An Internal Signal Sequence Directs Intramembrane Proteolysis of a Cellular Immunoglobulin Domain Protein*S⃞

PubMed Central

Robakis, Thalia; Bak, Beata; Lin, Shu-huei; Bernard, Daniel J.; Scheiffele, Peter

2008-01-01

Precursor proteolysis is a crucial mechanism for regulating protein structure and function. Signal peptidase (SP) is an enzyme with a well defined role in cleaving N-terminal signal sequences but no demonstrated function in the proteolysis of cellular precursor proteins. We provide evidence that SP mediates intraprotein cleavage of IgSF1, a large cellular Ig domain protein that is processed into two separate Ig domain proteins. In addition, our results suggest the involvement of signal peptide peptidase (SPP), an intramembrane protease, which acts on substrates that have been previously cleaved by SP. We show that IgSF1 is processed through sequential proteolysis by SP and SPP. Cleavage is directed by an internal signal sequence and generates two separate Ig domain proteins from a polytopic precursor. Our findings suggest that SP and SPP function are not restricted to N-terminal signal sequence cleavage but also contribute to the processing of cellular transmembrane proteins. PMID:18981173

Flanking signal and mature peptide residues influence signal peptide cleavage

PubMed Central

Choo, Khar Heng; Ranganathan, Shoba

2008-01-01

Background Signal peptides (SPs) mediate the targeting of secretory precursor proteins to the correct subcellular compartments in prokaryotes and eukaryotes. Identifying these transient peptides is crucial to the medical, food and beverage and biotechnology industries yet our understanding of these peptides remains limited. This paper examines the most common type of signal peptides cleavable by the endoprotease signal peptidase I (SPase I), and the residues flanking the cleavage sites of three groups of signal peptide sequences, namely (i) eukaryotes (Euk) (ii) Gram-positive (Gram+) bacteria, and (iii) Gram-negative (Gram-) bacteria. Results In this study, 2352 secretory peptide sequences from a variety of organisms with amino-terminal SPs are extracted from the manually curated SPdb database for analysis based on physicochemical properties such as pI, aliphatic index, GRAVY score, hydrophobicity, net charge and position-specific residue preferences. Our findings show that the three groups share several similarities in general, but they display distinctive features upon examination in terms of their amino acid compositions and frequencies, and various physico-chemical properties. Thus, analysis or prediction of their sequences should be separated and treated as distinct groups. Conclusion We conclude that the peptide segment recognized by SPase I extends to the start of the mature protein to a limited extent, upon our survey of the amino acid residues surrounding the cleavage processing site. These flanking residues possibly influence the cleavage processing and contribute to non-canonical cleavage sites. Our findings are applicable in defining more accurate prediction tools for recognition and identification of cleavage site of SPs. PMID:19091014

Collision-induced fragmentation of negative ions from N-linked glycans derivatized with 2-aminobenzoic acid.

PubMed

Harvey, David J

2005-05-01

N-Linked glycans from bovine ribonuclease B, chicken ovalbumin, bovine fetuin, porcine thyroglobulin and human alpha(1)-acid glycoprotein were derivatized with 2-aminobenzoic acid by reductive amination and their tandem mass spectra were recorded by negative ion electrospray ionization with a quadrupole time-of-flight mass spectrometer. Derivatives were also prepared from 2-amino-5-methyl- and 2-amino-4,5-dimethoxybenzoic acid in order to confirm the identity of fragment ions containing the reducing terminus. Major fragments from the [M - H](-) ions from the neutral glycans retained the derivative (Y-type cleavages) and provided information on sequence and branching. Other major fragments were products of A-type cross-ring cleavages giving information on antenna structure. Singly doubly and triply charged ions were formed from sialylated glycans. They produced major fragments by loss of sialic acid and a series of singly charged ions that were similar to those from the neutral analogues. Doubly charge ions were also produced by the neutral glycans and were fragmented to form product ions with one and two charges. Again, the fragment ions with a single charge were similar to those from the singly charged parents, but branching information was less obvious because of the occurrence of more abundant ions produced by multiple cleavages. Detection limits were around 200 fmol (3 : 1 signal-to-noise ratio). Copyright 2005 John Wiley & Sons, Ltd.

Top-Down Charge Transfer Dissociation (CTD) of Gas-Phase Insulin: Evidence of a One-Step, Two-Electron Oxidation Mechanism

NASA Astrophysics Data System (ADS)

Li, Pengfei; Kreft, Iris; Jackson, Glen P.

2018-02-01

Top-down analyses of protonated insulin cations of charge states of 4+, 5+, or 6+ were performed by exposing the isolated precursor ions to a beam of helium cations with kinetic energy of more than 6 keV, in a technique termed charge transfer dissociation (CTD). The 100 ms charge transfer reaction resulted in approximately 20% conversion efficiency to other intact charge exchange products (CTnoD), and a range of low abundance fragment ions. To increase backbone and sulfide cleavages, and to provide better structural information than straightforward MS2 CTD, the CTnoD oxidized products were isolated and subjected to collisional activation at the MS3 level. The MS3 CTD/CID reaction effectively broke the disulfide linkages, separated the two chains, and yielded more structurally informative fragment ions within the inter-chain cyclic region. CTD also provided doubly oxidized intact product ions at the MS2 level, and resonance ejection of the singly oxidized product ion revealed that the doubly oxidized product originates directly from the isolated precursor ion and not from consecutive CTD reactions of a singly oxidized intermediate. MS4 experiments were employed to help identify potential radical cations and diradical cations, but the results were negative or inconclusive. Nonetheless, the two-electron oxidation process is a demonstration of the very large potential energy (>20 eV) available through CTD, and is a notable capability for a 3D ion trap platform.

Proton affinity determinations and proton-bound dimer structure indications in C2 to C15, (alpha),(omega)-alkyldiamines

NASA Technical Reports Server (NTRS)

Karpas, Z.; Harden, C. S.; Smith, P. B. W.

1995-01-01

The 'kinetic method' was used to determine the proton affinity (PA) of a,coalkyldiamines from collision induced dissociation (CID) studies of protonated heterodimers. These PA values were consistently lower than those reported in the proton affinity scale. The apparent discrepancy was rationalized in terms of differences in the conformation of the protonated diamine monomers. The minimum energy species, formed by equilibrium proton transfer processes, have a cyclic conformation and the ion charge is shared by both amino-groups which are bridged by the proton. On the other hand, the species formed through dissociation of protonated dimers have a linear structure and the charge is localized on one of the amino-groups. Thus, the difference in the PA values obtained by both methods is a measure of the additional stability acquired by the protonated diamines through cyclization and charge delocalization. The major collision dissociation pathway of the protonated diamine monomers involved elimination of an ammonia moiety. Other reactions observed included loss of the second amino-group and several other bond cleavages. CID of the protonated dimers involved primarily formation of a protonated monomer through cleavage of the weaker hydrogen bond and subsequently loss of ammonia at higher collision energies. As observed from the CID studies, doubly charged ions were also formed from the diamines under conditions of the electrospray ionization.

Hierarchical recruitment of ribosomal proteins and assembly factors remodels nucleolar pre-60S ribosomes.

PubMed

Biedka, Stephanie; Micic, Jelena; Wilson, Daniel; Brown, Hailey; Diorio-Toth, Luke; Woolford, John L

2018-04-24

Ribosome biogenesis involves numerous preribosomal RNA (pre-rRNA) processing events to remove internal and external transcribed spacer sequences, ultimately yielding three mature rRNAs. Removal of the internal transcribed spacer 2 spacer RNA is the final step in large subunit pre-rRNA processing and begins with endonucleolytic cleavage at the C 2 site of 27SB pre-rRNA. C 2 cleavage requires the hierarchical recruitment of 11 ribosomal proteins and 14 ribosome assembly factors. However, the function of these proteins in C 2 cleavage remained unclear. In this study, we have performed a detailed analysis of the effects of depleting proteins required for C 2 cleavage and interpreted these results using cryo-electron microscopy structures of assembling 60S subunits. This work revealed that these proteins are required for remodeling of several neighborhoods, including two major functional centers of the 60S subunit, suggesting that these remodeling events form a checkpoint leading to C 2 cleavage. Interestingly, when C 2 cleavage is directly blocked by depleting or inactivating the C 2 endonuclease, assembly progresses through all other subsequent steps. © 2018 Biedka et al.

Development and application of bond cleavage reactions in bioorthogonal chemistry.

PubMed

Li, Jie; Chen, Peng R

2016-03-01

Bioorthogonal chemical reactions are a thriving area of chemical research in recent years as an unprecedented technique to dissect native biological processes through chemistry-enabled strategies. However, current concepts of bioorthogonal chemistry have largely centered on 'bond formation' reactions between two mutually reactive bioorthogonal handles. Recently, in a reverse strategy, a collection of 'bond cleavage' reactions has emerged with excellent biocompatibility. These reactions have expanded our bioorthogonal chemistry repertoire, enabling an array of exciting new biological applications that range from the chemically controlled spatial and temporal activation of intracellular proteins and small-molecule drugs to the direct manipulation of intact cells under physiological conditions. Here we highlight the development and applications of these bioorthogonal cleavage reactions. Furthermore, we lay out challenges and propose future directions along this appealing avenue of research.

Transparent Flexible Electronics By Directed Integration of Inorganic Micro and Nanomaterials

NASA Astrophysics Data System (ADS)

Cole, Jesse J.

This thesis focuses on nanomanufacturing processes for the heterogeneous integration of nanomaterials. Our approaches involved local adjustment of electrostatics at the surfaces to control material flux. Templating of surface electrostatics was implemented differently for three broad concepts resulting in control over nanomaterial synthesis, deposition, and printing. These three general concepts are: (A) Tailored ZnO nanowire synthesis and integration out of the liquid phase; (B) Arc discharge synthesis and continuous nanocluster deposition from the gas phase; (C) Contact electrification and xerographic printing of nanoparticles from the gas phase. Concept (A): We report a method to fabricate and transfer crystalline ZnO with control over location, orientation, size, and shape. The process uses an oxygen plasma treatment in combination with a photoresist pattern on Magnesium-doped GaN substrates to define narrow nucleation regions and attachment points with 100 nanometer scale dimensions. Lateral epitaxial overgrowth follows nucleation to produce single crystalline ZnO which were fabricated into LEDs and photovoltaic cells. Concept (B): We report a gas phase nanoparticle deposition system which shares characteristics with liquid phase electrodeposition. Clusters of charged nanoparticles selectively deposit onto electrically grounded surfaces. Similar to electroplating, the continued deposition of Au nanoparticles onto underlying resistive traces increased overall line conductivity. Alternatively, semiconducting ZnO and Ge nanomaterial sequentially deposited between interdigitated electrodes and served as addressable sensor active areas. Concept (C): We report patterned transfer of charge between conformal material interfaces through a concept referred to as nanocontact electrification. Nanocontacts of different size and shape are formed between surface functionalized polydimethylsiloxane (PDMS) stamps and other dielectric materials (PMMA, SiO 2). Forced delamination and cleavage of the interface yields a well defined charge pattern with a minimal feature size of 100 nm. The process produces charged surfaces and associated fields that exceed the breakdown strength of air leading to strong long range adhesive forces and force distance curves which are recorded over macroscopic distances. The process is applied to fabricate charge patterned surfaces for nanoxerography demonstrating 200 nm resolution nanoparticle prints and applied to thin film electronics where the patterned charges are used to shift the threshold voltages of underlying transistors by over 500 mV.

Regioselectivity of enzymatic and photochemical single electron transfer promoted carbon-carbon bond fragmentation reactions of tetrameric lignin model compounds.

PubMed

Cho, Dae Won; Latham, John A; Park, Hea Jung; Yoon, Ung Chan; Langan, Paul; Dunaway-Mariano, Debra; Mariano, Patrick S

2011-04-15

New types of tetrameric lignin model compounds, which contain the common β-O-4 and β-1 structural subunits found in natural lignins, have been prepared and carbon-carbon bond fragmentation reactions of their cation radicals, formed by photochemical (9,10-dicyanoanthracene) and enzymatic (lignin peroxidase) SET-promoted methods, have been explored. The results show that cation radical intermediates generated from the tetrameric model compounds undergo highly regioselective C-C bond cleavage in their β-1 subunits. The outcomes of these processes suggest that, independent of positive charge and odd-electron distributions, cation radicals of lignins formed by SET to excited states of sensitizers or heme-iron centers in enzymes degrade selectively through bond cleavage reactions in β-1 vs β-O-4 moieties. In addition, the findings made in the enzymatic studies demonstrate that the sterically large tetrameric lignin model compounds undergo lignin peroxidase-catalyzed cleavage via a mechanism involving preliminary formation of an enzyme-substrate complex.

Solid State Research.

DTIC Science & Technology

1982-11-22

48 Fabricated in Zone-Melting-Recrystallized Si Films on Si0 2-Coated Si Substrates V 4. MICROELECTRONICS 55 4.1 Charge-Coupled Devices: Time...OMCVD to the CLEFT (cleavage of lateral epitaxial films for transfer) process, a continuous epitaxial GaAs layer 3 Ym thick has been grown over a...complete-island-etch or local-oxidation-of-Si isolation, that were fabricated in zone-melting-recrystallized Si films on Si02-coated Si substrates. As

Gas phase reactions of doubly charged alkaline earth and transition metal(II)-ligand complexes generated by electrospray ionization

NASA Astrophysics Data System (ADS)

Kohler, Martin; Leary, Julie A.

1997-03-01

Doubly charged metal(II)-complexes of [alpha] 1-3, [alpha] 1-6 mannotriose and the conserved trimannosyl core pentasaccharide as well as doubly charged complexes of Co(II), Mn(II), Ca(II) and Sr(II) with acetonitrile generated by electrospray ionization were studied by low energy collision induced dissociation (CID). Two main fragmentation pathways were observed for the metal(II)-oligosaccharide complexes. Regardless of the coordinating metal, loss of a neutral dehydrohexose residue (162 Da) from the doubly charged precursor ion is observed, forming a doubly charged product ion. However, if the oligosaccharide is coordinated to Co(II) or Mn(II), loss of a dehydroxyhexose cation is also observed. Investigation of the low mass region of the mass spectra of the metal coordinated oligosaccharides revealed intense signals corresponding to [metal(II) + (CH3CN)n2+ (where n = 1-6) species which were being formed by the metal(II) ions and the acetonitrile present in the sample. Analysis of these metal(II)-acetonitrile complexes provided further insight into the processes occurring upon low energy CID of doubly charged metal complexes. The metal(II)-acetonitrile system showed neutral loss and ligand cleavage as observed with the oligosaccharide complexes, as well as a series of six different dissociation mechanisms, most notable among them reduction from [metal(II) + (CH3CN)n2+ to the bare [metal(I)]+ species by electron transfer. Depending on the metal and collision gas chosen, one observes electron transfer from the ligand to the metal, electron transfer from the collision gas to the metal, proton transfer between ligands, heterolytic cleavage of the ligands, reactive collisions and loss of neutral ligands.

DNA-binding and oxidative properties of cationic phthalocyanines and their dimeric complexes with anionic phthalocyanines covalently linked to oligonucleotides.

PubMed

Kuznetsova, A A; Lukyanets, E A; Solovyeva, L I; Knorre, D G; Fedorova, O S

2008-12-01

Design of chemically modified oligonucleotides for regulation of gene expression has attracted considerable attention over the past decades. One actively pursued approach involves antisense or antigene oligonucleotide constructs carrying reactive groups, many of these based on transition metal complexes. The complexes of Fe(II) and Co(II) with phthalocyanines are extremely good catalysts of oxidation of organic compounds with molecular oxygen and hydrogen peroxide. The binding of positively charged Fe(II) and Co(II) phthalocyanines with single- and double-stranded DNA was investigated. It was shown that these phthalocyanines interact with nucleic acids through an outside binding mode. The site-directed modification of single-stranded DNA by O2 and H2O2 in the presence of dimeric complexes of negatively and positively charged Fe(II) and Co(II) phthalocyanines was investigated. These complexes were formed directly on single-stranded DNA through interaction between negatively charged phthalocyanine in conjugate and positively charged phthalocyanine in solution. The resulting oppositely charged phthalocyanine complexes showed significant increase of catalytic activity compared with monomeric forms of phthalocyanines Fe(II) and Co(II). These complexes catalyzed the DNA oxidation with high efficacy and led to direct DNA strand cleavage. It was determined that oxidation of DNA by molecular oxygen catalyzed by complex of Fe(II)-phthalocyanines proceeds with higher rate than in the case of Co(II)-phthalocyanines but the latter led to a greater extent of target DNA modification.

Nanocomplexes of Photolabile Polyelectrolyte and Upconversion Nanoparticles for Near-Infrared Light-Triggered Payload Release.

PubMed

Xiang, Jun; Ge, Feijie; Yu, Bing; Yan, Qiang; Shi, Feng; Zhao, Yue

2018-06-07

A new approach to encapsulating charged cargo molecules into a nanovector and subsequently using near-infrared (NIR) light to trigger the release is demonstrated. NIR light-responsive nanovector was prepared through electrostatic interaction-driven complexation between negatively charged silica-coated upconversion nanoparticles (UCNP@silica, 87 nm hydrodynamic diameter, polydispersity index ∼0.05) and a positively charged UV-labile polyelectrolyte bearing pendants of poly(ethylene glycol) and o-nitrobenzyl side groups; whereas charged fluorescein (FLU) was loaded through a co-complexation process. By controlling the amount of polyelectrolyte, UCNP@silica can be covered by the polymer, whereas remaining dispersed in aqueous solution. Under 980 nm laser excitation, UV light emitted by UCNP is absorbed by photolytic side groups within polyelectrolyte, which results in cleavage of o-nitrobenzyl groups and formation of carboxylic acid groups. Such NIR light-induced partial reversal of positive charge to negative charge on the polyelectrolyte layer disrupts the equilibrium among UCNP@silica, polyelectrolyte, and FLU and, consequently, leads to release of FLU molecules.

Generalized Two-Dimensional Perturbation Correlation Infrared Spectroscopy reveals Mechanisms for the Development of Surface Charge and Recalcitrance in Plant-derived Biochars

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

Harvey, Omar R.; Herbert, Bruce; Kuo, Li-Jung

2012-09-05

Fundamental knowledge of how biochars develop surface-charge and resistance to environmental degradation (or recalcitrance) is crucial to their production for customized applications or, understanding their functions in the environment. Two-dimensional perturbation-based correlation infrared spectroscopy (2D-PCIS) was used to study the biochar formation process in three taxonomically-different plant biomass, under oxygen-limited conditions along a heat-treatment-temperature gradient (HTT; 200-650 oC). Results from 2D-PCIS pointed to the systematic, HTT-induced defragmenting of lignocellulose H-bonding network, and demethylenation/demethylation, oxidation or dehydroxylation/dehydrogenation of lignocellulose fragments as the primary reactions controlling biochar properties along the HTT gradient. The cleavage of OH O-type H-bonds, oxidation of free primarymore » hydroxyls (HTT≤500 oC), and their subsequent dehydrogenation/dehydroxylation (HTT>500 oC) controlled surface charge on the biochars; while the dehydrogenation of methylene groups, which yielded increasingly condensed structures (R-CH2-R →R=CH-R →R=C=R), controlled biochar recalcitrance. Variations in biochar properties across plant biomass type were attributable to taxa-specific transformations. For example, apparent inefficiencies in the cleavage of wood-specific H-bonds, and their subsequent oxidation to carboxyls, lead to lower surface charge in wood biochars (compared to grass biochars). Both non-taxa and taxa-specific transformations highlighted by 2D-PCIS could have significant implications for biochar functioning in fire-impacted or biochar-amended systems.« less

Active site electrostatics protect genome integrity by blocking abortive hydrolysis during DNA recombination

PubMed Central

Ma, Chien-Hui; Rowley, Paul A; Macieszak, Anna; Guga, Piotr; Jayaram, Makkuni

2009-01-01

Water, acting as a rogue nucleophile, can disrupt transesterification steps of important phosphoryl transfer reactions in DNA and RNA. We have unveiled this risk, and identified safeguards instituted against it, during strand cleavage and joining by the tyrosine site-specific recombinase Flp. Strand joining is threatened by a latent Flp endonuclease activity (type I) towards the 3′-phosphotyrosyl intermediate resulting from strand cleavage. This risk is not alleviated by phosphate electrostatics; neutralizing the negative charge on the scissile phosphate through methylphosphonate (MeP) substitution does not stimulate type I endonuclease. Rather, protection derives from the architecture of the recombination synapse and conformational dynamics within it. Strand cleavage is protected against water by active site electrostatics. Replacement of the catalytic Arg-308 of Flp by alanine, along with MeP substitution, elicits a second Flp endonuclease activity (type II) that directly targets the scissile phosphodiester bond in DNA. MeP substitution, combined with appropriate active site mutations, will be useful in revealing anti-hydrolytic mechanisms engendered by systems that mediate DNA relaxation, DNA transposition, site-specific recombination, telomere resolution, RNA splicing and retrohoming of mobile introns. PMID:19440204

Multiple nucleotide preferences determine cleavage-site recognition by the HIV-1 and M-MuLV RNases H.

PubMed

Schultz, Sharon J; Zhang, Miaohua; Champoux, James J

2010-03-19

The RNase H activity of reverse transcriptase is required during retroviral replication and represents a potential target in antiviral drug therapies. Sequence features flanking a cleavage site influence the three types of retroviral RNase H activity: internal, DNA 3'-end-directed, and RNA 5'-end-directed. Using the reverse transcriptases of HIV-1 (human immunodeficiency virus type 1) and Moloney murine leukemia virus (M-MuLV), we evaluated how individual base preferences at a cleavage site direct retroviral RNase H specificity. Strong test cleavage sites (designated as between nucleotide positions -1 and +1) for the HIV-1 and M-MuLV enzymes were introduced into model hybrid substrates designed to assay internal or DNA 3'-end-directed cleavage, and base substitutions were tested at specific nucleotide positions. For internal cleavage, positions +1, -2, -4, -5, -10, and -14 for HIV-1 and positions +1, -2, -6, and -7 for M-MuLV significantly affected RNase H cleavage efficiency, while positions -7 and -12 for HIV-1 and positions -4, -9, and -11 for M-MuLV had more modest effects. DNA 3'-end-directed cleavage was influenced substantially by positions +1, -2, -4, and -5 for HIV-1 and positions +1, -2, -6, and -7 for M-MuLV. Cleavage-site distance from the recessed end did not affect sequence preferences for M-MuLV reverse transcriptase. Based on the identified sequence preferences, a cleavage site recognized by both HIV-1 and M-MuLV enzymes was introduced into a sequence that was otherwise resistant to RNase H. The isolated RNase H domain of M-MuLV reverse transcriptase retained sequence preferences at positions +1 and -2 despite prolific cleavage in the absence of the polymerase domain. The sequence preferences of retroviral RNase H likely reflect structural features in the substrate that favor cleavage and represent a novel specificity determinant to consider in drug design. Copyright (c) 2010 Elsevier Ltd. All rights reserved.

Nop9 is a PUF-like protein that prevents premature cleavage to correctly process pre-18S rRNA

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

Zhang, Jun; McCann, Kathleen L.; Qiu, Chen

Numerous factors direct eukaryotic ribosome biogenesis, and defects in a single ribosome assembly factor may be lethal or produce tissue-specific human ribosomopathies. Pre-ribosomal RNAs (pre-rRNAs) must be processed stepwise and at the correct subcellular locations to produce the mature rRNAs. Nop9 is a conserved small ribosomal subunit biogenesis factor, essential in yeast. Here we report a 2.1-Å crystal structure of Nop9 and a small-angle X-ray-scattering model of a Nop9:RNA complex that reveals a ‘C’-shaped fold formed from 11 Pumilio repeats. We show that Nop9 recognizes sequence and structural features of the 20S pre-rRNA near the cleavage site of the nuclease,more » Nob1. We further demonstrate that Nop9 inhibits Nob1 cleavage, the final processing step to produce mature small ribosomal subunit 18S rRNA. Together, our results suggest that Nop9 is critical for timely cleavage of the 20S pre-rRNA. Moreover, the Nop9 structure exemplifies a new class of Pumilio repeat proteins.« less

A manganese-dependent ribozyme in the 3'-untranslated region of Xenopus Vg1 mRNA.

PubMed

Kolev, Nikolay G; Hartland, Emilia I; Huber, Paul W

2008-10-01

The smallest catalytic RNA identified to date is a manganese-dependent ribozyme that requires only a complex between GAAA and UUU to effect site-specific cleavage. We show here that this ribozyme occurs naturally in the 3'-UTR of Vg1 and beta-actin mRNAs. In accord with earlier studies with model RNAs, cleavage occurs only in the presence of manganese or cadmium ions and proceeds optimally near 30 degrees C and physiological pH. The time course of cleavage in Vg1 mRNA best fits a two-step process in which both steps are first-order. In Vg1 mRNA, the ribozyme is positioned adjacent to a polyadenylation signal, but has no influence on translation of the mRNA in Xenopus oocytes. Putative GAAA ribozyme structures are also near polyadenylation sites in yeast and rat actin mRNAs. Analysis of sequences in the PolyA Cleavage Site and 3'-UTR Database (PACdb) revealed no particular bias in the frequency or distribution of the GAAA motif that would suggest that this ribozyme is currently or was recently used for cleavage to generate processed transcripts. Nonetheless, we speculate that the complementary strands that comprise the ribozyme may account for the origin of sequence elements that direct present-day 3'-end processing of eukaryotic mRNAs.

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

Huang, Yingying; Triscari, Joseph M.; Tseng, George C.

Data mining was performed on 28 330 unique peptide tandem mass spectra for which sequences were assigned with high confidence. By dividing the spectra into different sets based on structural features and charge states of the corresponding peptides, chemical interactions involved in promoting specific cleavage patterns in gas-phase peptides were characterized. Pairwise fragmentation maps describing cleavages at all Xxx-Zzz residue combinations for b and y ions reveal that the difference in basicity between Arg and Lys results in different dissociation patterns for singly charged Arg- and Lys-ending tryptic peptides. While one dominant protonation form (proton localized) exists for Arg-ending peptides,more » a heterogeneous population of different protonated forms or more facile interconversion of protonated forms (proton partially mobile) exists for Lys-ending peptides. Cleavage C-terminal to acidic residues dominates spectra from peptides that have a localized proton and cleavage N-terminal to Pro dominates those that have a mobile or partially mobile proton. When Pro is absent from peptides that have a mobile or partially mobile proton, cleavage at each peptide bond becomes much more prominent. Whether the above patterns can be found in b ions, y ions, or both depends on the location of the proton holder(s). Enhanced cleavages C-terminal to branched aliphatic residues (Ile, Val, Leu) are observed in both b and y ions from peptides that have a mobile proton, as well as in y ions from peptides that have a partially mobile proton; enhanced cleavages N-terminal to these residues are observed in b ions from peptides that have a partially mobile proton. Statistical tools have been designed to visualize the fragmentation maps and measure the similarity between them. The pairwise cleavage patterns observed expand our knowledge of peptide gas-phase fragmentation behaviors and should be useful in algorithm development that employs improved models to predict fragment ion intensities.« less

Protein composition of catalytically active U7-dependent processing complexes assembled on histone pre-mRNA containing biotin and a photo-cleavable linker

PubMed Central

Skrajna, Aleksandra; Yang, Xiao-cui; Dadlez, Michał; Marzluff, William F; Dominski, Zbigniew

2018-01-01

Abstract 3′ end cleavage of metazoan replication-dependent histone pre-mRNAs requires the multi-subunit holo-U7 snRNP and the stem–loop binding protein (SLBP). The exact composition of the U7 snRNP and details of SLBP function in processing remain unclear. To identify components of the U7 snRNP in an unbiased manner, we developed a novel approach for purifying processing complexes from Drosophila and mouse nuclear extracts. In this method, catalytically active processing complexes are assembled in vitro on a cleavage-resistant histone pre-mRNA containing biotin and a photo-sensitive linker, and eluted from streptavidin beads by UV irradiation for direct analysis by mass spectrometry. In the purified processing complexes, Drosophila and mouse U7 snRNP have a remarkably similar composition, always being associated with CPSF73, CPSF100, symplekin and CstF64. Many other proteins previously implicated in the U7-dependent processing are not present. Drosophila U7 snRNP bound to histone pre-mRNA in the absence of SLBP contains the same subset of polyadenylation factors but is catalytically inactive and addition of recombinant SLBP is sufficient to trigger cleavage. This result suggests that Drosophila SLBP promotes a structural rearrangement of the processing complex, resulting in juxtaposition of the CPSF73 endonuclease with the cleavage site in the pre-mRNA substrate. PMID:29529248

Probing surface charge potentials of clay basal planes and edges by direct force measurements.

PubMed

Zhao, Hongying; Bhattacharjee, Subir; Chow, Ross; Wallace, Dean; Masliyah, Jacob H; Xu, Zhenghe

2008-11-18

The dispersion and gelation of clay suspensions have major impact on a number of industries, such as ceramic and composite materials processing, paper making, cement production, and consumer product formulation. To fundamentally understand controlling mechanisms of clay dispersion and gelation, it is necessary to study anisotropic surface charge properties and colloidal interactions of clay particles. In this study, a colloidal probe technique was employed to study the interaction forces between a silica probe and clay basal plane/edge surfaces. A muscovite mica was used as a representative of 2:1 phyllosilicate clay minerals. The muscovite basal plane was prepared by cleavage, while the edge surface was obtained by a microtome cutting technique. Direct force measurements demonstrated the anisotropic surface charge properties of the basal plane and edge surface. For the basal plane, the long-range forces were monotonically repulsive within pH 6-10 and the measured forces were pH-independent, thereby confirming that clay basal planes have permanent surface charge from isomorphic substitution of lattice elements. The measured interaction forces were fitted well with the classical DLVO theory. The surface potentials of muscovite basal plane derived from the measured force profiles were in good agreement with those reported in the literature. In the case of edge surfaces, the measured forces were monotonically repulsive at pH 10, decreasing with pH, and changed to be attractive at pH 5.6, strongly suggesting that the charge on the clay edge surfaces is pH-dependent. The measured force profiles could not be reasonably fitted with the classical DLVO theory, even with very small surface potential values, unless the surface roughness was considered. The surface element integration (SEI) method was used to calculate the DLVO forces to account for the surface roughness. The surface potentials of the muscovite edges were derived by fitting the measured force profiles with the surface element integrated DLVO model. The point of zero charge of the muscovite edge surface was estimated to be pH 7-8.

Improved design of hammerhead ribozyme for selective digestion of target RNA through recognition of site-specific adenosine-to-inosine RNA editing

PubMed Central

Fukuda, Masatora; Kurihara, Kei; Yamaguchi, Shota; Oyama, Yui; Deshimaru, Masanobu

2014-01-01

Adenosine-to-inosine (A-to-I) RNA editing is an endogenous regulatory mechanism involved in various biological processes. Site-specific, editing-state–dependent degradation of target RNA may be a powerful tool both for analyzing the mechanism of RNA editing and for regulating biological processes. Previously, we designed an artificial hammerhead ribozyme (HHR) for selective, site-specific RNA cleavage dependent on the A-to-I RNA editing state. In the present work, we developed an improved strategy for constructing a trans-acting HHR that specifically cleaves target editing sites in the adenosine but not the inosine state. Specificity for unedited sites was achieved by utilizing a sequence encoding the intrinsic cleavage specificity of a natural HHR. We used in vitro selection methods in an HHR library to select for an extended HHR containing a tertiary stabilization motif that facilitates HHR folding into an active conformation. By using this method, we successfully constructed highly active HHRs with unedited-specific cleavage. Moreover, using HHR cleavage followed by direct sequencing, we demonstrated that this ribozyme could cleave serotonin 2C receptor (HTR2C) mRNA extracted from mouse brain, depending on the site-specific editing state. This unedited-specific cleavage also enabled us to analyze the effect of editing state at the E and C sites on editing at other sites by using direct sequencing for the simultaneous quantification of the editing ratio at multiple sites. Our approach has the potential to elucidate the mechanism underlying the interdependencies of different editing states in substrate RNA with multiple editing sites. PMID:24448449

Driving Protein Conformational Changes with Light: Photoinduced Structural Rearrangement in a Heterobimetallic Oxidase.

PubMed

Maugeri, Pearson T; Griese, Julia J; Branca, Rui M; Miller, Effie K; Smith, Zachary R; Eirich, Jürgen; Högbom, Martin; Shafaat, Hannah S

2018-01-31

The heterobimetallic R2lox protein binds both manganese and iron ions in a site-selective fashion and activates oxygen, ultimately performing C-H bond oxidation to generate a tyrosine-valine cross-link near the active site. In this work, we demonstrate that, following assembly, R2lox undergoes photoinduced changes to the active site geometry and metal coordination motif. Through spectroscopic, structural, and mass spectrometric characterization, the photoconverted species is found to consist of a tyrosinate-bound iron center following light-induced decarboxylation of a coordinating glutamate residue and cleavage of the tyrosine-valine cross-link. This process occurs with high quantum efficiencies (Φ = 3%) using violet and near-ultraviolet light, suggesting that the photodecarboxylation is initiated via ligand-to-metal charge transfer excitation. Site-directed mutagenesis and structural analysis suggest that the cross-linked tyrosine-162 is the coordinating residue. One primary product is observed following irradiation, indicating potential use of this class of proteins, which contains a putative substrate channel, for controlled photoinduced decarboxylation processes, with relevance for in vivo functionality of R2lox as well as application in environmental remediation.

Kinetics of hairpin ribozyme cleavage in yeast.

PubMed Central

Donahue, C P; Fedor, M J

1997-01-01

Hairpin ribozymes catalyze a self-cleavage reaction that provides a simple model for quantitative analyses of intracellular mechanisms of RNA catalysis. Decay rates of chimeric mRNAs containing self-cleaving ribozymes give a direct measure of intracellular cleavage kinetics in yeast. Intracellular ribozyme-mediated cleavage occurs at similar rates and shows similar inhibition by ribozyme mutations as ribozyme-mediated reactions in vitro, but only when ribozymes are located in a favorable mRNA sequence context. The impact of cleavage on mRNA abundance is shown to depend directly on intrinsic mRNA stability. Surprisingly, cleavage products are no more labile than uncleaved mRNAs despite the loss of terminal cap structures or poly (A). PMID:9292496

Elucidating Direct Photolysis Mechanisms of Different Dissociation Species of Norfloxacin in Water and Mg2+ Effects by Quantum Chemical Calculations.

