sequence in the ACEI/BPP-CNP spacer. Fractionation of the crude venom by reverse phase HPLC (C(18)), and analysis of the fractions by mass spectrometry (MS) indicated a component of 1020.5 Da. Amino acid sequencing by MS/MS confirmed that C. d. collilineatus venom contains the peptide TPPAGPDGGPR. Its high proline content and paired proline residues are typical of venom hypotensive peptides, although it lacks the usual N-terminal pyroglutamate. It has no demonstrable hypotensive activity when injected intravenously in rats; however, its occurrence in the venoms of dissimilar species suggests that its presence is not accidental. Evidence suggests that these novel toxins probably activate anaphylatoxin C3a receptors.

A role for hippocampal gastrin-releasing peptide receptors in extinction of aversive memory.

PubMed

Luft, Tatiana; Flores, Debora G; Vianna, Monica R M; Schwartsmann, Gilberto; Roesler, Rafael; Izquierdo, Ivan

2006-06-26

Although the gastrin-releasing peptide receptor has been implicated in memory consolidation, previous studies have not examined whether it is involved in extinction. Here we show that gastrin-releasing peptide receptor blockade in the hippocampus disrupts extinction of aversive memory. Male rats were trained in inhibitory avoidance conditioning and then returned repeatedly to the training context without shock on a daily basis for 3 days. Infusion of a gastrin-releasing peptide receptor antagonist or the protein synthesis inhibitor anisomycin into the dorsal hippocampus immediately after the first extinction session blocked extinction. These drugs did not affect performance in subsequent sessions when the first extinction session (1 day after training) was omitted. The results indicate that hippocampal gastrin-releasing peptide receptors are involved in memory extinction.

Transient formation of nano-crystalline structures during fibrillation of an Aβ-like peptide

PubMed Central

Otzen, Daniel E.; Oliveberg, Mikael

2004-01-01

During the first few minutes of fibrillation of a 14-residue peptide homologous to the hydrophobic C-terminal part of the Aβ-peptide, EM micrographs reveal small crystalline areas (100 to 150 nm, repeating unit 47 Å) scattered in more amorphous material. On a longer time scale, these crystalline areas disappear and are replaced by tangled clusters resembling protofilaments (hours), and eventually by more regular amyloid fibrils of 60 Å to 120 Å diameter (days). The transient population of the crystalline areas indicates the presence of ordered substructures in the early fibrillation process, the diameter of which matches the length of the 14-mer peptide in an extended β-strand conformation. PMID:15096642

Efficient production of artificially designed gelatins with a Bacillus brevis system.

PubMed

Kajino, T; Takahashi, H; Hirai, M; Yamada, Y

2000-01-01

Artificially designed gelatins comprising tandemly repeated 30-amino-acid peptide units derived from human alphaI collagen were successfully produced with a Bacillus brevis system. The DNA encoding the peptide unit was synthesized by taking into consideration the codon usage of the host cells, but no clones having a tandemly repeated gene were obtained through the above-mentioned strategy. Minirepeat genes could be selected in vivo from a mixture of every possible sequence encoding an artificial gelatin by randomly ligating the mixed sequence unit and transforming it into Escherichia coli. Larger repeat genes constructed by connecting minirepeat genes obtained by in vivo selection were also stable in the expression host cells. Gelatins derived from the eight-unit and six-unit repeat genes were extracellularly produced at the level of 0.5 g/liter and easily purified by ammonium sulfate fractionation and anion-exchange chromatography. The purified artificial gelatins had the predicted N-terminal sequences and amino acid compositions and a solgel property similar to that of the native gelatin. These results suggest that the selection of a repeat unit sequence stable in an expression host is a shortcut for the efficient production of repetitive proteins and that it can conveniently be achieved by the in vivo selection method. This study revealed the possible industrial application of artificially designed repetitive proteins.

Consequences of non-uniformity in the stoichiometry of component fractions within one and two loops models of alpha-helical peptides

USDA-ARS?s Scientific Manuscript database

Atoms in biomolecular structures like alpha helices contain an array of distances and angles which include abundant multiple patterns of redundancies. Thus all peptides backbones contain the three atom sequence N-C*C, whereas the repeating set of a four atom sequences (N-C*C-N, C*-C-N-C*, and C-N-C...

Artificial 64-Residue HIV-1 Enhancer-Binding Peptide Is a Potent Inhibitor of Viral Replication in HIV-1-Infected Cells.

PubMed

Oufir, Mouhssin; Bisset, Leslie R; Hoffmann, Stefan R K; Xue, Gongda; Klauser, Stephan; Bergamaschi, Bianca; Gervaix, Alain; Böni, Jürg; Schüpbach, Jörg; Gutte, Bernd

2011-01-01

An artificial HIV-1 enhancer-binding peptide was extended by nine consecutive arginine residues at the C-terminus and by the nuclear localization signal of SV40 large T antigen at the N-terminus. The resulting synthetic 64-residue peptide was found to bind to the two enhancers of the HIV-1 long terminal repeat, cross the plasma membrane and the nuclear envelope of human cells, and suppress the HIV-1 enhancer-controlled expression of a green fluorescent protein reporter gene. Moreover, HIV-1 replication is inhibited by this peptide in HIV-1-infected CEM-GFP cells as revealed by HIV-1 p24 ELISA and real-time RT-PCR of HIV-1 RNA. Rapid uptake of this intracellular stable and inhibitory peptide into the cells implies that this peptide may have the potential to attenuate HIV-1 replication in vivo.

Artificial 64-Residue HIV-1 Enhancer-Binding Peptide Is a Potent Inhibitor of Viral Replication in HIV-1-Infected Cells

PubMed Central

Oufir, Mouhssin; Bisset, Leslie R.; Hoffmann, Stefan R. K.; Xue, Gongda; Klauser, Stephan; Bergamaschi, Bianca; Gervaix, Alain; Böni, Jürg; Schüpbach, Jörg; Gutte, Bernd

2011-01-01

An artificial HIV-1 enhancer-binding peptide was extended by nine consecutive arginine residues at the C-terminus and by the nuclear localization signal of SV40 large T antigen at the N-terminus. The resulting synthetic 64-residue peptide was found to bind to the two enhancers of the HIV-1 long terminal repeat, cross the plasma membrane and the nuclear envelope of human cells, and suppress the HIV-1 enhancer-controlled expression of a green fluorescent protein reporter gene. Moreover, HIV-1 replication is inhibited by this peptide in HIV-1-infected CEM-GFP cells as revealed by HIV-1 p24 ELISA and real-time RT-PCR of HIV-1 RNA. Rapid uptake of this intracellular stable and inhibitory peptide into the cells implies that this peptide may have the potential to attenuate HIV-1 replication in vivo. PMID:22312334

Unique phase identification of trimetallic copper iron manganese oxygen carrier using simultaneous differential scanning calorimetry/thermogravimetric analysis during chemical looping combustion reactions with methane

DOE PAGES

Benincosa, William; Siriwardane, Ranjani; Tian, Hanjing; ...

2017-07-05

Chemical looping combustion (CLC) is a promising combustion technology that generates heat and sequestration-ready carbon dioxide that is undiluted by nitrogen from the combustion of carbonaceous fuels with an oxygen carrier, or metal oxide. This process is highly dependent on the reactivity and stability of the oxygen carrier. The development of oxygen carriers remains one of the major barriers for commercialization of CLC. Synthetic oxygen carriers, consisting of multiple metal components, have demonstrated enhanced performance and improved CLC operation compared to single metal oxides. However, identification of the complex mixed metal oxide phases that form after calcination or during CLCmore » reactions has been challenging. Without an understanding of the dominant metal oxide phase, it is difficult to determine reaction parameters and the oxygen carrier reduction pathway, which are necessary for CLC reactor design. This is particularly challenging for complex multi-component oxygen carriers such as copper iron manganese oxide (CuFeMnO 4). This study aims to differentiate the unique phase formation of a highly reactive, complex trimetallic oxygen carrier, CuFeMnO 4, from its single and bimetallic counterparts using thermochemical and reaction data obtained from simultaneous differential scanning calorimetry (DSC) and thermogravimetric analysis (TGA) during temperature programmed reductions (TPR) with methane. DSC/TGA experiments during TPR with methane provides heat flow data and corresponding reaction rate data that can be used to determine reaction routes and mechanisms during methane reduction. Furthermore, non-isothermal TPR data provides the advantage of distinguishing reactions that may not be observable in isothermal analysis. The detailed thermochemical and reaction data, obtained during TPR with methane, distinguished a unique reduction pathway for CuFeMnO 4 that differed from its single and bimetallic counterparts. This is remarkable since X-ray diffraction (XRD) data alone could not be used to distinguish the reactive trimetallic oxide phase due to overlapping peaks from various single and mixed metal oxides. The unique reduction pathway of CuFeMnO 4 was further characterized in this study using in-situ XRD TPR with methane to determine changes in the dominant trimetallic phase that influenced the thermochemical and reaction rate data.« less

Unique phase identification of trimetallic copper iron manganese oxygen carrier using simultaneous differential scanning calorimetry/thermogravimetric analysis during chemical looping combustion reactions with methane

DOE Office of Scientific and Technical Information (OSTI.GOV)

Benincosa, William; Siriwardane, Ranjani; Tian, Hanjing

Chemical looping combustion (CLC) is a promising combustion technology that generates heat and sequestration-ready carbon dioxide that is undiluted by nitrogen from the combustion of carbonaceous fuels with an oxygen carrier, or metal oxide. This process is highly dependent on the reactivity and stability of the oxygen carrier. The development of oxygen carriers remains one of the major barriers for commercialization of CLC. Synthetic oxygen carriers, consisting of multiple metal components, have demonstrated enhanced performance and improved CLC operation compared to single metal oxides. However, identification of the complex mixed metal oxide phases that form after calcination or during CLCmore » reactions has been challenging. Without an understanding of the dominant metal oxide phase, it is difficult to determine reaction parameters and the oxygen carrier reduction pathway, which are necessary for CLC reactor design. This is particularly challenging for complex multi-component oxygen carriers such as copper iron manganese oxide (CuFeMnO 4). This study aims to differentiate the unique phase formation of a highly reactive, complex trimetallic oxygen carrier, CuFeMnO 4, from its single and bimetallic counterparts using thermochemical and reaction data obtained from simultaneous differential scanning calorimetry (DSC) and thermogravimetric analysis (TGA) during temperature programmed reductions (TPR) with methane. DSC/TGA experiments during TPR with methane provides heat flow data and corresponding reaction rate data that can be used to determine reaction routes and mechanisms during methane reduction. Furthermore, non-isothermal TPR data provides the advantage of distinguishing reactions that may not be observable in isothermal analysis. The detailed thermochemical and reaction data, obtained during TPR with methane, distinguished a unique reduction pathway for CuFeMnO 4 that differed from its single and bimetallic counterparts. This is remarkable since X-ray diffraction (XRD) data alone could not be used to distinguish the reactive trimetallic oxide phase due to overlapping peaks from various single and mixed metal oxides. The unique reduction pathway of CuFeMnO 4 was further characterized in this study using in-situ XRD TPR with methane to determine changes in the dominant trimetallic phase that influenced the thermochemical and reaction rate data.« less

Constitutive expression of transgenes encoding derivatives of the synthetic antimicrobial peptide BP100: impact on rice host plant fitness

PubMed Central

2012-01-01

Background The Biopeptide BP100 is a synthetic and strongly cationic α-helical undecapeptide with high, specific antibacterial activity against economically important plant-pathogenic bacteria, and very low toxicity. It was selected from a library of synthetic peptides, along with other peptides with activities against relevant bacterial and fungal species. Expression of the BP100 series of peptides in plants is of major interest to establish disease-resistant plants and facilitate molecular farming. Specific challenges were the small length, peptide degradation by plant proteases and toxicity to the host plant. Here we approached the expression of the BP100 peptide series in plants using BP100 as a proof-of-concept. Results Our design considered up to three tandemly arranged BP100 units and peptide accumulation in the endoplasmic reticulum (ER), analyzing five BP100 derivatives. The ER retention sequence did not reduce the antimicrobial activity of chemically synthesized BP100 derivatives, making this strategy possible. Transformation with sequences encoding BP100 derivatives (bp100der) was over ten-fold less efficient than that of the hygromycin phosphotransferase (hptII) transgene. The BP100 direct tandems did not show higher antimicrobial activity than BP100, and genetically modified (GM) plants constitutively expressing them were not viable. In contrast, inverted repeats of BP100, whether or not elongated with a portion of a natural antimicrobial peptide (AMP), had higher antimicrobial activity, and fertile GM rice lines constitutively expressing bp100der were produced. These GM lines had increased resistance to the pathogens Dickeya chrysanthemi and Fusarium verticillioides, and tolerance to oxidative stress, with agronomic performance comparable to untransformed lines. Conclusions Constitutive expression of transgenes encoding short cationic α-helical synthetic peptides can have a strong negative impact on rice fitness. However, GM plants expressing, for example, BP100 based on inverted repeats, have adequate agronomic performance and resistant phenotypes as a result of a complex equilibrium between bp100der toxicity to plant cells, antimicrobial activity and transgene-derived plant stress response. It is likely that these results can be extended to other peptides with similar characteristics. PMID:22947243

Constitutive expression of transgenes encoding derivatives of the synthetic antimicrobial peptide BP100: impact on rice host plant fitness.

PubMed

Nadal, Anna; Montero, Maria; Company, Nuri; Badosa, Esther; Messeguer, Joaquima; Montesinos, Laura; Montesinos, Emilio; Pla, Maria

2012-09-04

The Biopeptide BP100 is a synthetic and strongly cationic α-helical undecapeptide with high, specific antibacterial activity against economically important plant-pathogenic bacteria, and very low toxicity. It was selected from a library of synthetic peptides, along with other peptides with activities against relevant bacterial and fungal species. Expression of the BP100 series of peptides in plants is of major interest to establish disease-resistant plants and facilitate molecular farming. Specific challenges were the small length, peptide degradation by plant proteases and toxicity to the host plant. Here we approached the expression of the BP100 peptide series in plants using BP100 as a proof-of-concept. Our design considered up to three tandemly arranged BP100 units and peptide accumulation in the endoplasmic reticulum (ER), analyzing five BP100 derivatives. The ER retention sequence did not reduce the antimicrobial activity of chemically synthesized BP100 derivatives, making this strategy possible. Transformation with sequences encoding BP100 derivatives (bp100der) was over ten-fold less efficient than that of the hygromycin phosphotransferase (hptII) transgene. The BP100 direct tandems did not show higher antimicrobial activity than BP100, and genetically modified (GM) plants constitutively expressing them were not viable. In contrast, inverted repeats of BP100, whether or not elongated with a portion of a natural antimicrobial peptide (AMP), had higher antimicrobial activity, and fertile GM rice lines constitutively expressing bp100der were produced. These GM lines had increased resistance to the pathogens Dickeya chrysanthemi and Fusarium verticillioides, and tolerance to oxidative stress, with agronomic performance comparable to untransformed lines. Constitutive expression of transgenes encoding short cationic α-helical synthetic peptides can have a strong negative impact on rice fitness. However, GM plants expressing, for example, BP100 based on inverted repeats, have adequate agronomic performance and resistant phenotypes as a result of a complex equilibrium between bp100der toxicity to plant cells, antimicrobial activity and transgene-derived plant stress response. It is likely that these results can be extended to other peptides with similar characteristics.

DOE Office of Scientific and Technical Information (OSTI.GOV)

Hattori, Masaki; Sugino, Etsuko; Minoura, Katsuhiko

One of the priorities in Alzheimer research is to develop a compound that inhibits the filament formation of tau protein. Since the three- or four-repeat microtubule-binding domain (MBD) in tau protein plays an essential role in filament formation, the inhibitory behavior of cyanidin (Cy) and methylene blue (MB) with respect to heparin-induced filament formation of MBD in a neutral solution (pH 7.6) was characterized by fluorescence, circular dichroism, and electron microscopy measurements. The planar aromatic ring of Cy and the N-unsubstituted phenothiazine ring of MB were shown to be necessary for the inhibition. However, the inhibitory responses with respect tomore » heparin-induced filament formation to the second and third repeat peptides of MBD were different: Cy suppresses the formation and MB does not prevent the formation. This suggests the importance of the first and fourth repeat peptides in the inhibitory activity of MB for MBD filament formation. In this study, we showed that the decrease of thioflavin S fluorescence intensity is not always linked to inhibition of filament formation.« less

Full-length model of the human galectin-4 and insights into dynamics of inter-domain communication

NASA Astrophysics Data System (ADS)

Rustiguel, Joane K.; Soares, Ricardo O. S.; Meisburger, Steve P.; Davis, Katherine M.; Malzbender, Kristina L.; Ando, Nozomi; Dias-Baruffi, Marcelo; Nonato, Maria Cristina

2016-09-01

Galectins are proteins involved in diverse cellular contexts due to their capacity to decipher and respond to the information encoded by β-galactoside sugars. In particular, human galectin-4, normally expressed in the healthy gastrointestinal tract, displays differential expression in cancerous tissues and is considered a potential drug target for liver and lung cancer. Galectin-4 is a tandem-repeat galectin characterized by two carbohydrate recognition domains connected by a linker-peptide. Despite their relevance to cell function and pathogenesis, structural characterization of full-length tandem-repeat galectins has remained elusive. Here, we investigate galectin-4 using X-ray crystallography, small- and wide-angle X-ray scattering, molecular modelling, molecular dynamics simulations, and differential scanning fluorimetry assays and describe for the first time a structural model for human galectin-4. Our results provide insight into the structural role of the linker-peptide and shed light on the dynamic characteristics of the mechanism of carbohydrate recognition among tandem-repeat galectins.

Novel TPR-containing subunit of TOM complex functions as cytosolic receptor for Entamoeba mitosomal transport

PubMed Central

Makiuchi, Takashi; Mi-ichi, Fumika; Nakada-Tsukui, Kumiko; Nozaki, Tomoyoshi

2013-01-01

Under anaerobic environments, the mitochondria have undergone remarkable reduction and transformation into highly reduced structures, referred as mitochondrion-related organelles (MROs), which include mitosomes and hydrogenosomes. In agreement with the concept of reductive evolution, mitosomes of Entamoeba histolytica lack most of the components of the TOM (translocase of the outer mitochondrial membrane) complex, which is required for the targeting and membrane translocation of preproteins into the canonical aerobic mitochondria. Here we showed, in E. histolytica mitosomes, the presence of a 600-kDa TOM complex composed of Tom40, a conserved pore-forming subunit, and Tom60, a novel lineage-specific receptor protein. Tom60, containing multiple tetratricopeptide repeats, is localized to the mitosomal outer membrane and the cytosol, and serves as a receptor of both mitosomal matrix and membrane preproteins. Our data indicate that Entamoeba has invented a novel lineage-specific shuttle receptor of the TOM complex as a consequence of adaptation to an anaerobic environment. PMID:23350036

Novel TPR-containing subunit of TOM complex functions as cytosolic receptor for Entamoeba mitosomal transport.

PubMed

Makiuchi, Takashi; Mi-ichi, Fumika; Nakada-Tsukui, Kumiko; Nozaki, Tomoyoshi

2013-01-01

Under anaerobic environments, the mitochondria have undergone remarkable reduction and transformation into highly reduced structures, referred as mitochondrion-related organelles (MROs), which include mitosomes and hydrogenosomes. In agreement with the concept of reductive evolution, mitosomes of Entamoeba histolytica lack most of the components of the TOM (translocase of the outer mitochondrial membrane) complex, which is required for the targeting and membrane translocation of preproteins into the canonical aerobic mitochondria. Here we showed, in E. histolytica mitosomes, the presence of a 600-kDa TOM complex composed of Tom40, a conserved pore-forming subunit, and Tom60, a novel lineage-specific receptor protein. Tom60, containing multiple tetratricopeptide repeats, is localized to the mitosomal outer membrane and the cytosol, and serves as a receptor of both mitosomal matrix and membrane preproteins. Our data indicate that Entamoeba has invented a novel lineage-specific shuttle receptor of the TOM complex as a consequence of adaptation to an anaerobic environment.

Characterization of the transcriptional regulator Rv3124 of Mycobacterium tuberculosis identifies it as a positive regulator of molybdopterin biosynthesis and defines the functional consequences of a non-synonymous SNP in the Mycobacterium bovis BCG orthologue

PubMed Central

Mendoza Lopez, Pablo; Golby, Paul; Wooff, Esen; Garcia, Javier Nunez; Garcia Pelayo, M. Carmen; Conlon, Kevin; Gema Camacho, Ana; Hewinson, R. Glyn; Polaina, Julio; Suárez García, Antonio; Gordon, Stephen V.

2010-01-01

A number of single-nucleotide polymorphisms (SNPs) have been identified in the genome of Mycobacterium bovis BCG Pasteur compared with the sequenced strain M. bovis 2122/97. The functional consequences of many of these mutations remain to be described; however, mutations in genes encoding regulators may be particularly relevant to global phenotypic changes such as loss of virulence, since alteration of a regulator's function will affect the expression of a wide range of genes. One such SNP falls in bcg3145, encoding a member of the AfsR/DnrI/SARP class of global transcriptional regulators, that replaces a highly conserved glutamic acid residue at position 159 (E159G) with glycine in a tetratricopeptide repeat (TPR) located in the bacterial transcriptional activation (BTA) domain of BCG3145. TPR domains are associated with protein–protein interactions, and a conserved core (helices T1–T7) of the BTA domain seems to be required for proper function of SARP-family proteins. Structural modelling predicted that the E159G mutation perturbs the third α-helix of the BTA domain and could therefore have functional consequences. The E159G SNP was found to be present in all BCG strains, but absent from virulent M. bovis and Mycobacterium tuberculosis strains. By overexpressing BCG3145 and Rv3124 in BCG and H37Rv and monitoring transcriptome changes using microarrays, we determined that BCG3145/Rv3124 acts as a positive transcriptional regulator of the molybdopterin biosynthesis moa1 locus, and we suggest that rv3124 be renamed moaR1. The SNP in bcg3145 was found to have a subtle effect on the activity of MoaR1, suggesting that this mutation is not a key event in the attenuation of BCG. PMID:20378651

Structural characterization of the Yersinia pestis type III secretion system needle protein YscF in complex with its heterodimeric chaperone YscE/YscG

PubMed Central

Sun, Ping; Tropea, Joseph E.; Austin, Brian P.; Cherry, Scott; Waugh, David S.

2008-01-01

Summary The plague-causing bacterium Yersinia pestis utilizes a Type III Secretion System (T3SS) to deliver effector proteins into mammalian cells where they interfere with signal transduction pathways that mediate phagocytosis and the inflammatory response. Effector proteins are injected through a hollow needle structure composed of the protein YscF. YscG and YscE act as "chaperones" to prevent premature polymerization of YscF in the cytosol of the bacterium prior to assembly of the needle. Here, we report the crystal structure of the YscEFG protein complex at 1.8 Å resolution. Overall, the structure is similar to that of the analogous PscEFG complex from the Pseudomonas aeruginosa T3SS, but there are noteworthy differences. The structure confirms that, like PscG, YscG is a member of the tetratricopeptide repeat (TPR) family of proteins. YscG binds tightly to the C-terminal half of YscF, implying that it is this region of YscF that controls its polymerization into the needle structure. YscE interacts with the N-terminal TPR motif of YscG but makes very little direct contact with YscF. Its function may be to stabilize the structure of YscG and/or to participate in recruiting the complex to the secretion apparatus. No electron density could be observed for the N-terminal 49 residues of YscF. This and additional evidence suggest that the N-terminus of YscF is disordered in the complex with YscE and YscG. As expected, conserved residues in the C-terminal half of YscF mediate important intra- and intermolecular interactions in the complex. Moreover, the phenotypes of some previously characterized mutations in the C-terminal half of YscF can be rationalized in terms of the structure of the heterotrimeric YscEFG complex. PMID:18281060

A new method to compare statistical tree growth curves: the PL-GMANOVA model and its application with dendrochronological data.

PubMed

Ricker, Martin; Peña Ramírez, Víctor M; von Rosen, Dietrich

2014-01-01

Growth curves are monotonically increasing functions that measure repeatedly the same subjects over time. The classical growth curve model in the statistical literature is the Generalized Multivariate Analysis of Variance (GMANOVA) model. In order to model the tree trunk radius (r) over time (t) of trees on different sites, GMANOVA is combined here with the adapted PL regression model Q = A · T+E, where for b ≠ 0 : Q = Ei[-b · r]-Ei[-b · r1] and for b = 0 : Q  = Ln[r/r1], A =  initial relative growth to be estimated, T = t-t1, and E is an error term for each tree and time point. Furthermore, Ei[-b · r]  = ∫(Exp[-b · r]/r)dr, b = -1/TPR, with TPR being the turning point radius in a sigmoid curve, and r1 at t1 is an estimated calibrating time-radius point. Advantages of the approach are that growth rates can be compared among growth curves with different turning point radiuses and different starting points, hidden outliers are easily detectable, the method is statistically robust, and heteroscedasticity of the residuals among time points is allowed. The model was implemented with dendrochronological data of 235 Pinus montezumae trees on ten Mexican volcano sites to calculate comparison intervals for the estimated initial relative growth A. One site (at the Popocatépetl volcano) stood out, with A being 3.9 times the value of the site with the slowest-growing trees. Calculating variance components for the initial relative growth, 34% of the growth variation was found among sites, 31% among trees, and 35% over time. Without the Popocatépetl site, the numbers changed to 7%, 42%, and 51%. Further explanation of differences in growth would need to focus on factors that vary within sites and over time.

Proline-rich antimicrobial peptides: converging to a non-lytic mechanism of action.

PubMed

Scocchi, Marco; Tossi, Alessandro; Gennaro, Renato

2011-07-01

Proline-rich antimicrobial peptides are a group of cationic host defense peptides of vertebrates and invertebrates characterized by a high content of proline residues, often associated with arginine residues in repeated motifs. Those isolated from some mammalian and insect species, although not evolutionarily related, use a similar mechanism to selectively kill Gram-negative bacteria, with a low toxicity to animals. Unlike other types of antimicrobial peptides, their mode of action does not involve the lysis of bacterial membranes but entails penetration into susceptible cells, where they then act intracellularly. Some aspects of the transport system and cytoplasmic targets have been elucidated. These features make them attractive both as anti-infective lead compounds and as a new class of potential cell-penetrating peptides capable of internalising membrane-impermeant drugs into both bacterial and eukaryotic cells.

Action of Phα1β, a peptide from the venom of the spider Phoneutria nigriventer, on the analgesic and adverse effects caused by morphine in mice.

PubMed

Tonello, Raquel; Rigo, Flávia; Gewehr, Camila; Trevisan, Gabriela; Pereira, Elizete Maria Rita; Gomez, Marcus Vinicius; Ferreira, Juliano

2014-06-01

Opioids are standard therapy for the treatment of pain; however, adverse effects limit their use. Voltage-gated calcium channel blockers may be used to increase opioid analgesia, but their effect on opioid-induced side effects is little known. Thus, the goal of this study was to evaluate the action of the peptide Phα1β, a voltage-gated calcium channel blocker, on the antinociceptive and adverse effects produced by morphine in mice. A single administration of morphine (3-10 mg/kg) was able to reduce heat nociception as well as decrease gastrointestinal transit. The antinociception caused by a single injection of morphine was slightly increased by an intrathecal injection of Phα1β (30 pmol/site). Repeated treatment with morphine caused tolerance, hyperalgesia, withdrawal syndrome, and constipation, and the Phα1β (.1-30 pmol/site, intrathecal) was able to reverse these effects. Finally, the effects produced by the native form of Phα1β were fully mimicked by a recombinant version of this peptide. Taken together, these data show that Phα1β was effective in potentiating the analgesia caused by a single dose of morphine as well as in reducing tolerance and the adverse effects induced by repeated administration of morphine, indicating its potential use as an adjuvant drug in combination with opioids. This article presents preclinical evidence for a useful adjuvant drug in opioid treatment. Phα1β, a peptide calcium channel blocker, could be used not only to potentiate morphine analgesia but also to reduce the adverse effects caused by repeated administration of morphine. Copyright © 2014. Published by Elsevier Inc.

Evidence that C9ORF72 Dipeptide Repeat Proteins Associate with U2 snRNP to Cause Mis-splicing in ALS/FTD Patients.

PubMed

Yin, Shanye; Lopez-Gonzalez, Rodrigo; Kunz, Ryan C; Gangopadhyay, Jaya; Borufka, Carl; Gygi, Steven P; Gao, Fen-Biao; Reed, Robin

2017-06-13

Hexanucleotide repeat expansion in the C9ORF72 gene results in production of dipeptide repeat (DPR) proteins that may disrupt pre-mRNA splicing in amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD) patients. At present, the mechanisms underlying this mis-splicing are not understood. Here, we show that addition of proline-arginine (PR) and glycine-arginine (GR) toxic DPR peptides to nuclear extracts blocks spliceosome assembly and splicing, but not other types of RNA processing. Proteomic and biochemical analyses identified the U2 small nuclear ribonucleoprotein particle (snRNP) as a major interactor of PR and GR peptides. In addition, U2 snRNP, but not other splicing factors, mislocalizes from the nucleus to the cytoplasm both in C9ORF72 patient induced pluripotent stem cell (iPSC)-derived motor neurons and in HeLa cells treated with the toxic peptides. Bioinformatic studies support a specific role for U2-snRNP-dependent mis-splicing in C9ORF72 patient brains. Together, our data indicate that DPR-mediated dysfunction of U2 snRNP could account for as much as ∼44% of the mis-spliced cassette exons in C9ORF72 patient brains. Copyright © 2017 The Author(s). Published by Elsevier Inc. All rights reserved.

Development of bisphenol A-removing recombinant Escherichia coli by monomeric and dimeric surface display of bisphenol A-binding peptide.

PubMed

Maruthamuthu, Murali Kannan; Hong, Jiyeon; Arulsamy, Kulandaisamy; Somasundaram, Sivachandiran; Hong, SoonHo; Choe, Woo-Seok; Yoo, Ik-Keun

2018-04-01

Peptide-displaying Escherichia coli cells were investigated for use in adsorptive removal of bisphenol A (BPA) both in Luria-Bertani medium including BPA or ATM thermal paper eluted wastewater. Two recombinant strains were constructed with monomeric and dimeric repeats of the 7-mer BPA-binding peptide (KSLENSY), respectively. Greater than threefold increased adsorption of BPA [230.4 µmol BPA per g dry cell weight (DCW)] was found in dimeric peptide-displaying cells compared to monomeric strains (63.4 µmol per g DCW) in 15 ppm BPA solution. The selective removal of BPA from a mixture of BPA analogs (bisphenol F and bisphenol S) was verified in both monomeric and dimeric peptide-displaying cells. The binding chemistry of BPA with the peptide was assumed, based on molecular docking analysis, to be the interaction of BPA with serine and asparagine residues within the 7-mer peptide sequence. The peptide-displaying cells also functioned efficiently in thermal paper eluted wastewater containing 14.5 ppm BPA.

Catalytic Activities Of [GADV]-Peptides

NASA Astrophysics Data System (ADS)

Oba, Takae; Fukushima, Jun; Maruyama, Masako; Iwamoto, Ryoko; Ikehara, Kenji

2005-10-01

We have previously postulated a novel hypothesis for the origin of life, assuming that life on the earth originated from “[GADV]-protein world”, not from the “RNA world” (see Ikehara's review, 2002). The [GADV]-protein world is constituted from peptides and proteins with random sequences of four amino acids (glycine [G], alanine [A], aspartic acid [D] and valine [V]), which accumulated by pseudo-replication of the [GADV]-proteins. To obtain evidence for the hypothesis, we produced [GADV]-peptides by repeated heat-drying of the amino acids for 30 cycles ([GADV]-P30) and examined whether the peptides have some catalytic activities or not. From the results, it was found that the [GADV]-P30 can hydrolyze several kinds of chemical bonds in molecules, such as umbelliferyl-β-D-galactoside, glycine-p-nitroanilide and bovine serum albumin. This suggests that [GADV]-P30 could play an important role in the accumulation of [GADV]-proteins through pseudo-replication, leading to the emergence of life. We further show that [GADV]-octapaptides with random sequences, but containing no cyclic compounds as diketepiperazines, have catalytic activity, hydrolyzing peptide bonds in a natural protein, bovine serum albumin. The catalytic activity of the octapeptides was much higher than the [GADV]-P30 produced through repeated heat-drying treatments. These results also support the [GADV]-protein-world hypothesis of the origin of life (see Ikehara's review, 2002). Possible steps for the emergence of life on the primitive earth are presented.

Phase Variation of NadA in Invasive Neisseria meningitidis Isolates Impacts on Coverage Estimates for 4C-MenB, a MenB vaccine.

PubMed

Green, Luke R; Lucidarme, Jay; Dave, Neelam; Chan, Hannah; Clark, Stephen; Borrow, Ray; Bayliss, Christopher D

2018-06-27

A recombinant NadA protein is one of the four major protective antigens of 4C-MenB (Bexsero®), a vaccine developed for serogroup B Neisseria meningitidis (MenB). The Meningococcal Antigen Typing System (MATS) is utilised as a high throughput assay for assessing the invasive MenB strain coverage of 4C-MenB. Where present, the nadA gene is subject to phase variable changes in transcription due to a 5'TAAA repeat tract located in a regulatory region. The promoter-containing intergenic region sequences (IGR) and 5'TAAA repeat numbers were determined for 906 invasive meningococcal disease isolates possessing the nadA gene. Exclusion of the 5'TAAA repeats reduced the number of IGR alleles from 82 to 23. Repeat numbers were associated with low and high levels of NadA expression by Western blotting and ELISA. Low expression repeat numbers were present in 83% of 179 MenB isolates with NadA-2/3 or Nad-1 peptide variants and 68% of 480 MenW ST-11 complex isolates with Nad-2/3 peptide variants. For isolates with vaccine-compatible NadA variants, 93% of MATS negative isolates were associated with low expression repeat numbers whereas 63% of isolates with MATS RP scores above the 95% confidence interval for the positive bactericidal threshold had high expression repeat numbers. Analysis of the 5'TAAA repeat number has potential as a rapid, high throughput method for assessing strain coverage for the NadA-component of 4C-MenB. A key application will be assessing coverage in meningococcal disease cases where confirmation is by PCR only and MATS cannot be applied. Copyright © 2018 Green et al.

Antihypertensive activity of transgenic rice seed containing an 18-repeat novokinin peptide localized in the nucleolus of endosperm cells.

PubMed

Wakasa, Yuhya; Zhao, Hui; Hirose, Sakiko; Yamauchi, Daiki; Yamada, Yuko; Yang, Lijun; Ohinata, Kousaku; Yoshikawa, Masaaki; Takaiwa, Fumio

2011-09-01

Novokinin (Arg-Pro-Leu-Lys-Pro-Trp, RPLKPW) is a new potent antihypertensive peptide based on the sequence of ovokinin (2-7) derived from ovalbumin. We previously generated transgenic rice seeds in which eight novokinin were fused to storage protein glutelins (GluA2 and GluC) for expression. Oral administration of these seeds to spontaneously hypertensive rats (SHRs) reduced systolic blood pressures at a dose of 1 g seed/kg of SHR. Here, 10- or 18-tandem repeats of novokinin with an endoplasmic reticulum (ER) retention signal (Lys-Asp-Glu-Leu, KDEL) at the C terminus were directly expressed in rice under the control of the glutelin promoter containing its signal peptide. Only small amounts of the 18-repeat novokinin accumulated, and it was unexpectedly deposited in the nucleolus. This abnormal intracellular localization was explained by an endogenous signal for nuclear localization. The GFP reporter protein fused to this sequence targeted to nuclei by a transient assay using onion epidermal cells. Transgenic seed expressing the 18-repeat novokinin exhibited significantly higher antihypertensive activity after a single oral dose to SHR even at one-quarter the amount (0.25 g/kg) of the transgenic rice seed expressing the fusion construct; though, its novokinin content was much lower (1/5). Furthermore, in a long-term administration for 5 weeks, even a smaller dose (0.0625 g/kg) of transgenic seeds could confer antihypertensive activity. This high antihypertensive activity may be attributed to differences in digestibility of expressed products by gastrointestinal enzymes and the unique intracellular localization. These results indicate that accumulation of novokinin as a tandemly repeated structure in transgenic rice is more effective than as a fusion-type structure. © 2010 The Authors. Plant Biotechnology Journal © 2010 Society for Experimental Biology and Blackwell Publishing Ltd.

Structure of the large FK506-binding protein FKBP51, an Hsp90-binding protein and a component of steroid receptor complexes

PubMed Central

Sinars, Cindy R.; Cheung-Flynn, Joyce; Rimerman, Ronald A.; Scammell, Jonathan G.; Smith, David F.; Clardy, Jon

2003-01-01

The ability to bind immunosuppressive drugs such as cyclosporin and FK506 defines the immunophilin family of proteins, and the FK506-binding proteins form the FKBP subfamily of immunophilins. Some FKBPs, notably FKBP12 (the 12-kDa FK506-binding protein), have defined roles in regulating ion channels or cell signaling, and well established structures. Other FKBPs, especially the larger ones, participate in important biological processes, but their exact roles and the structural bases for these roles are poorly defined. FKBP51 (the 51-kDa FKBP) associates with heat shock protein 90 (Hsp90) and appears in functionally mature steroid receptor complexes. In New World monkeys, FKBP51 has been implicated in cortisol resistance. We report here the x-ray structures of human FKBP51, to 2.7 Å, and squirrel monkey FKBP51, to 2.8 Å, by using multiwavelength anomalous dispersion phasing. FKBP51 is composed of three domains: two consecutive FKBP domains and a three-unit repeat of the TPR (tetratricopeptide repeat) domain. This structure of a multi-FKBP domain protein clarifies the arrangement of these domains and their possible interactions with other proteins. The two FKBP domains differ by an insertion in the second that affects the formation of the progesterone receptor complex. PMID:12538866

Synaptic Targeting and Function of SAPAPs Mediated by Phosphorylation-Dependent Binding to PSD-95 MAGUKs.

PubMed

Zhu, Jinwei; Zhou, Qingqing; Shang, Yuan; Li, Hao; Peng, Mengjuan; Ke, Xiao; Weng, Zhuangfeng; Zhang, Rongguang; Huang, Xuhui; Li, Shawn S C; Feng, Guoping; Lu, Youming; Zhang, Mingjie

2017-12-26

The PSD-95/SAPAP/Shank complex functions as the major scaffold in orchestrating the formation and plasticity of the post-synaptic densities (PSDs). We previously demonstrated that the exquisitely specific SAPAP/Shank interaction is critical for Shank synaptic targeting and Shank-mediated synaptogenesis. Here, we show that the PSD-95/SAPAP interaction, SAPAP synaptic targeting, and SAPAP-mediated synaptogenesis require phosphorylation of the N-terminal repeat sequences of SAPAPs. The atomic structure of the PSD-95 guanylate kinase (GK) in complex with a phosphor-SAPAP repeat peptide, together with biochemical studies, reveals the molecular mechanism underlying the phosphorylation-dependent PSD-95/SAPAP interaction, and it also provides an explanation of a PSD-95 mutation found in patients with intellectual disabilities. Guided by the structural data, we developed potent non-phosphorylated GK inhibitory peptides capable of blocking the PSD-95/SAPAP interaction and interfering with PSD-95/SAPAP-mediated synaptic maturation and strength. These peptides are genetically encodable for investigating the functions of the PSD-95/SAPAP interaction in vivo. Copyright © 2017 The Author(s). Published by Elsevier Inc. All rights reserved.

Epitope mapping of PR81 anti-MUC1 monoclonal antibody following PEPSCAN and phage display techniques.

PubMed

Mohammadi, Mohammad; Rasaee, Mohammad Javad; Rajabibazl, Masoumeh; Paknejad, Malihe; Zare, Mehrak; Mohammadzadeh, Sara

2007-08-01

PR81 is an anti-MUC1 monoclonal antibody (MAb) which was generated against human MUC1 mucin that reacted with breast cancerous tissue, MUC1 positive cell line (MCF-7, BT-20, and T-4 7 D), and synthetic peptide, including the tandem repeat sequence of MUC1. Here we characterized the binding properties of PR81 against the tandem repeat of MUC1 by two different epitope mapping techniques, namely, PEPSCAN and phage display. Epitope mapping of PR81 MAb by PEPSCAN revealed a minimal consensus binding sequence, PDTRP, which is found on MUC1 peptide as the most important epitope. Using the phage display peptide library, we identified the motif PD(T/S/G)RP as an epitope and the motif AVGLSPDGSRGV as a mimotope recognized by PR81. Results of these two methods showed that the two residues, arginine and aspartic acid, have important roles in antibody binding and threonine can be substituted by either glycine or serine. These results may be of importance in tailor making antigens used in immunoassay.

Killing of melanoma cells and their metastases by human lactoferricin derivatives requires interaction with the cancer marker phosphatidylserine.

PubMed

Riedl, Sabrina; Rinner, Beate; Schaider, Helmut; Lohner, Karl; Zweytick, Dagmar

2014-10-01

Despite favorable advancements in therapy cancer is still not curative in many cases, which is often due to inadequate specificity for tumor cells. In this study derivatives of a short cationic peptide derived from the human host defense peptide lactoferricin were optimized in their selective toxicity towards cancer cells. We proved that the target of these peptides is the negatively charged membrane lipid phosphatidylserine (PS), specifically exposed on the surface of cancer cells. We have studied the membrane interaction of three peptides namely LF11-322, its N-acyl derivative 6-methyloctanoyl-LF11-322 and its retro repeat derivative R(etro)-DIM-P-LF11-322 with liposomes mimicking cancerous and non-cancerous cell membranes composed of PS and phosphatidylcholine (PC), respectively. Calorimetric and permeability studies showed that N-acylation and even more the repeat derivative of LF11-322 leads to strongly improved interaction with the cancer mimic PS, whereas only the N-acyl derivative also slightly affects PC. Tryptophan fluorescence of selective peptide R-DIM-P-LF11-322 revealed specific peptide penetration into the PS membrane interface and circular dichroism showed change of its secondary structure by increase of proportion of β-sheets just in the presence of the cancer mimic. Data correlated with in vitro studies with cell lines of human melanomas, their metastases and melanocytes, revealing R-DIM-P-LF11-322 to exhibit strongly increased specificity for cancer cells. This indicates the need of high affinity to the target PS, a minimum length and net positive charge, an adequate but moderate hydrophobicity, and capability of adoption of a defined structure exclusively in presence of the target membrane for high antitumor activity.

No effect of the altered peptide ligand NBI-6024 on beta-cell residual function and insulin needs in new-onset type 1 diabetes.

PubMed

Walter, Markus; Philotheou, Areti; Bonnici, François; Ziegler, Anette-G; Jimenez, Roland

2009-11-01

This randomized, four-arm, placebo-controlled, dose-ranging phase 2 trial was conducted to determine whether repeated subcutaneous injections of the altered peptide ligand, NBI-6024, designed to inhibit autoreactive T-cells, improves beta-cell function in patients with recently diagnosed type 1 diabetes. A total of 188 patients, aged 10-35 years, with recently diagnosed type 1 diabetes were randomly assigned for a treatment consisting of the subcutaneous administration of placebo or 1, 0.5, or 0.1 mg NBI-6024 at baseline, weeks 2 and 4, and then monthly until month 24. Fasting, peak, and area under the curve (AUC) C-peptide concentrations during a 2-h mixed-meal tolerance test were measured at 3-month intervals during treatment. Immune function parameters (islet antibodies and CD4 and CD8 T-cells) were also studied. The mean peak C-peptide concentration at 24 months after study entry showed no significant difference between the groups treated with 0.1 mg (0.59 pmol/ml), 0.5 mg (0.57 pmol/ml), and 1.0 mg NBI-6024 (0.48 pmol/ml) and the placebo group (0.54 pmol/ml). Fasting, stimulated peak, and AUC C-peptide concentrations declined linearly in all groups by approximately 60% over the 24-month treatment period. The average daily insulin needs at month 24 were also comparable between the four groups. No treatment-related changes in islet antibodies and T cell numbers were observed. Treatment with altered peptide ligand NBI-6024 at repeated doses of 0.1, 0.5, or 1.0 mg did not improve or maintain beta-cell function.

Nuclear pore complex evolution: a trypanosome Mlp analogue functions in chromosomal segregation but lacks transcriptional barrier activity

PubMed Central

Holden, Jennifer M.; Koreny, Ludek; Obado, Samson; Ratushny, Alexander V.; Chen, Wei-Ming; Chiang, Jung-Hsien; Kelly, Steven; Chait, Brian T.; Aitchison, John D.; Rout, Michael P.; Field, Mark C.

2014-01-01

The nuclear pore complex (NPC) has dual roles in nucleocytoplasmic transport and chromatin organization. In many eukaryotes the coiled-coil Mlp/Tpr proteins of the NPC nuclear basket have specific functions in interactions with chromatin and defining specialized regions of active transcription, whereas Mlp2 associates with the mitotic spindle/NPC in a cell cycle–dependent manner. We previously identified two putative Mlp-related proteins in African trypanosomes, TbNup110 and TbNup92, the latter of which associates with the spindle. We now provide evidence for independent ancestry for TbNup92/TbNup110 and Mlp/Tpr proteins. However, TbNup92 is required for correct chromosome segregation, with knockout cells exhibiting microaneuploidy and lowered fidelity of telomere segregation. Further, TbNup92 is intimately associated with the mitotic spindle and spindle anchor site but apparently has minimal roles in control of gene transcription, indicating that TbNup92 lacks major barrier activity. TbNup92 therefore acts as a functional analogue of Mlp/Tpr proteins, and, together with the lamina analogue NUP-1, represents a cohort of novel proteins operating at the nuclear periphery of trypanosomes, uncovering complex evolutionary trajectories for the NPC and nuclear lamina. PMID:24600046

Abnormal Magnetic Field Effects on Electrogenerated Chemiluminescence

NASA Astrophysics Data System (ADS)

Pan, Haiping; Shen, Yan; Wang, Hongfeng; He, Lei; Hu, Bin

2015-03-01

We report abnormal magnetic field effects on electrogenerated chemiluminescence (MFEECL) based on triplet emission from the Ru(bpy)3Cl2-TPrA electrochemical system: the appearance of MFEECL after magnetic field ceases. In early studies the normal MFEECL have been observed from electrochemical systems during the application of magnetic field. Here, the abnormal MFEECL suggest that the activated charge-transfer [Ru(bpy)33+ … TPrA•] complexes may become magnetized in magnetic field and experience a long magnetic relaxation after removing magnetic field. Our analysis indicates that the magnetic relaxation can gradually increase the density of charge-transfer complexes within reaction region due to decayed magnetic interactions, leading to a positive component in the abnormal MFEECL. On the other hand, the magnetic relaxation facilitates an inverse conversion from triplets to singlets within charge-transfer complexes. The inverse triplet --> singlet conversion reduces the density of triplet light-emitting states through charge-transfer complexes and gives rise to a negative component in the abnormal MFEECL. The combination of positive and negative components can essentially lead to a non-monotonic profile in the abnormal MFEECL after ceasing magnetic field. Nevertheless, our experimental studies may reveal un-usual magnetic behaviors with long magnetic relaxation from the activated charge-transfer [Ru(bpy)33+ … TPrA•] complexes in solution at room temperature.

ST proteins, a new family of plant tandem repeat proteins with a DUF2775 domain mainly found in Fabaceae and Asteraceae.

PubMed

Albornos, Lucía; Martín, Ignacio; Iglesias, Rebeca; Jiménez, Teresa; Labrador, Emilia; Dopico, Berta

2012-11-07

Many proteins with tandem repeats in their sequence have been described and classified according to the length of the repeats: I) Repeats of short oligopeptides (from 2 to 20 amino acids), including structural cell wall proteins and arabinogalactan proteins. II) Repeats that range in length from 20 to 40 residues, including proteins with a well-established three-dimensional structure often involved in mediating protein-protein interactions. (III) Longer repeats in the order of 100 amino acids that constitute structurally and functionally independent units. Here we analyse ShooT specific (ST) proteins, a family of proteins with tandem repeats of unknown function that were first found in Leguminosae, and their possible similarities to other proteins with tandem repeats. ST protein sequences were only found in dicotyledonous plants, limited to several plant families, mainly the Fabaceae and the Asteraceae. ST mRNAs accumulate mainly in the roots and under biotic interactions. Most ST proteins have one or several Domain(s) of Unknown Function 2775 (DUF2775). All deduced ST proteins have a signal peptide, indicating that these proteins enter the secretory pathway, and the mature proteins have tandem repeat oligopeptides that share a hexapeptide (E/D)FEPRP followed by 4 partially conserved amino acids, which could determine a putative N-glycosylation signal, and a fully conserved tyrosine. In a phylogenetic tree, the sequences clade according to taxonomic group. A possible involvement in symbiosis and abiotic stress as well as in plant cell elongation is suggested, although different STs could play different roles in plant development. We describe a new family of proteins called ST whose presence is limited to the plant kingdom, specifically to a few families of dicotyledonous plants. They present 20 to 40 amino acid tandem repeat sequences with different characteristics (signal peptide, DUF2775 domain, conservative repeat regions) from the described group of 20 to 40 amino acid tandem repeat proteins and also from known cell wall proteins with repeat sequences. Several putative roles in plant physiology can be inferred from the characteristics found.

ST proteins, a new family of plant tandem repeat proteins with a DUF2775 domain mainly found in Fabaceae and Asteraceae

PubMed Central

2012-01-01

Background Many proteins with tandem repeats in their sequence have been described and classified according to the length of the repeats: I) Repeats of short oligopeptides (from 2 to 20 amino acids), including structural cell wall proteins and arabinogalactan proteins. II) Repeats that range in length from 20 to 40 residues, including proteins with a well-established three-dimensional structure often involved in mediating protein-protein interactions. (III) Longer repeats in the order of 100 amino acids that constitute structurally and functionally independent units. Here we analyse ShooT specific (ST) proteins, a family of proteins with tandem repeats of unknown function that were first found in Leguminosae, and their possible similarities to other proteins with tandem repeats. Results ST protein sequences were only found in dicotyledonous plants, limited to several plant families, mainly the Fabaceae and the Asteraceae. ST mRNAs accumulate mainly in the roots and under biotic interactions. Most ST proteins have one or several Domain(s) of Unknown Function 2775 (DUF2775). All deduced ST proteins have a signal peptide, indicating that these proteins enter the secretory pathway, and the mature proteins have tandem repeat oligopeptides that share a hexapeptide (E/D)FEPRP followed by 4 partially conserved amino acids, which could determine a putative N-glycosylation signal, and a fully conserved tyrosine. In a phylogenetic tree, the sequences clade according to taxonomic group. A possible involvement in symbiosis and abiotic stress as well as in plant cell elongation is suggested, although different STs could play different roles in plant development. Conclusions We describe a new family of proteins called ST whose presence is limited to the plant kingdom, specifically to a few families of dicotyledonous plants. They present 20 to 40 amino acid tandem repeat sequences with different characteristics (signal peptide, DUF2775 domain, conservative repeat regions) from the described group of 20 to 40 amino acid tandem repeat proteins and also from known cell wall proteins with repeat sequences. Several putative roles in plant physiology can be inferred from the characteristics found. PMID:23134664

Reductionist Approach in Peptide-Based Nanotechnology.

PubMed

Gazit, Ehud

2018-06-20

The formation of ordered nanostructures by molecular self-assembly of proteins and peptides represents one of the principal directions in nanotechnology. Indeed, polyamides provide superior features as materials with diverse physical properties. A reductionist approach allowed the identification of extremely short peptide sequences, as short as dipeptides, which could form well-ordered amyloid-like β-sheet-rich assemblies comparable to supramolecular structures made of much larger proteins. Some of the peptide assemblies show remarkable mechanical, optical, and electrical characteristics. Another direction of reductionism utilized a natural noncoded amino acid, α-aminoisobutryic acid, to form short superhelical assemblies. The use of this exceptional helix inducer motif allowed the fabrication of single heptad repeats used in various biointerfaces, including their use as surfactants and DNA-binding agents. Two additional directions of the reductionist approach include the use of peptide nucleic acids (PNAs) and coassembly techniques. The diversified accomplishments of the reductionist approach, as well as the exciting future advances it bears, are discussed.

GRIM REAPER peptide binds to receptor kinase PRK5 to trigger cell death in Arabidopsis

PubMed Central

Wrzaczek, Michael; Vainonen, Julia P; Stael, Simon; Tsiatsiani, Liana; Help-Rinta-Rahko, Hanna; Gauthier, Adrien; Kaufholdt, David; Bollhöner, Benjamin; Lamminmäki, Airi; Staes, An; Gevaert, Kris; Tuominen, Hannele; Van Breusegem, Frank; Helariutta, Ykä; Kangasjärvi, Jaakko

2015-01-01

Recognition of extracellular peptides by plasma membrane-localized receptor proteins is commonly used in signal transduction. In plants, very little is known about how extracellular peptides are processed and activated in order to allow recognition by receptors. Here, we show that induction of cell death in planta by a secreted plant protein GRIM REAPER (GRI) is dependent on the activity of the type II metacaspase METACASPASE-9. GRI is cleaved by METACASPASE-9 in vitro resulting in the release of an 11 amino acid peptide. This peptide bound in vivo to the extracellular domain of the plasma membrane-localized, atypical leucine-rich repeat receptor-like kinase POLLEN-SPECIFIC RECEPTOR-LIKE KINASE 5 (PRK5) and was sufficient to induce oxidative stress/ROS-dependent cell death. This shows a signaling pathway in plants from processing and activation of an extracellular protein to recognition by its receptor. PMID:25398910

GRIM REAPER peptide binds to receptor kinase PRK5 to trigger cell death in Arabidopsis.

PubMed

Wrzaczek, Michael; Vainonen, Julia P; Stael, Simon; Tsiatsiani, Liana; Help-Rinta-Rahko, Hanna; Gauthier, Adrien; Kaufholdt, David; Bollhöner, Benjamin; Lamminmäki, Airi; Staes, An; Gevaert, Kris; Tuominen, Hannele; Van Breusegem, Frank; Helariutta, Ykä; Kangasjärvi, Jaakko

2015-01-02

Recognition of extracellular peptides by plasma membrane-localized receptor proteins is commonly used in signal transduction. In plants, very little is known about how extracellular peptides are processed and activated in order to allow recognition by receptors. Here, we show that induction of cell death in planta by a secreted plant protein GRIM REAPER (GRI) is dependent on the activity of the type II metacaspase METACASPASE-9. GRI is cleaved by METACASPASE-9 in vitro resulting in the release of an 11 amino acid peptide. This peptide bound in vivo to the extracellular domain of the plasma membrane-localized, atypical leucine-rich repeat receptor-like kinase POLLEN-SPECIFIC RECEPTOR-LIKE KINASE 5 (PRK5) and was sufficient to induce oxidative stress/ROS-dependent cell death. This shows a signaling pathway in plants from processing and activation of an extracellular protein to recognition by its receptor. © 2014 The Authors.

Quantification of Cell-Penetrating Peptide Associated with Polymeric Nanoparticles Using Isobaric-Tagging and MALDI-TOF MS/MS

NASA Astrophysics Data System (ADS)

Chiu, Jasper Z. S.; Tucker, Ian G.; McDowell, Arlene

2016-11-01

High sensitivity quantification of the putative cell-penetrating peptide di-arginine-histidine (RRH) associated with poly (ethyl-cyanoacrylate) (PECA) nanoparticles was achieved without analyte separation, using a novel application of isobaric-tagging and high matrix-assisted laser desorption/ionization coupled to time-of-flight (MALDI-TOF) mass spectrometry. Isobaric-tagging reaction equilibrium was reached after 5 min, with 90% or greater RRH peptide successfully isobaric-tagged after 60 min. The accuracy was greater than 90%, which indicates good reliability of using isobaric-tagged RRH as an internal standard for RRH quantification. The sample intra- and inter-spot coefficients of variations were less than 11%, which indicate good repeatability. The majority of RRH peptides in the nanoparticle formulation were physically associated with the nanoparticles (46.6%), whereas only a small fraction remained unassociated (13.7%). The unrecovered RRH peptide (~40%) was assumed to be covalently associated with PECA nanoparticles.

Design of a minimal protein oligomerization domain by a structural approach.

PubMed

Burkhard, P; Meier, M; Lustig, A

2000-12-01

Because of the simplicity and regularity of the alpha-helical coiled coil relative to other structural motifs, it can be conveniently used to clarify the molecular interactions responsible for protein folding and stability. Here we describe the de novo design and characterization of a two heptad-repeat peptide stabilized by a complex network of inter- and intrahelical salt bridges. Circular dichroism spectroscopy and analytical ultracentrifugation show that this peptide is highly alpha-helical and 100% dimeric tinder physiological buffer conditions. Interestingly, the peptide was shown to switch its oligomerization state from a dimer to a trimer upon increasing ionic strength. The correctness of the rational design principles used here is supported by details of the atomic structure of the peptide deduced from X-ray crystallography. The structure of the peptide shows that it is not a molten globule but assumes a unique, native-like conformation. This de novo peptide thus represents an attractive model system for the design of a molecular recognition system.

Design of a minimal protein oligomerization domain by a structural approach.

PubMed Central

Burkhard, P.; Meier, M.; Lustig, A.

2000-01-01

Because of the simplicity and regularity of the alpha-helical coiled coil relative to other structural motifs, it can be conveniently used to clarify the molecular interactions responsible for protein folding and stability. Here we describe the de novo design and characterization of a two heptad-repeat peptide stabilized by a complex network of inter- and intrahelical salt bridges. Circular dichroism spectroscopy and analytical ultracentrifugation show that this peptide is highly alpha-helical and 100% dimeric tinder physiological buffer conditions. Interestingly, the peptide was shown to switch its oligomerization state from a dimer to a trimer upon increasing ionic strength. The correctness of the rational design principles used here is supported by details of the atomic structure of the peptide deduced from X-ray crystallography. The structure of the peptide shows that it is not a molten globule but assumes a unique, native-like conformation. This de novo peptide thus represents an attractive model system for the design of a molecular recognition system. PMID:11206050

Cardiovascular regulation in humans in response to oscillatory lower body negative pressure

NASA Technical Reports Server (NTRS)

Levenhagen, D. K.; Evans, J. M.; Wang, M.; Knapp, C. F.

1994-01-01

The frequency response characteristics of human cardiovascular regulation during hypotensive stress have not been determined. We therefore exposed 10 male volunteers to seven frequencies (0.004-0.1 Hz) of oscillatory lower body negative pressure (OLBNP; 0-50 mmHg). Fourier spectra of arterial pressure (AP), central venous pressure (CVP), stroke volume (SV), cardiac output (CO), heart rate (HR), and total peripheral resistance (TPR) were determined and first harmonic mean, amplitude, and phase angles with respect to OLBNP are presented. AP was relatively well regulated as demonstrated by small oscillations in half amplitude (3.5 mmHg) that were independent of OLBNP frequency and similar to unstressed control spectra. Due to the biomechanics of the system, the magnitudes of oscillations in calf circumference (CC) and CVP decreased with increasing frequency; therefore, we normalized responses by these indexes of the fluid volume shifted. The ratios of oscillations in AP to oscillations in CC increased by an order of magnitude, whereas oscillations in CVP to oscillations in CC and oscillations in AP to oscillations in CVP both tripled between 0.004 and 0.1 Hz. Therefore, even though the amount of fluid shifted by OLBNP decreased with increasing frequency, the magnitude of both CVP and AP oscillations per volume of fluid shifted increased (peaking at 0.08 Hz). The phase relationships between variables, particularly the increasing lags in SV and TPR, but not CVP, indicated that efferent responses with lags of 5-6 s could account for the observed responses. We conclude that, at frequencies below 0.02 Hz, the neural system of humans functioned optimally in regulating AP; OLBNP-induced decreases in SV (by as much as 50%) were counteracted by appropriate oscillations in HR and TPR responses. As OLBNP frequency increased, SV, TPR, and HR oscillations increasingly lagged the input and became less optimally timed for AP regulation.