PubMed

Wang, Se; Wang, Zhuang

2017-11-11

The study of pollution due to combined antibiotics and metals is urgently needed. Photochemical processes are an important transformation pathway for antibiotics in the environment. The mechanisms underlying the effects of metal-ion complexation on the aquatic photochemical transformation of antibiotics in different dissociation forms are crucial problems in science, and beg solutions. Herein, we investigated the mechanisms of direct photolysis of norfloxacin (NOR) in different dissociation forms in water and metal ion Mg 2+ effects using quantum chemical calculations. Results show that different dissociation forms of NOR had different maximum electronic absorbance wavelengths (NOR 2+ < NOR⁰ < NOR⁺) and showed different photolysis reactivity. Analysis of transition states (TS) and reaction activation energies ( E a ) indicated NOR⁺ generally underwent loss of the piperazine ring (C10-N13 bond cleavage) and damage to piperazine ring (N13-C14 bond cleavage). For NOR 2+ , the main direct photolysis pathways were de-ethylation (N7-C8 bond cleavage) and decarboxylation (C2-C5 bond cleavage). Furthermore, the presence of Mg 2+ changed the order of the wavelength at maximum electronic absorbance (NOR⁺-Mg 2+ < NOR⁰-Mg 2+ < NOR 2+ -Mg 2+ ) and increased the intensities of absorbance peaks of all three dissociation species of NOR, implying that Mg 2+ played an important role in the direct photolysis of NOR⁰, NOR⁺, and NOR 2+ . The calculated TS results indicated that the presence of Mg 2+ increased E a for most direct photolysis pathways of NOR, while it decreased E a for some direct photolysis pathways such as the loss of the piperazine ring and the damage of the piperazine ring of NOR⁰ and the defluorination of NOR⁺.

Leveraging Electron Transfer Dissociation for Site Selective Radical Generation: Applications for Peptide Epimer Analysis

NASA Astrophysics Data System (ADS)

Lyon, Yana A.; Beran, Gregory; Julian, Ryan R.

2017-07-01

Traditional electron-transfer dissociation (ETD) experiments operate through a complex combination of hydrogen abundant and hydrogen deficient fragmentation pathways, yielding c and z ions, side-chain losses, and disulfide bond scission. Herein, a novel dissociation pathway is reported, yielding homolytic cleavage of carbon-iodine bonds via electronic excitation. This observation is very similar to photodissociation experiments where homolytic cleavage of carbon-iodine bonds has been utilized previously, but ETD activation can be performed without addition of a laser to the mass spectrometer. Both loss of iodine and loss of hydrogen iodide are observed, with the abundance of the latter product being greatly enhanced for some peptides after additional collisional activation. These observations suggest a novel ETD fragmentation pathway involving temporary storage of the electron in a charge-reduced arginine side chain. Subsequent collisional activation of the peptide radical produced by loss of HI yields spectra dominated by radical-directed dissociation, which can be usefully employed for identification of peptide isomers, including epimers.

Formation of Pmel17 Amyloid Is Regulated by Juxtamembrane Metalloproteinase Cleavage, and the Resulting C-terminal Fragment Is a Substrate for γ-Secretase*

PubMed Central

Kummer, Markus P.; Maruyama, Hiroko; Huelsmann, Claudia; Baches, Sandra; Weggen, Sascha; Koo, Edward H.

2009-01-01

The formation of insoluble cross β-sheet amyloid is pathologically associated with disorders such as Alzheimer, Parkinson, and Huntington diseases. One exception is the nonpathological amyloid derived from the protein Pmel17 within melanosomes to generate melanin pigment. Here we show that the formation of insoluble MαC intracellular fragments of Pmel17, which are the direct precursors to Pmel17 amyloid, depends on a novel juxtamembrane cleavage at amino acid position 583 between the furin-like proprotein convertase cleavage site and the transmembrane domain. The resulting Pmel17 C-terminal fragment is then processed by the γ-secretase complex to release a short-lived intracellular domain fragment. Thus, by analogy to the Notch receptor, we designate this cleavage the S2 cleavage site, whereas γ-secretase mediates proteolysis at the intramembrane S3 site. Substitutions or deletions at this S2 cleavage site, the use of the metalloproteinase inhibitor TAPI-2, as well as small interfering RNA-mediated knock-down of the metalloproteinases ADAM10 and 17 reduced the formation of insoluble Pmel17 fragments. These results demonstrate that the release of the Pmel17 ectodomain, which is critical for melanin amyloidogenesis, is initiated by S2 cleavage at a juxtamembrane position. PMID:19047044

alpha-Putrescinylthymine and the sensitivity of bacteriophage phi W-14 DNA to restriction endonucleases.

PubMed Central

Miller, P B; Wakarchuk, W W; Warren, R A

1985-01-01

The modified base alpha-putrescinylthymine (putT) in phi W-14 DNA blocks cleavage of the DNA by 17 of 32 Type II restriction endonucleases. The enzymes cleaving the DNA do so to widely varying extents. The frequencies of cleavage of three altered forms of the DNA show that putT blocks recognition sites either when it occurs within the site or when it is in a sequence flanking the site. The blocking is dependent on both charge and steric factors. The charge effects can be greater than the steric effects for some of the enzymes tested. All the enzymes cleaving phi W-14 DNA release discrete fragments, showing that the distribution of putT is ordered. The cleavage frequencies for different enzymes suggest that the sequence CAputTG occurs frequently in the DNA. Only TaqI of the enzymes tested appeared not to be blocked by putT, but it was slowed down. TaqI generated fragments are joinable by T4 DNA ligase. PMID:2987859

Detection of nucleic acids by multiple sequential invasive cleavages

DOEpatents

Hall, Jeff G.; Lyamichev, Victor I.; Mast, Andrea L.; Brow, Mary Ann D.

1999-01-01

The present invention relates to means for the detection and characterization of nucleic acid sequences, as well as variations in nucleic acid sequences. The present invention also relates to methods for forming a nucleic acid cleavage structure on a target sequence and cleaving the nucleic acid cleavage structure in a site-specific manner. The structure-specific nuclease activity of a variety of enzymes is used to cleave the target-dependent cleavage structure, thereby indicating the presence of specific nucleic acid sequences or specific variations thereof. The present invention further relates to methods and devices for the separation of nucleic acid molecules based on charge. The present invention also provides methods for the detection of non-target cleavage products via the formation of a complete and activated protein binding region. The invention further provides sensitive and specific methods for the detection of human cytomegalovirus nucleic acid in a sample.

Detection of nucleic acids by multiple sequential invasive cleavages 02

DOEpatents

Hall, Jeff G.; Lyamichev, Victor I.; Mast, Andrea L.; Brow, Mary Ann D.

2002-01-01

The present invention relates to means for the detection and characterization of nucleic acid sequences, as well as variations in nucleic acid sequences. The present invention also relates to methods for forming a nucleic acid cleavage structure on a target sequence and cleaving the nucleic acid cleavage structure in a site-specific manner. The structure-specific nuclease activity of a variety of enzymes is used to cleave the target-dependent cleavage structure, thereby indicating the presence of specific nucleic acid sequences or specific variations thereof. The present invention further relates to methods and devices for the separation of nucleic acid molecules based on charge. The present invention also provides methods for the detection of non-target cleavage products via the formation of a complete and activated protein binding region. The invention further provides sensitive and specific methods for the detection of human cytomegalovirus nucleic acid in a sample.

Detection of nucleic acids by multiple sequential invasive cleavages

DOEpatents

Hall, Jeff G; Lyamichev, Victor I; Mast, Andrea L; Brow, Mary Ann D

2012-10-16

The present invention relates to means for the detection and characterization of nucleic acid sequences, as well as variations in nucleic acid sequences. The present invention also relates to methods for forming a nucleic acid cleavage structure on a target sequence and cleaving the nucleic acid cleavage structure in a site-specific manner. The structure-specific nuclease activity of a variety of enzymes is used to cleave the target-dependent cleavage structure, thereby indicating the presence of specific nucleic acid sequences or specific variations thereof. The present invention further relates to methods and devices for the separation of nucleic acid molecules based on charge. The present invention also provides methods for the detection of non-target cleavage products via the formation of a complete and activated protein binding region. The invention further provides sensitive and specific methods for the detection of human cytomegalovirus nucleic acid in a sample.

Sequences downstream of AAUAAA signals affect pre-mRNA cleavage and polyadenylation in vitro both directly and indirectly.

PubMed Central

Ryner, L C; Takagaki, Y; Manley, J L

1989-01-01

To investigate the role of sequences lying downstream of the conserved AAUAAA hexanucleotide in pre-mRNA cleavage and polyadenylation, deletions or substitutions were constructed in polyadenylation signals from simian virus 40 and adenovirus, and their effects were assayed in both crude and fractionated HeLa cell nuclear extracts. As expected, these sequences influenced the efficiency of both cleavage and polyadenylation as well as the accuracy of the cleavage reaction. Sequences near or upstream of the actual site of poly(A) addition appeared to specify a unique cleavage site, since their deletion resulted, in some cases, in heterogeneous cleavage. Furthermore, the sequences that allowed the simian virus 40 late pre-RNA to be cleaved preferentially by partially purified cleavage activity were also those at the cleavage site itself. Interestingly, sequences downstream of the cleavage site interacted with factors not directly involved in catalyzing cleavage and polyadenylation, since the effects of deletions were substantially diminished when partially purified components were used in assays. In addition, these sequences contained elements that could affect 3'-end formation both positively and negatively. Images PMID:2566911

Directed evolution of an RNA enzyme

NASA Technical Reports Server (NTRS)

Beaudry, Amber A.; Joyce, Gerald F.

1992-01-01

An in vitro evolution procedures was used to obtain RNA enzymes with a particular catalytic function. A population of 10 exp 13 variants of the Tetrahymena ribozyme, a group I ribozyme that catalyzes sequence-specific cleavage of RNA via a phosphoester transfer mechanism, was generated. This enzyme has a limited ability to cleave DNA under conditions of high temperature or high MgCl2 concentration, or both. A selection constraint was imposed on the population of ribozyme variants such that only those individuals that carried out DNA cleavage under physiologic conditions were amplified to produce 'progeny' ribozymes. Mutations were introduced during amplification to maintain heterogeneity in the population. This process was repeated for ten successive generations, resulting in enhanced (100 times) DNA cleavage activity.

Metastable Atom-Activated Dissociation Mass Spectrometry of Phosphorylated and Sulfonated Peptides in Negative Ion Mode

NASA Astrophysics Data System (ADS)

Cook, Shannon L.; Jackson, Glen P.

2011-06-01

The dissociation behavior of phosphorylated and sulfonated peptide anions was explored using metastable atom-activated dissociation mass spectrometry (MAD-MS) and collision-induced dissociation (CID). A beam of high kinetic energy helium (He) metastable atoms was exposed to isolated phosphorylated and sulfonated peptides in the 3- and 2- charge states. Unlike CID, where phosphate losses are dominant, the major dissociation channels observed using MAD were Cα - C peptide backbone cleavages and neutral losses of CO2, H2O, and [CO2 + H2O] from the charge reduced (oxidized) product ion, consistent with an electron detachment dissociation (EDD) mechanism such as Penning ionization. Regardless of charge state or modification, MAD provides ample backbone cleavages with little modification loss, which allows for unambiguous PTM site determination. The relative abundance of certain fragment ions in MAD is also demonstrated to be somewhat sensitive to the number and location of deprotonation sites, with backbone cleavage somewhat favored adjacent to deprotonated sites like aspartic acid residues. MAD provides a complementary dissociation technique to CID, ECD, ETD, and EDD for peptide sequencing and modification identification. MAD offers the unique ability to analyze highly acidic peptides that contain few to no basic amino acids in either negative or positive ion mode.

The Dimer Interfaces of Protease and Extra-Protease Domains Influence the Activation of Protease and the Specificity of GagPol Cleavage

PubMed Central

Pettit, Steven C.; Gulnik, Sergei; Everitt, Lori; Kaplan, Andrew H.

2003-01-01

Activation of the human immunodeficiency virus type 1 (HIV-1) protease is an essential step in viral replication. As is the case for all retroviral proteases, enzyme activation requires the formation of protease homodimers. However, little is known about the mechanisms by which retroviral proteases become active within their precursors. Using an in vitro expression system, we have examined the determinants of activation efficiency and the order of cleavage site processing for the protease of HIV-1 within the full-length GagPol precursor. Following activation, initial cleavage occurs between the viral p2 and nucleocapsid proteins. This is followed by cleavage of a novel site located in the transframe domain. Mutational analysis of the dimer interface of the protease produced differential effects on activation and specificity. A subset of mutations produced enhanced cleavage at the amino terminus of the protease, suggesting that, in the wild-type precursor, cleavages that liberate the protease are a relatively late event. Replacement of the proline residue at position 1 of the protease dimer interface resulted in altered cleavage of distal sites and suggests that this residue functions as a cis-directed specificity determinant. In summary, our studies indicate that interactions within the protease dimer interface help determine the order of precursor cleavage and contribute to the formation of extended-protease intermediates. Assembly domains within GagPol outside the protease domain also influence enzyme activation. PMID:12477841

The dimer interfaces of protease and extra-protease domains influence the activation of protease and the specificity of GagPol cleavage.

PubMed

Pettit, Steven C; Gulnik, Sergei; Everitt, Lori; Kaplan, Andrew H

2003-01-01

Activation of the human immunodeficiency virus type 1 (HIV-1) protease is an essential step in viral replication. As is the case for all retroviral proteases, enzyme activation requires the formation of protease homodimers. However, little is known about the mechanisms by which retroviral proteases become active within their precursors. Using an in vitro expression system, we have examined the determinants of activation efficiency and the order of cleavage site processing for the protease of HIV-1 within the full-length GagPol precursor. Following activation, initial cleavage occurs between the viral p2 and nucleocapsid proteins. This is followed by cleavage of a novel site located in the transframe domain. Mutational analysis of the dimer interface of the protease produced differential effects on activation and specificity. A subset of mutations produced enhanced cleavage at the amino terminus of the protease, suggesting that, in the wild-type precursor, cleavages that liberate the protease are a relatively late event. Replacement of the proline residue at position 1 of the protease dimer interface resulted in altered cleavage of distal sites and suggests that this residue functions as a cis-directed specificity determinant. In summary, our studies indicate that interactions within the protease dimer interface help determine the order of precursor cleavage and contribute to the formation of extended-protease intermediates. Assembly domains within GagPol outside the protease domain also influence enzyme activation.

Crystal structures of yellowtail ascites virus VP4 protease: trapping an internal cleavage site trans acyl-enzyme complex in a native Ser/Lys dyad active site.

PubMed

Chung, Ivy Yeuk Wah; Paetzel, Mark

2013-05-03

Yellowtail ascites virus (YAV) is an aquabirnavirus that causes ascites in yellowtail, a fish often used in sushi. Segment A of the YAV genome codes for a polyprotein (pVP2-VP4-VP3), where processing by its own VP4 protease yields the capsid protein precursor pVP2, the ribonucleoprotein-forming VP3, and free VP4. VP4 protease utilizes the rarely observed serine-lysine catalytic dyad mechanism. Here we have confirmed the existence of an internal cleavage site, preceding the VP4/VP3 cleavage site. The resulting C-terminally truncated enzyme (ending at Ala(716)) is active, as shown by a trans full-length VP4 cleavage assay and a fluorometric peptide cleavage assay. We present a crystal structure of a native active site YAV VP4 with the internal cleavage site trapped as trans product complexes and trans acyl-enzyme complexes. The acyl-enzyme complexes confirm directly the role of Ser(633) as the nucleophile. A crystal structure of the lysine general base mutant (K674A) reveals the acyl-enzyme and empty binding site states of VP4, which allows for the observation of structural changes upon substrate or product binding. These snapshots of three different stages in the VP4 protease reaction mechanism will aid in the design of anti-birnavirus compounds, provide insight into previous site-directed mutagenesis results, and contribute to understanding of the serine-lysine dyad protease mechanism. In addition, we have discovered that this protease contains a channel that leads from the enzyme surface (adjacent to the substrate binding groove) to the active site and the deacylating water.

Cleavage-site specificity of prolyl endopeptidase FAP investigated with a full-length protein substrate.

PubMed

Huang, Chih-Hsiang; Suen, Ching-Shu; Lin, Ching-Ting; Chien, Chia-Hui; Lee, Hsin-Ying; Chung, Kuei-Min; Tsai, Ting-Yueh; Jiaang, Weir-Tong; Hwang, Ming-Jing; Chen, Xin

2011-06-01

Fibroblast activation protein (FAP) is a prolyl-cleaving endopeptidase proposed as an anti-cancer drug target. It is necessary to define its cleavage-site specificity to facilitate the identification of its in vivo substrates and to understand its biological functions. We found that the previously identified substrate of FAP, α(2)-anti-plasmin, is not a robust substrate in vitro. Instead, an intracellular protein, SPRY2, is cleavable by FAP and more suitable for investigation of its substrate specificity in the context of the full-length globular protein. FAP prefers uncharged residues, including small or bulky hydrophobic amino acids, but not charged amino acids, especially acidic residue at P1', P3 and P4 sites. Molecular modelling analysis shows that the substrate-binding site of FAP is surrounded by multiple tyrosine residues and some negatively charged residues, which may exert least preference for substrates with acidic residues. This provides an explanation why FAP cannot cleave interleukins, which have a glutamate at either P4 or P2', despite their P3-P2-P1 sites being identical to SPRY2 or α-AP. Our study provided new information on FAP cleavage-site specificity, which differs from the data obtained by profiling with a peptide library or with the denatured protein, gelatin, as the substrate. Furthermore, our study suggests that negatively charged residues should be avoided when designing FAP inhibitors.

Domainal cleavage as an Anisotropic Reaction-diffusion Process

NASA Astrophysics Data System (ADS)

Mulchrone, Kieran; Meere, Patrick

2017-04-01

Domainal cleavage comprises zones dominated by quartz and feldspar (QF-domains) and zones dominated by Mica (M-domains) which form at low metamorphic grades. The protolith is typically fairly homogeneous mudstone, siltstone, sandstone or limestone. Wet diffusion or pressure solution along grain boundaries is a key mechanism in the development of domanial cleavage. However, this does not explain why M-domains become sub-regularly spaced, visually evident in coarser-grained rocks, and take on an anastomising morphology. The ratio of M to QF-domains by volume can range from 1 to 0.1 and lower i.e. in extreme cases M-domains are intermittent but regularly spaced. It is suggested here that an anisotropic reaction-diffusion process model can explain these features. The imposed stress field instantaneously leads to anisotropy of diffusion by narrowing intergranular channels perpendicular to the principal stress. This leads to a preferred diffusion of chemicals parallel to the principal stress direction and lower diffusion rates in the normal direction. Combining this with the chemical reaction of pressure solution produces an anisotropic reaction-diffusion system. Both isotropic and anistropic reaction diffusion systems lead to pattern formation as discovered by Alan Turing on the 1950's as an explanation for patterns found in animal skins such as spots and stripes. Thus domanial cleavage is a striped pattern induced by diffusion anisotropy combined with a chemical reaction. Furthermore, rates of chemical reaction in intergranular fluids is likely to be many orders of magnitude greater that rates of deformation. Therefore we expect domanial cleavage to form relatively rapidly. As deformation progresses the M-domains behave less competently and may be the site of enhanced shearing. An example from Co. Cork, Ireland demonstrates shear folding in low-grade metasedimentary rocks with reverse shear along M-domains at a high angle to the maximum compressive stress.

Investigation of the Mechanism of Electron Capture and Electron Transfer Dissociation of Peptides with a Covalently Attached Free Radical Hydrogen Atom Scavenger.

PubMed

Sohn, Chang Ho; Yin, Sheng; Peng, Ivory; Loo, Joseph A; Beauchamp, J L

2015-11-15

The mechanisms of electron capture and electron transfer dissociation (ECD and ETD) are investigated by covalently attaching a free-radical hydrogen atom scavenger to a peptide. The 2,2,6,6-tetramethylpiperidin-l-oxyl (TEMPO) radical was chosen as the scavenger due to its high hydrogen atom affinity (ca. 280 kJ/mol) and low electron affinity (ca. 0.45 ev), and was derivatized to the model peptide, FQX TEMPO EEQQQTEDELQDK. The X TEMPO residue represents a cysteinyl residue derivatized with an acetamido-TEMPO group. The acetamide group without TEMPO was also examined as a control. The gas phase proton affinity (882 kJ/mol) of TEMPO is similar to backbone amide carbonyls (889 kJ/mol), minimizing perturbation to internal solvation and sites of protonation of the derivatized peptides. Collision induced dissociation (CID) of the TEMPO tagged peptide dication generated stable odd-electron b and y type ions without indication of any TEMPO radical induced fragmentation initiated by hydrogen abstraction. The type and abundance of fragment ions observed in the CID spectra of the TEMPO and acetamide tagged peptides are very similar. However, ECD of the TEMPO labeled peptide dication yielded no backbone cleavage. We propose that a labile hydrogen atom in the charge reduced radical ions is scavenged by the TEMPO radical moiety, resulting in inhibition of N-C α backbone cleavage processes. Supplemental activation after electron attachment (ETcaD) and CID of the charge-reduced precursor ion generated by electron transfer of the TEMPO tagged peptide dication produced a series of b + H (b H ) and y + H (y H ) ions along with some c ions having suppressed intensities, consistent with stable O-H bond formation at the TEMPO group. In summary, the results indicate that ECD and ETD backbone cleavage processes are inhibited by scavenging of a labile hydrogen atom by the localized TEMPO radical moiety. This observation supports the conjecture that ECD and ETD processes involve long-lived intermediates formed by electron capture/transfer in which a labile hydrogen atom is present and plays a key role with low energy processes leading to c and z ion formation. Ab initio and density functional calculations are performed to support our conclusion, which depends most importantly on the proton affinity, electron affinity and hydrogen atom affinity of the TEMPO moiety.

Phosphoinositide function in cytokinesis.

PubMed

Brill, Julie A; Wong, Raymond; Wilde, Andrew

2011-11-22

In systems as diverse as yeast, slime mold and animal cells, the levels and distribution of phosphatidylinositol phosphates (PIPs) must be strictly regulated for successful cell cleavage. The precise mechanism by which PIPs function in this process remains unknown. Recent experiments are beginning to shed light on the cellular pathways in which PIPs make key contributions during cytokinesis. In particular, PIPs promote proper actin cytoskeletal organization and direct membrane trafficking in dividing cells. Future research will uncover temporal and spatial regulation of the different PIPs, thus elucidating their role in cytoskeletal and membrane events that drive cell cleavage. Copyright © 2011 Elsevier Ltd. All rights reserved.

Dipole and Coulomb forces in electron capture dissociation and electron transfer dissociation mass spectroscopy.

PubMed

Świerszcz, Iwona; Skurski, Piotr; Simons, Jack

2012-02-23

Ab initio electronic structure calculations were performed on a doubly charged polypeptide model H(+)-Lys(Ala)(19)-CO-CH(NH(2))-CH(2)-SS-CH(2)-(NH(2))CH-CO-(Ala)(19)-Lys-H(+) consisting of a C-terminal protonated Lys followed by a 19-Ala α-helix with a 20th Ala-like unit whose side chain is linked by a disulfide bond to a corresponding Ala-like unit connected to a second 19-Ala α-helix terminated by a second C-terminal-protonated Lys. The Coulomb potentials arising from the two charged Lys residues and dipole potentials arising from the two oppositely directed 72 D dipoles of the α-helices act to stabilize the SS bond's σ* orbital. The Coulomb potentials provide stabilization of 1 eV, while the two large dipoles generate an additional 4 eV. Such stabilization allows the SS σ* orbital to attach an electron and thereby generate disulfide bond cleavage products. Although calculations are performed only on SS bond cleavage, discussion of N-C(α) bond cleavage caused by electron attachment to amide π* orbitals is also presented. The magnitudes of the stabilization energies as well as the fact that they arise from Coulomb and dipole potentials are supported by results on a small model system consisting of a H(3)C-SS-CH(3) molecule with positive and negative fractional point charges to its left and right designed to represent (i) two positive charges ca. 32 Å distant (i.e., the two charged Lys sites of the peptide model) and (ii) two 72 D dipoles (i.e., the two α-helices). Earlier workers suggested that internal dipole forces in polypeptides could act to guide incoming free electrons (i.e., in electron capture dissociation (ECD)) toward the positive end of the dipole and thus affect the branching ratios for cleaving various bonds. Those workers argued that, because of the huge mass difference between an anion donor and a free electron, internal dipole forces would have a far smaller influence over the trajectory of a donor (i.e., in electron transfer dissociation (ETD)). The present findings suggest that, in addition to their effects on guiding electron or donor trajectories, dipole potentials (in combination with Coulomb potentials) also alter the energies of SS σ* and amide π* orbitals, which then affects the ability of these orbitals to bind an electron. Thus, both by trajectory-guiding and by orbital energy stabilization, Coulomb and dipole potentials can have significant influences on the branching ratios of ECD and ETC in which disulfide or N-C(α) bonds are cleaved. © 2012 American Chemical Society

Photo- and radiation chemical induced degradation of lignin model compounds.

PubMed

Lanzalunga; Bietti, M

2000-07-01

The basic mechanistic aspects of the photo- and radiation chemistry of lignin model compounds (LMCs) are discussed with respect to important processes related to lignin degradation. Several reactions occur after direct irradiation, photosensitized or radiation chemically induced oxidation of LMCs. Direct irradiation studies on LMCs have provided supportive evidence for the involvement of hydrogen abstraction reactions from phenols, beta-cleavage of substituted alpha-aryloxyacetophenones and cleavage of ketyl radicals (formed by photoreduction of aromatic ketones or hydrogen abstraction from arylglycerol beta-aryl ethers) in the photoyellowing of lignin rich pulps. Photosensitized and radiation chemically induced generation of reactive oxygen species and their reaction with LMCs are reviewed. The side-chain reactivity of LMC radical cations, generated by radiation chemical means, is also discussed in relation with the enzymatic degradation of lignin.

A Nanoindentation Study of the Plastic Deformation and Fracture Mechanisms in Single-Crystalline CaFe2As2

NASA Astrophysics Data System (ADS)

Frawley, Keara G.; Bakst, Ian; Sypek, John T.; Vijayan, Sriram; Weinberger, Christopher R.; Canfield, Paul C.; Aindow, Mark; Lee, Seok-Woo

2018-04-01

The plastic deformation and fracture mechanisms in single-crystalline CaFe2As2 has been studied using nanoindentation and density functional theory simulations. CaFe2As2 single crystals were grown in a Sn-flux, resulting in homogeneous and nearly defect-free crystals. Nanoindentation along the [001] direction produces strain bursts, radial cracking, and lateral cracking. Ideal cleavage simulations along the [001] and [100] directions using density functional theory calculations revealed that cleavage along the [001] direction requires a much lower stress than cleavage along the [100] direction. This strong anisotropy of cleavage strength implies that CaFe2As2 has an atomic-scale layered structure, which typically exhibits lateral cracking during nanoindentation. This special layered structure results from weak atomic bonding between the (001) Ca and Fe2As2 layers.

A Nanoindentation Study of the Plastic Deformation and Fracture Mechanisms in Single-Crystalline CaFe 2As 2

DOE PAGES

Frawley, Keara G.; Bakst, Ian; Sypek, John T.; ...

2018-04-10

In this paper, the plastic deformation and fracture mechanisms in single-crystalline CaFe 2As 2 has been studied using nanoindentation and density functional theory simulations. CaFe 2As 2 single crystals were grown in a Sn-flux, resulting in homogeneous and nearly defect-free crystals. Nanoindentation along the [001] direction produces strain bursts, radial cracking, and lateral cracking. Ideal cleavage simulations along the [001] and [100] directions using density functional theory calculations revealed that cleavage along the [001] direction requires a much lower stress than cleavage along the [100] direction. This strong anisotropy of cleavage strength implies that CaFe 2As 2 has an atomic-scalemore » layered structure, which typically exhibits lateral cracking during nanoindentation. This special layered structure results from weak atomic bonding between the (001) Ca and Fe 2As 2 layers.« less

A Nanoindentation Study of the Plastic Deformation and Fracture Mechanisms in Single-Crystalline CaFe 2As 2

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

Frawley, Keara G.; Bakst, Ian; Sypek, John T.

In this paper, the plastic deformation and fracture mechanisms in single-crystalline CaFe 2As 2 has been studied using nanoindentation and density functional theory simulations. CaFe 2As 2 single crystals were grown in a Sn-flux, resulting in homogeneous and nearly defect-free crystals. Nanoindentation along the [001] direction produces strain bursts, radial cracking, and lateral cracking. Ideal cleavage simulations along the [001] and [100] directions using density functional theory calculations revealed that cleavage along the [001] direction requires a much lower stress than cleavage along the [100] direction. This strong anisotropy of cleavage strength implies that CaFe 2As 2 has an atomic-scalemore » layered structure, which typically exhibits lateral cracking during nanoindentation. This special layered structure results from weak atomic bonding between the (001) Ca and Fe 2As 2 layers.« less

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

Manolopoulou, Marika; Guo, Qing; Malito, Enrico

Insulin is a hormone vital for glucose homeostasis, and insulin-degrading enzyme (IDE) plays a key role in its clearance. IDE exhibits a remarkable specificity to degrade insulin without breaking the disulfide bonds that hold the insulin A and B chains together. Using Fourier transform ion cyclotron resonance (FTICR) mass spectrometry to obtain high mass accuracy, and electron capture dissociation (ECD) to selectively break the disulfide bonds in gas phase fragmentation, we determined the cleavage sites and composition of human insulin fragments generated by human IDE. Our time-dependent analysis of IDE-digested insulin fragments reveals that IDE is highly processive in itsmore » initial cleavage at the middle of both the insulin A and B chains. This ensures that IDE effectively splits insulin into inactive N- and C-terminal halves without breaking the disulfide bonds. To understand the molecular basis of the recognition and unfolding of insulin by IDE, we determined a 2.6-A resolution insulin-bound IDE structure. Our structure reveals that IDE forms an enclosed catalytic chamber that completely engulfs and intimately interacts with a partially unfolded insulin molecule. This structure also highlights how the unique size, shape, charge distribution, and exosite of the IDE catalytic chamber contribute to its high affinity ( approximately 100 nm) for insulin. In addition, this structure shows how IDE utilizes the interaction of its exosite with the N terminus of the insulin A chain as well as other properties of the catalytic chamber to guide the unfolding of insulin and allowing for the processive cleavages.« less

Copper-catalyzed transformation of ketones to amides via C(CO)-C(alkyl) bond cleavage directed by picolinamide.

PubMed

Ma, Haojie; Zhou, Xiaoqiang; Zhan, Zhenzhen; Wei, Daidong; Shi, Chong; Liu, Xingxing; Huang, Guosheng

2017-09-13

Copper catalyzed chemoselective cleavage of the C(CO)-C(alkyl) bond leading to C-N bond formation with chelation assistance of N-containing directing groups is described. Inexpensive Cu(ii)-acetate serves as a convenient catalyst for this transformation. This method highlights the emerging strategy to transform unactivated alkyl ketones into amides in organic synthesis and provides a new strategy for C-C bond cleavage.

Coat protein expression strategy of oat blue dwarf virus.

PubMed

Edwards, Michael C; Weiland, John J

2014-02-01

Oat blue dwarf virus (OBDV) is a member of the genus Marafivirus whose genome encodes a 227 kDa polyprotein (p227) ostensibly processed post-translationally into its functional components. Encoded near the 3' terminus and coterminal with the p227 ORF are ORFs specifying major and minor capsid proteins (CP). Since the CP expression strategy of marafiviruses has not been thoroughly investigated, we produced a series of point mutants in the OBDV CP encoding gene and examined expression in protoplasts. Results support a model in which the 21 kDa major CP is the product of direct translation of a sgRNA, while the 24 kDa minor CP is a cleavage product derived from both the polyprotein and a larger ~26 kDa precursor translated directly from the sgRNA. Cleavage occurs at an LXG[G/A] motif conserved in many viruses that use papain-like proteases for polyprotein processing and protection against degradation via the ubiquitin-proteasome system. Published by Elsevier Inc.

Herpes simplex virus DNA packaging sequences adopt novel structures that are specifically recognized by a component of the cleavage and packaging machinery.

PubMed

Adelman, K; Salmon, B; Baines, J D

2001-03-13

The product of the herpes simplex virus type 1 U(L)28 gene is essential for cleavage of concatemeric viral DNA into genome-length units and packaging of this DNA into viral procapsids. To address the role of U(L)28 in this process, purified U(L)28 protein was assayed for the ability to recognize conserved herpesvirus DNA packaging sequences. We report that DNA fragments containing the pac1 DNA packaging motif can be induced by heat treatment to adopt novel DNA conformations that migrate faster than the corresponding duplex in nondenaturing gels. Surprisingly, these novel DNA structures are high-affinity substrates for U(L)28 protein binding, whereas double-stranded DNA of identical sequence composition is not recognized by U(L)28 protein. We demonstrate that only one strand of the pac1 motif is responsible for the formation of novel DNA structures that are bound tightly and specifically by U(L)28 protein. To determine the relevance of the observed U(L)28 protein-pac1 interaction to the cleavage and packaging process, we have analyzed the binding affinity of U(L)28 protein for pac1 mutants previously shown to be deficient in cleavage and packaging in vivo. Each of the pac1 mutants exhibited a decrease in DNA binding by U(L)28 protein that correlated directly with the reported reduction in cleavage and packaging efficiency, thereby supporting a role for the U(L)28 protein-pac1 interaction in vivo. These data therefore suggest that the formation of novel DNA structures by the pac1 motif confers added specificity on recognition of DNA packaging sequences by the U(L)28-encoded component of the herpesvirus cleavage and packaging machinery.

Nucleic acid detection kits

DOEpatents

Hall, Jeff G.; Lyamichev, Victor I.; Mast, Andrea L.; Brow, Mary Ann; Kwiatkowski, Robert W.; Vavra, Stephanie H.

2005-03-29

The present invention relates to means for the detection and characterization of nucleic acid sequences, as well as variations in nucleic acid sequences. The present invention also relates to methods for forming a nucleic acid cleavage structure on a target sequence and cleaving the nucleic acid cleavage structure in a site-specific manner. The structure-specific nuclease activity of a variety of enzymes is used to cleave the target-dependent cleavage structure, thereby indicating the presence of specific nucleic acid sequences or specific variations thereof. The present invention further relates to methods and devices for the separation of nucleic acid molecules based on charge. The present invention also provides methods for the detection of non-target cleavage products via the formation of a complete and activated protein binding region. The invention further provides sensitive and specific methods for the detection of nucleic acid from various viruses in a sample.