Cerebrospinal Fluid Biomarkers in Highly Exposed PM2.5 Urbanites: The Risk of Alzheimer's and Parkinson's Diseases in Young Mexico City Residents.

PubMed

Calderón-Garcidueñas, Lilian; Avila-Ramírez, José; Calderón-Garcidueñas, Ana; González-Heredia, Tonatiuh; Acuña-Ayala, Hilda; Chao, Chih-Kai; Thompson, Charles; Ruiz-Ramos, Rubén; Cortés-González, Victor; Martínez-Martínez, Luz; García-Pérez, Mario Alberto; Reis, Jacques; Mukherjee, Partha S; Torres-Jardón, Ricardo; Lachmann, Ingolf

2016-09-06

Exposure to fine particulate matter (PM2.5) and ozone (O3) above US EPA standards is associated with Alzheimer's disease (AD) risk, while Mn toxicity induces parkinsonism. Mexico City Metropolitan Area (MCMA) children have pre- and postnatal sustained and high exposures to PM2.5, O3, polycyclic aromatic hydrocarbons, and metals. Young MCMA residents exhibit frontal tau hyperphosphorylation and amyloid-β (Aβ)1 - 42 diffuse plaques, and aggregated and hyperphosphorylated α-synuclein in olfactory nerves and key brainstem nuclei. We measured total prion protein (TPrP), total tau (T-tau), tau phosphorylated at threonine 181 (P-Tau), Aβ1-42, α-synuclein (t-α-syn and d-α-synuclein), BDNF, insulin, leptin, and/or inflammatory mediators, in 129 normal CSF samples from MCMA and clean air controls. Aβ1-42 and BDNF concentrations were significantly lower in MCMA children versus controls (p = 0.005 and 0.02, respectively). TPrP increased with cumulative PM2.5 up to 5 μg/m3 and then decreased, regardless of cumulative value or age (R2 = 0.56). TPrP strongly correlated with T-Tau and P-Tau, while d-α-synuclein showed a significant correlation with TNFα, IL10, and IL6 in MCMA children. Total synuclein showed an increment in childhood years related to cumulated PM2.5, followed by a decrease after age 12 years (R2 = 0.47), while d-α-synuclein exhibited a tendency to increase with cumulated PM2.5 (R2 = 0.30). CSF Aβ1-42, BDNF, α-synuclein, and TPrP changes are evolving in young MCMA urbanites historically showing underperformance in cognitive processes, odor identification deficits, downregulation of frontal cellular PrP, and neuropathological AD and PD hallmarks. Neuroprotection of young MCMA residents ought to be a public health priority.

Influence of physicochemical treatments on iron-based spent catalyst for catalytic oxidation of toluene.

PubMed

Kim, Sang Chai; Shim, Wang Geun

2008-06-15

The catalytic oxidation of toluene was studied over an iron-based spent and regenerated catalysts. Air, hydrogen, or four different acid solutions (oxalic acid (C2H2O4), citric acid (C6H8O7), acetic acid (CH3COOH), and nitric acid (HNO3)) were employed to regenerate the spent catalyst. The properties of pretreated spent catalyst were characterized by the Brunauer Emmett Teller (BET), inductively coupled plasma (ICP), temperature programmed reduction (TPR), and X-ray diffraction (XRD) analyses. The air pretreatment significantly enhanced the catalytic activity of the spent catalyst in the pretreatment temperature range of 200-400 degrees C, but its catalytic activity diminished at the pretreatment temperature of 600 degrees C. The catalytic activity sequence with respect to the air pretreatment temperatures was 400 degrees C>200 degrees C>parent>600 degrees C. The TPR results indicated that the catalytic activity was correlated with both the oxygen mobility and the amount of available oxygen on the catalyst. In contrast, the hydrogen pretreatment had a negative effect on the catalytic activity, and toluene conversion decreased with increasing pretreatment temperatures (200-600 degrees C). The XRD and TPR results confirmed the formation of metallic iron which had a negative effect on the catalytic activity with increasing pretreatment temperature. The acid pretreatment improved the catalytic activity of the spent catalyst. The catalytic activity sequence with respect to different acids pretreatment was found to be oxalic acid>citric acid>acetic acid>or=nitric acid>parent. The TPR results of acid pretreated samples showed an increased amount of available oxygen which gave a positive effect on the catalytic activity. Accordingly, air or acid pretreatments were more promising methods of regenerating the iron-based spent catalyst. In particular, the oxalic acid pretreatment was found to be most effective in the formation of FeC2O4 species which contributed highly to the catalytic combustion of toluene.

The Receptor That Tames the Innate Immune Response

PubMed Central

Brines, Michael; Cerami, Anthony

2012-01-01

Tissue injury, hypoxia and significant metabolic stress activate innate immune responses driven by tumor necrosis factor (TNF)-α and other proinflammatory cytokines that typically increase damage surrounding a lesion. In a compensatory protective response, erythropoietin (EPO) is synthesized in surrounding tissues, which subsequently triggers antiinflammatory and antiapoptotic processes that delimit injury and promote repair. What we refer to as the sequelae of injury or disease are often the consequences of this intentionally discoordinated, primitive system that uses a “scorched earth” strategy to rid the invader at the expense of a serious lesion. The EPO-mediated tissue-protective system depends on receptor expression that is upregulated by inflammation and hypoxia in a distinctive temporal and spatial pattern. The tissue-protective receptor (TPR) is generally not expressed by normal tissues but becomes functional immediately after injury. In contrast to robust and early receptor expression within the immediate injury site, EPO production is delayed, transient and relatively weak. The functional EPO receptor that attenuates tissue injury is distinct from the hematopoietic receptor responsible for erythropoiesis. On the basis of current evidence, the TPR is composed of the β common receptor subunit (CD131) in combination with the same EPO receptor subunit that is involved in erythropoiesis. Additional receptors, including that for the vascular endothelial growth factor, also appear to be a component of the TPR in some tissues, for example, the endothelium. The discoordination of the EPO response system and its relative weakness provide a window of opportunity to intervene with the exogenous ligand. Recently, molecules were designed that preferentially activate only the TPR and thus avoid the potential adverse consequences of activating the hematopoietic receptor. On administration, these agents successfully substitute for a relative deficiency of EPO production in damaged tissues in multiple animal models of disease and may pave the way to effective treatment of a wide variety of insults that cause tissue injury, leading to profoundly expanded lesions and attendant, irreversible sequelae. PMID:22183892

The receptor that tames the innate immune response.

PubMed

Brines, Michael; Cerami, Anthony

2012-05-09

Tissue injury, hypoxia and significant metabolic stress activate innate immune responses driven by tumor necrosis factor (TNF)-α and other proinflammatory cytokines that typically increase damage surrounding a lesion. In a compensatory protective response, erythropoietin (EPO) is synthesized in surrounding tissues, which subsequently triggers antiinflammatory and antiapoptotic processes that delimit injury and promote repair. What we refer to as the sequelae of injury or disease are often the consequences of this intentionally discoordinated, primitive system that uses a "scorched earth" strategy to rid the invader at the expense of a serious lesion. The EPO-mediated tissue-protective system depends on receptor expression that is upregulated by inflammation and hypoxia in a distinctive temporal and spatial pattern. The tissue-protective receptor (TPR) is generally not expressed by normal tissues but becomes functional immediately after injury. In contrast to robust and early receptor expression within the immediate injury site, EPO production is delayed, transient and relatively weak. The functional EPO receptor that attenuates tissue injury is distinct from the hematopoietic receptor responsible for erythropoiesis. On the basis of current evidence, the TPR is composed of the β common receptor subunit (CD131) in combination with the same EPO receptor subunit that is involved in erythropoiesis. Additional receptors, including that for the vascular endothelial growth factor, also appear to be a component of the TPR in some tissues, for example, the endothelium. The discoordination of the EPO response system and its relative weakness provide a window of opportunity to intervene with the exogenous ligand. Recently, molecules were designed that preferentially activate only the TPR and thus avoid the potential adverse consequences of activating the hematopoietic receptor. On administration, these agents successfully substitute for a relative deficiency of EPO production in damaged tissues in multiple animal models of disease and may pave the way to effective treatment of a wide variety of insults that cause tissue injury, leading to profoundly expanded lesions and attendant, irreversible sequelae.

Structurally Ordered Nanowire Formation from Co-Assembly of DNA Origami and Collagen-Mimetic Peptides

DOE Office of Scientific and Technical Information (OSTI.GOV)

Jiang, Tao; Meyer, Travis A.; Modlin, Charles

In this paper, we describe the co-assembly of two different building units: collagen-mimetic peptides and DNA origami. Two peptides CP ++ and sCP ++ are designed with a sequence comprising a central block (Pro-Hyp-Gly) and two positively charged domains (Pro-Arg-Gly) at both N- and C-termini. Co-assembly of peptides and DNA origami two-layer (TL) nanosheets affords the formation of one-dimensional nanowires with repeating periodicity of similar to 10 nm. Structural analyses suggest a face-to-face stacking of DNA nanosheets with peptides aligned perpendicularly to the sheet surfaces. We demonstrate the potential of selective peptide-DNA association between face-to-face and edge-to-edge packing by tailoringmore » the size of DNA nanostructures. Finally, this study presents an attractive strategy to create hybrid biomolecular assemblies from peptide and DNA-based building blocks that takes advantage of the intrinsic chemical and physical properties of the respective components to encode structural and, potentially, functional complexity within readily accessible biomimetic materials.« less

Structurally Ordered Nanowire Formation from Co-Assembly of DNA Origami and Collagen-Mimetic Peptides

DOE PAGES

Jiang, Tao; Meyer, Travis A.; Modlin, Charles; ...

2017-09-26

In this paper, we describe the co-assembly of two different building units: collagen-mimetic peptides and DNA origami. Two peptides CP ++ and sCP ++ are designed with a sequence comprising a central block (Pro-Hyp-Gly) and two positively charged domains (Pro-Arg-Gly) at both N- and C-termini. Co-assembly of peptides and DNA origami two-layer (TL) nanosheets affords the formation of one-dimensional nanowires with repeating periodicity of similar to 10 nm. Structural analyses suggest a face-to-face stacking of DNA nanosheets with peptides aligned perpendicularly to the sheet surfaces. We demonstrate the potential of selective peptide-DNA association between face-to-face and edge-to-edge packing by tailoringmore » the size of DNA nanostructures. Finally, this study presents an attractive strategy to create hybrid biomolecular assemblies from peptide and DNA-based building blocks that takes advantage of the intrinsic chemical and physical properties of the respective components to encode structural and, potentially, functional complexity within readily accessible biomimetic materials.« less

Polyclonal cell activity of a repeat peptide derived from the sequence of an 85-kilodalton surface protein of Trypanosoma cruzi trypomastigotes.

PubMed Central

Pestel, J; Defoort, J P; Gras-Masse, H; Afchain, D; Capron, A; Tartar, A; Ouaissi, A

1992-01-01

Some in vitro and in vivo biological activities of an octadecapeptide derived from an 85-kDa surface protein of Trypanosoma cruzi trypomastigote were studied. The peptide coupled to a carrier protein induced the proliferative response of lymph node cells from mice immunized with various antigens. Moreover, sera from mice immunized with the coupled peptide were found to contain antibodies against a number of self and nonself antigens: fibronectin, bovine serum albumin, myosin, tetanus toxoid, ovalbumin, keyhole limpet hemocyanin, and DNA. These results are discussed in the context of Chagas' disease immunopathology. PMID:1730508

C-peptide inhibitors of Ebola virus glycoprotein-mediated cell entry: effects of conjugation to cholesterol and side chain-side chain crosslinking.

PubMed

Higgins, Chelsea D; Koellhoffer, Jayne F; Chandran, Kartik; Lai, Jonathan R

2013-10-01

We previously described potent inhibition of Ebola virus entry by a 'C-peptide' based on the GP2 C-heptad repeat region (CHR) targeted to endosomes ('Tat-Ebo'). Here, we report the synthesis and evaluation of C-peptides conjugated to cholesterol, and Tat-Ebo analogs containing covalent side chain-side chain crosslinks to promote α-helical conformation. We found that the cholesterol-conjugated C-peptides were potent inhibitors of Ebola virus glycoprotein (GP)-mediated cell entry (~10(3)-fold reduction in infection at 40 μM). However, this mechanism of inhibition is somewhat non-specific because the cholesterol-conjugated peptides also inhibited cell entry mediated by vesicular stomatitis virus glycoprotein G. One side chain-side chain crosslinked peptide had moderately higher activity than the parent compound Tat-Ebo. Circular dichroism revealed that the cholesterol-conjugated peptides unexpectedly formed a strong α-helical conformation that was independent of concentration. Side chain-side chain crosslinking enhanced α-helical stability of the Tat-Ebo variants, but only at neutral pH. These result provide insight into mechanisms of C-peptide inhibiton of Ebola virus GP-mediated cell entry. Copyright © 2013 Elsevier Ltd. All rights reserved.

Sel1-like repeat proteins in signal transduction.

PubMed

Mittl, Peer R E; Schneider-Brachert, Wulf

2007-01-01

Solenoid proteins, which are distinguished from general globular proteins by their modular architectures, are frequently involved in signal transduction pathways. Proteins from the tetratricopeptide repeat (TPR) and Sel1-like repeat (SLR) families share similar alpha-helical conformations but different consensus sequence lengths and superhelical topologies. Both families are characterized by low sequence similarity levels, rendering the identification of functional homologous difficult. Therefore current knowledge of the molecular and cellular functions of the SLR proteins Sel1, Hrd3, Chs4, Nif1, PodJ, ExoR, AlgK, HcpA, Hsp12, EnhC, LpnE, MotX, and MerG has been reviewed. Although SLR proteins possess different cellular functions they all seem to serve as adaptor proteins for the assembly of macromolecular complexes. Sel1, Hrd3, Hsp12 and LpnE are activated under cellular stress. The eukaryotic Sel1 and Hrd3 proteins are involved in the ER-associated protein degradation, whereas the bacterial LpnE, EnhC, HcpA, ExoR, and AlgK proteins mediate the interactions between bacterial and eukaryotic host cells. LpnE and EnhC are responsible for the entry of L. pneumophila into epithelial cells and macrophages. ExoR from the symbiotic microorganism S. melioti and AlgK from the pathogen P. aeruginosa regulate exopolysaccaride synthesis. Nif1 and Chs4 from yeast are responsible for the regulation of mitosis and septum formation during cell division, respectively, and PodJ guides the cellular differentiation during the cell cycle of the bacterium C. crescentus. Taken together the SLR motif establishes a link between signal transduction pathways from eukaryotes and bacteria. The SLR motif is so far absent from archaea. Therefore the SLR could have developed in the last common ancestor between eukaryotes and bacteria.

Characterization of a possible amyloidogenic precursor in glutamine-repeat neurodegenerative diseases.

PubMed

Armen, Roger S; Bernard, Brady M; Day, Ryan; Alonso, Darwin O V; Daggett, Valerie

2005-09-20

Several neurodegenerative diseases are linked to expanded repeats of glutamine residues, which lead to the formation of amyloid fibrils and neuronal death. The length of the repeats correlates with the onset of Huntington's disease, such that healthy individuals have <38 residues and individuals with >38 repeats exhibit symptoms. Because it is difficult to obtain atomic-resolution structural information for poly(l-glutamine) (polyQ) in aqueous solution experimentally, we performed molecular dynamics simulations to investigate the conformational behavior of this homopolymer. In simulations of 20-, 40-, and 80-mer polyQ, we observed the formation of the "alpha-extended chain" conformation, which is characterized by alternating residues in the alpha(L) and alpha(R) conformations to yield a sheet. The structural transition from disordered random-coil conformations to the alpha-extended chain conformation exhibits modest length and temperature dependence, in agreement with the experimental observation that aggregation depends on length and temperature. We propose that fibril formation in polyQ may occur through an alpha-sheet structure, which was proposed by Pauling and Corey. Also, we propose an atomic-resolution model of how the inhibitory peptide QBP1 (polyQ-binding peptide 1) may bind to polyQ in an alpha-extended chain conformation to inhibit fibril formation.

Molecular characterization of long direct repeat (LDR) sequences expressing a stable mRNA encoding for a 35-amino-acid cell-killing peptide and a cis-encoded small antisense RNA in Escherichia coli.

PubMed

Kawano, Mitsuoki; Oshima, Taku; Kasai, Hiroaki; Mori, Hirotada

2002-07-01

Genome sequence analyses of Escherichia coli K-12 revealed four copies of long repetitive elements. These sequences are designated as long direct repeat (LDR) sequences. Three of the repeats (LDR-A, -B, -C), each approximately 500 bp in length, are located as tandem repeats at 27.4 min on the genetic map. Another copy (LDR-D), 450 bp in length and nearly identical to LDR-A, -B and -C, is located at 79.7 min, a position that is directly opposite the position of LDR-A, -B and -C. In this study, we demonstrate that LDR-D encodes a 35-amino-acid peptide, LdrD, the overexpression of which causes rapid cell killing and nucleoid condensation of the host cell. Northern blot and primer extension analysis showed constitutive transcription of a stable mRNA (approximately 370 nucleotides) encoding LdrD and an unstable cis-encoded antisense RNA (approximately 60 nucleotides), which functions as a trans-acting regulator of ldrD translation. We propose that LDR encodes a toxin-antitoxin module. LDR-homologous sequences are not pre-sent on any known plasmids but are conserved in Salmonella and other enterobacterial species.

High-resolution structures of a heterochiral coiled coil

DOE PAGES

Mortenson, David E.; Steinkruger, Jay D.; Kreitler, Dale F.; ...

2015-10-12

Interactions between polypeptide chains containing amino acid residues with opposite absolute configurations have long been a source of interest and speculation, but there is very little structural information for such heterochiral associations. The need to address this lacuna has grown in recent years because of increasing interest in the use of peptides generated from D amino acids (D peptides) as specific ligands for natural proteins, e.g., to inhibit deleterious protein–protein interactions. Coiled–coil interactions, between or among α-helices, represent the most common tertiary and quaternary packing motif in proteins. Heterochiral coiled–coil interactions were predicted over 50 years ago by Crick, andmore » limited experimental data obtained in solution suggest that such interactions can indeed occur. To address the dearth of atomic-level structural characterization of heterochiral helix pairings, we report in this paper two independent crystal structures that elucidate coiled-coil packing between L- and D-peptide helices. Both structures resulted from racemic crystallization of a peptide corresponding to the transmembrane segment of the influenza M2 protein. Networks of canonical knobs-into-holes side-chain packing interactions are observed at each helical interface. Finally, however, the underlying patterns for these heterochiral coiled coils seem to deviate from the heptad sequence repeat that is characteristic of most homochiral analogs, with an apparent preference for a hendecad repeat pattern.« less

Identification of a "glycine-loop"-like coiled structure in the 34 AA Pro,Gly,Met repeat domain of the biomineral-associated protein, PM27.

PubMed

Wustman, Brandon A; Santos, Rudolpho; Zhang, Bo; Evans, John Spencer

2002-12-05

Fracture resistance in biomineralized structures has been linked to the presence of proteins, some of which possess sequences that are associated with elastic behavior. One such protein superfamily, the Pro,Gly-rich sea urchin intracrystalline spicule matrix proteins, form protein-protein supramolecular assemblies that modify the microstructure and fracture-resistant properties of the calcium carbonate mineral phase within embryonic sea urchin spicules and adult sea urchin spines. In this report, we detail the identification of a repetitive keratin-like "glycine-loop"- or coil-like structure within the 34-AA (AA: amino acid) N-terminal domain, (PGMG)(8)PG, of the spicule matrix protein, PM27. The identification of this repetitive structural motif was accomplished using two capped model peptides: a 9-AA sequence, GPGMGPGMG, and a 34-AA peptide representing the entire motif. Using CD, NMR spectrometry, and molecular dynamics simulated annealing/minimization simulations, we have determined that the 9-AA model peptide adopts a loop-like structure at pH 7.4. The structure of the 34-AA polypeptide resembles a coil structure consisting of repeating loop motifs that do not exhibit long-range ordering. Given that loop structures have been associated with protein elastic behavior and protein motion, it is plausible that the 34-AA Pro,Gly,Met repeat sequence motif in PM27 represents a putative elastic or mobile domain. Copyright 2002 Wiley Periodicals, Inc.

CALCULATIONS OF SHUTDOWN DOSE RATE FOR THE TPR SPECTROMETER OF THE HIGH-RESOLUTION NEUTRON SPECTROMETER FOR ITER.

PubMed

Wójcik-Gargula, A; Tracz, G; Scholz, M

2017-12-13

This work presents results of the calculations performed in order to predict the neutron-induced activity in structural materials that are considered to be using at the TPR spectrometer-one of the detection system of the High-Resolution Neutron Spectrometer for ITER. An attempt has been made to estimate the shutdown dose rates in a Cuboid #1 and to check if they satisfy ICRP regulatory requirements for occupational exposure to radiation and ITER nuclear safety regulations for areas with personal access. The results were obtained by the MCNP and FISPACT-II calculations. © The Author 2017. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com.

Discovery of a polystyrene binding peptide isolated from phage display library and its application in peptide immobilization.

PubMed

Qiang, Xu; Sun, Keyong; Xing, Lijun; Xu, Yifeng; Wang, Hong; Zhou, Zhengpin; Zhang, Juan; Zhang, Fang; Caliskan, Bilgen; Wang, Min; Qiu, Zheng

2017-06-01

Phage peptide display is a powerful technique for discovery of various target-specific ligands. However, target-unrelated peptides can often be obtained and cause ambiguous results. Peptide PB-TUP has been isolated repeatedly in our laboratory on different targets and we conducted a research on PB-TUP phage to investigate their binding properties and rate of propagation. ELISA and phage recovery assay demonstrated that PB-TUP phage had a significant superior affinity to polystyrene solid surface compared with control phage clones. In this study, some incidental bindings are excluded like blocking agents and non-specific binding of secondary antibodies. Propagation rate assays of the selected phage clones showed that the growth rate of PB-TUP phage was not superior to the control phages. Furthermore, the binding of PB-TUB to polystyrene was concentration dependent and varied with solution pH. Molecular modeling revealed that stable structures of α-helix and β-turn may contribute to the binding of PB-TUP to polystyrene plate. The PB-TUP sequence was fused to the N-terminus of peptide P2 and the fusion peptide significantly increased the binding affinity to polystyrene. The fusion peptide also enhanced the cell adhesion ability of peptide P2 with human umbilical vein endothelial cell (HUVEC). The addition of the polystyrene binding peptide provided a convenient method for peptide immobilization.

Removing Cross-Linked Telopeptides Enhances the Production of Low-Molecular-Weight Collagen Peptides from Spent Hens.

PubMed

Hong, Hui; Chaplot, Shreyak; Chalamaiah, Meram; Roy, Bimol C; Bruce, Heather L; Wu, Jianping

2017-08-30

The low-molecular-weight (LMW) peptides derived from collagen have shown a potential for various nutritional and pharmaceutical applications. However, production of LMW peptides from vertebrate collagen remains a challenge. Herein, we report a new method to produce LMW collagen peptides using pepsin pretreatment that removed cross-linked telopeptides in collagen molecules. After the pretreatment, the proportion of LMW collagen peptides (<1.4 kDa) that were obtained from pepsin-soluble collagen increased to 32.59% compared to heat-soluble collagen peptides (16.10%). Fourier transform infrared spectroscopy results indicated that telopeptide cleavage retained the triple-helical conformation of collagen. Liquid chromatography-tandem mass spectrometry analysis suggested that Gly-X-Y (X is often proline, while Y is either hydroxyproline or hydroxylysine) repeats were not the main factors that hindered the enzymatic hydrolysis of collagen molecules. However, cross-link quantification demonstrated that trivalent cross-links that included pyridinolines and pyrroles were the primary obstacles to producing small peptides from collagen of spent hens. This study demonstrated for the first time that removing cross-linked telopeptides could enhance the production of LMW peptides from spent hen collagen, which is also of interest to manufacturers who produce LMW collagen peptides from other vertebrate animals, such as bovids and porcids.

Inhibition of Nipah virus infection in vivo: targeting an early stage of paramyxovirus fusion activation during viral entry.

PubMed

Porotto, Matteo; Rockx, Barry; Yokoyama, Christine C; Talekar, Aparna; Devito, Ilaria; Palermo, Laura M; Liu, Jie; Cortese, Riccardo; Lu, Min; Feldmann, Heinz; Pessi, Antonello; Moscona, Anne

2010-10-28

In the paramyxovirus cell entry process, receptor binding triggers conformational changes in the fusion protein (F) leading to viral and cellular membrane fusion. Peptides derived from C-terminal heptad repeat (HRC) regions in F have been shown to inhibit fusion by preventing formation of the fusogenic six-helix bundle. We recently showed that the addition of a cholesterol group to HRC peptides active against Nipah virus targets these peptides to the membrane where fusion occurs, dramatically increasing their antiviral effect. In this work, we report that unlike the untagged HRC peptides, which bind to the postulated extended intermediate state bridging the viral and cell membranes, the cholesterol tagged HRC-derived peptides interact with F before the fusion peptide inserts into the target cell membrane, thus capturing an earlier stage in the F-activation process. Furthermore, we show that cholesterol tagging renders these peptides active in vivo: the cholesterol-tagged peptides cross the blood brain barrier, and effectively prevent and treat in an established animal model what would otherwise be fatal Nipah virus encephalitis. The in vivo efficacy of cholesterol-tagged peptides, and in particular their ability to penetrate the CNS, suggests that they are promising candidates for the prevention or therapy of infection by Nipah and other lethal paramyxoviruses.

Unstable power threatens the powerful and challenges the powerless: evidence from cardiovascular markers of motivation.

PubMed

Scheepers, Daan; Röell, Charlotte; Ellemers, Naomi

2015-01-01

Possessing social power has psychological and biological benefits. For example, during task interactions, people high in power are more likely to display a benign cardiovascular (CV) response pattern indicative of "challenge" whereas people low in power are more likely to display a maladaptive CV pattern indicative of "threat" (Scheepers et al., 2012). Challenge is marked by high cardiac output (CO) and low total peripheral resistance (TPR), while threat is marked by low CO and high TPR (Blascovich and Mendes, 2010). In the current work we addressed a possible moderator of the power-threat/challenge relationship, namely the stability of power. We examined the influence of the stability of power (roles could or could not change) on CV responses during a dyadic task where one person was the "chief designer" (high power) and one person was the "assistant" (low power). During the task, different CV-measures were taken [CO, TPR, heart rate, pre-ejection period). Whereas participants in the unstable low power condition showed a stronger tendency toward challenge, participants in the unstable high power condition showed a stronger tendency toward threat. Moreover, participants in the stable low power condition showed CV signs of task disengagement. Results are discussed in terms of the importance of contextual variables in shaping the relationship between power and benign/maladaptive physiological responses.

Unstable power threatens the powerful and challenges the powerless: evidence from cardiovascular markers of motivation

PubMed Central

Scheepers, Daan; Röell, Charlotte; Ellemers, Naomi

2015-01-01

Possessing social power has psychological and biological benefits. For example, during task interactions, people high in power are more likely to display a benign cardiovascular (CV) response pattern indicative of “challenge” whereas people low in power are more likely to display a maladaptive CV pattern indicative of “threat” (Scheepers et al., 2012). Challenge is marked by high cardiac output (CO) and low total peripheral resistance (TPR), while threat is marked by low CO and high TPR (Blascovich and Mendes, 2010). In the current work we addressed a possible moderator of the power-threat/challenge relationship, namely the stability of power. We examined the influence of the stability of power (roles could or could not change) on CV responses during a dyadic task where one person was the “chief designer” (high power) and one person was the “assistant” (low power). During the task, different CV-measures were taken [CO, TPR, heart rate, pre-ejection period). Whereas participants in the unstable low power condition showed a stronger tendency toward challenge, participants in the unstable high power condition showed a stronger tendency toward threat. Moreover, participants in the stable low power condition showed CV signs of task disengagement. Results are discussed in terms of the importance of contextual variables in shaping the relationship between power and benign/maladaptive physiological responses. PMID:26074860

NUCLEAR PORE ANCHOR, the Arabidopsis Homolog of Tpr/Mlp1/Mlp2/Megator, Is Involved in mRNA Export and SUMO Homeostasis and Affects Diverse Aspects of Plant Development[W

PubMed Central

Xu, Xianfeng Morgan; Rose, Annkatrin; Muthuswamy, Sivaramakrishnan; Jeong, Sun Yong; Venkatakrishnan, Sowmya; Zhao, Qiao; Meier, Iris

2007-01-01

Vertebrate Tpr and its yeast homologs Mlp1/Mlp2, long coiled-coil proteins of nuclear pore inner basket filaments, are involved in mRNA export, telomere organization, spindle pole assembly, and unspliced RNA retention. We identified Arabidopsis thaliana NUCLEAR PORE ANCHOR (NUA) encoding a 237-kD protein with similarity to Tpr. NUA is located at the inner surface of the nuclear envelope in interphase and in the vicinity of the spindle in prometaphase. Four T-DNA insertion lines were characterized, which comprise an allelic series of increasing severity for several correlating phenotypes, such as early flowering under short days and long days, increased abundance of SUMO conjugates, altered expression of several flowering regulators, and nuclear accumulation of poly(A)+ RNA. nua mutants phenocopy mutants of EARLY IN SHORT DAYS4 (ESD4), an Arabidopsis SUMO protease concentrated at the nuclear periphery. nua esd4 double mutants resemble nua and esd4 single mutants, suggesting that the two proteins act in the same pathway or complex, supported by yeast two-hybrid interaction. Our data indicate that NUA is a component of nuclear pore-associated steps of sumoylation and mRNA export in plants and that defects in these processes affect the signaling events of flowering time regulation and additional developmental processes. PMID:17513499

Multimerized CHR-derived peptides as HIV-1 fusion inhibitors.

PubMed

Nomura, Wataru; Hashimoto, Chie; Suzuki, Takaharu; Ohashi, Nami; Fujino, Masayuki; Murakami, Tsutomu; Yamamoto, Naoki; Tamamura, Hirokazu

2013-08-01

To date, several HIV-1 fusion inhibitors based on the carboxy-terminal leucine/isoleucine heptad repeat (CHR) region of an HIV-1 envelope protein gp41 have been discovered. We have shown that a synthetic peptide mimetic of a trimer form of the CHR-derived peptide C34 has potent inhibitory activity against the HIV-1 fusion mechanism, compared to a monomer C34 peptide. The present study revealed that a dimeric form of C34 is evidently structurally critical for fusion inhibitors, and that the activity of multimerized CHR-derived peptides in fusion inhibition is affected by the properties of the unit peptides C34, SC34EK, and T20. The fluorescence-based study suggested that the N36-interactive sites of the C34 trimer, including hydrophobic residues, are exposed outside the trimer and that trimerization of C34 caused a remarkable increase in fusion inhibitory activity. The present results could be useful in the design of fusion inhibitors against viral infections which proceed via membrane fusion with host cells. Copyright © 2013 The Authors. Published by Elsevier Ltd.. All rights reserved.

VH1/BRL2 receptor-like kinase interacts with vascular-specific adaptor proteins VIT and VIK to influence leaf venation.

PubMed

Ceserani, Teresa; Trofka, Anna; Gandotra, Neeru; Nelson, Timothy

2009-03-01

VH1/BRL2 is a receptor-like kinase of the BRI1 family with a role in vascular development. In developing Arabidopsis leaves it is expressed first in ground cells and then becomes restricted to provascular and procambial cells as venation forms. We isolated proteins interacting with the activated (phosphorylated) cytoplasmic domain of VH1/BRL2, and found that most belong to three processes: proteasome activity, vesicle traffic and intracellular signal transduction. Two adaptor proteins are included that we named VIT [VH1-interacting tetratricopeptide repeat (TPR)-containing protein] and VIK (VH1-interacting kinase), which are co-expressed in the same cells as VH1/BRL2 at two distinct time points in vein differentiation. Mutation of either adaptor or of VH1 results in vein pattern defects and in alterations in response to auxin and brassinosteroids. We propose that these two adaptors facilitate the diversification and amplification of a ligand signal perceived by VH1/BRL2 in multiple downstream pathways affecting venation.

A 31-residue peptide induces aggregation of tau's microtubule-binding region in cells

NASA Astrophysics Data System (ADS)

Stöhr, Jan; Wu, Haifan; Nick, Mimi; Wu, Yibing; Bhate, Manasi; Condello, Carlo; Johnson, Noah; Rodgers, Jeffrey; Lemmin, Thomas; Acharya, Srabasti; Becker, Julia; Robinson, Kathleen; Kelly, Mark J. S.; Gai, Feng; Stubbs, Gerald; Prusiner, Stanley B.; Degrado, William F.

2017-09-01

The self-propagation of misfolded conformations of tau underlies neurodegenerative diseases, including Alzheimer's. There is considerable interest in discovering the minimal sequence and active conformational nucleus that defines this self-propagating event. The microtubule-binding region, spanning residues 244-372, reproduces much of the aggregation behaviour of tau in cells and animal models. Further dissection of the amyloid-forming region to a hexapeptide from the third microtubule-binding repeat resulted in a peptide that rapidly forms fibrils in vitro. We show that this peptide lacks the ability to seed aggregation of tau244-372 in cells. However, as the hexapeptide is gradually extended to 31 residues, the peptides aggregate more slowly and gain potent activity to induce aggregation of tau244-372 in cells. X-ray fibre diffraction, hydrogen-deuterium exchange and solid-state NMR studies map the beta-forming region to a 25-residue sequence. Thus, the nucleus for self-propagating aggregation of tau244-372 in cells is packaged in a remarkably small peptide.

Determination of hydroxyaromatic compounds in water by solid-phase microextraction coupled to high-performance liquid chromatography.

PubMed

Wu, Y C; Huang, S D

1999-03-12

Solid-phase microextraction (SPME) coupled with high-performance liquid chromatography (HPLC) for the analysis of hydroxyaromatic compounds is described. Three kinds of fibers [50 microns carbowax-templated resin (CW-TPR), 60 microns polydimethylsiloxane-divinylbenzene (PDMS-DVB) and 85 microns polyacrylate (PA) fibers] were evaluated. CW-TPR and PDMS-DVB were selected for further study. The parameters of the desorption procedure (such as desorption mode, the composition of the solvent for desorption and the duration of fiber soaking) were studied and optimized. The effect of the structure and physical properties of analytes, carryover, duration of absorption, temperature of absorption, pH and ionic strength of samples were also investigated. The method was applied to environmental samples (lake water) using a simple calibration curve.

Co-induction of hepatic IGF-I and progranulin mRNA by growth hormone in tilapia, Oreochromis mossambiccus.

PubMed

Chen, Mark Hung-Chih; Li, Yen-Hsing; Chang, Yvonne; Hu, Shao-Yang; Gong, Hong-Yi; Lin, Gen-Hwa; Chen, Thomas T; Wu, Jen-Leih

2007-01-15

Like IGF-I, progranulin (pgrn) is a growth factor involved in tumorigenesis and wound healing. We report here the identification and characterization of pgrn cDNA in tilapia and the regulation of its expression by growth hormone (GH). The tilapia pgrn cDNA was cloned by RT-PCR amplification, using gene specific oligonucleotides as amplification primers. The cDNA contains an open reading frame encoding a peptide of 206 amino acid residues (aa) that contains a presumptive signal peptide (23 aa) and two repeat units of granulin (grn, 51 and 52 aa, respectively) franked by a GAP of 49 aa and the carboxyl terminus with 31 aa. The two predicted grn peptides are arranged in tandem repeats interrupted by a GAP peptide. RT-PCR analysis revealed that high levels of prgn mRNA were present in several tissues such as spleen, gastric cecum, intestine, fat tissue, gill, kidney, eye and pancreas, and lower levels in liver, muscle, heart, brain, skin and stomach. Administration of a single dose (500 ng/g body weight) of recombinant seabream growth hormone (rbGH) by intraperitoneal (ip) injection into one-month-old tilapia resulted in an obvious increase of IGF-I and pgrn mRNA (2.7-fold and 2.5-fold, respectively) in the liver at three hours post-GH treatment. The peptide levels of pgrn in the liver of GH-treated fish also were substantially induced over controls at 12h post-GH treatment as detected by western immuno-blot analysis. The co-induction of IGF-I and pgrn following GH treatment may suggest the involvement of pgrn in GH regulated growth in tilapia.

The interaction of polyglutamine peptides with lipid membranes is regulated by flanking sequences associated with huntingtin.

PubMed

Burke, Kathleen A; Kauffman, Karlina J; Umbaugh, C Samuel; Frey, Shelli L; Legleiter, Justin

2013-05-24

Huntington disease (HD) is caused by an expanded polyglutamine (poly(Q)) repeat near the N terminus of the huntingtin (htt) protein. Expanded poly(Q) facilitates formation of htt aggregates, eventually leading to deposition of cytoplasmic and intranuclear inclusion bodies containing htt. Flanking sequences directly adjacent to the poly(Q) domain, such as the first 17 amino acids on the N terminus (Nt17) and the polyproline (poly(P)) domain on the C-terminal side of the poly(Q) domain, heavily influence aggregation. Additionally, htt interacts with a variety of membraneous structures within the cell, and Nt17 is implicated in lipid binding. To investigate the interaction between htt exon1 and lipid membranes, a combination of in situ atomic force microscopy, Langmuir trough techniques, and vesicle permeability assays were used to directly monitor the interaction of a variety of synthetic poly(Q) peptides with different combinations of flanking sequences (KK-Q35-KK, KK-Q35-P10-KK, Nt17-Q35-KK, and Nt17-Q35-P10-KK) on model membranes and surfaces. Each peptide aggregated on mica, predominately forming extended, fibrillar aggregates. In contrast, poly(Q) peptides that lacked the Nt17 domain did not appreciably aggregate on or insert into lipid membranes. Nt17 facilitated the interaction of peptides with lipid surfaces, whereas the poly(P) region enhanced this interaction. The aggregation of Nt17-Q35-P10-KK on the lipid bilayer closely resembled that of a htt exon1 construct containing 35 repeat glutamines. Collectively, this data suggests that the Nt17 domain plays a critical role in htt binding and aggregation on lipid membranes, and this lipid/htt interaction can be further modulated by the presence of the poly(P) domain.

Development of a peptide substrate for detection of Sunn pest damage in wheat flour.

PubMed

Hançerlioğulları, Begüm Zeynep; Köksel, Hamit; Dudak, Fahriye Ceyda

2018-05-07

Since the common protease substrates did not give satisfactory results for the determination of Sunn pest protease activity in damaged wheat, different peptide substrates derived from the repeat sequences of high molecular weight glutenin subunits were synthesized. Hydrolysis of peptides by pest protease was determined by HPLC. Among three peptides having the same consensus motifs, peptide1 (PGQGQQGYYPTSPQQ) showed the best catalytic efficiency. A novel assay was described for monitoring the enzymatic activity of protease extracted from damaged wheat flour. The selected peptide was labeled with a fluorophore (EDANS) and quencher (Dabcyl) to display fluorescence resonance energy transfer (FRET). The proteolytic activity was measured by the change in fluorescence intensity that occurred when the protease cleaved the peptide substrate. Furthermore, the developed assay was modified for rapid and easy detection of bug damage in flour. Flour samples were suspended in water and mixed with fluorescence peptide substrate. After centrifugation, the fluorescence intensities of the supernatants were determined which is proportional with the protease content of the flour. The total analysis time for the developed assay is estimated as 15 minutes. The developed assay permits a significant decrease in time and labor, offering sensitive detection of Sunn pest damage in wheat flour. This article is protected by copyright. All rights reserved.

A target-unrelated peptide in an M13 phage display library traced to an advantageous mutation in the gene II ribosome-binding site.

PubMed

Brammer, Leighanne A; Bolduc, Benjamin; Kass, Jessica L; Felice, Kristin M; Noren, Christopher J; Hall, Marilena Fitzsimons

2008-02-01

Screening of the commercially available Ph.D.-7 phage-displayed heptapeptide library for peptides that bind immobilized Zn2+ resulted in the repeated selection of the peptide HAIYPRH, although binding assays indicated that HAIYPRH is not a zinc-binding peptide. HAIYPRH has also been selected in several other laboratories using completely different targets, and its ubiquity suggests that it is a target-unrelated peptide. We demonstrated that phage displaying HAIYPRH are enriched after serial amplification of the library without exposure to target. The amplification of phage displaying HAIYPRH was found to be dramatically faster than that of the library itself. DNA sequencing uncovered a mutation in the Shine-Dalgarno (SD) sequence for gIIp, a protein involved in phage replication, imparting to the SD sequence better complementarity to the 16S ribosomal RNA (rRNA). Introducing this mutation into phage lacking a displayed peptide resulted in accelerated propagation, whereas phage displaying HAIYPRH with a wild-type SD sequence were found to amplify normally. The SD mutation may alter gIIp expression and, consequently, the rate of propagation of phage. In the Ph.D.-7 library, the mutation is coincident with the displayed peptide HAIYPRH, accounting for the target-unrelated selection of this peptide in multiple reported panning experiments.

The central domain of bovine submaxillary mucin consists of over 50 tandem repeats of 329 amino acids. Chromosomal localization of the BSM1 gene and relations to ovine and porcine counterparts.

PubMed

Jiang, W; Gupta, D; Gallagher, D; Davis, S; Bhavanandan, V P

2000-04-01

We previously elucidated five distinct protein domains (I-V) for bovine submaxillary mucin, which is encoded by two genes, BSM1 and BSM2. Using Southern blot analysis, genomic cloning and sequencing of the BSM1 gene, we now show that the central domain (V) consists of approximately 55 tandem repeats of 329 amino acids and that domains III-V are encoded by a 58.4-kb exon, the largest exon known for all genes to date. The BSM1 gene was mapped by fluorescence in situ hybridization to the proximal half of chromosome 5 at bands q2. 2-q2.3. The amino-acid sequence of six tandem repeats (two full and four partial) were found to have only 92-94% identities. We propose that the variability in the amino-acid sequences of the mucin tandem repeat is important for generating the combinatorial library of saccharides that are necessary for the protective function of mucins. The deduced peptide sequences of the central domain match those determined from the purified bovine submaxillary mucin and also show 68-94% identity to published peptide sequences of ovine submaxillary mucin. This indicates that the core protein of ovine submaxillary mucin is closely related to that of bovine submaxillary mucin and contains similar tandem repeats in the central domain. In contrast, the central domain of porcine submaxillary mucin is reported to consist of 81-amino-acid tandem repeats. However, both bovine submaxillary mucin and porcine submaxillary mucin contain similar N-terminal and C-terminal domains and the corresponding genes are in the conserved linkage regions of the respective genomes.

Genetic and physical interactions between factors involved in both cell cycle progression and pre-mRNA splicing in Saccharomyces cerevisiae.

PubMed Central

Ben-Yehuda, S; Dix, I; Russell, C S; McGarvey, M; Beggs, J D; Kupiec, M

2000-01-01

The PRP17/CDC40 gene of Saccharomyces cerevisiae functions in two different cellular processes: pre-mRNA splicing and cell cycle progression. The Prp17/Cdc40 protein participates in the second step of the splicing reaction and, in addition, prp17/cdc40 mutant cells held at the restrictive temperature arrest in the G2 phase of the cell cycle. Here we describe the identification of nine genes that, when mutated, show synthetic lethality with the prp17/cdc40Delta allele. Six of these encode known splicing factors: Prp8p, Slu7p, Prp16p, Prp22p, Slt11p, and U2 snRNA. The other three, SYF1, SYF2, and SYF3, represent genes also involved in cell cycle progression and in pre-mRNA splicing. Syf1p and Syf3p are highly conserved proteins containing several copies of a repeated motif, which we term RTPR. This newly defined motif is shared by proteins involved in RNA processing and represents a subfamily of the known TPR (tetratricopeptide repeat) motif. Using two-hybrid interaction screens and biochemical analysis, we show that the SYF gene products interact with each other and with four other proteins: Isy1p, Cef1p, Prp22p, and Ntc20p. We discuss the role played by these proteins in splicing and cell cycle progression. PMID:11102353

Genetic and physical interactions between factors involved in both cell cycle progression and pre-mRNA splicing in Saccharomyces cerevisiae.

PubMed

Ben-Yehuda, S; Dix, I; Russell, C S; McGarvey, M; Beggs, J D; Kupiec, M

2000-12-01

The PRP17/CDC40 gene of Saccharomyces cerevisiae functions in two different cellular processes: pre-mRNA splicing and cell cycle progression. The Prp17/Cdc40 protein participates in the second step of the splicing reaction and, in addition, prp17/cdc40 mutant cells held at the restrictive temperature arrest in the G2 phase of the cell cycle. Here we describe the identification of nine genes that, when mutated, show synthetic lethality with the prp17/cdc40Delta allele. Six of these encode known splicing factors: Prp8p, Slu7p, Prp16p, Prp22p, Slt11p, and U2 snRNA. The other three, SYF1, SYF2, and SYF3, represent genes also involved in cell cycle progression and in pre-mRNA splicing. Syf1p and Syf3p are highly conserved proteins containing several copies of a repeated motif, which we term RTPR. This newly defined motif is shared by proteins involved in RNA processing and represents a subfamily of the known TPR (tetratricopeptide repeat) motif. Using two-hybrid interaction screens and biochemical analysis, we show that the SYF gene products interact with each other and with four other proteins: Isy1p, Cef1p, Prp22p, and Ntc20p. We discuss the role played by these proteins in splicing and cell cycle progression.

Sensitivity of immune response quality to influenza helix 190 antigen structure displayed on a modular virus-like particle.

PubMed

Anggraeni, Melisa R; Connors, Natalie K; Wu, Yang; Chuan, Yap P; Lua, Linda H L; Middelberg, Anton P J

2013-09-13

Biomolecular engineering enables synthesis of improved proteins through synergistic fusion of modules from unrelated biomolecules. Modularization of peptide antigen from an unrelated pathogen for presentation on a modular virus-like particle (VLP) represents a new and promising approach to synthesize safe and efficacious vaccines. Addressing a key knowledge gap in modular VLP engineering, this study investigates the underlying fundamentals affecting the ability of induced antibodies to recognize the native pathogen. Specifically, this quality of immune response is correlated to the peptide antigen module structure. We modularized a helical peptide antigen element, helix 190 (H190) from the influenza hemagglutinin (HA) receptor binding region, for presentation on murine polyomavirus VLP, using two strategies aimed to promote H190 helicity on the VLP. In the first strategy, H190 was flanked by GCN4 structure-promoting elements within the antigen module; in the second, dual H190 copies were arrayed as tandem repeats in the module. Molecular dynamics simulation predicted that tandem repeat arraying would minimize secondary structural deviation of modularized H190 from its native conformation. In vivo testing supported this finding, showing that although both modularization strategies conferred high H190-specific immunogenicity, tandem repeat arraying of H190 led to a strikingly higher immune response quality, as measured by ability to generate antibodies recognizing a recombinant HA domain and split influenza virion. These findings provide new insights into the rational engineering of VLP vaccines, and could ultimately enable safe and efficacious vaccine design as an alternative to conventional approaches necessitating pathogen cultivation. Copyright © 2013 Elsevier Ltd. All rights reserved.

Production of novel recombinant single-domain antibodies against tandem repeat region of MUC1 mucin.

PubMed

Rahbarizadeh, F; Rasaee, M J; Forouzandeh Moghadam, M; Allameh, A A; Sadroddiny, E

2004-06-01

Recently, the existence of "heavy-chain" antibody in Camelidae has been described. However, as yet there is no data on the binding of this type of antibody to peptides. In addition, there was not any report of production of single-domain antibodies in two-humped camels (Camelus bactrianus). In the present study, these questions are addressed. We showed the feasibility of immunizing old world camels, cloning the repertoire of the variable domain of their heavy-chain antibodies, panning and selection, leading to the successful identification of minimum-sized antigen binders. Antigen-specific fragments of the heavy-chain IgGs (V(HH)) are of great interest in biotechnology because they are very stable, highly soluble, and react specifically and with high affinity to the antigens. In this study, we immunized two camels (Camelus dromedarius and Camelus bactrianus) with homogenized cancerous tissues, synthetic peptide, and human milk fat globule membrane (HMFG), and generated two V(HH) libraries displayed on phage particles. Some single-domain antibody fragments have been isolated that specifically recognize the tandem repeat region of MUC1. The camels' single-domain V(HH) harbor the original, intact antigen binding site and reacted specifically and with high affinity to the tandem repeat region of MUC1. Indeed soluble, specific antigen binders and good affinities (in the range of 0.2 x 10(9) M(-1) to 0.6 x 10(9) M(-1)) were identified from these libraries. This is the first example of the isolation of camel anti-peptide V(HH) domains.

Mass Spectrometric Determination of ILPR G-quadruplex Binding Sites in Insulin and IGF-2

PubMed Central

Xiao, JunFeng

2009-01-01

The insulin-linked polymorphic region (ILPR) of the human insulin gene promoter region forms G-quadruplex structures in vitro. Previous studies show that insulin and insulin-like growth factor-2 (IGF-2) exhibit high affinity binding in vitro to 2-repeat sequences of ILPR variants a and h, but negligible binding to variant i. Two-repeat sequences of variants a and h form intramolecular G-quadruplex structures that are not evidenced for variant i. Here we report on the use of protein digestion combined with affinity capture and MALDI-MS detection to pinpoint ILPR binding sites in insulin and IGF-2. Peptides captured by ILPR variants a and h were sequenced by MALDI-MS/MS, LC-MS and in silico digestion. On-bead digestion of insulin-ILPR variant a complexes supported the conclusions. The results indicate that the sequence VCG(N)RGF is generally present in the captured peptides and is likely involved in the affinity binding interactions of the proteins with the ILPR G-quadruplexes. The significance of arginine in the interactions was studied by comparing the affinities of synthesized peptides VCGERGF and VCGEAGF with ILPR variant a. Peptides from other regions of the proteins that are connected through disulfide linkages were also detected in some capture experiments. Identification of binding sites could facilitate design of DNA binding ligands for capture and detection of insulin and IGF-2. The interactions may have biological significance as well. PMID:19747845

Characterization of a possible amyloidogenic precursor in glutamine-repeat neurodegenerative diseases

PubMed Central

Armen, Roger S.; Bernard, Brady M.; Day, Ryan; Alonso, Darwin O. V.; Daggett, Valerie

2005-01-01

Several neurodegenerative diseases are linked to expanded repeats of glutamine residues, which lead to the formation of amyloid fibrils and neuronal death. The length of the repeats correlates with the onset of Huntington's disease, such that healthy individuals have <38 residues and individuals with >38 repeats exhibit symptoms. Because it is difficult to obtain atomic-resolution structural information for poly(l-glutamine) (polyQ) in aqueous solution experimentally, we performed molecular dynamics simulations to investigate the conformational behavior of this homopolymer. In simulations of 20-, 40-, and 80-mer polyQ, we observed the formation of the “α-extended chain” conformation, which is characterized by alternating residues in the αL and αR conformations to yield a sheet. The structural transition from disordered random-coil conformations to the α-extended chain conformation exhibits modest length and temperature dependence, in agreement with the experimental observation that aggregation depends on length and temperature. We propose that fibril formation in polyQ may occur through an α-sheet structure, which was proposed by Pauling and Corey [Pauling, L. & Corey, R. B. (1951) Proc. Natl. Acad. Sci. USA 37, 251-256]. Also, we propose an atomic-resolution model of how the inhibitory peptide QBP1 (polyQ-binding peptide 1) may bind to polyQ in an α-extended chain conformation to inhibit fibril formation. PMID:16157882

Site-Specific Modulation of Charge Controls the Structure and Stimulus Responsiveness of Intrinsically Disordered Peptide Brushes.

PubMed

Bhagawati, Maniraj; Rubashkin, Matt G; Lee, Jessica P; Ananthanarayanan, Badriprasad; Weaver, Valerie M; Kumar, Sanjay

2016-06-14

Intrinsically disordered proteins (IDPs) are an important and emerging class of materials for tailoring biointerfaces. While the importance of chain charge and resultant electrostatic interactions in controlling conformational properties of IDPs is beginning to be explored through in silico approaches, there is a dearth of experimental studies motivated toward a systematic study of these effects. In an effort to explore this relationship, we measured the conformations of two peptides derived from the intrinsically disordered neurofilament (NF) side arm domain: one depicting the wild-type sequence with four lysine-serine-proline repeats (KSP peptide) and another in which the serine residues were replaced with aspartates (KDP peptide), a strategy sometimes used to mimic phosphorylation. Using a variety of biophysical measurements including a novel application of scanning angle interference microscopy, we demonstrate that the KDP peptide assumes comparatively more expanded conformations in solution and forms significantly thicker brushes when immobilized on planar surfaces at high densities. In both settings, the peptides respond to changes in ambient ionic strength, with each peptide showing distinct stimulus-responsive characteristics. While the KDP peptide undergoes compaction with increasing ionic strength as would be expected for a polyampholyte, the KSP peptide shows biphasic behavior, with an initial compaction followed by an expanded state at a higher ionic strength. Together these results support the notion that modulation of charge on IDPs can regulate conformational and interfacial properties.

Inhibition of Nipah Virus Infectin In Vivo: Targeting an Early Stage of Paramyxovirus Fusion Activation during Viral Entry

DOE Office of Scientific and Technical Information (OSTI.GOV)

M Porotto; B Rockx; C Yokoyama

2011-12-31

In the paramyxovirus cell entry process, receptor binding triggers conformational changes in the fusion protein (F) leading to viral and cellular membrane fusion. Peptides derived from C-terminal heptad repeat (HRC) regions in F have been shown to inhibit fusion by preventing formation of the fusogenic six-helix bundle. We recently showed that the addition of a cholesterol group to HRC peptides active against Nipah virus targets these peptides to the membrane where fusion occurs, dramatically increasing their antiviral effect. In this work, we report that unlike the untagged HRC peptides, which bind to the postulated extended intermediate state bridging the viralmore » and cell membranes, the cholesterol tagged HRC-derived peptides interact with F before the fusion peptide inserts into the target cell membrane, thus capturing an earlier stage in the F-activation process. Furthermore, we show that cholesterol tagging renders these peptides active in vivo: the cholesterol-tagged peptides cross the blood brain barrier, and effectively prevent and treat in an established animal model what would otherwise be fatal Nipah virus encephalitis. The in vivo efficacy of cholesterol-tagged peptides, and in particular their ability to penetrate the CNS, suggests that they are promising candidates for the prevention or therapy of infection by Nipah and other lethal paramyxoviruses.« less

Formation of peptides from amino acids by single or multiple additions of ATP to suspensions of nucleoproteinoid microparticles

NASA Technical Reports Server (NTRS)

Nakashima, T.; Fox, S. W.

1981-01-01

The synthesis of peptides from individual amino acids or pairs of amino acids and ATP in the presence of catalysis by nucleoproteinoid microparticles is investigated. Experiments were performed with suspensions formed from the condensation of lysine-rich and acidic proteinoids with polyadenylic acid, to which were added glycine, phenylalanine, proline, lysine or glycine-phenylalanine mixtures, and ATP either at once or serially. Peptide yields are found to be greatest for equal amounts of acidic and basic proteinoids. The addition of imidazole is found to alter the preference of glycine-phenylalanine mixtures to form mixed heteropeptides rather than homopeptides. A rapid ATP decay in the peptide synthesis reaction is observed, and a greater yield is obtained for repeated small additions than for a single addition of ATP. The experimental system has properties similar to modern cells, and represents an organizational unit ready for the evolution of associated biochemical pathways.

Folding control in cyclic peptides through N-methylation pattern selection: formation of antiparallel beta-sheet dimers, double reverse turns and supramolecular helices by 3alpha,gamma cyclic peptides.

PubMed

Amorín, Manuel; Castedo, Luis; Granja, Juan R

2008-01-01

Peptide foldamers constitute a growing class of nanomaterials with potential applications in a wide variety of chemical, medical and technological fields. Here we describe the preparation and structural characteristics of a new class of cyclic peptide foldamers (3alpha,gamma-CPs) that, depending on their backbone N-methylation patterns and the medium, can either remain as flat rings that dimerize through arrays of hydrogen bonds of antiparallel beta-sheet type, or can fold into twisted double reverse turns that, in the case of double gamma-turns, associate in nonpolar solvents to form helical supramolecular structures. A 3alpha,gamma-CP consists of a number of multiples of a repeat unit made up of four amino acid residues of alternating chirality: three corresponding to alpha-amino acids and one to a gamma-amino acid (a cis-3-aminocycloalkanecarboxylic acid).

Catalysis of peptide bond formation by histidyl-histidine in a fluctuating clay environment

NASA Technical Reports Server (NTRS)

White, D. H.; Erickson, J. C.

1980-01-01

The condensation of glycine to form oligoglycines during wet-dry fluctuations on clay surfaces was enhanced up to threefold or greater by small amounts of histidyl-histidine. In addition, higher relative yields of the longer oligomers were produced. Other specific dipeptides tested gave no enhancement, and imidazole, histidine, and N-acetylhistidine gave only slight enhancements. Histidyl-histidine apparently acts as a true catalyst (in the sense of repeatedly catalyzing the reaction), since up to 52 nmol of additional glycine were incorporated into oligoglycine for each nmol of catalyst added. This is the first known instance of a peptide or similar molecule demonstrating a catalytic turnover number greater than unity in a prebiotic oligomer synthesis reaction, and suggests that histidyl-histidine is a model for a primitive prebiotic proto-enzyme. Catalysis of peptide bond synthesis by a molecule which is itself a peptide implies that related systems may be capable of exhibiting autocatalytic growth.

Comparative characterization of short monomeric polyglutamine peptides by replica exchange molecular dynamics simulation.

PubMed

Nakano, Miki; Watanabe, Hirofumi; Rothstein, Stuart M; Tanaka, Shigenori

2010-05-27

Polyglutamine (polyQ) diseases are caused by an abnormal expansion of CAG repeats. While their detailed structure remains unclear, polyQ peptides assume beta-sheet structures when they aggregate. To investigate the conformational ensemble of short, monomeric polyQ peptides, which consist of 15 glutamine residues (Q(15)), we performed replica exchange molecular dynamics (REMD) simulations. We found that Q(15) can assume multiple configurations due to all of the residues affecting the formation of side-chain hydrogen bonds. Analysis of the free energy landscape reveals that Q(15) has a basin for random-coil structures and another for alpha-helix or beta-turn structures. To investigate properties of aggregated polyQ peptides, we performed multiple molecular dynamics (MMD) simulations for monomeric and oligomeric Q(15). MMD revealed that the formation of oligomers stabilizes the beta-turn structure by increasing the number of hydrogen bonds between the main chains.

AJIPHASE®: A Highly Efficient Synthetic Method for One-Pot Peptide Elongation in the Solution Phase by an Fmoc Strategy.

PubMed

Takahashi, Daisuke; Inomata, Tatsuji; Fukui, Tatsuya

2017-06-26

We previously reported an efficient peptide synthesis method, AJIPHASE®, that comprises repeated reactions and isolations by precipitation. This method utilizes an anchor molecule with long-chain alkyl groups as a protecting group for the C-terminus. To further improve this method, we developed a one-pot synthesis of a peptide sequence wherein the synthetic intermediates were isolated by solvent extraction instead of precipitation. A branched-chain anchor molecule was used in the new process, significantly enhancing the solubility of long peptides and the operational efficiency compared with the previous method, which employed precipitation for isolation and a straight-chain aliphatic group. Another prerequisite for this solvent-extraction-based strategy was the use of thiomalic acid and DBU for Fmoc deprotection, which facilitates the removal of byproducts, such as the fulvene adduct. © 2017 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.

A peptide hormone required for Casparian strip diffusion barrier formation in Arabidopsis roots.