Cleavage crystallography of liquid metal embrittled aluminum alloys

NASA Technical Reports Server (NTRS)

Reynolds, A. P.; Stoner, G. E.

1991-01-01

The crystallography of liquid metal-induced transgranular cleavage in six aluminum alloys having a variety of microstructures has been determined via Laue X-ray back reflection. The cleavage crystallography was independent of alloy microstructure, and the cleavage plane was 100-plane oriented in all cases. It was further determined that the cleavage crystallography was not influenced by alloy texture. Examination of the fracture surface indicated that there was not a unique direction of crack propagation. In addition, the existence of 100-plane cleavage on alloy 2024 fracture surfaces was inferred by comparison of secondary cleavage crack intersection geometry on the 2024 surfaces with the geometry of secondary cleavage crack intersections on the test alloys.

Diastereoselective DNA Cleavage Recognition by Ni(II)•Gly-Gly-His Derived Metallopeptides

PubMed Central

Fang, Ya-Yin; Claussen, Craig A.; Lipkowitz, Kenny B.; Long, Eric C.

2008-01-01

Site-selective DNA cleavage by diastereoisomers of Ni(II)•Gly-Gly-His-derived metallopeptides was investigated through high-resolution gel analyses and molecular dynamics simulations. Ni(II)•L-Arg-Gly-His and Ni(II)•D-Arg-Gly-His (and their respective Lys analogues) targeted A/T-rich regions; however, the L-isomers consistently modified a sub-set of available nucleotides within a given minor groove site while the D-isomers differed in both their sites of preference and ability to target individual nucleotides within some sites. In comparison, Ni(II)•L-Pro-Gly-His and Ni(II)•D-Pro-Gly-His were unable to exhibit a similar diastereoselectivity. Simulations of the above systems, along with Ni(II)•Gly-Gly-His, indicated that the stereochemistry of the amino-terminal amino acid produces either an isohelical metallopeptide that associates stably at individual DNA sites (L-Arg or L-Lys) or, with D-Arg and D-Lys, a non-complementary metallopeptide structure that cannot fully employ its side chain nor amino-terminal amine as a positional stabilizing moiety. In contrast, amino-terminal Pro-containing metallopeptides of either stereochemistry, lacking an extended side chain directed toward the minor groove, did not exhibit a similar diastereoselectivity. While the identity and stereochemistry of amino acids located in the amino-terminal peptide position influenced DNA cleavage, metallopeptide diastereoisomers containing L- and D-Arg (or Lys) within the second peptide position did not exhibit diastereoselective DNA cleavage patterns; simulations indicated that a positively-charged amino acid in this location alters the interaction of the metallopeptide equatorial plane and the minor groove leading to an interaction similar to Ni(II)•Gly-Gly-His. PMID:16522100

A two-metal ion mechanism operates in the hammerhead ribozyme-mediated cleavage of an RNA substrate

PubMed Central

Lott, William B.; Pontius, Brian W.; von Hippel, Peter H.

1998-01-01

Evidence for a two-metal ion mechanism for cleavage of the HH16 hammerhead ribozyme is provided by monitoring the rate of cleavage of the RNA substrate as a function of La3+ concentration in the presence of a constant concentration of Mg2+. We show that a bell-shaped curve of cleavage activation is obtained as La3+ is added in micromolar concentrations in the presence of 8 mM Mg2+, with a maximal rate of cleavage being attained in the presence of 3 μM La3+. These results show that two-metal ion binding sites on the ribozyme regulate the rate of the cleavage reaction and, on the basis of earlier estimates of the Kd values for Mg2+ of 3.5 mM and >50 mM, that these sites bind La3+ with estimated Kd values of 0.9 and >37.5 μM, respectively. Furthermore, given the very different effects of these metal ions at the two binding sites, with displacement of Mg2+ by La3+ at the stronger (relative to Mg2+) binding site activating catalysis and displacement of Mg2+ by La3+ at the weaker (relative to Mg2+) (relative to Mg2+) binding site inhibiting catalysis, we show that the metal ions at these two sites play very different roles. We argue that the metal ion at binding site 1 coordinates the attacking 2′-oxygen species in the reaction and lowers the pKa of the attached proton, thereby increasing the concentration of the attacking alkoxide nucleophile in an equilibrium process. In contrast, the role of the metal ion at binding site 2 is to catalyze the reaction by absorbing the negative charge that accumulates at the leaving 5′-oxygen in the transition state. We suggest structural reasons why the Mg2+–La3+ ion combination is particularly suited to demonstrating these different roles of the two-metal ions in the ribozyme cleavage reaction. PMID:9435228

Genetic and mechanistic diversity of piRNA 3' end formation

PubMed Central

Handler, Dominik; Mohn, Fabio; Ameres, Stefan L.; Brennecke, Julius

2016-01-01

Small regulatory RNAs guide Argonaute (Ago) proteins in a sequence-specific manner to their targets and thereby play important roles in eukaryotic gene silencing1. Of the three small RNA classes, microRNAs and siRNAs are processed from double-stranded precursors into defined 21- to 23-mers by Dicer, an endoribonuclease with intrinsic ruler function. piRNAs—the 22-30 nt long guides for PIWI-clade Ago proteins that silence transposons in animal gonads—are generated Dicer-independently from single-stranded precursors2,3. piRNA 5' ends are defined either by Zucchini, a mitochondria-anchored endonuclease4,5, or by piRNA-guided target cleavage6,7. Formation of piRNA 3' ends is poorly understood. Here, we find that two genetically and mechanistically distinct pathways generate piRNA 3' ends in Drosophila. The initiating nucleases are either Zucchini or the PIWI-clade proteins Aubergine (Aub)/Ago3. While Zucchini-mediated cleavages directly define mature piRNA 3' ends8,9, Aub/Ago3-mediated cleavages liberate pre-piRNAs that require extensive resection by the 3'-to-5' exoribonuclease Nibbler/Mut-710–13. The relative activity of these two pathways dictates the extent to which piRNAs are fueled into cytoplasmic or nuclear PIWI-clade proteins and thereby sets the balance between post-transcriptional and transcriptional silencing. Strikingly, loss of both Zucchini and Nibbler reveals a minimal, Argonaute-driven small RNA biogenesis pathway where piRNA 5' and 3' ends are directly produced by closely spaced Aub/Ago3-mediated cleavage events. Our data establish a coherent blueprint for piRNA biogenesis, and set the stage for the mechanistic dissection of the processes that govern piRNA 3' end formation. PMID:27851737

Transition State Charge Stabilization and Acid-Base Catalysis of mRNA Cleavage by the Endoribonuclease RelE

PubMed Central

Dunican, Brian F.; Hiller, David A.; Strobel, Scott A.

2015-01-01

The bacterial toxin RelE is a ribosome-dependent endoribonuclease. It is part of a type II toxin-antitoxin system that contributes to antibiotic resistance and biofilm formation. During amino acid starvation RelE cleaves mRNA in the ribosomal A-site, globally inhibiting protein translation. RelE is structurally similar to microbial RNases that employ general acid-base catalysis to facilitate RNA cleavage. The RelE active-site is atypical for acid-base catalysis, in that it is enriched for positively charged residues and lacks the prototypical histidine-glutamate catalytic pair, making the mechanism of mRNA cleavage unclear. In this study we use a single-turnover kinetic analysis to measure the effect of pH and phosphorothioate substitution on the rate constant for cleavage of mRNA by wild-type RelE and seven active-site mutants. Mutation and thio-effects indicate a major role for stabilization of increased negative change in the transition state by arginine 61. The wild-type RelE cleavage rate constant is pH-independent, but the reaction catalyzed by many of the mutants is strongly pH dependent, suggestive of general acid-base catalysis. pH-rate curves indicate that wild-type RelE operates with the pKa of at least one catalytic residue significantly downshifted by the local environment. Mutation of any single active-site residue is sufficient to disrupt this microenvironment and revert the shifted pKa back above neutrality. pH-rate curves are consistent with K54 functioning as a general base and R81 as a general acid. The capacity of RelE to effect a large pKa shift and facilitate a common catalytic mechanism by uncommon means furthers our understanding of other atypical enzymatic active sites. PMID:26535789

Matriptase shedding is closely coupled with matriptase zymogen activation and requires de novo proteolytic cleavage likely involving its own activity

PubMed Central

Barndt, Robert; Gu, Yayun; Chen, Chien-Yu; Tseng, I-Chu; Su, Sheng-Fang; Wang, Jehng-Kang; Johnson, Michael D.

2017-01-01

The type 2 transmembrane serine protease matriptase is involved in many pathophysiological processes probably via its enzymatic activity, which depends on the dynamic relationship between zymogen activation and protease inhibition. Matriptase shedding can prolong the life of enzymatically active matriptase and increase accessibility to substrates. We show here that matriptase shedding occurs via a de novo proteolytic cleavage at sites located between the SEA domain and the CUB domain. Point or combined mutations at the four positively charged amino acid residues in the region following the SEA domain allowed Arg-186 to be identified as the primary cleavage site responsible for matriptase shedding. Kinetic studies further demonstrate that matriptase shedding is temporally coupled with matriptase zymogen activation. The onset of matriptase shedding lags one minute behind matriptase zymogen activation. Studies with active site triad Ser-805 point mutated matriptase, which no longer undergoes zymogen activation or shedding, further suggests that matriptase shedding depends on matriptase zymogen activation, and that matriptase proteolytic activity may be involved in its own shedding. Our studies uncover an autonomous mechanism coupling matriptase zymogen activation, proteolytic activity, and shedding such that a proportion of newly generated active matriptase escapes HAI-1-mediated rapid inhibition by shedding into the extracellular milieu. PMID:28829816

Coupled transcription and processing of mouse ribosomal RNA in a cell-free system.

PubMed Central

Mishima, Y; Mitsuma, T; Ogata, K

1985-01-01

An in vitro processing system of mouse rRNA was achieved using an RNA polymerase I-specific transcription system, (S100) and recombinant plasmids consisting of mouse rRNA gene (rDNA) segments containing the transcription initiation and 5'-terminal region of 18S (or 41S) rRNA. Pulse-chase experiments showed that a specific processing occurred with transcripts of the plasmid DNAs when the direction of transcription was the correct orientation relative to the 18S rRNA coding sequence, but not with transcripts of the DNA templates in which this coding sequence was in the opposite orientation. From the S1 nuclease protection analyses, we concluded that there are several steps of endonucleolytic cleavage including one 105 nucleotides upstream from the 5' end of 18S rRNA. Intermediates cleaved at this site were identified in in vivo processing of rRNA. This result indicates that endonucleolytic cleavage takes place 105 nucleotides upstream from the 5' terminus of 18S rRNA prior to the formation of mature 18S rRNA. Trimming or cleavage of the 105 nucleotides may be involved in the formation of the 5' terminus of mature 18S rRNA. Images Fig. 2. Fig. 3. Fig. 4. Fig. 5. Fig. 6. PMID:3004977

The impact of hydrogen on the formability of AHSS in Nakajima tests

NASA Astrophysics Data System (ADS)

Gao, Qi; Han, Fei; Wortberg, Daniel; Bleck, Wolfgang; Liewald, Mathias

2016-10-01

The application of Advanced High Strength Steels (AHSS) in the design of automotive body-in-white structures meets the requirement of light weight construction. The susceptibility to Hydrogen Embrittlement however, limits the wide application of steel grades over 1000 MPa. Up to now, various published studies focused on the hydrogen effects on mechanical properties; the impact of hydrogen on formability was not clarified. The present work aims to evaluate the influence of hydrogen on forming limit and fracture behavior in different strain states during the forming process. The hydrogen concentration in the material was modified by cathodic electro-charging. Nakajima tests in three strain conditions on uncharged and pre-charged samples were carried out. The localized necking was determined on the basis of the representative thinning rate in necking areas. The results revealed the shift of forming limits and critical strains to fracture owing to the presence of massive hydrogen. The fractography by Scanning Electron Microscope gave evidence of typical hydrogen-induced cleavage fracture on pre-charged samples.

Dehydrogenation involved Coulomb explosion of molecular C2H4FBr in an intense laser field

NASA Astrophysics Data System (ADS)

Pei, Minjie; Yang, Yan; Zhang, Jian; Sun, Zhenrong

2018-04-01

The dissociative double ionization (DDI) of molecular 1-fluo-2-bromoethane (FBE) in an intense laser field has been investigated by dc-slice imaging technology. The DDI channels involved with dehydrogenation are revealed and it's believed both the charge distribution and the bound character of real potential energy surfaces of parent ions play important roles in the dissociation process. The relationship between the potential energy surfaces of the precursor species and the photofragment ejection angles are also discussed and analyzed. Furthermore, the competition between the DDI channels has been studied and the Csbnd C bond cleavages dominate the DDI process at relative higher laser intensity.

Blackbody infrared radiative dissociation of oligonucleotide anions.

PubMed

Klassen, J S; Schnier, P D; Williams, E R

1998-11-01

The dissociation kinetics of a series of doubly deprotonated oligonucleotide 7-mers [d(A)7(2-), d(AATTAAT)2-, d(TTAATTA)2-, and d(CCGGCCG)2-] were measured using blackbody infrared radiative dissociation in a Fourier-transform mass spectrometer. The oligonucleotides dissociate first by cleavage at the glycosidic bond leading to the loss of a neutral nucleobase, followed by cleavage at the adjacent (5') phosphodiester bond to produce structurally informative a-base and w type ions. From the temperature dependence of the unimolecular dissociation rate constants, Arrhenius activation parameters in the zero-pressure limit are obtained for the loss of base. The measured Arrhenius parameters are dependent on the identity of the nucleobase. The process involving the loss of an adenine base from the dianions, d(A)7(2-), d(AATTAAT)2-, and d(TTAATTA)2- has an average activation energy (Ea) of approximately 1.0 eV and a preexponential factor (A) of 10(10) s-1. Both guanine and cytosine base loss occurs for d(CCGGCCG)2-. The average Arrhenius parameters for the loss of cytosine and guanine are Ea = 1.32 +/- 0.03 eV and A = 10(13.3 +/- 0.3) s-1. No loss of thymine was observed for mixed adenine-thymine oligonucleotides. Neither base loss nor any other fragmentation reactions occur for d(T)7(2-) over a 600 s reaction delay at 207 degrees C, a temperature close to the upper limit accessible with our instrument. The Arrhenius parameters indicate that the preferred cleavage sites for mixed oligonucleotides of similar mass-to-charge ratio will be strongly dependent on the internal energy of the precursor ions. At low internal energies (effective temperatures below 475 K), loss of adenine and subsequent cleavage of the adjacent phosphoester bonds will dominate, whereas at higher energies, preferential cleavage at C and G residues will occur. The magnitude of the A factors < or = 10(13) s-1 measured for the loss of the three nucleobases (A, G, and C) is indicative of an entropically neutral or disfavored process as the rate limiting step for this reaction.

Blackbody Infrared Radiative Dissociation of Oligonucleotide Anions

PubMed Central

Klassen, John S.; Schnier, Paul D.; Williams, Evan R.

2005-01-01

The dissociation kinetics of a series of doubly deprotonated oligonucleotide 7-mers [ d(A)72-, d(AATTAAT)2−, d(TTAATTA)2−, and d(CCGGCCG)2−] were measured using blackbody infrared radiative dissociation in a Fourier-transform mass spectrometer. The oligonucleotides dissociate first by cleavage at the glycosidic bond leading to the loss of a neutral nucleobase, followed by cleavage at the adjacent (5′) phosphodiester bond to produce structurally informative a-base and w type ions. From the temperature dependence of the unimolecular dissociation rate constants, Arrhenius activation parameters in the zero-pressure limit are obtained for the loss of base. The measured Arrhenius parameters are dependent on the identity of the nucleobase. The process involving the loss of an adenine base from the dianions, d(A)72-, d(AATTAAT)2−, and d(TTAATTA)2− has an average activation energy (Ea) of ~1.0 eV and a preexponential factor (A) of 1010 s−1. Both guanine and cytosine base loss occurs for d(CCGGCCG)2−. The average Arrhenius parameters for the loss of cytosine and guanine are Ea = 1.32 ± 0.03 eV and A = 1013.3±0.3 s−1. No loss of thymine was observed for mixed adenine–thymine oligonucleotides. Neither base loss nor any other fragmentation reactions occur for d(T)72- over a 600 s reaction delay at 207 °C, a temperature close to the upper limit accessible with our instrument. The Arrhenius parameters indicate that the preferred cleavage sites for mixed oligonucleotides of similar mass-to-charge ratio will be strongly dependent on the internal energy of the precursor ions. At low internal energies (effective temperatures below 475 K), loss of adenine and subsequent cleavage of the adjacent phosphoester bonds will dominate, whereas at higher energies, preferential cleavage at C and G residues will occur. The magnitude of the A factors ≤1013 s−1 measured for the loss of the three nucleobases (A, G, and C) is indicative of an entropically neutral or disfavored process as the rate limiting step for this reaction. PMID:9794082

Electron Transfer Dissociation: Effects of Cation Charge State on Product Partitioning in Ion/Ion Electron Transfer to Multiply Protonated Polypeptides

PubMed Central

Liu, Jian; McLuckey, Scott A.

2012-01-01

The effect of cation charge state on product partitioning in the gas-phase ion/ion electron transfer reactions of multiply protonated tryptic peptides, model peptides, and relatively large peptides with singly charged radical anions has been examined. In particular, partitioning into various competing channels, such as proton transfer (PT) versus electron transfer (ET), electron transfer with subsequent dissociation (ETD) versus electron transfer with no dissociation (ET,noD), and fragmentation of backbone bonds versus fragmentation of side chains, was measured quantitatively as a function of peptide charge state to allow insights to be drawn about the fundamental aspects of ion/ion reactions that lead to ETD. The ET channel increases relative to the PT channel, ETD increases relative to ET,noD, and fragmentation at backbone bonds increases relative to side-chain cleavages as cation charge state increases. The increase in ET versus PT with charge state is consistent with a Landau-Zener based curve-crossing model. An optimum charge state for ET is predicted by the model for the ground state-to-ground state reaction. However, when the population of excited product ion states is considered, it is possible that a decrease in ET efficiency as charge state increases will not be observed due to the possibility of the population of excited electronic states of the products. Several factors can contribute to the increase in ETD versus ET,noD and backbone cleavage versus side-chain losses. These factors include an increase in reaction exothermicity and charge state dependent differences in precursor and product ion structures, stabilities, and sites of protonation. PMID:23264749

Long-range sclerotome induction by sonic hedgehog: direct role of the amino-terminal cleavage product and modulation by the cyclic AMP signaling pathway.

PubMed

Fan, C M; Porter, J A; Chiang, C; Chang, D T; Beachy, P A; Tessier-Lavigne, M

1995-05-05

A long-range signal encoded by the Sonic hedgehog (Shh) gene has been implicated as the ventral patterning influence from the notochord that induces sclerotome and represses dermomyotome in somite differentiation. Long-range effects of hedgehog (hh) signaling have been suggested to result either from local induction of a secondary diffusible signal or from the direct action of the highly diffusible carboxy-terminal product of HH autoproteolytic cleavage. Here we provide evidence that the long-range somite patterning effects of SHH are instead mediated by a direct action of the amino-terminal cleavage product. We also show that pharmacological manipulations to increase the activity of cyclic AMP-dependent protein kinase A can selectively antagonize the effects of the amino-terminal cleavage product. Our results support the operation of a single evolutionarily conserved signaling pathway for both local and direct long-range inductive actions of HH family members.

Cleavage fracture in pearlitic eutectoid steel

NASA Astrophysics Data System (ADS)

Alexander, D. J.; Bernstein, I. M.

1989-11-01

The effect of microstructure on flow and fracture properties of fully pearlitic steel has been studied by independently varying the prior austenite grain size and the pearlite interlamellar spacing through appropriate heat treatments. The yield strength is independent of the prior austenite grain size but increases as the interlamellar spacing or the temperature decreases. The microstructural dependence can be explained by using a model which assumes that yielding is controlled by dislocation motion in the ferrite lamellae. The critical tensile stress for cleavage fracture is found to be independent of prior austenite grain size, increasing as the interlamellar spacing decreases. The cleavage fracture stress is independent of temperature for fine pearlite but increases as the temperature decreases for coarse pearlite. The associated fracture in blunt notch specimens initiates at inclusions beneath notch surface near the location of maximum tensile stress. From the size of such inclusions, the effective surface energy for cleavage fracture can be directly calculated and is found to be independent of temperature and prior austenite grain size but to increase as the interlamellar spacing decreases, from about 5 to 13 J/m2 for the range of microstructures and temperatures used in this study. Additional measurements of the effective surface energy and further theoretical analyses of the cleavage process are needed.

The rRNA methyltransferase Bud23 shows functional interaction with components of the SSU processome and RNase MRP.

PubMed

Sardana, Richa; White, Joshua P; Johnson, Arlen W

2013-06-01

Bud23 is responsible for the conserved methylation of G1575 of 18S rRNA, in the P-site of the small subunit of the ribosome. bud23Δ mutants have severely reduced small subunit levels and show a general failure in cleavage at site A2 during rRNA processing. Site A2 is the primary cleavage site for separating the precursors of 18S and 25S rRNAs. Here, we have taken a genetic approach to identify the functional environment of BUD23. We found mutations in UTP2 and UTP14, encoding components of the SSU processome, as spontaneous suppressors of a bud23Δ mutant. The suppressors improved growth and subunit balance and restored cleavage at site A2. In a directed screen of 50 ribosomal trans-acting factors, we identified strong positive and negative genetic interactions with components of the SSU processome and strong negative interactions with components of RNase MRP. RNase MRP is responsible for cleavage at site A3 in pre-rRNA, an alternative cleavage site for separating the precursor rRNAs. The strong negative genetic interaction between RNase MRP mutants and bud23Δ is likely due to the combined defects in cleavage at A2 and A3. Our results suggest that Bud23 plays a role at the time of A2 cleavage, earlier than previously thought. The genetic interaction with the SSU processome suggests that Bud23 could be involved in triggering disassembly of the SSU processome, or of particular subcomplexes of the processome.

Crystal Structures of Yellowtail Ascites Virus VP4 Protease

PubMed Central

Chung, Ivy Yeuk Wah; Paetzel, Mark

2013-01-01

Yellowtail ascites virus (YAV) is an aquabirnavirus that causes ascites in yellowtail, a fish often used in sushi. Segment A of the YAV genome codes for a polyprotein (pVP2-VP4-VP3), where processing by its own VP4 protease yields the capsid protein precursor pVP2, the ribonucleoprotein-forming VP3, and free VP4. VP4 protease utilizes the rarely observed serine-lysine catalytic dyad mechanism. Here we have confirmed the existence of an internal cleavage site, preceding the VP4/VP3 cleavage site. The resulting C-terminally truncated enzyme (ending at Ala716) is active, as shown by a trans full-length VP4 cleavage assay and a fluorometric peptide cleavage assay. We present a crystal structure of a native active site YAV VP4 with the internal cleavage site trapped as trans product complexes and trans acyl-enzyme complexes. The acyl-enzyme complexes confirm directly the role of Ser633 as the nucleophile. A crystal structure of the lysine general base mutant (K674A) reveals the acyl-enzyme and empty binding site states of VP4, which allows for the observation of structural changes upon substrate or product binding. These snapshots of three different stages in the VP4 protease reaction mechanism will aid in the design of anti-birnavirus compounds, provide insight into previous site-directed mutagenesis results, and contribute to understanding of the serine-lysine dyad protease mechanism. In addition, we have discovered that this protease contains a channel that leads from the enzyme surface (adjacent to the substrate binding groove) to the active site and the deacylating water. PMID:23511637

A density functional theory study on peptide bond cleavage at aspartic residues: direct vs cyclic intermediate hydrolysis.

PubMed

Sang-aroon, Wichien; Amornkitbamrung, Vittaya; Ruangpornvisuti, Vithaya

2013-12-01

In this work, peptide bond cleavages at carboxy- and amino-sides of the aspartic residue in a peptide model via direct (concerted and step-wise) and cyclic intermediate hydrolysis reaction pathways were explored computationally. The energetics, thermodynamic properties, rate constants, and equilibrium constants of all hydrolysis reactions, as well as their energy profiles were computed at the B3LYP/6-311++G(d,p) level of theory. The result indicated that peptide bond cleavage of the Asp residue occurred most preferentially via the cyclic intermediate hydrolysis pathway. In all reaction pathways, cleavage of the peptide bond at the amino-side occurred less preferentially than at the carboxy-side. The overall reaction rate constants of peptide bond cleavage of the Asp residue at the carboxy-side for the assisted system were, in increasing order: concerted < step-wise < cyclic intermediate.

Two Divalent Metal Ions and Conformational Changes Play Roles in the Hammerhead Ribozyme Cleavage Reaction

PubMed Central

Mir, Aamir; Chen, Ji; Robinson, Kyle; Lendy, Emma; Goodman, Jaclyn; Neau, David; Golden, Barbara L.

2016-01-01

The hammerhead ribozyme is a self-cleaving RNA broadly dispersed across all kingdoms of life. Although it was the first of the small, nucleolytic ribozymes discovered, the mechanism by which it catalyzes its reaction remains elusive. The nucleobase of G12 is well positioned to be a general base, but it is unclear if or how this guanine base becomes activated for proton transfer. Metal ions have been implicated in the chemical mechanism, but no interactions between divalent metal ions and the cleavage site have been observed crystallographically. To better understand how this ribozyme functions, we have solved crystal structures of wild-type and G12A mutant ribozymes. We observe a pH-dependent conformational change centered around G12, consistent with this nucleotide becoming deprotonated. Crystallographic and kinetic analysis of the G12A mutant reveals a Zn2+ specificity switch suggesting a direct interaction between a divalent metal ion and the purine at position 12. The metal ion specificity switch and the pH–rate profile of the G12A mutant suggest that the minor imino tautomer of A12 serves as the general base in the mutant ribozyme. We propose a model in which the hammerhead ribozyme rearranges prior to the cleavage reaction, positioning two divalent metal ions in the process. The first metal ion, positioned near G12, becomes directly coordinated to the O6 keto oxygen, to lower the pKa of the general base and organize the active site. The second metal ion, positioned near G10.1, bridges the N7 of G10.1 and the scissile phosphate and may participate directly in the cleavage reaction. PMID:26398724

Two Divalent Metal Ions and Conformational Changes Play Roles in the Hammerhead Ribozyme Cleavage Reaction.

PubMed

Mir, Aamir; Chen, Ji; Robinson, Kyle; Lendy, Emma; Goodman, Jaclyn; Neau, David; Golden, Barbara L

2015-10-20

The hammerhead ribozyme is a self-cleaving RNA broadly dispersed across all kingdoms of life. Although it was the first of the small, nucleolytic ribozymes discovered, the mechanism by which it catalyzes its reaction remains elusive. The nucleobase of G12 is well positioned to be a general base, but it is unclear if or how this guanine base becomes activated for proton transfer. Metal ions have been implicated in the chemical mechanism, but no interactions between divalent metal ions and the cleavage site have been observed crystallographically. To better understand how this ribozyme functions, we have solved crystal structures of wild-type and G12A mutant ribozymes. We observe a pH-dependent conformational change centered around G12, consistent with this nucleotide becoming deprotonated. Crystallographic and kinetic analysis of the G12A mutant reveals a Zn(2+) specificity switch suggesting a direct interaction between a divalent metal ion and the purine at position 12. The metal ion specificity switch and the pH-rate profile of the G12A mutant suggest that the minor imino tautomer of A12 serves as the general base in the mutant ribozyme. We propose a model in which the hammerhead ribozyme rearranges prior to the cleavage reaction, positioning two divalent metal ions in the process. The first metal ion, positioned near G12, becomes directly coordinated to the O6 keto oxygen, to lower the pKa of the general base and organize the active site. The second metal ion, positioned near G10.1, bridges the N7 of G10.1 and the scissile phosphate and may participate directly in the cleavage reaction.

Rapid Detection of Thrombin and Other Protease Activity Directly in Whole Blood

NASA Astrophysics Data System (ADS)

Yu, Johnson Chung Sing

Thrombin is a serine protease that plays a key role in the clotting cascade to promote hemostasis following injury to the endothelium. From a clinical diagnostic perspective, in-vivo thrombin activity is linked to various blood clotting disorders, as well as cardiovascular disease (DVT, arteriosclerosis, etc). Thus, the ability to rapidly measure protease activity directly in whole blood will provide important new diagnostics, and clinical researchers with a powerful tool to further elucidate the relationship between circulating protease levels and disease. The ultimate goal is to design novel point of care (POC) diagnostic devices that are capable of monitoring protease activities directly in whole blood and biological sample. A charge-changing substrate specific to the thrombin enzyme was engineered and its functionality was confirmed by a series of experiments. This led to the preliminary design, construction, and testing of two device platforms deemed fully functional for the electrophoretic separation and focusing of charged peptide fragments. The concept of using the existing charge-changing substrate platform for bacterial protease detection was also investigated. Certain strains of E coli are associated with severe symptoms such as abdominal cramps, bloody diarrhea, and vomiting. The OmpT protease is expressed on the outer membrane of E coli and plays a role in the cleavage of antimicrobial peptides, the degradation of recombinant heterologous proteins, and the activation of plasminogen in the host. Thus, a synthetic peptide substrate specific to the OmpT protease was designed and modeled for the purpose of detecting E coli in biological sample.

HIV-1 matrix domain removal ameliorates virus assembly and processing defects incurred by positive nucleocapsid charge elimination.

PubMed

Ko, Li-Jung; Yu, Fu-Hsien; Huang, Kuo-Jung; Wang, Chin-Tien

2015-01-01

Human immunodeficiency virus type 1 nucleocapsid (NC) basic residues presumably contribute to virus assembly via RNA, which serves as a scaffold for Gag-Gag interaction during particle assembly. To determine whether NC basic residues play a role in Gag cleavage (thereby impacting virus assembly), Gag processing efficiency and virus particle production were analyzed for an HIV-1 mutant NC15A, with alanine serving as a substitute for all NC basic residues. Results indicate that NC15A significantly impaired virus maturation in addition to significantly affecting Gag membrane binding and assembly. Interestingly, removal of the matrix (MA) central globular domain ameliorated the NC15A assembly and processing defects, likely through enhancement of Gag multimerization and membrane binding capacities.

The Efficiency of Dentin Sialoprotein-Phosphophoryn Processing Is Affected by Mutations Both Flanking and Distant from the Cleavage Site*

PubMed Central

Yang, Robert T.; Lim, Glendale L.; Dong, Zhihong; Lee, Arthur M.; Yee, Colin T.; Fuller, Robert S.; Ritchie, Helena H.

2013-01-01

Normal dentin mineralization requires two highly acidic proteins, dentin sialoprotein (DSP) and phosphophoryn (PP). DSP and PP are synthesized as part of a single secreted precursor, DSP-PP, which is conserved in marsupial and placental mammals. Using a baculovirus expression system, we previously found that DSP-PP is accurately cleaved into DSP and PP after secretion into medium by an endogenous, secreted, zinc-dependent Sf9 cell activity. Here we report that mutation of conserved residues near and distant from the G447↓D448 cleavage site in DSP-PP240 had dramatic effects on cleavage efficiency by the endogenous Sf9 cell processing enzyme. We found that: 1) mutation of residues flanking the cleavage site from P4 to P4′ blocked, impaired, or enhanced DSP-PP240 cleavage; 2) certain conserved amino acids distant from the cleavage site were important for precursor cleavage; 3) modification of the C terminus by appending a C-terminal tag altered the pattern of processing; and 4) mutations in DSP-PP240 had similar effects on cleavage by recombinant human BMP1, a candidate physiological processing enzyme, as was seen with the endogenous Sf9 cell activity. An analysis of a partial TLR1 cDNA from Sf9 cells indicates that residues that line the substrate-binding cleft of Sf9 TLR1 and human BMP1 are nearly perfectly conserved, offering an explanation of why Sf9 cells so accurately process mammalian DSP-PP. The fact that several mutations in DSP-PP240 significantly modified the amount of PP240 product generated from DSP-PP240 precursor protein cleavage suggests that such mutation may affect the mineralization process. PMID:23297400

The efficiency of dentin sialoprotein-phosphophoryn processing is affected by mutations both flanking and distant from the cleavage site.

PubMed

Yang, Robert T; Lim, Glendale L; Dong, Zhihong; Lee, Arthur M; Yee, Colin T; Fuller, Robert S; Ritchie, Helena H

2013-02-22

Normal dentin mineralization requires two highly acidic proteins, dentin sialoprotein (DSP) and phosphophoryn (PP). DSP and PP are synthesized as part of a single secreted precursor, DSP-PP, which is conserved in marsupial and placental mammals. Using a baculovirus expression system, we previously found that DSP-PP is accurately cleaved into DSP and PP after secretion into medium by an endogenous, secreted, zinc-dependent Sf9 cell activity. Here we report that mutation of conserved residues near and distant from the G(447)↓D(448) cleavage site in DSP-PP(240) had dramatic effects on cleavage efficiency by the endogenous Sf9 cell processing enzyme. We found that: 1) mutation of residues flanking the cleavage site from P(4) to P(4)' blocked, impaired, or enhanced DSP-PP(240) cleavage; 2) certain conserved amino acids distant from the cleavage site were important for precursor cleavage; 3) modification of the C terminus by appending a C-terminal tag altered the pattern of processing; and 4) mutations in DSP-PP(240) had similar effects on cleavage by recombinant human BMP1, a candidate physiological processing enzyme, as was seen with the endogenous Sf9 cell activity. An analysis of a partial TLR1 cDNA from Sf9 cells indicates that residues that line the substrate-binding cleft of Sf9 TLR1 and human BMP1 are nearly perfectly conserved, offering an explanation of why Sf9 cells so accurately process mammalian DSP-PP. The fact that several mutations in DSP-PP(240) significantly modified the amount of PP(240) product generated from DSP-PP(240) precursor protein cleavage suggests that such mutation may affect the mineralization process.

Analysis of RNA Processing Reactions Using Cell Free Systems: 3' End Cleavage of Pre-mRNA Substrates in vitro

PubMed Central

Jablonski, Joseph; Clementz, Mark; Ryan, Kevin; Valente, Susana T.

2014-01-01

The 3’ end of mammalian mRNAs is not formed by abrupt termination of transcription by RNA polymerase II (RNPII). Instead, RNPII synthesizes precursor mRNA beyond the end of mature RNAs, and an active process of endonuclease activity is required at a specific site. Cleavage of the precursor RNA normally occurs 10-30 nt downstream from the consensus polyA site (AAUAAA) after the CA dinucleotides. Proteins from the cleavage complex, a multifactorial protein complex of approximately 800 kDa, accomplish this specific nuclease activity. Specific RNA sequences upstream and downstream of the polyA site control the recruitment of the cleavage complex. Immediately after cleavage, pre-mRNAs are polyadenylated by the polyA polymerase (PAP) to produce mature stable RNA messages. Processing of the 3’ end of an RNA transcript may be studied using cellular nuclear extracts with specific radiolabeled RNA substrates. In sum, a long 32P-labeled uncleaved precursor RNA is incubated with nuclear extracts in vitro, and cleavage is assessed by gel electrophoresis and autoradiography. When proper cleavage occurs, a shorter 5’ cleaved product is detected and quantified. Here, we describe the cleavage assay in detail using, as an example, the 3’ end processing of HIV-1 mRNAs. PMID:24835792

The roles of SSU processome components and surveillance factors in the initial processing of human ribosomal RNA

PubMed Central

Sloan, Katherine E.; Bohnsack, Markus T.; Schneider, Claudia; Watkins, Nicholas J.

2014-01-01

During eukaryotic ribosome biogenesis, three of the mature ribosomal (r)RNAs are released from a single precursor transcript (pre-rRNA) by an ordered series of endonucleolytic cleavages and exonucleolytic processing steps. Production of the 18S rRNA requires the removal of the 5′ external transcribed spacer (5′ETS) by endonucleolytic cleavages at sites A0 and A1/site 1. In metazoans, an additional cleavage in the 5′ETS, at site A′, upstream of A0, has also been reported. Here, we have investigated how A′ processing is coordinated with assembly of the early preribosomal complex. We find that only the tUTP (UTP-A) complex is critical for A′ cleavage, while components of the bUTP (UTP-B) and U3 snoRNP are important, but not essential, for efficient processing at this site. All other factors involved in the early stages of 18S rRNA processing that were tested here function downstream from this processing step. Interestingly, we show that the RNA surveillance factors XRN2 and MTR4 are also involved in A′ cleavage in humans. A′ cleavage is largely bypassed when XRN2 is depleted, and we also discover that A′ cleavage is not always the initial processing event in all cell types. Together, our data suggest that A′ cleavage is not a prerequisite for downstream pre-rRNA processing steps and may, in fact, represent a quality control step for initial pre-rRNA transcripts. Furthermore, we show that components of the RNA surveillance machinery, including the exosome and TRAMP complexes, also play key roles in the recycling of excised spacer fragments and degradation of aberrant pre-rRNAs in human cells. PMID:24550520

Dendritic polymer imaging systems for the evaluation of conjugate uptake and cleavage

NASA Astrophysics Data System (ADS)

Krüger, Harald R.; Nagel, Gregor; Wedepohl, Stefanie; Calderón, Marcelo

2015-02-01

Fluorescent turn-on probes combined with polymers have a broad range of applications, e.g. for intracellular sensing of ions, small molecules, or DNA. In the field of polymer therapeutics, these probes can be applied to extend the in vitro characterization of novel conjugates beyond cytotoxicity and cellular uptake studies. This is particularly true in cases in which polymer conjugates contain drugs attached by cleavable linkers. Better information on the intracellular linker cleavage and drug release would allow a faster evaluation and optimization of novel polymer therapeutic concepts. We therefore developed a fluorescent turn-on probe that enables direct monitoring of pH-mediated cleavage processes over time. This is achieved by exploiting the fluorescence resonance energy transfer (FRET) between two dyes that have been coupled to a dendritic polymer. We demonstrate the use of this probe to evaluate polymer uptake and intracellular release of cargo in a cell based microplate assay that is suitable for high throughput screening.Fluorescent turn-on probes combined with polymers have a broad range of applications, e.g. for intracellular sensing of ions, small molecules, or DNA. In the field of polymer therapeutics, these probes can be applied to extend the in vitro characterization of novel conjugates beyond cytotoxicity and cellular uptake studies. This is particularly true in cases in which polymer conjugates contain drugs attached by cleavable linkers. Better information on the intracellular linker cleavage and drug release would allow a faster evaluation and optimization of novel polymer therapeutic concepts. We therefore developed a fluorescent turn-on probe that enables direct monitoring of pH-mediated cleavage processes over time. This is achieved by exploiting the fluorescence resonance energy transfer (FRET) between two dyes that have been coupled to a dendritic polymer. We demonstrate the use of this probe to evaluate polymer uptake and intracellular release of cargo in a cell based microplate assay that is suitable for high throughput screening. Electronic supplementary information (ESI) available: Including detailed synthetic procedures of the dye and conjugate synthesis, as well as cellular uptake and inhibitor studies. See DOI: 10.1039/c4nr04467c

Solution structure of conserved AGNN tetraloops: insights into Rnt1p RNA processing

PubMed Central

Lebars, Isabelle; Lamontagne, Bruno; Yoshizawa, Satoko; Abou Elela, Sherif; Fourmy, Dominique

2001-01-01

Rnt1p, the yeast orthologue of RNase III, cleaves rRNAs, snRNAs and snoRNAs at a stem capped with conserved AGNN tetraloop. Here we show that 9 bp long stems ending with AGAA or AGUC tetraloops bind to Rnt1p and direct specific but sequence-independent RNA cleavage when provided with stems longer than 13 bp. The solution structures of these two tetraloops reveal a common fold for the terminal loop stabilized by non-canonical A–A or A–C pairs and extensive base stacking. The conserved nucleotides are stacked at the 5′ side of the loop, exposing their Watson–Crick and Hoogsteen faces for recognition by Rnt1p. These results indicate that yeast RNase III recognizes the fold of a conserved single-stranded tetraloop to direct specific dsRNA cleavage. PMID:11743001

Preferential apelin-13 production by the proprotein convertase PCSK3 is implicated in obesity☆

PubMed Central

Shin, Kyungsoo; Pandey, Aditya; Liu, Xiang-Qin; Anini, Younes; Rainey, Jan K.

2013-01-01

The peptide hormone apelin is translated as a 77-residue preproprotein, truncated to the 55-residue proapelin and, subsequently, to 13–36-residue bioactive isoforms named apelin-13 to -36. Proapelin is hypothesized to be cleaved to apelin-36 and then to the shorter isoforms. However, neither the mechanism of proapelin processing nor the endoproteases involved have been determined. We show direct cleavage of proapelin to apelin-13 by proprotein convertase subtilisin/kexin 3 (PCSK3, or furin) in vitro, with no production of longer isoforms. Conversely, neither PCSK1 nor PCSK7 has appreciable proapelin cleavage activity. Furthermore, we show that both proapelin and PCSK3 transcript expression levels are increased in adipose tissue with obesity and during adipogenesis, suggesting that PCSK3 is responsible for proapelin processing in adipose tissue. PMID:24251091

Charge-reversal nanoparticles: novel targeted drug delivery carriers.

PubMed

Chen, Xinli; Liu, Lisha; Jiang, Chen

2016-07-01

Spurred by significant progress in materials chemistry and drug delivery, charge-reversal nanocarriers are being developed to deliver anticancer formulations in spatial-, temporal- and dosage-controlled approaches. Charge-reversal nanoparticles can release their drug payload in response to specific stimuli that alter the charge on their surface. They can elude clearance from the circulation and be activated by protonation, enzymatic cleavage, or a molecular conformational change. In this review, we discuss the physiological basis for, and recent advances in the design of charge-reversal nanoparticles that are able to control drug biodistribution in response to specific stimuli, endogenous factors (changes in pH, redox gradients, or enzyme concentration) or exogenous factors (light or thermos-stimulation).

High-energy Collision-induced Dissociation by MALDI TOF/TOF Causes Charge-Remote Fragmentation of Steroid Sulfates

PubMed Central

Yan, Yuetian; Ubukata, Masaaki; Cody, Robert B.; Holy, Timothy E.; Gross, Michael L.

2014-01-01

A method for structural elucidation of biomolecules dating to the 1980s utilized high-energy collisions (~10 keV, laboratory frame) that induced charge-remote fragmentations (CRF), a class of fragmentations particularly informative for lipids, steroids, surfactants, and peptides. Unfortunately, the capability for high-energy activation has largely disappeared with the demise of magnetic sector instruments. With the latest designs of tandem time-of-flight mass spectrometers (TOF/TOF), however, this capability is now being restored to coincide with the renewed interest in metabolites and lipids including steroid-sulfates and other steroid metabolites. For these metabolites, structure determinations are required at concentration levels below that appropriate for NMR. To meet this need, we explored CRF with TOF/TOF mass spectrometry for two groups of steroid sulfates, 3-sulfates and 21-sulfates. We demonstrated that the current generation of MALDI TOF/TOF instruments can generate charge-remote-fragmentations for these materials. The resulting collision-induced dissociation (CID) spectra are useful for positional isomer differentiation and very often allow the complete structure determination of the steroid. We also propose a new nomenclature that directly indicates the cleavage sites on the steroid ring with carbon numbers. PMID:24781458

High-energy collision-induced dissociation by MALDI TOF/TOF causes charge-remote fragmentation of steroid sulfates.

PubMed

Yan, Yuetian; Ubukata, Masaaki; Cody, Robert B; Holy, Timothy E; Gross, Michael L

2014-08-01

A method for structural elucidation of biomolecules dating to the 1980s utilized high-energy collisions (~10 keV, laboratory frame) that induced charge-remote fragmentations (CRF), a class of fragmentations particularly informative for lipids, steroids, surfactants, and peptides. Unfortunately, the capability for high-energy activation has largely disappeared with the demise of magnetic sector instruments. With the latest designs of tandem time-of-flight mass spectrometers (TOF/TOF), however, this capability is now being restored to coincide with the renewed interest in metabolites and lipids, including steroid-sulfates and other steroid metabolites. For these metabolites, structure determinations are required at concentration levels below that appropriate for NMR. To meet this need, we explored CRF with TOF/TOF mass spectrometry for two groups of steroid sulfates, 3-sulfates and 21-sulfates. We demonstrated that the current generation of MALDI TOF/TOF instruments can generate charge-remote fragmentations for these materials. The resulting collision-induced dissociation (CID) spectra are useful for positional isomer differentiation and very often allow the complete structure determination of the steroid. We also propose a new nomenclature that directly indicates the cleavage sites on the steroid ring with carbon numbers.

Metal-Free Multiple Carbon-Carbon and Carbon-Hydrogen Bond Activations via Charge-Switching Mechanism in Unstrained Diindolylmethanes.

PubMed

Challa, Chandrasekhar; Varughese, Sunil; Suresh, Cherumuttathu H; Lankalapalli, Ravi S

2017-08-18

A transformation of the unstrained phenol substituted 3,3'-diindolylmethanes (DIPMs) to 2,3'-diindolylketones (DIKs) by double C-C single bond cleavage with associated rearrangements, triggered by phenyliodine(III) diacetate (PIDA), is reported. Density functional theory studies reveal a mechanism involving multiple "charge-switching" steps by synergistic involvement of the two indole units with overall low activation energy. The indole 'charge-switching' mechanism in DIPMs was further extended toward synthesis of a natural product motif cyclohepta[b]indole from biaryl appended DIBM.

The role of collagen charge clusters in the modulation of matrix metalloproteinase activity.

PubMed

Lauer, Janelle L; Bhowmick, Manishabrata; Tokmina-Roszyk, Dorota; Lin, Yan; Van Doren, Steven R; Fields, Gregg B

2014-01-24

Members of the matrix metalloproteinase (MMP) family selectively cleave collagens in vivo. Several substrate structural features that direct MMP collagenolysis have been identified. The present study evaluated the role of charged residue clusters in the regulation of MMP collagenolysis. A series of 10 triple-helical peptide (THP) substrates were constructed in which either Lys-Gly-Asp or Gly-Asp-Lys motifs replaced Gly-Pro-Hyp (where Hyp is 4-hydroxy-L-proline) repeats. The stabilities of THPs containing the two different motifs were analyzed, and kinetic parameters for substrate hydrolysis by six MMPs were determined. A general trend for virtually all enzymes was that, as Gly-Asp-Lys motifs were moved from the extreme N and C termini to the interior next to the cleavage site sequence, kcat/Km values increased. Additionally, all Gly-Asp-Lys THPs were as good or better substrates than the parent THP in which Gly-Asp-Lys was not present. In turn, the Lys-Gly-Asp THPs were also always better substrates than the parent THP, but the magnitude of the difference was considerably less compared with the Gly-Asp-Lys series. Of the MMPs tested, MMP-2 and MMP-9 most greatly favored the presence of charged residues with preference for the Gly-Asp-Lys series. Lys-Gly-(Asp/Glu) motifs are more commonly found near potential MMP cleavage sites than Gly-(Asp/Glu)-Lys motifs. As Lys-Gly-Asp is not as favored by MMPs as Gly-Asp-Lys, the Lys-Gly-Asp motif appears advantageous over the Gly-Asp-Lys motif by preventing unwanted MMP hydrolysis. More specifically, the lack of Gly-Asp-Lys clusters may diminish potential MMP-2 and MMP-9 collagenolytic activity. The present study indicates that MMPs have interactions spanning the P23-P23' subsites of collagenous substrates.

Applying simulation model to uniform field space charge distribution measurements by the PEA method

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

Liu, Y.; Salama, M.M.A.

1996-12-31

Signals measured under uniform fields by the Pulsed Electroacoustic (PEA) method have been processed by the deconvolution procedure to obtain space charge distributions since 1988. To simplify data processing, a direct method has been proposed recently in which the deconvolution is eliminated. However, the surface charge cannot be represented well by the method because the surface charge has a bandwidth being from zero to infinity. The bandwidth of the charge distribution must be much narrower than the bandwidths of the PEA system transfer function in order to apply the direct method properly. When surface charges can not be distinguished frommore » space charge distributions, the accuracy and the resolution of the obtained space charge distributions decrease. To overcome this difficulty a simulation model is therefore proposed. This paper shows their attempts to apply the simulation model to obtain space charge distributions under plane-plane electrode configurations. Due to the page limitation for the paper, the charge distribution originated by the simulation model is compared to that obtained by the direct method with a set of simulated signals.« less

Bcl2-independent chromatin cleavage is a very early event during induction of apoptosis in mouse thymocytes after treatment with either dexamethasone or ionizing radiation.

PubMed

Hahn, Peter J; Lai, Zhi-Wei; Nevaldine, Barbara; Schiff, Ninel; Fiore, Nancy C; Silverstone, Allen E

2003-11-01

We have quantified the emergence of early chromatin breaks during the signal transduction phase of apoptosis in mouse thymocytes after treatment with either ionizing radiation or dexamethasone. Dexamethasone at 1 microM can induce significant levels of DNA breaks (equivalent to the amount induced directly by 7.5 Gy ionizing radiation) within 0.5 h of treatment. The execution phase of apoptosis was not observed until 4-6 h after the same treatment. The presence of the Bcl2 transgene under the control of the p56lck promoter almost completely inhibited apoptosis up to 24 h after treatment, but it had virtually no effect on the early chromatin cleavage occurring in the first 6 h. Ionizing radiation induced chromatin cleavage both directly by damaging DNA and indirectly with kinetics similar to the induction of chromatin cleavage by dexamethasone. The presence of the Bcl2 transgene had no effect on the direct or indirect radiation-induced cleavage in the first 6 h, but after the first 6 h, the Bcl2 gene inhibited further radiation-induced chromatin cleavage. These results suggest that endonucleases are activated within minutes of treatment with either dexamethasone or ionizing radiation as part of the very early signal transduction phase of apoptosis, and prior to the irreversible commitment to cell death.

Proteolytic processing of poliovirus polypeptides: antibodies to polypeptide P3-7c inhibit cleavage at glutamine-glycine pairs

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

Hanecak, R.; Semler, B.L.; Anderson, C.W.

1982-07-01

Proteolytic processing of poliovirus polypeptides was examined by the addition of antibodies directed against the viral proteins P3-7c and P2-X to a cell-free translation extract prepared from infected HeLa cells. Antisera to P3-7c specifically inhibited in vitro processing at Gln-Gly pairs. Partial amino acid sequence analysis revealed a second Tyr-Gly pair that is utilized in protein processing. Neither Tyr-Gly cleavage is affected by antibody to P3-7C. Anti-P3-7c antibodies react not only with P3-7c but also with P3-6a and P3-2, two viral polypeptides NH/sub 2/-coterminal with P3-7c. Preimmune and anti-P2-X antibodies had no effect on the processing of poliovirus proteins inmore » vitro. The authors conclude that the activity responsible for processing poliovirus polypeptides at Gln-Gly pairs resides in the primary structure of P3-7c and not in P2-X.« less

Structural asymmetry in the Thermus thermophilus RuvC dimer suggests a basis for sequential strand cleavages during Holliday junction resolution.

PubMed

Chen, Luan; Shi, Ke; Yin, Zhiqi; Aihara, Hideki

2013-01-07

Holliday junction (HJ) resolvases are structure-specific endonucleases that cleave four-way DNA junctions (HJs) generated during DNA recombination and repair. Bacterial RuvC, a prototypical HJ resolvase, functions as homodimer and nicks DNA strands precisely across the junction point. To gain insights into the mechanisms underlying symmetrical strand cleavages by RuvC, we performed crystallographic and biochemical analyses of RuvC from Thermus thermophilus (T.th. RuvC). The crystal structure of T.th. RuvC shows an overall protein fold similar to that of Escherichia coli RuvC, but T.th. RuvC has a more tightly associated dimer interface possibly reflecting its thermostability. The binding mode of a HJ-DNA substrate can be inferred from the shape/charge complementarity between the T.th. RuvC dimer and HJ-DNA, as well as positions of sulfate ions bound on the protein surface. Unexpectedly, the structure of T.th. RuvC homodimer refined at 1.28 Å resolution shows distinct asymmetry near the dimer interface, in the region harboring catalytically important aromatic residues. The observation suggests that the T.th. RuvC homodimer interconverts between two asymmetric conformations, with alternating subunits switched on for DNA strand cleavage. This model provides a structural basis for the 'nick-counter-nick' mechanism in HJ resolution, a mode of HJ processing shared by prokaryotic and eukaryotic HJ resolvases.

Mechanism of C-C and C-H bond cleavage in ethanol oxidation reaction on Cu2O(111): a DFT-D and DFT+U study.

PubMed

Xu, Han; Miao, Bei; Zhang, Minhua; Chen, Yifei; Wang, Lichang

2017-10-04

The performance of transition metal catalysts for ethanol oxidation reaction (EOR) in direct ethanol fuel cells (DEFCs) may be greatly affected by their oxidation. However, the specific effect and catalytic mechanism for EOR of transition metal oxides are still unclear and deserve in-depth exploitation. Copper as a potential anode catalyst can be easily oxidized in air. Thus, in this study, we investigated C-C and C-H bond cleavage reactions of CH x CO (x = 1, 2, 3) species in EOR on Cu 2 O(111) using PBE+U calculations, as well as the specific effect of +U correction on the process of adsorption and reaction on Cu 2 O(111). It was revealed that the catalytic performance of Cu 2 O(111) for EOR was restrained compared with that of Cu(100). Except for the C-H cleavage of CH 2 CO, all the reaction barriers for C-C and C-H cleavage were higher than those on Cu(100). The most probable pathway for CH 3 CO to CHCO on Cu 2 O(111) was the continuous dehydrogenation reaction. Besides, the barrier for C-C bond cleavage increased due to the loss of H atoms in the intermediate. Moreover, by the comparison of the traditional GGA/PBE method and the PBE+U method, it could be concluded that C-C cleavage barriers would be underestimated without +U correction, while C-H cleavage barriers would be overestimated. +U correction was proved to be necessary, and the reaction barriers and the values of the Hubbard U parameter had a proper linear relationship.

Structure of the cleavage-activated prefusion form of the parainfluenza virus 5 fusion protein.

PubMed

Welch, Brett D; Liu, Yuanyuan; Kors, Christopher A; Leser, George P; Jardetzky, Theodore S; Lamb, Robert A

2012-10-09

The paramyxovirus parainfluenza virus 5 (PIV5) enters cells by fusion of the viral envelope with the plasma membrane through the concerted action of the fusion (F) protein and the receptor binding protein hemagglutinin-neuraminidase. The F protein folds initially to form a trimeric metastable prefusion form that is triggered to undergo large-scale irreversible conformational changes to form the trimeric postfusion conformation. It is thought that F refolding couples the energy released with membrane fusion. The F protein is synthesized as a precursor (F0) that must be cleaved by a host protease to form a biologically active molecule, F1,F2. Cleavage of F protein is a prerequisite for fusion and virus infectivity. Cleavage creates a new N terminus on F1 that contains a hydrophobic region, known as the FP, which intercalates target membranes during F protein refolding. The crystal structure of the soluble ectodomain of the uncleaved form of PIV5 F is known; here we report the crystal structure of the cleavage-activated prefusion form of PIV5 F. The structure shows minimal movement of the residues adjacent to the protease cleavage site. Most of the hydrophobic FP residues are buried in the uncleaved F protein, and only F103 at the newly created N terminus becomes more solvent-accessible after cleavage. The conformational freedom of the charged arginine residues that compose the protease recognition site increases on cleavage of F protein.

Structure of the cleavage-activated prefusion form of the parainfluenza virus 5 fusion protein

PubMed Central

Welch, Brett D.; Liu, Yuanyuan; Kors, Christopher A.; Leser, George P.; Jardetzky, Theodore S.; Lamb, Robert A.

2012-01-01

The paramyxovirus parainfluenza virus 5 (PIV5) enters cells by fusion of the viral envelope with the plasma membrane through the concerted action of the fusion (F) protein and the receptor binding protein hemagglutinin-neuraminidase. The F protein folds initially to form a trimeric metastable prefusion form that is triggered to undergo large-scale irreversible conformational changes to form the trimeric postfusion conformation. It is thought that F refolding couples the energy released with membrane fusion. The F protein is synthesized as a precursor (F0) that must be cleaved by a host protease to form a biologically active molecule, F1,F2. Cleavage of F protein is a prerequisite for fusion and virus infectivity. Cleavage creates a new N terminus on F1 that contains a hydrophobic region, known as the FP, which intercalates target membranes during F protein refolding. The crystal structure of the soluble ectodomain of the uncleaved form of PIV5 F is known; here we report the crystal structure of the cleavage-activated prefusion form of PIV5 F. The structure shows minimal movement of the residues adjacent to the protease cleavage site. Most of the hydrophobic FP residues are buried in the uncleaved F protein, and only F103 at the newly created N terminus becomes more solvent-accessible after cleavage. The conformational freedom of the charged arginine residues that compose the protease recognition site increases on cleavage of F protein. PMID:23012473

Surface-induced dissociation: a unique tool for studying energetics and kinetics of the gas-phase fragmentation of large ions

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

Laskin, Julia

2015-01-01

Surface-induced dissociation (SID) is valuable tool for investigating activation and dissociation of large ions in tandem mass spectrometry. This account summarizes key findings from studies of the energetics and mechanisms of complex ion dissociation, in which SID experiments were combined with Rice-Ramsperger-Kassel-Marcus (RRKM) modeling of the experimental data. These studies used time- and collision-energy-resolved SID experiments and SID combined with resonant ejection of selected fragment ions on a specially designed Fourier transform ion cyclotron resonance mass spectrometer (FT-ICR MS). Fast ion activation by collision with a surface combined with the long and variable timescale of a FT-ICR MS is perfectlymore » suited for studying the energetics and dynamics of complex ion dissociation in the gas phase. Modeling of time- and collision-energy-resolved SID enables accurate determination of energy and entropy effects in the dissociation process. It has been demonstrated that entropy effects play an important role in determining the dissociation rates of both covalent and non-covalent bonds in large gaseous ions. SID studies have provided important insights on the competition between charge-directed and charge-remote fragmentation in even-electron peptide ions and the role of charge and radical site on the energetics of the dissociation of odd-electron peptide ions. Furthermore, this work examined factors that affect the strength of non-covalent binding, as well as the competition between covalent and non-covalent bond cleavages and between proton and electron transfer in model systems. Finally, SID studies have been used to understand the factors affecting nucleation and growth of clusters in solution and the gas phase.« less

100 M.a. remagnetization as a dating tool for deformation and cleavage in the Central High Atlas (Morocco)

NASA Astrophysics Data System (ADS)

Calvin, P.; Casas, A. M.; Villalain, J. J.; Moussaid, B.

2015-12-01

The High Atlas is an intracontinental chain developed as a result of the inversion of Mesozoic basins during the Cenozoic. Its structure is characterized by ENE-WSW tight anticlines limited by wide synclines. In the central sector of the chain, a pervasive tectonic foliation affects Jurassic limestones and marls. There is a controversy about the age of this tectonic foliation and its relationship with either a Late Jurassic or Cenozoic compressional events. The Jurassic carbonates of the Central High Atlas (CHA) show a widespread syntectonic remagnetization carried by magnetite and dated at 100 M.a. by comparing the paleomagnetic direction obtained by small circle intersection (SCI) method with the apparent polar wander path in NW Africa coordinates. Once the 100 M.a. paleomagnetic direction is known, the obtained paleomagnetic direction remagnetization in each site can be used to restore the bedding at the time of the acquisition. In each site, the in situ mean direction of remagnetization defines a small circle (SC) in a complete rotation about the strike of the bedding. This SC gives all possible original directions of the magnetization and if all deformation events are coaxial and without vertical rotation (as it is the case in the study area) all the SC contains the 100 M.a. expected direction. Then, the angle between the 100 M.a. and the in situ paleomagnetic direction along the small circle, equals the angle of rotation of each bed to ultimately find their dips (paleodip) at 100 M.a. Since the consistency between folding and cleavage can be examined from their geometrical relationship, and bedding can be restored to its 100 M.a. geometry (paleodips obtained from paleomagnetic analysis), two end-members exist for the different examined folds: (i) cleavage is consistent with present-day bedding orientation and attitude of bedding was acquired after remagnetization (Cenozoic cleavage), (ii) cleavage is consistent with bedding, but dip of bedding was acquired during the pre-remagnetization stage, as inferred from paleomagnetic analysis (Mesozoic cleavage). Intermediate possibilities, having cleavage developed during one of the two stages with intermediate dips between present-day orientation and the horizontal, are also possible, and are analyzed in different examples of the Atlas chain.

Phosphorylation-dependent cleavage regulates von Hippel Lindau proteostasis and function

PubMed Central

German, Peter; Bai, Shanshan; Liu, Xian-De; Sun, Mianen; Zhou, Lijun; Kalra, Sarathi; Zhang, Xuesong; Minelli, Rosalba; Scott, Kenneth L.; Mills, Gordon B.; Jonasch, Eric; Ding, Zhiyong

2016-01-01

Loss of von Hippel Lindau (VHL) protein function is a key driver of VHL diseases, including sporadic and inherited clear cell renal cell carcinoma. Modulation of the proteostasis of VHL, especially missense point-mutated VHL, is a promising approach to augmenting VHL levels and function. VHL proteostasis is regulated by multiple mechanisms including folding, chaperone binding, complex formation, and phosphorylation. Nevertheless, many details underlying the regulations of VHL proteostasis are unknown. VHL is expressed as two variants, VHL30 and VHL19. Furthermore, the long form variant of VHL was often detected as multiple bands by Western blotting. However, how these multiple species of VHL are generated and whether the process regulates VHL proteostasis and function are unknown. We hypothesized that the two major species are generated by VHL protein cleavage, and the cleavage regulates VHL proteostasis and subsequent function. We characterized VHL species using genetic and pharmacologic approaches and showed that VHL was first cleaved at the N-terminus by chymotrypsin C before being directed for proteasomal degradation. Casein kinase 2-mediated phosphorylation at VHL N-terminus was required for the cleavage. Furthermore, inhibition of cleavage stabilized VHL protein, thereby promoting HIF downregulation. Our study reveals a novel mechanism regulating VHL proteostasis and function, which is significant for identifying new drug targets and developing new therapeutic approaches targeting VHL deficiency in VHL diseases. PMID:26973240

Catalytic Arylation and Vinylation Reactions Directed by Anionic Oxygen Functions via Cleavage of C - H and C - C Bonds

NASA Astrophysics Data System (ADS)

Satoh, Tetsuya; Miura, Masahiro

Aromatic compounds having oxygen-containing substituents such as phenols, phenyl ketones, benzyl alcohols, and benzoic acids undergo regioselective arylation and vinylation via C-H bond cleavage in the presence of transition-metal catalysts. The latter two substrates are also arylated and vinylated via C-C bond cleavage accompanied by liberation of ketones and CO2, respectively. Coordination of their anionic oxygen to the metal center is the key to activate the inert bonds effectively and regioselectively. The recent progress of these oxygen-directed reactions is summarized herein.

Photodissociation of aligned CH3I and C6H3F2I molecules probed with time-resolved Coulomb explosion imaging by site-selective extreme ultraviolet ionization

PubMed Central

Amini, Kasra; Savelyev, Evgeny; Brauße, Felix; Berrah, Nora; Bomme, Cédric; Brouard, Mark; Burt, Michael; Christensen, Lauge; Düsterer, Stefan; Erk, Benjamin; Höppner, Hauke; Kierspel, Thomas; Krecinic, Faruk; Lauer, Alexandra; Lee, Jason W. L.; Müller, Maria; Müller, Erland; Mullins, Terence; Redlin, Harald; Schirmel, Nora; Thøgersen, Jan; Techert, Simone; Toleikis, Sven; Treusch, Rolf; Trippel, Sebastian; Ulmer, Anatoli; Vallance, Claire; Wiese, Joss; Johnsson, Per; Küpper, Jochen; Rudenko, Artem; Rouzée, Arnaud; Stapelfeldt, Henrik; Rolles, Daniel; Boll, Rebecca

2018-01-01

We explore time-resolved Coulomb explosion induced by intense, extreme ultraviolet (XUV) femtosecond pulses from a free-electron laser as a method to image photo-induced molecular dynamics in two molecules, iodomethane and 2,6-difluoroiodobenzene. At an excitation wavelength of 267 nm, the dominant reaction pathway in both molecules is neutral dissociation via cleavage of the carbon–iodine bond. This allows investigating the influence of the molecular environment on the absorption of an intense, femtosecond XUV pulse and the subsequent Coulomb explosion process. We find that the XUV probe pulse induces local inner-shell ionization of atomic iodine in dissociating iodomethane, in contrast to non-selective ionization of all photofragments in difluoroiodobenzene. The results reveal evidence of electron transfer from methyl and phenyl moieties to a multiply charged iodine ion. In addition, indications for ultrafast charge rearrangement on the phenyl radical are found, suggesting that time-resolved Coulomb explosion imaging is sensitive to the localization of charge in extended molecules. PMID:29430482

Photodissociation of aligned CH3I and C6H3F2I molecules probed with time-resolved Coulomb explosion imaging by site-selective extreme ultraviolet ionization.

PubMed

Amini, Kasra; Savelyev, Evgeny; Brauße, Felix; Berrah, Nora; Bomme, Cédric; Brouard, Mark; Burt, Michael; Christensen, Lauge; Düsterer, Stefan; Erk, Benjamin; Höppner, Hauke; Kierspel, Thomas; Krecinic, Faruk; Lauer, Alexandra; Lee, Jason W L; Müller, Maria; Müller, Erland; Mullins, Terence; Redlin, Harald; Schirmel, Nora; Thøgersen, Jan; Techert, Simone; Toleikis, Sven; Treusch, Rolf; Trippel, Sebastian; Ulmer, Anatoli; Vallance, Claire; Wiese, Joss; Johnsson, Per; Küpper, Jochen; Rudenko, Artem; Rouzée, Arnaud; Stapelfeldt, Henrik; Rolles, Daniel; Boll, Rebecca

2018-01-01

We explore time-resolved Coulomb explosion induced by intense, extreme ultraviolet (XUV) femtosecond pulses from a free-electron laser as a method to image photo-induced molecular dynamics in two molecules, iodomethane and 2,6-difluoroiodobenzene. At an excitation wavelength of 267 nm, the dominant reaction pathway in both molecules is neutral dissociation via cleavage of the carbon-iodine bond. This allows investigating the influence of the molecular environment on the absorption of an intense, femtosecond XUV pulse and the subsequent Coulomb explosion process. We find that the XUV probe pulse induces local inner-shell ionization of atomic iodine in dissociating iodomethane, in contrast to non-selective ionization of all photofragments in difluoroiodobenzene. The results reveal evidence of electron transfer from methyl and phenyl moieties to a multiply charged iodine ion. In addition, indications for ultrafast charge rearrangement on the phenyl radical are found, suggesting that time-resolved Coulomb explosion imaging is sensitive to the localization of charge in extended molecules.

tRNA biology charges to the front

PubMed Central

Phizicky, Eric M.; Hopper, Anita K.

2010-01-01

tRNA biology has come of age, revealing an unprecedented level of understanding and many unexpected discoveries along the way. This review highlights new findings on the diverse pathways of tRNA maturation, and on the formation and function of a number of modifications. Topics of special focus include the regulation of tRNA biosynthesis, quality control tRNA turnover mechanisms, widespread tRNA cleavage pathways activated in response to stress and other growth conditions, emerging evidence of signaling pathways involving tRNA and cleavage fragments, and the sophisticated intracellular tRNA trafficking that occurs during and after biosynthesis. PMID:20810645

Bacterial Hsp70 (DnaK) and mammalian Hsp70 interact differently with lipid membranes.

PubMed

Lopez, Victor; Cauvi, David M; Arispe, Nelson; De Maio, Antonio

2016-07-01

The cellular response to stress is orchestrated by the expression of a family of proteins termed heat shock proteins (hsp) that are involved in the stabilization of basic cellular processes to preserve cell viability and homeostasis. The bulk of hsp function occurs within the cytosol and subcellular compartments. However, some hsp have also been found outside cells released by an active mechanism independent of cell death. Extracellular hsp act as signaling molecules directed at activating a systemic response to stress. The export of hsp requires the translocation from the cytosol into the extracellular milieu across the plasma membrane. We have proposed that membrane insertion is the initial step in this export process. We investigated the interaction of the major inducible hsp from mammalian (Hsp70) and bacterial (DnaK) species with liposomes. We found that mammalian Hsp70 displayed a high specificity for negatively charged phospholipids, such as phosphatidyl serine, whereas DnaK interacted with all lipids tested regardless of the charge. Both proteins were inserted into the lipid bilayer as demonstrated by resistance to acid or basic washes that was confirmed by partial protection from proteolytic cleavage. Several regions of mammalian Hsp70 were inserted into the membrane with a small portion of the N-terminus end exposed to the outer phase of the liposome. In contrast, the N-terminus end of DnaK was inserted into the membrane, exposing the C-terminus end outside the liposome. Mammalian Hsp70 was found to make high oligomeric complexes upon insertion into the membranes whereas DnaK only formed dimers within the lipid bilayer. These observations suggest that both Hsp70s interact with lipids, but mammalian Hsp70 displays a high degree of specificity and structure as compared with the bacterial form.