PubMed

Nakayama, Takuya; Shinohara, Hidefumi; Tanaka, Mina; Baba, Koki; Ogawa-Ohnishi, Mari; Matsubayashi, Yoshikatsu

2017-01-20

Plants achieve mineral ion homeostasis by means of a hydrophobic barrier on endodermal cells called the Casparian strip, which restricts lateral diffusion of ions between the root vascular bundles and the soil. We identified a family of sulfated peptides required for contiguous Casparian strip formation in Arabidopsis roots. These peptide hormones, which we named Casparian strip integrity factor 1 (CIF1) and CIF2, are expressed in the root stele and specifically bind the endodermis-expressed leucine-rich repeat receptor kinase GASSHO1 (GSO1)/SCHENGEN3 and its homolog, GSO2. A mutant devoid of CIF peptides is defective in ion homeostasis in the xylem. CIF genes are environmentally responsive. Casparian strip regulation is not merely a passive process driven by root developmental cues; it also serves as an active strategy to cope with adverse soil conditions. Copyright © 2017, American Association for the Advancement of Science.

Peptide Modified ZnO Nanoparticles as Gas Sensors Array for Volatile Organic Compounds (VOCs)

PubMed Central

Mascini, Marcello; Gaggiotti, Sara; Della Pelle, Flavio; Di Natale, Corrado; Qakala, Sinazo; Iwuoha, Emmanuel; Pittia, Paola; Compagnone, Dario

2018-01-01

In this work a peptide based gas sensor array based of ZnO nanoparticles (ZnONPs) has been realized. Four different pentapeptides molecularly modeled for alcohols and esters having cysteine as a common spacer have been immobilized onto ZnONPs. ZnONPs have been morphologically and spectroscopically characterized. Modified nanoparticles have been then deposited onto quartz crystal microbalances (QCMs) and used as gas sensors with nitrogen as carrier gas. Analysis of the pure compounds modeled demonstrated a nice fitting of modeling with real data. The peptide based ZnONPs had very low sensitivity to water, compared to previously studied AuNPs peptide based gas sensors allowing the use of the array on samples with high water content. Real samples of fruit juices have been assayed; stability of the signal, good repeatability, and discrimination ability of the array was achieved. PMID:29713626

Peptide Modified ZnO Nanoparticles as Gas Sensors Array for Volatile Organic Compounds (VOCs).

PubMed

Mascini, Marcello; Gaggiotti, Sara; Della Pelle, Flavio; Di Natale, Corrado; Qakala, Sinazo; Iwuoha, Emmanuel; Pittia, Paola; Compagnone, Dario

2018-01-01

In this work a peptide based gas sensor array based of ZnO nanoparticles (ZnONPs) has been realized. Four different pentapeptides molecularly modeled for alcohols and esters having cysteine as a common spacer have been immobilized onto ZnONPs. ZnONPs have been morphologically and spectroscopically characterized. Modified nanoparticles have been then deposited onto quartz crystal microbalances (QCMs) and used as gas sensors with nitrogen as carrier gas. Analysis of the pure compounds modeled demonstrated a nice fitting of modeling with real data. The peptide based ZnONPs had very low sensitivity to water, compared to previously studied AuNPs peptide based gas sensors allowing the use of the array on samples with high water content. Real samples of fruit juices have been assayed; stability of the signal, good repeatability, and discrimination ability of the array was achieved.

Peptide modified ZnO nanoparticles as gas sensors array for volatile organic compounds (VOCs)

NASA Astrophysics Data System (ADS)

Mascini, Marcello; Gaggiotti, Sara; Della Pelle, Flavio; Di Natale, Corrado; Qakala, Sinazo; Iwuoha, Emmanuel; Pittia, Paola; Compagnone, Dario

2018-04-01

In this work a peptide based gas sensor array based of ZnO nanoparticles (ZnONPs) has been realized. Four different pentapeptides molecularly modelled for alcohols and esters having cysteine as a common spacer have been immobilized onto ZnONPs. ZnONPs have been morphologically and spectroscopically characterized. Modified nanoparticles have been then deposited onto quartz crystal microbalances (QCMs) and used as gas sensors with nitrogen as carrier gas. Analysis of the pure compounds modelled demonstrated a nice fitting of modelling with real data. The peptide based ZnONPs had very low sensitivity to water, compared to previously studied AuNPs peptide based gas sensors allowing the use of the array on samples with high water content. Real samples of fruit juices have been assayed; stability of the signal, good repeatability and discrimination ability of the array was achieved.

SU-E-T-252: Developing a Pencil Beam Dose Calculation Algorithm for CyberKnife System

DOE Office of Scientific and Technical Information (OSTI.GOV)

Liang, B; Duke University Medical Center, Durham, NC; Liu, B

2015-06-15

Purpose: Currently there are two dose calculation algorithms available in the Cyberknife planning system: ray-tracing and Monte Carlo, which is either not accurate or time-consuming for irregular field shaped by the MLC that was recently introduced. The purpose of this study is to develop a fast and accurate pencil beam dose calculation algorithm which can handle irregular field. Methods: A pencil beam dose calculation algorithm widely used in Linac system is modified. The algorithm models both primary (short range) and scatter (long range) components with a single input parameter: TPR{sub 20}/{sub 10}. The TPR{sub 20}/{sub 20}/{sub 10} value was firstmore » estimated to derive an initial set of pencil beam model parameters (PBMP). The agreement between predicted and measured TPRs for all cones were evaluated using the root mean square of the difference (RMSTPR), which was then minimized by adjusting PBMPs. PBMPs are further tuned to minimize OCR RMS (RMSocr) by focusing at the outfield region. Finally, an arbitrary intensity profile is optimized by minimizing RMSocr difference at infield region. To test model validity, the PBMPs were obtained by fitting to only a subset of cones (4) and applied to all cones (12) for evaluation. Results: With RMS values normalized to the dmax and all cones combined, the average RMSTPR at build-up and descending region is 2.3% and 0.4%, respectively. The RMSocr at infield, penumbra and outfield region is 1.5%, 7.8% and 0.6%, respectively. Average DTA in penumbra region is 0.5mm. There is no trend found in TPR or OCR agreement among cones or depths. Conclusion: We have developed a pencil beam algorithm for Cyberknife system. The prediction agrees well with commissioning data. Only a subset of measurements is needed to derive the model. Further improvements are needed for TPR buildup region and OCR penumbra. Experimental validations on MLC shaped irregular field needs to be performed. This work was partially supported by the National Natural Science Foundation of China (61171005) and the China Scholarship Council (CSC)« less

The Implicit Positive and Negative Affect Test: Validity and Relationship with Cardiovascular Stress-Responses

PubMed Central

van der Ploeg, Melanie M.; Brosschot, Jos F.; Thayer, Julian F.; Verkuil, Bart

2016-01-01

Self-report, i.e., explicit, measures of affect cannot fully explain the cardiovascular (CV) responses to stressors. Measuring affect beyond self-report, i.e., using implicit measures, could add to our understanding of stress-related CV activity. The Implicit Positive and Negative Affect Test (IPANAT) was administered in two studies to test its ecological validity and relation with CV responses and self-report measures of affect. In Study 1 students (N = 34) viewed four film clips inducing anger, happiness, fear, or no emotion, and completed the IPANAT and the Positive And Negative Affect Scale at baseline and after each clip. Implicit negative affect (INA) was higher and implicit positive affect (IPA) was lower after the anger inducing clip and vice versa after the happiness inducing clip. In Study 2 students performed a stressful math task with (n = 14) or without anger harassment (n = 15) and completed the IPANAT and a Visual Analog Scale as an explicit measure afterwards. Systolic (SBP), diastolic (DBP) blood pressure, heart rate (HR), heart rate variability (HRV), and total peripheral resistance (TPR) were recorded throughout. SBP and DBP were higher and TPR was lower in the harassment condition during the task with a prolonged effect on SBP and DBP during recovery. As expected, explicit negative affect (ENA) was higher and explicit positive affect (EPA) lower after harassment, but ENA and EPA were not related to CV activity. Although neither INA nor IPA differed between the tasks, during both tasks higher INA was related to higher SBP, lower HRV and lower TPR and to slower recovery of DBP after both tasks. Low IPA was related to slower recovery of SBP and DBP after the tasks. Implicit affect was not related to recovery of HR, HRV, and TPR. In conclusion, the IPANAT seems to respond to film clip-induced negative and positive affect and was related to CV activity during and after stressful tasks. These findings support the theory that implicitly measured affect can add to the explanation of prolonged stress-related CV responses that influence CV health. PMID:27065908

The Implicit Positive and Negative Affect Test: Validity and Relationship with Cardiovascular Stress-Responses.

PubMed

van der Ploeg, Melanie M; Brosschot, Jos F; Thayer, Julian F; Verkuil, Bart

2016-01-01

Self-report, i.e., explicit, measures of affect cannot fully explain the cardiovascular (CV) responses to stressors. Measuring affect beyond self-report, i.e., using implicit measures, could add to our understanding of stress-related CV activity. The Implicit Positive and Negative Affect Test (IPANAT) was administered in two studies to test its ecological validity and relation with CV responses and self-report measures of affect. In Study 1 students (N = 34) viewed four film clips inducing anger, happiness, fear, or no emotion, and completed the IPANAT and the Positive And Negative Affect Scale at baseline and after each clip. Implicit negative affect (INA) was higher and implicit positive affect (IPA) was lower after the anger inducing clip and vice versa after the happiness inducing clip. In Study 2 students performed a stressful math task with (n = 14) or without anger harassment (n = 15) and completed the IPANAT and a Visual Analog Scale as an explicit measure afterwards. Systolic (SBP), diastolic (DBP) blood pressure, heart rate (HR), heart rate variability (HRV), and total peripheral resistance (TPR) were recorded throughout. SBP and DBP were higher and TPR was lower in the harassment condition during the task with a prolonged effect on SBP and DBP during recovery. As expected, explicit negative affect (ENA) was higher and explicit positive affect (EPA) lower after harassment, but ENA and EPA were not related to CV activity. Although neither INA nor IPA differed between the tasks, during both tasks higher INA was related to higher SBP, lower HRV and lower TPR and to slower recovery of DBP after both tasks. Low IPA was related to slower recovery of SBP and DBP after the tasks. Implicit affect was not related to recovery of HR, HRV, and TPR. In conclusion, the IPANAT seems to respond to film clip-induced negative and positive affect and was related to CV activity during and after stressful tasks. These findings support the theory that implicitly measured affect can add to the explanation of prolonged stress-related CV responses that influence CV health.

The PDZ and band 4.1 containing protein Frmpd1 regulates the subcellular location of activator of G-protein signaling 3 and its interaction with G-proteins.

PubMed

An, Ningfei; Blumer, Joe B; Bernard, Michael L; Lanier, Stephen M

2008-09-05

Activator of G-protein signaling 3 (AGS3) is one of nine mammalian proteins containing one or more G-protein regulatory (GPR) motifs that stabilize the GDP-bound conformation of Galphai. Such proteins have revealed unexpected functional diversity for the "G-switch" in the control of events within the cell independent of the role of heterotrimeric G-proteins as transducers for G-protein-coupled receptors at the cell surface. A key question regarding this class of proteins is what controls their subcellular positioning and interaction with G-proteins. We conducted a series of yeast two-hybrid screens to identify proteins interacting with the tetratricopeptide repeat (TPR) of AGS3, which plays an important role in subcellular positioning of the protein. We report the identification of Frmpd1 (FERM and PDZ domain containing 1) as a regulatory binding partner of AGS3. Frmpd1 binds to the TPR domain of AGS3 and coimmunoprecipitates with AGS3 from cell lysates. Cell fractionation indicated that Frmpd1 stabilizes AGS3 in a membrane fraction. Upon cotransfection of COS7 cells with Frmpd1-GFP and AGS3-mRFP, AGS3-mRFP is observed in regions of the cell cortex and also in membrane extensions or processes where it appears to be colocalized with Frmpd1-GFP based upon the merged fluorescent signals. Frmpd1 knockdown (siRNA) in Cath.a-differentiated neuronal cells decreased the level of endogenous AGS3 in membrane fractions by approximately 50% and enhanced the alpha2-adrenergic receptor-mediated inhibition of forskolin-induced increases in cAMP. The coimmunoprecipitation of Frmpd1 with AGS3 is lost as the amount of Galphai3 in the cell is increased and AGS3 apparently switches its binding partner from Frmpd1 to Galphai3 indicating that the interaction of AGS3 with Frmpd1 and Galphai3 is mutually exclusive. Mechanistically, Frmpd1 may position AGS3 in a membrane environment where it then interacts with Galphai in a regulated manner.

Breast-feeding and maternal cardiovascular function.

PubMed

Mezzacappa, E S; Kelsey, R M; Myers, M M; Katkin, E S

2001-11-01

Two studies examined the effects of breast-feeding on maternal cardiovascular function. In the first experiment, groups of breast-feeding and bottle-feeding women were compared on preejection period (PEP), heart rate (HR), cardiac output (CO), and total peripheral resistance (TPR) recorded for 1-min periods before and during standard laboratory stressors. Compared with bottle-feeders, breast-feeders had higher CO throughout the session, and greater decreases in CO and increases in TPR during cold pressor. In the second experiment, HR and blood pressure (BP) were compared before and after one breast-feeding and one bottle-feeding session in a within-subjects design. Both feeding methods increased BP but decreased HR, and systolic BP was higher for the breast-feeding than the bottle-feeding condition. Both studies support the notion that breast-feeding alters maternal cardiovascular function, possibly through the actions of oxytocin.

Enhanced electrochemiluminescence sensor from tris(2,2'-bipyridyl)ruthenium(II) incorporated into MCM-41 and an ionic liquid-based carbon paste electrode.

PubMed

Li, Jing; Huang, Minghua; Liu, Xiaoqing; Wei, Hui; Xu, Yuanhong; Xu, Guobao; Wang, Erkang

2007-07-01

The electrochemiluminescence (ECL) of tris(2,2'-bipyridyl)ruthenium(II) [Ru(bpy)(3)(2+)] ion-exchanged in the sulfonic-functionalized MCM-41 silicas was developed with tripropylamine (TPrA) as a co-reactant in a carbon paste electrode (CPE) using a room temperature ionic liquid (IL) as a binder. The sulfonic-functionalized silicas MCM-41 were used for preparing an ECL sensor by the electrostatic interactions between Ru(bpy)(3)(2+) cations and sulfonic acid groups. We used the IL as a binder to construct the CPE (IL-CPE) to replace the traditional binder of the CPE (T-CPE)--silicone oil. The results indicated that the MCM-41-modified IL-CPE had more open structures to allow faster diffusion of Ru(bpy)(3)(2+) and that the ionic liquid also acted as a conducting bridge to connect TPrA with Ru(bpy)(3)(2+) sites immobilized in the electrode, resulting in a higher ECL intensity compared with the MCM-41-modified T-CPE. Herein, the detection limit for TPrA of the MCM-41-modified IL-CPE was 7.2 nM, which was two orders of magnitude lower than that observed at the T-CPE. When this new sensor was used in flow injection analysis (FIA), the MCM-41-modified IL-CPE ECL sensor also showed good reproducibility. Furthermore, the sensor could also be renewed easily by mechanical polishing whenever needed.

HEMATOLOGY AND PLASMA CHEMISTRY OF THE PLOUGHSHARE TORTOISE (ASTROCHELYS YNIPHORA) IN A CAPTIVE BREEDING PROGRAM.

PubMed

López, Javier; Waters, Michael; Routh, Andrew; Rakotonanahary, Tsanta F; Woolaver, Lance; Thomasson, Ann; Holmes, Emma; Steinmetz, Hanspeter W

2017-03-01

Blood samples from 172 captive and 40 wild, healthy, juvenile and adult, ploughshare tortoises ( Astrochelys yniphora ) were analyzed to determine hematological and biochemical reference intervals. Hematological analytes included packed cell volume (PCV), white blood cell count (WBC), and WBC differential estimates. Biochemical analysis included total protein measured by photometry (TP) and by refractometry (TPr), albumin (ALB), creatine kinase (CK), aspartate aminotransferase (AST), glutamate dehydrogenase (GLDH), bile acids (BA), calcium (Ca), phosphorus (P), urea (UREA), and uric acid (UA). The jugular vein was identified as the preferred venipuncture site as subcarapacial vein venipuncture resulted in regular hemodilution. In due consideration of small sample sizes in some of the groups studied, adult tortoises had significantly higher plasma GLDH activity and TPr, TP, ALB, BA, and UREA concentrations and significantly lower AST activity and P concentration than juveniles. Captivity had a significant influence in some reference intervals, with captive adults presenting significantly higher WBC, and estimated counts of all white cell types as well as UREA and TPr than wild counterparts. Captive juveniles also showed significantly higher estimated monocyte and lower estimated eosinophil and basophil counts. Although these differences most likely reflect local environmental or dietary differences, without representing pathology or a deviation from the normal, they question the applicability of reference values from captive animals to wild animals and vice versa. Significant sex differences were only observed for PCV and UA. The reported reference intervals may serve as benchmarks for clinical assessment and conservation of this critically endangered species.

Comparative peptidomics analysis of neural adaptations in rats repeatedly exposed to amphetamine.

PubMed

Romanova, Elena V; Lee, Ji Eun; Kelleher, Neil L; Sweedler, Jonathan V; Gulley, Joshua M

2012-10-01

Repeated exposure to amphetamine (AMPH) induces long-lasting behavioral changes, referred to as sensitization, that are accompanied by various neuroadaptations in the brain. To investigate the chemical changes that occur during behavioral sensitization, we applied a comparative proteomics approach to screen for neuropeptide changes in a rodent model of AMPH-induced sensitization. By measuring peptide profiles with matrix-assisted laser desorption/ionization time-of-flight mass spectrometry and comparing signal intensities using principal component analysis and variance statistics, subsets of peptides are found with significant differences in the dorsal striatum, nucleus accumbens, and medial prefrontal cortex of AMPH-sensitized male Sprague-Dawley rats. These biomarker peptides, identified in follow-up analyses using liquid chromatography and tandem mass spectrometry, suggest that behavioral sensitization to AMPH is associated with complex chemical adaptations that regulate energy/metabolism, neurotransmission, apoptosis, neuroprotection, and neuritogenesis, as well as cytoskeleton integrity and neuronal morphology. Our data contribute to a growing number of reports showing that in addition to the mesolimbic dopamine system, which is the best known signaling pathway involved with reinforcing the effect of psychostimulants, concomitant chemical changes in other pathways and in neuronal organization may play a part in the overall effect of chronic AMPH exposure on behavior. © 2012 The Authors Journal of Neurochemistry © 2012 International Society for Neurochemistry.

The properties conferred upon triple-helical collagen-mimetic peptides by the presence of cysteine residues

PubMed Central

Slatter, David A.; Bihan, Dominique G.; Jarvis, Gavin E.; Stone, Rachael; Pugh, Nicholas; Giddu, Sumana; Farndale, Richard W.

2012-01-01

Recently, the ability of polymeric collagen-like peptides to regulate cell behavior has generated great interest. A triple-helical peptide known as collagen-related peptide (CRP) contains the sequence (Gly-Pro-Hyp)10. With Gly-Pro-Cys triplets appended to both of its termini, designated CRPcys, chemical cross-linking using heterobifunctional reagents generates CRPcys-XL, a potent, widely used, polymeric agonist for platelet Glycoprotein VI, whereas non-cross-linked, monomeric CRPcys antagonizes Glycoprotein VI. Here, we describe how cysteine in these triplets may also undergo random air-induced oxidation, especially upon prolonged storage or repeated freeze–thawing, to form disulphide bonds, resulting in a lesser degree of polymerization than with chemical cross-linking. We investigated the monomeric and polymeric states of these and other cysteine-containing collagen-derived peptides, using gel filtration and dynamic light scattering, allowing the size of a CRP-XL aggregate to be estimated. The effect of cysteine thiols upon peptide adsorption to surfaces and subsequent platelet responses was investigated. This demonstrated that cysteine is required for strong binding to glass coverslips and to plastic plates used in ELISA assays. PMID:22555281

The properties conferred upon triple-helical collagen-mimetic peptides by the presence of cysteine residues.

PubMed

Slatter, David A; Bihan, Dominique G; Jarvis, Gavin E; Stone, Rachael; Pugh, Nicholas; Giddu, Sumana; Farndale, Richard W

2012-07-01

Recently, the ability of polymeric collagen-like peptides to regulate cell behavior has generated great interest. A triple-helical peptide known as collagen-related peptide (CRP) contains the sequence (Gly-Pro-Hyp)(10). With Gly-Pro-Cys triplets appended to both of its termini, designated CRP(cys), chemical cross-linking using heterobifunctional reagents generates CRP(cys)-XL, a potent, widely used, polymeric agonist for platelet Glycoprotein VI, whereas non-cross-linked, monomeric CRP(cys) antagonizes Glycoprotein VI. Here, we describe how cysteine in these triplets may also undergo random air-induced oxidation, especially upon prolonged storage or repeated freeze-thawing, to form disulphide bonds, resulting in a lesser degree of polymerization than with chemical cross-linking. We investigated the monomeric and polymeric states of these and other cysteine-containing collagen-derived peptides, using gel filtration and dynamic light scattering, allowing the size of a CRP-XL aggregate to be estimated. The effect of cysteine thiols upon peptide adsorption to surfaces and subsequent platelet responses was investigated. This demonstrated that cysteine is required for strong binding to glass coverslips and to plastic plates used in ELISA assays. Copyright © 2012 Elsevier Inc. All rights reserved.

Chain length effect on the structure and stability of antimicrobial peptides of the (RW)n series.

PubMed

Phambu, Nsoki; Almarwani, Bashiyar; Garcia, Arlette M; Hamza, Nafisa S; Muhsen, Amira; Baidoo, Jacqueline E; Sunda-Meya, Anderson

2017-08-01

Three peptides containing (RW) n -NH 2 units (where n=4, 6, and 8) have been chosen to study the effect of the chain length on the structure and stability of the peptide using Fourier transform infrared (FTIR), scanning electron microscopy (SEM), thermogravimetric analysis (TGA), and differential scanning calorimetry (DSC) techniques. Their interactions with Escherichia coli (E. coli) membrane mimetic vesicles are discussed. Infrared results indicate that addition of (RW) n -NH 2 units increases intermolecular H bonds with antiparallel orientation. TGA and DSC results reveal that (RW) 6 -NH 2 shows the optimal chain length in terms of stability and all three peptides show a preferential interaction with one of the anionic lipids in E. coli membranes. SEM images of (RW) 4 -NH 2 present large aggregates while those of (RW) 6 -NH 2 and (RW) 8 -NH 2 present layers of sheet-like structure. In the presence of model membranes, (RW) n -NH 2 show fibrillar peptide superstructures. This study suggests that repeating structures of (RW) n -NH 2 promotes lateral assembly. Copyright © 2017 Elsevier B.V. All rights reserved.

The CLAVATA receptor FASCIATED EAR2 responds to distinct CLE peptides by signaling through two downstream effectors.

PubMed

Je, Byoung Il; Xu, Fang; Wu, Qingyu; Liu, Lei; Meeley, Robert; Gallagher, Joseph P; Corcilius, Leo; Payne, Richard J; Bartlett, Madelaine E; Jackson, David

2018-03-15

Meristems contain groups of indeterminate stem cells, which are maintained by a feedback loop between CLAVATA ( CLV ) and WUSCHEL ( WUS ) signaling. CLV signaling involves the secretion of the CLV3 peptide and its perception by a number of Leucine-Rich-Repeat (LRR) receptors, including the receptor-like kinase CLV1 and the receptor-like protein CLV2 coupled with the CORYNE (CRN) pseudokinase. CLV2, and its maize ortholog FASCIATED EAR2 (FEA2) appear to function in signaling by CLV3 and several related CLV3/EMBRYO-SURROUNDING REGION (CLE) peptide ligands. Nevertheless, how signaling specificity is achieved remains unknown. Here we show that FEA2 transmits signaling from two distinct CLE peptides, the maize CLV3 ortholog ZmCLE7 and ZmFON2-LIKE CLE PROTEIN1 (ZmFCP1) through two different candidate downstream effectors, the alpha subunit of the maize heterotrimeric G protein COMPACT PLANT2 (CT2), and ZmCRN. Our data provide a novel framework to understand how diverse signaling peptides can activate different downstream pathways through common receptor proteins. © 2018, Je et al.

A 31-residue peptide induces aggregation of tau’s microtubule-binding region in cells

PubMed Central

Stöhr, Jan; Wu, Haifan; Nick, Mimi; Wu, Yibing; Bhate, Manasi; Condello, Carlo; Johnson, Noah; Rodgers, Jeffrey; Lemmin, Thomas; Achyraya, Srabasti; Becker, Julia; Robinson, Kathleen; Kelly, Mark J.S.; Gai, Feng; Stubbs, Gerald; Prusiner, Stanley B.; DeGrado, William F.

2018-01-01

The self-propagation of misfolded conformations of tau underlies neurodegenerative diseases, including Alzheimer’s disease. There is considerable interest in discovering the minimal sequence and active conformational nucleus that defines this self-propagating event. The microtubule-binding region, spanning residues 244-372, reproduces much of the aggregation behavior of tau in cells and animal models. Further dissection of the amyloid-forming region to a hexapeptide from the third microtubule-binding repeat resulted in a peptide that rapidly forms fibrils in vitro. We show here that this peptide lacks the ability to seed aggregation of tau244-372 in cells. However, as the hexapeptide is gradually extended to 31 residues, the peptides aggregate more slowly and gain potent activity to induce aggregation of tau244-372 in cells. X-ray fiber diffraction, hydrogen-deuterium exchange and solids NMR studies map the beta-forming region to a 25-residue sequence. Thus, the nucleus for self-propagating aggregation of tau244-372 in cells is packaged in a remarkably small peptide. PMID:28837163

Characterization of the branched antimicrobial peptide M6 by analyzing its mechanism of action and in vivo toxicity.

PubMed

Pini, Alessandro; Giuliani, Andrea; Falciani, Chiara; Fabbrini, Monica; Pileri, Silvia; Lelli, Barbara; Bracci, Luisa

2007-06-01

We analyzed functional activity of the antimicrobial peptide M6 in vitro and in vivo. The peptide was identified by our group by phage library selection, rational modification and synthesis in a tetrabranched form (Pini et al., Antimicrob. Agents Chemother. 2005; 49: 2665-72). We found that it binds lipopolysaccharide, causes perforation of cell membranes without destroying external cell morphology and strongly binds DNA. The latter feature suggests that it could inhibit metabolic pathways, blocking DNA replication and/or transcription. We also observed that M6 does not stimulate humoral immune response when repeatedly administered to animals. We also analyzed M6 toxicity when administered to animals by intraperitoneal or by intravenous injection, determining a preliminary LD50 (125 and 37.5 mg/kg, respectively), which suggested that M6 could be used in vivo. These features make the antimicrobial branched peptide M6 a promising candidate for the development of a new antibacterial drug. Copyright (c) 2007 European Peptide Society and John Wiley & Sons, Ltd.

Ga + TOF-SIMS lineshape analysis for resolution enhancement of MALDI MS spectra of a peptide mixture

NASA Astrophysics Data System (ADS)

Malyarenko, D. I.; Chen, H.; Wilkerson, A. L.; Tracy, E. R.; Cooke, W. E.; Manos, D. M.; Sasinowski, M.; Semmes, O. J.

2004-06-01

The use of mass spectrometry to obtain molecular profiles indicative of alteration of concentrations of peptides in body fluids is currently the subject of intense investigation. For surface-based time-of-flight mass spectrometry the reliability and specificity of such profiling methods depend both on the resolution of the measuring instrument and on the preparation of samples. The present work is a part of a program to use Ga + beam TOF-SIMS alone, and as an adjunct to MALDI, in the development of reliable protein and peptide markers for diseases. Here, we describe techniques to prepare samples of relatively high-mass peptides, which serve as calibration standards and proxies for biomarkers. These are: Arg8-vasopressin, human angiotensin II, and somatostatin. Their TOF-SIMS spectra show repeatable characteristic features, with mass resolution exceeding 2000, including parent peaks and chemical adducts. The lineshape analysis for high-resolution parent peaks is shown to be useful for filter construction and deconvolution of inferior resolution SELDI-TOF spectra of calibration peptide mixture.

Peptidergic contribution to posttetanic potentiation at a central synapse of aplysia.

PubMed

Koh, Hae-Young; Weiss, Klaudiusz R

2005-08-01

Posttetanic potentiation (PTP)-like phenomena appear to be mediated by a variety of mechanisms. Although neuropeptides are located in a large number of neurons and many neuropeptides, like PTP, can enhance synaptic transmission, there is a paucity of studies indicating that peptides may actually participate in PTP. Here, we utilize a single central synapse in the feeding circuit of Aplysia to investigate a possible peptidergic contribution to PTP in the CNS. The cholinergic command-like interneuron, cerebral-buccal interneuron 2 (CBI-2), contains two neuropeptides, feeding circuit activating peptide (FCAP) and cerebral peptide 2 (CP2). Previous studies showed that tetanic prestimulation or repeated stimulation of CBI-2, as well as perfusion of FCAP and CP2, increase the size of the cholinergic excitatory postsynaptic potentials (EPSPs) that CBI-2 evokes in the motoneurons B61/62 and shorten the latency to initiate B61/62 firing in response to CBI-2 stimulation. We used temperature-dependent suppression of peptide release and occlusion experiments to examine the possible contribution of FCAP and CP2 to PTP at the CBI-2 to B61/62 synapse. When peptide release was suppressed, perfusion of exogenous peptides increased the size of posttetanic EPSPs. In contrast, when peptide release was not suppressed, exogenous peptides did not enhance the size of posttetanic EPSPs, thus indicating occlusion. Temperature manipulation and occlusion experiments also indicated that peptides extend PTP duration. This peptide-dependent prolongation of PTP has functional consequences in that it extends the duration of time during which the latency to initiate B61/62 firing in response to CBI-2 stimulation is shortened.

An online x-ray based position validation system for prostate hypofractionated radiotherapy

DOE Office of Scientific and Technical Information (OSTI.GOV)

Arumugam, Sankar, E-mail: Sankar.Arumugam@sswahs.nsw.gov.au; Xing, Aitang; Sidhom, Mark

Purpose: Accurate positioning of the target volume during treatment is paramount for stereotactic body radiation therapy (SBRT). In this work, the authors present the development of an in-house software tool to verify target position with an Elekta-Synergy linear accelerator using kV planar images acquired during treatment delivery. Methods: In-house software, SeedTracker, was developed in MATLAB to perform the following three functions: 1. predict intended seed positions in a planar view perpendicular to any gantry angle, simulating a portal imaging device, from the 3D seed co-ordinates derived from the treatment planning system; 2. autosegment seed positions in kV planar images; andmore » 3. report the position shift based on the seed positions in the projection images. The performance of SeedTracker was verified using a CIRS humanoid phantom (CIRS, VA, USA) implanted with three Civco gold seed markers (Civco, IA, USA) in the prostate. The true positive rate of autosegmentation (TPR{sub seg}) and the accuracy of the software in alerting the user when the isocenter position was outside the tolerance (TPR{sub trig}) were studied. Two-dimensional and 3D static position offsets introduced to the humanoid phantom and 3D dynamic offsets introduced to a gel phantom containing gold seeds were used for evaluation of the system. Results: SeedTracker showed a TPR{sub seg} of 100% in the humanoid phantom for projection images acquired at all angles except in the ranges of 80°–100° and 260°–280° where seeds are obscured by anatomy. This resulted in a TPR{sub trig} of 88% over the entire treatment range for considered 3D static offsets introduced to the phantom. For 2D static offsets where the position offsets were only introduced in the anterior–posterior and lateral directions, the TPR{sub trig} of SeedTracker was limited by both seed detectability and positional offset. SeedTracker showed a false positive trigger in the projection angle range between 130°–170° and 310°–350° (a maximum of 24% of treatment time) due to limited information that can be derived from monoscopic images. The system accurately determined the dynamic trajectory of the isocenter position in the superior and inferior direction for the studied dynamic offset scenarios based on the seed position in monoscopic images. Conclusions: The developed software has been shown to accurately autosegment the seed positions in kV planar images except for two 20° arcs where seeds are obscured by anatomical structures. The isocenter trajectories determined by the system, based on the monoscopic images, provide useful information for monitoring the prostate position. The developed system has potential application for monitoring prostate position during treatment delivery in linear accelerator based SBRT.« less

REVIEW: ASSESSING THE POTENTIAL TO INDUCE RESPIRATORY HYPERSENSITIVITY

EPA Science Inventory

The respiratory tract has been long recognized as an important target organ in the safety assessment of drugs and chemicals, as well as protein- or peptide-based products. Indeed, acute and repeat dose inhalation studies have been an important part of guideline studies throughou...

Group B Streptococcal Serine-Rich Repeat Proteins Promote Interaction With Fibrinogen and Vaginal Colonization

PubMed Central

Wang, Nai-Yu; Patras, Kathryn A.; Seo, Ho Seong; Cavaco, Courtney K.; Rösler, Berenice; Neely, Melody N.; Sullam, Paul M.; Doran, Kelly S.

2014-01-01

Group B streptococcus (GBS) can cause severe disease in susceptible hosts, including newborns, pregnant women, and the elderly. GBS serine-rich repeat (Srr) surface glycoproteins are important adhesins/invasins in multiple host tissues, including the vagina. However, exact molecular mechanisms contributing to their importance in colonization are unknown. We have recently determined that Srr proteins contain a fibrinogen-binding region (BR) and hypothesize that Srr-mediated fibrinogen binding may contribute to GBS cervicovaginal colonization. In this study, we observed that fibrinogen enhanced wild-type GBS attachment to cervical and vaginal epithelium, and that this was dependent on Srr1. Moreover, purified Srr1-BR peptide bound directly to host cells, and peptide administration in vivo reduced GBS recovery from the vaginal tract. Furthermore, a GBS mutant strain lacking only the Srr1 “latching” domain exhibited decreased adherence in vitro and decreased persistence in a mouse model of GBS vaginal colonization, suggesting the importance of Srr–fibrinogen interactions in the female reproductive tract. PMID:24620021

Uranium oxide catalysts: environmental applications for treatment of chlorinated organic waste from nuclear industry.

PubMed

Lazareva, Svetlana; Ismagilov, Zinfer; Kuznetsov, Vadim; Shikina, Nadezhda; Kerzhentsev, Mikhail

2018-02-05

Huge amounts of nuclear waste, including depleted uranium, significantly contribute to the adverse environmental situation throughout the world. An approach to the effective use of uranium oxides in catalysts for the deep oxidation of chlorine-containing hydrocarbons is suggested. Investigation of the catalytic activity of the synthesized supported uranium oxide catalysts doped with Cr, Mn and Co transition metals in the chlorobenzene oxidation showed that these catalysts are comparable with conventional commercial ones. Physicochemical properties of the catalysts were studied by X-ray diffraction, temperature-programmed reduction with hydrogen (H 2 -TPR), and Fourier transform infrared spectroscopy. The higher activity of Mn- and Co-containing uranium oxide catalysts in the H 2 -TPR and oxidation of chlorobenzene in comparison with non-uranium catalysts may be related to the formation of a new disperse phase represented by uranates. The study of chlorobenzene adsorption revealed that the surface oxygen is involved in the catalytic process.

Solid-phase microextraction coupled with high-performance liquid chromatography for the determination of phenylurea herbicides in aqueous samples.

PubMed

Lin, Hsin-Hang; Sung, Yu-Hsiang; Huang, Shang-Da

2003-09-12

Solid-phase microextraction coupled with high-performance liquid chromatography was successfully applied to the analysis of nine phenylurea herbicides (metoxuron, monuron, chlorotoluron, isoproturon, monolinuron, metobromuron, buturon, linuron, and chlorbromuron). Polydimethylsiloxane-divinylbenzene (PDMS-DVB, 60 microm) and Carbowax-templated resin (CW-TPR, 50 microm) fibers were selected from four commercial fibers for further study because of their better extraction efficiencies. The parameters of the desorption procedure were studied and optimized. The effects of the properties of analytes and fiber coatings, carryover, duration and temperature of absorption, pH, organic solvent and ionic strength of samples were also investigated. External calibration with an aqueous standard can be used for the analysis of environmental samples (lake water) using either PDMS-DVB or CW-TPR fibers. Good precisions (1.0-5.9%) are achieved for this method, and the detection limits are at the level of 0.5-5.1 ng/ml.

System identification of dynamic closed-loop control of total peripheral resistance by arterial and cardiopulmonary baroreceptors

NASA Astrophysics Data System (ADS)

Nikolai Aljuri, A.; Bursac, Nenad; Marini, Robert; Cohen, Richard J.

2001-08-01

Prolonged exposure to microgravity in space flight missions (days) impairs the mechanisms responsible for defense of arterial blood pressure (ABP) and cardiac output (CO) against orthostatic stress in the post-flight period. The mechanisms responsible for the observed orthostatic intolerance are not yet completely understood. Additionally, effective counter measures to attenuate this pathophysiological response are not available. The aim of this study was to investigate the ability of our proposed system identification method to predict closed-loop dynamic changes in TPR induced by changes in mean arterial pressure (MAP) and right atrial pressure (RAP). For this purpose we designed and employed a novel experimental animal model for the examination of arterial and cardiopulmonary baroreceptors in the dynamic closed-loop control of total peripheral resistance (TPR), and applied system identification to the analysis of beat-to-beat fluctuations in the measured signals.

CCl 4 chemistry on the magnetite selvedge of single-crystal hematite: competitive surface reactions

NASA Astrophysics Data System (ADS)

Adib, K.; Camillone, N., III; Fitts, J. P.; Rim, K. T.; Flynn, G. W.; Joyce, S. A.; Osgood, R. M., Jr.

2002-01-01

Temperature programmed reaction/desorption (TPR/D) studies were undertaken to characterize the surface chemistry which occurs between CCl 4 and the Fe 3O 4 (1 1 1) selvedge of single crystal α-Fe 2O 3 (0 0 0 1). Six separate desorption events are clearly observed and four desorbing species are identified: CCl 4, OCCl 2, C 2Cl 4 and FeCl 2. It is proposed that OCCl 2, CCl 4 and C 2Cl 4 are produced in reactions involving the same precursor, CCl 2. Three reaction paths compete for the CCl 2 precursor: oxygen atom abstraction (for OCCl 2), molecular recombinative desorption (for CCl 4) and associative desorption (for C 2Cl 4). During the TPR/D temperature ramp, the branching ratio is observed to depend upon temperature and the availability of reactive sites. The data are consistent with a rich site-dependent chemistry.

Preclinical profiling of the immunogenicity of a two-component subunit malaria vaccine candidate based on virosome technology.

PubMed

Okitsu, Shinji L; Mueller, Markus S; Amacker, Mario; Vogel, Denise; Westerfeld, Nicole; Robinson, John A; Zurbriggen, Rinaldo; Pluschke, Gerd

2008-01-01

Presentation of synthetic peptides on immunopotentiating reconstituted influenza virosomes is a promising technology for subunit vaccine development. An optimized virosomally delivered peptide representing 5 NPNA repeats of P. falciparum circumsporozoite protein is highly immunogenic in mice. Antibodies against this peptide (UK-39) inhibit sporozoite invasion of human hepatocytes. A second peptide (AMA49-C1) based on domain III of apical membrane antigen 1, induces antibodies that inhibit blood-stage parasite growth in vitro. Here we show a detailed pre-clinical profiling of these virosomally formulated peptides alone and in combination in mice and rabbits. Two immunizations with virosomally formulated UK-39 or AMA49-C1 were enough to elicit high titers of parasite cross-reactive antibodies in both species. A low dose of 10 microg UK-39 was enough to induce maximal titers in rabbits. Higher doses of peptide did not increase antibody titers. In contrast, AMA49-C1 induced higher antibody titers with 25 and 50 microg peptide. Combination of UK-39 and AMA49- C1 on separate virosomes did not have any negative effect on anti-peptide antibody titers in mice or rabbits. No MHC restriction was observed in the development of humoral responses in outbred rabbits with different immunogenetic backgrounds. All vaccine formulations were safe in toxicity studies in rabbits and rats. Taken together, low amounts of synthetic peptides delivered on virosomes induced high antibody titers in mice and rabbits. Moreover, different peptides could be combined without interfering with individual anti-peptide responses, augmenting the value of this system for the development of a multivalent malaria vaccine.

Production of monoclonal antibody, PR81, recognizing the tandem repeat region of MUC1 mucin.

PubMed

Paknejad, M; Rasaee, M J; Tehrani, F Karami; Kashanian, S; Mohagheghi, M A; Omidfar, K; Bazl, M Rajabi

2003-06-01

A monoclonal antibody (MAb) was generated by immunizing BALB/c mice with homogenized breast cancerous tissues. This antibody (PR81) was found to be of IgG(1) class and subclass, containing kappa light chain. PR81 reacted with either the membrane extracts of several breast cancerous tissues or the cell surface of some MUC1 positive cell lines (MCF-7, BT-20 and T-47D) tested by enzyme immunoassay and for MCF-7 by immunofluorescence method. PR81 also reacted with two synthetic 27 and 16-amino acid peptides, TSA-P1-24 and A-P1-15, respectively, which included the core tandem repeat sequence of MUC1. However, this antibody did not react with a synthetic 14 amino acid peptide that has no similarity with tandem repeat found in MUC1. The generated antibody had good and similar affinities (2.19 x 10(8) M(-1)) toward TSA-P1-24 and A-P1-15, which are mainly shared in the hydrophilic sequence of PDTRPAP. Through Western blot analysis of homogenized breast tissues, PR81 recognized only a major band of 250 kDa. This band is stronger in malignant tissue than benign and normal tissues.

Structure determination of a peptide model of the repeated helical domain in Samia cynthia ricini silk fibroin before spinning by a combination of advanced solid-state NMR methods.

PubMed

Nakazawa, Yasumoto; Asakura, Tetsuo

2003-06-18

Fibrous proteins unlike globular proteins, contain repetitive amino acid sequences, giving rise to very regular secondary protein structures. Silk fibroin from a wild silkworm, Samia cynthia ricini, consists of about 100 repeats of alternating polyalanine (poly-Ala) regions of 12-13 residues in length and Gly-rich regions. In this paper, the precise structure of the model peptide, GGAGGGYGGDGG(A)(12)GGAGDGYGAG, which is a typical repeated sequence of the silk fibroin, was determined using a combination of three kinds of solid-state NMR studies; a quantitative use of (13)C CP/MAS NMR chemical shift with conformation-dependent (13)C chemical shift contour plots, 2D spin diffusion (13)C solid-state NMR under off magic angle spinning and rotational echo double resonance. The structure of the model peptide corresponding to the silk fibroin structure before spinning was determined. The torsion angles of the central Ala residue, Ala(19), in the poly-Ala region were determined to be (phi, psi) = (-59 degrees, -48 degrees ) which are values typically associated with alpha-helical structures. However, the torsion angles of the Gly(25) residue adjacent to the C-terminal side of the poly-Ala chain were determined to be (phi, psi) = (-66 degrees, -22 degrees ) and those of Gly(12) and Ala(13) residues at the N-terminal of the poly-Ala chain to be (phi, psi) = (-70 degrees, -30 degrees ). In addition, REDOR experiments indicate that the torsion angles of the two C-terminal Ala residues, Ala(23) and Ala(24), are (phi, psi) = (-66 degrees, -22 degrees ) and those of N-terminal two Ala residues, Ala(13) and Ala(14) are (phi, psi) = (-70 degrees, -30 degrees ). Thus, the local structure of N-terminal and C-terminal residues, and also the neighboring residues of alpha-helical poly-Ala chain in the model peptide is a more strongly wound structure than found in typical alpha-helix structures.

A high-throughput LC-MS/MS screen for GHRP in equine and human urine, featuring peptide derivatization for improved chromatography.

PubMed

Timms, Mark; Hall, Nikki; Levina, Vita; Vine, John; Steel, Rohan

2014-10-01

The growth hormone releasing peptides (GHRPs) hexarelin, ipamorelin, alexamorelin, GHRP-1, GHRP-2, GHRP-4, GHRP-5, and GHRP-6 are all synthetic met-enkephalin analogues that include unnatural D-amino acids. They were designed specifically for their ability to stimulate growth hormone release and may serve as performance enhancing drugs. To regulate the use of these peptides within the horse racing industry and by human athletes, a method is presented for the extraction, derivatization, and detection of GHRPs from equine and human urine. This method takes advantage of a highly specific solid-phase extraction combined with a novel derivatization method to improve the chromatography of basic peptides. The method was validated with respect to linearity, repeatability, intermediate precision, specificity, limits of detection, limits of confirmation, ion suppression, and stability. As proof of principle, all eight GHRPs or their metabolites could be detected in urine collected from rats after intravenous administration. Copyright © 2014 John Wiley & Sons, Ltd.

Ghrelin, the proglucagon-derived peptides and peptide YY in nutrient homeostasis.

PubMed

Dong, Charlotte X; Brubaker, Patricia L

2012-12-01

Dysregulation of nutrient homeostasis is implicated in the current epidemics of obesity and type 2 diabetes mellitus. The maintenance of homeostasis in the setting of repeated cycles of feeding and fasting occurs through complex interactions between metabolic, hormonal and neural factors. Although pancreatic islets, the liver, muscle, adipocytes and the central nervous system are all key players in this network, the gastrointestinal tract is the first tissue exposed to ingested nutrients and thus has an important role. This Review focuses on several of the endocrine hormones released by the gastrointestinal tract prior to or during nutrient ingestion that have key roles in maintaining energy balance. These hormones include the gastric orexigenic hormone, ghrelin, and the distal L cell anorexigenic and metabolic hormones, glucagon-like peptide (GLP)-1, GLP-2, oxyntomodulin and peptide YY. Each of these hormones exerts a distinct set of biological actions to maintain nutrient homeostasis, the properties of which are currently, or might soon be, exploited in the clinic for the treatment of obesity and type 2 diabetes mellitus.

Postural Change Alters Autonomic Responses to Breath-Holding

PubMed Central

Taneja, Indu; Medow, Marvin S.; Clarke, Debbie; Ocon, Anthony; Stewart, Julian M.

2011-01-01

We used breath-holding during inspiration as a model to study the effect of pulmonary stretch on sympathetic nerve activity. Twelve healthy subjects (7 females, 5 males; 19–27 yrs) were tested while they performed an inspiratory breath-hold, both supine and during a 60° head-up tilt (HUT 60). Heart rate (HR), mean arterial blood pressure (MAP), respiration, muscle sympathetic nerve activity (MSNA), oxygen saturation (SaO2) and end tidal carbon dioxide (ETCO2) were recorded. Cardiac output (CO) and total peripheral resistance (TPR) were calculated. While breath-holding, ETCO2 increased significantly from 41±2 to 60±2 Torr during supine (p<0.05) and 38±2 Torr to 58±2 during HUT60 (p<0.05); SaO2 decreased from 98±1.5% to 95±1.4% supine, and from 97±1.5% to 94±1.7% during HUT60 (p=NS). MSNA showed three distinctive phases - a quiescent phase due to pulmonary stretch associated with decreased MAP, HR, CO and TPR; a second phase of baroreflex-mediated elevated MSNA which was associated with recovery of MAP and HR only during HUT60; CO and peripheral resistance returned to baseline while supine and HUT60; a third phase of further increased MSNA activity related to hypercapnia and associated with increased TPR. Breath-holding results in initial reductions of MSNA, MAP and HR by the pulmonary stretch reflex followed by increased sympathetic activity related to the arterial baroreflex and chemoreflex. PMID:20012144

Postexercise hypotension in an endurance-trained population of men and women following high-intensity interval and steady-state cycling.

PubMed

Rossow, Lindy; Yan, Huimin; Fahs, Christopher A; Ranadive, Sushant M; Agiovlasitis, Stamatis; Wilund, Kenneth R; Baynard, Tracy; Fernhall, Bo

2010-04-01

The acute effect of high-intensity interval exercise (HI) on blood pressure (BP) is unknown although this type of exercise has similar or greater cardiovascular benefits compared to steady-state aerobic exercise (SS). This study examined postexercise hypotension (PEH) and potential mechanisms of this response in endurance-trained subjects following acute SS and HI. Sex differences were also evaluated. A total of 25 endurance-trained men (n = 15) and women (n = 10) performed a bout of HI and a bout of SS cycling in randomized order on separate days. Before exercise, 30 min postexercise, and 60 min postexercise, we measured brachial and aortic BP. Cardiac output (CO), stroke volume (SV), end diastolic volume (EDV), end systolic volume (ESV), and left ventricular wall-velocities were measured using ultrasonography with tissue Doppler capabilities. Ejection fraction and fractional shortening (FS), total peripheral resistance (TPR), and calf vascular resistance were calculated from the above variables and measures of leg blood flow. BP, ejection fraction, and FS decreased by a similar magnitude following both bouts but changes in CO, heart rate (HR), TPR, and calf vascular resistance were greater in magnitude following HI than following SS. Men and women responded similarly to HI. Although men and women exhibited a similar PEH following SS, they showed differential changes in SV, EDV, and TPR. HI acutely reduces BP similarly to SS. The mechanistic response to HI appears to differ from that of SS, and endurance-trained men and women may exhibit differential mechanisms for PEH following SS but not HI.

Attenuation of Exaggerated Exercise Blood Pressure Response in African-American Women by Regular Aerobic Physical Activity

PubMed Central

Bond, Vernon; Millis, Richard M.; Adams, R. George; Oke, Luc M.; Enweze, Larry; Blakely, Raymond; Banks, Marshall; Thompson, Terry; Obisesan, Thomas; Sween, Jennifer C.

2011-01-01

Introduction A hyperreactive blood pressure response to exercise is a predictor of developing hypertension. The present study determined the influence of physical activity on an exaggerated exercise blood pressure response (EEBPR) in normotensive African-American women. Methods We screened 36 women 18–26 years of age for EEBPR defined as a ≥50 mm Hg difference in systolic blood pressure at rest and during exercise at 50% peak oxygen uptake (VO2peak). Seven subjects demonstrated an EEBPR and participated in the study. Study participants trained for eight weeks on a bicycle ergometer at a work intensity of 70% VO2peak. Blood pressure, heart rate, cardiac output (CO), stroke volume (SV), and total peripheral vascular resistance (TPR) were determined at baseline and during submaximal exercise at power outputs of 30 W and 50% VO2peak. Subjects served as their own controls, and data were evaluated by using a paired t test at P<.05. Results Effectiveness of the intervention was shown by a significantly greater VO2peak associated with significant decrements in systolic and mean arterial pressures at power outputs of 30 W and 50% VO2peak. A significant decrement in heart rate was observed during exercise at 30 W. Significant increments in CO and SV and decrement in TPR were found during exercise at 50% VO2peak. Conclusion The reduction in TPR associated with regular aerobic physical activity may attenuate the EEBPR and decrease the risk for hypertension in normotensive, young-adult, African-American women. PMID:16315376

Development of hypertension in animals with reduced total peripheral resistance.

PubMed

Huang, M; Hester, R L; Coleman, T G; Smith, M J; Guyton, A C

1992-12-01

The object of the present study was to determine whether deoxycorticosterone acetate (DOCA)-salt hypertension can be produced in rats in the presence of low total peripheral resistance (TPR) induced by long-term administration of minoxidil, a vasodilator. The rats were divided into four groups: sham-control, DOCA-salt, minoxidil, and DOCA-salt with minoxidil. The rats in both DOCA groups had DOCA pellets implanted subcutaneously and were given saline to drink. The rats in both minoxidil groups were given minoxidil (3 mg/day) in the drinking water throughout the experiment. Final measurements, including mean arterial blood pressure, cardiac index, and renal blood flow were made after 4-6 weeks. Flow measurements were made using radioactive microspheres. Cardiac index (ml.min-1.100 g-1) in sham-control rats averaged 18 +/- 2 and was higher in the other groups: 23 +/- 4 (DOCA-salt), 25 +/- 2 (minoxidil), and 30 +/- 2 (DOCA-salt plus minoxidil). Mean arterial pressure (mm Hg) was increased in both DOCA-salt rats (160 +/- 8) and DOCA-salt plus minoxidil rats (153 +/- 5) as compared with sham-control (116 +/- 2) and minoxidil (113 +/- 3) rats. There was no significant difference in TPR between the sham-control and DOCA-salt rats, but TPR in minoxidil and DOCA-salt plus minoxidil rats was 30% and 28% lower than that in untreated sham-control and DOCA-salt hypertensive rats, respectively. In contrast, renal vascular resistance was significantly increased in both DOCA-salt groups as compared with non-DOCA-salt groups.(ABSTRACT TRUNCATED AT 250 WORDS)

Pharmacological and Toxicological Properties of the Potent Oral γ-Secretase Modulator BPN-15606.

PubMed

Wagner, Steven L; Rynearson, Kevin D; Duddy, Steven K; Zhang, Can; Nguyen, Phuong D; Becker, Ann; Vo, Uyen; Masliah, Deborah; Monte, Louise; Klee, Justin B; Echmalian, Corinne M; Xia, Weiming; Quinti, Luisa; Johnson, Graham; Lin, Jiunn H; Kim, Doo Y; Mobley, William C; Rissman, Robert A; Tanzi, Rudolph E

2017-07-01

Alzheimer's disease (AD) is characterized neuropathologically by an abundance of 1) neuritic plaques, which are primarily composed of a fibrillar 42-amino-acid amyloid- β peptide (A β ), as well as 2) neurofibrillary tangles composed of aggregates of hyperphosporylated tau. Elevations in the concentrations of the A β 42 peptide in the brain, as a result of either increased production or decreased clearance, are postulated to initiate and drive the AD pathologic process. We initially introduced a novel class of bridged aromatics referred t γ -secretase modulatoro as γ -secretase modulators that inhibited the production of the A β 42 peptide and to a lesser degree the A β 40 peptide while concomitantly increasing the production of the carboxyl-truncated A β 38 and A β 37 peptides. These modulators potently lower A β 42 levels without inhibiting the γ -secretase-mediated proteolysis of Notch or causing accumulation of carboxyl-terminal fragments of APP. In this study, we report a large number of pharmacological studies and early assessment of toxicology characterizing a highly potent γ -secretase modulator (GSM), ( S )- N -(1-(4-fluorophenyl)ethyl)-6-(6-methoxy-5-(4-methyl-1 H -imidazol-1-yl)pyridin-2-yl)-4-methylpyridazin-3-amine (BPN-15606). BPN-15606 displayed the ability to significantly lower A β 42 levels in the central nervous system of rats and mice at doses as low as 5-10 mg/kg, significantly reduce A β neuritic plaque load in an AD transgenic mouse model, and significantly reduce levels of insoluble A β 42 and pThr181 tau in a three-dimensional human neural cell culture model. Results from repeat-dose toxicity studies in rats and dose escalation/repeat-dose toxicity studies in nonhuman primates have designated this GSM for 28-day Investigational New Drug-enabling good laboratory practice studies and positioned it as a candidate for human clinical trials. Copyright © 2017 by The Author(s).

Characterization of toxin-producing cyanobacteria by using an oligonucleotide probe containing a tandemly repeated heptamer.

PubMed Central

Rouhiainen, L; Sivonen, K; Buikema, W J; Haselkorn, R

1995-01-01

Cyanobacteria produce toxins that kill animals. The two main classes of cyanobacterial toxins are cyclic peptides that cause liver damage and alkaloids that block nerve transmission. Many toxin-producing strains from Finnish lakes were brought into axenic culture, and their toxins were characterized. Restriction fragment length polymorphism analysis, probing with a short tandemly repeated DNA sequence found at many locations in the chromosome of Anabaena sp. strain PCC 7120, distinguishes hepatotoxic Anabaena isolates from neurotoxin-producing strains and from Nostoc spp. PMID:7592362

Immobilization methods for the rapid total chemical synthesis of proteins on microtiter plates.

PubMed

Zitterbart, Robert; Krumrey, Michael; Seitz, Oliver

2017-07-01

The chemical synthesis of proteins typically involves the solid-phase peptide synthesis of unprotected peptide fragments that are stitched together in solution by native chemical ligation (NCL). The process is slow, and throughput is limited because of the need for repeated high performance liquid chromatography purification steps after both solid-phase peptide synthesis and NCL. With an aim to provide faster access to functional proteins and to accelerate the functional analysis of synthetic proteins by parallelization, we developed a method for the high performance liquid chromatography-free synthesis of proteins on the surface of microtiter plates. The method relies on solid-phase synthesis of unprotected peptide fragments, immobilization of the C-terminal fragment and on-surface NCL with an unprotected peptide thioester in crude form. Herein, we describe the development of a suitable immobilization chemistry. We compared (i) formation of nickel(II)-oligohistidine complexes, (ii) Cu-based [2 + 3] alkine-azide cycloaddition and (iii) hydrazone ligation. The comparative study identified the hydrazone ligation as most suitable. The sequence of immobilization via hydrazone ligation, on-surface NCL and radical desulfurization furnished the targeted SH3 domains in near quantitative yield. The synthetic proteins were functional as demonstrated by an on-surface fluorescence-based saturation binding analysis. Copyright © 2017 European Peptide Society and John Wiley & Sons, Ltd. Copyright © 2017 European Peptide Society and John Wiley & Sons, Ltd.

Synthesis, characterization and assembly of metal pnictide nanoparticles, and evaluation of their physicochemical (catalytic, magnetic, and semiconducting) properties

NASA Astrophysics Data System (ADS)

Senevirathne, Keerthisinghe

Synthesis of transition metal phosphide (Ni2P) and arsenide (MnAs) discrete nanoparticles was conducted by following a solution-phase arrested precipitation route and the size- and structure-dependent physicochemical properties of these materials were explored. Furthermore, the assembly of metal phosphide nanoparticles into a network structure via a sol-gel process and the evaluation of their structure related properties also was conducted. The surface ligation chemistry of unsupported Ni2P nanoparticles prepared by arrested precipitation was found to strongly impact the structural integrity and the hydrodesulfurization (HDS) catalytic activity of Ni 2P nanoparticles. The HDS activity of unsupported surface modified Ni2P nanoparticles is higher than that of unsupported Ni2P prepared by temperature programmed reduction (TPR) but considerably lower than silica-supported Ni2P prepared by TPR. However, by supporting the pre-formed Ni 2P nanoparticles on silica, activity comparable to that of silica-supported Ni2P prepared by TPR can be achieved. The synthetic control offered by the Ni2P nanoparticle preparation, not achieved by TPR methods, is expected to enable a systematic study of particle size and shape effects on HDS activity. By using arrested precipitation reactions, for the first time, discrete and dispersible MnAs nanoparticles have been prepared and their magnetic properties evaluated. Syntheses were developed to target both the thermodynamically stable alpha-type (hexagonal) and the metastable beta-type (orthorhombic) MnAs nanoparticles. Surprisingly, both types of ˜25 nm particles exhibit nearly identical ferromagnetic behavior with blocking temperatures, T B, in the region ˜275-310 K, TC's of 315 K and room temperature coercivities of HC ˜ 190-320 Oe. No evidence of the expected structural transition from alpha to beta-MnAs at TC is observed. Oxidative sol-gel assembly of nanoparticles to make nanoparticulate gels was successfully employed to Ni2P nanoparticles, and further extended to MnP and InP nanoparticles, for the first time. The gels were transformed into highly porous, high surface area (175-270 m2/g) 3-D structures (aerogels) via CO2 supercritical drying. Relative to discrete nanoparticles, Ni2P aerogels are less active to HDS, MnP aerogels have similar magnetic properties, and InP aerogels exhibit a greater degree of quantum confinement.

Kv7(KCNQ)-K+-Channels Influence Total Peripheral Resistance in Female but Not Male Rats, and Hamper Catecholamine Release in Hypertensive Rats of Both Sexes

PubMed Central

Berg, Torill

2018-01-01

K+-channels of the Kv7/KCNQ-family hyperpolarize and stabilize excitable cells such as autonomic neurons and vascular smooth muscle cells (VSMC). Kv7 may therefore play a role in blood pressure (BP) homeostasis, and prevent a high total peripheral vascular resistance (TPR), a hallmark of hypertensive disease. The present study analyzed if Kv7 channels influence catecholamine release and TPR in normotensive (WKY) and spontaneously hypertensive rats (SHR), and if they may contribute to the antihypertensive protection seen in young, female SHR. Tyramine-stimulated norepinephrine release evokes an adrenergic cardiovascular response, and also allows modulation of release to be reflected in the overflow to plasma. The experiment itself activated some secretion of epinephrine. The results show: (1) XE-991 (Kv7.1-7.4-inhibitor), but not chromanol 293B (Kv7.1-inhibitor), increased tyramine-stimulated norepinephrine overflow and epinephrine secretion in both sexes in SHR, but not WKY. (2) Surprisingly, the Kv7-openers retigabine (Kv7.2-7.5) and ICA-27243 (Kv7.2-7.3-preferring) increased catecholamine release in female SHR. (3) The rise in TPR following tyramine-stimulated norepinephrine release was increased by XE-991 but not chromanol in the female WKY only. (4) Retigabine and ICA-27243 reduced the TPR-response to tyramine in the female SHR only. These results suggested: (1) Up-regulation of Kv7.2-7.3 function in sympathetic neurons and chromaffin cells hampered catecholamine release in SHR of both sexes. (2) The increase catecholamine release observed after channel openers in the female SHR may possibly involve reduced transmission in cholinergic neurons which hamper catecholamine release. These two mechanisms may serve to counter-act the hyperadrenergic state in SHR. (3) Kv7.4, most likely in the vasculature, opposed the tension-response to norepinephrine in the female WKY. (4) Vascular Kv7.4-7.5 could be stimulated and then opposed norepinephrine-induced vasoconstriction in the female SHR. (5) Vascular Kv7 channels did not counter-act norepinephrine induced vasoconstriction in male rats, possibly due to different Kv7 channel regulation. Kv7 channels may represent a novel target for antihypertensive therapy. PMID:29515459

Kv7(KCNQ)-K+-Channels Influence Total Peripheral Resistance in Female but Not Male Rats, and Hamper Catecholamine Release in Hypertensive Rats of Both Sexes.

PubMed

Berg, Torill

2018-01-01

K + -channels of the Kv7/KCNQ-family hyperpolarize and stabilize excitable cells such as autonomic neurons and vascular smooth muscle cells (VSMC). Kv7 may therefore play a role in blood pressure (BP) homeostasis, and prevent a high total peripheral vascular resistance (TPR), a hallmark of hypertensive disease. The present study analyzed if Kv7 channels influence catecholamine release and TPR in normotensive (WKY) and spontaneously hypertensive rats (SHR), and if they may contribute to the antihypertensive protection seen in young, female SHR. Tyramine-stimulated norepinephrine release evokes an adrenergic cardiovascular response, and also allows modulation of release to be reflected in the overflow to plasma. The experiment itself activated some secretion of epinephrine. The results show: (1) XE-991 (Kv7.1-7.4-inhibitor), but not chromanol 293B (Kv7.1-inhibitor), increased tyramine-stimulated norepinephrine overflow and epinephrine secretion in both sexes in SHR, but not WKY. (2) Surprisingly, the Kv7-openers retigabine (Kv7.2-7.5) and ICA-27243 (Kv7.2-7.3-preferring) increased catecholamine release in female SHR. (3) The rise in TPR following tyramine-stimulated norepinephrine release was increased by XE-991 but not chromanol in the female WKY only. (4) Retigabine and ICA-27243 reduced the TPR-response to tyramine in the female SHR only. These results suggested: (1) Up-regulation of Kv7.2-7.3 function in sympathetic neurons and chromaffin cells hampered catecholamine release in SHR of both sexes. (2) The increase catecholamine release observed after channel openers in the female SHR may possibly involve reduced transmission in cholinergic neurons which hamper catecholamine release. These two mechanisms may serve to counter-act the hyperadrenergic state in SHR. (3) Kv7.4, most likely in the vasculature, opposed the tension-response to norepinephrine in the female WKY. (4) Vascular Kv7.4-7.5 could be stimulated and then opposed norepinephrine-induced vasoconstriction in the female SHR. (5) Vascular Kv7 channels did not counter-act norepinephrine induced vasoconstriction in male rats, possibly due to different Kv7 channel regulation. Kv7 channels may represent a novel target for antihypertensive therapy.

Study of the aggregation mechanism of polyglutamine peptides using replica exchange molecular dynamics simulations.

PubMed

Nakano, Miki; Ebina, Kuniyoshi; Tanaka, Shigenori

2013-04-01

Polyglutamine (polyQ, a peptide) with an abnormal repeat length is the causative agent of polyQ diseases, such as Huntington's disease. Although glutamine is a polar residue, polyQ peptides form insoluble aggregates in water, and the mechanism for this aggregation is still unclear. To elucidate the detailed mechanism for the nucleation and aggregation of polyQ peptides, replica exchange molecular dynamics simulations were performed for monomers and dimers of polyQ peptides with several chain lengths. Furthermore, to determine how the aggregation mechanism of polyQ differs from those of other peptides, we compared the results for polyQ with those of polyasparagine and polyleucine. The energy barrier between the monomeric and dimeric states of polyQ was found to be relatively low, and it was observed that polyQ dimers strongly favor the formation of antiparallel β-sheet structures. We also found a characteristic behavior of the monomeric polyQ peptide: a turn at the eighth residue is always present, even when the chain length is varied. We previously showed that a structure including more than two sets of β-turns is stable, so a long monomeric polyQ chain can act as an aggregation nucleus by forming several pairs of antiparallel β-sheet structures within a single chain. Since the aggregation of polyQ peptides has some features in common with an amyloid fibril, our results shed light on the mechanism for the aggregation of polyQ peptides as well as the mechanism for the formation of general amyloid fibrils, which cause the onset of amyloid diseases.

Self-Assembly of an α-Helical Peptide into a Crystalline Two-Dimensional Nanoporous Framework

DOE Office of Scientific and Technical Information (OSTI.GOV)

Magnotti, Elizabeth L.; Hughes, Spencer A.; Dillard, Rebecca S.

Sequence-specific peptides have been demonstrated to self-assemble into structurally defined nanoscale objects including nanofibers, nanotubes, and nanosheets. The latter structures display significant promise for the construction of hybrid materials for functional devices due to their extended planar geometry. Realization of this objective necessitates the ability to control the structural features of the resultant assemblies through the peptide sequence. The design of a amphiphilic peptide, 3FD-IL, is described that comprises two repeats of a canonical 18 amino acid sequence associated with straight α-helical structures. Peptide 3FD-IL displays 3-fold screw symmetry in a helical conformation and self-assembles into nanosheets based on hexagonalmore » packing of helices. Biophysical evidence from TEM, cryo-TEM, SAXS, AFM, and STEM measurements on the 3FD-IL nanosheets support a structural model based on a honeycomb lattice, in which the length of the peptide determines the thickness of the nanosheet and the packing of helices defines the presence of nanoscale channels that permeate the sheet. The honeycomb structure can be rationalized on the basis of geometrical packing frustration in which the channels occupy defect sites that define a periodic superlattice. In conclusion, the resultant 2D materials may have potential as materials for nanoscale transport and controlled release applications.« less

Peptide YY: a gut hormone associated with anorexia during infectious diarrhea in children.

PubMed

Beck, Amy L; Cabrera, Lilia; Pan, William K Y; Cama, Vitaliano; Friedland, Jon S; Ghatei, Mohammad A; Bloom, Stephen R; Lewis, Judy; Gilman, Robert H

2008-11-01

To evaluate the effects of diarrhea on appetite among Peruvian children age 12 to 71 months and to assess whether elevated plasma levels of peptide YY, tumor necrosis factor (TNF)-alpha, and interleukin (IL)-1beta contribute to anorexia in this population. A total of 46 Peruvian children with diarrhea and 46 healthy controls underwent an observed feeding trial that was repeated when cases were healthy. Blood samples were obtained from 30 cases and 30 controls at the first trial and from 30 cases at the second trial and assayed for peptide YY, TNF-alpha, and IL-1beta. In the cases, mean consumption was less when sick than when healthy. The mean plasma level of peptide YY was higher for cases than controls and higher for cases when sick than when healthy. TNF-alpha levels were higher in cases than controls at visit 1 and also higher in cases when sick than when healthy. There were no differences in IL-1beta levels between cases and controls or between cases when sick and healthy. Peptide YY levels in children with diarrhea correlated with the likelihood of them eating less when sick than when healthy. Elevated serum peptide YY may be a mechanism for anorexia in children with diarrhea.

Self-Assembly of an α-Helical Peptide into a Crystalline Two-Dimensional Nanoporous Framework

DOE PAGES

Magnotti, Elizabeth L.; Hughes, Spencer A.; Dillard, Rebecca S.; ...

2016-11-22

Sequence-specific peptides have been demonstrated to self-assemble into structurally defined nanoscale objects including nanofibers, nanotubes, and nanosheets. The latter structures display significant promise for the construction of hybrid materials for functional devices due to their extended planar geometry. Realization of this objective necessitates the ability to control the structural features of the resultant assemblies through the peptide sequence. The design of a amphiphilic peptide, 3FD-IL, is described that comprises two repeats of a canonical 18 amino acid sequence associated with straight α-helical structures. Peptide 3FD-IL displays 3-fold screw symmetry in a helical conformation and self-assembles into nanosheets based on hexagonalmore » packing of helices. Biophysical evidence from TEM, cryo-TEM, SAXS, AFM, and STEM measurements on the 3FD-IL nanosheets support a structural model based on a honeycomb lattice, in which the length of the peptide determines the thickness of the nanosheet and the packing of helices defines the presence of nanoscale channels that permeate the sheet. The honeycomb structure can be rationalized on the basis of geometrical packing frustration in which the channels occupy defect sites that define a periodic superlattice. In conclusion, the resultant 2D materials may have potential as materials for nanoscale transport and controlled release applications.« less

Identification and application of self-binding zipper-like sequences in SARS-CoV spike protein.

PubMed

Zhang, Si Min; Liao, Ying; Neo, Tuan Ling; Lu, Yanning; Liu, Ding Xiang; Vahlne, Anders; Tam, James P

2018-05-22

Self-binding peptides containing zipper-like sequences, such as the Leu/Ile zipper sequence within the coiled coil regions of proteins and the cross-β spine steric zippers within the amyloid-like fibrils, could bind to the protein-of-origin through homophilic sequence-specific zipper motifs. These self-binding sequences represent opportunities for the development of biochemical tools and/or therapeutics. Here, we report on the identification of a putative self-binding β-zipper-forming peptide within the severe acute respiratory syndrome-associated coronavirus spike (S) protein and its application in viral detection. Peptide array scanning of overlapping peptides covering the entire length of S protein identified 34 putative self-binding peptides of six clusters, five of which contained octapeptide core consensus sequences. The Cluster I consensus octapeptide sequence GINITNFR was predicted by the Eisenberg's 3D profile method to have high amyloid-like fibrillation potential through steric β-zipper formation. Peptide C6 containing the Cluster I consensus sequence was shown to oligomerize and form amyloid-like fibrils. Taking advantage of this, C6 was further applied to detect the S protein expression in vitro by fluorescence staining. Meanwhile, the coiled-coil-forming Leu/Ile heptad repeat sequences within the S protein were under-represented during peptide array scanning, in agreement with that long peptide lengths were required to attain high helix-mediated interaction avidity. The data suggest that short β-zipper-like self-binding peptides within the S protein could be identified through combining the peptide scanning and predictive methods, and could be exploited as biochemical detection reagents for viral infection. Copyright © 2018. Published by Elsevier Ltd.

The major genetic determinants of HIV-1 control affect HLA class I peptide presentation.

PubMed

Pereyra, Florencia; Jia, Xiaoming; McLaren, Paul J; Telenti, Amalio; de Bakker, Paul I W; Walker, Bruce D; Ripke, Stephan; Brumme, Chanson J; Pulit, Sara L; Carrington, Mary; Kadie, Carl M; Carlson, Jonathan M; Heckerman, David; Graham, Robert R; Plenge, Robert M; Deeks, Steven G; Gianniny, Lauren; Crawford, Gabriel; Sullivan, Jordan; Gonzalez, Elena; Davies, Leela; Camargo, Amy; Moore, Jamie M; Beattie, Nicole; Gupta, Supriya; Crenshaw, Andrew; Burtt, Noël P; Guiducci, Candace; Gupta, Namrata; Gao, Xiaojiang; Qi, Ying; Yuki, Yuko; Piechocka-Trocha, Alicja; Cutrell, Emily; Rosenberg, Rachel; Moss, Kristin L; Lemay, Paul; O'Leary, Jessica; Schaefer, Todd; Verma, Pranshu; Toth, Ildiko; Block, Brian; Baker, Brett; Rothchild, Alissa; Lian, Jeffrey; Proudfoot, Jacqueline; Alvino, Donna Marie L; Vine, Seanna; Addo, Marylyn M; Allen, Todd M; Altfeld, Marcus; Henn, Matthew R; Le Gall, Sylvie; Streeck, Hendrik; Haas, David W; Kuritzkes, Daniel R; Robbins, Gregory K; Shafer, Robert W; Gulick, Roy M; Shikuma, Cecilia M; Haubrich, Richard; Riddler, Sharon; Sax, Paul E; Daar, Eric S; Ribaudo, Heather J; Agan, Brian; Agarwal, Shanu; Ahern, Richard L; Allen, Brady L; Altidor, Sherly; Altschuler, Eric L; Ambardar, Sujata; Anastos, Kathryn; Anderson, Ben; Anderson, Val; Andrady, Ushan; Antoniskis, Diana; Bangsberg, David; Barbaro, Daniel; Barrie, William; Bartczak, J; Barton, Simon; Basden, Patricia; Basgoz, Nesli; Bazner, Suzane; Bellos, Nicholaos C; Benson, Anne M; Berger, Judith; Bernard, Nicole F; Bernard, Annette M; Birch, Christopher; Bodner, Stanley J; Bolan, Robert K; Boudreaux, Emilie T; Bradley, Meg; Braun, James F; Brndjar, Jon E; Brown, Stephen J; Brown, Katherine; Brown, Sheldon T; Burack, Jedidiah; Bush, Larry M; Cafaro, Virginia; Campbell, Omobolaji; Campbell, John; Carlson, Robert H; Carmichael, J Kevin; Casey, Kathleen K; Cavacuiti, Chris; Celestin, Gregory; Chambers, Steven T; Chez, Nancy; Chirch, Lisa M; Cimoch, Paul J; Cohen, Daniel; Cohn, Lillian E; Conway, Brian; Cooper, David A; Cornelson, Brian; Cox, David T; Cristofano, Michael V; Cuchural, George; Czartoski, Julie L; Dahman, Joseph M; Daly, Jennifer S; Davis, Benjamin T; Davis, Kristine; Davod, Sheila M; DeJesus, Edwin; Dietz, Craig A; Dunham, Eleanor; Dunn, Michael E; Ellerin, Todd B; Eron, Joseph J; Fangman, John J W; Farel, Claire E; Ferlazzo, Helen; Fidler, Sarah; Fleenor-Ford, Anita; Frankel, Renee; Freedberg, Kenneth A; French, Neel K; Fuchs, Jonathan D; Fuller, Jon D; Gaberman, Jonna; Gallant, Joel E; Gandhi, Rajesh T; Garcia, Efrain; Garmon, Donald; Gathe, Joseph C; Gaultier, Cyril R; Gebre, Wondwoosen; Gilman, Frank D; Gilson, Ian; Goepfert, Paul A; Gottlieb, Michael S; Goulston, Claudia; Groger, Richard K; Gurley, T Douglas; Haber, Stuart; Hardwicke, Robin; Hardy, W David; Harrigan, P Richard; Hawkins, Trevor N; Heath, Sonya; Hecht, Frederick M; Henry, W Keith; Hladek, Melissa; Hoffman, Robert P; Horton, James M; Hsu, Ricky K; Huhn, Gregory D; Hunt, Peter; Hupert, Mark J; Illeman, Mark L; Jaeger, Hans; Jellinger, Robert M; John, Mina; Johnson, Jennifer A; Johnson, Kristin L; Johnson, Heather; Johnson, Kay; Joly, Jennifer; Jordan, Wilbert C; Kauffman, Carol A; Khanlou, Homayoon; Killian, Robert K; Kim, Arthur Y; Kim, David D; Kinder, Clifford A; Kirchner, Jeffrey T; Kogelman, Laura; Kojic, Erna Milunka; Korthuis, P Todd; Kurisu, Wayne; Kwon, Douglas S; LaMar, Melissa; Lampiris, Harry; Lanzafame, Massimiliano; Lederman, Michael M; Lee, David M; Lee, Jean M L; Lee, Marah J; Lee, Edward T Y; Lemoine, Janice; Levy, Jay A; Llibre, Josep M; Liguori, Michael A; Little, Susan J; Liu, Anne Y; Lopez, Alvaro J; Loutfy, Mono R; Loy, Dawn; Mohammed, Debbie Y; Man, Alan; Mansour, Michael K; Marconi, Vincent C; Markowitz, Martin; Marques, Rui; Martin, Jeffrey N; Martin, Harold L; Mayer, Kenneth Hugh; McElrath, M Juliana; McGhee, Theresa A; McGovern, Barbara H; McGowan, Katherine; McIntyre, Dawn; Mcleod, Gavin X; Menezes, Prema; Mesa, Greg; Metroka, Craig E; Meyer-Olson, Dirk; Miller, Andy O; Montgomery, Kate; Mounzer, Karam C; Nagami, Ellen H; Nagin, Iris; Nahass, Ronald G; Nelson, Margret O; Nielsen, Craig; Norene, David L; O'Connor, David H; Ojikutu, Bisola O; Okulicz, Jason; Oladehin, Olakunle O; Oldfield, Edward C; Olender, Susan A; Ostrowski, Mario; Owen, William F; Pae, Eunice; Parsonnet, Jeffrey; Pavlatos, Andrew M; Perlmutter, Aaron M; Pierce, Michael N; Pincus, Jonathan M; Pisani, Leandro; Price, Lawrence Jay; Proia, Laurie; Prokesch, Richard C; Pujet, Heather Calderon; Ramgopal, Moti; Rathod, Almas; Rausch, Michael; Ravishankar, J; Rhame, Frank S; Richards, Constance Shamuyarira; Richman, Douglas D; Rodes, Berta; Rodriguez, Milagros; Rose, Richard C; Rosenberg, Eric S; Rosenthal, Daniel; Ross, Polly E; Rubin, David S; Rumbaugh, Elease; Saenz, Luis; Salvaggio, Michelle R; Sanchez, William C; Sanjana, Veeraf M; Santiago, Steven; Schmidt, Wolfgang; Schuitemaker, Hanneke; Sestak, Philip M; Shalit, Peter; Shay, William; Shirvani, Vivian N; Silebi, Vanessa I; Sizemore, James M; Skolnik, Paul R; Sokol-Anderson, Marcia; Sosman, James M; Stabile, Paul; Stapleton, Jack T; Starrett, Sheree; Stein, Francine; Stellbrink, Hans-Jurgen; Sterman, F Lisa; Stone, Valerie E; Stone, David R; Tambussi, Giuseppe; Taplitz, Randy A; Tedaldi, Ellen M; Telenti, Amalio; Theisen, William; Torres, Richard; Tosiello, Lorraine; Tremblay, Cecile; Tribble, Marc A; Trinh, Phuong D; Tsao, Alice; Ueda, Peggy; Vaccaro, Anthony; Valadas, Emilia; Vanig, Thanes J; Vecino, Isabel; Vega, Vilma M; Veikley, Wenoah; Wade, Barbara H; Walworth, Charles; Wanidworanun, Chingchai; Ward, Douglas J; Warner, Daniel A; Weber, Robert D; Webster, Duncan; Weis, Steve; Wheeler, David A; White, David J; Wilkins, Ed; Winston, Alan; Wlodaver, Clifford G; van't Wout, Angelique; Wright, David P; Yang, Otto O; Yurdin, David L; Zabukovic, Brandon W; Zachary, Kimon C; Zeeman, Beth; Zhao, Meng

2010-12-10

Infectious and inflammatory diseases have repeatedly shown strong genetic associations within the major histocompatibility complex (MHC); however, the basis for these associations remains elusive. To define host genetic effects on the outcome of a chronic viral infection, we performed genome-wide association analysis in a multiethnic cohort of HIV-1 controllers and progressors, and we analyzed the effects of individual amino acids within the classical human leukocyte antigen (HLA) proteins. We identified >300 genome-wide significant single-nucleotide polymorphisms (SNPs) within the MHC and none elsewhere. Specific amino acids in the HLA-B peptide binding groove, as well as an independent HLA-C effect, explain the SNP associations and reconcile both protective and risk HLA alleles. These results implicate the nature of the HLA-viral peptide interaction as the major factor modulating durable control of HIV infection.

Leucine-rich Repeats of Bacterial Surface Proteins Serve as Common Pattern Recognition Motifs of Human Scavenger Receptor gp340*

PubMed Central

Loimaranta, Vuokko; Hytönen, Jukka; Pulliainen, Arto T.; Sharma, Ashu; Tenovuo, Jorma; Strömberg, Nicklas; Finne, Jukka

2009-01-01

Scavenger receptors are innate immune molecules recognizing and inducing the clearance of non-host as well as modified host molecules. To recognize a wide pattern of invading microbes, many scavenger receptors bind to common pathogen-associated molecular patterns, such as lipopolysaccharides and lipoteichoic acids. Similarly, the gp340/DMBT1 protein, a member of the human scavenger receptor cysteine-rich protein family, displays a wide ligand repertoire. The peptide motif VEVLXXXXW derived from its scavenger receptor cysteine-rich domains is involved in some of these interactions, but most of the recognition mechanisms are unknown. In this study, we used mass spectrometry sequencing, gene inactivation, and recombinant proteins to identify Streptococcus pyogenes protein Spy0843 as a recognition receptor of gp340. Antibodies against Spy0843 are shown to protect against S. pyogenes infection, but no function or host receptor have been identified for the protein. Spy0843 belongs to the leucine-rich repeat (Lrr) family of eukaryotic and prokaryotic proteins. Experiments with truncated forms of the recombinant proteins confirmed that the Lrr region is needed in the binding of Spy0843 to gp340. The same motif of two other Lrr proteins, LrrG from the Gram-positive S. agalactiae and BspA from the Gram-negative Tannerella forsythia, also mediated binding to gp340. Moreover, inhibition of Spy0843 binding occurred with peptides containing the VEVLXXXXW motif, but also peptides devoid of the XXXXW motif inhibited binding of Lrr proteins. These results thus suggest that the conserved Lrr motif in bacterial proteins serves as a novel pattern recognition motif for unique core peptides of human scavenger receptor gp340. PMID:19465482

Biochemistry and Biophysics of HIV-1 gp41 – membrane interactions

PubMed Central

Cai, Lifeng; Gochin, Miriam; Liu, Keliang

2011-01-01

Human immunodeficiency virus type 1 (HIV-1), the pathogen of acquired immunodeficiency syndrome (AIDS), causes ~2 millions death every year and still defies an effective vaccine. HIV-1 infects host cells through envelope protein – mediated virus-cell fusion. The transmembrane subunit of envelope protein, gp41, is the molecular machinery which facilitates fusion. Its ectodomain contains several distinguishing functional domains, fusion peptide (FP), N-terminal heptad repeat (NHR), C-terminal heptad repeat (CHR) and membrane proximal extracellular region (MPER). During the fusion process, FP inserts into the host cell membrane, and an extended gp41 prehairpin conformation bridges the viral and cell membranes through MPER and FP respectively. Subsequent conformational change of the unstable prehairpin results in a coiled-coil 6-helix bundle (6HB) structure formed between NHR and CHR. The energetics of 6HB formation drives membrane apposition and fusion. Drugs targeting gp41 functional domains to prevent 6HB formation inhibit HIV-1 infection. T20 (enfuvirtide, Fuzeon) was approved by the US FDA in 2003 as the first fusion inhibitor. It is a 36-residue peptide from the gp41 CHR, and it inhibits 6HB formation by targeting NHR and lipids. Development of new fusion inhibitors, especially small molecule drugs, is encouraged to overcome the shortcomings of T20 as a peptide drug. Hydrophobic characteristics and membrane association are critical for gp41 function and mechanism of action. Research in gp41-membrane interactions, using peptides corresponding to specific functional domains, or constructs including several interactive domains, are reviewed here to get a better understanding of gp41 mediated virus-cell fusion that can inform or guide the design of new HIV-1 fusion inhibitors. PMID:22044229

Biochemistry and biophysics of HIV-1 gp41 - membrane interactions and implications for HIV-1 envelope protein mediated viral-cell fusion and fusion inhibitor design.

PubMed

Cai, Lifeng; Gochin, Miriam; Liu, Keliang

2011-12-01

Human immunodeficiency virus type 1 (HIV-1), the pathogen of acquired immunodeficiency syndrome (AIDS), causes ~2 millions death every year and still defies an effective vaccine. HIV-1 infects host cells through envelope protein - mediated virus-cell fusion. The transmembrane subunit of envelope protein, gp41, is the molecular machinery which facilitates fusion. Its ectodomain contains several distinguishing functional domains, fusion peptide (FP), Nterminal heptad repeat (NHR), C-terminal heptad repeat (CHR) and membrane proximal extracellular region (MPER). During the fusion process, FP inserts into the host cell membrane, and an extended gp41 prehairpin conformation bridges the viral and cell membranes through MPER and FP respectively. Subsequent conformational change of the unstable prehairpin results in a coiled-coil 6-helix bundle (6HB) structure formed between NHR and CHR. The energetics of 6HB formation drives membrane apposition and fusion. Drugs targeting gp41 functional domains to prevent 6HB formation inhibit HIV-1 infection. T20 (enfuvirtide, Fuzeon) was approved by the US FDA in 2003 as the first fusion inhibitor. It is a 36-residue peptide from the gp41 CHR, and it inhibits 6HB formation by targeting NHR and lipids. Development of new fusion inhibitors, especially small molecule drugs, is encouraged to overcome the shortcomings of T20 as a peptide drug. Hydrophobic characteristics and membrane association are critical for gp41 function and mechanism of action. Research in gp41-membrane interactions, using peptides corresponding to specific functional domains, or constructs including several interactive domains, are reviewed here to get a better understanding of gp41 mediated virus-cell fusion that can inform or guide the design of new HIV-1 fusion inhibitors.

Deleting the Redundant TSH Receptor C-Peptide Region Permits Generation of the Conformationally Intact Extracellular Domain by Insect Cells.

PubMed

Chen, Chun-Rong; Salazar, Larry M; McLachlan, Sandra M; Rapoport, Basil

2015-07-01

The TSH receptor (TSHR) extracellular domain (ECD) comprises a N-terminal leucine-rich repeat domain and an hinge region (HR), the latter contributing to ligand binding and critical for receptor activation. The crystal structure of the leucine-rich repeat domain component has been solved, but previous attempts to generate conformationally intact complete ECD or the isolated HR component for structural analysis have failed. The TSHR HR contains a C-peptide segment that is removed during spontaneous TSHR intramolecular cleavage into disulfide linked A- and B-subunits. We hypothesized that deletion of the redundant C-peptide would overcome the obstacle to generating conformationally intact TSHR ECD protein. Indeed, lacking the C-peptide region, the TSHR ECD (termed ECD-D1) and the isolated HR (termed HR-D1) were secreted into medium of insect cells infected with baculoviruses coding for these modified proteins. The identities of TSHR ECD-D1 and HR-D1 were confirmed by ELISA and immunoblotting using TSHR-specific monoclonal antibodies. The TSHR-ECD-D1 in conditioned medium was folded correctly, as demonstrated by its ability to inhibit radiolabeled TSH binding to the TSH holoreceptor. The TSHR ECD-D1 purification was accomplished in a single step using a TSHR monoclonal antibody affinity column, whereas the HR-D1 required a multistep protocol with a low yield. In conclusion, we report a novel approach to generate the TSHR ECD, as well as the isolated HR in insect cells, the former in sufficient amounts for structural studies. However, such studies will require previous complexing of the ECD with a ligand such as TSH or a thyroid-stimulating antibody.

ABT-510, a modified type 1 repeat peptide of thrombospondin, inhibits malignant glioma growth in vivo by inhibiting angiogenesis.

PubMed

Anderson, Joshua C; Grammer, J Robert; Wang, Wenquan; Nabors, L Burton; Henkin, Jack; Stewart, Jerry E; Gladson, Candece L

2007-03-01

Anti-angiogenic therapies would be particularly beneficial in the treatment of malignant gliomas. Peptides derived from the second type 1 repeat (TSR) of thrombospondin-1 (TSP-1) have been shown to inhibit angiogenesis in non-glioma tumor models and a modified TSR peptide, ABT-510, has now entered into Phase II clinical trials of its efficacy in non-glioma tumors. As microvascular endothelial cells (MvEC) exhibit heterogeneity, we evaluated the ability of the modified TSR peptide (NAcSarGlyValDallolleThrNvalleArgProNHE, ABT-510) to inhibit malignant glioma growth in vivo and to induce apoptosis of brain microvessel endothelial cells (MvEC) propagated in vitro. We found that daily administration of ABT-510 until euthanasia (days 7 to 19), significantly inhibited the growth of human malignant astrocytoma tumors established in the brain of athymic nude mice. The microvessel density was significantly lower and the number of apoptotic MvEC was significantly higher (3-fold) in the tumors of the ABT-510-treated animals. Similar results were found using a model in which the established tumor is an intracerebral malignant glioma propagated in a syngeneic mouse model. ABT-510 treatment of primary human brain MvEC propagated as a monolayer resulted in induction of apoptosis in a dose- and time-dependent manner through a caspase-8-dependent mechanism. It also inhibited tubular morphogenesis of MvEC propagated in collagen gels in a dose- and caspase-8 dependent manner through a mechanism that requires the TSP-1 receptor (CD36) on the MvEC. These findings indicate that ABT-510 should be evaluated as a therapeutic option for patients with malignant glioma.

Development of a group contribution method for estimating free energy of peptides in a dodecane-water system via molecular dynamic simulations.

PubMed

Mora Osorio, Camilo Andrés; González Barrios, Andrés Fernando

2016-12-07

Calculation of the Gibbs free energy changes of biological molecules at the oil-water interface is commonly performed with Molecular Dynamics simulations (MD). It is a process that could be performed repeatedly in order to find some molecules of high stability in this medium. Here, an alternative method of calculation has been proposed: a group contribution method (GCM) for peptides based on MD of the twenty classic amino acids to obtain free energy change during the insertion of any peptide chain in water-dodecane interfaces. Multiple MD of the twenty classic amino acids located at the interface of rectangular simulation boxes with a dodecane-water medium were performed. A GCM to calculate the free energy of entire peptides is then proposed. The method uses the summation of the Gibbs free energy of each amino acid adjusted in function of its presence or absence in the chain as well as its hydrophobic characteristics. Validation of the equation was performed with twenty-one peptides all simulated using MD in dodecane-water rectangular boxes in previous work, obtaining an average relative error of 16%.

NMR based solvent exchange experiments to understand the conformational preference of intrinsically disordered proteins using FG-nucleoporin peptide as a model

PubMed Central

Heisel, Kurt A.; Krishnan, V. V.

2014-01-01

The conformational preference of a peptide with three phenylalanine-glycine (FG) repeats from the intrinsically disordered domain of nucleoporin 159 (nup159) from the yeast nucleopore complex (NPC) is studied. Conformational states of this FG-peptide in dimethyl sulfoxide (DMSO), a non-native solvent are first studied. A solvent exchange scheme is designed and performed to understand how the conformational preferences of the peptide are altered as the solvent shifts from DMSO to water. An ensemble of structures of a 19-residue peptide is determined based on 13Cα, 1Hα, and 1HN chemical shifts and with inter-proton distances. An experimental model is then presented where chemical shifts and amide-proton temperature dependence is probed at changing DMSO to water ratios. These co-solvent experiments provide evidence of a conformational change as the fraction of water increases by the stark change in the behavior of amide protons under varied temperature. This investigation provides a NMR based experimental method in the field of intrinsically disordered proteins to realize conformational transitions from a non-native set of structures (in DMSO) to a native set of disordered conformers (in water). PMID:24037535

Graphene Nanopores for Protein Sequencing.

PubMed

Wilson, James; Sloman, Leila; He, Zhiren; Aksimentiev, Aleksei

2016-07-19

An inexpensive, reliable method for protein sequencing is essential to unraveling the biological mechanisms governing cellular behavior and disease. Current protein sequencing methods suffer from limitations associated with the size of proteins that can be sequenced, the time, and the cost of the sequencing procedures. Here, we report the results of all-atom molecular dynamics simulations that investigated the feasibility of using graphene nanopores for protein sequencing. We focus our study on the biologically significant phenylalanine-glycine repeat peptides (FG-nups)-parts of the nuclear pore transport machinery. Surprisingly, we found FG-nups to behave similarly to single stranded DNA: the peptides adhere to graphene and exhibit step-wise translocation when subject to a transmembrane bias or a hydrostatic pressure gradient. Reducing the peptide's charge density or increasing the peptide's hydrophobicity was found to decrease the translocation speed. Yet, unidirectional and stepwise translocation driven by a transmembrane bias was observed even when the ratio of charged to hydrophobic amino acids was as low as 1:8. The nanopore transport of the peptides was found to produce stepwise modulations of the nanopore ionic current correlated with the type of amino acids present in the nanopore, suggesting that protein sequencing by measuring ionic current blockades may be possible.

Dynamic reassembly of peptide RADA16 nanofiber scaffold

NASA Astrophysics Data System (ADS)

Yokoi, Hidenori; Kinoshita, Takatoshi; Zhang, Shuguang

2005-06-01

Nanofiber structures of some peptides and proteins as biological materials have been studied extensively, but their molecular mechanism of self-assembly and reassembly still remains unclear. We report here the reassembly of an ionic self-complementary peptide RADARADARADARADA (RADA16-I) that forms a well defined nanofiber scaffold. The 16-residue peptide forms stable -sheet structure and undergoes molecular self-assembly into nanofibers and eventually a scaffold hydrogel consisting of >99.5% water. In this study, the nanofiber scaffold was sonicated into smaller fragments. Circular dichroism, atomic force microscopy, and rheology were used to follow the kinetics of the reassembly. These sonicated fragments not only quickly reassemble into nanofibers that were indistinguishable from the original material, but their reassembly also correlated with the rheological analyses showing an increase of scaffold rigidity as a function of nanofiber length. The disassembly and reassembly processes were repeated four times and, each time, the reassembly reached the original length. We proposed a plausible sliding diffusion model to interpret the reassembly involving complementary nanofiber cohesive ends. This reassembly process is important for fabrication of new scaffolds for 3D cell culture, tissue repair, and regenerative medicine. atomic force microscopy | circular dichroism | dynamic behaviors | ionic self-complementary peptides | nanofiber hydrogels

Development of a Recombinant Multifunctional Biomacromolecule for Targeted Gene Transfer to Prostate Cancer Cells.

PubMed

Hatefi, Arash; Karjoo, Zahra; Nomani, Alireza

2017-09-11

The objective of this study was to genetically engineer a fully functional single chain fusion peptide composed of motifs from diverse biological and synthetic origins that can perform multiple tasks including DNA condensation, cell targeting, cell transfection, particle shielding from immune system and effective gene transfer to prostate tumors. To achieve the objective, a single chain biomacromolecule (vector) consisted of four repeatative units of histone H2A peptide, fusogenic peptide GALA, short elastin-like peptide, and PC-3 cell targeting peptide was designed. To examine the functionality of each motif in the vector sequence, it was characterized in terms of size and zeta potential by Zetasizer, PC-3 cell targeting and transfection by flowcytometry, IgG induction by immunogenicity assay, and PC-3 tumor transfection by quantitative live animal imaging. Overall, the results of this study showed the possibility of using genetic engineering techniques to program various functionalities into one single chain vector and create a multifunctional nonimmunogenic biomacromolecule for targeted gene transfer to prostate cancer cells. This proof-of-concept study is a significant step forward toward creating a library of vectors for targeted gene transfer to any cancer cell type at both in vitro and in vivo levels.

Inhibition of Henipavirus fusion and infection by heptad-derived peptides of the Nipah virus fusion glycoprotein

PubMed Central

Bossart, Katharine N; Mungall, Bruce A; Crameri, Gary; Wang, Lin-Fa; Eaton, Bryan T; Broder, Christopher C

2005-01-01

Background The recent emergence of four new members of the paramyxovirus family has heightened the awareness of and re-energized research on new and emerging diseases. In particular, the high mortality and person to person transmission associated with the most recent Nipah virus outbreaks, as well as the very recent re-emergence of Hendra virus, has confirmed the importance of developing effective therapeutic interventions. We have previously shown that peptides corresponding to the C-terminal heptad repeat (HR-2) of the fusion envelope glycoprotein of Hendra virus and Nipah virus were potent inhibitors of both Hendra virus and Nipah virus-mediated membrane fusion using recombinant expression systems. In the current study, we have developed shorter, second generation HR-2 peptides which include a capped peptide via amidation and acetylation and two poly(ethylene glycol)-linked (PEGylated) peptides, one with the PEG moity at the C-terminus and the other at the N-terminus. Here, we have evaluated these peptides as well as the corresponding scrambled peptide controls in Nipah virus and Hendra virus-mediated membrane fusion and against infection by live virus in vitro. Results Unlike their predecessors, the second generation HR-2 peptides exhibited high solubility and improved synthesis yields. Importantly, both Nipah virus and Hendra virus-mediated fusion as well as live virus infection were potently inhibited by both capped and PEGylated peptides with IC50 concentrations similar to the original HR-2 peptides, whereas the scrambled modified peptides had no inhibitory effect. These data also indicate that these chemical modifications did not alter the functional properties of the peptides as inhibitors. Conclusion Nipah virus and Hendra virus infection in vitro can be potently blocked by specific HR-2 peptides. The improved synthesis and solubility characteristics of the second generation HR-2 peptides will facilitate peptide synthesis for pre-clinical trial application in an animal model of Henipavirus infection. The applied chemical modifications are also predicted to increase the serum half-life in vivo and should increase the chance of success in the development of an effective antiviral therapy. PMID:16026621

Effect of noncovalent interaction on the self-assembly of a designed peptide and its potential use as a carrier for controlled bFGF release

PubMed Central

Liu, Yanfei; Zhang, Ling; Wei, Wei

2017-01-01

Peptide self-assembly is one of the promising bottom-up approaches for creating synthetic supermolecular architectures. Noncovalent interactions such as hydrophobic packing, electrostatic interaction, and polypeptide chain entropy (ΔSC) are the most relevant factors that affect the folding and self-assembly of peptides and the stability of supermolecular structures. The GVGV tetrapeptide is an abundant repeat in elastin, an extracellular matrix protein. In this study, four GVGV-containing peptides were designed with the aim of understanding the effects of these weak interactions on peptide self-assembly. Transmission electron microscopy, circular dichroism spectroscopy, dynamic light scattering measurements, and rheometry assays were used to study the structural features of the peptides. Because self-assembling peptides with different amino acid sequences may significantly affect protein release, basic fibroblast growth factor (bFGF) was used as a model molecule and encapsulated within the P2 (RLDLGVGVRLDLGVGV) hydrogel to study the release kinetics. The results showed that the balance among hydrophobic effects, electrostatic interactions, and chain entropy determined the molecular state and self-assembly of the peptide. Moreover, encapsulation of bFGF within the P2 hydrogel allowed its sustained release without causing changes in the secondary structure. The release profiles could be tuned by adjusting the P2 hydrogel concentration. Cell Counting Kit-8 and Western blot assays demonstrated that the encapsulated and released bFGFs were biologically active and capable of promoting the proliferation of murine fibroblast NIH-3T3 cells, most likely due to the activation of downstream signaling pathways. PMID:28176898

The SysteMHC Atlas project

PubMed Central

Shao, Wenguang; Pedrioli, Patrick G A; Wolski, Witold; Scurtescu, Cristian; Schmid, Emanuel; Courcelles, Mathieu; Schuster, Heiko; Kowalewski, Daniel; Marino, Fabio; Arlehamn, Cecilia S L; Vaughan, Kerrie; Peters, Bjoern; Sette, Alessandro; Ottenhoff, Tom H M; Meijgaarden, Krista E; Nieuwenhuizen, Natalie; Kaufmann, Stefan H E; Schlapbach, Ralph; Castle, John C; Nesvizhskii, Alexey I; Nielsen, Morten; Deutsch, Eric W; Campbell, David S; Moritz, Robert L; Zubarev, Roman A; Ytterberg, Anders Jimmy; Purcell, Anthony W; Marcilla, Miguel; Paradela, Alberto; Wang, Qi; Costello, Catherine E; Ternette, Nicola; van Veelen, Peter A; van Els, Cécile A C M; de Souza, Gustavo A; Sollid, Ludvig M; Admon, Arie; Stevanovic, Stefan; Rammensee, Hans-Georg; Thibault, Pierre; Perreault, Claude; Bassani-Sternberg, Michal

2018-01-01

Abstract Mass spectrometry (MS)-based immunopeptidomics investigates the repertoire of peptides presented at the cell surface by major histocompatibility complex (MHC) molecules. The broad clinical relevance of MHC-associated peptides, e.g. in precision medicine, provides a strong rationale for the large-scale generation of immunopeptidomic datasets and recent developments in MS-based peptide analysis technologies now support the generation of the required data. Importantly, the availability of diverse immunopeptidomic datasets has resulted in an increasing need to standardize, store and exchange this type of data to enable better collaborations among researchers, to advance the field more efficiently and to establish quality measures required for the meaningful comparison of datasets. Here we present the SysteMHC Atlas (https://systemhcatlas.org), a public database that aims at collecting, organizing, sharing, visualizing and exploring immunopeptidomic data generated by MS. The Atlas includes raw mass spectrometer output files collected from several laboratories around the globe, a catalog of context-specific datasets of MHC class I and class II peptides, standardized MHC allele-specific peptide spectral libraries consisting of consensus spectra calculated from repeat measurements of the same peptide sequence, and links to other proteomics and immunology databases. The SysteMHC Atlas project was created and will be further expanded using a uniform and open computational pipeline that controls the quality of peptide identifications and peptide annotations. Thus, the SysteMHC Atlas disseminates quality controlled immunopeptidomic information to the public domain and serves as a community resource toward the generation of a high-quality comprehensive map of the human immunopeptidome and the support of consistent measurement of immunopeptidomic sample cohorts. PMID:28985418

A software suite for the generation and comparison of peptide arrays from sets of data collected by liquid chromatography-mass spectrometry.

PubMed

Li, Xiao-jun; Yi, Eugene C; Kemp, Christopher J; Zhang, Hui; Aebersold, Ruedi

2005-09-01

There is an increasing interest in the quantitative proteomic measurement of the protein contents of substantially similar biological samples, e.g. for the analysis of cellular response to perturbations over time or for the discovery of protein biomarkers from clinical samples. Technical limitations of current proteomic platforms such as limited reproducibility and low throughput make this a challenging task. A new LC-MS-based platform is able to generate complex peptide patterns from the analysis of proteolyzed protein samples at high throughput and represents a promising approach for quantitative proteomics. A crucial component of the LC-MS approach is the accurate evaluation of the abundance of detected peptides over many samples and the identification of peptide features that can stratify samples with respect to their genetic, physiological, or environmental origins. We present here a new software suite, SpecArray, that generates a peptide versus sample array from a set of LC-MS data. A peptide array stores the relative abundance of thousands of peptide features in many samples and is in a format identical to that of a gene expression microarray. A peptide array can be subjected to an unsupervised clustering analysis to stratify samples or to a discriminant analysis to identify discriminatory peptide features. We applied the SpecArray to analyze two sets of LC-MS data: one was from four repeat LC-MS analyses of the same glycopeptide sample, and another was from LC-MS analysis of serum samples of five male and five female mice. We demonstrate through these two study cases that the SpecArray software suite can serve as an effective software platform in the LC-MS approach for quantitative proteomics.

High-Resolution Structure of a Self-Assembly-Competent Form of a Hydrophobic Peptide Captured in a Soluble [beta]-Sheet Scaffold

DOE Office of Scientific and Technical Information (OSTI.GOV)

Makabe, Koki; Biancalana, Matthew; Yan, Shude

2010-02-08

{beta}-Rich self-assembly is a major structural class of polypeptides, but still little is known about its atomic structures and biophysical properties. Major impediments for structural and biophysical studies of peptide self-assemblies include their insolubility and heterogeneous composition. We have developed a model system, termed peptide self-assembly mimic (PSAM), based on the single-layer {beta}-sheet of Borrelia outer surface protein A. PSAM allows for the capture of a defined number of self-assembly-like peptide repeats within a water-soluble protein, making structural and energetic studies possible. In this work, we extend our PSAM approach to a highly hydrophobic peptide sequence. We show that amore » penta-Ile peptide (Ile{sub 5}), which is insoluble and forms {beta}-rich self-assemblies in aqueous solution, can be captured within the PSAM scaffold in a form capable of self-assembly. The 1.1-{angstrom} crystal structure revealed that the Ile{sub 5} stretch forms a highly regular {beta}-strand within this flat {beta}-sheet. Self-assembly models built with multiple copies of the crystal structure of the Ile5 peptide segment showed no steric conflict, indicating that this conformation represents an assembly-competent form. The PSAM retained high conformational stability, suggesting that the flat {beta}-strand of the Ile{sub 5} stretch primed for self-assembly is a low-energy conformation of the Ile{sub 5} stretch and rationalizing its high propensity for self-assembly. The ability of the PSAM to 'solubilize' an otherwise insoluble peptide stretch suggests the potential of the PSAM approach to the characterization of self-assembling peptides.« less

Effects of pre-existing anti-carrier immunity and antigenic element multiplicity on efficacy of a modular virus-like particle vaccine.

PubMed

Chuan, Yap P; Rivera-Hernandez, Tania; Wibowo, Nani; Connors, Natalie K; Wu, Yang; Hughes, Fiona K; Lua, Linda H L; Middelberg, Anton P J

2013-09-01

Modularization of a peptide antigen for presentation on a microbially synthesized murine polyomavirus (MuPyV) virus-like particle (VLP) offers a new alternative for rapid and low-cost vaccine delivery at a global scale. In this approach, heterologous modules containing peptide antigenic elements are fused to and displayed on the VLP carrier, allowing enhancement of peptide immunogenicity via ordered and densely repeated presentation of the modules. This study addresses two key engineering questions pertaining to this platform, exploring the effects of (i) pre-existing carrier-specific immunity on modular VLP vaccine effectiveness and (ii) increase in the antigenic element number per VLP on peptide-specific immune response. These effects were studied in a mouse model and with modular MuPyV VLPs presenting a group A streptococcus (GAS) peptide antigen, J8i. The data presented here demonstrate that immunization with a modular VLP could induce high levels of J8i-specific antibodies despite a strong pre-existing anti-carrier immune response. Doubling of the J8i antigenic element number per VLP did not enhance J8i immunogenicity at a constant peptide dose. However, the strategy, when used in conjunction with increased VLP dose, could effectively increase the peptide dose up to 10-fold, leading to a significantly higher J8i-specific antibody titer. This study further supports feasibility of the MuPyV modular VLP vaccine platform by showing that, in the absence of adjuvant, modularized GAS antigenic peptide at a dose as low as 150 ng was sufficient to raise a high level of peptide-specific IgGs indicative of bactericidal activity. Copyright © 2013 Wiley Periodicals, Inc.

Production of double repeated B subunit of Shiga toxin 2e at high levels in transgenic lettuce plants as vaccine material for porcine edema disease.

PubMed

Matsui, Takeshi; Takita, Eiji; Sato, Toshio; Aizawa, Michie; Ki, Misa; Kadoyama, Yumiko; Hirano, Kenji; Kinjo, Satoko; Asao, Hiroshi; Kawamoto, Keiko; Kariya, Haruko; Makino, Sou-Ichi; Hamabata, Takashi; Sawada, Kazutoshi; Kato, Ko

2011-08-01

Pig edema disease is a bacterial disease caused by enterohemorrhagic Escherichia coli. E. coli produces Shiga toxin 2e (Stx2e), which is composed of one A subunit (Stx2eA) and five B subunits (Stx2eB). We previously reported production of Stx2eB in lettuce plants as a potential edible vaccine (Matsui et al. in Biosci Biotechnol Biochem 73:1628-1634, 2009). However, the accumulation level was very low, and it was necessary to improve expression of Stx2eB for potential use of this plant-based vaccine. Therefore, in this study, we optimized the Stx2eB expression cassette and found that a double repeated Stx2eB (2× Stx2eB) accumulates to higher levels than a single Stx2eB in cultured tobacco cells. Furthermore, a linker peptide between the two Stx2eB moieties played an important role in maximizing the effects of the double repeat. Finally, we generated transgenic lettuce plants expressing 2× Stx2eB with a suitable linker peptide that accumulate as much as 80 mg per 100 g fresh weight, a level that will allow us to use these transgenic lettuce plants practically to generate vaccine material.

A receptor-targeted nanocomplex vector system optimized for respiratory gene transfer.

PubMed

Tagalakis, Aristides D; McAnulty, Robin J; Devaney, James; Bottoms, Stephen E; Wong, John B; Elbs, Martin; Writer, Michele J; Hailes, Helen C; Tabor, Alethea B; O'Callaghan, Christopher; Jaffe, Adam; Hart, Stephen L

2008-05-01

Synthetic vectors for cystic fibrosis (CF) gene therapy are required that efficiently and safely transfect airway epithelial cells, rather than alveolar epithelial cells or macrophages, and that are nonimmunogenic, thus allowing for repeated delivery. We have compared several vector systems against these criteria including GL67, polyethylenimine (PEI) 22 and 25 kd and two new, synthetic vector formulations, comprising a cationic, receptor-targeting peptide K(16)GACSERSMNFCG (E), and the cationic liposomes (L) DHDTMA/DOPE or DOSEP3/DOPE. The lipid and peptide formulations self assemble into receptor-targeted nanocomplexes (RTNs) LED-1 and LED-2, respectively, on mixing with plasmid (D). LED-1 transfected airway epithelium efficiently, while LED-2 and GL67 preferentially transfected alveolar cells. PEI transfected airway epithelial cells with high efficiency, but was more toxic to the mice than the other formulations. On repeat dosing, LED-1 was equally as effective as the single dose, while GL67 was 30% less effective and PEI 22 kd displayed a 90% reduction of efficiency on repeated delivery. LED-1 thus was the only formulation that fulfilled the criteria for a CF gene therapy vector while GL67 and LED-2 may be appropriate for other respiratory diseases. Opportunities for PEI depend on a solution to its toxicity problems. LED-1 formulations were stable to nebulization, the most appropriate delivery method for CF.

Regional Blood Volume and Peripheral Blood Flow in the Postural Tachycardia Syndrome

PubMed Central

Stewart, Julian M.; Montgomery, Leslie D.

2015-01-01

Variants of postural tachycardia syndrome (POTS) are associated with increased (“high flow” POTS, HFP), decreased (“low flow POTS”, LFP) and normal (“normal flow POTS”, NFP) blood flow measured in the lower extremities while supine. We propose that postural tachycardia is related to thoracic hypovolemia during orthostasis but that the patterns of peripheral blood flow relate to different mechanisms for thoracic hypovolemia. We studied 37 POTS patients aged 14-21 years: 14 LFP, 15 NFP and 8 HFP patients and 12 healthy control subjects. Peripheral blood flow was measured supine by venous occlusion strain gauge plethysmography of the forearm and calf in order to subgroup patients. Using indocyanine green techniques we showed decreased cardiac index (CI) and increased total peripheral resistance (TPR) in LFP, increased CI and decreased TPR in HFP, and unchanged CI and TPR in NFP while supine compared to control subjects. Blood volume tended to be decreased in LFP compared to control subjects. We used impedance plethysmography to assess regional blood volume redistribution during upright tilt. Thoracic blood volume decreased while splanchnic, pelvic and leg blood volumes increased for all subjects during orthostasis, but were markedly lower than control for all POTS groups. Splanchnic volume was increased in NFP and LFP. Pelvic blood volume was increased in HFP only. Calf volume was increased above control in HFP and LFP. The results support the hypothesis of [at least] three pathophysiologic variants of POTS distinguished by peripheral blood flow related to characteristic changes in regional circulations. The data demonstrate enhanced thoracic hypovolemia during upright tilt and confirm that POTS is related to inadequate cardiac venous return during orthostasis. PMID:15117717

No association between ACE I/D polymorphism and cardiovascular hemodynamics during exercise in young women.

PubMed

Roltsch, M H; Brown, M D; Hand, B D; Kostek, M C; Phares, D A; Huberty, A; Douglass, L W; Ferrell, R E; Hagberg, J M

2005-10-01

The ACE I/D polymorphism has been shown to interact with habitual physical activity levels in postmenopausal women to associate with submaximal and with maximal exercise hemodynamics. This investigation was designed to assess the potential relationships between ACE genotype and oxygen consumption (VO2), cardiac output (Q), stroke volume (SV), heart rate (HR), blood pressure (BP), total peripheral resistance (TPR), and arteriovenous oxygen difference ([a-v]O2 diff) during submaximal and maximal exercise in young sedentary and endurance-trained women. Seventy-seven 18-35-yr-old women underwent a maximal exercise test and a number of cardiac output tests on a treadmill using the acetylene rebreathing technique. ACE genotype was not significantly associated with VO2max (II 41.4+/-1.2, ID 39.8+/-0.9, DD 39.8+/-1.1 ml/kg/min, p=ns) or maximal HR (II 191+/-2, ID 191+/-1, DD 193+/-2 bpm, p=ns). In addition, systolic and diastolic BP, (a-v)O2 diff, TPR, SV, and Q during maximal exercise were not significantly associated with ACE genotype. During submaximal exercise, SBP, Q, SV, HR, TPR, and (a-v)O2 diff were not significantly associated with ACE genotype. However, the association between diastolic BP during submaximal exercise and ACE genotype approached significance (p=0.08). In addition, there were no statistically significant interactions between ACE genotype and habitual physical activity (PA) levels for any of the submaximal or the maximal exercise hemodynamic variables. We conclude that the ACE I/D polymorphism was not associated, independently or interacting with habitual PA levels, submaximal, or maximal cardiovascular hemodynamics in young women.

Poundbury Camp in Context-a new Perspective on the Lives of Children from urban and rural Roman England.

PubMed

Rohnbogner, Anna; Lewis, Mary Elizabeth

2017-02-01

The current understanding of child morbidity in Roman England is dominated by studies of single sites/regions. Much of the data are derived from third to fifth century AD Poundbury Camp, Dorchester, Dorset, considered an unusual site due to high levels of non-adult morbidity. There is little understanding of children in rural areas, and whether Poundbury Camp was representative of Romano-British childhood. The study provides the first large scale analysis of child health in urban and rural Roman England, adding to the previously published intra-site analysis of non-adult paleopathology at Poundbury Camp. Age-at-death and pathology prevalence rates were reassessed for 953 non-adults (0-17 years) from five major urban, six minor urban, and four rural sites (first to fifth century AD). The data were compared to the results from 364 non-adults from Poundbury Camp. Rural sites demonstrated higher levels of infant burials, and greater prevalence of cribra orbitalia in the 1.1-2.5 year (TPR 64.3%), and 6.6-10.5 year cohorts (TPR 66.7%). Endocranial lesions were more frequent in the minor urban sample (TPR 15.9%). Three new cases of tuberculosis were identified in urban contexts. Vitamin D deficiency was most prevalent at Poundbury Camp (CPR 18.8%), vitamin C deficiency was identified more frequently in rural settlements (CPR 5.9%). The Poundbury Camp data on morbidity and mortality are not representative of patterns in Roman England and other major urban sites. Rural children suffered from a distinct set of pathologies described as diseases of deprivation, prompting reconsideration of how Romano-British land management affected those at the bottom of the social hierarchy. © 2016 Wiley Periodicals, Inc.

SU-E-T-525: Ionization Chamber Perturbation in Flattening Filter Free Beams

DOE Office of Scientific and Technical Information (OSTI.GOV)

Czarnecki, D; Voigts-Rhetz, P von; Zink, K

2015-06-15

Purpose: Changing the characteristic of a photon beam by mechanically removing the flattening filter may impact the dose response of ionization chambers. Thus, perturbation factors of cylindrical ionization chambers in conventional and flattening filter free photon beams were calculated by Monte Carlo simulations. Methods: The EGSnrc/BEAMnrc code system was used for all Monte Carlo calculations. BEAMnrc models of nine different linear accelerators with and without flattening filter were used to create realistic photon sources. Monte Carlo based calculations to determine the fluence perturbations due to the presens of the chambers components, the different material of the sensitive volume (air insteadmore » of water) as well as the volume effect were performed by the user code egs-chamber. Results: Stem, central electrode, wall, density and volume perturbation factors for linear accelerators with and without flattening filter were calculated as a function of the beam quality specifier TPR{sub 20/10}. A bias between the perturbation factors as a function of TPR{sub 20/10} for flattening filter free beams and conventional linear accelerators could not be observed for the perturbations caused by the components of the ionization chamber and the sensitive volume. Conclusion: The results indicate that the well-known small bias between the beam quality correction factor as a function of TPR20/10 for the flattening filter free and conventional linear accelerators is not caused by the geometry of the detector but rather by the material of the sensitive volume. This suggest that the bias for flattening filter free photon fields is only caused by the different material of the sensitive volume (air instead of water)« less

Pulmonary hemodynamics responses to hypoxia and/or CO2 inhalation during moderate exercise in humans.

PubMed

Doutreleau, Stéphane; Enache, Irina; Pistea, Cristina; Geny, Bernard; Charloux, Anne

2018-03-03

In this study, we hypothesized that adding CO 2 to an inhaled hypoxic gas mixture will limit the rise of pulmonary artery pressure (PAP) induced by a moderate exercise. Eight 20-year-old males performed four constant-load exercise tests on cycle at 40% of maximal oxygen consumption in four conditions: ambient air, normobaric hypoxia (12.5% O 2 ), inhaled CO 2 (4.5% CO 2 ), and combination of hypoxia and inhaled CO 2 . Doppler echocardiography was used to measure systolic (s)PAP, cardiac output (CO). Total pulmonary resistance (TPR) was calculated. Arterialized blood pH was 7.40 at exercise in ambient and hypoxia conditions, whereas CO 2 inhalation and combined conditions showed acidosis. sPAP increases from rest in ambient air to exercise ranged as follows: ambient + 110%, CO 2 inhalation + 135%, combined + 184%, hypoxia + 217% (p < 0.001). CO was higher when inhaling O 2 -poor gas mixtures with or without CO 2 (~ 17 L min -1 ) than in the other conditions (~ 14 L min -1 , p < 0.001). Exercise induced a significant decrease in TPR in the four conditions (p < 0.05) but less marked in hypoxia (- 19% of the resting value in ambient air) than in ambient (- 33%) and in both CO 2 inhalation and combined condition (- 29%). We conclude that (1) acute CO 2 inhalation did not significantly modify pulmonary hemodynamics during moderate exercise. (2) CO 2 adjunction to hypoxic gas mixture did not modify CO, despite a higher CaO 2 in combined condition than in hypoxia. (3) TPR was lower in combined than in hypoxia condition, limiting sPAP increase in combined condition.

Cyclooxygenase 2 inhibition suppresses tubuloglomerular feedback: roles of thromboxane receptors and nitric oxide

PubMed Central

Araujo, Magali; Welch, William J.