Structural studies and molecular dynamics simulations suggest a processive mechanism of exolytic lytic transglycosylase from Campylobacter jejuni.

PubMed

Vijayaraghavan, Jagamya; Kumar, Vijay; Krishnan, Nikhil P; Kaufhold, Ross T; Zeng, Ximin; Lin, Jun; van den Akker, Focco

2018-01-01

The bacterial soluble lytic transglycosylase (LT) breaks down the peptidoglycan (PG) layer during processes such as cell division. We present here crystal structures of the soluble LT Cj0843 from Campylobacter jejuni with and without bulgecin A inhibitor in the active site. Cj0843 has a doughnut shape similar but not identical to that of E. coli SLT70. The C-terminal catalytic domain is preceded by an L-domain, a large helical U-domain, a flexible linker, and a small N-terminal NU-domain. The flexible linker allows the NU-domain to reach over and complete the circular shape, using residues conserved in the Epsilonproteobacteria LT family. The inner surface of the Cj0843 doughnut is mostly positively charged including a pocket that has 8 Arg/Lys residues. Molecular dynamics simulations with PG strands revealed a potential functional role for this pocket in anchoring the negatively charged terminal tetrapeptide of the PG during several steps in the reaction including homing and aligning the PG strand for exolytic cleavage, and subsequent ratcheting of the PG strand to enhance processivity in degrading PG strands.

Direct Observation of Individual Charges and Their Dynamics on Graphene by Low-Energy Electron Holography.

PubMed

Latychevskaia, Tatiana; Wicki, Flavio; Longchamp, Jean-Nicolas; Escher, Conrad; Fink, Hans-Werner

2016-09-14

Visualizing individual charges confined to molecules and observing their dynamics with high spatial resolution is a challenge for advancing various fields in science, ranging from mesoscopic physics to electron transfer events in biological molecules. We show here that the high sensitivity of low-energy electrons to local electric fields can be employed to directly visualize individual charged adsorbates and to study their behavior in a quantitative way. This makes electron holography a unique probing tool for directly visualizing charge distributions with a sensitivity of a fraction of an elementary charge. Moreover, spatial resolution in the nanometer range and fast data acquisition inherent to lens-less low-energy electron holography allows for direct visual inspection of charge transfer processes.

The endosomal protein CHARGED MULTIVESICULAR BODY PROTEIN1 regulates the autophagic turnover of plastids in Arabidopsis.

PubMed

Spitzer, Christoph; Li, Faqiang; Buono, Rafael; Roschzttardtz, Hannetz; Chung, Taijoon; Zhang, Min; Osteryoung, Katherine W; Vierstra, Richard D; Otegui, Marisa S

2015-02-01

Endosomal Sorting Complex Required for Transport (ESCRT)-III proteins mediate membrane remodeling and the release of endosomal intraluminal vesicles into multivesicular bodies. Here, we show that the ESCRT-III subunit paralogs CHARGED MULTIVESICULAR BODY PROTEIN1 (CHMP1A) and CHMP1B are required for autophagic degradation of plastid proteins in Arabidopsis thaliana. Similar to autophagy mutants, chmp1a chmp1b (chmp1) plants hyperaccumulated plastid components, including proteins involved in plastid division. The autophagy machinery directed the release of bodies containing plastid material into the cytoplasm, whereas CHMP1A and B were required for delivery of these bodies to the vacuole. Autophagy was upregulated in chmp1 as indicated by an increase in vacuolar green fluorescent protein (GFP) cleavage from the autophagic reporter GFP-ATG8. However, autophagic degradation of the stromal cargo RECA-GFP was drastically reduced in the chmp1 plants upon starvation, suggesting that CHMP1 mediates the efficient delivery of autophagic plastid cargo to the vacuole. Consistent with the compromised degradation of plastid proteins, chmp1 plastids show severe morphological defects and aberrant division. We propose that CHMP1 plays a direct role in the autophagic turnover of plastid constituents. © 2015 American Society of Plant Biologists. All rights reserved.

Specific detection of the cleavage activity of mycobacterial enzymes using a quantum dot based DNA nanosensor

NASA Astrophysics Data System (ADS)

Jepsen, Morten Leth; Harmsen, Charlotte; Godbole, Adwait Anand; Nagaraja, Valakunja; Knudsen, Birgitta R.; Ho, Yi-Ping

2015-12-01

We present a quantum dot based DNA nanosensor specifically targeting the cleavage step in the reaction cycle of the essential DNA-modifying enzyme, mycobacterial topoisomerase I. The design takes advantages of the unique photophysical properties of quantum dots to generate visible fluorescence recovery upon specific cleavage by mycobacterial topoisomerase I. This report, for the first time, demonstrates the possibility to quantify the cleavage activity of the mycobacterial enzyme without the pre-processing sample purification or post-processing signal amplification. The cleavage induced signal response has also proven reliable in biological matrices, such as whole cell extracts prepared from Escherichia coli and human Caco-2 cells. It is expected that the assay may contribute to the clinical diagnostics of bacterial diseases, as well as the evaluation of treatment outcomes.We present a quantum dot based DNA nanosensor specifically targeting the cleavage step in the reaction cycle of the essential DNA-modifying enzyme, mycobacterial topoisomerase I. The design takes advantages of the unique photophysical properties of quantum dots to generate visible fluorescence recovery upon specific cleavage by mycobacterial topoisomerase I. This report, for the first time, demonstrates the possibility to quantify the cleavage activity of the mycobacterial enzyme without the pre-processing sample purification or post-processing signal amplification. The cleavage induced signal response has also proven reliable in biological matrices, such as whole cell extracts prepared from Escherichia coli and human Caco-2 cells. It is expected that the assay may contribute to the clinical diagnostics of bacterial diseases, as well as the evaluation of treatment outcomes. Electronic supplementary information (ESI) available: Characterization of the QD-based DNA Nanosensor. See DOI: 10.1039/c5nr06326d

Can sucrose cleavage enzymes serve as markers for sink strength and is sucrose a signal molecule during plant sink development?

Treesearch

C.C. Black; T. Lobodia; J.-Q Chen; Shi-Jean S. Sung

1995-01-01

Sucrose cleavage is an essential reaction for higher plant cells to initiate intermediary metabolism and to direct its carbon into the host of essential compounds derived therefrom for maintaining the cells of intact plants.This review will focus on: the concentrations of sucrose available to plant cells; some biochemical traits of sucrose cleavage enzymes; the...

Charge stabilization and entropy reduction of central lysine residues in fructose-bisphosphate aldolase.

PubMed

St-Jean, Miguel; Blonski, Casimir; Sygusch, Jurgen

2009-06-02

Fructose-1,6-bisphosphate muscle aldolase is an essential glycolytic enzyme that catalyzes reversible carbon-carbon bond formation by cleaving fructose 1,6-bisphosphate to yield dihydroxyacetone phosphate (DHAP) and d-glyceraldehyde phosphate. To elucidate the mechanistic role of conserved amino acid Asp-33, Asn-33 and Ser-33 mutants were examined by kinetic and structural analyses. The mutations significantly compromised enzymatic activity and carbanion oxidation in presence of DHAP. Detailed structural analysis demonstrated that, like native crystals, Asp-33 mutant crystals, soaked in DHAP solutions, trapped Schiff base-derived intermediates covalently attached to Lys-229. The mutant structures, however, exhibited an abridged conformational change with the helical region (34-65) flanking the active site as well as pK(a) reductions and increased side chain disorder by central lysine residues, Lys-107 and Lys-146. These changes directly affect their interaction with the C-terminal Tyr-363, consistent with the absence of active site binding by the C-terminal region in the presence of phosphate. Lys-146 pK(a) reduction and side chain disorder would further compromise charge stabilization during C-C bond cleavage and proton transfer during enamine formation. These mechanistic impediments explain diminished catalytic activity and a reduced level of carbanion oxidation and are consistent with rate-determining proton transfer observed in the Asn-33 mutant. Asp-33 reduces the entropic cost and augments the enthalpic gain during catalysis by rigidifying Lys-107 and Lys-146, stabilizing their protonated forms, and promoting a conformational change triggered by substrate or obligate product binding, which lower kinetic barriers in C-C bond cleavage and Schiff base-enamine interconversion.

Charged-particle emission tomography

PubMed Central

Ding, Yijun; Caucci, Luca; Barrett, Harrison H.

2018-01-01

Purpose Conventional charged-particle imaging techniques —such as autoradiography —provide only two-dimensional (2D) black ex vivo images of thin tissue slices. In order to get volumetric information, images of multiple thin slices are stacked. This process is time consuming and prone to distortions, as registration of 2D images is required. We propose a direct three-dimensional (3D) autoradiography technique, which we call charged-particle emission tomography (CPET). This 3D imaging technique enables imaging of thick tissue sections, thus increasing laboratory throughput and eliminating distortions due to registration. CPET also has the potential to enable in vivo charged-particle imaging with a window chamber or an endoscope. Methods Our approach to charged-particle emission tomography uses particle-processing detectors (PPDs) to estimate attributes of each detected particle. The attributes we estimate include location, direction of propagation, and/or the energy deposited in the detector. Estimated attributes are then fed into a reconstruction algorithm to reconstruct the 3D distribution of charged-particle-emitting radionuclides. Several setups to realize PPDs are designed. Reconstruction algorithms for CPET are developed. Results Reconstruction results from simulated data showed that a PPD enables CPET if the PPD measures more attributes than just the position from each detected particle. Experiments showed that a two-foil charged-particle detector is able to measure the position and direction of incident alpha particles. Conclusions We proposed a new volumetric imaging technique for charged-particle-emitting radionuclides, which we have called charged-particle emission tomography (CPET). We also proposed a new class of charged-particle detectors, which we have called particle-processing detectors (PPDs). When a PPD is used to measure the direction and/or energy attributes along with the position attributes, CPET is feasible. PMID:28370094

Charged-particle emission tomography.

PubMed

Ding, Yijun; Caucci, Luca; Barrett, Harrison H

2017-06-01

Conventional charged-particle imaging techniques - such as autoradiography - provide only two-dimensional (2D) black ex vivo images of thin tissue slices. In order to get volumetric information, images of multiple thin slices are stacked. This process is time consuming and prone to distortions, as registration of 2D images is required. We propose a direct three-dimensional (3D) autoradiography technique, which we call charged-particle emission tomography (CPET). This 3D imaging technique enables imaging of thick tissue sections, thus increasing laboratory throughput and eliminating distortions due to registration. CPET also has the potential to enable in vivo charged-particle imaging with a window chamber or an endoscope. Our approach to charged-particle emission tomography uses particle-processing detectors (PPDs) to estimate attributes of each detected particle. The attributes we estimate include location, direction of propagation, and/or the energy deposited in the detector. Estimated attributes are then fed into a reconstruction algorithm to reconstruct the 3D distribution of charged-particle-emitting radionuclides. Several setups to realize PPDs are designed. Reconstruction algorithms for CPET are developed. Reconstruction results from simulated data showed that a PPD enables CPET if the PPD measures more attributes than just the position from each detected particle. Experiments showed that a two-foil charged-particle detector is able to measure the position and direction of incident alpha particles. We proposed a new volumetric imaging technique for charged-particle-emitting radionuclides, which we have called charged-particle emission tomography (CPET). We also proposed a new class of charged-particle detectors, which we have called particle-processing detectors (PPDs). When a PPD is used to measure the direction and/or energy attributes along with the position attributes, CPET is feasible. © 2017 The Authors. Medical Physics published by Wiley Periodicals, Inc. on behalf of American Association of Physicists in Medicine.

Electron Detachment Dissociation (EDD) of Fluorescently Labeled Sialylated Oligosaccharides

PubMed Central

Zhou, Wen; Håkansson, Kristina

2012-01-01

We explored the application of electron detachment dissociation (EDD) and infrared multiphoton dissociation (IRMPD) tandem mass spectrometry to fluorescently labeled sialylated oligosaccharides. Standard sialylated oligosaccharides and a sialylated N-linked glycan released from human transferrin were investigated. EDD yielded extensive glycosidic cleavages and cross-ring cleavages in all cases studied, consistently providing complementary structural information compared to IRMPD. Neutral losses and satellite ions such as C – 2H ions were also observed following EDD. In addition, we examined the influence of different fluorescent labels. The acidic label 2-aminobenzoic acid (2-AA) enhanced signal abundance in negative-ion mode. However, few cross-ring fragments were observed for 2-AA labeled oligosaccharides. The neutral label 2-aminobenzamide (2-AB) resulted in more cross-ring cleavages compared to 2-AA labeled species, but not as extensive fragmentation as for native oligosaccharides, likely resulting from altered negative charge locations from introduction of the fluorescent tag. PMID:22120881

A deletion mutation in the 5' part of the pol gene of Moloney murine leukemia virus blocks proteolytic processing of the gag and pol polyproteins.

PubMed Central

Crawford, S; Goff, S P

1985-01-01

Deletion mutations in the 5' part of the pol gene of Moloney murine leukemia virus were generated by restriction enzyme site-directed mutagenesis of cloned proviral DNA. DNA sequence analysis indicated that one such deletion was localized entirely within the 5' part of the pol gene, did not affect the region encoding reverse transcriptase, and preserved the translational reading frame downstream of the mutation. The major viral precursor polyproteins (Pr65gag, Pr200gag-pol, and gPr80env) were synthesized at wild-type levels in cell lines carrying the mutant genome. These cell lines assembled and released wild-type levels of virion particles into the medium. Cleavage of both Pr65gag and Pr200gag-pol precursors to the mature proteins was completely blocked in the mutant virions. Surprisingly, these virions contained high levels of active reverse transcriptase; examination of the endogenous reverse transcription products synthesized by the mutant virions revealed normal amounts of minus-strand strong-stop DNA, indicating that the RNA genome was packaged and that reverse transcription in detergent-permeabilized virions was not significantly impaired. Processing of gPr80env to gP70env and P15E was not affected by the mutation, but cleavage of P15E to P12E was not observed. The mutant particles were poorly infectious; analysis indicated that infection was blocked at an early stage. The data are consistent with the idea that the 5' part of the pol gene encodes a protease directly responsible for processing Pr65gag, and possibly Pr200gag-pol, to the structural virion proteins. It appears that cleavage of the gag gene product is not required for budding and release of virions and that complete processing of the pol gene product to the mature form of reverse transcriptase is not required for its functional activation. Images PMID:3882995

Processing sites in the human immunodeficiency virus type 1 (HIV-1) Gag-Pro-Pol precursor are cleaved by the viral protease at different rates

PubMed Central

Pettit, Steve C; Lindquist, Jeffrey N; Kaplan, Andrew H; Swanstrom, Ronald

2005-01-01

We have examined the kinetics of processing of the HIV-1 Gag-Pro-Pol precursor in an in vitro assay with mature protease added in trans. The processing sites were cleaved at different rates to produce distinct intermediates. The initial cleavage occurred at the p2/NC site. Intermediate cleavages occurred at similar rates at the MA/CA and RT/IN sites, and to a lesser extent at sites upstream of RT. Late cleavages occurred at the sites flanking the protease (PR) domain, suggesting sequestering of these sites. We observed paired intermediates indicative of half- cleavage of RT/RH site, suggesting that the RT domain in Gag-Pro-Pol was in a dimeric form under these assay conditions. These results clarify our understanding of the processing kinetics of the Gag-Pro-Pol precursor and suggest regulated cleavage. Our results further suggest that early dimerization of the PR and RT domains may serve as a regulatory element to influence the kinetics of processing within the Pol domain. PMID:16262906

Processing sites in the human immunodeficiency virus type 1 (HIV-1) Gag-Pro-Pol precursor are cleaved by the viral protease at different rates.

PubMed

Pettit, Steve C; Lindquist, Jeffrey N; Kaplan, Andrew H; Swanstrom, Ronald

2005-11-01

We have examined the kinetics of processing of the HIV-1 Gag-Pro-Pol precursor in an in vitro assay with mature protease added in trans. The processing sites were cleaved at different rates to produce distinct intermediates. The initial cleavage occurred at the p2/NC site. Intermediate cleavages occurred at similar rates at the MA/CA and RT/IN sites, and to a lesser extent at sites upstream of RT. Late cleavages occurred at the sites flanking the protease (PR) domain, suggesting sequestering of these sites. We observed paired intermediates indicative of half- cleavage of RT/RH site, suggesting that the RT domain in Gag-Pro-Pol was in a dimeric form under these assay conditions. These results clarify our understanding of the processing kinetics of the Gag-Pro-Pol precursor and suggest regulated cleavage. Our results further suggest that early dimerization of the PR and RT domains may serve as a regulatory element to influence the kinetics of processing within the Pol domain.

Distinct requirements for signal peptidase processing and function in the stable signal peptide subunit of the Junin virus envelope glycoprotein

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

York, Joanne; Nunberg, Jack H.

2007-03-01

The arenavirus envelope glycoprotein (GP-C) retains a cleaved and stable signal peptide (SSP) as an essential subunit of the mature complex. This 58-amino-acid residue peptide serves as a signal sequence and is additionally required to enable transit of the assembled GP-C complex to the Golgi, and for pH-dependent membrane fusion activity. We have investigated the C-terminal region of the Junin virus SSP to study the role of the cellular signal peptidase (SPase) in generating SSP. Site-directed mutagenesis at the cleavage site (positions - 1 and - 3) reveals a pattern of side-chain preferences consistent with those of SPase. Although positionmore » - 2 is degenerate for SPase cleavage, this residue in the arenavirus SSP is invariably a cysteine. In the Junin virus, this cysteine is not involved in disulfide bonding. We show that replacement with alanine or serine is tolerated for SPase cleavage but prevents the mutant SSP from associating with GP-C and enabling transport to the cell surface. Conversely, an arginine mutation at position - 1 that prevents SPase cleavage is fully compatible with GP-C-mediated membrane fusion activity when the mutant SSP is provided in trans. These results point to distinct roles of SSP sequences in SPase cleavage and GP-C biogenesis. Further studies of the unique structural organization of the GP-C complex will be important in identifying novel opportunities for antiviral intervention against arenaviral hemorrhagic disease.« less

A molecular switch sensor for detection of PRSS1 genotype based on site-specific DNA cleavage of restriction endonuclease.

PubMed

Liu, Qicai; Gao, Feng; Weng, Shaohuang; Peng, Huaping; Lin, Liqing; Zhao, Chengfei; Lin, Xinhua

2015-01-01

PRSS1 mutations or polymorphism in the peripheral blood of patients can be used as susceptible molecular markers to pancreatic cancer. A sensor for selective electrochemical detection of PRSS1 genotypes was developed based on site-specific DNA cleavage of restriction endonuclease EcoRI. A mercapto-modified hairpin probe was immobilized on a gold electrode. The probe's neck can be cleaved by EcoRI in the absence of rs10273639 C/C of PRSS1 genotype, but it cannot be cleaved in the presence of T/T. The difference in quantity of electric charge was monitored by biosensors before and after enzymatic cleavage. Electrochemical signals are generated by differential pulse voltammetry interrogation of methylene blue (MB) that quantitatively binds to surface-confined hairpin probe via electrostatic interactions. The results suggested this method had a good specificity in distinguishing PRSS1 genotypes. There was a good linear relationship between the charge and the logarithmic function of PRSS1 rs10273639 T/T type DNA concentration (current=120.6303+8.8512log C, R=0.9942). The detection limit was estimated at 0.5 fM. The molecular switch sensor has several advantages, and it is possible to qualitatively, quantitatively, and noninvasively detect PRSS1 genotypes in the blood of patients with pancreatic cancer. © 2015 by the Association of Clinical Scientists, Inc.

Progressive engineering of a homing endonuclease genome editing reagent for the murine X-linked immunodeficiency locus

PubMed Central

Wang, Yupeng; Khan, Iram F.; Boissel, Sandrine; Jarjour, Jordan; Pangallo, Joseph; Thyme, Summer; Baker, David; Scharenberg, Andrew M.; Rawlings, David J.

2014-01-01

LAGLIDADG homing endonucleases (LHEs) are compact endonucleases with 20–22 bp recognition sites, and thus are ideal scaffolds for engineering site-specific DNA cleavage enzymes for genome editing applications. Here, we describe a general approach to LHE engineering that combines rational design with directed evolution, using a yeast surface display high-throughput cleavage selection. This approach was employed to alter the binding and cleavage specificity of the I-Anil LHE to recognize a mutation in the mouse Bruton tyrosine kinase (Btk) gene causative for mouse X-linked immunodeficiency (XID)—a model of human X-linked agammaglobulinemia (XLA). The required re-targeting of I-AniI involved progressive resculpting of the DNA contact interface to accommodate nine base differences from the native cleavage sequence. The enzyme emerging from the progressive engineering process was specific for the XID mutant allele versus the wild-type (WT) allele, and exhibited activity equivalent to WT I-AniI in vitro and in cellulo reporter assays. Fusion of the enzyme to a site-specific DNA binding domain of transcription activator-like effector (TALE) resulted in a further enhancement of gene editing efficiency. These results illustrate the potential of LHE enzymes as specific and efficient tools for therapeutic genome engineering. PMID:24682825

Tomato carotenoid cleavage dioxygenases 1A and 1B: Relaxed double bond specificity leads to a plenitude of dialdehydes, mono-apocarotenoids and isoprenoid volatiles

PubMed Central

Ilg, Andrea; Bruno, Mark; Beyer, Peter; Al-Babili, Salim

2014-01-01

The biosynthetic processes leading to many of the isoprenoid volatiles released by tomato fruits are still unknown, though previous reports suggested a clear correlation with the carotenoids contained within the fruit. In this study, we investigated the activity of the tomato (Solanum lycopersicum) carotenoid cleavage dioxygenase (SlCCD1B), which is highly expressed in fruits, and of its homolog SlCCD1A. Using in vitro assays performed with purified recombinant enzymes and by analyzing products formed by the two enzymes in carotene-accumulating Escherichia coli strains, we demonstrate that SlCCD1A and, to a larger extent, SlCCD1B, have a very relaxed specificity for both substrate and cleavage site, mediating the oxidative cleavage of cis- and all-trans-carotenoids as well as of different apocarotenoids at many more double bonds than previously reported. This activity gives rise to a plenitude of volatiles, mono-apocarotenoids and dialdehyde products, including cis-pseudoionone, neral, geranial, and farnesylacetone. Our results provide a direct evidence for a carotenoid origin of these compounds and point to CCD1s as the enzymes catalyzing the formation of the vast majority of tomato isoprenoid volatiles, many of which are aroma constituents. PMID:25057464

Imaging of Endogenous Metabolites of Plant Leaves by Mass Spectrometry Based on Laser Activated Electron Tunneling.

PubMed

Huang, Lulu; Tang, Xuemei; Zhang, Wenyang; Jiang, Ruowei; Chen, Disong; Zhang, Juan; Zhong, Hongying

2016-04-07

A new mass spectrometric imaging approach based on laser activated electron tunneling (LAET) was described and applied to analysis of endogenous metabolites of plant leaves. LAET is an electron-directed soft ionization technique. Compressed thin films of semiconductor nanoparticles of bismuth cobalt zinc oxide were placed on the sample plate for proof-of-principle demonstration because they can not only absorb ultraviolet laser but also have high electron mobility. Upon laser irradiation, electrons are excited from valence bands to conduction bands. With appropriate kinetic energies, photoexcited electrons can tunnel away from the barrier and eventually be captured by charge deficient atoms present in neutral molecules. Resultant unpaired electron subsequently initiates specific chemical bond cleavage and generates ions that can be detected in negative ion mode of the mass spectrometer. LAET avoids the co-crystallization process of routinely used organic matrix materials with analyzes in MALDI (matrix assisted-laser desorption ionization) analysis. Thus uneven distribution of crystals with different sizes and shapes as well as background peaks in the low mass range resulting from matrix molecules is eliminated. Advantages of LAET imaging technique include not only improved spatial resolution but also photoelectron capture dissociation which produces predictable fragment ions.

Controllable laser thermal cleavage of sapphire wafers

NASA Astrophysics Data System (ADS)

Xu, Jiayu; Hu, Hong; Zhuang, Changhui; Ma, Guodong; Han, Junlong; Lei, Yulin

2018-03-01

Laser processing of substrates for light-emitting diodes (LEDs) offers advantages over other processing techniques and is therefore an active research area in both industrial and academic sectors. The processing of sapphire wafers is problematic because sapphire is a hard and brittle material. Semiconductor laser scribing processing suffers certain disadvantages that have yet to be overcome, thereby necessitating further investigation. In this work, a platform for controllable laser thermal cleavage was constructed. A sapphire LED wafer was modeled using the finite element method to simulate the thermal and stress distributions under different conditions. A guide groove cut by laser ablation before the cleavage process was observed to guide the crack extension and avoid deviation. The surface and cross section of sapphire wafers processed using controllable laser thermal cleavage were characterized by scanning electron microscopy and optical microscopy, and their morphology was compared to that of wafers processed using stealth dicing. The differences in luminous efficiency between substrates prepared using these two processing methods are explained.

Molecular Mechanism of Processive 3' to 5' RNA Translocation in the Active Subunit of the RNA Exosome Complex.

PubMed

Vuković, Lela; Chipot, Christophe; Makino, Debora L; Conti, Elena; Schulten, Klaus

2016-03-30

Recent experimental studies revealed structural details of 3' to 5' degradation of RNA molecules, performed by the exosome complex. ssRNA is channeled through its multisubunit ring-like core into the active site tunnel of its key exonuclease subunit Rrp44, which acts both as an enzyme and a motor. Even in isolation, Rrp44 can pull and sequentially cleave RNA nucleotides, one at a time, without any external energy input and release a final 3-5 nucleotide long product. Using molecular dynamics simulations, we identify the main factors that control these processes. Our free energy calculations reveal that RNA transfer from solution into the active site of Rrp44 is highly favorable, but dependent on the length of the RNA strand. While RNA strands formed by 5 nucleotides or more correspond to a decreasing free energy along the translocation coordinate toward the cleavage site, a 4-nucleotide RNA experiences a free energy barrier along the same direction, potentially leading to incomplete cleavage of ssRNA and the release of short (3-5) nucleotide products. We provide new insight into how Rrp44 catalyzes a localized enzymatic reaction and performs an action distributed over several RNA nucleotides, leading eventually to the translocation of whole RNA segments into the position suitable for cleavage.

Different Requirements for Proteolytic Processing of Bone Morphogenetic Protein 5/6/7/8 Ligands in Drosophila melanogaster*

PubMed Central

Fritsch, Cornelia; Sawala, Annick; Harris, Robin; Maartens, Aidan; Sutcliffe, Catherine; Ashe, Hilary L.; Ray, Robert P.

2012-01-01

Bone morphogenetic proteins (BMPs) are synthesized as proproteins that undergo proteolytic processing by furin/subtilisin proprotein convertases to release the active ligand. Here we study processing of BMP5/6/7/8 proteins, including the Drosophila orthologs Glass Bottom Boat (Gbb) and Screw (Scw) and human BMP7. Gbb and Scw have three functional furin/subtilisin proprotein convertase cleavage sites; two between the prodomain and ligand domain, which we call the Main and Shadow sites, and one within the prodomain, which we call the Pro site. In Gbb each site can be cleaved independently, although efficient cleavage at the Shadow site requires cleavage at the Main site, and remarkably, none of the sites is essential for Gbb function. Rather, Gbb must be processed at either the Pro or Main site to produce a functional ligand. Like Gbb, the Pro and Main sites in Scw can be cleaved independently, but cleavage at the Shadow site is dependent on cleavage at the Main site. However, both Pro and Main sites are essential for Scw function. Thus, Gbb and Scw have different processing requirements. The BMP7 ligand rescues gbb mutants in Drosophila, but full-length BMP7 cannot, showing that functional differences in the prodomain limit the BMP7 activity in flies. Furthermore, unlike Gbb, cleavage-resistant BMP7, although non-functional in rescue assays, activates the downstream signaling cascade and thus retains some functionality. Our data show that cleavage requirements evolve rapidly, supporting the notion that changes in post-translational processing are used to create functional diversity between BMPs within and between species. PMID:22199351

cis-Apa: a practical linker for the microwave-assisted preparation of cyclic pseudopeptides via RCM cyclative cleavage.

PubMed

Baron, Alice; Verdié, Pascal; Martinez, Jean; Lamaty, Frédéric

2011-02-04

A new linker cis-5-aminopent-3-enoic acid (cis-Apa) was prepared for the synthesis of cyclic pseudopeptides by cyclization-cleavage by using ring-closing methatesis (RCM). We developed a new synthetic pathway for the preparation of the cis-Apa linker that was tested in the cyclization-cleavage process of different RGD peptide sequences. Different macrocyclic peptidomimetics were prepared by using this integrated microwave-assisted method, showing that the readily available cis-Apa amino acid is well adapted as a linker in the cyclization-cleavage process.

Role of mitochondrial processing peptidase and AAA proteases in processing of the yeast acetohydroxyacid synthase precursor.

PubMed

Dasari, Suvarna; Kölling, Ralf

2016-07-01

We studied presequence processing of the mitochondrial-matrix targeted acetohydroxyacid synthase (Ilv2). C-terminal 3HA-tagging altered the cleavage pattern from a single step to sequential two-step cleavage, giving rise to two Ilv2-3HA forms (A and B). Both cleavage events were dependent on the mitochondrial processing peptidase (MPP). We present evidence for the involvement of three AAA ATPases, m- and i-AAA proteases, and Mcx1, in Ilv2-3HA processing. Both, precursor to A-form and A-form to B-form cleavage were strongly affected in a ∆yme1 mutant. These defects could be suppressed by overexpression of MPP, suggesting that MPP activity is limiting in the ∆yme1 mutant. Our data suggest that for some substrates AAA ATPases could play an active role in the translocation of matrix-targeted proteins.

SWI/SNF interacts with cleavage and polyadenylation factors and facilitates pre-mRNA 3' end processing.

PubMed

Yu, Simei; Jordán-Pla, Antonio; Gañez-Zapater, Antoni; Jain, Shruti; Rolicka, Anna; Östlund Farrants, Ann-Kristin; Visa, Neus

2018-05-31

SWI/SNF complexes associate with genes and regulate transcription by altering the chromatin at the promoter. It has recently been shown that these complexes play a role in pre-mRNA processing by associating at alternative splice sites. Here, we show that SWI/SNF complexes are involved also in pre-mRNA 3' end maturation by facilitating 3' end cleavage of specific pre-mRNAs. Comparative proteomics show that SWI/SNF ATPases interact physically with subunits of the cleavage and polyadenylation complexes in fly and human cells. In Drosophila melanogaster, the SWI/SNF ATPase Brahma (dBRM) interacts with the CPSF6 subunit of cleavage factor I. We have investigated the function of dBRM in 3' end formation in S2 cells by RNA interference, single-gene analysis and RNA sequencing. Our data show that dBRM facilitates pre-mRNA cleavage in two different ways: by promoting the association of CPSF6 to the cleavage region and by stabilizing positioned nucleosomes downstream of the cleavage site. These findings show that SWI/SNF complexes play a role also in the cleavage of specific pre-mRNAs in animal cells.

Transition-metal-catalyzed direct arylation of (hetero)arenes by C-H bond cleavage.

PubMed

Ackermann, Lutz; Vicente, Rubén; Kapdi, Anant R

2009-01-01

The area of transition-metal-catalyzed direct arylation through cleavage of C-H bonds has undergone rapid development in recent years, and is becoming an increasingly viable alternative to traditional cross-coupling reactions with organometallic reagents. In particular, palladium and ruthenium catalysts have been described that enable the direct arylation of (hetero)arenes with challenging coupling partners--including electrophilic aryl chlorides and tosylates as well as simple arenes in cross-dehydrogenative arylations. Furthermore, less expensive copper, iron, and nickel complexes were recently shown to be effective for economically attractive direct arylations.

The RNA-induced silencing complex is a Mg2+-dependent endonuclease.

PubMed

Schwarz, Dianne S; Tomari, Yukihide; Zamore, Phillip D

2004-05-04

In the Drosophila and mammalian RNA interference (RNAi) pathways, target RNA destruction is catalyzed by the siRNA-guided, RNA-induced silencing complex (RISC). RISC has been proposed to be an siRNA-directed endonuclease, catalyzing cleavage of a single phosphodiester bond on the RNA target. Although 5' cleavage products are readily detected for RNAi in vitro, only 3' cleavage products have been observed in vivo. Proof that RISC acts as an endonuclease requires detection of both 5' and 3' cleavage products in a single experimental system. Here, we show that siRNA-programmed RISC generates both 5' and 3' cleavage products in vitro; cleavage requires Mg(2+), but not Ca(2+), and the cleavage product termini suggest a role for Mg(2+) in catalysis. Moreover, a single phosphorothioate in place of the scissile phosphate blocks cleavage; the phosphorothioate effect can be rescued by the thiophilic cation Mn(2+), but not by Ca(2+) or Mg(2+). We propose that during catalysis, a Mg(2+) ion is bound to the RNA substrate through a nonbridging oxygen of the scissile phosphate. The mechanism of endonucleolytic cleavage is not consistent with the mechanisms of the previously identified RISC nuclease, Tudor-SN. Thus, the RISC-component that mediates endonucleolytic cleavage of the target RNA remains to be identified.

Development of Quenching-qPCR (Q-Q) assay for measuring absolute intracellular cleavage efficiency of ribozyme.