2009-01-01

Thromboxane (TxA2) and nitric oxide (NO) are potent vasoactive autocoids that modulate tubuloglomerular feedback (TGF). Each is produced in the macula densa (MD) by cyclooxygenase-2 (COX-2) and neuronal nitric oxide synthase (nNOS), respectively. Both enzymes are similarly regulated in the MD and their interaction may be an important factor in the regulation of TGF and glomerular filtration rate. We tested the hypothesis that TGF is modified by the balance between MD nNOS-dependent NO and MD COX-2-dependent TxA2. We measured maximal TGF during perfusion of the loop of Henle (LH) by continuous recording of the proximal tubule stopped flow pressure response to LH perfusion of artificial tubular fluid (ATF) at 0 and 40 nl/min. The response to inhibitors of COX-1 (SC-560), COX-2 [parecoxib (Pxb)], and nNOS (l-NPA) added to the ATF solution was measured in separate nephrons. COX-2 inhibition with Pxb reduced TGF by 46% (ATF + vehicle vs. ATF + Pxb), whereas COX-1 inhibition with SC-560 reduced TGF by only 23%. Pretreatment with intravenous infusion of SQ-29,548, a selective thromboxone/PGH2 receptor (TPR) antagonist, blocked all of the SC-560 effect on TGF, suggesting that this effect was due to activation of TPR. However, SQ-29,548 only partially diminished the effect of Pxb (−66%). Specific inhibition of nNOS with l-NPA increased TGF, as expected. However, the ability of Pxb to reduce TGF was significantly impaired with comicroperfusion of l-NPA. These data suggest that COX-2 modulates TGF by two proconstrictive actions: generation of TxA2 acting on TPR and by simultaneous reduction of NO. PMID:19144694

Signal transduction and downregulation of C-MET in HGF stimulated low and highly metastatic human osteosarcoma cells

DOE Office of Scientific and Technical Information (OSTI.GOV)

Husmann, Knut, E-mail: khusmann@research.balgrist.ch; Ducommun, Pascal; Division of Plastic Surgery and Hand Surgery, Department of Surgery, University Hospital Zurich, Zurich

2015-09-04

The poor outcome of osteosarcoma (OS), particularly in patients with metastatic disease and a five-year survival rate of only 20%, asks for more effective therapeutic strategies targeting malignancy-promoting mechanisms. Dysregulation of C-MET, its ligand hepatocyte growth factor (HGF) and the fusion oncogene product TPR-MET, first identified in human MNNG-HOS OS cells, have been described as cancer-causing factors in human cancers. Here, the expression of these molecules at the mRNA and the protein level and of HGF-stimulated signaling and downregulation of C-MET was compared in the parental low metastatic HOS and MG63 cell lines and the respective highly metastatic MNNG-HOS andmore » 143B and the MG63-M6 and MG63-M8 sublines. Interestingly, expression of TPR-MET was only observed in MNNG-HOS cells. HGF stimulated the phosphorylation of Akt and Erk1/2 in all cell lines investigated, but phospho-Stat3 remained at basal levels. Downregulation of HGF-stimulated Akt and Erk1/2 phosphorylation was much faster in the HGF expressing MG63-M8 cells than in HOS cells. Degradation of HGF-activated C-MET occurred predominantly through the proteasomal and to a lesser extent the lysosomal pathway in the cell lines investigated. Thus, HGF-stimulated Akt and Erk1/2 signaling as well as proteasomal degradation of HGF activated C-MET are potential therapeutic targets in OS. - Highlights: • Expression of TPR-MET was only observed in MNNG-HOS cells. • HGF stimulated the phosphorylation of Akt and Erk1/2 but not of Stat3 in osteosarcoma cell lines. • Degradation of HGF-activated C-MET occurred predominantly through the proteasomal pathway.« less

Resurgence of Malaria Following Discontinuation of Indoor Residual Spraying of Insecticide in an Area of Uganda With Previously High-Transmission Intensity.

PubMed

Raouf, Saned; Mpimbaza, Arthur; Kigozi, Ruth; Sserwanga, Asadu; Rubahika, Denis; Katamba, Henry; Lindsay, Steve W; Kapella, Bryan K; Belay, Kassahun A; Kamya, Moses R; Staedke, Sarah G; Dorsey, Grant

2017-08-01

Indoor residual spraying (IRS) and long-lasting insecticidal nets (LLINs) are the primary tools for malaria prevention in Africa. It is not known whether reductions in malaria can be sustained after IRS is discontinued. Our aim in this study was to assess changes in malaria morbidity in an area of Uganda with historically high transmission where IRS was discontinued after a 4-year period followed by universal LLIN distribution. Individual-level malaria surveillance data were collected from 1 outpatient department and 1 inpatient setting in Apac District, Uganda, from July 2009 through November 2015. Rounds of IRS were delivered approximately every 6 months from February 2010 through May 2014 followed by universal LLIN distribution in June 2014. Temporal changes in the malaria test positivity rate (TPR) were estimated during and after IRS using interrupted time series analyses, controlling for age, rainfall, and autocorrelation. Data include 65 421 outpatient visits and 13 955 pediatric inpatient admissions for which a diagnostic test for malaria was performed. In outpatients aged <5 years, baseline TPR was 60%-80% followed by a rapid and then sustained decrease to 15%-30%. During the 4-18 months following discontinuation of IRS, absolute TPR values increased by an average of 3.29% per month (95% confidence interval, 2.01%-4.57%), returning to baseline levels. Similar trends were seen in outpatients aged ≥5 years and pediatric admissions. Discontinuation of IRS in an area with historically high transmission intensity was associated with a rapid increase in malaria morbidity to pre-IRS levels. © The Author 2017. Published by Oxford University Press for the Infectious Diseases Society of America. All rights reserved. For permissions, e-mail: journals.permissions@oup.com

Molecular biomimetics: utilizing nature's molecular ways in practical engineering.

PubMed

Tamerler, Candan; Sarikaya, Mehmet

2007-05-01

In nature, proteins are the machinery that accomplish many functions through their specific recognition and interactions in biological systems from single-celled to multicellular organisms. Biomolecule-material interaction is accomplished via molecular specificity, leading to the formation of controlled structures and functions at all scales of dimensional hierarchy. Through evolution, molecular recognition and, consequently, functions developed through successive cycles of mutation and selection. Using biology as a guide, we can now understand, engineer and control peptide-material interactions and exploit these to tailor novel materials and systems for practical applications. We adapted combinatorial biology protocols to display peptide libraries, either on the cell surface or on phages, to select short peptides specific to a variety of practical materials systems. Following the selection step, we determined the kinetics and stability of peptide binding experimentally to understand the bound peptide structure via modeling and its assembly via atomic force microscopy. The peptides were further engineered to have multiple repeats or their amino acid sequences varied to tailor their function. Both nanoparticles and flat inorganic substrates containing multimaterials patterned at the nano- and microscales were used for self-directed immobilization of molecular constructs. The molecular biomimetic approach opens up new avenues for the design and utilization of multifunctional molecular systems with wide ranging applications, from tissue engineering, drug delivery and biosensors, to nanotechnology and bioremediation. Here we give examples of protein-mediated functional materials in biology, peptide selection and engineering with affinity to inorganics, demonstrate potential utilizations in materials science, engineering and medicine, and describe future prospects.

Histidine-lysine peptides as carriers of nucleic acids.

PubMed

Leng, Qixin; Goldgeier, Lisa; Zhu, Jingsong; Cambell, Patricia; Ambulos, Nicholas; Mixson, A James

2007-03-01

With their biodegradability and diversity of permutations, peptides have significant potential as carriers of nucleic acids. This review will focus on the sequence and branching patterns of peptide carriers composed primarily of histidines and lysines. While lysines within peptides are important for binding to the negatively charged phosphates, histidines are critical for endosomal lysis enabling nucleic acids to reach the cytosol. Histidine-lysine (HK) polymers by either covalent or ionic bonds with liposomes augment transfection compared to liposome carriers alone. More recently, we have examined peptides as sole carriers of nucleic acids because of their intrinsic advantages compared to the bipartite HK/liposome carriers. With a protocol change and addition of a histidine-rich tail, HK peptides as sole carriers were more effective than liposomes alone in several cell lines. While four-branched polymers with a primary repeating sequence pattern of -HHK- were more effective as carriers of plasmids, eight-branched polymers with a sequence pattern of -HHHK- were more effective as carriers of siRNA. Compared to polyethylenimine, HK carriers of siRNA and plasmids had reduced toxicity. When injected intravenously, HK polymers in complex with plasmids encoding antiangiogenic proteins significantly decreased tumor growth. Furthermore, modification of HK polymers with polyethylene glycol and vascular-specific ligands increased specificity of the polyplex to the tumor by more than 40-fold. Together with further development and insight on the structure of HK polyplexes, HK peptides may prove to be useful as carriers of different forms of nucleic acids both in vitro and in vivo.

Complete covalent structure of statherin, a tyrosine-rich acidic peptide which inhibits calcium phosphate precipitation from human parotid saliva.

PubMed

Schlesinger, D H; Hay, D I

1977-03-10

The complete amino acid sequence of human salivary statherin, a peptide which strongly inhibits precipitation from supersaturated calcium phosphate solutions, and therefore stabilizes supersaturated saliva, has been determined. The NH2-terminal half of this Mr=5380 (43 amino acids) polypeptide was determined by automated Edman degradations (liquid phase) on native statherin. The peptide was digested separately with trypsin, chymotrypsin, and Staphylococcus aureus protease, and the resulting peptides were purified by gel filtration. Manual Edman degradations on purified peptide fragments yielded peptides that completed the amino acid sequence through the penultimate COOH-terminal residue. These analyses, together with carboxypeptidase digestion of native statherin and of peptide fragments of statherin, established the complete sequence of the molecule. The 2 serine residues (positions 2 and 3) in statherin were identified as phosphoserine. The amino acid sequence of human salivary statherin is striking in a number of ways. The NH2-terminal one-third is highly polar and includes three polar dipeptides: H2PO3-Ser-Ser-H2PO3-Arg-Arg-, and Glu-Glu-. The COOH-terminal two-thirds of the molecule is hydrophobic, containing several repeating dipeptides: four of -Gn-Pro-, three of -Tyr-Gln-, two of -Gly-Tyr-, two of-Gln-Tyr-, and two of the tetrapeptide sequence -Pro-Tyr-Gln-Pro-. Unusual cleavage sites in the statherin sequence obtained with chymotrypsin and S. aureus protease were also noted.

Cathepsins L and Z Are Critical in Degrading Polyglutamine-containing Proteins within Lysosomes*

PubMed Central

Bhutani, Nidhi; Piccirillo, Rosanna; Hourez, Raphael; Venkatraman, Prasanna; Goldberg, Alfred L.

2012-01-01

In neurodegenerative diseases caused by extended polyglutamine (polyQ) sequences in proteins, aggregation-prone polyQ proteins accumulate in intraneuronal inclusions. PolyQ proteins can be degraded by lysosomes or proteasomes. Proteasomes are unable to hydrolyze polyQ repeat sequences, and during breakdown of polyQ proteins, they release polyQ repeat fragments for degradation by other cellular enzymes. This study was undertaken to identify the responsible proteases. Lysosomal extracts (unlike cytosolic enzymes) were found to rapidly hydrolyze polyQ sequences in peptides, proteins, or insoluble aggregates. Using specific inhibitors against lysosomal proteases, enzyme-deficient extracts, and pure cathepsins, we identified cathepsins L and Z as the lysosomal cysteine proteases that digest polyQ proteins and peptides. RNAi for cathepsins L and Z in different cell lines and adult mouse muscles confirmed that they are critical in degrading polyQ proteins (expanded huntingtin exon 1) but not other types of aggregation-prone proteins (e.g. mutant SOD1). Therefore, the activities of these two lysosomal cysteine proteases are important in host defense against toxic accumulation of polyQ proteins. PMID:22451661

Molecular cloning of crustins from the hemocytes of Brazilian penaeid shrimps.

PubMed

Rosa, Rafael Diego; Bandeira, Paula Terra; Barracco, Margherita Anna

2007-09-01

Crustins are antimicrobial peptides initially identified in the hemocytes of the crab Carcinus maenas (11.5-kDa peptide or carcinin) and recently also recognized in penaeid shrimps and other crustacean species. The aim of this study was to identify sequences encoding for crustins from the hemocytes of four Brazilian penaeid species: Farfantepenaeus paulensis, Farfantepenaeus subtilis, Farfantepenaeus brasiliensis and Litopenaeus schmitti. Using primers based on consensus nucleotide alignment of crustins from different crustaceans, cDNA sequences coding for crustins in all indigenous penaeid species were amplified. The obtained four crustin sequences encoded for peptides containing a hydrophobic N-terminal region rich in glycine repeats and a C-terminal part with 12 cysteine residues and a conserved whey acidic protein domain. All obtained crustin sequences showed high amino acidic similarity among each other and with crustins from litopenaeid shrimps (76-98%). This is the first report of crustins in native Brazilian penaeid shrimps.

The Major Genetic Determinants of HIV-1 Control Affect HLA Class I Peptide Presentation

PubMed Central

Pereyra, Florencia; Jia, Xiaoming; McLaren, Paul J.; Telenti, Amalio; de Bakker, Paul I.W.; Walker, Bruce D.; Jia, Xiaoming; McLaren, Paul J.; Ripke, Stephan; Brumme, Chanson J.; Pulit, Sara L.; Telenti, Amalio; Carrington, Mary; Kadie, Carl M.; Carlson, Jonathan M.; Heckerman, David; de Bakker, Paul I.W.; Pereyra, Florencia; de Bakker, Paul I.W.; Graham, Robert R.; Plenge, Robert M.; Deeks, Steven G.; Walker, Bruce D.; Gianniny, Lauren; Crawford, Gabriel; Sullivan, Jordan; Gonzalez, Elena; Davies, Leela; Camargo, Amy; Moore, Jamie M.; Beattie, Nicole; Gupta, Supriya; Crenshaw, Andrew; Burtt, Noël P.; Guiducci, Candace; Gupta, Namrata; Carrington, Mary; Gao, Xiaojiang; Qi, Ying; Yuki, Yuko; Pereyra, Florencia; Piechocka-Trocha, Alicja; Cutrell, Emily; Rosenberg, Rachel; Moss, Kristin L.; Lemay, Paul; O’Leary, Jessica; Schaefer, Todd; Verma, Pranshu; Toth, Ildiko; Block, Brian; Baker, Brett; Rothchild, Alissa; Lian, Jeffrey; Proudfoot, Jacqueline; Alvino, Donna Marie L.; Vine, Seanna; Addo, Marylyn M.; Allen, Todd M.; Altfeld, Marcus; Henn, Matthew R.; Le Gall, Sylvie; Streeck, Hendrik; Walker, Bruce D.; Haas, David W.; Kuritzkes, Daniel R.; Robbins, Gregory K.; Shafer, Robert W.; Gulick, Roy M.; Shikuma, Cecilia M.; Haubrich, Richard; Riddler, Sharon; Sax, Paul E.; Daar, Eric S.; Ribaudo, Heather J.; Agan, Brian; Agarwal, Shanu; Ahern, Richard L.; Allen, Brady L.; Altidor, Sherly; Altschuler, Eric L.; Ambardar, Sujata; Anastos, Kathryn; Anderson, Ben; Anderson, Val; Andrady, Ushan; Antoniskis, Diana; Bangsberg, David; Barbaro, Daniel; Barrie, William; Bartczak, J.; Barton, Simon; Basden, Patricia; Basgoz, Nesli; Bazner, Suzane; Bellos, Nicholaos C.; Benson, Anne M.; Berger, Judith; Bernard, Nicole F.; Bernard, Annette M.; Birch, Christopher; Bodner, Stanley J.; Bolan, Robert K.; Boudreaux, Emilie T.; Bradley, Meg; Braun, James F.; Brndjar, Jon E.; Brown, Stephen J.; Brown, Katherine; Brown, Sheldon T.; Burack, Jedidiah; Bush, Larry M.; Cafaro, Virginia; Campbell, Omobolaji; Campbell, John; Carlson, Robert H.; Carmichael, J. Kevin; Casey, Kathleen K.; Cavacuiti, Chris; Celestin, Gregory; Chambers, Steven T.; Chez, Nancy; Chirch, Lisa M.; Cimoch, Paul J.; Cohen, Daniel; Cohn, Lillian E.; Conway, Brian; Cooper, David A.; Cornelson, Brian; Cox, David T.; Cristofano, Michael V.; Cuchural, George; Czartoski, Julie L.; Dahman, Joseph M.; Daly, Jennifer S.; Davis, Benjamin T.; Davis, Kristine; Davod, Sheila M.; Deeks, Steven G.; DeJesus, Edwin; Dietz, Craig A.; Dunham, Eleanor; Dunn, Michael E.; Ellerin, Todd B.; Eron, Joseph J.; Fangman, John J.W.; Farel, Claire E.; Ferlazzo, Helen; Fidler, Sarah; Fleenor-Ford, Anita; Frankel, Renee; Freedberg, Kenneth A.; French, Neel K.; Fuchs, Jonathan D.; Fuller, Jon D.; Gaberman, Jonna; Gallant, Joel E.; Gandhi, Rajesh T.; Garcia, Efrain; Garmon, Donald; Gathe, Joseph C.; Gaultier, Cyril R.; Gebre, Wondwoosen; Gilman, Frank D.; Gilson, Ian; Goepfert, Paul A.; Gottlieb, Michael S.; Goulston, Claudia; Groger, Richard K.; Gurley, T. Douglas; Haber, Stuart; Hardwicke, Robin; Hardy, W. David; Harrigan, P. Richard; Hawkins, Trevor N.; Heath, Sonya; Hecht, Frederick M.; Henry, W. Keith; Hladek, Melissa; Hoffman, Robert P.; Horton, James M.; Hsu, Ricky K.; Huhn, Gregory D.; Hunt, Peter; Hupert, Mark J.; Illeman, Mark L.; Jaeger, Hans; Jellinger, Robert M.; John, Mina; Johnson, Jennifer A.; Johnson, Kristin L.; Johnson, Heather; Johnson, Kay; Joly, Jennifer; Jordan, Wilbert C.; Kauffman, Carol A.; Khanlou, Homayoon; Killian, Robert K.; Kim, Arthur Y.; Kim, David D.; Kinder, Clifford A.; Kirchner, Jeffrey T.; Kogelman, Laura; Kojic, Erna Milunka; Korthuis, P. Todd; Kurisu, Wayne; Kwon, Douglas S.; LaMar, Melissa; Lampiris, Harry; Lanzafame, Massimiliano; Lederman, Michael M.; Lee, David M.; Lee, Jean M.L.; Lee, Marah J.; Lee, Edward T.Y.; Lemoine, Janice; Levy, Jay A.; Llibre, Josep M.; Liguori, Michael A.; Little, Susan J.; Liu, Anne Y.; Lopez, Alvaro J.; Loutfy, Mono R.; Loy, Dawn; Mohammed, Debbie Y.; Man, Alan; Mansour, Michael K.; Marconi, Vincent C.; Markowitz, Martin; Marques, Rui; Martin, Jeffrey N.; Martin, Harold L.; Mayer, Kenneth Hugh; McElrath, M. Juliana; McGhee, Theresa A.; McGovern, Barbara H.; McGowan, Katherine; McIntyre, Dawn; Mcleod, Gavin X.; Menezes, Prema; Mesa, Greg; Metroka, Craig E.; Meyer-Olson, Dirk; Miller, Andy O.; Montgomery, Kate; Mounzer, Karam C.; Nagami, Ellen H.; Nagin, Iris; Nahass, Ronald G.; Nelson, Margret O.; Nielsen, Craig; Norene, David L.; O’Connor, David H.; Ojikutu, Bisola O.; Okulicz, Jason; Oladehin, Olakunle O.; Oldfield, Edward C.; Olender, Susan A.; Ostrowski, Mario; Owen, William F.; Pae, Eunice; Parsonnet, Jeffrey; Pavlatos, Andrew M.; Perlmutter, Aaron M.; Pierce, Michael N.; Pincus, Jonathan M.; Pisani, Leandro; Price, Lawrence Jay; Proia, Laurie; Prokesch, Richard C.; Pujet, Heather Calderon; Ramgopal, Moti; Rathod, Almas; Rausch, Michael; Ravishankar, J.; Rhame, Frank S.; Richards, Constance Shamuyarira; Richman, Douglas D.; Robbins, Gregory K.; Rodes, Berta; Rodriguez, Milagros; Rose, Richard C.; Rosenberg, Eric S.; Rosenthal, Daniel; Ross, Polly E.; Rubin, David S.; Rumbaugh, Elease; Saenz, Luis; Salvaggio, Michelle R.; Sanchez, William C.; Sanjana, Veeraf M.; Santiago, Steven; Schmidt, Wolfgang; Schuitemaker, Hanneke; Sestak, Philip M.; Shalit, Peter; Shay, William; Shirvani, Vivian N.; Silebi, Vanessa I.; Sizemore, James M.; Skolnik, Paul R.; Sokol-Anderson, Marcia; Sosman, James M.; Stabile, Paul; Stapleton, Jack T.; Starrett, Sheree; Stein, Francine; Stellbrink, Hans-Jurgen; Sterman, F. Lisa; Stone, Valerie E.; Stone, David R.; Tambussi, Giuseppe; Taplitz, Randy A.; Tedaldi, Ellen M.; Telenti, Amalio; Theisen, William; Torres, Richard; Tosiello, Lorraine; Tremblay, Cecile; Tribble, Marc A.; Trinh, Phuong D.; Tsao, Alice; Ueda, Peggy; Vaccaro, Anthony; Valadas, Emilia; Vanig, Thanes J.; Vecino, Isabel; Vega, Vilma M.; Veikley, Wenoah; Wade, Barbara H.; Walworth, Charles; Wanidworanun, Chingchai; Ward, Douglas J.; Warner, Daniel A.; Weber, Robert D.; Webster, Duncan; Weis, Steve; Wheeler, David A.; White, David J.; Wilkins, Ed; Winston, Alan; Wlodaver, Clifford G.; Wout, Angelique van’t; Wright, David P.; Yang, Otto O.; Yurdin, David L.; Zabukovic, Brandon W.; Zachary, Kimon C.; Zeeman, Beth; Zhao, Meng

2011-01-01

Infectious and inflammatory diseases have repeatedly shown strong genetic associations within the major histocompatibility complex (MHC); however, the basis for these associations remains elusive. To define host genetic effects on the outcome of a chronic viral infection, we performed genome-wide association analysis in a multiethnic cohort of HIV-1 controllers and progressors, and we analyzed the effects of individual amino acids within the classical human leukocyte antigen (HLA) proteins. We identified >300 genome-wide significant single-nucleotide polymorphisms (SNPs) within the MHC and none elsewhere. Specific amino acids in the HLA-B peptide binding groove, as well as an independent HLA-C effect, explain the SNP associations and reconcile both protective and risk HLA alleles. These results implicate the nature of the HLA–viral peptide interaction as the major factor modulating durable control of HIV infection. PMID:21051598

Ghrelin, a novel growth hormone-releasing peptide, in the treatment of cardiopulmonary-associated cachexia.

PubMed

Nagaya, Noritoshi; Kojima, Masakazu; Kangawa, Kenji

2006-01-01

Ghrelin is a novel growth hormone (GH)-releasing peptide, isolated from the stomach, which has been identified as an endogenous ligand for GH secretagogue receptor. The discovery of ghrelin indicates that the release of GH from the pituitary might be regulated not only by hypothalamic GH-releasing hormone, but also by ghrelin derived from the stomach. This peptide also stimulates food intake and induces adiposity through GH-independent mechanisms. In addition, ghrelin acts directly on the central nervous system to decrease sympathetic nerve activity. Thus, ghrelin plays important roles for maintaining GH release and energy homeostasis. Repeated administration of ghrelin improves body composition, muscle wasting, functional capacity, and sympathetic augmentation in cachectic patients with heart failure or chronic obstructive pulmonary disease. These results suggest that ghrelin has anti-cachectic effects through GH-dependent and independent mechanisms. Thus, administration of ghrelin may be a new therapeutic strategy for the treatment of cardiopulmonary-associated cachexia.

The Design and Delivery of a Thermally Responsive Peptide to Inhibit S100B Mediated Neurodegeneration

PubMed Central

Hearst, Scoty M; Walker, Leslie R; Shao, Qingmei; Lopez, Mariper; Raucher, Drazen; Vig, Parminder J S

2011-01-01

S100B, a glial secreted protein is believed to play a major role in neurodegeneration in Alzheimer's disease, Down syndrome, traumatic brain injury and spinocerebellar ataxia type 1 (SCA1). SCA1 is a trinucleotide repeat disorder in which the expanded polyglutamine mutation in the protein ataxin-1 primarily targets Purkinje cells (PCs) of the cerebellum. Currently, the exact mechanism of S100B mediated PC damage in SCA1 is not clear. However, here we show that S100B may act via the activation of the RAGE signaling pathway resulting in oxidative stress mediated injury to mutant ataxin-1 expressing neurons. To combat S100B mediated neurodegeneration, we have designed a selective thermally responsive S100B inhibitory peptide, Synb1-ELP-TRTK. Our therapeutic polypeptide was developed using three key elements: (1) the elastin-like polypeptide (ELP), a thermally responsive polypeptide, (2) the TRTK12 peptide, a known S100B inhibitory peptide and (3) a cell penetrating peptide, Synb1, to enhance intracellular delivery. Binding studies revealed that our peptide, Synb1-ELP-TRTK, interacts with its molecular target S100B and maintains a high S100B binding affinity as comparable with the TRTK12 peptide alone. In addition, in vitro studies revealed that Synb1-ELP-TRTK treatment reduces S100B uptake in SHSY5Y cells. Furthermore, the Synb1-ELP-TRTK peptide decreased S100B induced oxidative damage to mutant ataxin-1 expressing neurons. To test the delivery capabilities of ELP based therapeutic peptides to the cerebellum; we treated mice with fluorescently labeled Synb1-ELP and observed that thermal targeting enhanced peptide delivery to the cerebellum. Here, we have laid the framework for thermal based therapeutic targeting to regions of the brain, particularly the cerebellum. Overall, our data suggests that thermal targeting of ELP based therapeutic peptides to the cerebellum is a novel treatment strategy for cerebellar neurodegenerative disorders. PMID:21958864

The functionalization of limonite to prepare NZVI and its application in decomposition of p-nitrophenol

NASA Astrophysics Data System (ADS)

Liu, Haibo; Chen, Tianhu; Xie, Qiaoqin; Zou, Xuehua; Chen, Chen; Frost, Ray L.

2015-09-01

Nano zero valent iron (NZVI) was prepared by reducing natural limonite using hydrogen. X-ray fluorescence, thermogravimetry, X-ray diffraction, transmission electron microscope, temperature programmed reduction (TPR), field emission scanning electron microscope/energy disperse spectroscopy (FESEM/EDS) were utilized to characterize the natural limonite and reduced limonite. The ratios of Fe:O before and after reducing was determined using EDS. The reactivity of the NZVI was assessed by decomposition of p-nitrophenol ( p-NP) and was compared with commercial iron powder. In this study, the results of TPR and FESEM/EDS indicated that NZVI can be prepared by reducing natural limonite using hydrogen. Most importantly, this NZVI was proved to have a good performance on decomposition of p-NP and the process of p-NP decomposition agreed well with the pseudo-first-order kinetic model. The reactivity of this NZVI for decomposition of p-NP was greatly superior to that of commercial iron powder.

Bioinformatic Analysis Reveals Conservation of Intrinsic Disorder in the Linker Sequences of Prokaryotic Dual-family Immunophilin Chaperones.

PubMed

Barik, Sailen

2018-01-01

The two classical immunophilin families, found essentially in all living cells, are: cyclophilin (CYN) and FK506-binding protein (FKBP). We previously reported a novel class of immunophilins that are natural chimera of these two, which we named dual-family immunophilin (DFI). The DFIs were found in either of two conformations: CYN-linker-FKBP (CFBP) or FKBP-3TPR-CYN (FCBP). While the 3TPR domain can serve as a flexible linker between the FKBP and CYN modules in the FCBP-type DFI, the linker sequences in the CFBP-type DFIs are relatively short, diverse in sequence, and contain no discernible motif or signature. Here, I present several lines of computational evidence that, regardless of their primary structure, these CFBP linkers are intrinsically disordered. This report provides the first molecular foundation for the model that the CFBP linker acts as an unstructured, flexible loop, allowing the two flanking chaperone modules function independently while linked in cis , likely to assist in the folding of multisubunit client complexes.

System identification of dynamic closed-loop control of total peripheral resistance by arterial and cardiopulmonary baroreceptors

NASA Technical Reports Server (NTRS)

Aljuri, A. N.; Bursac, N.; Marini, R.; Cohen, R. J.

2001-01-01

Prolonged exposure to microgravity in space flight missions (days) impairs the mechanisms responsible for defense of arterial blood pressure (ABP) and cardiac output (CO) against orthostatic stress in the post-flight period. The mechanisms responsible for the observed orthostatic intolerance are not yet completely understood. Additionally, effective counter measures to attenuate this pathophysiological response are not available. The aim of this study was to investigate the ability of our proposed system identification method to predict closed-loop dynamic changes in TPR induced by changes in mean arterial pressure (MAP) and right atrial pressure (RAP). For this purpose we designed and employed a novel experimental animal model for the examination of arterial and cardiopulmonary baroreceptors in the dynamic closed-loop control of total peripheral resistance (TPR), and applied system identification to the analysis of beat-to-beat fluctuations in the measured signals. Grant numbers: NAG5-4989. c 2001. Elsevier Science Ltd. All rights reserved.

Evolution in Action: N and C Termini of Subunits in Related T=4 Viruses Exchange Roles as Molecular Switches

PubMed Central

Speir, Jeffrey A.; Taylor, Derek J.; Natarajan, Padmaja; Pringle, Fiona M.; Ball, L. Andrew; Johnson, John E.

2010-01-01

Summary The T=4 tetravirus and T=3 nodavirus capsid proteins undergo closely similar autoproteolysis to produce the N-terminal ß and C-terminal, lipophilic γ polypeptides. The γ peptides and N-termini of ß also act as molecular switches that determine their quasi-equivalent capsid structures. The crystal structure of Providence virus (PrV), only the second of a tetravirus (the first was NωV), reveals conserved folds and cleavage sites, but the protein termini have completely different structures and the opposite functions of those in N⌉V. N-termini of ß form the molecular switch in PrV, while γ peptides have this role in N⌉V. PrV γ peptides instead interact with packaged RNA at the particle 2-folds using a repeating sequence pattern found in only four other RNA or membrane binding proteins. The disposition of peptide termini in PrV is closely related to those in nodaviruses suggesting that PrV may be closer to the primordial T=4 particle than NωV. PMID:20541507

Technical aspects and inter-laboratory variability in native peptide profiling: the CE-MS experience.

PubMed

Mischak, Harald; Vlahou, Antonia; Ioannidis, John P A

2013-04-01

Mass spectrometry platforms have attracted a lot of interest in the last 2 decades as profiling tools for native peptides and proteins with clinical potential. However, limitations associated with reproducibility and analytical robustness, especially pronounced with the initial SELDI systems, hindered the application of such platforms in biomarker qualification and clinical implementation. The scope of this article is to give a short overview on data available on performance and on analytical robustness of the different platforms for peptide profiling. Using the CE-MS platform as a paradigm, data on analytical performance are described including reproducibility (short-term and intermediate repeatability), stability, interference, quantification capabilities (limits of detection), and inter-laboratory variability. We discuss these issues by using as an example our experience with the development of a 273-peptide marker for chronic kidney disease. Finally, we discuss pros and cons and means for improvement and emphasize the need to test in terms of comparative clinical performance and impact, different platforms that pass reasonably well analytical validation tests. Copyright © 2012 The Canadian Society of Clinical Chemists. Published by Elsevier Inc. All rights reserved.

The Inherent Conformational Preferences of Glutamine-Containing Peptides: the Role for Side-Chain Backbone Hydrogen Bonds

NASA Astrophysics Data System (ADS)

Walsh, Patrick S.; McBurney, Carl; Gellman, Samuel H.; Zwier, Timothy S.

2015-06-01

Glutamine is widely known to be found in critical regions of peptides which readily fold into amyloid fibrils, the structures commonly associated with Alzheimer's disease and glutamine repeat diseases such as Huntington's disease. Building on previous single-conformation data on Gln-containing peptides containing an aromatic cap on the N-terminus (Z-Gln-OH and Z-Gln-NHMe), we present here single-conformation UV and IR spectra of Ac-Gln-NHBn and Ac-Ala-Gln-NHBn, with its C-terminal benzyl cap. These results point towards side-chain to backbone hydrogen bonds dominating the structures observed in the cold, isolated environment of a molecular beam. We have identified and assigned three main conformers for Ac-Gln-NHBn all involving primary side-chain to backbone interactions. Ac-Ala-Gln-NHBn extends the peptide chain by one amino acid, but affords an improvement in the conformational flexibility. Despite this increase in the flexibility, only a single conformation is observed in the gas-phase: a structure which makes use of both side-chain-to-backbone and backbone-to-backbone hydrogen bonds.

Hypothermic and antipyretic effects of ACTH (1-24) and alpha-melanotropin in guinea-pigs

NASA Technical Reports Server (NTRS)

Kandasamy, S. B.; Williams, B. A.

1984-01-01

Intracerebroventricular administration of adrenocorticotropin (ACTH 1-24) and alpha-melanotropin (alpha-MSH), peptides which occur naturally in brain induced dose-related hypothermia in guinea-pigs at room temperature (21 C) and also produced greater hypothermia at low (10 C) ambient temperature. However, when the experiments were repeated in a warm (30 C) environment, no effect on body temperature was observed. These results indicate that the peptides did not reduce the central set-point of temperature control. The hypothermia induced by ACTH and alpha-MSH was not mediated via histamine H1- or H2-receptors and serotonin since the H1-receptor antagonist, mepyramine, the H2-receptor antagonist, cimetidine, and the serotonin antagonist, methysergide, had no antagonistic effects. The peptides were antipyretic since they reduced pyrogen-induced-fever and hyperthermia due to prostaglandin E2, norepinephrine and dibutyryl cAMP, at a dose which did not affect normal body temperature. The powerful central effects of these peptides on normal body temperature, fever and hyperthermia, together with their presence of the brain regions important to temperature control, suggest that they participate in thermoregulation.

A substance P-opioid chimeric peptide as a unique nontolerance-forming analgesic

PubMed Central

Foran, Stacy E.; Carr, Daniel B.; Lipkowski, Andrzej W.; Maszczynska, Iwona; Marchand, James E.; Misicka, Aleksandra; Beinborn, Martin; Kopin, Alan S.; Kream, Richard M.

2000-01-01

To elucidate mechanisms of acute and chronic pain, it is important to understand how spinal excitatory systems influence opioid analgesia. The tachykinin substance P (SP) represents the prototypic spinal excitatory peptide neurotransmitter/neuromodulator, acting in concert with endogenous opioid systems to regulate analgesic responses to nociceptive stimuli. We have synthesized and pharmacologically characterized a chimeric peptide containing overlapping NH2- and COOH-terminal functional domains of the endogenous opioid endomorphin-2 (EM-2) and the tachykinin SP, respectively. Repeated administration of the chimeric molecule YPFFGLM-NH2, designated ESP7, into the rat spinal cord produces opioid-dependent analgesia without loss of potency over 5 days. In contrast, repeated administration of ESP7 with concurrent SP receptor (SPR) blockade results in a progressive loss of analgesic potency, consistent with the development of tolerance. Furthermore, tolerant animals completely regain opioid sensitivity after post hoc administration of ESP7 alone, suggesting that coactivation of SPRs is essential to maintaining opioid responsiveness. Radioligand binding and signaling assays, using recombinant receptors, confirm that ESP7 can coactivate μ-opioid receptors (MOR) and SPRs in vitro. We hypothesize that coincidental activation of the MOR- and SPR-expressing systems in the spinal cord mimics an ongoing state of reciprocal excitation and inhibition, which is normally encountered in nociceptive processing. Due to the ability of ESP7 to interact with both MOR and SPRs, it represents a unique prototypic, anti-tolerance-forming analgesic with future therapeutic potential. PMID:10852965

Regulation of Bacteriocin Production in Streptococcus mutans by the Quorum-Sensing System Required for Development of Genetic Competence

PubMed Central

van der Ploeg, Jan R.

2005-01-01

In Streptococcus mutans, competence for genetic transformation and biofilm formation are dependent on the two-component signal transduction system ComDE together with the inducer peptide pheromone competence-stimulating peptide (CSP) (encoded by comC). Here, it is shown that the same system is also required for expression of the nlmAB genes, which encode a two-peptide nonlantibiotic bacteriocin. Expression from a transcriptional nlmAB′-lacZ fusion was highest at high cell density and was increased up to 60-fold following addition of CSP, but it was abolished when the comDE genes were interrupted. Two more genes, encoding another putative bacteriocin and a putative bacteriocin immunity protein, were also regulated by this system. The regions upstream of these genes and of two further putative bacteriocin-encoding genes and a gene encoding a putative bacteriocin immunity protein contained a conserved 9-bp repeat element just upstream of the transcription start, which suggests that expression of these genes is also dependent on the ComCDE regulatory system. Mutations in the repeat element of the nlmAB promoter region led to a decrease in CSP-dependent expression of nlmAB′-lacZ. In agreement with these results, a comDE mutant and mutants unable to synthesize or export CSP did not produce bacteriocins. It is speculated that, at high cell density, bacteriocin production is induced to liberate DNA from competing streptococci. PMID:15937160

EGF-like peptide-enhanced cell motility in Dictyostelium functions independently of the cAMP-mediated pathway and requires active Ca2+/calmodulin signaling.

PubMed

Huber, Robert; O'Day, Danton H

2011-04-01

Current knowledge suggests that cell movement in the eukaryotic slime mold Dictyostelium discoideum is mediated by different signaling pathways involving a number of redundant components. Our previous research has identified a specific motility-enhancing function for epidermal growth factor-like (EGFL) repeats in Dictyostelium, specifically for the EGFL repeats of cyrA, a matricellular, calmodulin (CaM)-binding protein in Dictyostelium. Using mutants of cAMP signaling (carA(-), carC(-), gpaB(-), gpbA(-)), the endogenous calcium (Ca(2+)) release inhibitor TMB-8, the CaM antagonist W-7, and a radial motility bioassay, we show that DdEGFL1, a synthetic peptide whose sequence is obtained from the first EGFL repeat of cyrA, functions independently of the cAMP-mediated signaling pathways to enhance cell motility through a mechanism involving Ca(2+) signaling, CaM, and RasG. We show that DdEGFL1 increases the amounts of polymeric myosin II heavy chain and actin in the cytoskeleton by 24.1±10.7% and 25.9±2.1% respectively and demonstrate a link between Ca(2+)/CaM signaling and cytoskeletal dynamics. Finally, our findings suggest that carA and carC mediate a brake mechanism during chemotaxis since DdEGFL1 enhanced the movement of carA(-)/carC(-) cells by 844±136% compared to only 106±6% for parental DH1 cells. Based on our data, this signaling pathway also appears to involve the G-protein β subunit, RasC, RasGEFA, and protein kinase B. Together, our research provides insight into the functionality of EGFL repeats in Dictyostelium and the signaling pathways regulating cell movement in this model organism. It also identifies several mechanistic components of DdEGFL1-enhanced cell movement, which may ultimately provide a model system for understanding EGFL repeat function in higher organisms. Copyright © 2010 Elsevier Inc. All rights reserved.

Repeated measurements of NT-pro-B-type natriuretic peptide, troponin T or C-reactive protein do not predict future allograft rejection in heart transplant recipients.

PubMed

Battes, Linda C; Caliskan, Kadir; Rizopoulos, Dimitris; Constantinescu, Alina A; Robertus, Jan L; Akkerhuis, Martijn; Manintveld, Olivier C; Boersma, Eric; Kardys, Isabella

2015-03-01

Studies on the prognostic value of serial biomarker assays for future occurrence of allograft rejection (AR) are scarce. We examined whether repeated measurements of NT-pro-B-type natriuretic peptide (NT-proBNP), troponin T (TropT) and C-reactive protein (CRP) predict AR. From 2005 to 2010, 77 consecutive heart transplantation (HTx) recipients were included. The NT-proBNP, TropT, and CRP were measured at 16 ± 4 (mean ± standard deviation) consecutive routine endomyocardial biopsy surveillance visits during the first year of follow-up. Allograft rejection was defined as International Society for Heart and Lung Transplantation (ISHLT) grade 2R or higher at endomyocardial biopsy. Joint modeling was used to assess the association between repeated biomarker measurements and occurrence of future AR. Joint modeling accounts for dependence among repeated observations in individual patients. The mean age of the patients at HTx was 49 ± 9.2 years, and 68% were men. During the first year of follow-up, 1,136 biopsies and concurrent blood samples were obtained, and 56 patients (73%) experienced at least one episode of AR. All biomarkers were elevated directly after HTx and achieved steady-state after ∼ 12 weeks, both in patients with or without AR. No associations were present between the repeated measurements of NT-proBNP, TropT, or CRP and AR both early (weeks 0-12) and late (weeks 13-52) in the course after HTx (hazard ratios for weeks 13-52: 0.96 (95% confidence interval, 0.55-1.68), 0.67 (0.27-1.69), and 1.44 (0.90-2.30), respectively, per ln[unit]). Combining the three biomarkers in one model also rendered null results. The temporal evolution of NT-proBNP, TropT, and CRP before AR did not predict occurrence of acute AR both in the early and late course of the first year after HTx.

Comparison of k Q factors measured with a water calorimeter in flattening filter free (FFF) and conventional flattening filter (cFF) photon beams

NASA Astrophysics Data System (ADS)

de Prez, Leon; de Pooter, Jacco; Jansen, Bartel; Perik, Thijs; Wittkämper, Frits

2018-02-01

Recently flattening filter free (FFF) beams became available for application in modern radiotherapy. There are several advantages of FFF beams over conventional flattening filtered (cFF) beams, however differences in beam spectra at the point of interest in a phantom potentially affect the ion chamber response. Beams are also non-uniform over the length of a typical reference ion chamber and recombination is usually larger. Despite several studies describing FFF beam characteristics, only a limited number of studies investigated their effect on k Q factors. Some of those studies predicted significant discrepancies in k Q factors (0.4% up to 1.0%) if TPR20,10 based codes of practice (CoPs) were to be used. This study addresses the question to which extent k Q factors, based on a TPR20,10 CoP, can be applied in clinical reference dosimetry. It is the first study that compares k Q factors measured directly with an absorbed dose to water primary standard in FFF-cFF pairs of clinical photon beams. This was done with a transportable water calorimeter described elsewhere. The measurements corrected for recombination and beam radial non-uniformity were performed in FFF-cFF beam pairs at 6 MV and 10 MV of an Elekta Versa HD for a selection of three different Farmer-type ion chambers (eight serial numbers). The ratio of measured k Q factors of the FFF-cFF beam pairs were compared with the TPR20,10 CoPs of the NCS and IAEA and the %dd(10) x CoP of the AAPM. For the TPR20,10 based CoPs differences less than 0.23% were found in k Q factors between the corresponding FFF-cFF beams with standard uncertainties smaller than 0.35%, while for the %dd(10) x these differences were smaller than 0.46% and within the expanded uncertainty of the measurements. Based on the measurements made with the equipment described in this study the authors conclude that the k Q factors provided by the NCS-18 and IAEA TRS-398 codes of practice can be applied for flattening filter free beams without additional correction. However, existing codes of practice cannot be applied ignoring the significant volume averaging effect of the FFF beams over the ion chamber cavity. For this a corresponding volume averaging correction must be applied.

[Hemodynamic effects of sub-chronic NO synthase inhibition in conscious dogs: role of EDRF/NO in muscular exertion].

PubMed

Puybasset, L; Béa, M L; Simon, L; Ghaleh, B; Giudicelli, J F; Berdeaux, A

1995-08-01

Acute and chronic administration of nitric oxide (NO) synthase (NOS) inhibitors increase mean arterial blood pressure (MAP) in rats but their hemodynamic effects in other species remain unknown. Moreover, the role of NO in the control of exercise-induced vasodilation is still debated. To answer these questions, six dogs were instrumented for the continuous measurement of cardiac output (CO, electromagnetic flow probe on the aorta), MAP (aortic catheter) and left ventricular pressure (Konigsberg gauge). Total peripheral resistance (TPR) was calculated as MAP/CO ratio and dP/dt was used as an index of cardiac inotropism. The dogs were treated from day 0 (D0) to 7 (D7) by the NOS inhibitor, N omega-nitro-L-arginine (L-NNA), 20 mg/kg/day (IV). Such a dose regimen resulted in NOS inhibition evidenced (a) in vivo by a reduction of the hypotensive responses to graded doses of acetylcholine and bradykinin, (b) ex vivo by a decrease in the relaxation of the femoral artery to acetylcholine (EC 50 = 2.2 +/- 0.6 10(-7) M after L-NNA vs 2.2 +/- 0.8 10(-8) M in controls). One month after instrumentation, the dogs being conscious, MAP measured at rest remained unchanged following one week L-NNA treatment (from 90 +/- 2 at D0 to 91 +/- 5 mmHg at D7). However, TPR increased (from 3,600 +/- 290 at D0 to 6,300 +/- 510 dyn.s.cm-5 at D7) and CO decreased (from 2.1 +/- 0.2 at D0 to 1.2 +/- 0.1 l/min at D7) (all p < 0.01), partly as the result of a marked bradycardia (from 100 +/- 7 at D0 to 60 +/- 7 beats/min at D7). L-NNA induced-increase in TPR was completely reversed by a bolus injection of nitroglycerin (10 micrograms/kg). During treadmill exercise (12 km/h), heart rate (251 +/- 9 at D0 vs 226 +/- 11 beats/min at D7), CO (6.3 +/- 0.9 at D0 vs 4.3 +/- 0.7 l/min at D7) and stroke volume remained significantly lower, and TPR significantly higher (1,662 +/- 278 at D0 vs 2,621 +/- 489 dyn.s.cm-5 at D7) after L-NNA than in the control state. Thus, NOS inhibition in resting conscious dogs by L-NNA markedly increases peripheral resistance but does not increase arterial pressure. In addition, L-NNA blunts both exercise-induced peripheral vasodilation and increase in cardiac output, despite metabolic vasodilation.

[Study on the DNA vaccine against foot-and-mouth disease virus using the heavy chain constant region of swine IgG as the carrier for peptide epitopes].

PubMed

Li, G J; Yan, W Y; Xu, Q X; Sheng, Z T; Zheng, Z X

2001-05-01

The peptide of amino acids 141-160 of VP1 protein of foot-and-mouth disease virus (FMDV) is a major B cell epitope and the peptide of amino acids 21-40 is an important T cell epitope. In this study, the DNA fragments of 141-160 and 21-40 peptide epitopes of a strain of type O FMDV was chemically synthesized and arranged into a tandem repeat 141-160 (20AA)-21-40 (20AA)-141-160 (20AA). This tandem sequence was fused to the 3' end of the heavy chain constant region gene of swine immunoglobulin G and was then cloned into mammalian expression vector pCDM8 to form a recombinant plasmid pCDM8FZ3. After pCDM8FZ3 was inoculated intramuscularly into guinea pigs, it elicited a neutralizing antibody response and a specific spleen T cell proliferative response, and 66% of the vaccinated animals were protected from viral challenge. Our study indicated that the heavy chain constant region of swine IgG can act as the carrier protein for FMDV peptide epitopes, and pC-DM8FZ3 is a potential DNA vaccine candidate to prevent FMDV infection.

High precision quantification of human plasma proteins using the automated SISCAPA Immuno-MS workflow.

PubMed

Razavi, Morteza; Leigh Anderson, N; Pope, Matthew E; Yip, Richard; Pearson, Terry W

2016-09-25

Efficient robotic workflows for trypsin digestion of human plasma and subsequent antibody-mediated peptide enrichment (the SISCAPA method) were developed with the goal of improving assay precision and throughput for multiplexed protein biomarker quantification. First, an 'addition only' tryptic digestion protocol was simplified from classical methods, eliminating the need for sample cleanup, while improving reproducibility, scalability and cost. Second, methods were developed to allow multiplexed enrichment and quantification of peptide surrogates of protein biomarkers representing a very broad range of concentrations and widely different molecular masses in human plasma. The total workflow coefficients of variation (including the 3 sequential steps of digestion, SISCAPA peptide enrichment and mass spectrometric analysis) for 5 proteotypic peptides measured in 6 replicates of each of 6 different samples repeated over 6 days averaged 3.4% within-run and 4.3% across all runs. An experiment to identify sources of variation in the workflow demonstrated that MRM measurement and tryptic digestion steps each had average CVs of ∼2.7%. Because of the high purity of the peptide analytes enriched by antibody capture, the liquid chromatography step is minimized and in some cases eliminated altogether, enabling throughput levels consistent with requirements of large biomarker and clinical studies. Copyright © 2016 Elsevier B.V. All rights reserved.

Gluten-specific antibodies of celiac disease gut plasma cells recognize long proteolytic fragments that typically harbor T-cell epitopes.

PubMed

Dørum, Siri; Steinsbø, Øyvind; Bergseng, Elin; Arntzen, Magnus Ø; de Souza, Gustavo A; Sollid, Ludvig M

2016-05-05

This study aimed to identify proteolytic fragments of gluten proteins recognized by recombinant IgG1 monoclonal antibodies generated from single IgA plasma cells of celiac disease lesions. Peptides bound by monoclonal antibodies in complex gut-enzyme digests of gluten treated with the deamidating enzyme transglutaminase 2, were identified by mass spectrometry after antibody pull-down with protein G beads. The antibody bound peptides were long deamidated peptide fragments that contained the substrate recognition sequence of transglutaminase 2. Characteristically, the fragments contained epitopes with the sequence QPEQPFP and variants thereof in multiple copies, and they typically also harbored many different gluten T-cell epitopes. In the pull-down setting where antibodies were immobilized on a solid phase, peptide fragments with multivalent display of epitopes were targeted. This scenario resembles the situation of the B-cell receptor on the surface of B cells. Conceivably, B cells of celiac disease patients select gluten epitopes that are repeated multiple times in long peptide fragments generated by gut digestive enzymes. As the fragments also contain many different T-cell epitopes, this will lead to generation of strong antibody responses by effective presentation of several distinct T-cell epitopes and establishment of T-cell help to B cells.

Gluten-specific antibodies of celiac disease gut plasma cells recognize long proteolytic fragments that typically harbor T-cell epitopes

PubMed Central

Dørum, Siri; Steinsbø, Øyvind; Bergseng, Elin; Arntzen, Magnus Ø.; de Souza, Gustavo A.; Sollid, Ludvig M.

2016-01-01

This study aimed to identify proteolytic fragments of gluten proteins recognized by recombinant IgG1 monoclonal antibodies generated from single IgA plasma cells of celiac disease lesions. Peptides bound by monoclonal antibodies in complex gut-enzyme digests of gluten treated with the deamidating enzyme transglutaminase 2, were identified by mass spectrometry after antibody pull-down with protein G beads. The antibody bound peptides were long deamidated peptide fragments that contained the substrate recognition sequence of transglutaminase 2. Characteristically, the fragments contained epitopes with the sequence QPEQPFP and variants thereof in multiple copies, and they typically also harbored many different gluten T-cell epitopes. In the pull-down setting where antibodies were immobilized on a solid phase, peptide fragments with multivalent display of epitopes were targeted. This scenario resembles the situation of the B-cell receptor on the surface of B cells. Conceivably, B cells of celiac disease patients select gluten epitopes that are repeated multiple times in long peptide fragments generated by gut digestive enzymes. As the fragments also contain many different T-cell epitopes, this will lead to generation of strong antibody responses by effective presentation of several distinct T-cell epitopes and establishment of T-cell help to B cells. PMID:27146306

Gene Therapy Vectors with Enhanced Transfection Based on Hydrogels Modified with Affinity Peptides

PubMed Central

Shepard, Jaclyn A.; Wesson, Paul J.; Wang, Christine E.; Stevans, Alyson C.; Holland, Samantha J.; Shikanov, Ariella; Grzybowski, Bartosz A.; Shea, Lonnie D.

2011-01-01

Regenerative strategies for damaged tissue aim to present biochemical cues that recruit and direct progenitor cell migration and differentiation. Hydrogels capable of localized gene delivery are being developed to provide a support for tissue growth, and as a versatile method to induce the expression of inductive proteins; however, the duration, level, and localization of expression isoften insufficient for regeneration. We thus investigated the modification of hydrogels with affinity peptides to enhance vector retention and increase transfection within the matrix. PEG hydrogels were modified with lysine-based repeats (K4, K8), which retained approximately 25% more vector than control peptides. Transfection increased 5- to 15-fold with K8 and K4 respectively, over the RDG control peptide. K8- and K4-modified hydrogels bound similar quantities of vector, yet the vector dissociation rate was reduced for K8, suggesting excessive binding that limited transfection. These hydrogels were subsequently applied to an in vitro co-culture model to induce NGF expression and promote neurite outgrowth. K4-modified hydrogels promoted maximal neurite outgrowth, likely due to retention of both the vector and the NGF. Thus, hydrogels modified with affinity peptides enhanced vector retention and increased gene delivery, and these hydrogels may provide a versatile scaffold for numerous regenerative medicine applications. PMID:21514659

Evaluation of separation properties of a modified strong cation exchange material named MEX and its application in 2D-MEX × C18 system to separate peptides from scorpion venom.

PubMed

Chen, Bo; Xu, Junyan; Fu, Qing; Dong, Xuefang; Guo, Zhimou; Jin, Yu; Liang, Xinmiao

2015-07-07

Peptides from scorpion venom represent one of the most promising drug sources for drug discovery for some specific diseases. Current challenges in their separation include high complexity, high homologies and the huge range of peptides. In this paper, a modified strong cation exchange material, named MEX, was utilised for the two-dimensional separation of peptides from complex scorpion venom. The silica-based MEX column was bonded with two functional groups; benzenesulfonic acid and cyanopropyl. To better understand its separation mechanisms, seven standard peptides with different properties were employed in an evaluation study, the results of which showed that two interactions were involved in the MEX column: electrostatic interactions based on benzenesulfonic acid groups dominated the separation of peptides; weak hydrophobic interactions introduced by cyanopropyl groups increased the column's selectivity for peptides with the same charge. This characteristic allowed the MEX column to overcome some of the drawbacks of traditional strong cation exchange (SCX) columns. Furthermore, the study showed the great effects of the acetonitrile (ACN) content, the sodium perchlorate (NaClO4) concentration and the buffer pH in the mobile phase on the peptides' retention and separation selectivity on the MEX column. Subsequently, the MEX column was combined with a C18 column to establish an off-line 2D-MEX × C18 system to separate peptides from scorpion Buthus martensi Karsch (BmK) venom. Due to complementary separation mechanisms in each dimension, a high orthogonality of 47.62% was achieved. Moreover, a good loading capacity, excellent stability and repeatability were exhibited by the MEX column, which are beneficial for its use in future preparation experiments. Therefore, the MEX column could be an alternative to the traditional SCX columns for the separation of peptides from scorpion venom.

Effects on Hedonic Feeding, Energy Expenditure and Balance of the Non-opioid Peptide DYN-A2-17.

PubMed

Alvarez, B; Barrientos, T; Gac, L; Teske, J A; Perez-Leighton, C E

2018-02-10

The dynorphin (DYN) peptide family includes opioid and non-opioid peptides, yet the physiological role of the non-opioid DYN peptides remains poorly understood. Recent evidence shows that administering the non-opioid peptide DYN-A 2-17 into the paraventricular hypothalamic nucleus (PVN) simultaneously increased short-term intake of standard rodent chow and spontaneous physical activity (SPA). The present studies aimed to expand upon the mechanisms and role of DYN-A 2-17 on food intake and energy expenditure. Injection of DYN-A 2-17 in PVN increased SPA, energy expenditure and wheel running in the absence of food. Repeated DYN-A 2-17 injection in PVN increased short-term chow intake, but this effect habituated over time and failed to alter cumulative food intake, body weight or adiposity. Pre-treatment with a CRF receptor antagonist into PVN blocked the effects of DYN-A 2-17 on food intake while injection of DYN-A 2-17 in PVN increased plasma ACTH. Finally, as DYN peptides are co-released with orexin peptides, we compared the effects of DYN-A 2-17 to orexin-A and the opioid peptide DYN-A 1-13 on food choice and intake in PVN when palatable snacks and chow were available. DYN-A 1-13 selectively increased intake of palatable snacks. DYN-A 2-17 and orexin-A decreased palatable snack intake while orexin-A also increased chow intake. These findings demonstrate that the non-opioid peptide DYN-A 2-17 acutely regulates physical activity, energy expenditure and food intake without long-term effects on energy balance. These data also propose different roles of opioid, non-opioid DYN and orexin peptides on food choice and intake when palatable and non-palatable food options are available. Copyright © 2017 IBRO. Published by Elsevier Ltd. All rights reserved.

Chemical synthesis and characterization of peptides and oligomeric proteins designed to form transmembrane ion channels.

PubMed

Iwamoto, T; Grove, A; Montal, M O; Montal, M; Tomich, J M

1994-06-01

A strategy for the synthesis of peptides and oligomeric proteins designed to form transmembrane ion channels is described. A folding motif that exhibits a functional ionic pore encompasses amphipathic alpha-helices organized as a four-helix bundle around a central hydrophilic pore. The channel-forming activity of monomeric amphipathic peptides may be examined after reconstitution in lipid bilayers in which peptides self-assemble into conductive oligomers. The covalent attachment of channel-forming peptides to the lysine epsilon-amino groups of a template molecule (KKKPGKEKG) specifies oligomeric number and facilitates the study of ionic permeation and channel blockade. Here we describe detailed protocols for the total synthesis of peptides and template-assembled four-helix bundle proteins, exemplified with the sequence of M2 delta (EKM-STAISVLLAQAVFLLLTSQR), considered involved in lining the pore of the nicotinic acetylcholine receptor channel. For comparison, the synthesis of a second four-helix bundle, T4CaIVS3 with the sequence of predicted transmembrane segment S3 (DPWNVFDFLIVIGSIIDVILSE) of the fourth repeat of the L-type voltage-gated calcium channel, is included. Peptides and proteins are synthesized step-wise by solid-phase methods, purified by reversed-phase HPLC, and homogeneity ascertained by analytical HPLC, capillary zone electrophoresis, SDS/PAGE, amino acid analysis and sequencing. Optimization of synthetic procedures for hydrophobic molecules include reducing resin substitution to avoid steric hindrance and aggregation of the final product. Protocols for the preparation of the samples prior to HPLC purification as well as the conditions and columns required for successful purification are presented. The methods developed are generally applicable for the chemical synthesis, purification and characterization of amphipathic peptides and template directed helical bundle proteins.

In Vivo Efficacy of Measles Virus Fusion Protein-Derived Peptides Is Modulated by the Properties of Self-Assembly and Membrane Residence

PubMed Central

Figueira, T. N.; Palermo, L. M.; Veiga, A. S.; Huey, D.; Alabi, C. A.; Santos, N. C.; Welsch, J. C.; Mathieu, C.; Niewiesk, S.; Moscona, A.

2016-01-01

ABSTRACT Measles virus (MV) infection is undergoing resurgence and remains one of the leading causes of death among young children worldwide despite the availability of an effective measles vaccine. MV infects its target cells by coordinated action of the MV hemagglutinin (H) and fusion (F) envelope glycoproteins; upon receptor engagement by H, the prefusion F undergoes a structural transition, extending and inserting into the target cell membrane and then refolding into a postfusion structure that fuses the viral and cell membranes. By interfering with this structural transition of F, peptides derived from the heptad repeat (HR) regions of F can inhibit MV infection at the entry stage. In previous work, we have generated potent MV fusion inhibitors by dimerizing the F-derived peptides and conjugating them to cholesterol. We have shown that prophylactic intranasal administration of our lead fusion inhibitor efficiently protects from MV infection in vivo. We show here that peptides tagged with lipophilic moieties self-assemble into nanoparticles until they reach the target cells, where they are integrated into cell membranes. The self-assembly feature enhances biodistribution and the half-life of the peptides, while integration into the target cell membrane increases fusion inhibitor potency. These factors together modulate in vivo efficacy. The results suggest a new framework for developing effective fusion inhibitory peptides. IMPORTANCE Measles virus (MV) infection causes an acute illness that may be associated with infection of the central nervous system (CNS) and severe neurological disease. No specific treatment is available. We have shown that fusion-inhibitory peptides delivered intranasally provide effective prophylaxis against MV infection. We show here that specific biophysical properties regulate the in vivo efficacy of MV F-derived peptides. PMID:27733647

The SysteMHC Atlas project.