PubMed

Kim, Min Woo; Sun, Gwanggyu; Lee, Jung Hyuk; Kim, Byung-Gee

2018-06-01

Ribozyme (Rz) is a very attractive RNA molecule in metabolic engineering and synthetic biology fields where RNA processing is required as a control unit or ON/OFF signal for its cleavage reaction. In order to use Rz for such RNA processing, Rz must have highly active and specific catalytic activity. However, current methods for assessing the intracellular activity of Rz have limitations such as difficulty in handling and inaccuracies in the evaluation of correct cleavage activity. In this paper, we proposed a simple method to accurately measure the "intracellular cleavage efficiency" of Rz. This method deactivates unwanted activity of Rz which may consistently occur after cell lysis using DNA quenching method, and calculates the cleavage efficiency by analyzing the cleaved fraction of mRNA by Rz from the total amount of mRNA containing Rz via quantitative real-time PCR (qPCR). The proposed method was applied to measure "intracellular cleavage efficiency" of sTRSV, a representative Rz, and its mutant, and their intracellular cleavage efficiencies were calculated as 89% and 93%, respectively. Copyright © 2018 Elsevier Inc. All rights reserved.

Neutral Porphyrin Derivative Exerts Anticancer Activity by Targeting Cellular Topoisomerase I (Top1) and Promotes Apoptotic Cell Death without Stabilizing Top1-DNA Cleavage Complexes

PubMed Central

2017-01-01

Camptothecin (CPT) selectively traps topoisomerase 1-DNA cleavable complexes (Top1cc) to promote anticancer activity. Here, we report the design and synthesis of a new class of neutral porphyrin derivative 5,10-bis(4-carboxyphenyl)-15, 20-bis(4-dimethylaminophenyl)porphyrin (compound 8) as a potent catalytic inhibitor of human Top1. In contrast to CPT, compound 8 reversibly binds with the free enzyme and inhibits the formation of Top1cc and promotes reversal of the preformed Top1cc with CPT. Compound 8 induced inhibition of Top1cc formation in live cells was substantiated by fluorescence recovery after photobleaching (FRAP) assays. We established that MCF7 cells treated with compound 8 trigger proteasome-mediated Top1 degradation, accumulate higher levels of reactive oxygen species (ROS), PARP1 cleavage, oxidative DNA fragmentation, and stimulate apoptotic cell death without stabilizing apoptotic Top1-DNA cleavage complexes. Finally, compound 8 shows anticancer activity by targeting cellular Top1 and preventing the enzyme from directly participating in the apoptotic process. PMID:29290109

Regulator-dependent mechanisms of C3b processing by factor I allow differentiation of immune responses.

PubMed

Xue, Xiaoguang; Wu, Jin; Ricklin, Daniel; Forneris, Federico; Di Crescenzio, Patrizia; Schmidt, Christoph Q; Granneman, Joke; Sharp, Thomas H; Lambris, John D; Gros, Piet

2017-08-01

The complement system labels microbes and host debris for clearance. Degradation of surface-bound C3b is pivotal to direct immune responses and protect host cells. How the serine protease factor I (FI), assisted by regulators, cleaves either two or three distant peptide bonds in the CUB domain of C3b remains unclear. We present a crystal structure of C3b in complex with FI and regulator factor H (FH; domains 1-4 with 19-20). FI binds C3b-FH between FH domains 2 and 3 and a reoriented C3b C-terminal domain and docks onto the first scissile bond, while stabilizing its catalytic domain for proteolytic activity. One cleavage in C3b does not affect its overall structure, whereas two cleavages unfold CUB and dislodge the thioester-containing domain (TED), affecting binding of regulators and thereby determining the number of cleavages. These data explain how FI generates late-stage opsonins iC3b or C3dg in a context-dependent manner, to react to foreign, danger or healthy self signals.

Modeling Radial Holoblastic Cleavage: A Laboratory Activity for Developmental Biology.

ERIC Educational Resources Information Center

Ellis, Linda K.

2000-01-01

Introduces a laboratory activity designed for an undergraduate developmental biology course. Uses Play-Doh (plastic modeling clay) to build a multicellular embryo in order to provide a 3-D demonstration of cleavage. Includes notes for the instructor and student directions. (YDS)

A non-catalytic histidine residue influences the function of the metalloprotease of Listeria monocytogenes.

PubMed

Forster, Brian M; Bitar, Alan Pavinski; Marquis, Hélène

2014-01-01

Mpl, a thermolysin-like metalloprotease, and PC-PLC, a phospholipase C, are synthesized as proenzymes by the intracellular bacterial pathogen Listeria monocytogenes. During intracellular growth, L. monocytogenes is temporarily confined in a membrane-bound vacuole whose acidification leads to Mpl autolysis and Mpl-mediated cleavage of the PC-PLC N-terminal propeptide. Mpl maturation also leads to the secretion of both Mpl and PC-PLC across the bacterial cell wall. Previously, we identified negatively charged and uncharged amino acid residues within the N terminus of the PC-PLC propeptide that influence the ability of Mpl to mediate the maturation of PC-PLC, suggesting that these residues promote the interaction of the PC-PLC propeptide with Mpl. In the present study, we identified a non-catalytic histidine residue (H226) that influences Mpl secretion across the cell wall and its ability to process PC-PLC. Our results suggest that a positive charge at position 226 is required for Mpl functions other than autolysis. Based on the charge requirement at this position, we hypothesize that this residue contributes to the interaction of Mpl with the PC-PLC propeptide.

Electron Transfer Dissociation with Supplemental Activation to Differentiate Aspartic and Isoaspartic Residues in Doubly Charged Peptide Cations

PubMed Central

Chan, Wai Yi Kelly; Chan, T. W. Dominic; O’Connor, Peter B.

2011-01-01

Electron-transfer dissociation (ETD) with supplemental activation of the doubly charged deamidated tryptic digested peptide ions allows differentiation of isoaspartic acid and aspartic acid residues using c + 57 or z• − 57 peaks. The diagnostic peak clearly localizes and characterizes the isoaspartic acid residue. Supplemental activation in ETD of the doubly charged peptide ions involves resonant excitation of the charge reduced precursor radical cations and leads to further dissociation, including extra backbone cleavages and secondary fragmentation. Supplemental activation is essential to obtain a high quality ETD spectrum (especially for doubly charged peptide ions) with sequence information. Unfortunately, the low-resolution of the ion trap mass spectrometer makes detection of the diagnostic peak for the aspartic acid residue difficult due to interference with side-chain loss from arginine and glutamic acid residues. PMID:20304674

Propeptide cleavage conditions sortilin/neurotensin receptor-3 for ligand binding.

PubMed

Munck Petersen, C; Nielsen, M S; Jacobsen, C; Tauris, J; Jacobsen, L; Gliemann, J; Moestrup, S K; Madsen, P

1999-02-01

We recently reported the isolation and sequencing of sortilin, a new putative sorting receptor that binds receptor-associated protein (RAP). The luminal N-terminus of sortilin comprises a consensus sequence for cleavage by furin, R41WRR44, which precedes a truncation originally found in sortilin isolated from human brain. We now show that the truncation results from cellular processing. Sortilin is synthesized as a proform which, in late Golgi compartments, is converted to the mature receptor by furin-mediated cleavage of a 44 residue N-terminal propeptide. We further demonstrate that the propeptide exhibits pH-dependent high affinity binding to fully processed sortilin, that the binding is competed for by RAP and the newly discovered sortilin ligand neurotensin, and that prevention of propeptide cleavage essentially prevents binding of RAP and neurotensin. The findings evidence that the propeptide sterically hinders ligands from gaining access to overlapping binding sites in prosortilin, and that cleavage and release of the propeptide preconditions sortilin for full functional activity. Although proteolytic processing is involved in the maturation of several receptors, the described exposure of previously concealed ligand-binding sites after furin-mediated cleavage of propeptide represents a novel mechanism in receptor activation.

Propeptide cleavage conditions sortilin/neurotensin receptor-3 for ligand binding.

PubMed Central

Munck Petersen, C; Nielsen, M S; Jacobsen, C; Tauris, J; Jacobsen, L; Gliemann, J; Moestrup, S K; Madsen, P

1999-01-01

We recently reported the isolation and sequencing of sortilin, a new putative sorting receptor that binds receptor-associated protein (RAP). The luminal N-terminus of sortilin comprises a consensus sequence for cleavage by furin, R41WRR44, which precedes a truncation originally found in sortilin isolated from human brain. We now show that the truncation results from cellular processing. Sortilin is synthesized as a proform which, in late Golgi compartments, is converted to the mature receptor by furin-mediated cleavage of a 44 residue N-terminal propeptide. We further demonstrate that the propeptide exhibits pH-dependent high affinity binding to fully processed sortilin, that the binding is competed for by RAP and the newly discovered sortilin ligand neurotensin, and that prevention of propeptide cleavage essentially prevents binding of RAP and neurotensin. The findings evidence that the propeptide sterically hinders ligands from gaining access to overlapping binding sites in prosortilin, and that cleavage and release of the propeptide preconditions sortilin for full functional activity. Although proteolytic processing is involved in the maturation of several receptors, the described exposure of previously concealed ligand-binding sites after furin-mediated cleavage of propeptide represents a novel mechanism in receptor activation. PMID:9927419

Determinants of the VP1/2A junction cleavage by the 3C protease in foot-and-mouth disease virus-infected cells.

PubMed

Kristensen, Thea; Normann, Preben; Gullberg, Maria; Fahnøe, Ulrik; Polacek, Charlotta; Rasmussen, Thomas Bruun; Belsham, Graham J

2017-03-01

The foot-and-mouth disease virus (FMDV) capsid precursor, P1-2A, is cleaved by FMDV 3C protease to yield VP0, VP3, VP1 and 2A. Cleavage of the VP1/2A junction is the slowest. Serotype O FMDVs with uncleaved VP1-2A (having a K210E substitution in VP1; at position P2 in cleavage site) have been described previously and acquired a second site substitution (VP1 E83K) during virus rescue. Furthermore, introduction of the VP1 E83K substitution alone generated a second site change at the VP1/2A junction (2A L2P, position P2' in cleavage site). These virus adaptations have now been analysed using next-generation sequencing to determine sub-consensus level changes in the virus; this revealed other variants within the E83K mutant virus population that changed residue VP1 K210. The construction of serotype A viruses with a blocked VP1/2A cleavage site (containing K210E) has now been achieved. A collection of alternative amino acid substitutions was made at this site, and the properties of the mutant viruses were determined. Only the presence of a positively charged residue at position P2 in the cleavage site permitted efficient cleavage of the VP1/2A junction, consistent with analyses of diverse FMDV genome sequences. Interestingly, in contrast to the serotype O virus results, no second site mutations occurred within the VP1 coding region of serotype A viruses with the blocked VP1/2A cleavage site. However, some of these viruses acquired changes in the 2C protein that is involved in enterovirus morphogenesis. These results have implications for the testing of potential antiviral agents targeting the FMDV 3C protease.

Atypical cleavage of protonated N-fatty acyl amino acids derived from aspartic acid evidenced by sequential MS3 experiments.

PubMed

Boukerche, Toufik Taalibi; Alves, Sandra; Le Faouder, Pauline; Warnet, Anna; Bertrand-Michel, Justine; Bouchekara, Mohamed; Belbachir, Mohammed; Tabet, Jean-Claude

2016-12-01

Lipidomics calls for information on detected lipids and conjugates whose structural elucidation by mass spectrometry requires to rationalization of their gas phase dissociations toward collision-induced dissociation (CID) processes. This study focused on activated dissociations of two lipoamino acid (LAA) systems composed of N-palmitoyl acyl coupled with aspartic and glutamic acid mono ethyl esters (as LAA (*D) and LAA (*E) ). Although in MS/MS, their CID spectra show similar trends, e.g., release of water and ethanol, the [(LAA (*D/*E) +H)-C 2 H 5 OH] + product ions dissociate via distinct pathways in sequential MS 3 experiments. The formation of all the product ions is rationalized by charge-promoted cleavages often involving stepwise processes with ion isomerization into ion-dipole prior to dissociation. The latter explains the maleic anhydride or ketene neutral losses from N-palmitoyl acyl aspartate and glutamate anhydride fragment ions, respectively. Consequently, protonated palmitoyl acid amide is generated from LAA (*D), whereas LAA (*E) leads to the [*E+H-H 2 O] + anhydride. The former releases ammonia to provide acylium, which gives the C n H (2n-1) and C n H (2n-3) carbenium series. This should offer structural information, e.g., to locate either unsaturation(s) or alkyl group branching present on the various fatty acyl moieties of lipo-aspartic acid in further studies based on MS n experiments.

Two distinct RNase activities of CRISPR-C2c2 enable guide-RNA processing and RNA detection.

PubMed

East-Seletsky, Alexandra; O'Connell, Mitchell R; Knight, Spencer C; Burstein, David; Cate, Jamie H D; Tjian, Robert; Doudna, Jennifer A

2016-10-13

Bacterial adaptive immune systems use CRISPRs (clustered regularly interspaced short palindromic repeats) and CRISPR-associated (Cas) proteins for RNA-guided nucleic acid cleavage. Although most prokaryotic adaptive immune systems generally target DNA substrates, type III and VI CRISPR systems direct interference complexes against single-stranded RNA substrates. In type VI systems, the single-subunit C2c2 protein functions as an RNA-guided RNA endonuclease (RNase). How this enzyme acquires mature CRISPR RNAs (crRNAs) that are essential for immune surveillance and how it carries out crRNA-mediated RNA cleavage remain unclear. Here we show that bacterial C2c2 possesses a unique RNase activity responsible for CRISPR RNA maturation that is distinct from its RNA-activated single-stranded RNA degradation activity. These dual RNase functions are chemically and mechanistically different from each other and from the crRNA-processing behaviour of the evolutionarily unrelated CRISPR enzyme Cpf1 (ref. 11). The two RNase activities of C2c2 enable multiplexed processing and loading of guide RNAs that in turn allow sensitive detection of cellular transcripts.

DNA damage induced by ascorbate in the presence of Cu2+.

PubMed

Kobayashi, S; Ueda, K; Morita, J; Sakai, H; Komano, T

1988-01-25

DNA damage induced by ascorbate in the presence of Cu2+ was investigated by use of bacteriophage phi X174 double-stranded supercoiled DNA and linear restriction fragments as substrates. Single-strand cleavage was induced when supercoiled DNA was incubated with 5 microM-10 mM ascorbate and 50 microM Cu2+ at 37 degrees C for 10 min. The induced DNA damage was analyzed by sequencing of fragments singly labeled at their 5'- or 3'-end. DNA was cleaved directly and almost uniformly at every nucleotide by ascorbate and Cu2+. Piperidine treatment after the reaction showed that ascorbate and Cu2+ induced another kind of DNA damage different from the direct cleavage. The damage proceeded to DNA cleavage by piperidine treatment and was sequence-specific rather than random. These results indicate that ascorbate induces two classes of DNA damage in the presence of Cu2+, one being direct strand cleavage, probably via damage to the DNA backbone, and the other being a base modification labile to alkali treatment. These two classes of DNA damage were inhibited by potassium iodide, catalase and metal chelaters, suggesting the involvement of radicals generated from ascorbate hydroperoxide.

Increased Foxo3a Nuclear Translocation and Activity is an Early Neuronal Response to βγ-Secretase-Mediated Processing of the Amyloid-β Protein Precursor: Utility of an AβPP-GAL4 Reporter Assay.

PubMed

Law, Bernard M; Guest, Amy L; Pullen, Matthew W J; Perkinton, Michael S; Williams, Robert J

2018-01-01

Sequential cleavage of the amyloid-β protein precursor (AβPP) by BACE1 (β-secretase) followed by theγ-secretase complex, is strongly implicated in Alzheimer's disease (AD) but the initial cellular responses to these cleavage events are not fully defined. β-secretase-mediated AβPP processing yields an extracellular domain (sAβPPβ) and a C-terminal fragment of AβPP of 99 amino acids (C99). Subsequent cleavage by γ-secretase produces amyloid-β (Aβ) and an AβPP intracellular domain (AICD). A cellular screen based on the generation of AICD from an AβPP-Gal4 fusion protein was adapted by introducing familial AD (FAD) mutations into the AβPP sequence and linking the assay to Gal4-UAS driven luciferase and GFP expression, to identify responses immediately downstream of AβPP processing in neurons with a focus on the transcription factor Foxo3a which has been implicated in neurodegeneration. The K670N/M671L, E682K, E693G, and V717I FAD mutations and the A673T protective mutation, were introduced into the AβPP sequence by site directed mutagenesis. When expressed in mouse cortical neurons, AβPP-Gal4-UAS driven luciferase and GFP expression was substantially reduced by γ-secretase inhibitors, lowered by β-secretase inhibitors, and enhanced by α-secretase inhibitors suggesting that AICD is a product of the βγ-secretase pathway. AβPP-Gal4-UAS driven GFP expression was exploited to identify individual neurons undergoing amyloidogenic AβPP processing, revealing increased nuclear localization of Foxo3a and enhanced Foxo3a-mediated transcription downstream of AICD production. Foxo3a translocation was not driven by AICD directly but correlated with reduced Akt phosphorylation. Collectively this suggests that βγ-secretase-mediated AβPP processing couples to Foxo3a which could be an early neuronal signaling response in AD.

DELAY OF CLEAVAGE OF THE ARBACIA EGG BY ULTRAVIOLET RADIATION

PubMed Central

Blum, Harold F.; Price, Judith P.

1950-01-01

While our data do not permit us to state the exact locus or mode of action of ultraviolet radiation in the Arbacia egg, certain general conclusions may be reached. The amount of delay of cleavage of these eggs is determined by two principal factors: (1) The extent of an effect, resulting from photochemical action induced by ultraviolet radiation, which is reversible in a biological sense, the reversibility not being directly dependent upon the process of cell division. (2) The sensitivity of the cell division process to the effects of the ultraviolet-induced photochemical reaction. This factor varies with the stage of cell division, the cell being insensitive during a period corresponding to most of mitosis. It seems likely that these findings may apply to cell division in general, but, since the quantitative relationships observed must, in this case, reflect the integration of two semi-independent factors, the over-all picture may appear quite different for different kinds of cells. PMID:15410486

Measurement of laser activated electron tunneling from semiconductor zinc oxide to adsorbed organic molecules by a matrix assisted laser desorption ionization mass spectrometer.

PubMed

Zhong, Hongying; Fu, Jieying; Wang, Xiaoli; Zheng, Shi

2012-06-04

Measurement of light induced heterogeneous electron transfer is important for understanding of fundamental processes involved in chemistry, physics and biology, which is still challenging by current techniques. Laser activated electron tunneling (LAET) from semiconductor metal oxides was observed and characterized by a MALDI (matrix assisted laser desorption ionization) mass spectrometer in this work. Nanoparticles of ZnO were placed on a MALDI sample plate. Free fatty acids and derivatives were used as models of organic compounds and directly deposited on the surface of ZnO nanoparticles. Irradiation of UV laser (λ=355 nm) with energy more than the band gap of ZnO produces ions that can be detected in negative mode. When TiO(2) nanoparticles with similar band gap but much lower electron mobility were used, these ions were not observed unless the voltage on the sample plate was increased. The experimental results indicate that laser induced electron tunneling is dependent on the electron mobility and the strength of the electric field. Capture of low energy electrons by charge-deficient atoms of adsorbed organic molecules causes unpaired electron-directed cleavages of chemical bonds in a nonergodic pathway. In positive detection mode, electron tunneling cannot be observed due to the reverse moving direction of electrons. It should be able to expect that laser desorption ionization mass spectrometry is a new technique capable of probing the dynamics of electron tunneling. LAET offers advantages as a new ionization dissociation method for mass spectrometry. Copyright © 2012 Elsevier B.V. All rights reserved.

Characterization of plasma processing induced charging damage to MOS devices

NASA Astrophysics Data System (ADS)

Ma, Shawming

1997-12-01

Plasma processing has become an integral part of the fabrication of integrated circuits and takes at least 30% of whole process steps since it offers advantages in terms of directionality, low temperature and process convenience. However, wafer charging during plasma processes is a significant concern for both thin oxide damage and profile distortion. In this work, the factors affecting this damage will be explained by plasma issues, device structure and oxide quality. The SPORT (Stanford Plasma On-wafer Real Time) charging probe was developed to investigate the charging mechanism of different plasma processes including poly-Si etching, resist ashing and PECVD. The basic idea of this probe is that it simulates a real device structure in the plasma environment and allows measurement of plasma induced charging voltages and currents directly in real time. This measurement is fully compatible with other charging voltage measurement but it is the only one to do in real-time. Effect of magnetic field induced plasma nonuniformity on spatial dependent charging is well understood by this measurement. In addition, the plasma parameters including ion current density and electron temperature can also be extracted from the probe's plasma I-V characteristics using a dc Langmuir probe like theory. It will be shown that the MOS device tunneling current from charging, the dependence on antenna ratio and the etch uniformity can all be predicted by using this measurement. Moreover, the real-time measurement reveals transient and electrode edge effect during processing. Furthermore, high aspect ratio pattern induced electron shading effects can also be characterized by the probe. On the oxide quality issue, wafer temperature during plasma processing has been experimentally shown to be critical to charging damage. Finally, different MOS capacitor testing methods including breakdown voltage, charge-to-breakdown, gate leakage current and voltage-time at constant current bias were compared to find the optimum method for charging device reliability testing.

Roles of the redox-active disulfide and histidine residues forming a catalytic dyad in reactions catalyzed by 2-ketopropyl coenzyme M oxidoreductase/carboxylase.

PubMed

Kofoed, Melissa A; Wampler, David A; Pandey, Arti S; Peters, John W; Ensign, Scott A

2011-09-01

NADPH:2-ketopropyl-coenzyme M oxidoreductase/carboxylase (2-KPCC), an atypical member of the disulfide oxidoreductase (DSOR) family of enzymes, catalyzes the reductive cleavage and carboxylation of 2-ketopropyl-coenzyme M [2-(2-ketopropylthio)ethanesulfonate; 2-KPC] to form acetoacetate and coenzyme M (CoM) in the bacterial pathway of propylene metabolism. Structural studies of 2-KPCC from Xanthobacter autotrophicus strain Py2 have revealed a distinctive active-site architecture that includes a putative catalytic triad consisting of two histidine residues that are hydrogen bonded to an ordered water molecule proposed to stabilize enolacetone formed from dithiol-mediated 2-KPC thioether bond cleavage. Site-directed mutants of 2-KPCC were constructed to test the tenets of the mechanism proposed from studies of the native enzyme. Mutagenesis of the interchange thiol of 2-KPCC (C82A) abolished all redox-dependent reactions of 2-KPCC (2-KPC carboxylation or protonation). The air-oxidized C82A mutant, as well as wild-type 2-KPCC, exhibited the characteristic charge transfer absorbance seen in site-directed variants of other DSOR enzymes but with a pK(a) value for C87 (8.8) four units higher (i.e., four orders of magnitude less acidic) than that for the flavin thiol of canonical DSOR enzymes. The same higher pK(a) value was observed in native 2-KPCC when the interchange thiol was alkylated by the CoM analog 2-bromoethanesulfonate. Mutagenesis of the flavin thiol (C87A) also resulted in an inactive enzyme for steady-state redox-dependent reactions, but this variant catalyzed a single-turnover reaction producing a 0.8:1 ratio of product to enzyme. Mutagenesis of the histidine proximal to the ordered water (H137A) led to nearly complete loss of redox-dependent 2-KPCC reactions, while mutagenesis of the distal histidine (H84A) reduced these activities by 58 to 76%. A redox-independent reaction of 2-KPCC (acetoacetate decarboxylation) was not decreased for any of the aforementioned site-directed mutants. We interpreted and rationalized these results in terms of a mechanism of catalysis for 2-KPCC employing a unique hydrophobic active-site architecture promoting thioether bond cleavage and enolacetone formation not seen for other DSOR enzymes. Copyright © 2011, American Society for Microbiology. All Rights Reserved.

Use of a Designed Peptide Array To Infer Dissociation Trends for Nontryptic Peptides in Quadrupole Ion Trap and Quadrupole Time-of-Flight Mass Spectrometry

DOE PAGES

Gaucher, Sara P.; Morrow, Jeffrey A.; Faulon, Jean-Loup M.

2007-09-14

Observed peptide gas-phase fragmentation patterns are a complex function of many variables. In order to systematically probe this phenomenon, an array of 40 peptides was synthesized for study. The array of sequences was designed to hold certain variables (peptide length) constant and randomize or balance others (peptide amino acid distribution and position). A high-quality tandem mass spectrometry (MS/MS) data set was acquired for each peptide for all observed charge states on multiple MS instruments, quadrupole-time-of-flight and quadrupole ion trap. The data were analyzed as a function of total charge state and number of mobile protons. Previously known dissociation trends weremore » observed, validating our approach. In addition, the general influence of basic amino acids on dissociation could be determined because, in contrast to the more widely studied tryptic peptides, the amino acids H, K, and R were positionally distributed. Interestingly, our results suggest that cleavage at all basic amino acids is suppressed when a mobile proton is available. Cleavage at H becomes favored only under conditions where a partially mobile proton is present, a caveat to the previously reported trend of enhanced cleavage at H. In conclusion, all acquired data were used as a benchmark to determine how well these sequences would have been identified in a database search using a common algorithm, Mascot.« less

Modeling the MHC class I pathway by combining predictions of proteasomal cleavage, TAP transport and MHC class I binding.

PubMed

Tenzer, S; Peters, B; Bulik, S; Schoor, O; Lemmel, C; Schatz, M M; Kloetzel, P-M; Rammensee, H-G; Schild, H; Holzhütter, H-G

2005-05-01

Epitopes presented by major histocompatibility complex (MHC) class I molecules are selected by a multi-step process. Here we present the first computational prediction of this process based on in vitro experiments characterizing proteasomal cleavage, transport by the transporter associated with antigen processing (TAP) and MHC class I binding. Our novel prediction method for proteasomal cleavages outperforms existing methods when tested on in vitro cleavage data. The analysis of our predictions for a new dataset consisting of 390 endogenously processed MHC class I ligands from cells with known proteasome composition shows that the immunological advantage of switching from constitutive to immunoproteasomes is mainly to suppress the creation of peptides in the cytosol that TAP cannot transport. Furthermore, we show that proteasomes are unlikely to generate MHC class I ligands with a C-terminal lysine residue, suggesting processing of these ligands by a different protease that may be tripeptidyl-peptidase II (TPPII).

Electrochemistry-Assisted Top-Down Characterization of Disulfide-Containing Proteins

PubMed Central

Zhang, Yun; Cui, Weidong; Zhang, Hao; Dewald, Howard D.; Chen, Hao

2013-01-01

Covalent disulfide bond linkage in a protein represents an important challenge for mass spectrometry (MS)-based top-down protein structure analysis as it reduces the backbone cleavage efficiency for MS/MS dissociation. This study presents a strategy for solving this critical issue via integrating electrochemistry (EC) online with top-down MS approach. In this approach, proteins undergo electrolytic reduction in an electrochemical cell to break disulfide bonds and then online ionized into gaseous ions for analysis by electron-capture dissociation (ECD) and collision-induced dissociation (CID). The electrochemical reduction of proteins allows to remove disulfide bond constraints and also leads to increased charge numbers of the resulting protein ions. As a result, sequence coverage was significantly enhanced, as exemplified by β-lactoglobulin A (24 vs. 73 backbone cleavages before and after electrolytic reduction, respectively) and lysozyme (5 vs. 66 backbone cleavages before and after electrolytic reduction, respectively). This methodology is fast and does not need chemical reductants, which would have an important impact in high-throughput proteomics research. PMID:22448817

Electrochemistry-assisted top-down characterization of disulfide-containing proteins.

PubMed

Zhang, Yun; Cui, Weidong; Zhang, Hao; Dewald, Howard D; Chen, Hao

2012-04-17

Covalent disulfide bond linkage in a protein represents an important challenge for mass spectrometry (MS)-based top-down protein structure analysis as it reduces the backbone cleavage efficiency for MS/MS dissociation. This study presents a strategy for solving this critical issue via integrating electrochemistry (EC) online with a top-down MS approach. In this approach, proteins undergo electrolytic reduction in an electrochemical cell to break disulfide bonds and then undergo online ionization into gaseous ions for analysis by electron-capture dissociation (ECD) and collision-induced dissociation (CID). The electrochemical reduction of proteins allows one to remove disulfide bond constraints and also leads to increased charge numbers of the resulting protein ions. As a result, sequence coverage was significantly enhanced, as exemplified by β-lactoglobulin A (24 vs 75 backbone cleavages before and after electrolytic reduction, respectively) and lysozyme (5 vs 66 backbone cleavages before and after electrolytic reduction, respectively). This methodology is fast and does not need chemical reductants, which would have an important impact in high-throughput proteomics research.

Electron detachment dissociation of fluorescently labeled sialylated oligosaccharides.

PubMed

Zhou, Wen; Håkansson, Kristina

2011-12-01

We explored the application of electron detachment dissociation (EDD) and infrared multiphoton dissociation (IRMPD) tandem mass spectrometry to fluorescently labeled sialylated oligosaccharides. Standard sialylated oligosaccharides and a sialylated N-linked glycan released from human transferrin were investigated. EDD yielded extensive glycosidic cleavages and cross-ring cleavages in all cases studied, consistently providing complementary structural information compared with infrared multiphoton dissociation. Neutral losses and satellite ions such as C-2H ions were also observed following EDD. In addition, we examined the influence of different fluorescent labels. The acidic label 2-aminobenzoic acid (2-AA) enhanced signal abundance in negative-ion mode. However, few cross-ring fragments were observed for 2-AA-labeled oligosaccharides. The neutral label 2-aminobenzamide (2-AB) resulted in more cross-ring cleavages compared with 2-AA-labeled species, but not as extensive fragmentation as for native oligosaccharides, likely resulting from altered negative charge locations from introduction of the fluorescent tag. Copyright © 2011 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

The prototype HIV-1 maturation inhibitor, bevirimat, binds to the CA-SP1 cleavage site in immature Gag particles.

PubMed

Nguyen, Albert T; Feasley, Christa L; Jackson, Ken W; Nitz, Theodore J; Salzwedel, Karl; Air, Gillian M; Sakalian, Michael

2011-12-07

Bevirimat, the prototype Human Immunodeficiency Virus type 1 (HIV-1) maturation inhibitor, is highly potent in cell culture and efficacious in HIV-1 infected patients. In contrast to inhibitors that target the active site of the viral protease, bevirimat specifically inhibits a single cleavage event, the final processing step for the Gag precursor where p25 (CA-SP1) is cleaved to p24 (CA) and SP1. In this study, photoaffinity analogs of bevirimat and mass spectrometry were employed to map the binding site of bevirimat to Gag within immature virus-like particles. Bevirimat analogs were found to crosslink to sequences overlapping, or proximal to, the CA-SP1 cleavage site, consistent with previous biochemical data on the effect of bevirimat on Gag processing and with genetic data from resistance mutations, in a region predicted by NMR and mutational studies to have α-helical character. Unexpectedly, a second region of interaction was found within the Major Homology Region (MHR). Extensive prior genetic evidence suggests that the MHR is critical for virus assembly. This is the first demonstration of a direct interaction between the maturation inhibitor, bevirimat, and its target, Gag. Information gained from this study sheds light on the mechanisms by which the virus develops resistance to this class of drug and may aid in the design of next-generation maturation inhibitors.

Intermolecular cleavage by UmuD-like mutagenesis proteins

PubMed Central

McDonald, John P.; Frank, Ekaterina G.; Levine, Arthur S.; Woodgate, Roger

1998-01-01

The activity of a number of proteins is regulated by self-processing reactions. Elegant examples are the cleavage of the prokaryotic LexA and λCI transcriptional repressors and the UmuD-like mutagenesis proteins. Various studies support the hypothesis that LexA and λCI cleavage reactions are predominantly intramolecular in nature. The recently described crystal structure of the Escherichia coli UmuD′ protein (the posttranslational cleavage product of the UmuD protein) suggests, however, that the region of the protein corresponding to the cleavage site is at least 50 Å away from the catalytic active site. We considered the possibility, therefore, that the UmuD-like proteins might undergo self-processing that, in contrast to LexA and λCI, occurs via an intermolecular rather than intramolecular reaction. To test this hypothesis, we introduced into E. coli compatible plasmids with mutations at either the cleavage or the catalytic site of three UmuD-like proteins. Cleavage of these proteins only occurs in the presence of both plasmids, indicating that the reaction is indeed intermolecular in nature. Furthermore, this intermolecular reaction is completely dependent upon the multifunctional RecA protein and leads to the restoration of cellular mutagenesis in nonmutable E. coli strains. Intermolecular cleavage of a biotinylated UmuD active site mutant was also observed in vitro in the presence of the wild-type UmuD′ protein, indicating that in addition to the intact UmuD protein, the normal cleavage product (UmuD′) can also act as a classical enzyme. PMID:9465040

The capsid-spacer peptide 1 Gag processing intermediate is a dominant-negative inhibitor of HIV-1 maturation.

PubMed

Checkley, Mary Ann; Luttge, Benjamin G; Soheilian, Ferri; Nagashima, Kunio; Freed, Eric O

2010-04-25

The human immunodeficiency virus type 1 (HIV-1) maturation inhibitor bevirimat disrupts virus replication by inhibiting the cleavage of the capsid-spacer peptide 1 (CA-SP1) Gag processing intermediate to mature CA. The observation that bevirimat delays but does not completely block CA-SP1 processing suggests that the presence of uncleaved CA-SP1 may disrupt the maturation process in trans. In this study, we validate this hypothesis by using a genetic approach to demonstrate that a non-cleavable CA-SP1 mutant exerts a dominant-negative effect on maturation of wild-type HIV-1. In contrast, a mutant in which cleavage can occur internally within SP1 is significantly less potent as a dominant-negative inhibitor. We also show that bevirimat blocks processing at both the major CA-SP1 cleavage site and the internal site. These data underscore the importance of full CA-SP1 processing for HIV-1 maturation and highlight the therapeutic potential of inhibitors that target this Gag cleavage event. Published by Elsevier Inc.

Active site remodeling during the catalytic cycle in metal-dependent fructose-1,6-bisphosphate aldolases.