PubMed

Shao, Wenguang; Pedrioli, Patrick G A; Wolski, Witold; Scurtescu, Cristian; Schmid, Emanuel; Vizcaíno, Juan A; Courcelles, Mathieu; Schuster, Heiko; Kowalewski, Daniel; Marino, Fabio; Arlehamn, Cecilia S L; Vaughan, Kerrie; Peters, Bjoern; Sette, Alessandro; Ottenhoff, Tom H M; Meijgaarden, Krista E; Nieuwenhuizen, Natalie; Kaufmann, Stefan H E; Schlapbach, Ralph; Castle, John C; Nesvizhskii, Alexey I; Nielsen, Morten; Deutsch, Eric W; Campbell, David S; Moritz, Robert L; Zubarev, Roman A; Ytterberg, Anders Jimmy; Purcell, Anthony W; Marcilla, Miguel; Paradela, Alberto; Wang, Qi; Costello, Catherine E; Ternette, Nicola; van Veelen, Peter A; van Els, Cécile A C M; Heck, Albert J R; de Souza, Gustavo A; Sollid, Ludvig M; Admon, Arie; Stevanovic, Stefan; Rammensee, Hans-Georg; Thibault, Pierre; Perreault, Claude; Bassani-Sternberg, Michal; Aebersold, Ruedi; Caron, Etienne

2018-01-04

Mass spectrometry (MS)-based immunopeptidomics investigates the repertoire of peptides presented at the cell surface by major histocompatibility complex (MHC) molecules. The broad clinical relevance of MHC-associated peptides, e.g. in precision medicine, provides a strong rationale for the large-scale generation of immunopeptidomic datasets and recent developments in MS-based peptide analysis technologies now support the generation of the required data. Importantly, the availability of diverse immunopeptidomic datasets has resulted in an increasing need to standardize, store and exchange this type of data to enable better collaborations among researchers, to advance the field more efficiently and to establish quality measures required for the meaningful comparison of datasets. Here we present the SysteMHC Atlas (https://systemhcatlas.org), a public database that aims at collecting, organizing, sharing, visualizing and exploring immunopeptidomic data generated by MS. The Atlas includes raw mass spectrometer output files collected from several laboratories around the globe, a catalog of context-specific datasets of MHC class I and class II peptides, standardized MHC allele-specific peptide spectral libraries consisting of consensus spectra calculated from repeat measurements of the same peptide sequence, and links to other proteomics and immunology databases. The SysteMHC Atlas project was created and will be further expanded using a uniform and open computational pipeline that controls the quality of peptide identifications and peptide annotations. Thus, the SysteMHC Atlas disseminates quality controlled immunopeptidomic information to the public domain and serves as a community resource toward the generation of a high-quality comprehensive map of the human immunopeptidome and the support of consistent measurement of immunopeptidomic sample cohorts. © The Author(s) 2017. Published by Oxford University Press on behalf of Nucleic Acids Research.

In Vivo Efficacy of Measles Virus Fusion Protein-Derived Peptides Is Modulated by the Properties of Self-Assembly and Membrane Residence.

PubMed

Figueira, T N; Palermo, L M; Veiga, A S; Huey, D; Alabi, C A; Santos, N C; Welsch, J C; Mathieu, C; Horvat, B; Niewiesk, S; Moscona, A; Castanho, M A R B; Porotto, M

2017-01-01

Measles virus (MV) infection is undergoing resurgence and remains one of the leading causes of death among young children worldwide despite the availability of an effective measles vaccine. MV infects its target cells by coordinated action of the MV hemagglutinin (H) and fusion (F) envelope glycoproteins; upon receptor engagement by H, the prefusion F undergoes a structural transition, extending and inserting into the target cell membrane and then refolding into a postfusion structure that fuses the viral and cell membranes. By interfering with this structural transition of F, peptides derived from the heptad repeat (HR) regions of F can inhibit MV infection at the entry stage. In previous work, we have generated potent MV fusion inhibitors by dimerizing the F-derived peptides and conjugating them to cholesterol. We have shown that prophylactic intranasal administration of our lead fusion inhibitor efficiently protects from MV infection in vivo We show here that peptides tagged with lipophilic moieties self-assemble into nanoparticles until they reach the target cells, where they are integrated into cell membranes. The self-assembly feature enhances biodistribution and the half-life of the peptides, while integration into the target cell membrane increases fusion inhibitor potency. These factors together modulate in vivo efficacy. The results suggest a new framework for developing effective fusion inhibitory peptides. Measles virus (MV) infection causes an acute illness that may be associated with infection of the central nervous system (CNS) and severe neurological disease. No specific treatment is available. We have shown that fusion-inhibitory peptides delivered intranasally provide effective prophylaxis against MV infection. We show here that specific biophysical properties regulate the in vivo efficacy of MV F-derived peptides. Copyright © 2016 American Society for Microbiology.

Amino acid sequence preferences to control cell-specific organization of endothelial cells, smooth muscle cells, and fibroblasts.

PubMed

Kanie, Kei; Kato, Ryuji; Zhao, Yingzi; Narita, Yuji; Okochi, Mina; Honda, Hiroyuki

2011-06-01

Effective surface modification with biocompatible molecules is known to be effective in reducing the life-threatening risks related to artificial cardiovascular implants. In recent strategies in regenerative medicine, the enhancement and support of natural repair systems at the site of injury by designed biocompatible molecules have succeeded in rapid and effective injury repair. Therefore, such a strategy could also be effective for rapid endothelialization of cardiovascular implants to lower the risk of thrombosis and stenosis. To achieve this enhancement of the natural repair system, a biomimetic molecule that mimics proper cellular organization at the implant location is required. In spite of the fact that many reported peptides have cell-attracting properties on material surfaces, there have been few peptides that could control cell-specific adhesion. For the advanced cardiovascular implants, peptides that can mimic the natural mechanism that controls cell-specific organization have been strongly anticipated. To obtain such peptides, we hypothesized the cellular bias toward certain varieties of amino acids and examined the cell preference (in terms of adhesion, proliferation, and protein attraction) of varieties and of repeat length on SPOT peptide arrays. To investigate the role of specific peptides in controlling the organization of various cardiovascular-related cells, we compared endothelial cells (ECs), smooth muscle cells (SMCs), and fibroblasts (FBs). A clear, cell-specific preference was found for amino acids (longer than 5-mer) using three types of cells, and the combinational effect of the physicochemical properties of the residues was analyzed to interpret the mechanism. Copyright © 2011 European Peptide Society and John Wiley & Sons, Ltd.

Helical peptides with three pairs of Asp-Arg and Glu-Arg residues in different orientations and spacings.

PubMed Central

Huyghues-Despointes, B. M.; Scholtz, J. M.; Baldwin, R. L.

1993-01-01

The helix-stabilizing effects of repeating pairs of Asp-Arg and Glu-Arg residues have been characterized using a peptide system of the same design used earlier to study Glu-Lys (Marqusee, S. & Baldwin, R.L., 1987, Proc. Natl. Acad. Sci. USA 84, 8898-8902) and Asp-Lys ion pairs (Marqusee, S. & Baldwin, R.L., 1990, In Protein Folding [Gierasch, L.M. & King, J., Eds.], pp. 85-94, AAAS, Washington, D.C.). The consequences of breaking ion pair and charge-helix dipole interactions by titration to pH 2 have been compared with the results of screening these interactions with NaCl at pH 7.0 and pH 2.5. The four peptides in each set contain three pairs of acidic (A) and basic (B) residues spaced either i, i + 4 or i, i + 3 apart. In one peptide of each kind the pairwise order of residues is AB, with the charges oriented favorably to the helix macrodipole, and in the other peptide the order is BA. The results are as follows: (1) Remarkably, both Asp-Arg and Glu-Arg peptides show the same pattern of helix stabilization at pH 7.0 found earlier for Glu-Lys and Asp-Lys peptides: i + 4 AB > i + 4 BA approximately i + 3 AB > i + 3 BA. (2) The ion pairs and charge-helix dipole interactions cannot be cleanly separated, but the results suggest that both interactions make important contributions to helix stability.(ABSTRACT TRUNCATED AT 250 WORDS) PMID:8443591

PTA and TPR: A Comprehension Based Approach in a Public Elementary School.

ERIC Educational Resources Information Center

Schneider, Judith Morganroth

1984-01-01

Describes a pilot program offering conversational Spanish to elementary school students. The "Total Physical Response" technique was used initially, followed by an emphasis on comprehension and the creation of a natural language environment, all as means of facilitating second language acquisition at the children's level. (SL)

Theory and Practice in Participatory Research: Lessons from the Native Elder Care Study

ERIC Educational Resources Information Center

Goins, R. Turner; Garroutte, Eva Marie; Fox, Susan Leading; Geiger, Sarah Dee; Manson, Spero M.

2011-01-01

Models for community-based participatory research (CBPR) urge academic investigators to collaborate with communities to identify and pursue research questions, processes, and outcomes valuable to both partners. The tribal participatory research (TPR) conceptual model suggests modifications to CBPR to fit the special needs of American Indian…

WISE-2005: Changes in cardiovascular variables due to increasing levels of adrenergic stimulation in women according to phase of menstruation. H. Edgell, D. Greaves, P. Arbeille, M-A. Custaud, R.L. Hughson.

NASA Astrophysics Data System (ADS)

Edgell, Heather; Greaves, Danielle; Custaud, Marc-Antoine; Arbeille, Phillipe; Hughson, Richard L.

Women are more susceptible to orthostatic intolerance (OI) on return from space flight or after bed rest than men. The WISE campaigns of 2005 studied the effects of 60 days of 6o head-down bed rest (HDBR) on women. One of the protocols involved infusing the adrenergic agonists isoproterenol (ISO; beta-agonist) or norepinephrine (NOR; alphaand beta-agonist). At the time of the pre-HDBR test, 5 women were in the follicular phase (F) of the menstrual cycle (Day 7-12) and 5 women were in the luteal phase (L; Day 18-24). Mean arterial pressure (MAP), heart rate (HR), cardiac output (Q), stroke volume (SV) and total peripheral resistance (TPR) were all measured (Finometer and Doppler ultrasound) before and 3 minutes after the i.v. infusion of 2 doses of ISO (0.005 and 0.01µg/kg/min) or 2 doses of NOR (10 and 50ng/kg/min). As described previously in these women (Edgell et al.(2007) AJP 293:R2343-52), ISO resulted in significantly increasing HR, SV and Q with decreasing TPR while NOR resulted in decreasing HR and Q with increasing TPR. While HR, MAP and Q tended to be higher in L than F, there were no significant differences between responses to ISO or NOR (i.e. no group or interaction effects). ISO2 responses: HR: L: 76.8 ± 3.7 bpm; F: 71.1 ± 3.9 bpm, MAP: L: 94.5 ± 5.3 mmHg; F: 91.7 ± 3.7 mmHg, SV: L: 99.7 ± 15.2 mL; F: 92.2 ± 12.3 mL, Q: L: 7.8 ± 1.3 L/min; F: 6.6 ± 0.9 L/min, TPR: L: 13.2 ± 2.6 mmHg/L/min; F: 15.1 ± 2.3 mmHg/L/min. NOR2 responses: HR: L: 59.7 ± 3.3 bpm; F: 54.8 ± 4.0 bpm, MAP: L: 102.4 ± 4.7 mmHg; F: 97.1 ± 2.9 mmHg, SV: L: 77.5 ± 8.2 mL; F: 71.1 ± 7.2 mL, Q: L: 4.7 ± 0.7 L/min; F: 3.9 ± 0.5 L/min, TPR: L: 23.7 ± 3.2 mmHg/L/min; F: 26.2 ± 2.5 mmHg/L/min. Also, after ISO2 there appeared to be higher norepinephrine (NE) concentrations in follicular women (L: 161.6±61.1 pg/mL; F: 311.0±31.7 pg/mL; P=0.0817 (interaction effect)). These results suggest that women in follicular phase may exhibit different orthostatic tolerance than those in luteal phase, especially in light of data from Waters et al. (JAP 92(2):586-94; 2002) which indicate that subjects with lower tolerance had smaller increases of NE. These results are important to consider for all studies involving women. Funding provided by the CSA, NASA, ESA, and CNES.

Synthesis and characterization of Her2-NLP peptide conjugates targeting circulating breast cancer cells: cellular uptake and localization by fluorescent microscopic imaging.

PubMed

Cai, Huawei; Singh, Ajay N; Sun, Xiankai; Peng, Fangyu

2015-01-01

To synthesize a fluorescent Her2-NLP peptide conjugate consisting of Her2/neu targeting peptide and nuclear localization sequence peptide (NLP) and assess its cellular uptake and intracellular localization for radionuclide cancer therapy targeting Her2/neu-positive circulating breast cancer cells (CBCC). Fluorescent Cy5.5 Her2-NLP peptide conjugate was synthesized by coupling a bivalent peptide sequence, which consisted of a Her2-binding peptide (NH2-GSGKCCYSL) and an NLP peptide (CGYGPKKKRKVGG) linked by a polyethylene glycol (PEG) chain with 6 repeating units, with an activated Cy5.5 ester. The conjugate was separated and purified by HPLC and then characterized by Maldi-MS. The intracellular localization of fluorescent Cy5.5 Her2-NLP peptide conjugate was assessed by fluorescent microscopic imaging using a confocal microscope after incubation of Cy5.5-Her2-NLP with Her2/neu positive breast cancer cells and Her2/neu negative control breast cancer cells, respectively. Fluorescent signals were detected in cytoplasm of Her2/neu positive breast cancer cells (SKBR-3 and BT474 cell lines), but not or little in cytoplasm of Her2/neu negative breast cancer cells (MDA-MB-231), after incubation of the breast cancer cells with Cy5.5-Her2-NLP conjugates in vitro. No fluorescent signals were detected within the nuclei of Her2/neu positive SKBR-3 and BT474 breast cancer cells, neither Her2/neu negative MDA-MB-231 cells, incubated with the Cy5.5-Her2-NLP peptide conjugates, suggesting poor nuclear localization of the Cy5.5-Her2-NLP conjugates localized within the cytoplasm after their cellular uptake and internalization by the Her2/neu positive breast cancer cells. Her2-binding peptide (KCCYSL) is a promising agent for radionuclide therapy of Her2/neu positive breast cancer using a β(-) or α emitting radionuclide, but poor nuclear localization of the Her2-NLP peptide conjugates may limit its use for eradication of Her2/neu-positive CBCC using I-125 or other Auger electron emitting radionuclide.

Bioengineered Chimeric Spider Silk-Uranium Binding Proteins

PubMed Central

Krishnaji, Sreevidhya Tarakkad; Kaplan, David L.

2014-01-01

Heavy metals constitute a source of environmental pollution. Here, novel functional hybrid biomaterials for specific interactions with heavy metals are designed by bioengineering consensus sequence repeats from spider silk of Nephila clavipes with repeats of a uranium peptide recognition motif from a mutated 33-residue of calmodulin protein from Paramecium tetraurelia. The self-assembly features of the silk to control nanoscale organic/inorganic material interfaces provides new biomaterials for uranium recovery. With subsequent enzymatic digestion of the silk to concentrate the sequestered metals, options can be envisaged to use these new chimeric protein systems in environmental engineering, including to remediate environments contaminated by uranium. PMID:23212989

ACCA phosphopeptide recognition by the BRCT repeats of BRCA1.

PubMed

Ray, Hind; Moreau, Karen; Dizin, Eva; Callebaut, Isabelle; Venezia, Nicole Dalla

2006-06-16

The tumour suppressor gene BRCA1 encodes a 220 kDa protein that participates in multiple cellular processes. The BRCA1 protein contains a tandem of two BRCT repeats at its carboxy-terminal region. The majority of disease-associated BRCA1 mutations affect this region and provide to the BRCT repeats a central role in the BRCA1 tumour suppressor function. The BRCT repeats have been shown to mediate phospho-dependant protein-protein interactions. They recognize phosphorylated peptides using a recognition groove that spans both BRCT repeats. We previously identified an interaction between the tandem of BRCA1 BRCT repeats and ACCA, which was disrupted by germ line BRCA1 mutations that affect the BRCT repeats. We recently showed that BRCA1 modulates ACCA activity through its phospho-dependent binding to ACCA. To delineate the region of ACCA that is crucial for the regulation of its activity by BRCA1, we searched for potential phosphorylation sites in the ACCA sequence that might be recognized by the BRCA1 BRCT repeats. Using sequence analysis and structure modelling, we proposed the Ser1263 residue as the most favourable candidate among six residues, for recognition by the BRCA1 BRCT repeats. Using experimental approaches, such as GST pull-down assay with Bosc cells, we clearly showed that phosphorylation of only Ser1263 was essential for the interaction of ACCA with the BRCT repeats. We finally demonstrated by immunoprecipitation of ACCA in cells, that the whole BRCA1 protein interacts with ACCA when phosphorylated on Ser1263.

Epitope mapping of the variable repetitive region with the MB antigen of Ureaplasma urealyticum.

PubMed Central

Zheng, X; Lau, K; Frazier, M; Cassell, G H; Watson, H L

1996-01-01

One of the major surface structures of Ureaplasma urealyticum recognized by antibodies of patients during infection is the MB antigen. Previously, we showed by Western blot (immunoblot) analysis that any one of the anti-MB monoclonal antibodies (MAbs) 3B1.5, 5B1.1, and 10C6.6 could block the binding of patient antibodies to MB. Subsequent DNA sequencing revealed that a unique six-amino-acid direct tandem repeat region composed the carboxy two-thirds of this antigen. In the present study, using antibody-reactive peptide scanning of this repeat region, we demonstrated that the amino acids defining the epitopes for MAbs 3B1.5 5B1.1 and 10C6.6 are EQP, GK, and KEQPA, respectively. Peptide scanning analysis of an infected patient's serum antibody response showed that the dominant epitope was defined by the sequence PAGK. Mapping of these continuous epitopes revealed overlap between all MAb and patient polyclonal antibody binding sites, thus explaining the ability of a single MAb to apparently block all polyclonal antibody binding sites. We also show that a single amino acid difference in the sequence of the repeats of serovars 3 and 14 accounts for the lack of reactivity with serovar 14 of two of the serovar 3-specific MAbs. Finally, the data demonstrate the need to obtain the sequences of the mba genes of all serovars before an effective serovar-specific antibody detection method can be developed. PMID:8914774

Molecular cloning and sequence analysis of the gene coding for the 57kDa soluble antigen of the salmonid fish pathogen Renibacterium salmoninarum

USGS Publications Warehouse

Chien, Maw-Sheng; Gilbert , Teresa L.; Huang, Chienjin; Landolt, Marsha L.; O'Hara, Patrick J.; Winton, James R.

1992-01-01

The complete sequence coding for the 57-kDa major soluble antigen of the salmonid fish pathogen, Renibacterium salmoninarum, was determined. The gene contained an opening reading frame of 1671 nucleotides coding for a protein of 557 amino acids with a calculated Mr value of 57190. The first 26 amino acids constituted a signal peptide. The deduced sequence for amino acid residues 27–61 was in agreement with the 35 N-terminal amino acid residues determined by microsequencing, suggesting the protein in synthesized as a 557-amino acid precursor and processed to produce a mature protein of Mr 54505. Two regions of the protein contained imperfect direct repeats. The first region contained two copies of an 81-residue repeat, the second contained five copies of an unrelated 25-residue repeat. Also, a perfect inverted repeat (including three in-frame UAA stop codons) was observed at the carboxyl-terminus of the gene.

Cas9 in Genetically Modified Food Is Unlikely to Cause Food Allergy.

PubMed

Nakajima, Osamu; Nishimaki-Mogami, Tomoko; Kondo, Kazunari

2016-01-01

Genome editing has undergone rapid development during the last three years. It is anticipated that genetically modified organisms (GMOs) for food purposes will be widely produced using the clustered regularly interspaced short palindromic repeat/Cas9 (CRISPR)/Cas9 system in the near future. However, the Cas9 gene may then enter the genomes of GMOs for food if the breeding process is not strictly managed, which could lead to the Cas9 protein or associated peptides being produced within these organisms. A variety of peptides could theoretically be produced from the Cas9 gene by using open reading frames different from that of Cas9 in the GMOs. In this study, Cas9 and the peptides potentially encoded by Cas9 genes were studied regarding their immunogenicity, in terms of the digestibility of Cas9 and the homology of the peptides to food allergens. First, the digestibility and thermal stability of Cas9 were studied. Digestibility was tested with natural or heat-denatured Cas9 in simulated gastric fluid in vitro. The two types of Cas9 were digested rapidly. Cas9 was also gradually degraded during heat treatment. Second, the peptides potentially encoded by Cas9 genes were examined for their homology to food allergens. Specifically, an 8-mer exact match search and a sliding 80-mer window search were performed using allergen databases. One of the peptides was found to have homology with a food allergen.

The HIV-1 gp41 ectodomain is cleaved by matriptase to produce a chemotactic peptide that acts through FPR2

PubMed Central

Wood, Matthew P; Cole, Amy L; Eade, Colleen R; Chen, Li-Mei; Chai, Karl X; Cole, Alexander M

2014-01-01

Several aspects of HIV-1 virulence and pathogenesis are mediated by the envelope protein gp41. Additionally, peptides derived from the gp41 ectodomain have been shown to induce chemotaxis in monocytes and neutrophils. Whereas this chemotactic activity has been reported, it is not known how these peptides could be produced under biological conditions. The heptad repeat 1 (HR1) region of gp41 is exposed to the extracellular environment and could therefore be susceptible to proteolytic processing into smaller peptides. Matriptase is a serine protease expressed at the surface of most epithelia, including the prostate and mucosal surfaces. Here, we present evidence that matriptase efficiently cleaves the HR1 portion of gp41 into a 22-residue chemotactic peptide MAT-1, the sequence of which is highly conserved across HIV-1 clades. We found that MAT-1 induced migration of primary neutrophils and monocytes, the latter of which act as a cellular reservoir of HIV during early stage infection. We then used formyl peptide receptor 1 (FPR1) and FPR2 inhibitors, along with HEK 293 cells, to demonstrate that MAT-1 can induce chemotaxis specifically using FPR2, a receptor found on the surface of monocytes, macrophages and neutrophils. These findings are the first to identify a proteolytic cleavage product of gp41 with chemotactic activity and highlight a potential role for matriptase in HIV-1 transmission and infection at epithelial surfaces and within tissue reservoirs of HIV-1. PMID:24617769

Amyloid-like self-assembly of peptide sequences from the adenovirus fiber shaft: insights from molecular dynamics simulations.

PubMed

Tamamis, Phanourios; Kasotakis, Emmanouil; Mitraki, Anna; Archontis, Georgios

2009-11-26

The self-assembly of peptides and proteins into nanostructures is related to the fundamental problems of protein folding and misfolding and has potential applications in medicine, materials science and nanotechnology. Natural peptides, corresponding to sequence repeats from self-assembling proteins, may constitute elementary building blocks of such nanostructures. In this work, we study by implicit-solvent replica-exchange simulations the self-assembly of two amyloidogenic sequences derived from the naturally occurring fiber shaft of the adenovirus, the octapeptide NSGAITIG (asparagine-serine-glycine-alanine-isoleucine-threonine-isoleucine-glycine) and its hexapeptide counterpart, GAITIG. In accordance with their amyloidogenic capacity, both peptides form readily intermolecular beta-sheets, stabilized by extensive main- and side-chain contacts involving the C-terminal moieties (segments 3-8 and 2-6, respectively). The structural and energetic properties of these sheets are analyzed extensively. The N-terminal residues Asn1 and Ser2 of the octapeptide remain disordered in the sheets, suggesting that these residues are exposed at the exterior of the fibrils and accessible. On the basis of insight provided by the simulations, cysteine residues were recently substituted at positions 1 and 2 of NSGAITIG; the newly designed peptides maintain their amyloidogenic properties and can bind to silver, gold and platinum nanoparticles [Kasotakis et al. Biopolymers 2009, 92, 164-172]. Computational investigation can identify suitable positions for rational modification of peptide building blocks, aiming at the fabrication of novel biomaterials.

E3 ubiquitin ligase CHIP interacts with C-type lectin-like receptor CLEC-2 and promotes its ubiquitin-proteasome degradation.

PubMed

Shao, Miaomiao; Li, Lili; Song, Shushu; Wu, Weicheng; Peng, Peike; Yang, Caiting; Zhang, Mingming; Duan, Fangfang; Jia, Dongwei; Zhang, Jie; Wu, Hao; Zhao, Ran; Wang, Lan; Ruan, Yuanyuan; Gu, Jianxin

2016-10-01

C-type lectin-like receptor 2 (CLEC-2) was originally identified as a member of non-classical C-type lectin-like receptors in platelets and immune cells. Activation of CLEC-2 is involved in thrombus formation, lymphatic/blood vessel separation, platelet-mediated tumor metastasis and immune response. Nevertheless, the regulation of CLEC-2 expression is little understood. In this study, we identified that the C terminus of Hsc70-interacting protein (CHIP) interacted with CLEC-2 by mass spectrometry analysis, and CHIP decreased the protein expression of CLEC-2 through lysine-48-linked ubiquitination and proteasomal degradation. Deleted and point mutation also revealed that CHIP controlled CLEC-2 protein expression via both tetratricopeptide repeats (TPR) domain and Ubox domain in a HSP70/90-independent manner. Moreover, reduced CHIP expression was associated with decreased CLEC-2 polyubiquitination and increased CLEC-2 protein levels in PMA-induced differentiation of THP-1 monocytes into macrophages. These results indicate that CLEC-2 is the target substrate of E3 ubiquitin ligase CHIP, and suggest that the CHIP/CLEC-2 axis may play an important role in the modulation of immune response. Copyright © 2016 Elsevier Inc. All rights reserved.

Loss of function of a rice TPR-domain RNA-binding protein confers broad-spectrum disease resistance.

PubMed

Zhou, Xiaogang; Liao, Haicheng; Chern, Mawsheng; Yin, Junjie; Chen, Yufei; Wang, Jianping; Zhu, Xiaobo; Chen, Zhixiong; Yuan, Can; Zhao, Wen; Wang, Jing; Li, Weitao; He, Min; Ma, Bingtian; Wang, Jichun; Qin, Peng; Chen, Weilan; Wang, Yuping; Liu, Jiali; Qian, Yangwen; Wang, Wenming; Wu, Xianjun; Li, Ping; Zhu, Lihuang; Li, Shigui; Ronald, Pamela C; Chen, Xuewei

2018-03-20

Crops carrying broad-spectrum resistance loci provide an effective strategy for controlling infectious disease because these loci typically confer resistance to diverse races of a pathogen or even multiple species of pathogens. Despite their importance, only a few crop broad-spectrum resistance loci have been reported. Here, we report the identification and characterization of the rice bsr-k1 (broad-spectrum resistance Kitaake-1) mutant, which confers broad-spectrum resistance against Magnaporthe oryzae and Xanthomonas oryzae pv oryzae with no major penalty on key agronomic traits. Map-based cloning reveals that Bsr-k1 encodes a tetratricopeptide repeats (TPRs)-containing protein, which binds to mRNAs of multiple OsPAL ( OsPAL1-7 ) genes and promotes their turnover. Loss of function of the Bsr-k1 gene leads to accumulation of OsPAL1-7 mRNAs in the bsr-k1 mutant. Furthermore, overexpression of OsPAL1 in wild-type rice TP309 confers resistance to M. oryzae , supporting the role of OsPAL1 Our discovery of the bsr-k1 allele constitutes a significant conceptual advancement and provides a valuable tool for breeding broad-spectrum resistant rice. Copyright © 2018 the Author(s). Published by PNAS.

Hsp70 Protein Complexes as Drug Targets

PubMed Central

Assimon, Victoria A.; Gillies, Anne T.; Rauch, Jennifer N.; Gestwicki, Jason E.

2013-01-01

Heat shock protein 70 (Hsp70) plays critical roles in proteostasis and is an emerging target for multiple diseases. However, competitive inhibition of the enzymatic activity of Hsp70 has proven challenging and, in some cases, may not be the most productive way to redirect Hsp70 function. Another approach is to inhibit Hsp70’s interactions with important co-chaperones, such as J proteins, nucleotide exchange factors (NEFs) and tetratricopeptide repeat (TPR) domain-containing proteins. These co-chaperones normally bind Hsp70 and guide its many diverse cellular activities. Complexes between Hsp70 and co-chaperones have been shown to have specific functions, such as pro-folding, pro-degradation and pro-trafficking. Thus, a promising strategy may be to block protein-protein interactions between Hsp70 and its co-chaperones or to target allosteric sites that disrupt these contacts. Such an approach might shift the balance of Hsp70 complexes and re-shape the proteome and it has the potential to restore healthy proteostasis. In this review, we discuss specific challenges and opportunities related to those goals. By pursuing Hsp70 complexes as drug targets, we might not only develop new leads for therapeutic development, but also discover new chemical probes for use in understanding Hsp70 biology. PMID:22920901

Cloning and characterization of carboxyl terminus of heat shock cognate 70-interacting protein gene from the silkworm, Bombyx mori.

PubMed

Ohsawa, Takeshi; Fujimoto, Shota; Tsunakawa, Akane; Shibano, Yuka; Kawasaki, Hideki; Iwanaga, Masashi

2016-11-01

Carboxyl terminus of heat shock cognate 70-interacting protein (CHIP) is an evolutionarily conserved E3 ubiquitin ligase across different eukaryotic species and is known to play a key role in protein quality control. CHIP has two distinct functional domains, an N-terminal tetratricopeptide repeat (TPR) and a C-terminal U-box domain, which are required for the ubiquitination of numerous labile client proteins that are chaperoned by heat shock proteins (HSPs) and heat shock cognate proteins (HSCs). During our screen for CHIP-like proteins in the Bombyx mori databases, we found a novel silkworm gene, Bombyx mori CHIP. Phylogenetic analysis showed that BmCHIP belongs to Lepidopteran lineages. Quantitative reverse transcription-PCR analysis indicated that BmCHIP was relatively highly expressed in the gonad and fat body. A pull-down experiment and auto-ubiquitination assay showed that BmCHIP interacted with BmHSC70 and had E3 ligase activity. Additionally, immunohistochemical analysis revealed that BmCHIP was partially co-localized with ubiquitin in BmN4 cells. These data support that BmCHIP plays an important role in the ubiquitin proteasome system as an E3 ubiquitin ligase in B. mori. Copyright © 2016 Elsevier Inc. All rights reserved.

Incorporating electron-transfer functionality into synthetic metalloproteins from the bottom-up.

PubMed

Hong, Jing; Kharenko, Olesya A; Ogawa, Michael Y

2006-12-11

The alpha-helical coiled-coil motif serves as a robust scaffold for incorporating electron-transfer (ET) functionality into synthetic metalloproteins. These structures consist of a supercoiling of two or more aplha helices that are formed by the self-assembly of individual polypeptide chains whose sequences contain a repeating pattern of hydrophobic and hydrophilic residues. Early work from our group attached abiotic Ru-based redox sites to the most surface-exposed positions of two stranded coiled-coils and used electron-pulse radiolysis to study both intra- and intermolecular ET reactions in these systems. Later work used smaller metallopeptides to investigate the effects of conformational gating within electrostatic peptide-protein complexes. We have recently designed the C16C19-GGY peptide, which contains Cys residues located at both the "a" and "d" positions of its third heptad repeat in order to construct a nativelike metal-binding domain within its hydrophobic core. It was shown that the binding of both Cd(II) and Cu(I) ions induces the peptide to undergo a conformational change from a disordered random coil to a metal-bridged coiled-coil. However, whereas the Cd(II)-protein exists as a two-stranded coiled-coil, the Cu(I) derivative exists as a four-stranded coiled-coil. Upon the incorporation of other metal ions, metal-bridged peptide dimers, tetramers, and hexamers are formed. The Cu(I)-protein is of particular interest because it exhibits a long-lived (microsecond) room-temperature luminescence at 600 nm. The luminophore in this protein is thought to be a multinuclear CuI4Cys4(N/O)4 cage complex, which can be quenched by exogenous electron acceptors in solution, as shown by emission-lifetime and transient-absorption experiments. It is anticipated that further investigation into these systems will contribute to the expanding effort of bioinorganic chemists to prepare new kinds of functionally active synthetic metalloproteins.

Signal peptide cleavage is essential for surface expression of a regulatory T cell surface protein, leucine rich repeat containing 32 (LRRC32)

PubMed Central

2011-01-01

Background Elevated numbers of regulatory T cells (Tregs) have been implicated in certain cancers. Depletion of Tregs has been shown to increase anti-tumor immunity. Tregs also play a critical role in the suppression of autoimmune responses. The study of Tregs has been hampered by a lack of adequate surface markers. Leucine Rich Repeat Containing 32 (LRRC32), also known as Glycoprotein A Repetitions Predominant (GARP), has been postulated as a novel surface marker of activated Tregs. However, there is limited information regarding the processing of LRRC32 or the regulatory phenotype and functional activity of Tregs expressing LRRC32. Results Using naturally-occurring freshly isolated Tregs, we demonstrate that low levels of LRRC32 are present intracellularly prior to activation and that freshly isolated LRRC32+ Tregs are distinct from LRRC32- Tregs with respect to the expression of surface CD62L. Using LRRC32 transfectants of HEK cells, we demonstrate that the N-terminus of LRRC32 is cleaved prior to expression of the protein at the cell surface. Furthermore, we demonstrate using a construct containing a deleted putative signal peptide region that the presence of a signal peptide region is critical to cell surface expression of LRRC32. Finally, mixed lymphocyte assays demonstrate that LRRC32+ Tregs are more potent suppressors than LRRC32- Tregs. Conclusions A cleaved signal peptide site in LRRC32 is necessary for surface localization of native LRRC32 following activation of naturally-occurring freshly-isolated regulatory T cells. LRRC32 expression appears to alter the surface expression of activation markers of T cells such as CD62L. LRRC32 surface expression may be useful as a marker that selects for more potent Treg populations. In summary, understanding the processing and expression of LRRC32 may provide insight into the mechanism of action of Tregs and the refinement of immunotherapeutic strategies aimed at targeting these cells. PMID:21615933

Inhibition of Human Papillomavirus DNA Replication by an E1-Derived p80/UAF1-Binding Peptide

PubMed Central

Lehoux, Michaël; Fradet-Turcotte, Amélie; Lussier-Price, Mathieu; Omichinski, James G.

2012-01-01

The papillomavirus E1 helicase is recruited by E2 to the viral origin, where it assembles into a double hexamer that orchestrates replication of the viral genome. We previously identified the cellular WD40 repeat-containing protein p80/UAF1 as a novel interaction partner of E1 from anogenital human papillomavirus (HPV) types. p80 was found to interact with the first 40 residues of HPV type 31 (HPV31) E1, and amino acid substitutions within this domain abrogated the maintenance of the viral episome in keratinocytes. In this study, we report that these p80-binding substitutions reduce by 70% the ability of E1 to support transient viral DNA replication without affecting its interaction with E2 and assembly at the origin in vivo. Microscopy studies revealed that p80 is relocalized from the cytoplasm to discrete subnuclear foci by E1 and E2. Chromatin immunoprecipitation assays further revealed that p80 is recruited to the viral origin in an E1- and E2-dependent manner. Interestingly, overexpression of a 40-amino-acid-long p80-binding peptide, derived from HPV31 E1, was found to inhibit viral DNA replication by preventing the recruitment of endogenous p80 to the origin. Mutant peptides defective for p80 interaction were not inhibitory, demonstrating the specificity of this effect. Characterization of this E1 peptide by nuclear magnetic resonance (NMR) showed that it is intrinsically disordered in solution, while mapping studies indicated that the WD repeats of p80 are required for E1 interaction. These results provide additional evidence for the requirement for p80 in anogenital HPV DNA replication and highlight the potential of E1-p80 interaction as a novel antiviral target. PMID:22278251

A brush-polymer conjugate of exendin-4 reduces blood glucose for up to five days and eliminates poly(ethylene glycol) antigenicity

PubMed Central

Qi, Yizhi; Simakova, Antonina; Ganson, Nancy J.; Li, Xinghai; Luginbuhl, Kelli M.; Özer, Imran; Liu, Wenge; Hershfield, Michael S.; Matyjaszewski, Krzysztof; Chilkoti, Ashutosh

2017-01-01

The delivery of therapeutic peptides and proteins is often challenged by a short half-life, and thus the need for frequent injections that limit efficacy, reduce patient compliance and increase treatment cost. Here, we demonstrate that a single subcutaneous injection of site-specific (C-terminal) conjugates of exendin-4 (exendin) — a therapeutic peptide that is clinically used to treat type 2 diabetes — and poly[oligo(ethylene glycol) methyl ether methacrylate] (POEGMA) with precisely controlled molecular weights lowered blood glucose for up to 120 h in fed mice. Most notably, we show that an exendin-C-POEGMA conjugate with an average of 9 side-chain ethylene glycol (EG) repeats exhibits significantly lower reactivity towards patient-derived anti-poly(ethylene glycol) (PEG) antibodies than two FDA-approved PEGylated drugs, and that reducing the side-chain length to 3 EG repeats completely eliminates PEG antigenicity without compromising in vivo efficacy. Our findings establish the site-specific conjugation of POEGMA as a next-generation PEGylation technology for improving the pharmacological performance of traditional PEGylated drugs, whose safety and efficacy are hindered by pre-existing anti-PEG antibodies in patients. PMID:28989813

Group B streptococcal serine-rich repeat proteins promote interaction with fibrinogen and vaginal colonization.

PubMed

Wang, Nai-Yu; Patras, Kathryn A; Seo, Ho Seong; Cavaco, Courtney K; Rösler, Berenice; Neely, Melody N; Sullam, Paul M; Doran, Kelly S

2014-09-15

Group B streptococcus (GBS) can cause severe disease in susceptible hosts, including newborns, pregnant women, and the elderly. GBS serine-rich repeat (Srr) surface glycoproteins are important adhesins/invasins in multiple host tissues, including the vagina. However, exact molecular mechanisms contributing to their importance in colonization are unknown. We have recently determined that Srr proteins contain a fibrinogen-binding region (BR) and hypothesize that Srr-mediated fibrinogen binding may contribute to GBS cervicovaginal colonization. In this study, we observed that fibrinogen enhanced wild-type GBS attachment to cervical and vaginal epithelium, and that this was dependent on Srr1. Moreover, purified Srr1-BR peptide bound directly to host cells, and peptide administration in vivo reduced GBS recovery from the vaginal tract. Furthermore, a GBS mutant strain lacking only the Srr1 "latching" domain exhibited decreased adherence in vitro and decreased persistence in a mouse model of GBS vaginal colonization, suggesting the importance of Srr-fibrinogen interactions in the female reproductive tract. © The Author 2014. Published by Oxford University Press on behalf of the Infectious Diseases Society of America. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com.

Cyclic Peptides Arising by Evolutionary Parallelism via Asparaginyl-Endopeptidase–Mediated Biosynthesis[C][W

PubMed Central

Mylne, Joshua S.; Chan, Lai Yue; Chanson, Aurelie H.; Daly, Norelle L.; Schaefer, Hanno; Bailey, Timothy L.; Nguyencong, Philip; Cascales, Laura; Craik, David J.

2012-01-01

The cyclic miniprotein Momordica cochinchinensis Trypsin Inhibitor II (MCoTI-II) (34 amino acids) is a potent trypsin inhibitor (TI) and a favored scaffold for drug design. We have cloned the corresponding genes and determined that each precursor protein contains a tandem series of cyclic TIs terminating with the more commonly known, and potentially ancestral, acyclic TI. Expression of the precursor protein in Arabidopsis thaliana showed that production of the cyclic TIs, but not the terminal acyclic TI, depends on asparaginyl endopeptidase (AEP) for maturation. The nature of their repetitive sequences and the almost identical structures of emerging TIs suggest these cyclic peptides evolved by internal gene amplification associated with recruitment of AEP for processing between domain repeats. This is the third example of similar AEP-mediated processing of a class of cyclic peptides from unrelated precursor proteins in phylogenetically distant plant families. This suggests that production of cyclic peptides in angiosperms has evolved in parallel using AEP as a constraining evolutionary channel. We believe this is evolutionary evidence that, in addition to its known roles in proteolysis, AEP is especially suited to performing protein cyclization. PMID:22822203

Non-linearity of the collagen triple helix in solution and implications for collagen function.

PubMed

Walker, Kenneth T; Nan, Ruodan; Wright, David W; Gor, Jayesh; Bishop, Anthony C; Makhatadze, George I; Brodsky, Barbara; Perkins, Stephen J

2017-06-16

Collagen adopts a characteristic supercoiled triple helical conformation which requires a repeating (Xaa-Yaa-Gly) n sequence. Despite the abundance of collagen, a combined experimental and atomistic modelling approach has not so far quantitated the degree of flexibility seen experimentally in the solution structures of collagen triple helices. To address this question, we report an experimental study on the flexibility of varying lengths of collagen triple helical peptides, composed of six, eight, ten and twelve repeats of the most stable Pro-Hyp-Gly (POG) units. In addition, one unblocked peptide, (POG) 10unblocked , was compared with the blocked (POG) 10 as a control for the significance of end effects. Complementary analytical ultracentrifugation and synchrotron small angle X-ray scattering data showed that the conformations of the longer triple helical peptides were not well explained by a linear structure derived from crystallography. To interpret these data, molecular dynamics simulations were used to generate 50 000 physically realistic collagen structures for each of the helices. These structures were fitted against their respective scattering data to reveal the best fitting structures from this large ensemble of possible helix structures. This curve fitting confirmed a small degree of non-linearity to exist in these best fit triple helices, with the degree of bending approximated as 4-17° from linearity. Our results open the way for further studies of other collagen triple helices with different sequences and stabilities in order to clarify the role of molecular rigidity and flexibility in collagen extracellular and immune function and disease. © 2017 The Author(s).

Multiple conserved domains of the nucleoporin Nup124p and its orthologs Nup1p and Nup153 are critical for nuclear import and activity of the fission yeast Tf1 retrotransposon.

PubMed

Sistla, Srivani; Pang, Junxiong Vincent; Wang, Cui Xia; Balasundaram, David

2007-09-01

The nucleoporin Nup124p is a host protein required for the nuclear import of both, retrotransposon Tf1-Gag as well as the retroviral HIV-1 Vpr in fission yeast. The human nucleoporin Nup153 and the Saccharomyces cerevisiae Nup1p were identified as orthologs of Nup124p. In this study, we show that all three nucleoporins share a large FG/FXFG-repeat domain and a C-terminal peptide sequence, GRKIxxxxxRRKx, that are absolutely essential for Tf1 retrotransposition. Though the FXFG domain was essential, the FXFG repeats themselves could be eliminated without loss of retrotransposon activity, suggesting the existence of a common element unrelated to FG/FXFG motifs. The Nup124p C-terminal peptide, GRKIAVPRSRRKR, was extremely sensitive to certain single amino acid changes within stretches of the basic residues. On the basis of our comparative study of Nup124p, Nup1p, and Nup153 domains, we have developed peptides that specifically knockdown retrotransposon activity by disengaging the Tf1-Gag from its host nuclear transport machinery without any harmful consequence to the host itself. Our results imply that those domains challenged a specific pathway affecting Tf1 transposition. Although full-length Nup1p or Nup153 does not complement Nup124p, the functionality of their conserved domains with reference to Tf1 activity suggests that these three proteins evolved from a common ancestor.

Multiple Conserved Domains of the Nucleoporin Nup124p and Its Orthologs Nup1p and Nup153 Are Critical for Nuclear Import and Activity of the Fission Yeast Tf1 Retrotransposon

PubMed Central

Sistla, Srivani; Pang, Junxiong Vincent; Wang, Cui Xia

2007-01-01

The nucleoporin Nup124p is a host protein required for the nuclear import of both, retrotransposon Tf1-Gag as well as the retroviral HIV-1 Vpr in fission yeast. The human nucleoporin Nup153 and the Saccharomyces cerevisiae Nup1p were identified as orthologs of Nup124p. In this study, we show that all three nucleoporins share a large FG/FXFG-repeat domain and a C-terminal peptide sequence, GRKIxxxxxRRKx, that are absolutely essential for Tf1 retrotransposition. Though the FXFG domain was essential, the FXFG repeats themselves could be eliminated without loss of retrotransposon activity, suggesting the existence of a common element unrelated to FG/FXFG motifs. The Nup124p C-terminal peptide, GRKIAVPRSRRKR, was extremely sensitive to certain single amino acid changes within stretches of the basic residues. On the basis of our comparative study of Nup124p, Nup1p, and Nup153 domains, we have developed peptides that specifically knockdown retrotransposon activity by disengaging the Tf1-Gag from its host nuclear transport machinery without any harmful consequence to the host itself. Our results imply that those domains challenged a specific pathway affecting Tf1 transposition. Although full-length Nup1p or Nup153 does not complement Nup124p, the functionality of their conserved domains with reference to Tf1 activity suggests that these three proteins evolved from a common ancestor. PMID:17615301

Rational design of alpha-helical tandem repeat proteins with closed architectures

PubMed Central

Doyle, Lindsey; Hallinan, Jazmine; Bolduc, Jill; Parmeggiani, Fabio; Baker, David; Stoddard, Barry L.; Bradley, Philip

2015-01-01

Tandem repeat proteins, which are formed by repetition of modular units of protein sequence and structure, play important biological roles as macromolecular binding and scaffolding domains, enzymes, and building blocks for the assembly of fibrous materials1,2. The modular nature of repeat proteins enables the rapid construction and diversification of extended binding surfaces by duplication and recombination of simple building blocks3,4. The overall architecture of tandem repeat protein structures – which is dictated by the internal geometry and local packing of the repeat building blocks – is highly diverse, ranging from extended, super-helical folds that bind peptide, DNA, and RNA partners5–9, to closed and compact conformations with internal cavities suitable for small molecule binding and catalysis10. Here we report the development and validation of computational methods for de novo design of tandem repeat protein architectures driven purely by geometric criteria defining the inter-repeat geometry, without reference to the sequences and structures of existing repeat protein families. We have applied these methods to design a series of closed alpha-solenoid11 repeat structures (alpha-toroids) in which the inter-repeat packing geometry is constrained so as to juxtapose the N- and C-termini; several of these designed structures have been validated by X-ray crystallography. Unlike previous approaches to tandem repeat protein engineering12–20, our design procedure does not rely on template sequence or structural information taken from natural repeat proteins and hence can produce structures unlike those seen in nature. As an example, we have successfully designed and validated closed alpha-solenoid repeats with a left-handed helical architecture that – to our knowledge – is not yet present in the protein structure database21. PMID:26675735

A replaceable microreactor for on-line protein digestion in a two-dimensional capillary electrophoresis system with tandem mass spectrometry detection

PubMed Central

Li, Yihan; Wojcik, Roza; Dovichi, Norman J.

2010-01-01

We describe a two-dimensional capillary electrophoresis system that incorporates a replaceable enzymatic microreactor for on-line protein digestion. In this system, trypsin is immobilized on magnetic beads. At the start of each experiment, old beads are flushed to waste and replaced with a fresh plug of beads, which is captured by a pair of magnets at the distal tip of the first capillary. For analysis, proteins are separated in the first capillary. A fraction is then parked in the reactor to create peptides. Digested peptides are periodically transferred to the second capillary for separation; a fresh protein fraction is simultaneously moved to the reactor for digestion. An electrospray interface is used to introduce peptides into a mass spectrometer for analysis. This procedure is repeated for several dozen fractions under computer control. The system was demonstrated by the separation and digestion of insulin chain b oxidized and β-casein as model proteins. PMID:21030030

Total Physical Response Storytelling: A Communicative Approach to Language Learning.

ERIC Educational Resources Information Center

Marsh, Valeri

1998-01-01

Describes total physical response storytelling, which provides the critical vehicle--storytelling--for utilizing and expanding vocabulary. High-interest stories contextualize the vocabulary, enabling students to hear and see a story and then to act out, revise, and rewrite. A brief outline of the sequence of steps for using TPR storytelling in…

Using "Total Physical Response" with Young Learners in Oman

ERIC Educational Resources Information Center

Al Harrasi, Kothar Talib Sulaiman

2014-01-01

Among several approaches to teaching and learning a foreign language, Total Physical Response, or TPR, is one that simulates the way children naturally acquire their mother tongue. Instructors give commands to students in the new language, and students respond through gestures. This article showcases a language learning project that the Ministry…

The TIE1 Transcriptional Repressor Links TCP Transcription Factors with TOPLESS/TOPLESS-RELATED Corepressors and Modulates Leaf Development in Arabidopsis[W

PubMed Central

Tao, Qing; Guo, Dongshu; Wei, Baoye; Zhang, Fan; Pang, Changxu; Jiang, Hao; Zhang, Jinzhe; Wei, Tong; Gu, Hongya; Qu, Li-Jia; Qin, Genji

2013-01-01

Leaf size and shape are mainly determined by coordinated cell division and differentiation in lamina. The CINCINNATA (CIN)-like TEOSINTE BRANCHED1/CYCLOIDEA/PCF (TCP) transcription factors are key regulators of leaf development. However, the mechanisms that control TCP activities during leaf development are largely unknown. We identified the TCP Interactor containing EAR motif protein1 (TIE1), a novel transcriptional repressor, as a major modulator of TCP activities during leaf development. Overexpression of TIE1 leads to hyponastic and serrated leaves, whereas disruption of TIE1 causes epinastic leaves. TIE1 is expressed in young leaves and encodes a transcriptional repressor containing a C-terminal EAR motif, which mediates interactions with the TOPLESS (TPL)/TOPLESS-RELATED (TPR) corepressors. In addition, TIE1 physically interacts with CIN-like TCPs. We propose that TIE1 regulates leaf size and morphology by inhibiting the activities of TCPs through recruiting the TPL/TPR corepressors to form a tertiary complex at early stages of leaf development. PMID:23444332

Nup133 Is Required for Proper Nuclear Pore Basket Assembly and Dynamics in Embryonic Stem Cells.

PubMed

Souquet, Benoit; Freed, Ellen; Berto, Alessandro; Andric, Vedrana; Audugé, Nicolas; Reina-San-Martin, Bernardo; Lacy, Elizabeth; Doye, Valérie

2018-05-22

Nup133 belongs to the Y-complex, a key component of the nuclear pore complex (NPC) scaffold. Studies on a null mutation in mice previously revealed that Nup133 is essential for embryonic development but not for mouse embryonic stem cell (mESC) proliferation. Using single-pore detection and average NE-fluorescence intensity, we find that Nup133 is dispensable for interphase and postmitotic NPC scaffold assembly in pluripotent mESCs. However, loss of Nup133 specifically perturbs the formation of the nuclear basket as manifested by the absence of Tpr in about half of the NPCs combined with altered dynamics of Nup153. We further demonstrate that its central domain mediates Nup133's role in assembling Tpr and Nup153 into a properly configured nuclear basket. Our findings thus revisit the role of the Y-complex in pore biogenesis and provide insights into the interplay between NPC scaffold architecture, nuclear basket assembly, and the generation of heterogeneity among NPCs. Copyright © 2018 The Authors. Published by Elsevier Inc. All rights reserved.

Machine-learning-based real-bogus system for the HSC-SSP moving object detection pipeline

NASA Astrophysics Data System (ADS)

Lin, Hsing-Wen; Chen, Ying-Tung; Wang, Jen-Hung; Wang, Shiang-Yu; Yoshida, Fumi; Ip, Wing-Huen; Miyazaki, Satoshi; Terai, Tsuyoshi

2018-01-01

Machine-learning techniques are widely applied in many modern optical sky surveys, e.g., Pan-STARRS1, PTF/iPTF, and the Subaru/Hyper Suprime-Cam survey, to reduce human intervention in data verification. In this study, we have established a machine-learning-based real-bogus system to reject false detections in the Subaru/Hyper-Suprime-Cam Strategic Survey Program (HSC-SSP) source catalog. Therefore, the HSC-SSP moving object detection pipeline can operate more effectively due to the reduction of false positives. To train the real-bogus system, we use stationary sources as the real training set and "flagged" data as the bogus set. The training set contains 47 features, most of which are photometric measurements and shape moments generated from the HSC image reduction pipeline (hscPipe). Our system can reach a true positive rate (tpr) ˜96% with a false positive rate (fpr) ˜1% or tpr ˜99% at fpr ˜5%. Therefore, we conclude that stationary sources are decent real training samples, and using photometry measurements and shape moments can reject false positives effectively.

Judicial Reliance on Parental IQ in Appellate-Level Child Welfare Cases Involving Parents with Intellectual and Developmental Disabilities.

PubMed

Callow, Ella; Tahir, Munazza; Feldman, Maurice

2017-05-01

Parents with intellectual and developmental disabilities (IDDs) are over-represented in child welfare cases. Although IQ per se is an invalid indicator of parenting abilities, this study examined the prevalence of judicial consideration of parental IQ test evidence in US appellate cases. The present authors conducted Boolean searches of Westlaw Corporation's case database since 1999. The present authors used a six-question checklist to survey the 42 most recent American appellate cases involving termination of parental rights (TPR) decisions that included evidence of parental intellectual and developmental disabilities based on IQ. In 86% of cases, parental low IQ was presented as a barrier to parenting competence. Higher courts uphold TPR decision in 81% of cases involving parents with intellectual and developmental disabilities. Parental IQ scores are routinely relied upon to judge parenting capacity in custody cases where parents have intellectual and developmental disabilities. The present authors recommend more comprehensive assessments examining a broader range of contextual variable that may impact on parenting abilities. © 2016 John Wiley & Sons Ltd.

In situ TPR XANES study of the partial oxidation of methane using a Ni-substituted hexaaluminate catalyst

DOE Office of Scientific and Technical Information (OSTI.GOV)

Kugler, E.L.; Gardner, T.H.; Campos, Andrew

2008-04-01

Metallic Ni formation near the mirror cation site, Ba in this study, is believed to cause the partial oxidation activity observed in Ni-substituted hexaaluminate catalysts. The BaNi1.0Al11.6O19-d catalyst was prepared by coprecipitation with nitrate salt precursors; following the coprecipitation procedure, the catalyst was calcined at 1400°C to create the hexaaluminate structure. TPR XANES in fluorescence was used to probe the local structure of the BaNi1.0Al11.6O19-d catalyst to determine whether metallic nickel forms at different temperatures: 825°C, 875°C, 925°C. The XANES results indicate that the Ni in the hexaaluminate catalyst only reduces if the temperature is maintained at 925°C. Once themore » metallic state is formed, the oxidation state is stable; even in the POX environment. Future work using a theoretical approach to the XANES data using FEFF 8.4 gives information on the interactions between Ni and Ba, which will be used to further optimize the catalyst.« less

UV-light-induced one-color and two-color photorefractive effects in congruent and near-stoichiometric LiNbO 3:Mg crystals

NASA Astrophysics Data System (ADS)

Qiao, Haijun; Xu, Jingjun; Tomita, Yasuo; Zhu, Dengsong; Fu, Bo; Zhang, Guoquan; Zhang, Guangyin

2007-03-01

We describe the ultraviolet-light one-color photorefraction (UV-OPR) at 351 nm in LiNbO3 crystals with different Mg-doping concentrations and [Li]/[Nb] ratios. It is shown that as the Mg-doping concentration and/or the [Li]/[Nb] ratio increase, the refractive index change and the two-beam coupling gain increase but the response time decreases. It is also shown that the recording sensitivity as large as ∼27 cm/J is obtainable at a recording intensity of ∼1 W/cm2 in near-stoichiometric LiNbO3 doped with 2 mol% Mg. This sensitivity is approximately one order of magnitude higher than those for other LiNbO3 crystals. We also describe the ultraviolet-light-gating two-color photorefraction (UV-TPR) using 365 nm gating and 633 nm recording beams in LiNbO3 crystals with different Mg-doping concentrations and [Li]/[Nb] ratios. It is shown that UV-TPR is only observed in near-stoichiometric crystals and the grating-formation dynamics strongly depend on the Mg concentration.

Signature motif-guided identification of receptors for peptide hormones essential for root meristem growth.

PubMed

Song, Wen; Liu, Li; Wang, Jizong; Wu, Zhen; Zhang, Heqiao; Tang, Jiao; Lin, Guangzhong; Wang, Yichuan; Wen, Xing; Li, Wenyang; Han, Zhifu; Guo, Hongwei; Chai, Jijie

2016-06-01

Peptide-mediated cell-to-cell signaling has crucial roles in coordination and definition of cellular functions in plants. Peptide-receptor matching is important for understanding the mechanisms underlying peptide-mediated signaling. Here we report the structure-guided identification of root meristem growth factor (RGF) receptors important for plant development. An assay based on a signature ligand recognition motif (Arg-x-Arg) conserved in a subfamily of leucine-rich repeat receptor kinases (LRR-RKs) identified the functionally uncharacterized LRR-RK At4g26540 as a receptor of RGF1 (RGFR1). We further solved the crystal structure of RGF1 in complex with the LRR domain of RGFR1 at a resolution of 2.6 Å, which reveals that the Arg-x-Gly-Gly (RxGG) motif is responsible for specific recognition of the sulfate group of RGF1 by RGFR1. Based on the RxGG motif, we identified additional four RGFRs. Participation of the five RGFRs in RGF-induced signaling is supported by biochemical and genetic data. We also offer evidence showing that SERKs function as co-receptors for RGFs. Taken together, our study identifies RGF receptors and co-receptors that can link RGF signals with their downstream components and provides a proof of principle for structure-based matching of LRR-RKs with their peptide ligands.

Rational improvement of gp41-targeting HIV-1 fusion inhibitors: an innovatively designed Ile-Asp-Leu tail with alternative conformations.

PubMed

Zhu, Yun; Su, Shan; Qin, Lili; Wang, Qian; Shi, Lei; Ma, Zhenxuan; Tang, Jianchao; Jiang, Shibo; Lu, Lu; Ye, Sheng; Zhang, Rongguang

2016-09-26

Peptides derived from the C-terminal heptad repeat (CHR) of HIV gp41 have been developed as effective fusion inhibitors against HIV-1, but facing the challenges of enhancing potency and stability. Here, we report a rationally designed novel HIV-1 fusion inhibitor derived from CHR-derived peptide (Trp628~Gln653, named CP), but with an innovative Ile-Asp-Leu tail (IDL) that dramatically increased the inhibitory activity by up to 100 folds. We also determined the crystal structures of artificial fusion peptides N36- and N43-L6-CP-IDL. Although the overall structures of both fusion peptides share the canonical six-helix bundle (6-HB) configuration, their IDL tails adopt two different conformations: a one-turn helix with the N36, and a hook-like structure with the longer N43. Structural comparison showed that the hook-like IDL tail possesses a larger interaction interface with NHR than the helical one. Further molecular dynamics simulations of the two 6-HBs and isolated CP-IDL peptides suggested that hook-like form of IDL tail can be stabilized by its binding to NHR trimer. Therefore, CP-IDL has potential for further development as a new HIV fusion inhibitor, and this strategy could be widely used in developing artificial fusion inhibitors against HIV and other enveloped viruses.

Crystal and Molecular Structure of a Collagen-Like Peptide at 1.9 overset{circ}{A} Resolution

NASA Astrophysics Data System (ADS)

Bella, Jordi; Eaton, Mark; Brodsky, Barbara; Berman, Helen M.

1994-10-01

The structure of a protein triple helix has been determined at 1.9 angstrom resolution by x-ray crystallographic studies of a collagen-like peptide containing a single substitution of the consensus sequence. This peptide adopts a triple-helical structure that confirms the basic features determined from fiber diffraction studies on collagen: supercoiling of polyproline II helices and interchain hydrogen bonding that follows the model II of Rich and Crick. In addition, the structure provides new information concerning the nature of this protein fold. Each triple helix is surrounded by a cylinder of hydration, with an extensive hydrogen bonding network between water molecules and peptide acceptor groups. Hydroxyproline residues have a critical role in this water network. The interaxial spacing of triple helices in the crystal is similar to that in collagen fibrils, and the water networks linking adjacent triple helices in the crystal structure are likely to be present in connective tissues. The breaking of the repeating (X-Y-Gly)_n pattern by a Gly-->Ala substitution results in a subtle alteration of the conformation, with a local untwisting of the triple helix. At the substitution site, direct interchain hydrogen bonds are replaced with interstitial water bridges between the peptide groups. Similar conformational changes may occur in Gly-->X mutated collagens responsible for the diseases osteogenesis imperfecta, chondrodysplasias, and Ehlers-Danlos syndrome IV.

Puromycin-sensitive aminopeptidase is the major peptidase responsible for digesting polyglutamine sequences released by proteasomes during protein degradation

PubMed Central

Bhutani, N; Venkatraman, P; Goldberg, A L

2007-01-01

Long stretches of glutamine (Q) residues are found in many cellular proteins. Expansion of these polyglutamine (polyQ) sequences is the underlying cause of several neurodegenerative diseases (e.g. Huntington's disease). Eukaryotic proteasomes have been found to digest polyQ sequences in proteins very slowly, or not at all, and to release such potentially toxic sequences for degradation by other peptidases. To identify these key peptidases, we investigated the degradation in cell extracts of model Q-rich fluorescent substrates and peptides containing 10–30 Q's. Their degradation at neutral pH was due to a single aminopeptidase, the puromycin-sensitive aminopeptidase (PSA, cytosol alanyl aminopeptidase). No other known cytosolic aminopeptidase or endopeptidase was found to digest these polyQ peptides. Although tripeptidyl peptidase II (TPPII) exhibited limited activity, studies with specific inhibitors, pure enzymes and extracts of cells treated with siRNA for TPPII or PSA showed PSA to be the rate-limiting activity against polyQ peptides up to 30 residues long. (PSA digests such Q sequences, shorter ones and typical (non-repeating) peptides at similar rates.) Thus, PSA, which is induced in neurons expressing mutant huntingtin, appears critical in preventing the accumulation of polyQ peptides in normal cells, and its activity may influence susceptibility to polyQ diseases. PMID:17318184

Cocaine- and amphetamine-regulated transcript peptide in the nucleus accumbens shell inhibits cocaine-induced locomotor sensitization to transient over-expression of α-Ca2+ /calmodulin-dependent protein kinase II.

PubMed

Xiong, Lixia; Meng, Qing; Sun, Xi; Lu, Xiangtong; Fu, Qiang; Peng, Qinghua; Yang, Jianhua; Oh, Ki-Wan; Hu, Zhenzhen

2018-01-04

Cocaine- and amphetamine-regulated transcript (CART) peptide is a widely distributed neurotransmitter that attenuates cocaine-induced locomotor activity when injected into the nucleus accumbens (NAc). Our previous work first confirmed that the inhibitory mechanism of the CART peptide on cocaine-induced locomotor activity is related to a reduction in cocaine-enhanced phosphorylated Ca 2+ /calmodulin-dependent protein kinaseIIα (pCaMKIIα) and the enhancement of cocaine-induced D3R function. This study investigated whether CART peptide inhibited cocaine-induced locomotor activity via inhibition of interactions between pCaMKIIα and the D3 dopamine receptor (D3R). We demonstrated that lentivirus-mediated gene transfer transiently increased pCaMKIIα expression, which peaked at 10 days after microinjection into the rat NAc shell, and induced a significant increase in Ca 2+ influx along with greater behavioral sensitivity in the open field test after intraperitoneal injections of cocaine (15 mg/kg). However, western blot analysis and coimmunoprecipitation demonstrated that CART peptide treatment in lentivirus-transfected CaMKIIα-over-expressing NAc rat tissues or cells prior to cocaine administration inhibited the cocaine-induced Ca 2+ influx and attenuated the cocaine-increased pCaMKIIα expression in lentivirus-transfected CaMKIIα-over-expressing cells. CART peptide decreased the cocaine-enhanced phosphorylated cAMP response element binding protein (pCREB) expression via inhibition of the pCaMKIIα-D3R interaction, which may account for the prolonged locomotor sensitization induced by repeated cocaine treatment in lentivirus-transfected CaMKIIα-over-expressing cells. These results provide strong evidence for the inhibitory modulation of CART peptide in cocaine-induced locomotor sensitization. © 2018 International Society for Neurochemistry.

Relative and accurate measurement of protein abundance using 15N stable isotope labeling in Arabidopsis (SILIA).

PubMed

Guo, Guangyu; Li, Ning

2011-07-01

In the quantitative proteomic studies, numerous in vitro and in vivo peptide labeling strategies have been successfully applied to measure differentially regulated protein and peptide abundance. These approaches have been proven to be versatile and repeatable in biological discoveries. (15)N metabolic labeling is one of these widely adopted and economical methods. However, due to the differential incorporation rates of (15)N or (14)N, the labeling results produce imperfectly matched isotopic envelopes between the heavy and light nitrogen-labeled peptides. In the present study, we have modified the solid Arabidopsis growth medium to standardize the (15)N supply, which led to a uniform incorporation of (15)N into the whole plant protein complement. The incorporation rate (97.43±0.11%) of (15)N into (15)N-coded peptides was determined by correlating the intensities of peptide ions with the labeling efficiencies according to Gaussian distribution. The resulting actual incorporation rate (97.44%) and natural abundance of (15)N/(14)N-coded peptides are used to re-calculate the intensities of isotopic envelopes of differentially labeled peptides, respectively. A modified (15)N/(14)N stable isotope labeling strategy, SILIA, is assessed and the results demonstrate that this approach is able to differentiate the fold change in protein abundance down to 10%. The machine dynamic range limitation and purification step will make the precursor ion ratio deriving from the actual ratio fold change. It is suggested that the differentially mixed (15)N-coded and (14)N-coded plant protein samples that are used to establish the protein abundance standard curve should be prepared following a similar protein isolation protocol used to isolate the proteins to be quantitated. Crown Copyright © 2011. Published by Elsevier Ltd. All rights reserved.

Competitive antagonists discriminate between NK2 tachykinin receptor subtypes.

PubMed Central

Maggi, C. A.; Patacchini, R.; Giuliani, S.; Rovero, P.; Dion, S.; Regoli, D.; Giachetti, A.; Meli, A.

1990-01-01

1. We have compared the ability of various tachykinins and selective tachykinin receptor agonists to induce contraction of the endothelium-denuded rabbit pulmonary artery (RPA) and hamster trachea (HT) and have estimated the affinity of some newly developed NK2 selective antagonists in the same tissues. 2. In confirmation of previous findings, experiments with the agonists indicated that NK2 receptors are the main if not the sole mediators of the response to tachykinins in both RPA and HT. No evidence for significant degradation of neurokinin A (NKA) was found in either tissue when experiments were repeated in the presence of a mixture of peptidase inhibitors (thiorphan, captopril and bestatin, 1 microM each). 3. The peptide antagonists tested were: Peptide I = [Tyr5, D-Trp6,8,9, Arg10]-NKA(4-10); Peptide II = [Tyr5, D-Trp6,8,9, Arg10]-NKA(3-10); Peptide III = Ac-Leu-Asp-Gln-Trp-Phe-Gly-NH2. The three peptides produced a concentration-dependent rightward shift of the concentration-response curve to NKA in both RPA and HT with no significant depression of the maximal response attainable. The slopes of the Schild plots were not significantly different from unity, indicating a competitive antagonism. Peptides I and II were about 100 times more potent in the RPA than in the HT, while Peptide III was about 100 times more potent in the HT than RPA. 4. The pA2 values obtained in these two tissues with the three antagonists were not significantly different when tested in the absence or presence of peptidase inhibitors, or when a selective NK2 receptor agonist, [beta Ala8]-NKA(4-10) was used instead of NKA.(ABSTRACT TRUNCATED AT 250 WORDS) PMID:2167737

Genome-Wide Sensitivity Analysis of the Microsymbiont Sinorhizobium meliloti to Symbiotically Important, Defensin-Like Host Peptides

PubMed Central

Arnold, Markus F. F.; Shabab, Mohammed; Penterman, Jon; Boehme, Kevin L.; Griffitts, Joel S.

2017-01-01

ABSTRACT The model legume species Medicago truncatula expresses more than 700 nodule-specific cysteine-rich (NCR) signaling peptides that mediate the differentiation of Sinorhizobium meliloti bacteria into nitrogen-fixing bacteroids. NCR peptides are essential for a successful symbiosis in legume plants of the inverted-repeat-lacking clade (IRLC) and show similarity to mammalian defensins. In addition to signaling functions, many NCR peptides exhibit antimicrobial activity in vitro and in vivo. Bacterial resistance to these antimicrobial activities is likely to be important for symbiosis. However, the mechanisms used by S. meliloti to resist antimicrobial activity of plant peptides are poorly understood. To address this, we applied a global genetic approach using transposon mutagenesis followed by high-throughput sequencing (Tn-seq) to identify S. meliloti genes and pathways that increase or decrease bacterial competitiveness during exposure to the well-studied cationic NCR247 peptide and also to the unrelated model antimicrobial peptide polymyxin B. We identified 78 genes and several diverse pathways whose interruption alters S. meliloti resistance to NCR247. These genes encode the following: (i) cell envelope polysaccharide biosynthesis and modification proteins, (ii) inner and outer membrane proteins, (iii) peptidoglycan (PG) effector proteins, and (iv) non-membrane-associated factors such as transcriptional regulators and ribosome-associated factors. We describe a previously uncharacterized yet highly conserved peptidase, which protects S. meliloti from NCR247 and increases competitiveness during symbiosis. Additionally, we highlight a considerable number of uncharacterized genes that provide the basis for future studies to investigate the molecular basis of symbiotic development as well as chronic pathogenic interactions. PMID:28765224

Effect of physical countermaneuvers on orthostatic hypotension in familial dysautonomia.

PubMed

Tutaj, Marcin; Marthol, Harald; Berlin, Dena; Brown, Clive M; Axelrod, Felicia B; Hilz, Max J

2006-01-01

Familial dysautonomia (FD) patients frequently experience debilitating orthostatic hypotension. Since physical countermaneuvers can increase blood pressure (BP) in other groups of patients with orthostatic hypotension, we evaluated the effectiveness of countermaneuvers in FD patients. In 17 FD patients (26.4 +/- 12.4 years, eight female), we monitored heart rate (HR), blood pressure (BP), cardiac output (CO), total peripheral resistance (TPR) and calf volume while supine, during standing and during application of four countermaneuvers: bending forward, squatting, leg crossing, and abdominal compression using an inflatable belt. Countermaneuvers were initiated after standing up,when systolic BP had fallen by 40mmHg or diastolic BP by 30mmHg or presyncope had occurred. During active standing, blood pressure and TPR decreased, calf volume increased but CO remained stable. Mean BP increased significantly during bending forward (by 20.0 (17 - 28.5) mmHg; P = 0.005) (median (25(th) - 75(th) quartile)), squatting (by 50.8 (33.5 - 56) mmHg; P = 0.002), and abdominal compression (by 5.8 (-1 - 34.7) mmHg; P = 0.04) - but not during leg-crossing. Squatting and abdominal compression also induced a significant increase in CO (by 18.1 (-1.3 - 47.9) % during squatting (P = 0.02) and by 7.6 (0.4 - 19.6) % during abdominal compression (P=0.014)). HR did not change significantly during the countermaneuvers. TPR increased significantly only during squatting (by 37.2 (11.8 - 48.2) %; P = 0.01). However, orthopedic problems or ataxia prevented several patients from performing some of the countermaneuvers. Additionally, many patients required assistance with the maneuvers. Squatting, bending forward and abdominal compression can improve orthostatic BP in FD patients, which is achieved mainly by an increased cardiac output. Squatting has the greatest effect on orthostatic blood pressure in FD patients. Suitability and effectiveness of a specific countermaneuver depends on the orthopedic or neurological complications of each FD patient and must be individually tested before a therapeutic recommendation can be given.

WE-H-BRC-05: Catastrophic Error Metrics for Radiation Therapy

DOE Office of Scientific and Technical Information (OSTI.GOV)

Murphy, S; Molloy, J

Purpose: Intuitive evaluation of complex radiotherapy treatments is impractical, while data transfer anomalies create the potential for catastrophic treatment delivery errors. Contrary to prevailing wisdom, logical scrutiny can be applied to patient-specific machine settings. Such tests can be automated, applied at the point of treatment delivery and can be dissociated from prior states of the treatment plan, potentially revealing errors introduced early in the process. Methods: Analytical metrics were formulated for conventional and intensity modulated RT (IMRT) treatments. These were designed to assess consistency between monitor unit settings, wedge values, prescription dose and leaf positioning (IMRT). Institutional metric averages formore » 218 clinical plans were stratified over multiple anatomical sites. Treatment delivery errors were simulated using a commercial treatment planning system and metric behavior assessed via receiver-operator-characteristic (ROC) analysis. A positive result was returned if the erred plan metric value exceeded a given number of standard deviations, e.g. 2. The finding was declared true positive if the dosimetric impact exceeded 25%. ROC curves were generated over a range of metric standard deviations. Results: Data for the conventional treatment metric indicated standard deviations of 3%, 12%, 11%, 8%, and 5 % for brain, pelvis, abdomen, lung and breast sites, respectively. Optimum error declaration thresholds yielded true positive rates (TPR) between 0.7 and 1, and false positive rates (FPR) between 0 and 0.2. Two proposed IMRT metrics possessed standard deviations of 23% and 37%. The superior metric returned TPR and FPR of 0.7 and 0.2, respectively, when both leaf position and MUs were modelled. Isolation to only leaf position errors yielded TPR and FPR values of 0.9 and 0.1. Conclusion: Logical tests can reveal treatment delivery errors and prevent large, catastrophic errors. Analytical metrics are able to identify errors in monitor units, wedging and leaf positions with favorable sensitivity and specificity. In part by Varian.« less

Sequence heuristics to encode phase behaviour in intrinsically disordered protein polymers

PubMed Central

Quiroz, Felipe García; Chilkoti, Ashutosh

2015-01-01

Proteins and synthetic polymers that undergo aqueous phase transitions mediate self-assembly in nature and in man-made material systems. Yet little is known about how the phase behaviour of a protein is encoded in its amino acid sequence. Here, by synthesizing intrinsically disordered, repeat proteins to test motifs that we hypothesized would encode phase behaviour, we show that the proteins can be designed to exhibit tunable lower or upper critical solution temperature (LCST and UCST, respectively) transitions in physiological solutions. We also show that mutation of key residues at the repeat level abolishes phase behaviour or encodes an orthogonal transition. Furthermore, we provide heuristics to identify, at the proteome level, proteins that might exhibit phase behaviour and to design novel protein polymers consisting of biologically active peptide repeats that exhibit LCST or UCST transitions. These findings set the foundation for the prediction and encoding of phase behaviour at the sequence level. PMID:26390327

Evaluating seroprevalence to circumsporozoite protein to estimate exposure to three species of Plasmodium in the Brazilian Amazon.

PubMed

Pereira, Virginia Araujo; Sánchez-Arcila, Juan Camilo; Vasconcelos, Mariana Pinheiro Alves; Ferreira, Amanda Ribeiro; de Souza Videira, Lorene; Teva, Antonio; Perce-da-Silva, Daiana; Marques, Maria Teresa Queiroz; de Carvalho, Luzia Helena; Banic, Dalma Maria; Pôrto, Luiz Cristóvão Sobrino; Oliveira-Ferreira, Joseli

2018-05-14

Brazil has seen a great decline in malaria and the country is moving towards elimination. However, for eventual elimination, the control program needs efficient tools in order to monitor malaria exposure and transmission. In this study, we aimed to evaluate whether seroprevalence to the circumsporozoite protein (CSP) is a good tool for monitoring the exposure to and/or evaluating the burden and distribution of Plasmodium species in the Brazilian Amazon. Cross-sectional surveys were conducted in a rural area of Porto Velho, Rondônia state. Parasite infection was detected by microscopy and polymerase chain reaction. Antibodies to the sporozoite CSP repeats of Plasmodium vivax, P. falciparum, and P. malariae (PvCS, PfCS, and PmCS) were detected using the enzyme-linked immunosorbent assay technique. Human leukocyte antigen (HLA)-DRB1 and DQB1 genes were typed using Luminex® xMAP® technology. The prevalence of immunoglobulin G against P. vivax CSP peptide (62%) was higher than P. falciparum (49%) and P. malariae (46%) CSP peptide. Most of the studied individuals had antibodies to at least one of the three peptides (72%), 34% had antibodies to all three peptides and 28% were non-responders. Although the majority of the population was not infected at the time of the survey, 74.3% of parasite-negative individuals had antibodies to at least one of the CSPs. Importantly, among individuals carrying the haplotypes DRB1*04~DQB1*03, there was a significantly higher frequency of PfCS responders, and DRB1*16~DQB1*03 haplotype for PvCS and PfCS responders. In contrast, HLA-DRB1*01 and HLA-DQB1*05 allelic groups were associated with a lack of antibodies to P. vivax and P. falciparum CSP repeats, and the haplotype DRB1*01~DQB1*05 was also associated with non-responders, including non-responders to P. malariae. Our results show that in low transmission settings, naturally acquired antibody responses against the CSP repeats of P. vivax, P. falciparum, and P. malariae in a single cross-sectional study may not represent a valuable marker for monitoring recent malaria exposure, especially in an area with a high prevalence of P. vivax. Furthermore, HLA class II molecules play an important role in antibody response and require further study with a larger sample size. It will be of interest to consider HLA analysis when using serosurveillance to monitor malaria exposure among genetically diverse populations.

TRMM- and GPM-based precipitation analysis and modelling in the Tropical Andes

NASA Astrophysics Data System (ADS)

Manz, Bastian; Buytaert, Wouter; Zulkafli, Zed; Onof, Christian

2016-04-01

Despite wide-spread applications of satellite-based precipitation products (SPPs) throughout the TRMM-era, the scarcity of ground-based in-situ data (high density gauge networks, rainfall radar) in many hydro-meteorologically important regions, such as tropical mountain environments, has limited our ability to evaluate both SPPs and individual satellite-based sensors as well as accurately model or merge rainfall at high spatial resolutions, particularly with respect to extremes. This has restricted both the understanding of sensor behaviour and performance controls in such regions as well as the accuracy of precipitation estimates and respective hydrological applications ranging from water resources management to early warning systems. Here we report on our recent research into precipitation analysis and modelling using various TRMM and GPM products (2A25, 3B42 and IMERG) in the tropical Andes. In an initial study, 78 high-frequency (10-min) recording gauges in Colombia and Ecuador are used to generate a ground-based validation dataset for evaluation of instantaneous TRMM Precipitation Radar (TPR) overpasses from the 2A25 product. Detection ability, precipitation time-series, empirical distributions and statistical moments are evaluated with respect to regional climatological differences, seasonal behaviour, rainfall types and detection thresholds. Results confirmed previous findings from extra-tropical regions of over-estimation of low rainfall intensities and under-estimation of the highest 10% of rainfall intensities by the TPR. However, in spite of evident regionalised performance differences as a function of local climatological regimes, the TPR provides an accurate estimate of climatological annual and seasonal rainfall means. On this basis, high-resolution (5 km) climatological maps are derived for the entire tropical Andes. The second objective of this work is to improve the local precipitation estimation accuracy and representation of spatial patterns of extreme rainfall probabilities over the region. For this purpose, an ensemble of high-resolution rainfall fields is generated by stochastic simulation using space-time averaged, coarse-scale (daily, 0.25°) satellite-based rainfall inputs (TRMM 3B42/ -RT) and the high-resolution climatological information derived from the TPR as spatial disaggregation proxies. For evaluation and merging, gridded ground-based rainfall fields are generated from gauge data using sequential simulation. Satellite and ground-based ensembles are subsequently merged using an inverse error weighting scheme. The model was tested over a case study in the Colombian Andes with optional coarse-scale bias correction prior to disaggregation and merging. The resulting outputs were assessed in the context of Generalized Extreme Value theory and showed improved estimation of extreme rainfall probabilities compared to the original TMPA inputs. Initial findings using GPM-IMERG inputs are also presented.

The Gluten-Free, Casein-Free Diet in Autism: Results of a Preliminary Double Blind Clinical Trial

ERIC Educational Resources Information Center

Elder, Jennifer Harrison; Shankar, Meena; Shuster, Jonathan; Theriaque, Douglas; Burns, Sylvia; Sherrill, Lindsay

2006-01-01

This study tested the efficacy of a gluten-free and casein-free (GFCF) diet in treating autism using a randomized, double blind repeated measures crossover design. The sample included 15 children aged 2-16 years with autism spectrum disorder. Data on autistic symptoms and urinary peptide levels were collected in the subjects' homes over the 12…

Total chemical synthesis of proteins without HPLC purification† †Electronic supplementary information (ESI) available. See DOI: 10.1039/c6sc01883a Click here for additional data file.

PubMed Central

Loibl, S. F.; Harpaz, Z.; Zitterbart, R.

2016-01-01

The total chemical synthesis of proteins is a tedious and time-consuming endeavour. The typical steps involve solid phase synthesis of peptide thioesters and cysteinyl peptides, native chemical ligation (NCL) in solution, desulfurization or removal of ligation auxiliaries in the case of extended NCL as well as many intermediary and final HPLC purification steps. With an aim to facilitate and improve the throughput of protein synthesis we developed the first method for the rapid chemical total on-resin synthesis of proteins that proceeds without a single HPLC-purification step. The method relies on the combination of three orthogonal protein tags that allow sequential immobilization (via the N-terminal and C-terminal ends), extended native chemical ligation and release reactions. The peptide fragments to be ligated are prepared by conventional solid phase synthesis and used as crude materials in the subsequent steps. An N-terminal His6 unit permits selective immobilization of the full length peptide thioester onto Ni-NTA agarose beads. The C-terminal peptide fragment carries a C-terminal peptide hydrazide and an N-terminal 2-mercapto-2-phenyl-ethyl ligation auxiliary, which serves as a reactivity tag for the full length peptide. As a result, only full length peptides, not truncation products, react in the subsequent on-bead extended NCL. After auxiliary removal the ligation product is liberated into solution upon treatment with mild acid, and is concomitantly captured by an aldehyde-modified resin. This step allows the removal of the most frequently observed by-product in NCL chemistry, i.e. the hydrolysed peptide thioester (which does not contain a C-terminal peptide hydrazide). Finally, the target protein is released with diluted hydrazine or acid. We applied the method in the synthesis of 46 to 126 amino acid long MUC1 proteins comprising 2–6 copies of a 20mer tandem repeat sequence. Only three days were required for the parallel synthesis of 9 MUC1 proteins which were obtained in 8–33% overall yield with 90–98% purity despite the omission of HPLC purification. PMID:28451120

Natural and synthetic cathelicidin peptides with anti-microbial and anti-biofilm activity against Staphylococcus aureus.

PubMed

Dean, Scott N; Bishop, Barney M; van Hoek, Monique L

2011-05-23

Chronic, infected wounds typically contain multiple genera of bacteria, including Staphylococcus aureus, many of which are strong biofilm formers. Bacterial biofilms are thought to be a direct impediment to wound healing. New therapies that focus on a biofilm approach may improve the recovery and healing rate for infected wounds. In this study, cathelicidins and related short, synthetic peptides were tested for their anti-microbial effectiveness as well as their ability to inhibit the ability of S. aureus to form biofilms. The helical human cathelicidin LL-37 was tested against S. aureus, and was found to exhibit effective anti-microbial, anti-attachment as well as anti-biofilm activity at concentrations in the low μg/ml range. The effect of peptide chirality and associated protease-resistance was explored through the use of an all-D amino acid peptide, D-LL-37, and in turn compared to scrambled LL-37. Helical cathelicidins have been identified in other animals such as the Chinese cobra, Naja atra (NA-CATH). We previously identified an 11-residue imperfectly repeated pattern (ATRA motif) within the sequence of NA-CATH. A series of short peptides (ATRA-1, -2, -1A), as well as a synthetic peptide, NA-CATH:ATRA1-ATRA1, were designed to explore the significance of the conserved residues within the ATRA motif for anti-microbial activity. The CD spectrum of NA-CATH and NA-CATH:ATRA1-ATRA1 revealed the structural properties of these peptides and suggested that helicity may factor into their anti-microbial and anti-biofilm activities. The NA-CATH:ATRA1-ATRA1 peptide inhibits the production of biofilm by S. aureus in the presence of salt, exhibiting anti-biofilm activity at lower peptide concentrations than NA-CATH, LL-37 and D-LL-37; and demonstrates low cytoxicity against host cells but does not affect bacterial attachment. The peptides utilized in this anti-biofilm approach may provide templates for a new group of anti-microbials and potential future topical therapeutics for treating chronic wound infections.

CXCL10 Controls Inflammatory Pain via Opioid Peptide-Containing Macrophages in Electroacupuncture

PubMed Central

Wang, Ying; Gehringer, Rebekka; Mousa, Shaaban A.; Hackel, Dagmar; Brack, Alexander; Rittner, Heike L.

2014-01-01

Acupuncture is widely used for pain treatment in patients with osteoarthritis or low back pain, but molecular mechanisms remain largely enigmatic. In the early phase of inflammation neutrophilic chemokines direct opioid-containing neutrophils in the inflamed tissue and stimulate opioid peptide release and antinociception. In this study the molecular pathway and neuroimmune connections in complete Freund's adjuvant (CFA)-induced hind paw inflammation and electroacupuncture for peripheral pain control were analyzed. Free moving Wistar rats with hind paw inflammation were treated twice with electroacupuncture at GB30 (Huan Tiao - gall bladder meridian) (day 0 and 1) and analyzed for mechanical and thermal nociceptive thresholds. The cytokine profiles as well as the expression of opioid peptides were quantified in the inflamed paw. Electroacupuncture elicited long-term antinociception blocked by local injection of anti-opioid peptide antibodies (beta-endorphin, met-enkephalin, dynorphin A). The treatment altered the cytokine profile towards an anti-inflammatory pattern but augmented interferon (IFN)-gamma and the chemokine CXCL10 (IP-10: interferon gamma-inducible protein) protein and mRNA expression with concomitant increased numbers of opioid peptide-containing CXCR3+ macrophages. In rats with CFA hind paw inflammation without acupuncture repeated injection of CXCL10 triggered opioid-mediated antinociception and increase opioid-containing macrophages. Conversely, neutralization of CXCL10 time-dependently decreased electroacupuncture-induced antinociception and the number of infiltrating opioid peptide-expressing CXCR3+ macrophages. In summary, we describe a novel function of the chemokine CXCL10 - as a regulator for an increase of opioid-containing macrophages and antinociceptive mediator in inflammatory pain and as a key chemokine regulated by electroacupuncture. PMID:24732949

Association of a Model Transmembrane Peptide Containing Gly in a Heptad Sequence Motif

PubMed Central

Lear, James D.; Stouffer, Amanda L.; Gratkowski, Holly; Nanda, Vikas; DeGrado, William F.

2004-01-01

A peptide containing glycine at a and d positions of a heptad motif was synthesized to investigate the possibility that membrane-soluble peptides with a Gly-based, left-handed helical packing motif would associate. Based on analytical ultracentrifugation in C14-betaine detergent micelles, the peptide did associate in a monomer-dimer equilibrium, although the association constant was significantly less than that reported for the right-handed dimer of the glycophorin A transmembrane peptide in similar detergents. Fluorescence resonance energy transfer (FRET) experiments conducted on peptides labeled at their N-termini with either tetramethylrhodamine (TMR) or 7-nitrobenz-2-oxa-1,3-diazole (NBD) also indicated association. However, analysis of the FRET data using the usual assumption of complete quenching for NBD-TMR pairs in the dimer could not be quantitatively reconciled with the analytical ultracentrifugation-measured dimerization constant. This led us to develop a general treatment for the association of helices to either parallel or antiparallel structures of any aggregation state. Applying this treatment to the FRET data, constraining the dimerization constant to be within experimental uncertainty of that measured by analytical ultracentrifugation, we found the data could be well described by a monomer-dimer equilibrium with only partial quenching of the dimer, suggesting that the helices are most probably antiparallel. These results also suggest that a left-handed Gly heptad repeat motif can drive membrane helix association, but the affinity is likely to be less strong than the previously reported right-handed motif described for glycophorin A. PMID:15315956

Shortening of the Lactobacillus paracasei subsp. paracasei BGNJ1-64 AggLb Protein Switches Its Activity from Auto-aggregation to Biofilm Formation.

PubMed

Miljkovic, Marija; Bertani, Iris; Fira, Djordje; Jovcic, Branko; Novovic, Katarina; Venturi, Vittorio; Kojic, Milan

2016-01-01

AggLb is the largest (318.6 kDa) aggregation-promoting protein of Lactobacillus paracasei subsp. paracasei BGNJ1-64 responsible for forming large cell aggregates, which causes auto-aggregation, collagen binding and pathogen exclusion in vitro. It contains an N-terminus leader peptide, followed by six successive collagen binding domains, 20 successive repeats (CnaB-like domains) and an LPXTG sorting signal at the C-terminus for cell wall anchoring. Experimental information about the roles of the domains of AggLb is currently unknown. To define the domain that confers cell aggregation and the key domains for interactions of specific affinity between AggLb and components of the extracellular matrix, we constructed a series of variants of the aggLb gene and expressed them in Lactococcus lactis subsp. lactis BGKP1-20 using a lactococcal promoter. All of the variants contained a leader peptide, an inter collagen binding-CnaB domain region (used to raise an anti-AggLb antibody), an anchor domain and a different number of collagen binding and CnaB-like domains. The role of the collagen binding repeats of the N-terminus in auto-aggregation and binding to collagen and fibronectin was confirmed. Deletion of the collagen binding repeats II, III, and IV resulted in a loss of the strong auto-aggregation, collagen and fibronectin binding abilities whereas the biofilm formation capability was increased. The strong auto-aggregation, collagen and fibronectin binding abilities of AggLb were negatively correlated to biofilm formation.

Partial MHC/Neuroantigen Peptide Constructs: A Potential Neuroimmune-Based Treatment for Methamphetamine Addiction

PubMed Central

Loftis, Jennifer M.; Wilhelm, Clare J.; Vandenbark, Arthur A.; Huckans, Marilyn

2013-01-01

Relapse rates following current methamphetamine abuse treatments are very high (∼40–60%), and the neuropsychiatric impairments (e.g., cognitive deficits, mood disorders) that arise and persist during remission from methamphetamine addiction likely contribute to these high relapse rates. Pharmacotherapeutic development of medications to treat addiction has focused on neurotransmitter systems with only limited success, and there are no Food and Drug Administration approved pharmacotherapies for methamphetamine addiction. A growing literature shows that methamphetamine alters peripheral and central immune functions and that immune factors such as cytokines, chemokines, and adhesion molecules play a role in the development and persistence of methamphetamine induced neuronal injury and neuropsychiatric impairments. The objective of this study was to evaluate the efficacy of a new immunotherapy, partial MHC/neuroantigen peptide construct (RTL551; pI-Ab/mMOG-35-55), in treating learning and memory impairments induced by repeated methamphetamine exposure. C57BL/6J mice were exposed to two different methamphetamine treatment regimens (using repeated doses of 4 mg/kg or 10 mg/kg, s.c.). Cognitive performance was assessed using the Morris water maze and CNS cytokine levels were measured by multiplex assay. Immunotherapy with RTL551 improved the memory impairments induced by repeated methamphetamine exposure in both mouse models of chronic methamphetamine addiction. Treatment with RTL551 also attenuated the methamphetamine induced increases in hypothalamic interleukin-2 (IL-2) levels. Collectively, these initial results indicate that neuroimmune targeted therapies, and specifically RTL551, may have potential as treatments for methamphetamine-induced neuropsychiatric impairments. PMID:23460798

Predictors of variation in serum IGF1 and IGFBP3 levels in healthy African American and white men.

PubMed

Hoyo, Cathrine; Grubber, Janet; Demark-Wahnefried, Wendy; Lobaugh, Bruce; Jeffreys, Amy S; Grambow, Steven C; Marks, Jeffrey R; Keku, Temitope O; Walther, Phillip J; Schildkraut, Joellen M

2009-07-01

Individual variation in circulating insulinlike growth factor-1 (IGF1) and its major binding protein, insulinlike growth factor binding protein-3 (IGFBP3), have been etiologically linked to several chronic diseases, including some cancers. Factors associated with variation in circulating levels of these peptide hormones remain unclear. Multiple linear regression models were used to determine the extent to which sociodemographic characteristics, lifestyle factors, personal and family history of chronic disease, and common genetic variants, the (CA)n repeat polymorphism in the IGF1 promoter and the IGFBP3-202 A/C polymorphism (rs2854744) predict variation in IGF1 or IGFBP3 serum levels in 33 otherwise healthy African American and 37 white males recruited from Durham Veterans Administration Medical Center. Predictors of serum IGF1, IGFBP3, and the IGF1:IGFBP3 molar ratio varied by race. In African Americans, 17% and 28% of the variation in serum IGF1 and the IGF1:IGFBP3 molar ratio, were explained by cigarette smoking and carrying the IGF1 (CA)19 repeat allele, respectively. Not carrying at least 1 IGF1 (CA)19 repeat allele and a high body mass index explained 8% and 14%, respectively, of the variation IGFBP3 levels. These factors did not predict variation of these peptides in whites. If successfully replicated in larger studies, these findings would add to recent evidence, suggesting known genetic and lifestyle chronic disease risk factors influence IGF1 and IGFBP3 circulating levels differently in African Americans and whites.

Predictors of variation in serum IGFI and IGFBP3 levels in healthy African-American and white men

PubMed Central

Grubber, Janet; Demark-Wahnefried, Wendy; Lobaugh, Bruce; Jeffreys, Amy S.; Grambow, Steven C.; Marks, Jeffrey R.; Keku, Temitope O.; Walther, Phillip J.; Schildkraut, Joellen M.

2010-01-01

Background Individual variation in circulating insulin-like growth factor-I (IGF1) and its major binding protein, insulin-like growth factor binding protein-3 (IGFBP3) have been etiologically linked to several chronic diseases, including some cancers. Factors associated with variation in circulating levels of these peptide hormones remain unclear. Methods Multiple linear regression models were used to determine the extent to which socio-demographic characteristics, lifestyle factors, personal and family history of chronic disease, and common genetic variants, the (CA)n repeat polymorphism in the IGF1 promoter and the IGFBP3 -202 A/C polymorphism (rs2854744) predict variation in IGF1 or IGFBP3 serum levels in 33 otherwise healthy African American and 37 white males recruited from Durham Veterans Administration Medical Center. Results Predictors of serum IGF1, IGFBP3 and the IGF1:IGFBP3 molar ratio varied by race. In African Americans, 17% and 28% of the variation in serum IGF1 and the IGF1:IGFBP3 molar ratio, respectively, was explained by cigarette smoking and carrying the IGF1 (CA)19 repeat allele, respectively. Not carrying at least one IGF1 (CA)19 repeat allele and a high BMI explained 8% and 14%, respectively, of the variation IGFBP3 levels. These factors did not predict variation of these peptides in whites. Conclusion If successfully replicated in larger studies, these findings add to recent evidence suggesting known genetic and lifestyle chronic disease risk factors influence IGF1 and IGFBP3 circulating levels differently in African Americans and whites. PMID:19634593

Improved prediction of peptide detectability for targeted proteomics using a rank-based algorithm and organism-specific data.

PubMed

Qeli, Ermir; Omasits, Ulrich; Goetze, Sandra; Stekhoven, Daniel J; Frey, Juerg E; Basler, Konrad; Wollscheid, Bernd; Brunner, Erich; Ahrens, Christian H

2014-08-28

The in silico prediction of the best-observable "proteotypic" peptides in mass spectrometry-based workflows is a challenging problem. Being able to accurately predict such peptides would enable the informed selection of proteotypic peptides for targeted quantification of previously observed and non-observed proteins for any organism, with a significant impact for clinical proteomics and systems biology studies. Current prediction algorithms rely on physicochemical parameters in combination with positive and negative training sets to identify those peptide properties that most profoundly affect their general detectability. Here we present PeptideRank, an approach that uses learning to rank algorithm for peptide detectability prediction from shotgun proteomics data, and that eliminates the need to select a negative dataset for the training step. A large number of different peptide properties are used to train ranking models in order to predict a ranking of the best-observable peptides within a protein. Empirical evaluation with rank accuracy metrics showed that PeptideRank complements existing prediction algorithms. Our results indicate that the best performance is achieved when it is trained on organism-specific shotgun proteomics data, and that PeptideRank is most accurate for short to medium-sized and abundant proteins, without any loss in prediction accuracy for the important class of membrane proteins. Targeted proteomics approaches have been gaining a lot of momentum and hold immense potential for systems biology studies and clinical proteomics. However, since only very few complete proteomes have been reported to date, for a considerable fraction of a proteome there is no experimental proteomics evidence that would allow to guide the selection of the best-suited proteotypic peptides (PTPs), i.e. peptides that are specific to a given proteoform and that are repeatedly observed in a mass spectrometer. We describe a novel, rank-based approach for the prediction of the best-suited PTPs for targeted proteomics applications. By building on methods developed in the field of information retrieval (e.g. web search engines like Google's PageRank), we circumvent the delicate step of selecting positive and negative training sets and at the same time also more closely reflect the experimentalist´s need for selecting e.g. the 5 most promising peptides for targeting a protein of interest. This approach allows to predict PTPs for not yet observed proteins or for organisms without prior experimental proteomics data such as many non-model organisms. Copyright © 2014 Elsevier B.V. All rights reserved.

The Inclusion of Disability as a Condition for Termination of Parental Rights

ERIC Educational Resources Information Center

Lightfoot, Elizabeth; Hill, Katharine; LaLiberte, Traci

2010-01-01

Objectives: All 50 states and the District of Columbia have statutes outlining the grounds for terminating parental rights (TPR) in relation to child abuse and neglect. Although recent research has found that parents with disabilities are not more likely to maltreat their children than parents without disabilities ([Glaun and Brown, 1999] and…

Expansion for the Brachylophosaurus canadensis Collagen I Sequence and Additional Evidence of the Preservation of Cretaceous Protein.

PubMed

Schroeter, Elena R; DeHart, Caroline J; Cleland, Timothy P; Zheng, Wenxia; Thomas, Paul M; Kelleher, Neil L; Bern, Marshall; Schweitzer, Mary H

2017-02-03

Sequence data from biomolecules such as DNA and proteins, which provide critical information for evolutionary studies, have been assumed to be forever outside the reach of dinosaur paleontology. Proteins, which are predicted to have greater longevity than DNA, have been recovered from two nonavian dinosaurs, but these results remain controversial. For proteomic data derived from extinct Mesozoic organisms to reach their greatest potential for investigating questions of phylogeny and paleobiology, it must be shown that peptide sequences can be reliably and reproducibly obtained from fossils and that fragmentary sequences for ancient proteins can be increasingly expanded. To test the hypothesis that peptides can be repeatedly detected and validated from fossil tissues many millions of years old, we applied updated extraction methodology, high-resolution mass spectrometry, and bioinformatics analyses on a Brachylophosaurus canadensis specimen (MOR 2598) from which collagen I peptides were recovered in 2009. We recovered eight peptide sequences of collagen I: two identical to peptides recovered in 2009 and six new peptides. Phylogenetic analyses place the recovered sequences within basal archosauria. When only the new sequences are considered, B. canadensis is grouped more closely to crocodylians, but when all sequences (current and those reported in 2009) are analyzed, B. canadensis is placed more closely to basal birds. The data robustly support the hypothesis of an endogenous origin for these peptides, confirm the idea that peptides can survive in specimens tens of millions of years old, and bolster the validity of the 2009 study. Furthermore, the new data expand the coverage of B. canadensis collagen I (a 33.6% increase in collagen I alpha 1 and 116.7% in alpha 2). Finally, this study demonstrates the importance of reexamining previously studied specimens with updated methods and instrumentation, as we obtained roughly the same amount of sequence data as the previous study with substantially less sample material. Data are available via ProteomeXchange with identifier PXD005087.

Trimeric, Coiled-coil Extension on Peptide Fusion Inhibitor of HIV-1 Influences Selection of Resistance Pathways*

PubMed Central

Zhuang, Min; Wang, Wei; De Feo, Christopher J.; Vassell, Russell; Weiss, Carol D.

2012-01-01

Peptides corresponding to N- and C-terminal heptad repeat regions (HR1 and HR2, respectively) of viral fusion proteins can block infection of viruses in a dominant negative manner by interfering with refolding of the viral HR1 and HR2 to form a six-helix bundle (6HB) that drives fusion between viral and host cell membranes. The 6HB of the HIV gp41 (endogenous bundle) consists of an HR1 coiled-coil trimer with grooves lined by antiparallel HR2 helices. HR1 peptides form coiled-coil oligomers that may bind to gp41 HR2 as trimers to form a heterologous 6HB (inhibitor bundle) or to gp41 HR1 as monomers or dimers to form a heterologous coiled coil. To gain insights into mechanisms of Env entry and inhibition by HR1 peptides, we compared resistance to a peptide corresponding to 36 residues in gp41 HR1 (N36) and the same peptide with a coiled-coil trimerization domain fused to its N terminus (IZN36) that stabilizes the trimer and increases inhibitor potency (Eckert, D. M., and Kim, P. S. (2001) Proc. Nat. Acad. Sci. U.S.A. 98, 11187–11192). Whereas N36 selected two genetic pathways with equal probability, each defined by an early mutation in either HR1 or HR2, IZN36 preferentially selected the HR1 pathway. Both pathways conferred cross-resistance to both peptides. Each HR mutation enhanced the thermostability of the endogenous 6HB, potentially allowing the virus to simultaneously escape inhibitors targeting either gp41 HR1 or HR2. These findings inform inhibitor design and identify regions of plasticity in the highly conserved gp41 that modulate virus entry and escape from HR1 peptide inhibitors. PMID:22235115

[The predictive value of plasma B-type natriuretic peptide levels on outcome in children with pulmonary hypertension undergoing congenital heart surgery].

PubMed

Baysal, Ayse; Saşmazel, Ahmet; Yildirim, Ayse; Ozyaprak, Buket; Gundogus, Narin; Kocak, Tuncer

2014-01-01

In children undergoing congenital heart surgery, plasma brain natriuretic peptide levels may have a role in development of low cardiac output syndrome that is defined as a combination of clinical findings and interventions to augment cardiac output in children with pulmonary hypertension. In a prospective observational study, fifty-one children undergoing congenital heart surgery with preoperative echocardiographic study showing pulmonary hypertension were enrolled. The plasma brain natriuretic peptide levels were collected before operation, 12, 24 and 48h after operation. The patients enrolled into the study were divided into two groups depending on: (1) Development of LCOS which is defined as a combination of clinical findings or interventions to augment cardiac output postoperatively; (2) Determination of preoperative brain natriuretic peptide cut-off value by receiver operating curve analysis for low cardiac output syndrome. The secondary end points were: (1) duration of mechanical ventilation ≥72h, (2) intensive care unit stay >7days, and (3) mortality. The differences in preoperative and postoperative brain natriuretic peptide levels of patients with or without low cardiac output syndrome (n=35, n=16, respectively) showed significant differences in repeated measurement time points (p=0.0001). The preoperative brain natriuretic peptide cut-off value of 125.5pgmL-1 was found to have the highest sensitivity of 88.9% and specificity of 96.9% in predicting low cardiac output syndrome in patients with pulmonary hypertension. A good correlation was found between preoperative plasma brain natriuretic peptide level and duration of mechanical ventilation (r=0.67, p=0.0001). In patients with pulmonary hypertension undergoing congenital heart surgery, 91% of patients with preoperative plasma brain natriuretic peptide levels above 125.5pgmL-1 are at risk of developing low cardiac output syndrome which is an important postoperative outcome. Copyright © 2013 Sociedade Brasileira de Anestesiologia. Publicado por Elsevier Editora Ltda. All rights reserved.

The first armadillo repeat is involved in the recognition and regulation of beta-catenin phosphorylation by protein kinase CK1.

PubMed

Bustos, Victor H; Ferrarese, Anna; Venerando, Andrea; Marin, Oriano; Allende, Jorge E; Pinna, Lorenzo A

2006-12-26

Multiple phosphorylation of beta-catenin by glycogen synthase kinase 3 (GSK3) in the Wnt pathway is primed by CK1 through phosphorylation of Ser-45, which lacks a typical CK1 canonical sequence. Synthetic peptides encompassing amino acids 38-64 of beta-catenin are phosphorylated by CK1 on Ser-45 with low affinity (K(m) approximately 1 mM), whereas intact beta-catenin is phosphorylated at Ser-45 with very high affinity (K(m) approximately 200 nM). Peptides extended to include a putative CK1 docking motif (FXXXF) at 70-74 positions or a F74AA mutation in full-length beta-catenin had no significant effect on CK1 phosphorylation efficiency. beta-Catenin C-terminal deletion mutants up to residue 181 maintained their high affinity, whereas removal of the 131-181 fragment, corresponding to the first armadillo repeat, was deleterious, resulting in a 50-fold increase in K(m) value. Implication of the first armadillo repeat in beta-catenin targeting by CK1 is supported in that the Y142E mutation, which mimics phosphorylation of Tyr-142 by tyrosine kinases and promotes dissociation of beta-catenin from alpha-catenin, further improves CK1 phosphorylation efficiency, lowering the K(m) value to <50 nM, approximating the physiological concentration of beta-catenin. In contrast, alpha-catenin, which interacts with the N-terminal region of beta-catenin, prevents Ser-45 phosphorylation of CK1 in a dose-dependent manner. Our data show that the integrity of the N-terminal region and the first armadillo repeat are necessary and sufficient for high-affinity phosphorylation by CK1 of Ser-45. They also suggest that beta-catenin association with alpha-catenin and beta-catenin phosphorylation by CK1 at Ser-45 are mutually exclusive.

Preclinical safety evaluation of human platelets treated with antimicrobial peptides in severe combined immunodeficient mice.

PubMed

Bosch-Marcé, Marta; Mohan, Ketha V K; Gelderman, Monique P; Ryan, Patricia L; Russek-Cohen, Estelle; Atreya, Chintamani D

2014-03-01

Bacterial sepsis is a complication attributed to room temperature (RT)-stored platelets (PLTs) in transfusion medicine. Antimicrobial peptides (AMPs) are emerging as new therapeutic agents against microbes. We had previously demonstrated bactericidal activity of select synthetic AMPs against six types of bacteria in stored PLTs. In this report, we tested these AMPs for their potential antibody response and interference with the recovery and survival of human PLTs in an animal model. Two separate studies were conducted to evaluate the safety of the synthetic AMPs. 1) Two AMPs (PD3 and PD4), derived from thrombin-induced human PLT microbicidal protein, and four repeats of arginine-tryptophan (RW), containing two to five repeats (RW2-RW5), were tested in rabbits for potential antibody response. 2) RT-stored human PLTs treated for 2 hours with each of the six AMPs individually or with phosphate-buffered saline (PBS) alone were infused into severe combined immunodeficient (SCID) mice to evaluate their in vivo recovery and survival by flow cytometry. Except for PD3, which showed a weak immune response, all other peptides did not induce any detectable antibodies in rabbits. Furthermore, all six AMPs tested did not significantly affect the in vivo recovery and survival of human PLTs in SCID mice compared to PBS alone-treated PLTs. Preclinical evaluation studies reported here demonstrate that the selected AMPs used in the study did not adversely affect the human PLT recovery and survival in the SCID mouse model, suggesting further study of AMPs toward addressing the bacterial contamination of PLTs. Published 2013. This article is a U.S. Government work and is in the public domain in the USA.

Role of the Simian Virus 5 Fusion Protein N-Terminal Coiled-Coil Domain in Folding and Promotion of Membrane Fusion

PubMed Central

West, Dava S.; Sheehan, Michael S.; Segeleon, Patrick K.; Dutch, Rebecca Ellis

2005-01-01

Formation of a six-helix bundle comprised of three C-terminal heptad repeat regions in antiparallel orientation in the grooves of an N-terminal coiled-coil is critical for promotion of membrane fusion by paramyxovirus fusion (F) proteins. We have examined the effect of mutations in four residues of the N-terminal heptad repeat in the simian virus 5 (SV5) F protein on protein folding, transport, and fusogenic activity. The residues chosen have previously been shown from study of isolated peptides to have differing effects on stability of the N-terminal coiled-coil and six-helix bundle (R. E. Dutch, G. P. Leser, and R. A. Lamb, Virology 254:147-159, 1999). The mutant V154M showed reduced proteolytic cleavage and surface expression, indicating a defect in intracellular transport, though this mutation had no effect when studied in isolated peptides. The mutation I137M, previously shown to lower thermostability of the six-helix bundle, resulted in an F protein which was properly processed and transported to the cell surface but which had reduced fusogenic activity. Finally, mutations at L140M and L161M, previously shown to disrupt α-helix formation of isolated N-1 peptides but not to affect six-helix bundle formation, resulted in F proteins that were properly processed. Interestingly, the L161M mutant showed increased syncytium formation and promoted fusion at lower temperatures than the wild-type F protein. These results indicate that interactions separate from formation of an N-terminal coiled-coil or six-helix bundle are important in the initial folding and transport of the SV5 F protein and that mutations that destabilize the N-terminal coiled-coil can result in stimulation of membrane fusion. PMID:15650180

Strong Electro‐Optic Effect and Spontaneous Domain Formation in Self‐Assembled Peptide Structures

PubMed Central

Lafargue, Clément; Handelman, Amir; Shimon, Linda J. W.; Rosenman, Gil; Zyss, Joseph

2017-01-01

Short peptides made from repeating units of phenylalanine self‐assemble into a remarkable variety of micro‐ and nanostructures including tubes, tapes, spheres, and fibrils. These bio‐organic structures are found to possess striking mechanical, electrical, and optical properties, which are rarely seen in organic materials, and are therefore shown useful for diverse applications including regenerative medicine, targeted drug delivery, and biocompatible fluorescent probes. Consequently, finding new optical properties in these materials can significantly advance their practical use, for example, by allowing new ways to visualize, manipulate, and utilize them in new, in vivo, sensing applications. Here, by leveraging a unique electro‐optic phase microscopy technique, combined with traditional structural analysis, it is measured in di‐ and triphenylalanine peptide structures a surprisingly large electro‐optic response of the same order as the best performing inorganic crystals. In addition, spontaneous domain formation is observed in triphenylalanine tapes, and the origin of their electro‐optic activity is unveiled to be related to a porous triclinic structure, with extensive antiparallel beta‐sheet arrangement. The strong electro‐optic response of these porous peptide structures with the capability of hosting guest molecules opens the door to create new biocompatible, environmental friendly functional materials for electro‐optic applications, including biomedical imaging, sensing, and optical manipulation. PMID:28932664

Two M-T hook residues greatly improve the antiviral activity and resistance profile of the HIV-1 fusion inhibitor SC29EK

PubMed Central

2014-01-01

Background Peptides derived from the C-terminal heptad repeat (CHR) of HIV-1 gp41 such as T20 (Enfuvirtide) and C34 are potent viral fusion inhibitors. We have recently found that two N-terminal residues (Met115 and Thr116) of CHR peptides form a unique M-T hook structure that can greatly enhance the binding and anti-HIV activity of inhibitors. Here, we applied two M-T hook residues to optimize SC29EK, an electrostatically constrained peptide inhibitor with a potent anti-HIV activity. Results The resulting peptide MT-SC29EK showed a dramatically increased binding affinity and could block the six-helical bundle (6-HB) formation more efficiently. As expected, MT-SC29EK potently inhibited HIV-1 entry and infection, especially against those T20- and SC29EK-resistant HIV-1 variants. More importantly, MT-SC29EK and its short form (MT-SC22EK) suffered from the difficulty to induce HIV-1 resistance during the in vitro selection, suggesting their high genetic barriers to the development of resistance. Conclusions Our studies have verified the M-T hook structure as a vital strategy to design novel HIV-1 fusion inhibitors and offered an ideal candidate for clinical development. PMID:24884671

Two neuropeptides colocalized in a command-like neuron use distinct mechanisms to enhance its fast synaptic connection.

PubMed

Koh, H Y; Vilim, F S; Jing, J; Weiss, K R

2003-09-01

In many neurons more than one peptide is colocalized with a classical neurotransmitter. The functional consequence of such an arrangement has been rarely investigated. Here, within the feeding circuit of Aplysia, we investigate at a single synapse the actions of two modulatory neuropeptides that are present in a cholinergic interneuron. In combination with previous work, our study shows that the command-like neuron for feeding, CBI-2, contains two neuropeptides, feeding circuit activating peptide (FCAP) and cerebral peptide 2 (CP2). Previous studies showed that high-frequency prestimulation or repeated stimulation of CBI-2 increases the size of CBI-2 to B61/62 excitatory postsynaptic potentials (EPSPs) and shortens the latency of firing of neuron B61/62 in response to CBI-2 stimulation. We find that both FCAP and CP2 mimic these two effects. The variance method of quantal analysis indicates that FCAP increases the calculated quantal size (q) and CP2 increases the calculated quantal content (m) of EPSPs. Since the PSP amplitude represents the product of q and m, the joint action of the two peptides is expected to be cooperative. This observation suggests a possible functional implication for multiple neuropeptides colocalized with a classical neurotransmitter in one neuron.

Strategies for the construction and use of peptide and antibody libraries displayed on phages.

PubMed

Pini, Alessandro; Giuliani, Andrea; Ricci, Claudia; Runci, Ylenia; Bracci, Luisa

2004-12-01

Combinatorial chemistry and biology have become popular methods for the identification of bio-active molecules in drug discovery. A widely used technique in combinatorial biology is "phage display", by which peptides, antibody fragments and enzymes are displayed on the surface of bacteriophages, and can be selected by simple procedures of biopanning. The construction of phage libraries of peptides or antibody fragments provides a huge source of ligands and bio-active molecules that can be isolated from the library without laborious studies on antigen characteristics and prediction of ligand structure. This "irrational" approach for the construction of new drugs is extremely rapid and is now used by thousands of laboratories world-wide. The bottleneck in this procedure is the availability of large reliable libraries that can be used repeatedly over the years without loss of ligand expression and diversity. Construction of personalized libraries is therefore important for public and private laboratories engaged in the isolation of specific molecules for therapeutic or diagnostic use. Here we report the general strategies for constructing large phage peptide and antibody libraries, based on the experience of researchers who built the world's most widely used libraries. Particular attention is paid to advanced strategies for the construction, preservation and panning.

Interaction of Huntingtin Exon-1 Peptides with Lipid-Based Micellar Nanoparticles Probed by Solution NMR and Q-Band Pulsed EPR.

PubMed

Ceccon, Alberto; Schmidt, Thomas; Tugarinov, Vitali; Kotler, Samuel A; Schwieters, Charles D; Clore, G Marius

2018-05-23

Lipid-based micellar nanoparticles promote aggregation of huntingtin exon-1 peptides. Here we characterize the interaction of two such peptides, htt NT Q  7 and htt NT Q  10 comprising the N-terminal amphiphilic domain of huntingtin followed by 7 and 10 glutamine repeats, respectively, with 8 nm lipid micelles using NMR chemical exchange saturation transfer (CEST), circular dichroism and pulsed Q-band EPR. Exchange between free and micelle-bound htt NT Q  n peptides occurs on the millisecond time scale with a K D ∼ 0.5-1 mM. Upon binding micelles, residues 1-15 adopt a helical conformation. Oxidation of Met 7 to a sulfoxide reduces the binding affinity for micelles ∼3-4-fold and increases the length of the helix by a further two residues. A structure of the bound monomer unit is calculated from the backbone chemical shifts of the micelle-bound state obtained from CEST. Pulsed Q-band EPR shows that a monomer-dimer equilibrium exists on the surface of the micelles and that the two helices of the dimer adopt a parallel orientation, thereby bringing two disordered polyQ tails into close proximity which may promote aggregation upon dissociation from the micelle surface.

Adenovirus Particles that Display the Plasmodium falciparum Circumsporozoite Protein NANP Repeat Induce Sporozoite-Neutralizing Antibodies in Mice

PubMed Central

Palma, Christopher; Overstreet, Michael G.; Guedon, Jean-Marc; Hoiczyk, Egbert; Ward, Cameron; Karen, Kasey A.; Zavala, Fidel; Ketner, Gary

2011-01-01

Adenovirus particles can be engineered to display exogenous peptides on their surfaces by modification of viral capsid proteins, and particles that display pathogen-derived peptides can induce protective immunity. We constructed viable recombinant adenoviruses that display B-cell epitopes from the Plasmodium falciparum circumsporozoite protein (PfCSP) in the major adenovirus capsid protein, hexon. Recombinants induced high-titer antibodies against CSP when injected intraperitoneally into mice. Serum obtained from immunized mice recognized both recombinant PfCSP protein and P. falciparum sporozoites, and neutralized P. falciparum sporozoites in vitro. Replicating adenovirus vaccines have provided economical protection against adenovirus disease for over three decades. The recombinants described here may provide a path to an affordable malaria vaccine in the developing world. PMID:21199707

Long-distance transport of signals during symbiosis

PubMed Central

Xie, Zhi-Ping; Illana, Antonio

2011-01-01

Legumes enter nodule symbioses with nitrogen-fixing bacteria (rhizobia), whereas most flowering plants establish symbiotic associations with arbuscular mycorrhizal (AM) fungi. Once first steps of symbiosis are initiated, nodule formation and mycorrhization in legumes is negatively controlled by a shoot-derived inhibitor (SDI), a phenomenon termed autoregulation. According to current views, autoregulation of nodulation and mycorrhization in legumes is regulated in a similar way. CLE peptides induced in response to rhizobial nodulation signals (Nod factors) have been proposed to represent the ascending long-distance signals to the shoot. Although not proven yet, these CLE peptides are likely perceived by leucine-rich repeat (LRR) autoregulation receptor kinases in the shoot. Autoregulation of mycorrhization in non-legumes is reminiscent to the phenomenon of “systemic acquired resistance” in plant-pathogen interactions. PMID:21455020

Insulin Mimetic Peptide Disrupts the Primary Binding Site of the Insulin Receptor*

PubMed Central

Lawrence, Callum F.; Margetts, Mai B.; Menting, John G.; Smith, Nicholas A.; Smith, Brian J.; Ward, Colin W.; Lawrence, Michael C.

2016-01-01

Sets of synthetic peptides that interact with the insulin receptor ectodomain have been discovered by phage display and reported in the literature. These peptides were grouped into three classes termed Site 1, Site 2, and Site 3 based on their mutual competition of binding to the receptor. Further refinement has yielded, in particular, a 36-residue Site 2-Site 1 fusion peptide, S519, that binds the insulin receptor with subnanomolar affinity and exhibits agonist activity in both lipogenesis and glucose uptake assays. Here, we report three-dimensional crystallographic detail of the interaction of the C-terminal, 16-residue Site 1 component (S519C16) of S519 with the first leucine-rich repeat domain (L1) of the insulin receptor. Our structure shows that S519C16 binds to the same site on the L1 surface as that occupied by a critical component of the primary binding site, namely the helical C-terminal segment of the insulin receptor α-chain (termed αCT). In particular, the two phenylalanine residues within the FYXWF motif of S519C16 are seen to engage the insulin receptor L1 domain surface in a fashion almost identical to the respective αCT residues Phe701 and Phe705. The structure provides a platform for the further development of peptidic and/or small molecule agents directed toward the insulin receptor and/or the type 1 insulin-like growth factor receptor. PMID:27281820

Crystal Structure of a Bacterial Signal Peptide Peptidase

DOE Office of Scientific and Technical Information (OSTI.GOV)

Kim,A.; Oliver, D.; Paetzel, M.

2008-01-01

Signal peptide peptidase (Spp) is the enzyme responsible for cleaving the remnant signal peptides left behind in the membrane following Sec-dependent protein secretion. Spp activity appears to be present in all cell types, eukaryotic, prokaryotic and archaeal. Here we report the first structure of a signal peptide peptidase, that of the Escherichia coli SppA (SppAEC). SppAEC forms a tetrameric assembly with a novel bowl-shaped architecture. The bowl has a dramatically hydrophobic interior and contains four separate active sites that utilize a Ser/Lys catalytic dyad mechanism. Our structural analysis of SppA reveals that while in many Gram-negative bacteria as well asmore » characterized plant variants, a tandem duplication in the protein fold creates an intact active site at the interface between the repeated domains, other species, particularly Gram-positive and archaeal organisms, encode half-size, unduplicated SppA variants that could form similar oligomers to their duplicated counterparts, but using an octamer arrangement and with the catalytic residues provided by neighboring monomers. The structure reveals a similarity in the protein fold between the domains in the periplasmic Ser/Lys protease SppA and the monomers seen in the cytoplasmic Ser/His/Asp protease ClpP. We propose that SppA may, in addition to its role in signal peptide hydrolysis, have a role in the quality assurance of periplasmic and membrane-bound proteins, similar to the role that ClpP plays for cytoplasmic proteins.« less

Identical IgM antibodies recognizing a glycine-alanine epitope are induced during acute infection with Epstein-Barr virus and cytomegalovirus.