PubMed

Jacques, Benoit; Coinçon, Mathieu; Sygusch, Jurgen

2018-03-28

Crystal structures of two bacterial metal (Zn) dependent D-fructose 1,6-bisphosphate (FBP) aldolases in complex with substrate, analogues, and triose-P reaction products were determined to 1.5-2.0 Å resolution. The ligand complexes cryotrapped in native or mutant H. pylori aldolase crystals enabled a novel mechanistic description of FBP C 3 -C 4 bond cleavage. The reaction mechanism uses active site remodelling during the catalytic cycle implicating relocation of the Zn cofactor that is mediated by conformational changes of active site loops. Substrate binding initiates conformational changes, triggered upon P 1 -phosphate binding, which liberates the Zn chelating His180, allowing it to act as a general base for the proton abstraction at the FBP C 4 -hydroxyl group. A second zinc chelating His83 hydrogen bonds the substrate C 4 - hydroxyl group and assists cleavage by stabilizing the developing negative charge during proton abstraction. Cleavage is concerted with relocation of the metal cofactor from an interior to a surface exposed site, thereby stabilizing the nascent enediolate form. Conserved residue Glu142 is essential for protonation of the enediolate form, prior to product release. A D-tagatose 1,6-bisphosphate enzymatic complex reveals how His180 mediated proton abstraction controls stereospecificity of the cleavage reaction. Recognition and discrimination of the reaction products, dihydroxyacetone-P and D-glyceraldehyde-3-P, occurs via charged hydrogen bonds between hydroxyl groups of the triose-Ps and conserved residues, Asp82 and Asp255, respectively, and are crucial aspects of the enzyme's role in gluconeogenesis. Conformational changes in mobile loops β5-α7 and β6-α8 (containing catalytic residues Glu142 and His180, respectively) drive active site remodelling enabling the relocation of the metal cofactor. Published under license by The American Society for Biochemistry and Molecular Biology, Inc.

Chemistry in acetone complexes of metal dications: a remarkable ethylene production pathway.

PubMed

Wu, Jianhua; Liu, Dan; Zhou, Jian-Ge; Hagelberg, Frank; Park, Sung Soo; Shvartsburg, Alexandre A

2007-06-07

Electrospray ionization can generate microsolvated multiply charged metal ions for various metals and ligands, allowing exploration of chemistry within such clusters. The finite size of these systems permits comparing experimental results with accurate calculations, creating a natural laboratory to research ion solvation. Mass spectrometry has provided much insight into the stability and dissociation of ligated metal cations. While solvated singly charged ions tend to shrink by ligand evaporation, solvated polycations below a certain size exhibit charge reduction and/or ligand fragmentation due to organometallic reactions. Here we investigate the acetone complexes of representative divalent metals (Ca, Mn, Co, Ni, and Cu), comparing the results of collision-induced dissociation with the predictions of density functional theory. As for other solvated dications, channels involving proton or electron transfer compete with ligand loss and become dominant for smaller complexes. The heterolytic C-C bond cleavage is common, like in DMSO and acetonitrile complexes. Of primary interest is the unanticipated neutral ethylene loss, found for all metals studied except Cu and particularly intense for Ca and Mn. We focus on understanding that process in the context of competing dissociation pathways, as a function of metal identity and number of ligands. According to first-principles modeling, ethylene elimination proceeds along a complex path involving two intermediates. These results suggest that chemistry in microsolvated multiply charged ions may still hold major surprises.

An autonomous chemically fuelled small-molecule motor

NASA Astrophysics Data System (ADS)

Wilson, Miriam R.; Solà, Jordi; Carlone, Armando; Goldup, Stephen M.; Lebrasseur, Nathalie; Leigh, David A.

2016-06-01

Molecular machines are among the most complex of all functional molecules and lie at the heart of nearly every biological process. A number of synthetic small-molecule machines have been developed, including molecular muscles, synthesizers, pumps, walkers, transporters and light-driven and electrically driven rotary motors. However, although biological molecular motors are powered by chemical gradients or the hydrolysis of adenosine triphosphate (ATP), so far there are no synthetic small-molecule motors that can operate autonomously using chemical energy (that is, the components move with net directionality as long as a chemical fuel is present). Here we describe a system in which a small molecular ring (macrocycle) is continuously transported directionally around a cyclic molecular track when powered by irreversible reactions of a chemical fuel, 9-fluorenylmethoxycarbonyl chloride. Key to the design is that the rate of reaction of this fuel with reactive sites on the cyclic track is faster when the macrocycle is far from the reactive site than when it is near to it. We find that a bulky pyridine-based catalyst promotes carbonate-forming reactions that ratchet the displacement of the macrocycle away from the reactive sites on the track. Under reaction conditions where both attachment and cleavage of the 9-fluorenylmethoxycarbonyl groups occur through different processes, and the cleavage reaction occurs at a rate independent of macrocycle location, net directional rotation of the molecular motor continues for as long as unreacted fuel remains. We anticipate that autonomous chemically fuelled molecular motors will find application as engines in molecular nanotechnology.

The Vps27/Hrs/STAM (VHS) Domain of the Signal-transducing Adaptor Molecule (STAM) Directs Associated Molecule with the SH3 Domain of STAM (AMSH) Specificity to Longer Ubiquitin Chains and Dictates the Position of Cleavage*

PubMed Central

Baiady, Nardeen; Padala, Prasanth; Mashahreh, Bayan; Cohen-Kfir, Einav; Todd, Emily A.; Du Pont, Kelly E.; Berndsen, Christopher E.; Wiener, Reuven

2016-01-01

The deubiquitinating enzyme associated molecule with the SH3 domain of STAM (AMSH) is crucial for the removal of ubiquitin molecules during receptor-mediated endocytosis and lysosomal receptor sorting. AMSH interacts with signal transducing adapter molecule (STAM) 1 or 2, which enhances the activity of AMSH through an unknown mechanism. This stimulation is dependent on the ubiquitin-interacting motif of STAM. Here we investigate the specific mechanism of AMSH stimulation by STAM proteins and the role of the STAM Vps27/Hrs/STAM domain. We show that, in the presence of STAM, the length of the ubiquitin chains affects the apparent cleavage rate. Through measurement of the chain cleavage kinetics, we found that, although the kcat of Lys63-linked ubiquitin chain cleavage was comparable for di- and tri-ubiquitin, the Km value was lower for tri-ubiquitin. This increased affinity for longer chains was dependent on the Vps27/Hrs/STAM domain of STAM and required that the substrate ubiquitin chain contain homogenous Lys63-linkages. In addition, STAM directed AMSH cleavage toward the distal isopeptide bond in tri-ubiquitin chains. Finally, we generated a structural model of AMSH-STAM to show how the complex binds Lys63-linked ubiquitin chains and cleaves at the distal end. These data show how a deubiquitinating enzyme-interacting protein dictates the efficiency and specificity of substrate cleavage. PMID:26601948

Directional charge separation in isolated organic semiconductor crystalline nanowires

DOE PAGES

Labastide, J. A.; Thompson, H. B.; Marques, S. R.; ...

2016-02-25

One of the fundamental design paradigms in organic photovoltaic device engineering is based on the idea that charge separation is an extrinsically driven process requiring an interface for exciton fission. This idea has driven an enormous materials science engineering effort focused on construction of domain sizes commensurate with a nominal exciton diffusion length of order 10 nm. Here, we show that polarized optical excitation of isolated pristine crystalline nanowires of a small molecule n-type organic semiconductor, 7,8,15,16-tetraazaterrylene, generates a significant population of charge-separated polaron pairs along the π-stacking direction. Charge separation was signalled by pronounced power-law photoluminescence decay polarized alongmore » the same axis. In the transverse direction, we observed exponential decay associated with excitons localized on individual monomers. We propose that this effect derives from an intrinsic directional charge-transfer interaction that can ultimately be programmed by molecular packing geometry.« less

A prohormone convertase cleavage site within a predicted alpha-helix mediates sorting of the neuronal and endocrine polypeptide VGF into the regulated secretory pathway.

PubMed

Garcia, Angelo L; Han, Shan-Kuo; Janssen, William G; Khaing, Zin Z; Ito, Timothy; Glucksman, Marc J; Benson, Deanna L; Salton, Stephen R J

2005-12-16

Distinct intracellular pathways are involved in regulated and constitutive protein secretion from neuronal and endocrine cells, yet the peptide signals and molecular mechanisms responsible for targeting and retention of soluble proteins in secretory granules are incompletely understood. By using confocal microscopy and subcellular fractionation, we examined trafficking of the neuronal and endocrine peptide precursor VGF that is stored in large dense core vesicles and undergoes regulated secretion. VGF cofractionated with secretory vesicle membranes but was not detected in detergent-resistant lipid rafts. Deletional analysis using epitope-tagged VGF suggested that the C-terminal 73-amino acid fragment of VGF, containing two predicted alpha-helical loops and four potential prohormone convertase (PC) cleavage sites, was necessary and sufficient with an N-terminal signal peptide-containing domain, for large dense core vesicle sorting and regulated secretion from PC12 and INS-1 cells. Further transfection analysis identified the sorting sequence as a compact C-terminal alpha-helix and embedded 564RRR566 PC cleavage site; mutation of the 564RRR566 PC site in VGF-(1-65): GFP:VGF-(545-617) blocked regulated secretion, whereas disruption of the alpha-helix had no effect. Mutation of the adjacent 567HFHH570 motif, a charged region that might enhance PC cleavage in acidic environments, also blocked regulated release. Finally, inhibition of PC cleavage in PC12 cells using the membrane-permeable synthetic peptide chloromethyl ketone (decanoyl-RVKR-CMK) blocked regulated secretion of VGF. Our studies define a critical RRR-containing C-terminal domain that targets VGF into the regulated pathway in neuronal PC12 and endocrine INS-1 cells, providing additional support for the proposed role that PCs and their cleavage sites play in regulated peptide secretion.

Identification of Caspase Cleavage Sites in KSHV Latency-Associated Nuclear Antigen and Their Effects on Caspase-Related Host Defense Responses.

PubMed

Davis, David A; Naiman, Nicole E; Wang, Victoria; Shrestha, Prabha; Haque, Muzammel; Hu, Duosha; Anagho, Holda A; Carey, Robert F; Davidoff, Katharine S; Yarchoan, Robert

2015-07-01

Kaposi's sarcoma-associated herpesvirus (KSHV), also known as human herpesvirus-8, is the causative agent of three hyperproliferative disorders: Kaposi's sarcoma, primary effusion lymphoma (PEL) and multicentric Castleman's disease. During viral latency a small subset of viral genes are produced, including KSHV latency-associated nuclear antigen (LANA), which help the virus thwart cellular defense responses. We found that exposure of KSHV-infected cells to oxidative stress, or other inducers of apoptosis and caspase activation, led to processing of LANA and that this processing could be inhibited with the pan-caspase inhibitor Z-VAD-FMK. Using sequence, peptide, and mutational analysis, two caspase cleavage sites within LANA were identified: a site for caspase-3 type caspases at the N-terminus and a site for caspase-1 and-3 type caspases at the C-terminus. Using LANA expression plasmids, we demonstrated that mutation of these cleavage sites prevents caspase-1 and caspase-3 processing of LANA. This indicates that these are the principal sites that are susceptible to caspase cleavage. Using peptides spanning the identified LANA cleavage sites, we show that caspase activity can be inhibited in vitro and that a cell-permeable peptide spanning the C-terminal cleavage site could inhibit cleavage of poly (ADP-ribose) polymerase and increase viability in cells undergoing etoposide-induced apoptosis. The C-terminal peptide of LANA also inhibited interleukin-1 beta (IL-1β) production from lipopolysaccharide-treated THP-1 cells by more than 50%. Furthermore, mutation of the two cleavage sites in LANA led to a significant increase in IL-1β production in transfected THP-1 cells; this provides evidence that these sites function to blunt the inflammasome, which is known to be activated in latently infected PEL cells. These results suggest that specific caspase cleavage sites in KSHV LANA function to blunt apoptosis as well as interfere with the caspase-1-mediated inflammasome, thus thwarting key cellular defense mechanisms.

Atomistic investigations on the mechanical properties and fracture mechanisms of indium phosphide nanowires.

PubMed

Pial, Turash Haque; Rakib, Tawfiqur; Mojumder, Satyajit; Motalab, Mohammad; Akanda, M A Salam

2018-03-28

The mechanical properties of indium phosphide (InP) nanowires are an emerging issue due to the promising applications of these nanowires in nanoelectromechanical and microelectromechanical devices. In this study, molecular dynamics simulations of zincblende (ZB) and wurtzite (WZ) crystal structured InP nanowires (NWs) are presented under uniaxial tension at varying sizes and temperatures. It is observed that the tensile strengths of both types of NWs show inverse relationships with temperature, but are independent of the size of the nanowires. Moreover, applied load causes brittle fracture by nucleating cleavage on ZB and WZ NWs. When the tensile load is applied along the [001] direction, the direction of the cleavage planes of ZB NWs changes with temperature. It is found that the {111} planes are the cleavage planes at lower temperatures; on the other hand, the {110} cleavage planes are activated at elevated temperatures. In the case of WZ NWs, fracture of the material is observed to occur by cleaving along the (0001) plane irrespective of temperature when the tensile load is applied along the [0001] direction. Furthermore, the WZ NWs of InP show considerably higher strength than their ZB counterparts. Finally, the impact of strain rate on the failure behavior of InP NWs is also studied, and higher fracture strengths and strains at higher strain rates are found. With increasing strain rate, the number of cleavages also increases in the NWs. This paper also provides in-depth understanding of the failure behavior of InP NWs, which will aid the design of efficient InP NWs-based devices.

P40 and P90 from Mpn142 are Targets of Multiple Processing Events on the Surface of Mycoplasma pneumoniae

PubMed Central

Widjaja, Michael; Berry, Iain J.; Pont, Elsa J.; Padula, Matthew P.; Djordjevic, Steven P.

2015-01-01

Mycoplasma pneumoniae is a significant cause of community acquired pneumonia globally. Despite having a genome less than 1 Mb in size, M. pneumoniae presents a structurally sophisticated attachment organelle that (i) provides cell polarity, (ii) directs adherence to receptors presented on respiratory epithelium, and (iii) plays a major role in cell motility. The major adhesins, P1 (Mpn141) and P30 (Mpn453), are localised to the tip of the attachment organelle by the surface accessible cleavage fragments P90 and P40 derived from Mpn142. Two events play a defining role in the formation of P90 and P40; removal of a leader peptide at position 26 (23SLA↓NTY28) during secretion to the cell surface and cleavage at amino acid 455 (452GPL↓RAG457) generating P40 and P90. Liquid Chromatography Tandem Mass Spectrometry (LC-MS/MS) analysis of tryptic peptides generated by digesting size-fractionated cell lysates of M. pneumoniae identified 15 cleavage fragments of Mpn142 ranging in mass from 9–84 kDa. Further evidence for the existence of cleavage fragments of Mpn142 was generated by mapping tryptic peptides to proteins recovered from size fractionated eluents from affinity columns loaded with heparin, fibronectin, fetuin, actin, plasminogen and A549 surface proteins as bait. To define the sites of cleavage in Mpn142, neo-N-termini in cell lysates of M. pneumoniae were dimethyl-labelled and characterised by LC-MS/MS. Our data suggests that Mpn142 is cleaved to generate adhesins that are auxiliary to P1 and P30. PMID:28248283

Specialization of the DNA-Cleaving Activity of a Group I Ribozyme Through In Vitro Evolution

NASA Technical Reports Server (NTRS)

Tsang, Joyce; Joyce, Gerald F.

1996-01-01

In an earlier study, an in vitro evolution procedure was applied to a large population of variants of the Tetrahymena group 1 ribozyme to obtain individuals with a 10(exp 5)-fold improved ability to cleave a target single-stranded DNA substrate under simulated physiological conditions. The evolved ribozymes also showed a twofold improvement, compared to the wild-type, in their ability to cleave a single-stranded RNA substrate. Here, we report continuation of the in vitro evolution process using a new selection strategy to achieve both enhanced DNA and diminished RNA-cleavage activity. Our strategy combines a positive selection for DNA cleavage with a negative selection against RNA binding. After 36 "generations" of in vitro evolution, the evolved population showed an approx. 100-fold increase in the ratio of DNA to RNA-cleavage activity. Site-directed mutagenesis experiment confirmed the selective advantage of two covarying mutations within the catalytic core of ribozyme that are largely responsible for this modified behavior. The population of ribozymes has now undergone a total of 63 successive generations of evolution, resulting in an average 28 mutations relative to the wild-type that are responsible for the altered phenotype.

Identification of the cleavage sites of the RNA2-encoded polyproteins for two members of the genus Torradovirus by N-terminal sequencing of the virion capsid proteins.

PubMed

Ferriol, I; Silva Junior, D M; Nigg, J C; Zamora-Macorra, E J; Falk, B W

2016-11-01

Torradoviruses, family Secoviridae, are emergent bipartite RNA plant viruses. RNA1 is ca. 7kb and has one open reading frame (ORF) encoding for the protease, helicase and RNA-dependent RNA polymerase (RdRp). RNA2 is ca. 5kb and has two ORFs. RNA2-ORF1 encodes for a putative protein with unknown function(s). RNA2-ORF2 encodes for a putative movement protein and three capsid proteins. Little is known about the replication and polyprotein processing strategies of torradoviruses. Here, the cleavage sites in the RNA2-ORF2-encoded polyproteins of two torradoviruses, Tomato marchitez virus isolate M (ToMarV-M) and tomato chocolate spot virus, were determined by N-terminal sequencing, revealing that the amino acid (aa) at the -1 position of the cleavage sites is a glutamine. Multiple aa sequence comparison confirmed that this glutamine is conserved among other torradoviruses. Finally, site-directed mutagenesis of conserved aas in the ToMarV-M RdRp and protease prevented substantial accumulation of viral coat proteins or RNAs. Copyright © 2016 Elsevier Inc. All rights reserved.

A novel processing system of sterol regulatory element-binding protein-1c regulated by polyunsaturated fatty acid.

PubMed

Nakakuki, Masanori; Kawano, Hiroyuki; Notsu, Tatsuto; Imada, Kazunori; Mizuguchi, Kiyoshi; Shimano, Hitoshi

2014-05-01

The proteolytic cascade is the key step in transactivation of sterol regulatory element-binding proteins (SREBPs), a transcriptional factor of lipid synthesis. Proteolysis of SREBP-2 is strictly regulated by sterols, but that of SREBP-1c was not strongly sterol-regulated, but inhibited by polyunsaturated fatty acids (PUFAs). In this study, the proteolytic processing of SREBP-1 and -2 was examined by transfection studies of cDNA-encoding mutants in which all the known cleavage sites were disrupted. In cultured cells, sterol-regulated SREBP-2 processing was completely eliminated by mutation of cleavage sites. In contrast, the corresponding SREBP-1c mutants as well as wild type exhibited large amounts of cleaved products in the nuclear extracts from culture cells and murine liver in vivo. The nuclear form of the mutant SREBP-1c was induced by delipidated condition and suppressed by eicosapentaenoic acid, an n-3 PUFA, but not by sterols. This novel processing mechanism was affected by neither SREBP cleavage-activating protein (SCAP) nor insulin-induced gene (Insig)-1, unlike SREBP-2, but abolished by a serine protease inhibitor. Through analysis of deletion mutant, a site-2 protease recognition sequence (DRSR) was identified to be involved in this novel processing. These findings suggest that SREBP-1c cleavage could be subjected to a novel PUFA-regulated cleavage system in addition to the sterol-regulatory SCAP/Insig system.

Inorganic phosphate blocks binding of pre-miRNA to Dicer-2 via its PAZ domain

PubMed Central

Fukunaga, Ryuya; Colpan, Cansu; Han, Bo W; Zamore, Phillip D

2014-01-01

In Drosophila, Dicer-1 produces microRNAs (miRNAs) from pre-miRNAs, whereas Dicer-2 generates small interfering RNAs from long double-stranded RNA (dsRNA), a process that requires ATP hydrolysis. We previously showed that inorganic phosphate inhibits Dicer-2 cleavage of pre-miRNAs, but not long dsRNAs. Here, we report that phosphate-dependent substrate discrimination by Dicer-2 reflects dsRNA substrate length. Efficient processing by Dicer-2 of short dsRNA requires a 5′ terminal phosphate and a two-nucleotide, 3′ overhang, but does not require ATP. Phosphate inhibits cleavage of such short substrates. In contrast, cleavage of longer dsRNA requires ATP but no specific end structure: phosphate does not inhibit cleavage of these substrates. Mutation of a pair of conserved arginine residues in the Dicer-2 PAZ domain blocked cleavage of short, but not long, dsRNA. We propose that inorganic phosphate occupies a PAZ domain pocket required to bind the 5′ terminal phosphate of short substrates, blocking their use and restricting pre-miRNA processing in flies to Dicer-1. Our study helps explain how a small molecule can alter the substrate specificity of a nucleic acid processing enzyme. PMID:24488111

Direct excitation luminescence spectroscopy of Eu(III) complexes of 1,4,7-tris(carbamoylmethyl)-1,4,7,10- tetraazacyclododecane derivatives and kinetic studies of their catalytic cleavage of an RNA analog.

PubMed

Nwe, Kido; Richard, John P; Morrow, Janet R

2007-11-28

The macrocycles 1,4,7-tris(carbamoylmethyl)-1,4,7,10-tetrazacyclododecane (1), 1,4,7-tris[(N-ethyl)carbamoylmethyl]-1,4,7,10-tetraazacyclododecane (2), 1,4,7-tris[(N,N-diethyl)carbamoylmethyl]-1,4,7,10-tetraazacyclododecane (3) and their Eu(III) complexes are prepared. Studies using direct Eu(III) excitation luminescence spectroscopy show that all three Eu(III) complexes exhibit only one predominant isomer with two bound waters under neutral to mildly basic conditions (Eu(X)(H(2)O)(2) for X = 1-3). There are no detectable ligand ionizations over the pH range 5.0-8.0 for Eu(3), 5.0-8.5 for Eu(2) or 5.0-9.5 for Eu(1). The three Eu(III) complexes show a linear dependence of second-order rate constants for the cleavage of 4-nitrophenyl-2-hydroxyethylphosphate (HpPNP) on pH in the range 6.5-8.0 for Eu(3), 7.0-8.5 for Eu(2) and 7.0-9.0 for Eu(1). This pH-rate profile is consistent with the Eu(III) complex-substrate complex being converted to the active form by loss of a proton and with Eu(III) water pK(a) values that are higher than 8.0 for Eu(3), 8.5 for Eu(2) and 9.0 for Eu(1). Inhibition studies show that Eu() binds strongly to the dianionic ligand methylphosphate (K(d) = 0.28 mM), and more weakly to diethylphosphate (K(d) = 7.5 mM), consistent with a catalytic role of the Eu(III) complexes in stabilizing the developing negative charge on the phosphorane transition state.

Two Distinct RNase Activities of CRISPR-C2c2 Enable Guide RNA Processing and RNA Detection

PubMed Central

East-Seletsky, Alexandra; O’Connell, Mitchell R.; Knight, Spencer C.; Burstein, David; Cate, Jamie H. D.; Tjian, Robert; Doudna, Jennifer A.

2017-01-01

Bacterial adaptive immune systems employ CRISPRs (clustered regularly interspaced short palindromic repeats) and CRISPR-associated (Cas) proteins for RNA-guided nucleic acid cleavage1,2. Although generally targeted to DNA substrates3–5, the Type III and Type VI CRISPR systems direct interference complexes against single-stranded RNA (ssRNA) substrates6–9. In Type VI systems, the single-subunit C2c2 protein functions as an RNA-guided RNA endonuclease9,10. How this enzyme acquires mature CRISPR RNAs (crRNAs) essential for immune surveillance and its mechanism of crRNA-mediated RNA cleavage remain unclear. Here we show that C2c2 possesses a unique ribonuclease activity responsible for CRISPR RNA maturation that is distinct from its RNA-activated ssRNA-degradation activity. These dual ribonuclease functions are chemically and mechanistically different from each other and from the crRNA-processing behavior of the evolutionarily unrelated CRISPR enzyme Cpf111. We show that the two ribonuclease activities of C2c2 enable multiplexed processing and loading of guide RNAs that in turn allow for sensitive cellular transcript detection. PMID:27669025

Surface functions during mitosis. III. Quantitative analysis of ligand- receptor movement into the cleavage furrow: diffusion vs. flow

PubMed Central

1982-01-01

The surface distribution of concanavalin A (Con A) bound to cell membrane receptors varies dramatically as a function of mitotic phase. The lectin is distributed diffusely on cells labeled and observed between mid-prophase and early anaphase, whereas cells observed in late anaphase or telophase demonstrate a marked accumulation of Con A- receptor complexes over the developing cleavage furrow (Berlin, Oliver, and Walter. 1978. Cell. 15:327-341). In this report, we first use a system based on video intensification fluorescence microscopy to describe the simultaneous changes in cell shape and in lectin-receptor complex topography during progression of single cells through the mitotic cycle. The video analysis establishes that fluorescein succinyl Con A (F-S Con A)-receptor complex redistribution begins coincident with the first appearance of the cleavage furrow and is essentially complete within 2-3 min. This remarkable redistribution of surface fluorescence occurs during only a modest change in cell shape from a sphere to a belted cylinder. It reflects the translocation of complexes and not the accumulation of excess labeled membrane in the cleavage furrow: first, bound fluorescent cholera toxin which faithfully outlines the plasma membrane is not accumulated in the cleavage furrow, and, second, electron microscopy of peroxidase-Con A labeled cells undergoing cleavage shows that there is a high linear density of lectin within the furrow while Con A is virtually eliminated from the poles. The rate of surface movement of F-S Con A was quantitated by photon counting during a repetitive series of laser-excited fluorescence scans across dividing cells. Results were analyzed in terms of two alternative models of movement: a flow model in which complexes moved unidirectionally at constant velocity, and a diffusion model in which complexes could diffuse freely but were trapped at the cleavage furrow. According to these models, the observed rates of accumulation were attainable at either an effective flow velocity of approximately 1 micron/min, or an effective diffusion coefficient of approximately 10(- 9) cm2/s. However, in separate experiments the lectin-receptor diffusion rate measured directly by the method of fluorescence recovery after photobleaching (FRAP) on metaphase cells was only approximately 10(-10) cm2/s. Most importantly, photobleaching experiments during the actual period of F-S Con A accumulation showed that lectin-receptor movement during cleavage occurs unidirectionally. These results rule out diffusion and make a process of oriented flow of ligand-receptor complexes the most likely mechanism for ligand-receptor accumulation in the cleavage furrow. PMID:7119007

Influence of hydrogen on formability and bendability of DP1180 steel for car body application

NASA Astrophysics Data System (ADS)

Gao, Q.; Han, F.; Wortberg, D.; Bleck, W.; Liewald, M.

2016-11-01

In order to reach future light weight targets, it is increasing necessary to use advanced high strength steels with tensile strength 980 MPa or higher in automotive body-inwhite structures. Due to the sensitivity to hydrogen embrittlement and the limited understanding of various aspects of hydrogen embrittlement on processing and function, the wide application of these steels is still limited. In the current work, the influence of hydrogen on the multiaxial forming behavior was investigated by determining the forming limit curve and bending limit curve of DP1180 steel. Hydrogen concentration in the material was modified by cathodic charging. Then Nakajima tests on hydrogen uncharged and pre-charged samples were carried out in order to adjust and study different strain states resulting in the forming limit curve. In the study of bending limit curve, the steel sheets were pre-strained by Marciniak test. Bending load on the uncharged and pre-charged samples was introduced by VDA238-100 bending tests. The experimental results indicated that the presence of hydrogen affected the formability and bendability of DP1180 steel. A clear difference in the influence of hydrogen at different strain states was observed. When formed in a biaxial strain state via the Nakajima test, the material showed the highest degradation in formability. Moreover, the samples with biaxial pre-loading showed more degradation in bendability comparing to those pre-strained in plane strain and uni-axial paths. Fractography by scanning electron microscope gave evidence of hydrogen-induced cleavage fracture on pre-charged Nakajima samples. Thus this investigation improves the understanding of influences of hydrogen on forming processes and provides important evidence for further studies on HE susceptibility of AHSS for the application on car body constructions.

Low-energy collision induced dissociation (low-energy CID), collision induced dissociation (CID) and higher-energy collision dissociation (HCD) mass spectrometry for structural elucidation of saccharides and clarification of their dissolution mechanism in DMAc/LiCl.

PubMed

Bayat, Parisa; Lesage, Denis; Cole, Richard B

2018-05-29

The dissolution mechanism of oligosaccharides in N,N-dimethylacetamide/lithium chloride (DMAc/LiCl), a solvent used for cellulose dissolution, and the capabilities of low-energy collision induced dissociation (low-energy CID), collision induced dissociation (CID) and higher-energy collision dissociation (HCD) for structural analysis of carbohydrates were investigated. Comparing the spectra obtained using three techniques shows that, generally, when working with mono-lithiated sugars, CID spectra provide more structurally informative fragments, and glycosidic bond cleavage is the main pathway. However, when working with di-lithiated sugars, HCD spectra can be more informative providing predominately cross-ring cleavage fragments. This is because HCD is a non-resonant activation technique and it allows a higher amount of energy to be deposited in a short time, giving access to more endothermic decomposition pathways as well as consecutive fragmentations. The difference in preferred dissociation pathways of mono-lithiated and di-lithiated sugars indicates that the presence of the second lithium strongly influences the relative rate constants for cross-ring cleavages (rearrangement) vs. direct glycosidic bond cleavages, and disfavors the latter. Regarding the dissolution mechanism of sugars in DMAc/LiCl, CID and HCD experiments on di-lithiated and tri-lithiated sugars reveal that intensities of product ions containing two Li + or three Li + , respectively, are higher than those bearing only one Li + . In addition, comparing the fragmentation spectra (both HCD and CID) of LiCl adducted lithiated sugar and NaCl adducted sodiated sugar shows that while, in the latter case, loss of NaCl is dominant, in the former case, loss of HCl occurs preferentially. The compiled evidence implies that there is a strong and direct interaction between lithium and the saccharide during the dissolution process in the DMAc/LiCl solvent system. This article is protected by copyright. All rights reserved.

The Maternal to Zygotic Transition in Mammals

PubMed Central

Li, Lei; Lu, Xukun; Dean, Jurrien

2013-01-01

Prior to activation of the embryonic genome, the initiating events of mammalian development are under maternal control and include fertilization, the block to polyspermy and processing sperm DNA. Following gamete union, the transcriptionally inert sperm DNA is repackaged into the male pronucleus which fuses with the female pronucleus to form a 1-cell zygote. Embryonic transcription begins during the maternal to zygotic transfer of control in directing development. This transition occurs at species-specific times after one or several rounds of blastomere cleavage and is essential for normal development. However, even after activation of the embryonic genome, successful development relies on stored maternal components without which embryos fail to progress beyond initial cell divisions. Better understanding of the molecular basis of maternal to zygotic transition including fertilization, the activation of the embryonic genome and cleavage-stage development will provide insight into early human development that should translate into clinical applications for regenerative medicine and assisted reproductive technologies. PMID:23352575

The soluble leptin receptor.

PubMed

Schaab, Michael; Kratzsch, Juergen

2015-10-01

The adipokine leptin realizes signal transduction via four different leptin receptor (OB-R) isoforms. The amount of functionally active OB-R, however, is affected by constitutive shedding of the extracellular domain. The product of the cleavage process, the so-called soluble leptin receptor (sOB-R), is the main binding protein for leptin in human blood and modulates its bioavailability. Concentrations of sOB-R are differentially regulated in metabolic disorders, such as type 1 diabetes mellitus or obesity, and can, therefore, enhance or reduce leptin sensitivity. Lipotoxicity and apoptosis increase OB-R cleavage via ADAM10-dependent mechanisms. In contrast, although increased sOB-R concentrations seem to directly inhibit leptin effects, reduced amounts of sOB-R may reflect decreased membrane expression of OB-R. These findings, in part, explain alterations of leptin sensitivity that are associated with changes in serum sOB-R concentrations seen in metabolic disorders. Copyright © 2015 Elsevier Ltd. All rights reserved.

Fracture of single crystals of the nickel-base superalloy PWA 1480E in helium at 22 C

NASA Technical Reports Server (NTRS)

Chen, P. S.; Wilcox, R. C.

1991-01-01

The fracture behavior and deformation of He-charged (at 22 C) single crystals of PWA 1480E Ni-base superalloy were investigated using SEM and TEM techniques to observe the behavior of tensile fractures in notched single crystals with seven different crystal growth orientations: 100-line, 110-line, 111-line, 013-line, 112-line, 123-line, and 223-line. To identify the cleavage plane orientation, a stereoscopic technique, combined with the use of planar gamma-prime morphologies, was applied. It was found that gamma-prime particles were orderly and closely aligned with edges along the 100-line, 010-line, and 001-line-oriented directions of the gamma matrix. Different crystal growth orientations were found not to affect the morphology of gamma-prime particles. The accumulation of dislocations around gamma/gamma-prime interfaces formed strong barriers to subsequent dislocation movement and was the primary strengthening mechanism at room temperature.

The decolouration of methyl orange using aluminum foam, ultrasound and direct electric current

NASA Astrophysics Data System (ADS)

Liu, C. M.; Huang, X. Y.; Zhang, H. Y.; Dai, J. D.; Ning, C. C.

2018-01-01

The decolouration of methyl orange (MO) using aluminum (Al) foam, ultrasound and direct electric current (DC) is investigated. The decolouration rate (DR) of MO using only Al foam is low because there is a passivation oxide layer on the Al foam surface. Due to the low utilization of ultrasound in MO water solution medium, the DR of MO using only ultrasonic irradiation is also poor. The DR of MO is greatly increased when Al foam, ultrasonic irradiation and DC are used together. There is good synergistic effect between Al foam, ultrasound and DC in decolouration of MO. This enhancement of DR may be related to the cavitation, cleaning of Al foam surface and water electrolysis. Due to the surface charge on wire carrying stationary current, Al foam with DC acts like a serious anodes and cathodes and makes water electrolysis giving hydrogen gas to cleavage azo bond. The DC applied on Al foam is beneficial for reductive decolouration of MO. Our results show that DC is a new way for the reductive decolouration MO in water.