PubMed

Rhodes, G; Smith, R S; Rubin, R E; Vaughan, J; Horwitz, C A

1990-01-01

We studied antibody production in serial serum samples from patients with acute Epstein-Barr virus (EBV) and cytomegalovirus (CMV) infections. Sera were analyzed both by enzyme-linked immunosorbent assay (ELISA) using a synthetic peptide (P62) derived from the glycine-alanine repeating region of the Epstein-Barr nuclear antigen (EBNA-1) and by immunoblotting. In prior studies, we have shown that patients with acute EBV infection make IgM antibodies that react with this peptide, that recognize a viral-specific protein (EBNA-1), and that bind with a number of proteins present in uninfected cells; however, antibody binding to these autoantigens was inhibited by the peptide. IgG antibodies reactive with the peptide did not appear until months after the disease and were specific for the EBNA-1 protein. We now find that patients with acute CMV infection but not those with acute infections from a variety of other nonherpes organisms also produce IgM antibodies that recognize the EBV-derived peptide P62. These antibodies also appear to recognize the same cellular proteins as the EBV-induced IgM antibodies. The IgM antibodies appeared in all acutely infected CMV patients studied and occurred both in patients with previous EBV infections and in one patient studied who had not previously been exposed to EBV. It appears that infection with EBV or CMV can induce the synthesis of a very similar or identical set of IgM antibodies.

Immune Responses and Protection of Aotus Monkeys Immunized with Irradiated Plasmodium vivax Sporozoites

DTIC Science & Technology

2011-01-01

The experimental protocol was approved by the Animal Ethical Committee of the Universidad del Valle (Cali). Parasite and irradiation. Plasmodium...three repeats ofpll (GDRADGPA) and (ANGAGNQPG) sequences, derived from VK210 and VK247 CS variants, respectively. A non-malaria related peptide Ptt30...Universidad del Valle and Centro Internacional de Vacunas, Cali, Colombia, E-mails: alejovi@hotmail.com, anbonelo@yahoo.com, alejcaste@yahoo.com

Mussel glue protein has an open conformation.

PubMed

Williams, T; Marumo, K; Waite, J H; Henkens, R W

1989-03-01

Both native glue protein from marine mussels and a synthetic nonhydroxylated analog were analyzed by far-uv CD under a variety of conditions. Analysis of the CD spectra using various models strongly suggest a primarily random coil structure for both forms of the protein, a fact also supported by the absence of spectral change for the glue protein upon dilution into 6 M guanidine hydrochloride. The nonhydroxylated analog, which consists of 20 repeats of the peptide sequence Ala-Lys-Pro-Ser-Tyr-Pro-Pro-Thr-Tyr-Lys, was further characterized by enzyme modification using mushroom tyrosinase. Enzymatic hydroxylation of tyrosines was found to be best fit by a model containing two rate constants, 5.6 (+/- 0.6) X 10(-3) and 7.2 (+/- 0.3) X 10(-2) min-1. At equilibrium, HPLC analysis of digests showed nearly 100% conversion of Tyr-9 and only 15 to 35% conversion of Tyr-5. The Chou and Fasman rules for predicting structure were applied to the repeat sequence listed above. The rules predict the absence of alpha helix and beta pleated sheets in the structure of this peptide. On the other hand, beta turns are predicted to be present with Tyr-5 being in the region of highest probability. These data suggest that the protein in solution has only a small amount of secondary structure.

DOE Office of Scientific and Technical Information (OSTI.GOV)

Chang, Yanqi; Levy, Dan; Horton, John R.

SET domain containing 6 (SETD6) monomethylates the RelA subunit of nuclear factor kappa B (NF-{kappa}B). The ankyrin repeats of G9a-like protein (GLP) recognizes RelA monomethylated at Lys310. Adjacent to Lys310 is Ser311, a known phosphorylation site of RelA. Ser311 phosphorylation inhibits Lys310 methylation by SETD6 as well as binding of Lys310me1 by GLP. The structure of SETD6 in complex with RelA peptide containing the methylation site, in the presence of S-adenosyl-l-methionine, reveals a V-like protein structure and suggests a model for NF-{kappa}B binding to SETD6. In addition, structural modeling of the GLP ankyrin repeats bound to Lys310me1 peptide provides insightmore » into the molecular basis for inhibition of Lys310me1 binding by Ser311 phosphorylation. Together, these findings provide a structural explanation for a key cellular signaling pathway centered on RelA Lys310 methylation, which is generated by SETD6 and recognized by GLP, and incorporate a methylation-phosphorylation switch of adjacent lysine and serine residues. Finally, SETD6 is structurally similar to the Rubisco large subunit methyltransferase. Given the restriction of Rubisco to plant species, this particular appearance of the protein lysine methyltransferase has been evolutionarily well conserved.« less

Genetic determinants of mate recognition in Brachionus manjavacas (Rotifera)

PubMed Central

Snell, Terry W; Shearer, Tonya L; Smith, Hilary A; Kubanek, Julia; Gribble, Kristin E; Welch, David B Mark

2009-01-01

Background Mate choice is of central importance to most animals, influencing population structure, speciation, and ultimately the survival of a species. Mating behavior of male brachionid rotifers is triggered by the product of a chemosensory gene, a glycoprotein on the body surface of females called the mate recognition pheromone. The mate recognition pheromone has been biochemically characterized, but little was known about the gene(s). We describe the isolation and characterization of the mate recognition pheromone gene through protein purification, N-terminal amino acid sequence determination, identification of the mate recognition pheromone gene from a cDNA library, sequencing, and RNAi knockdown to confirm the functional role of the mate recognition pheromone gene in rotifer mating. Results A 29 kD protein capable of eliciting rotifer male circling was isolated by high-performance liquid chromatography. Two transcript types containing the N-terminal sequence were identified in a cDNA library; further characterization by screening a genomic library and by polymerase chain reaction revealed two genes belonging to each type. Each gene begins with a signal peptide region followed by nearly perfect repeats of an 87 to 92 codon motif with no codons between repeats and the final motif prematurely terminated by the stop codon. The two Type A genes contain four and seven repeats and the two Type B genes contain three and five repeats, respectively. Only the Type B gene with three repeats encodes a peptide with a molecular weight of 29 kD. Each repeat of the Type B gene products contains three asparagines as potential sites for N-glycosylation; there are no asparagines in the Type A genes. RNAi with Type A double-stranded RNA did not result in less circling than in the phosphate-buffered saline control, but transfection with Type B double-stranded RNA significantly reduced male circling by 17%. The very low divergence between repeat units, even at synonymous positions, suggests that the repeats are kept nearly identical through a process of concerted evolution. Information-rich molecules like surface glycoproteins are well adapted for chemical communication and aquatic animals may have evolved signaling systems based on these compounds, whereas insects use cuticular hydrocarbons. Conclusion Owing to its critical role in mating, the mate recognition pheromone gene will be a useful molecular marker for exploring the mechanisms and rates of selection and the evolution of reproductive isolation and speciation using rotifers as a model system. The phylogenetic variation in the mate recognition pheromone gene can now be studied in conjunction with the large amount of ecological and population genetic data being gathered for the Brachionus plicatilis species complex to understand better the evolutionary drivers of cryptic speciation. PMID:19740420

Genetic determinants of mate recognition in Brachionus manjavacas (Rotifera).

PubMed

Snell, Terry W; Shearer, Tonya L; Smith, Hilary A; Kubanek, Julia; Gribble, Kristin E; Welch, David B Mark

2009-09-09

Mate choice is of central importance to most animals, influencing population structure, speciation, and ultimately the survival of a species. Mating behavior of male brachionid rotifers is triggered by the product of a chemosensory gene, a glycoprotein on the body surface of females called the mate recognition pheromone. The mate recognition pheromone has been biochemically characterized, but little was known about the gene(s). We describe the isolation and characterization of the mate recognition pheromone gene through protein purification, N-terminal amino acid sequence determination, identification of the mate recognition pheromone gene from a cDNA library, sequencing, and RNAi knockdown to confirm the functional role of the mate recognition pheromone gene in rotifer mating. A 29 kD protein capable of eliciting rotifer male circling was isolated by high-performance liquid chromatography. Two transcript types containing the N-terminal sequence were identified in a cDNA library; further characterization by screening a genomic library and by polymerase chain reaction revealed two genes belonging to each type. Each gene begins with a signal peptide region followed by nearly perfect repeats of an 87 to 92 codon motif with no codons between repeats and the final motif prematurely terminated by the stop codon. The two Type A genes contain four and seven repeats and the two Type B genes contain three and five repeats, respectively. Only the Type B gene with three repeats encodes a peptide with a molecular weight of 29 kD. Each repeat of the Type B gene products contains three asparagines as potential sites for N-glycosylation; there are no asparagines in the Type A genes. RNAi with Type A double-stranded RNA did not result in less circling than in the phosphate-buffered saline control, but transfection with Type B double-stranded RNA significantly reduced male circling by 17%. The very low divergence between repeat units, even at synonymous positions, suggests that the repeats are kept nearly identical through a process of concerted evolution. Information-rich molecules like surface glycoproteins are well adapted for chemical communication and aquatic animals may have evolved signaling systems based on these compounds, whereas insects use cuticular hydrocarbons. Owing to its critical role in mating, the mate recognition pheromone gene will be a useful molecular marker for exploring the mechanisms and rates of selection and the evolution of reproductive isolation and speciation using rotifers as a model system. The phylogenetic variation in the mate recognition pheromone gene can now be studied in conjunction with the large amount of ecological and population genetic data being gathered for the Brachionus plicatilis species complex to understand better the evolutionary drivers of cryptic speciation.

Major antigenic determinants of F and ColB2 pili.

PubMed Central

Finlay, B B; Frost, L S; Paranchych, W; Parker, J M; Hodges, R S

1985-01-01

F-like conjugative pili are expressed by plasmids with closely related transfer systems. They are tubular filaments that are composed of repeating pilin subunits arranged in a helical array. Both F and ColB2 pilin have nearly identical protein sequences, and both contain an acetylated amino-terminal alanine residue. However, they differ by a few amino acid residues at their amino termini. Rabbit antisera raised against purified F and ColB2 pili are immunologically cross-reactive by only 25%, as measured by a competition enzyme-linked immunosorbent assay (ELISA). A tryptic peptide corresponding to the first 15 amino acid residues of ColB2 pilin was isolated and found to remove nearly 80% of ColB2 pilus-directed rabbit antibodies. The corresponding tryptic peptide from F pilin, which reacted with anti-F pilus antibodies to remove 80%, was less than 20% reactive with anti-ColB2 pilus antiserum. Cleavage of these peptides with cyanogen bromide (at a methionine residue approximately in the middle of the peptide) did not affect the antigenicity of these peptides. Synthetic N alpha-acetylated peptides corresponding to the first eight amino acids of F pilin (Ac-Ala-Gly-Ser-Ser-Gly-Gln-Asp-Leu-COOH) and the first six amino acids of ColB2 pilin (Ac-Ala-Gln-Gly-Gln-Asp-Leu-COOH) were prepared and tested by competition ELISA with homologous and heterologous anti-pilus antisera. The F peptide F(1-8) inhibited the interaction of F pili and anti-F pilus antiserum to 80%, while the ColB2 peptide ColB2(1-6) inhibited anti-ColB2 pilus antiserum reacting with ColB2 pili by greater than 60%. The two peptides F(1-8) and ColB2(1-6) were inactive by competition ELISAs with heterologous antisera. These results suggest that the major antigenic determinant of both F and ColB2 pili is at the amino terminus of the pilin subunit and that 80% of antibodies raised against these pili are specific for this region of the pilin molecule. PMID:2409073

Radioimmunotherapy of MCF7 breast cancer cell line with 131I-PR81 monoclonal antibody against MUC1: comparison of direct and indirect radioiodination methods.

PubMed

Mohammadnejad, Javad; Rasaee, Mohammad Javad; Babaei, Mohammad Hosein; Paknejad, Malihe; Hasan, Zahir Mohammad; Salouti, Mojtaba; Gandomkar, Mostafa; Sadri, Keyvan

2010-01-01

PR81 is a monoclonal antibody that binds with high affinity to MUC1, which is over expressed on breast and other tumors. The objective of this study was to compare the two labeling methods (direct and indirect radioiodination) for application of this antibody against MUC1 as a radioimmunotherapeutical agent.Monoclonal antibody (PR81) against the tandem repeat of the core protein (MUC1) was prepared, characterized, purified, and labeled with 131I using the direct (chloramin-T) and indirect (Fmoc-D-Tyr (tBu)-D-Tyr (tBu)-D-Lys (Boc)-OH (YYK) attached to N-hydroxysuccinimide as a linker between PR81 and 131I) methods. The immunoreactivity of 131I-PR81 and 131I-TP-PR81 complexes with MUC1 (the native protein), BSA-P20 (a 20 amino acid corresponding the tandem repeat of MUC1) and MCF7 cell line were performed by RIA. In vitro stability of 131I-PR81 and 131I-YYK-peptide-PR81 complexes in human serum was determined by thin layer chromatography (TLC). Cell toxicity and in vitro internalization studies were performed with the MCF7 cell line, and the tissue biodistribution of the 131I-PR81 and 131I- YYK-peptide -PR81 complexes was evaluated in normal BALB/c mice at 4, 24 and 48 hrs. The labeling efficiency was determined by measuring the percentage recovery of radioactivity in the final product relative to the initial activity in the shipment vial, was found to be 59.9% +/- 7.9% for direct and 50% +/- 3.2% for indirect methods. 131I-PR81 and 131I- YYK- peptide -PR81 complexes showed high immunoreactivity towards MUC1 protein, BSA-P20 and MCF7 cell line. In vitro stability of the labeled products in human serum which was measured by thin layer chromatography (TLC) was found to be more than 50% over 24 hr for 131I-PR81 and 70% for 131I- YYK-peptide -PR81 complexes. Cell toxicity and in vitro internalization studies showed that the 131I-PR81 and 131I- YYK-peptide -PR81 complexes inhibited 80% growth of the MCF7 cultured cell lines in vitro in a high concentration and up to 40% of the 131I-PR81 and 60% of the 131I- YYK-peptide -PR81 complexes internalized after 24 h. Biodistribution studies were performed in normal BALB/c mice at 4, 24 and 48 hrs post-injection. Thyroid and stomach levels from PR81 labeled with 131I- YYK-peptide were two- to three- fold less than those with directly labeled 131I-PR81, suggesting low recognition of its D-iodotyrosine residue by endogenous deiodinase. These results show that the indirect labeling was better than the indirect labeling and 131I- YYK-peptide -PR81 may be considered as a promising candidate for therapy of breast cancer.

Attitudes of Teachers of Arabic as a Foreign Language toward Methods of Foreign Language Teaching

ERIC Educational Resources Information Center

Seraj, Sami A.

2010-01-01

This study examined the attitude of teachers of Arabic as a foreign language toward some of the most well known teaching methods. For this reason the following eight methods were selected: (1) the Grammar-Translation Method (GTM), (2) the Direct Method (DM), (3) the Audio-Lingual Method (ALM), (4) Total Physical Response (TPR), (5) Community…

76 FR 45638 - Self-Regulatory Organizations; Fixed Income Clearing Corporation; Notice of Filing of Proposed...

Federal Register 2010, 2011, 2012, 2013, 2014

2011-07-29

... the TPR, an industry group formed and sponsored by the Federal Reserve Bank of New York.\\5\\ Because... that GSCC would likely have unbalanced net GCF securities and cash positions within each clearing bank... clearing bank will be net funds borrowers, while the dealers at the other clearing bank will be net funds...

Silver-palladium catalysts for the direct synthesis of hydrogen peroxide

NASA Astrophysics Data System (ADS)

Khan, Zainab; Dummer, Nicholas F.; Edwards, Jennifer K.

2017-11-01

A series of bimetallic silver-palladium catalysts supported on titania were prepared by wet impregnation and assessed for the direct synthesis of hydrogen peroxide, and its subsequent side reactions. The addition of silver to a palladium catalyst was found to significantly decrease hydrogen peroxide productivity and hydrogenation, but crucially increase the rate of decomposition. The decomposition product, which is predominantly hydroxyl radicals, can be used to decrease bacterial colonies. The interaction between silver and palladium was characterized using scanning electron microscopy, X-ray diffraction, X-ray photoelectron spectroscopy (XPS) and temperature programmed reduction (TPR). The results of the TPR and XPS indicated the formation of a silver-palladium alloy. The optimal 1% Ag-4% Pd/TiO2 bimetallic catalyst was able to produce approximately 200 ppm of H2O2 in 30 min. The findings demonstrate that AgPd/TiO2 catalysts are active for the synthesis of hydrogen peroxide and its subsequent decomposition to reactive oxygen species. The catalysts are promising for use in wastewater treatment as they combine the disinfectant properties of silver, hydrogen peroxide production and subsequent decomposition. This article is part of a discussion meeting issue 'Providing sustainable catalytic solutions for a rapidly changing world'.

Solid solutions of gadolinium doped zinc oxide nanorods by combined microwave-ultrasonic irradiation assisted crystallization

NASA Astrophysics Data System (ADS)

Kiani, Armin; Dastafkan, Kamran; Obeydavi, Ali; Rahimi, Mohammad

2017-12-01

Nanocrystalline solid solutions consisting of un-doped and gadolinium doped zinc oxide nanorods were fabricated by a modified sol-gel process utilizing combined ultrasonic-microwave irradiations. Polyvinylpyrrolidone, diethylene glycol, and triethylenetetramine respectively as capping, structure directing, and complexing agents were used under ultrasound dynamic aging and microwave heating to obtain crystalline nanorods. Crystalline phase monitoring, lattice parameters and variation, morphology and shape, elemental analysis, functional groups, reducibility, and the oxidation state of emerged species were examined by PXRD, FESEM, TEM, EDX, FTIR, micro Raman, H2-TPR, and EPR techniques. Results have verified that irradiation mechanism of gelation and crystallization reduces the reaction time, augments the crystal quality, and formation of hexagonal close pack structure of Wurtzite morphology. Besides, dissolution of gadolinium within host lattice involves lattice deformation, unit cell distortion, and angular position variation. Structure related shape and growth along with compositional purity were observed through microscopic and spectroscopic surveys. Furthermore, TPR and EPR studies elucidated more detailed behavior upon exposure to the exerted irradiations and subsequent air-annealing including the formed oxidation states and electron trapping centers, presence of gadolinium, zinc, and oxygen disarrays and defects, as well as alteration in the host unit cell via gadolinium addition.

Regulation of RNA-binding proteins affinity to export receptors enables the nuclear basket proteins to distinguish and retain aberrant mRNAs

PubMed Central

Soheilypour, M.; Mofrad, M. R. K.

2016-01-01

Export of messenger ribonucleic acids (mRNAs) into the cytoplasm is a fundamental step in gene regulation processes, which is meticulously quality controlled by highly efficient mechanisms in eukaryotic cells. Yet, it remains unclear how the aberrant mRNAs are recognized and retained inside the nucleus. Using a new modelling approach for complex systems, namely the agent-based modelling (ABM) approach, we develop a minimal model of the mRNA quality control (QC) mechanism. Our results demonstrate that regulation of the affinity of RNA-binding proteins (RBPs) to export receptors along with the weak interaction between the nuclear basket protein (Mlp1 or Tpr) and RBPs are the minimum requirements to distinguish and retain aberrant mRNAs. Our results show that the affinity between Tpr and RBPs is optimized to maximize the retention of aberrant mRNAs. In addition, we demonstrate how the length of mRNA affects the QC process. Since longer mRNAs spend more time in the nuclear basket to form a compact conformation and initiate their export, nuclear basket proteins could more easily capture and retain them inside the nucleus. PMID:27805000

Regulation of RNA-binding proteins affinity to export receptors enables the nuclear basket proteins to distinguish and retain aberrant mRNAs.

PubMed

Soheilypour, M; Mofrad, M R K

2016-11-02

Export of messenger ribonucleic acids (mRNAs) into the cytoplasm is a fundamental step in gene regulation processes, which is meticulously quality controlled by highly efficient mechanisms in eukaryotic cells. Yet, it remains unclear how the aberrant mRNAs are recognized and retained inside the nucleus. Using a new modelling approach for complex systems, namely the agent-based modelling (ABM) approach, we develop a minimal model of the mRNA quality control (QC) mechanism. Our results demonstrate that regulation of the affinity of RNA-binding proteins (RBPs) to export receptors along with the weak interaction between the nuclear basket protein (Mlp1 or Tpr) and RBPs are the minimum requirements to distinguish and retain aberrant mRNAs. Our results show that the affinity between Tpr and RBPs is optimized to maximize the retention of aberrant mRNAs. In addition, we demonstrate how the length of mRNA affects the QC process. Since longer mRNAs spend more time in the nuclear basket to form a compact conformation and initiate their export, nuclear basket proteins could more easily capture and retain them inside the nucleus.

Preparation of Hollow CuO@SiO2 Spheres and Its Catalytic Performances for the NO + CO and CO Oxidation

PubMed Central

Niu, Xiaoyu; Zhao, Tieying; Yuan, Fulong; Zhu, Yujun

2015-01-01

The hollow CuO@SiO2 spheres with a mean diameter of 240 nm and a thin shell layer of about 30 nm in thickness was synthesized using an inorganic SiO2 shell coating on the surface of Cu@C composite that was prepared by a two-step hydrothermal method. The obtained hollow CuO@SiO2 spheres were characterized by ICP-AES, nitrogen adsorption-desorption, SEM, TEM, XRD, H2-TPR, CO-TPR, CO-TPD and NO-TPD. The results revealed that the hollow CuO@SiO2 spheres consist of CuO uniformly inserted into SiO2 layer. The CuO@SiO2 sample exhibits particular catalytic activities for CO oxidation and NO + CO reactions compared with CuO supported on SiO2 (CuO/SiO2). The higher catalytic activity is attributed to the special hollow shell structure that possesses much more highly dispersed CuO nanocluster that can be easy toward the CO and NO adsorption and the oxidation of CO on its surface. PMID:25777579

Influence of metal substrates on the detection of explosive residues with laser-induced breakdown spectroscopy.

PubMed

Gottfried, Jennifer L

2013-02-01

Laser-induced breakdown spectroscopy is a promising approach for explosive residue detection, but several limitations to its widespread use remain. One issue is that the emission spectra of the residues are dependent on the substrate composition because some of the substrate is usually entrained in the laser-induced plasma and the laser-material interaction can be significantly affected by the substrate type. Here, we have demonstrated that despite the strong spectral variation in cyclotrimethylenetrinitramine (RDX) residues applied to various metal substrates, classification of the RDX residue independent of substrate type is feasible. Several approaches to improving the chemometric models based on partial least squares discriminant analysis (PLS-DA) have been described: classifying the RDX residue spectra together in one class independent of substrate, using selected emission intensities and ratios to increase the true positive rate (TPR) and decrease the false positive rate (FPR), and fusing the results from two PLS-DA models generated using the full broadband spectra and selected intensities and ratios. The combination of these approaches resulted in a TPR of 97.5% and a FPR of 1.0% for RDX classification on metal substrates.

Ion Pair Formation between Tertiary Aliphatic Amines and Perchlorate in the Biphasic Water/Dichloromethane System.

PubMed

Badocco, Denis; Di Marco, Valerio; Venzo, Alfonso; Frasconi, Marco; Frezzato, Diego; Pastore, Paolo

2017-10-12

The ability of aliphatic amines (AAs), namely, tripropylamine (TPrA), trisobutylamine (TisoBuA), and tributylamine (TBuA), to form ion pairs with perchlorate anion (ClO 4 - ) in biphasic aqueous/dichloromethane (CH 2 Cl 2 ) mixtures containing ClO 4 - 0.1 M has been demonstrated by GC with flame ionization (FID) and mass detectors (MS) and by NMR measurements. The extraction efficiency of the AAs to the organic phase was modeled by equations that were used to fit the experimental GC data, allowing us to determine values for K P (partition constant of the free AA), K IP (formation constant of the ion pair), and K P IP (partition constant of the ion pair) for TPrA, TisoBuA, and TBuA at 25 °C. Ion pairs were shown to form in CH 2 Cl 2 also when ClO 4 - is replaced by other inorganic anions, like NO 3 - , ClO 3 - , Cl - , H 2 PO 4 - , and IO 3 - . No ion pairs formed when CH 2 Cl 2 was replaced by n-hexane, suggesting that aliphatic amine ion pairs can form in polar organic solvents but not in nonpolar ones.

α-Conotoxin Decontamination Protocol Evaluation: What Works and What Doesn’t

PubMed Central

Turner, Matthew W.; Cort, John R.; McDougal, Owen M.

2017-01-01

Nine publically available biosafety protocols for safely handling conotoxin peptides were tested to evaluate their decontamination efficacy. Circular dichroism (CD) spectroscopy and mass spectrometry (MS) were used to assess the effect of each chemical treatment on the secondary and primary structure of α-CTx MII (L10V, E11A). Of the nine decontamination methods tested, treatment with 1% (m/v) solution of the enzymatic detergent Contrex™ EZ resulted in a 76.8% decrease in α-helical content as assessed by the mean residue ellipticity at 222 nm, and partial peptide digestion was demonstrated using high performance liquid chromatography mass spectrometry (HPLC-MS). Additionally, treatment with 6% sodium hypochlorite (m/v) resulted in 80.5% decrease in α-helical content and complete digestion of the peptide. The Contrex™ EZ treatment was repeated with three additional α-conotoxins (α-CTxs), α-CTxs LvIA, ImI and PeIA, which verified the decontamination method was reasonably robust. These results support the use of either 1% Contrex™ EZ solution or 6% sodium hypochlorite in biosafety protocols for the decontamination of α-CTxs in research laboratories. PMID:28906461

Study of Binding Interaction between Pif80 Protein Fragment and Aragonite

NASA Astrophysics Data System (ADS)

Du, Yuan-Peng; Chang, Hsun-Hui; Yang, Sheng-Yu; Huang, Shing-Jong; Tsai, Yu-Ju; Huang, Joseph Jen-Tse; Chan, Jerry Chun Chung

2016-08-01

Pif is a crucial protein for the formation of the nacreous layer in Pinctada fucata. Three non-acidic peptide fragments of the aragonite-binding domain (Pif80) are selected, which contain multiple copies of the repeat sequence DDRK, to study the interaction between non-acidic peptides and aragonite. The polypeptides DDRKDDRKGGK (Pif80-11) and DDRKDDRKGGKDDRKDDRKGGK (Pif80-22) have similar binding affinity to aragonite. Solid-state NMR data indicate that the backbones of Pif80-11 and Pif80-22 peptides bound on aragonite adopt a random-coil conformation. Pif80-11 is a lot more effective than Pif80-22 in promoting the nucleation of aragonite on the substrate of β-chitin. Our results suggest that the structural arrangement at a protein-mineral interface depends on the surface structure of the mineral substrate and the protein sequence. The side chains of the basic residues, which function as anchors to the aragonite surface, have uniform structures. The role of basic residues as anchors in protein-mineral interaction may play an important role in biomineralization.

Midbody Targeting of the ESCRT Machinery by a Noncanonical Coiled Coil in CEP55

DOE Office of Scientific and Technical Information (OSTI.GOV)

Lee, Hyung Ho; Elia, Natalie; Ghirlando, Rodolfo

2008-11-14

The ESCRT (endosomal sorting complex required for transport) machinery is required for the scission of membrane necks in processes including the budding of HIV-1 and cytokinesis. An essential step in cytokinesis is recruitment of the ESCRT-I complex and the ESCRT-associated protein ALIX to the midbody (the structure that tethers two daughter cells) by the protein CEP55. Biochemical experiments show that peptides from ALIX and the ESCRT-I subunit TSG101 compete for binding to the ESCRT and ALIX-binding region (EABR) of CEP55. We solved the crystal structure of EABR bound to an ALIX peptide at a resolution of 2.0 angstroms. The structuremore » shows that EABR forms an aberrant dimeric parallel coiled coil. Bulky and charged residues at the interface of the two central heptad repeats create asymmetry and a single binding site for an ALIX or TSG101 peptide. Both ALIX and ESCRT-I are required for cytokinesis, which suggests that multiple CEP55 dimers are required for function.« less

Integrated enzyme reactor and high resolving chromatography in "sub-chip" dimensions for sensitive protein mass spectrometry.

PubMed

Hustoft, Hanne Kolsrud; Brandtzaeg, Ole Kristian; Rogeberg, Magnus; Misaghian, Dorna; Torsetnes, Silje Bøen; Greibrokk, Tyge; Reubsaet, Léon; Wilson, Steven Ray; Lundanes, Elsa

2013-12-16

Reliable, sensitive and automatable analytical methodology is of great value in e.g. cancer diagnostics. In this context, an on-line system for enzymatic cleavage of proteins, subsequent peptide separation by liquid chromatography (LC) with mass spectrometric detection has been developed using "sub-chip" columns (10-20 μm inner diameter, ID). The system could detect attomole amounts of isolated cancer biomarker progastrin-releasing peptide (ProGRP), in a more automatable fashion compared to previous methods. The workflow combines protein digestion using an 20 μm ID immobilized trypsin reactor with a polymeric layer of 2-hydroxyethyl methacrylate-vinyl azlactone (HEMA-VDM), desalting on a polystyrene-divinylbenzene (PS-DVB) monolithic trap column, and subsequent separation of resulting peptides on a 10 μm ID (PS-DVB) porous layer open tubular (PLOT) column. The high resolution of the PLOT columns was maintained in the on-line system, resulting in narrow chromatographic peaks of 3-5 seconds. The trypsin reactors provided repeatable performance and were compatible with long-term storage.

[Preparation and activity validation of PP7 bacteriophage-like particles displaying PAP114-128 peptide].

PubMed

Sun, Yanli; Sun, Yanhua

2016-10-01

Objective To obtain the PP7 bacteriophage-like particles carrying the peptide of prostatic acid phosphatase PAP 114-128 , and prove that they retain the original biological activity. Methods First, the plasmid pETDuet-2PP7 was constructed as follows: the gene of PP7 coat protein dimer was amplified by gene mutation combined with overlapping PCR technology, and inserted into the vector pETDuet-1. Following that, the plasmid pETDuet-2PP7-PAP 114-128 was constructed as follows: the PP7 coat protein gene carrying the coding gene of PAP 114-128 peptide was amplified using PCR, and then inserted into the vector pETDuet-2PP7. Both pETDuet-2PP7 and pETDuet-2PP7-PAP 114-128 were transformed into E.coli and expressed. The expression product was verified by SDS-PAGE, double immunodiffusion assay and ELISA. Results The gene fragment of PP7 coat protein dimer was obtained by overlapping PCR using Ex Taq DNA polymerase, and the antigenicity of its expression product was the same as that of the coat protein of wild-type PP7 bacteriophage. Moreover, the PAP 114-128 peptide epitope that was displayed on the surface of PP7 bacteriophage was identical with the corresponding epitope of natural human PAP, and it was able to induce high levels of antibodies. Conclusion The gene of PP7 coat protein dimer with repeated sequences can be prepared by gene mutation combined with overlapping PCR. Based on this, PP7 bacteriophage-like particles carrying PAP peptide can be prepared, which not only solves the problem of the instability of the peptides, but also lays a foundation for the study on their delivery and function.

mapDIA: Preprocessing and statistical analysis of quantitative proteomics data from data independent acquisition mass spectrometry.

PubMed

Teo, Guoshou; Kim, Sinae; Tsou, Chih-Chiang; Collins, Ben; Gingras, Anne-Claude; Nesvizhskii, Alexey I; Choi, Hyungwon

2015-11-03

Data independent acquisition (DIA) mass spectrometry is an emerging technique that offers more complete detection and quantification of peptides and proteins across multiple samples. DIA allows fragment-level quantification, which can be considered as repeated measurements of the abundance of the corresponding peptides and proteins in the downstream statistical analysis. However, few statistical approaches are available for aggregating these complex fragment-level data into peptide- or protein-level statistical summaries. In this work, we describe a software package, mapDIA, for statistical analysis of differential protein expression using DIA fragment-level intensities. The workflow consists of three major steps: intensity normalization, peptide/fragment selection, and statistical analysis. First, mapDIA offers normalization of fragment-level intensities by total intensity sums as well as a novel alternative normalization by local intensity sums in retention time space. Second, mapDIA removes outlier observations and selects peptides/fragments that preserve the major quantitative patterns across all samples for each protein. Last, using the selected fragments and peptides, mapDIA performs model-based statistical significance analysis of protein-level differential expression between specified groups of samples. Using a comprehensive set of simulation datasets, we show that mapDIA detects differentially expressed proteins with accurate control of the false discovery rates. We also describe the analysis procedure in detail using two recently published DIA datasets generated for 14-3-3β dynamic interaction network and prostate cancer glycoproteome. The software was written in C++ language and the source code is available for free through SourceForge website http://sourceforge.net/projects/mapdia/.This article is part of a Special Issue entitled: Computational Proteomics. Copyright © 2015 Elsevier B.V. All rights reserved.

Thermal and adsorbate effects on the activity and morphology of size-selected Pdn/TiO2 model catalysts

NASA Astrophysics Data System (ADS)

Kaden, William E.; Kunkel, William A.; Roberts, F. Sloan; Kane, Matthew; Anderson, Scott L.

2014-03-01

Model catalysts containing size-selected Pdn (n = 1,2,4,7,10,16,20,25) deposited on rutile TiO2(110) deactivate during repeated CO oxidation temperature-programmed reaction (TPR) cycles, and the deactivation process has been probed using a combination of X-ray photoelectron spectroscopy (XPS), ultraviolet photoelectron spectroscopy (UPS), low-energy ion scattering (ISS), temperature-dependent ion scattering (TD-ISS), annealing experiments, and temperature-programmed desorption following exposure to CO and O2 reactants. Results from such experiments suggest the cluster deactivation proceeds via an alloy-like, strong metal-support interaction (SMSI) effect that chemically modifies the clusters via electronic interactions between the supported metal atoms and Ti from the support. Threshold measurements show that this effect detrimentally affects CO-oxidation activity prior to the formation of an encapsulating overlayer by severely weakening the COPd bond strengths for binding configurations on top of the clusters. Oxidation appears to provide means of partially restoring the clusters to their initial state, but after sufficient exposure to reducing environments and elevated temperatures, all Pdn become covered by an overlayer and begin to electronically and chemically resemble freshly deposited atoms, which are completely inactive towards the probe reaction. In addition, we find evidence of oxygen spillover induced by co-adsorbed CO during TPRs for all active Pdn clusters.

Ordered Mesoporous NiCeAl Containing Catalysts for Hydrogenolysis of Sorbitol to Glycols

NASA Astrophysics Data System (ADS)

Zhou, Zhiwei; Zhang, Jiaqi; Qin, Juan; Li, Dong; Wu, Wenliang

2018-03-01

Cellulose-derived sorbitol is emerging as a feasible and renewable feedstock for the production of value-added chemicals. Highly active and stable catalyst is essential for sorbitol hydrogenolysis. Ordered mesoporous M- xNi yCeAl catalysts with different loadings of nickel and cerium species were successfully synthesized via one-pot evaporation-induced self-assembly strategy (EISA) and their catalytic performance were tested in the hydrogenolysis of sorbitol. The physical chemical properties for the catalysts were characterized by XRD, N2 physisorption, H2-TPR, H2 impulse chemisorption, ICP and TEM techniques. The results showed that the ordered mesopores with uniform pore sizes can be obtained and the Ni nanoparticles around 6 nm in size were homogeneously dispersed in the mesopore channels. A little amount of cerium species introduced would be beneficial to their textural properties resulting in higher Ni dispersion, metal area and smaller size of Ni nanoparticles. The M-10Ni2CeAl catalyst with Ni and Ce loading of 10.9 and 6.3 wt % shows better catalytic performance than other catalysts, and the yield of 1,2-PG and EG can reach 56.9% at 493 K and 6 MPa pressure for 8 h after repeating reactions for 12 times without obvious deterioration of physical and chemical properties. Ordered mesoporous M-NiCeAl catalysts are active and stable in sorbitol hydrogenolysis.

Lineage-specific expansion of IFIT gene family: an insight into coevolution with IFN gene family.

PubMed

Liu, Ying; Zhang, Yi-Bing; Liu, Ting-Kai; Gui, Jian-Fang

2013-01-01

In mammals, IFIT (Interferon [IFN]-induced proteins with Tetratricopeptide Repeat [TPR] motifs) family genes are involved in many cellular and viral processes, which are tightly related to mammalian IFN response. However, little is known about non-mammalian IFIT genes. In the present study, IFIT genes are identified in the genome databases from the jawed vertebrates including the cartilaginous elephant shark but not from non-vertebrates such as lancelet, sea squirt and acorn worm, suggesting that IFIT gene family originates from a vertebrate ancestor about 450 million years ago. IFIT family genes show conserved gene structure and gene arrangements. Phylogenetic analyses reveal that this gene family has expanded through lineage-specific and species-specific gene duplication. Interestingly, IFN gene family seem to share a common ancestor and a similar evolutionary mechanism; the function link of IFIT genes to IFN response is present early since the origin of both gene families, as evidenced by the finding that zebrafish IFIT genes are upregulated by fish IFNs, poly(I:C) and two transcription factors IRF3/IRF7, likely via the IFN-stimulated response elements (ISRE) within the promoters of vertebrate IFIT family genes. These coevolution features creates functional association of both family genes to fulfill a common biological process, which is likely selected by viral infection during evolution of vertebrates. Our results are helpful for understanding of evolution of vertebrate IFN system.

Signal peptide cleavage is essential for surface expression of a regulatory T cell surface protein, leucine rich repeat containing 32 (LRRC32).

PubMed

Chan, Derek V; Somani, Ally-Khan; Young, Andrew B; Massari, Jessica V; Ohtola, Jennifer; Sugiyama, Hideaki; Garaczi, Edina; Babineau, Denise; Cooper, Kevin D; McCormick, Thomas S

2011-05-26

Elevated numbers of regulatory T cells (T(regs)) have been implicated in certain cancers. Depletion of T(regs) has been shown to increase anti-tumor immunity. T(regs) also play a critical role in the suppression of autoimmune responses. The study of T(regs) has been hampered by a lack of adequate surface markers. Leucine Rich Repeat Containing 32 (LRRC32), also known as Glycoprotein A Repetitions Predominant (GARP), has been postulated as a novel surface marker of activated T(regs). However, there is limited information regarding the processing of LRRC32 or the regulatory phenotype and functional activity of T(regs) expressing LRRC32. Using naturally-occurring freshly isolated T(regs), we demonstrate that low levels of LRRC32 are present intracellularly prior to activation and that freshly isolated LRRC32+ T(regs) are distinct from LRRC32- T(regs) with respect to the expression of surface CD62L. Using LRRC32 transfectants of HEK cells, we demonstrate that the N-terminus of LRRC32 is cleaved prior to expression of the protein at the cell surface. Furthermore, we demonstrate using a construct containing a deleted putative signal peptide region that the presence of a signal peptide region is critical to cell surface expression of LRRC32. Finally, mixed lymphocyte assays demonstrate that LRRC32+ T(regs) are more potent suppressors than LRRC32- T(regs). A cleaved signal peptide site in LRRC32 is necessary for surface localization of native LRRC32 following activation of naturally-occurring freshly-isolated regulatory T cells. LRRC32 expression appears to alter the surface expression of activation markers of T cells such as CD62L. LRRC32 surface expression may be useful as a marker that selects for more potent T(reg) populations. In summary, understanding the processing and expression of LRRC32 may provide insight into the mechanism of action of T(regs) and the refinement of immunotherapeutic strategies aimed at targeting these cells.

Impact of the excision of an ancient repeat insertion on Rickettsia conorii guanylate kinase activity.

PubMed

Abergel, Chantal; Blanc, Guillaume; Monchois, Vincent; Renesto, Patricia; Sigoillot, Cécile; Ogata, Hiroyuki; Raoult, Didier; Claverie, Jean-Michel

2006-11-01

The genomic sequencing of Rickettsia conorii revealed a new family of Rickettsia-specific palindromic elements (RPEs) capable of in-frame insertion in preexisting open reading frames (ORFs). Many of these altered ORFs correspond to proteins with well-characterized or essential functions in other microorganisms. Previous experiments indicated that RPE-containing genes are normally transcribed and that no excision of the repeat occurs at the mRNA level. Using mass spectrometry, we now confirmed the retention of the RPE-derived amino acid residues in 4 proteins successfully expressed in Escherichia coli, raising the general question of the consequences of this common insertion event on the fitness of Rickettsia enzymes. The predicted guanylate kinase activity of the R. conorii gmk gene product was measured both on the RPE-containing and RPE-excised recombinant proteins. We show that the 2 proteins are active but exhibit substantial differences in their affinity for adenosine triphosphate, guanosine monophosphate, and catalytic constants. The distribution of the RPEgmk insert among Rickettsia species indicates that the insertion event is ancient and occurred after the divergence of Rickettsia felis and R. conorii but before that of Rickettsia helvetica and R. conorii. We found no evidence that the gmk gene fixed adaptive changes to compensate the RPE peptide insertion. Furthermore, the analysis of the rates of divergence in 23 RPE-containing genes indicates that coding RPE repeats tend to evolve under weak selective constraint, at a rate similar to intergenic noncoding RPE sequences. Altogether, these results suggest that the insertion of RPE-encoded "selfish peptides," although respecting the original fold and activity of the host proteins, might be slightly detrimental to the enzyme efficiency within limits tolerable for slow-growing intracellular parasites such as Rickettsia.

An additional function of the rough endoplasmic reticulum protein complex prolyl 3-hydroxylase 1·cartilage-associated protein·cyclophilin B: the CXXXC motif reveals disulfide isomerase activity in vitro.

PubMed

Ishikawa, Yoshihiro; Bächinger, Hans Peter

2013-11-01

Collagen biosynthesis occurs in the rough endoplasmic reticulum, and many molecular chaperones and folding enzymes are involved in this process. The folding mechanism of type I procollagen has been well characterized, and protein disulfide isomerase (PDI) has been suggested as a key player in the formation of the correct disulfide bonds in the noncollagenous carboxyl-terminal and amino-terminal propeptides. Prolyl 3-hydroxylase 1 (P3H1) forms a hetero-trimeric complex with cartilage-associated protein and cyclophilin B (CypB). This complex is a multifunctional complex acting as a prolyl 3-hydroxylase, a peptidyl prolyl cis-trans isomerase, and a molecular chaperone. Two major domains are predicted from the primary sequence of P3H1: an amino-terminal domain and a carboxyl-terminal domain corresponding to the 2-oxoglutarate- and iron-dependent dioxygenase domains similar to the α-subunit of prolyl 4-hydroxylase and lysyl hydroxylases. The amino-terminal domain contains four CXXXC sequence repeats. The primary sequence of cartilage-associated protein is homologous to the amino-terminal domain of P3H1 and also contains four CXXXC sequence repeats. However, the function of the CXXXC sequence repeats is not known. Several publications have reported that short peptides containing a CXC or a CXXC sequence show oxido-reductase activity similar to PDI in vitro. We hypothesize that CXXXC motifs have oxido-reductase activity similar to the CXXC motif in PDI. We have tested the enzyme activities on model substrates in vitro using a GCRALCG peptide and the P3H1 complex. Our results suggest that this complex could function as a disulfide isomerase in the rough endoplasmic reticulum.

Plasmodium falciparum Hop (PfHop) Interacts with the Hsp70 Chaperone in a Nucleotide-Dependent Fashion and Exhibits Ligand Selectivity.

PubMed

Zininga, Tawanda; Makumire, Stanely; Gitau, Grace Wairimu; Njunge, James M; Pooe, Ofentse Jacob; Klimek, Hanna; Scheurr, Robina; Raifer, Hartmann; Prinsloo, Earl; Przyborski, Jude M; Hoppe, Heinrich; Shonhai, Addmore

2015-01-01

Heat shock proteins (Hsps) play an important role in the development and pathogenicity of malaria parasites. One of the most prominent functions of Hsps is to facilitate the folding of other proteins. Hsps are thought to play a crucial role when malaria parasites invade their host cells and during their subsequent development in hepatocytes and red blood cells. It is thought that Hsps maintain proteostasis under the unfavourable conditions that malaria parasites encounter in the host environment. Although heat shock protein 70 (Hsp70) is capable of independent folding of some proteins, its functional cooperation with heat shock protein 90 (Hsp90) facilitates folding of some proteins such as kinases and steroid hormone receptors into their fully functional forms. The cooperation of Hsp70 and Hsp90 occurs through an adaptor protein called Hsp70-Hsp90 organising protein (Hop). We previously characterised the Hop protein from Plasmodium falciparum (PfHop). We observed that the protein co-localised with the cytosol-localised chaperones, PfHsp70-1 and PfHsp90 at the blood stages of the malaria parasite. In the current study, we demonstrated that PfHop is a stress-inducible protein. We further explored the direct interaction between PfHop and PfHsp70-1 using far Western and surface plasmon resonance (SPR) analyses. The interaction of the two proteins was further validated by co-immunoprecipitation studies. We observed that PfHop and PfHsp70-1 associate in the absence and presence of either ATP or ADP. However, ADP appears to promote the association of the two proteins better than ATP. In addition, we investigated the specific interaction between PfHop TPR subdomains and PfHsp70-1/ PfHsp90, using a split-GFP approach. This method allowed us to observe that TPR1 and TPR2B subdomains of PfHop bind preferentially to the C-terminus of PfHsp70-1 compared to PfHsp90. Conversely, the TPR2A motif preferentially interacted with the C-terminus of PfHsp90. Finally, we observed that recombinant PfHop occasionally eluted as a protein species of twice its predicted size, suggesting that it may occur as a dimer. We conducted SPR analysis which suggested that PfHop is capable of self-association in presence or absence of ATP/ADP. Overall, our findings suggest that PfHop is a stress-inducible protein that directly associates with PfHsp70-1 and PfHsp90. In addition, the protein is capable of self-association. The findings suggest that PfHop serves as a module that brings these two prominent chaperones (PfHsp70-1 and PfHsp90) into a functional complex. Since PfHsp70-1 and PfHsp90 are essential for parasite growth, findings from this study are important towards the development of possible antimalarial inhibitors targeting the cooperation of these two chaperones.

Monte Carlo-based investigations on the impact of removing the flattening filter on beam quality specifiers for photon beam dosimetry.

PubMed

Czarnecki, Damian; Poppe, Björn; Zink, Klemens

2017-06-01

The impact of removing the flattening filter in clinical electron accelerators on the relationship between dosimetric quantities such as beam quality specifiers and the mean photon and electron energies of the photon radiation field was investigated by Monte Carlo simulations. The purpose of this work was to determine the uncertainties when using the well-known beam quality specifiers or energy-based beam specifiers as predictors of dosimetric photon field properties when removing the flattening filter. Monte Carlo simulations applying eight different linear accelerator head models with and without flattening filter were performed in order to generate realistic radiation sources and calculate field properties such as restricted mass collision stopping power ratios (L¯/ρ)airwater, mean photon and secondary electron energies. To study the impact of removing the flattening filter on the beam quality correction factors k Q , this factor for detailed ionization chamber models was calculated by Monte Carlo simulations. Stopping power ratios (L¯/ρ)airwater and k Q values for different ionization chambers as a function of TPR1020 and %dd(10) x were calculated. Moreover, mean photon energies in air and at the point of measurement in water as well as mean secondary electron energies at the point of measurement were calculated. The results revealed that removing the flattening filter led to a change within 0.3% in the relationship between %dd(10) x and (L¯/ρ)airwater, whereby the relationship between TPR1020 and (L¯/ρ)airwater changed up to 0.8% for high energy photon beams. However, TPR1020 was a good predictor of (L¯/ρ)airwater for both types of linear accelerator with energies < 10 MeV with a maximal deviation between both types of accelerators of 0.23%. According to the results, the mean photon energy below the linear accelerators head as well as at the point of measurement may not be suitable as a predictor of (L¯/ρ)airwater and k Q to merge the dosimetry of both linear accelerator types. It was possible to derive (L¯/ρ)airwater using the mean secondary electron energy at the point of measurement as a predictor with an accuracy of 0.17%. A bias between k Q for linear accelerators with and without flattening filter within 1.1% and 1.6% was observed for TPR1020 and %dd(10) x respectively. The results of this study have shown that removing the flattening filter led to a change in the relationship between the well-known beam quality specifiers and dosimetric quantities at the point of measurement, namely (L¯/ρ)airwater, mean photon and electron energy. Furthermore, the results show that a beam profile correction is important for dose measurements with large ionization chambers in flattening filter free beams. © 2017 American Association of Physicists in Medicine.

Disruption of Higher Order DNA Structures in Friedreich’s Ataxia (GAA)n Repeats by PNA or LNA Targeting

PubMed Central

Bergquist, Helen; Rocha, Cristina S. J.; Álvarez-Asencio, Rubén; Nguyen, Chi-Hung; Rutland, Mark. W.; Smith, C. I. Edvard; Good, Liam; Nielsen, Peter E.; Zain, Rula

2016-01-01

Expansion of (GAA)n repeats in the first intron of the Frataxin gene is associated with reduced mRNA and protein levels and the development of Friedreich’s ataxia. (GAA)n expansions form non-canonical structures, including intramolecular triplex (H-DNA), and R-loops and are associated with epigenetic modifications. With the aim of interfering with higher order H-DNA (like) DNA structures within pathological (GAA)n expansions, we examined sequence-specific interaction of peptide nucleic acid (PNA) with (GAA)n repeats of different lengths (short: n=9, medium: n=75 or long: n=115) by chemical probing of triple helical and single stranded regions. We found that a triplex structure (H-DNA) forms at GAA repeats of different lengths; however, single stranded regions were not detected within the medium size pathological repeat, suggesting the presence of a more complex structure. Furthermore, (GAA)4-PNA binding of the repeat abolished all detectable triplex DNA structures, whereas (CTT)5-PNA did not. We present evidence that (GAA)4-PNA can invade the DNA at the repeat region by binding the DNA CTT strand, thereby preventing non-canonical-DNA formation, and that triplex invasion complexes by (CTT)5-PNA form at the GAA repeats. Locked nucleic acid (LNA) oligonucleotides also inhibited triplex formation at GAA repeat expansions, and atomic force microscopy analysis showed significant relaxation of plasmid morphology in the presence of GAA-LNA. Thus, by inhibiting disease related higher order DNA structures in the Frataxin gene, such PNA and LNA oligomers may have potential for discovery of drugs aiming at recovering Frataxin expression. PMID:27846236

Identification of metalloprotease/disintegrins in Xenopus laevis testis with a potential role in fertilization.

PubMed

Shilling, F M; Krätzschmar, J; Cai, H; Weskamp, G; Gayko, U; Leibow, J; Myles, D G; Nuccitelli, R; Blobel, C P

1997-06-15

Proteins containing a membrane-anchored metalloprotease domain, a disintegrin domain, and a cysteine-rich region (MDC proteins) are thought to play an important role in mammalian fertilization, as well as in somatic cell-cell interactions. We have identified PCR sequence tags encoding the disintegrin domain of five distinct MDC proteins from Xenopus laevis testis cDNA. Four of these sequence tags (xMDC9, xMDC11.1, xMDC11.2, and xMDC13) showed strong similarity to known mammalian MDC proteins, whereas the fifth (xMDC16) apparently represents a novel family member. Northern blot analysis revealed that the mRNA for xMDC16 was only expressed in testis, and not in heart, muscle, liver, ovaries, or eggs, whereas the mRNAs corresponding to the four other PCR products were expressed in testis and in some or all somatic tissues tested. The xMDC16 protein sequence, as predicted from the full-length cDNA, contains a metalloprotease domain with the active-site sequence HEXXH, a disintegrin domain, a cysteine-rich region, an EGF repeat, a transmembrane domain, and a short cytoplasmic tail. To study a potential role for these xMDC proteins in fertilization, peptides corresponding to the predicted integrin-binding domain of each protein were tested for their ability to inhibit X. laevis fertilization. Cyclic and linear xMDC16 peptides inhibited fertilization in a concentration-dependent manner, whereas xMDC16 peptides that were scrambled or had certain amino acid replacements in the predicted integrin-binding domain did not affect fertilization. Cyclic and linear xMDC9 peptides and linear xMDC13 peptides also inhibited fertilization similarly to xMDC16 peptides, whereas peptides corresponding to the predicted integrin-binding site of xMDC11.1 and xMDC11.2 did not. These results are discussed in the context of a model in which multiple MDC protein-receptor interactions are necessary for fertilization to occur.

Highly Selective End-Tagged Antimicrobial Peptides Derived from PRELP

PubMed Central

Malmsten, Martin; Kasetty, Gopinath; Pasupuleti, Mukesh; Alenfall, Jan; Schmidtchen, Artur

2011-01-01

Background Antimicrobial peptides (AMPs) are receiving increasing attention due to resistance development against conventional antibiotics. Pseudomonas aeruginosa and Staphylococcus aureus are two major pathogens involved in an array of infections such as ocular infections, cystic fibrosis, wound and post-surgery infections, and sepsis. The goal of the study was to design novel AMPs against these pathogens. Methodology and Principal Findings Antibacterial activity was determined by radial diffusion, viable count, and minimal inhibitory concentration assays, while toxicity was evaluated by hemolysis and effects on human epithelial cells. Liposome and fluorescence studies provided mechanistic information. Protease sensitivity was evaluated after subjection to human leukocyte elastase, staphylococcal aureolysin and V8 proteinase, as well as P. aeruginosa elastase. Highly active peptides were evaluated in ex vivo skin infection models. C-terminal end-tagging by W and F amino acid residues increased antimicrobial potency of the peptide sequences GRRPRPRPRP and RRPRPRPRP, derived from proline arginine-rich and leucine-rich repeat protein (PRELP). The optimized peptides were antimicrobial against a range of Gram-positive S. aureus and Gram-negative P. aeruginosa clinical isolates, also in the presence of human plasma and blood. Simultaneously, they showed low toxicity against mammalian cells. Particularly W-tagged peptides displayed stability against P. aeruginosa elastase, and S. aureus V8 proteinase and aureolysin, and the peptide RRPRPRPRPWWWW-NH2 was effective against various “superbugs” including vancomycin-resistant enterococci, multi-drug resistant P. aeruginosa, and methicillin-resistant S. aureus, as well as demonstrated efficiency in an ex vivo skin wound model of S. aureus and P. aeruginosa infection. Conclusions/Significance Hydrophobic C-terminal end-tagging of the cationic sequence RRPRPRPRP generates highly selective AMPs with potent activity against multiresistant bacteria and efficiency in ex vivo wound infection models. A precise “tuning” of toxicity and proteolytic stability may be achieved by changing tag-length and adding W- or F-amino acid tags. PMID:21298015

Synthesis and Characterization of High Molecular Weight Peptide Polymers and Copolymers Containing L-Dopa Residues

DTIC Science & Technology

1988-07-01

decapeptide (GLUE-12) with blocked lysines with DPPA for 24 hours. A parallel reaction was carried out to polymerize E- aminocaproic acid with DPPA for...shear adhesive strength tests. OPPA has also been used to prepare block copolymers between GLUE polypeptides and poly(e-amino caproic acid ). Concurrent...amino acid residues towards intramolecular or intermolecular bond formation. Polypeptides with repeating amino acid sequences have also been produced

Small leucine-rich repeat proteoglycans associated with mature insoluble elastin serve as binding sites for galectins.

PubMed

Itoh, Aiko; Nonaka, Yasuhiro; Ogawa, Takashi; Nakamura, Takanori; Nishi, Nozomu

2017-11-01

We previously reported that galectin-9 (Gal-9), an immunomodulatory animal lectin, could bind to insoluble collagen preparations and exerted direct cytocidal effects on immune cells. In the present study, we found that mature insoluble elastin is capable of binding Gal-9 and other members of the human galectin family. Lectin blot analysis of a series of commercial water-soluble elastin preparations, PES-(A) ~ PES-(E), revealed that only PES-(E) contained substances recognized by Gal-9. Gal-9-interacting substances in PES-(E) were affinity-purified, digested with trypsin and then analyzed by reversed-phase HPLC. Peptide fragments derived from five members of the small leucine-rich repeat proteoglycan family, versican, lumican, osteoglycin/mimecan, prolargin, and fibromodulin, were identified by N-terminal amino acid sequence analysis. The results indicate that Gal-9 and possibly other galectins recognize glycans attached to small leucine-rich repeat proteoglycans associated with insoluble elastin and also indicate the possibility that mature insoluble elastin serves as an extracellular reservoir for galectins.

Thermal denaturation of the BRCT tandem repeat region of human tumour suppressor gene product BRCA1.

PubMed

Pyrpassopoulos, Serapion; Ladopoulou, Angela; Vlassi, Metaxia; Papanikolau, Yannis; Vorgias, Constantinos E; Yannoukakos, Drakoulis; Nounesis, George

2005-04-01

Reduced stability of the tandem BRCT domains of human BReast CAncer 1 (BRCA1) due to missense mutations may be critical for loss of function in DNA repair and damage-induced checkpoint control. In the present thermal denaturation study of the BRCA1 BRCT region, high-precision differential scanning calorimetry (DSC) and circular dichroism (CD) spectroscopy provide evidence for the existence of a denatured state that is structurally very similar to the native. Consistency between theoretical structure-based estimates of the enthalpy (DeltaH) and heat capacity change (DeltaCp) and the calorimetric results is obtained when considering partial thermal unfolding contained in the region of the conserved hydrophobic pocket formed at the interface of the two BRCT repeats. The structural integrity of this region has been shown to be crucial for the interaction of BRCA1 with phosphorylated peptides. In addition, cancer-causing missense mutations located at the inter-BRCT-repeat interface have been linked to the destabilization of the tandem BRCT structure.

Insight into Amyloid Structure Using Chemical Probes

PubMed Central

Reinke, Ashley A.; Gestwicki, Jason E.

2011-01-01

Alzheimer’s disease (AD) is a common neurodegenerative disorder characterized by the deposition of amyloids in the brain. One prominent form of amyloid is composed of repeating units of the amyloid-β (Aβ) peptide. Over the past decade, it has become clear that these Aβ amyloids are not homogeneous; rather, they are composed of a series of structures varying in their overall size and shape and the number of Aβ peptides they contain. Recent theories suggest that these different amyloid conformations may play distinct roles in disease, although their relative contributions are still being discovered. Here, we review how chemical probes, such as congo red, thioflavin T and their derivatives, have been powerful tools for better understanding amyloid structure and function. Moreover, we discuss how design and deployment of conformationally selective probes might be used to test emerging models of AD. PMID:21457473

Effects of various doses of selenite on stinging nettle (Urtica dioica L.).

PubMed

Krystofova, Olga; Adam, Vojtech; Babula, Petr; Zehnalek, Josef; Beklova, Miroslava; Havel, Ladislav; Kizek, Rene

2010-10-01

The aim of this study was to investigate the effects of selenium (Se) on the growth, accumulation and possible mechanisms of Se transport in certain parts (roots, leaves, stamp and apex) of nettle (Urtica dioica L.) plants. Se was supplemented by one-shot and two repeated doses to the soil (2.0 and 4.0 mg Se per kg of substrate). Selenium content in roots increased linearly with dose and was significantly higher compared to other plant parts of interest. However, growth of the above-ground parts of plant as well as roots was slightly inhibited with increasing selenium concentration in comparison to the untreated plants. The content of phytochelatin2, a low molecular mass peptide containing a sulfhydryl group, correlated well with the Se content. This suggests a possible stimulation of synthesis of this plant peptide by Se.

Teaching English through Action: Total Physical Response (T.P.R.). A Right-Brain/Left-Brain Approach to Language Acquisition. A Workshop.

ERIC Educational Resources Information Center

Segal, Bertha E.

Materials from a teacher workshop on the Total Physical Response method for teaching English as a second language are presented. The technique describes the process of first language acquisition, uses physical activities in the classroom to reinforce learning, and allows a long period of receptive language learning before requiring production. The…