Amyloid Precursor Protein (APP) Mediated Regulation of Ganglioside Homeostasis Linking Alzheimer's Disease Pathology with Ganglioside Metabolism

PubMed Central

Grimm, Marcus O. W.; Zinser, Eva G.; Grösgen, Sven; Hundsdörfer, Benjamin; Rothhaar, Tatjana L.; Burg, Verena K.; Kaestner, Lars; Bayer, Thomas A.; Lipp, Peter; Müller, Ulrike; Grimm, Heike S.; Hartmann, Tobias

2012-01-01

Gangliosides are important players for controlling neuronal function and are directly involved in AD pathology. They are among the most potent stimulators of Aβ production, are enriched in amyloid plaques and bind amyloid beta (Aβ). However, the molecular mechanisms linking gangliosides with AD are unknown. Here we identified the previously unknown function of the amyloid precursor protein (APP), specifically its cleavage products Aβ and the APP intracellular domain (AICD), of regulating GD3-synthase (GD3S). Since GD3S is the key enzyme converting a- to b-series gangliosides, it therefore plays a major role in controlling the levels of major brain gangliosides. This regulation occurs by two separate and additive mechanisms. The first mechanism directly targets the enzymatic activity of GD3S: Upon binding of Aβ to the ganglioside GM3, the immediate substrate of the GD3S, enzymatic turnover of GM3 by GD3S was strongly reduced. The second mechanism targets GD3S expression. APP cleavage results, in addition to Aβ release, in the release of AICD, a known candidate for gene transcriptional regulation. AICD strongly down regulated GD3S transcription and knock-in of an AICD deletion mutant of APP in vivo, or knock-down of Fe65 in neuroblastoma cells, was sufficient to abrogate normal GD3S functionality. Equally, knock-out of the presenilin genes, presenilin 1 and presenilin 2, essential for Aβ and AICD production, or of APP itself, increased GD3S activity and expression and consequently resulted in a major shift of a- to b-series gangliosides. In addition to GD3S regulation by APP processing, gangliosides in turn altered APP cleavage. GM3 decreased, whereas the ganglioside GD3, the GD3S product, increased Aβ production, resulting in a regulatory feedback cycle, directly linking ganglioside metabolism with APP processing and Aβ generation. A central aspect of this homeostatic control is the reduction of GD3S activity via an Aβ-GM3 complex and AICD-mediated repression of GD3S transcription. PMID:22470521

Hydrogen-Bonded Organic Semiconductor Micro- And Nanocrystals: From Colloidal Syntheses to (Opto-)Electronic Devices

PubMed Central

2014-01-01

Organic pigments such as indigos, quinacridones, and phthalocyanines are widely produced industrially as colorants for everyday products as various as cosmetics and printing inks. Herein we introduce a general procedure to transform commercially available insoluble microcrystalline pigment powders into colloidal solutions of variously sized and shaped semiconductor micro- and nanocrystals. The synthesis is based on the transformation of the pigments into soluble dyes by introducing transient protecting groups on the secondary amine moieties, followed by controlled deprotection in solution. Three deprotection methods are demonstrated: thermal cleavage, acid-catalyzed deprotection, and amine-induced deprotection. During these processes, ligands are introduced to afford colloidal stability and to provide dedicated surface functionality and for size and shape control. The resulting micro- and nanocrystals exhibit a wide range of optical absorption and photoluminescence over spectral regions from the visible to the near-infrared. Due to excellent colloidal solubility offered by the ligands, the achieved organic nanocrystals are suitable for solution processing of (opto)electronic devices. As examples, phthalocyanine nanowire transistors as well as quinacridone nanocrystal photodetectors, with photoresponsivity values by far outperforming those of vacuum deposited reference samples, are demonstrated. The high responsivity is enabled by photoinduced charge transfer between the nanocrystals and the directly attached electron-accepting vitamin B2 ligands. The semiconducting nanocrystals described here offer a cheap, nontoxic, and environmentally friendly alternative to inorganic nanocrystals as well as a new paradigm for obtaining organic semiconductor materials from commercial colorants. PMID:25253644

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

Hamilton, Brian S.; Sun, Xiangjie; Chung, Changik

A critical feature of highly pathogenic avian influenza viruses (H5N1 and H7N7) is the efficient intracellular cleavage of the hemagglutinin (HA) protein. H7N7 viruses also exist in equine species, and a unique feature of the equine H7N7 HA is the presence of an eleven amino acid insertion directly N-terminal to a tetrabasic cleavage site. Here, we show that three histidine residues within the unique insertion of the equine H7N7 HA are essential for intracellular cleavage. An asparagine residue within the insertion-derived glycosylation site was also found to be essential for intracellular cleavage. The presence of the histidine residues also appearmore » to be involved in triggering fusion, since mutation of the histidine residues resulted in a destabilizing effect. Importantly, the addition of a tetrabasic site and the eleven amino acid insertion conferred efficient intracellular cleavage to the HA of an H7N3 low pathogenicity avian influenza virus. Our studies show that acquisition of the eleven amino acid insertion offers an alternative mechanism for intracellular cleavage of influenza HA.« less

21 CFR 1401.10 - Fees to be charged-general.

Code of Federal Regulations, 2011 CFR

2011-04-01

... search. (b) Computerized search for records. ONDCP will charge 116% of the salary of the programmer/operator and the apportionable time of the central processing unit directly attributed to the search. (c...

21 CFR 1401.10 - Fees to be charged-general.

Code of Federal Regulations, 2010 CFR

2010-04-01

... search. (b) Computerized search for records. ONDCP will charge 116% of the salary of the programmer/operator and the apportionable time of the central processing unit directly attributed to the search. (c...

Amplified QCM biosensor for type IV collagenase based on collagenase-cleavage of gold nanoparticles functionalized peptide.

PubMed

Dong, Zong-Mu; Jin, Xin; Zhao, Guang-Chao

2018-05-30

The present study develops a rapid, simple and efficient method for the determination of type IV collagenase by using a specific peptide-modified quartz crystal microbalance (QCM). A small peptide (P1), contains a specific sequence (Pro-Gly) and a terminal cysteine, was synthetized and immobilized to the surface of QCM electrode via the reaction between Au and thiol of the cysteine. The peptide bond between proline and glycine can be specific hydrolyzed cleavage by type IV collagenase, which enabled the modified electrode with a high selectivity toward type IV collagenase. The cleaving process caused a frequency change of QCM to give a signal related to the concentration of type IV collagenase. The morphologies of the modified electrodes were characterized by scanning electron microscope (SEM) and the specific hydrolyzed cleavage process was monitored by QCM. When P1 was modified with gold nanoparticles (P1-Au NPs), the signal could be amplified to further enhance the sensitivity of the designed sensor due to the high-mass of the modified Au NPs. Compared the direct unamplified assay, the values obtained for the limit of detection for type IV collagenase was 0.96 ng mL -1 , yielding about 6.5 times of magnitude improvement in sensitivity. This signal enhanced peptide based QCM biosensor for type IV collagenase also showed good selectivity and sensitivity in complex matrix. Copyright © 2018 Elsevier B.V. All rights reserved.

Unfolding the HIV-1 reverse transcriptase RNase H domain – how to lose a molecular tug-of-war

DOE PAGES

Zheng, Xunhai; Pedersen, Lars C.; Gabel, Scott A.; ...

2016-01-14

Formation of the mature HIV-1 reverse transcriptase (RT) p66/p51 heterodimer requires subunit-specific processing of the p66/p66' homodimer precursor. Since the ribonuclease H (RH) domain contains an occult cleavage site located near its center, cleavage must occur either prior to folding or subsequent to unfolding. Recent NMR studies have identified a slow, subunit-specific RH domain unfolding process proposed to result from a residue tug-of-war between the polymerase and RH domains on the functionally inactive, p66' subunit. Here, we describe a structural comparison of the isolated RH domain with a domain swapped RH dimer that reveals several intrinsically destabilizing characteristics of themore » isolated domain that facilitate excursions of Tyr427 from its binding pocket and separation of helices B and D. These studies provide independent support for the subunit-selective RH domain unfolding pathway in which instability of the Tyr427 binding pocket facilitates its release followed by domain transfer, acting as a trigger for further RH domain destabilization and subsequent unfolding. As further support for this pathway, NMR studies demonstrate that addition of an RH active site-directed isoquinolone ligand retards the subunit-selective RH' domain unfolding behavior of the p66/p66' homodimer. As a result, this study demonstrates the feasibility of directly targeting RT maturation with therapeutics.« less

Unfolding the HIV-1 reverse transcriptase RNase H domain – how to lose a molecular tug-of-war

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

Zheng, Xunhai; Pedersen, Lars C.; Gabel, Scott A.

Formation of the mature HIV-1 reverse transcriptase (RT) p66/p51 heterodimer requires subunit-specific processing of the p66/p66' homodimer precursor. Since the ribonuclease H (RH) domain contains an occult cleavage site located near its center, cleavage must occur either prior to folding or subsequent to unfolding. Recent NMR studies have identified a slow, subunit-specific RH domain unfolding process proposed to result from a residue tug-of-war between the polymerase and RH domains on the functionally inactive, p66' subunit. Here, we describe a structural comparison of the isolated RH domain with a domain swapped RH dimer that reveals several intrinsically destabilizing characteristics of themore » isolated domain that facilitate excursions of Tyr427 from its binding pocket and separation of helices B and D. These studies provide independent support for the subunit-selective RH domain unfolding pathway in which instability of the Tyr427 binding pocket facilitates its release followed by domain transfer, acting as a trigger for further RH domain destabilization and subsequent unfolding. As further support for this pathway, NMR studies demonstrate that addition of an RH active site-directed isoquinolone ligand retards the subunit-selective RH' domain unfolding behavior of the p66/p66' homodimer. As a result, this study demonstrates the feasibility of directly targeting RT maturation with therapeutics.« less

Hybridizing energy conversion and storage in a mechanical-to-electrochemical process for self-charging power cell.

PubMed

Xue, Xinyu; Wang, Sihong; Guo, Wenxi; Zhang, Yan; Wang, Zhong Lin

2012-09-12

Energy generation and energy storage are two distinct processes that are usually accomplished using two separated units designed on the basis of different physical principles, such as piezoelectric nanogenerator and Li-ion battery; the former converts mechanical energy into electricity, and the latter stores electric energy as chemical energy. Here, we introduce a fundamental mechanism that directly hybridizes the two processes into one, in which the mechanical energy is directly converted and simultaneously stored as chemical energy without going through the intermediate step of first converting into electricity. By replacing the polyethylene (PE) separator as for conventional Li battery with a piezoelectric poly(vinylidene fluoride) (PVDF) film, the piezoelectric potential from the PVDF film as created by mechanical straining acts as a charge pump to drive Li ions to migrate from the cathode to the anode accompanying charging reactions at electrodes. This new approach can be applied to fabricating a self-charging power cell (SCPC) for sustainable driving micro/nanosystems and personal electronics.

Cleavage fracture in high strength low alloy weld metal

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

Bose, W.W.; Bowen, P.; Strangwood, M.

1996-12-31

The present investigation gives an evaluation of the effect of microstructure on the cleavage fracture process of High Strength Low Alloy (HSLA) multipass weld metals. With additions of alloying elements, such as Ti, Ni, Mo and Cr, the microstructure of C-Mn weld metal changes from the classical composition, i.e., allotriomorphic ferrite with acicular ferrite and Widmanstaetten ferrite, to bainite and low carbon martensite. Although the physical metallurgy of some HSLA weld metals has been studied before, more work is necessary to correlate the effect of the microstructure on the fracture behavior of such weld metals. In this work detailed microstructuralmore » analysis was carried out using optical and electron (SEM and TEM) microscopy. Single edge notched (SEN) bend testpieces were used to assess the cleavage fracture stress, {sigma}{sub F}. Inclusions beneath the notch surface were identified as the crack initiators of unstable cleavage fracture. From the size of such inclusions and the value of tensile stress predicted at the initiation site, the effective surface energy for cleavage was calculated using a modified Griffth energy balance for a penny shape crack. The results suggest that even though inclusions initiate cleavage fracture, the local microstructure may play an important role in the fracture process of these weld metals. The implications of these observations for a quantitative theory of the cleavage fracture of ferritic steels is discussed.« less

Enhancing Interleukin-6 and Interleukin-11 receptor cleavage.

PubMed

Lokau, Juliane; Wandel, Marieke; Garbers, Christoph

2017-04-01

Proteolytic cleavage of the membrane-bound Interleukin-6 receptor (IL-6R) by the metalloprotease ADAM17 releases an agonistic soluble form of the IL-6R (sIL-6R), which is responsible for the pro-inflammatory trans-signaling branch of the cytokine's activities. This proteolytic step, which is also called ectodomain shedding, is critically regulated by the cleavage site within the IL-6R stalk, because mutations or small deletions within this region are known to render the IL-6R irresponsive towards proteolysis. In the present study, we employed cleavage site profiling data of ADAM17 to generate an IL-6R with increased cleavage susceptibility. Using site-directed mutagenesis, we showed that the non-prime sites P3 and P2 and the prime site P1' were critical for this increase in proteolysis, whereas other positions within the cleavage site were of minor importance. Insertion of this optimized cleavage site into the stalk of the Interleukin-11 receptor (IL-11R) was not sufficient to enable ADAM17-mediated proteolysis, but transfer of different parts of the IL-6R stalk enabled shedding by ADAM17. These findings shed light on the cleavage site specificities of ADAM17 using a native substrate and reveal further differences in the proteolysis of IL-6R and IL-11R. Copyright © 2017 Elsevier Ltd. All rights reserved.

DFT study on the interaction of TiO2 (001) surface with HCHO molecules

NASA Astrophysics Data System (ADS)

Wu, Guofei; Zhao, Cuihua; Guo, Changqing; Chen, Jianhua; Zhang, Yibing; Li, Yuqiong

2018-01-01

The interactions of formaldehyde (HCHO) molecule with TiO2 (001) surface were studied using density functional theory calculations. HCHO molecules are dissociated by the cleavage of Csbnd H bonds after adsorption on TiO2 surface. The strong interactions between HCHO melecules and TiO2 surface are largely attributed to the bonding of hydrogen of HCHO and oxygen of TiO2 surface, which is mainly from the hybridization of the H 1s, O 2p and O 2s. The newly formed Hsbnd O bonds cause the structure changes of TiO2 surface, and lead to the cleavage of Osbnd Ti bond of TiO2 surface. The Csbnd O bond that the dissociated remains of HCHO and newly formed Hsbnd O bond can be oxidized to form carbon dioxide and water in subsequent action by oxygen from the atomosphere. The charges transfer from HCHO to TiO2 surface, and the sum amount of the charges transferred from four HCHO molecules to TiO2 surface is bigger than that from one HCHO molecule to TiO2 surface due to the combined interaction of four HCHO molecules with TiO2 surface.

Unusual enzymatic glycoside cleavage mechanisms.

PubMed

Jongkees, Seino A K; Withers, Stephen G

2014-01-21

Over the sixty years since Koshland initially formulated the classical mechanisms for retaining and inverting glycosidases, researchers have assembled a large body of supporting evidence and have documented variations of these mechanisms. Recently, however, researchers have uncovered a number of completely distinct mechanisms for enzymatic cleavage of glycosides involving elimination and/or hydration steps. In family GH4 and GH109 glycosidases, the reaction proceeds via transient NAD(+)-mediated oxidation at C3, thereby acidifying the proton at C2 and allowing for elimination across the C1-C2 bond. Subsequent Michael-type addition of water followed by reduction at C3 generates the hydrolyzed product. Enzymes employing this mechanism can hydrolyze thioglycosides as well as both anomers of activated substrates. Sialidases employ a conventional retaining mechanism in which a tyrosine functions as the nucleophile, but in some cases researchers have observed off-path elimination end products. These reactions occur via the normal covalent intermediate, but instead of an attack by water on the anomeric center, the catalytic acid/base residue abstracts an adjacent proton. These enzymes can also catalyze hydration of the enol ether via the reverse pathway. Reactions of α-(1,4)-glucan lyases also proceed through a covalent intermediate with subsequent abstraction of an adjacent proton to give elimination. However, in this case, the departing carboxylate "nucleophile" serves as the base in a concerted but asynchronous syn-elimination process. These enzymes perform only elimination reactions. Polysaccharide lyases, which act on uronic acid-containing substrates, also catalyze only elimination reactions. Substrate binding neutralizes the charge on the carboxylate, which allows for abstraction of the proton on C5 and leads to an elimination reaction via an E1cb mechanism. These enzymes can also cleave thioglycosides, albeit slowly. The unsaturated product of polysaccharide lyases can then serve as a substrate for a hydration reaction carried out by unsaturated glucuronyl hydrolases. This hydration is initiated by protonation at C4 and proceeds in a Markovnikov fashion rather than undergoing a Michael-type addition, giving a hemiketal at C5. This hemiketal then undergoes a rearrangement that results in cleavage of the anomeric bond. These enzymes can also hydrolyze thioglycosides efficiently and slowly turn over substrates with inverted anomeric configuration. The mechanisms discussed in this Account proceed through transition states that involve either positive or negative charges, unlike the exclusively cationic transition states of the classical Koshland retaining and inverting glycosidases. In addition, the distribution of this charge throughout the substrate can vary substantially. The nature of these mechanisms and their transition states means that any inhibitors or inactivators of these unusual enzymes probably differ from those presently used for Koshland retaining or inverting glycosidases.

Membrane microdomain switching: a regulatory mechanism of amyloid precursor protein processing.

PubMed

Sakurai, Takashi; Kaneko, Kumi; Okuno, Misako; Wada, Koji; Kashiyama, Taku; Shimizu, Hideaki; Akagi, Takumi; Hashikawa, Tsutomu; Nukina, Nobuyuki

2008-10-20

Neuronal activity has an impact on beta cleavage of amyloid precursor protein (APP) by BACE1 to generate amyloid-beta peptide (Abeta). However, the molecular mechanisms underlying this effect remain to be elucidated. Cholesterol dependency of beta cleavage prompted us to analyze immunoisolated APP-containing detergent-resistant membranes from rodent brains. We found syntaxin 1 as a key molecule for activity-dependent regulation of APP processing in cholesterol-dependent microdomains. In living cells, APP associates with syntaxin 1-containing microdomains through X11-Munc18, which inhibits the APP-BACE1 interaction and beta cleavage via microdomain segregation. Phosphorylation of Munc18 by cdk5 causes a shift of APP to BACE1-containing microdomains. Neuronal hyperactivity, implicated in Abeta overproduction, promotes the switching of APP microdomain association as well as beta cleavage in a partially cdk5-dependent manner. We propose that microdomain switching is a mechanism of cholesterol- and activity-dependent regulation of APP processing in neurons.

Membrane microdomain switching: a regulatory mechanism of amyloid precursor protein processing

PubMed Central

Sakurai, Takashi; Kaneko, Kumi; Okuno, Misako; Wada, Koji; Kashiyama, Taku; Shimizu, Hideaki; Akagi, Takumi; Hashikawa, Tsutomu; Nukina, Nobuyuki

2008-01-01

Neuronal activity has an impact on β cleavage of amyloid precursor protein (APP) by BACE1 to generate amyloid-β peptide (Aβ). However, the molecular mechanisms underlying this effect remain to be elucidated. Cholesterol dependency of β cleavage prompted us to analyze immunoisolated APP-containing detergent-resistant membranes from rodent brains. We found syntaxin 1 as a key molecule for activity-dependent regulation of APP processing in cholesterol-dependent microdomains. In living cells, APP associates with syntaxin 1–containing microdomains through X11–Munc18, which inhibits the APP–BACE1 interaction and β cleavage via microdomain segregation. Phosphorylation of Munc18 by cdk5 causes a shift of APP to BACE1-containing microdomains. Neuronal hyperactivity, implicated in Aβ overproduction, promotes the switching of APP microdomain association as well as β cleavage in a partially cdk5-dependent manner. We propose that microdomain switching is a mechanism of cholesterol- and activity-dependent regulation of APP processing in neurons. PMID:18936252

GPS-CCD: A Novel Computational Program for the Prediction of Calpain Cleavage Sites

PubMed Central

Gao, Xinjiao; Ma, Qian; Ren, Jian; Xue, Yu

2011-01-01

As one of the most essential post-translational modifications (PTMs) of proteins, proteolysis, especially calpain-mediated cleavage, plays an important role in many biological processes, including cell death/apoptosis, cytoskeletal remodeling, and the cell cycle. Experimental identification of calpain targets with bona fide cleavage sites is fundamental for dissecting the molecular mechanisms and biological roles of calpain cleavage. In contrast to time-consuming and labor-intensive experimental approaches, computational prediction of calpain cleavage sites might more cheaply and readily provide useful information for further experimental investigation. In this work, we constructed a novel software package of GPS-CCD (Calpain Cleavage Detector) for the prediction of calpain cleavage sites, with an accuracy of 89.98%, sensitivity of 60.87% and specificity of 90.07%. With this software, we annotated potential calpain cleavage sites for hundreds of calpain substrates, for which the exact cleavage sites had not been previously determined. In this regard, GPS-CCD 1.0 is considered to be a useful tool for experimentalists. The online service and local packages of GPS-CCD 1.0 were implemented in JAVA and are freely available at: http://ccd.biocuckoo.org/. PMID:21533053

Dynamics of charge-transfer excitons in type-II semiconductor heterostructures

NASA Astrophysics Data System (ADS)

Stein, M.; Lammers, C.; Richter, P.-H.; Fuchs, C.; Stolz, W.; Koch, M.; Vänskä, O.; Weseloh, M. J.; Kira, M.; Koch, S. W.

2018-03-01

The formation, decay, and coherence properties of charge-transfer excitons in semiconductor heterostructures are investigated by applying four-wave-mixing and terahertz spectroscopy in combination with a predictive microscopic theory. A charge-transfer process is identified where the optically induced coherences decay directly into a charge-transfer electron-hole plasma and exciton states. It is shown that charge-transfer excitons are more sensitive to the fermionic electron-hole substructure than regular excitons.

Highly Chemoselective Reduction of Amides (Primary, Secondary, Tertiary) to Alcohols using SmI2/Amine/H2O under Mild Conditions

PubMed Central

2014-01-01

Highly chemoselective direct reduction of primary, secondary, and tertiary amides to alcohols using SmI2/amine/H2O is reported. The reaction proceeds with C–N bond cleavage in the carbinolamine intermediate, shows excellent functional group tolerance, and delivers the alcohol products in very high yields. The expected C–O cleavage products are not formed under the reaction conditions. The observed reactivity is opposite to the electrophilicity of polar carbonyl groups resulting from the nX → π*C=O (X = O, N) conjugation. Mechanistic studies suggest that coordination of Sm to the carbonyl and then to Lewis basic nitrogen in the tetrahedral intermediate facilitate electron transfer and control the selectivity of the C–N/C–O cleavage. Notably, the method provides direct access to acyl-type radicals from unactivated amides under mild electron transfer conditions. PMID:24460078

Microbial cleavage of organic C-S bonds

DOEpatents

Kilbane, J.J. II.

1994-10-25

A microbial process is described for selective cleavage of organic C-S bonds which may be used for reducing the sulfur content of sulfur-containing organic carbonaceous materials. Microorganisms of Rhodococcus rhodochrous and Bacillus sphaericus have been found which have the ability of selective cleavage of organic C-S bonds. Particularly preferred microorganisms are Rhodococcus rhodochrous strain ATCC 53968 and Bacillus sphaericus strain ATCC 53969 and their derivatives.

Microbial cleavage of organic C-S bonds

DOEpatents

Kilbane, II, John J.

1994-01-01

A microbial process for selective cleavage of organic C--S bonds which may be used for reducing the sulfur content of sulfur-containing organic carbonaceous materials, Microorganisms of Rhodococcus rhodochrous and Bacillus sphaericus have been found which have the ability of selective cleavage of organic C--S bonds. Particularly preferred microorganisms are Rhodococcus rhodochrous strain ATCC 53968 and Bacillus sphaericus strain ATCC 53969 and their derivatives.

Differences and Comparisons of the Properties and Reactivities of Iron(III)–hydroperoxo Complexes with Saturated Coordination Sphere

PubMed Central

Faponle, Abayomi S; Quesne, Matthew G; Sastri, Chivukula V; Banse, Frédéric; de Visser, Sam P

2015-01-01

Heme and nonheme monoxygenases and dioxygenases catalyze important oxygen atom transfer reactions to substrates in the body. It is now well established that the cytochrome P450 enzymes react through the formation of a high-valent iron(IV)–oxo heme cation radical. Its precursor in the catalytic cycle, the iron(III)–hydroperoxo complex, was tested for catalytic activity and found to be a sluggish oxidant of hydroxylation, epoxidation and sulfoxidation reactions. In a recent twist of events, evidence has emerged of several nonheme iron(III)–hydroperoxo complexes that appear to react with substrates via oxygen atom transfer processes. Although it was not clear from these studies whether the iron(III)–hydroperoxo reacted directly with substrates or that an initial O–O bond cleavage preceded the reaction. Clearly, the catalytic activity of heme and nonheme iron(III)–hydroperoxo complexes is substantially different, but the origins of this are still poorly understood and warrant a detailed analysis. In this work, an extensive computational analysis of aromatic hydroxylation by biomimetic nonheme and heme iron systems is presented, starting from an iron(III)–hydroperoxo complex with pentadentate ligand system (L52). Direct C–O bond formation by an iron(III)–hydroperoxo complex is investigated, as well as the initial heterolytic and homolytic bond cleavage of the hydroperoxo group. The calculations show that [(L52)FeIII(OOH)]2+ should be able to initiate an aromatic hydroxylation process, although a low-energy homolytic cleavage pathway is only slightly higher in energy. A detailed valence bond and thermochemical analysis rationalizes the differences in chemical reactivity of heme and nonheme iron(III)–hydroperoxo and show that the main reason for this particular nonheme complex to be reactive comes from the fact that they homolytically split the O–O bond, whereas a heterolytic O–O bond breaking in heme iron(III)–hydroperoxo is found. PMID:25399782

Cleavage-Independent HIV-1 Trimers From CHO Cell Lines Elicit Robust Autologous Tier 2 Neutralizing Antibodies

PubMed Central

Bale, Shridhar; Martiné, Alexandra; Wilson, Richard; Behrens, Anna-Janina; Le Fourn, Valérie; de Val, Natalia; Sharma, Shailendra K.; Tran, Karen; Torres, Jonathan L.; Girod, Pierre-Alain; Ward, Andrew B.; Crispin, Max; Wyatt, Richard T.

2018-01-01

Native flexibly linked (NFL) HIV-1 envelope glycoprotein (Env) trimers are cleavage-independent and display a native-like, well-folded conformation that preferentially displays broadly neutralizing determinants. The NFL platform simplifies large-scale production of Env by eliminating the need to co-transfect the precursor-cleaving protease, furin that is required by the cleavage-dependent SOSIP trimers. Here, we report the development of a CHO-M cell line that expressed BG505 NFL trimers at a high level of homogeneity and yields of ~1.8 g/l. BG505 NFL trimers purified by single-step lectin-affinity chromatography displayed a native-like closed structure, efficient recognition by trimer-preferring bNAbs, no recognition by non-neutralizing CD4 binding site-directed and V3-directed antibodies, long-term stability, and proper N-glycan processing. Following negative-selection, formulation in ISCOMATRIX adjuvant and inoculation into rabbits, the trimers rapidly elicited potent autologous tier 2 neutralizing antibodies. These antibodies targeted the N-glycan “hole” naturally present on the BG505 Env proximal to residues at positions 230, 241, and 289. The BG505 NFL trimers that did not expose V3 in vitro, elicited low-to-no tier 1 virus neutralization in vivo, indicating that they remained intact during the immunization process, not exposing V3. In addition, BG505 NFL and BG505 SOSIP trimers expressed from 293F cells, when formulated in Adjuplex adjuvant, elicited equivalent BG505 tier 2 autologous neutralizing titers. These titers were lower in potency when compared to the titers elicited by CHO-M cell derived trimers. In addition, increased neutralization of tier 1 viruses was detected. Taken together, these data indicate that both adjuvant and cell-type expression can affect the elicitation of tier 2 and tier 1 neutralizing responses in vivo.

γ-Secretase Modulators and APH1 Isoforms Modulate γ-Secretase Cleavage but Not Position of ε-Cleavage of the Amyloid Precursor Protein (APP).

PubMed

Lessard, Christian B; Cottrell, Barbara A; Maruyama, Hiroko; Suresh, Suraj; Golde, Todd E; Koo, Edward H

2015-01-01

The relative increase in Aβ42 peptides from familial Alzheimer disease (FAD) linked APP and PSEN mutations can be related to changes in both ε-cleavage site utilization and subsequent step-wise cleavage. Cleavage at the ε-site releases the amyloid precursor protein (APP) intracellular domain (AICD), and perturbations in the position of ε-cleavage are closely associated with changes in the profile of amyloid β-protein (Aβ) species that are produced and secreted. The mechanisms by which γ-secretase modulators (GSMs) or FAD mutations affect the various γ-secretase cleavages to alter the generation of Aβ peptides have not been fully elucidated. Recent studies suggested that GSMs do not modulate ε-cleavage of APP, but the data were derived principally from recombinant truncated epitope tagged APP substrate. Here, using full length APP from transfected cells, we investigated whether GSMs modify the ε-cleavage of APP under more native conditions. Our results confirmed the previous findings that ε-cleavage is insensitive to GSMs. In addition, fenofibrate, an inverse GSM (iGSM), did not alter the position or kinetics of ε-cleavage position in vitro. APH1A and APH1B, a subunit of the γ-secretase complex, also modulated Aβ42/Aβ40 ratio without any alterations in ε-cleavage, a result in contrast to what has been observed with PS1 and APP FAD mutations. Consequently, GSMs and APH1 appear to modulate γ-secretase activity and Aβ42 generation by altering processivity but not ε-cleavage site utilization.

Bovine viral diarrhea virus NS3 serine proteinase: polyprotein cleavage sites, cofactor requirements, and molecular model of an enzyme essential for pestivirus replication.

PubMed Central

Xu, J; Mendez, E; Caron, P R; Lin, C; Murcko, M A; Collett, M S; Rice, C M

1997-01-01

Members of the Flaviviridae encode a serine proteinase termed NS3 that is responsible for processing at several sites in the viral polyproteins. In this report, we show that the NS3 proteinase of the pestivirus bovine viral diarrhea virus (BVDV) (NADL strain) is required for processing at nonstructural (NS) protein sites 3/4A, 4A/4B, 4B/5A, and 5A/5B but not for cleavage at the junction between NS2 and NS3. Cleavage sites of the proteinase were determined by amino-terminal sequence analysis of the NS4A, NS4B, NS5A, and NS5B proteins. A conserved leucine residue is found at the P1 position of all four cleavage sites, followed by either serine (3/4A, 4B/5A, and 5A/5B sites) or alanine (4A/4B site) at the P1' position. Consistent with this cleavage site preference, a structural model of the pestivirus NS3 proteinase predicts a highly hydrophobic P1 specificity pocket. trans-Processing experiments implicate the 64-residue NS4A protein as an NS3 proteinase cofactor required for cleavage at the 4B/5A and 5A/5B sites. Finally, using a full-length functional BVDV cDNA clone, we demonstrate that a catalytically active NS3 serine proteinase is essential for pestivirus replication. PMID:9188600

The caspase-generated cleavage product of Ets-1 p51 and Ets-1 p27, Cp17, induces apoptosis.

PubMed

Choul-Li, Souhaila; Tulasne, David; Aumercier, Marc

2016-11-04

The transcription factor Ets-1 is involved in various physiological processes and invasive pathologies. Human Ets-1 exists under three isoforms: p51, the predominant full-length isoform, p42 and p27, shorter alternatively spliced isoforms. We have previously demonstrated that Ets-1 p51, but not the spliced variant Ets-1 p42, is processed by caspases in vitro and during apoptosis. However, the caspase cleavage of the second spliced variant Ets-1 p27 remains to investigate. In the present study, we demonstrate that Ets-1 p27 is a cleavage substrate of caspases. We show that Ets-1 p27 is processed in vitro by caspase-3, resulting in three C-terminal fragments Cp20, Cp17 and Cp14. Similarly, Ets-1 p27 was cleaved during apoptotic cell death induced by anisomycin, producing fragments consistent with those observed in in vitro cleavage assay. These fragments are generated by cleavage at three sites located in the exon VII-encoded region of Ets-1 p27. As a functional consequences, Cp17 fragment, the major cleavage product generated during apoptosis, induced itself apoptosis when transfected into cells. Our results show that Ets-1 p27 is cleaved in the same manner as Ets-1 p51 within the exon VII-encoded region, thus generating a stable C-terminal fragment that induces cell death by initiating apoptosis. Copyright © 2016 Elsevier Inc. All rights reserved.

Positive and negative ion mode comparison for the determination of DNA/peptide noncovalent binding sites through the formation of "three-body" noncovalent fragment ions.

PubMed

Brahim, Bessem; Tabet, Jean-Claude; Alves, Sandra

2018-02-01

Gas-phase fragmentation of single strand DNA-peptide noncovalent complexes is investigated in positive and negative electrospray ionization modes.Collision-induced dissociation experiments, performed on the positively charged noncovalent complex precursor ions, have confirmed the trend previously observed in negative ion mode, i.e. a high stability of noncovalent complexes containing very basic peptidic residues (i.e. R > K) and acidic nucleotide units (i.e. Thy units), certainly incoming from the existence of salt bridge interactions. Independent of the ion polarity, stable noncovalent complex precursor ions were found to dissociate preferentially through covalent bond cleavages of the partners without disrupting noncovalent interactions. The resulting DNA fragment ions were found to be still noncovalently linked to the peptides. Additionally, the losses of an internal nucleic fragment producing "three-body" noncovalent fragment ions were also observed in both ion polarities, demonstrating the spectacular salt bridge interaction stability. The identical fragmentation patterns (regardless of the relative fragment ion abundances) observed in both polarities have shown a common location of salt bridge interaction certainly preserved from solution. Nonetheless, most abundant noncovalent fragment ions (and particularly three-body ones) are observed from positively charged noncovalent complexes. Therefore, we assume that, independent of the preexisting salt bridge interaction and zwitterion structures, multiple covalent bond cleavages from single-stranded DNA/peptide complexes rely on an excess of positive charges in both electrospray ionization ion polarities.

Unusually short chalcogen bonds involving organoselenium: insights into the Se-N bond cleavage mechanism of the antioxidant ebselen and analogues.

PubMed

Thomas, Sajesh P; Satheeshkumar, K; Mugesh, Govindasamy; Guru Row, T N

2015-04-27

Structural studies on the polymorphs of the organoselenium antioxidant ebselen and its derivative show the potential of organic selenium to form unusually short Se⋅⋅⋅O chalcogen bonds that lead to conserved supramolecular recognition units. Se⋅⋅⋅O interactions observed in these polymorphs are the shortest such chalcogen bonds known for organoselenium compounds. The FTIR spectral evolution characteristics of this interaction from solution state to solid crystalline state further validates the robustness of this class of supramolecular recognition units. The strength and electronic nature of the Se⋅⋅⋅O chalcogen bonds were explored using high-resolution X-ray charge density analysis and atons-in-molecules (AIM) theoretical analysis. A charge density study unravels the strong electrostatic nature of Se⋅⋅⋅O chalcogen bonding and soft-metal-like behavior of organoselenium. An analysis of the charge density around Se-N and Se-C covalent bonds in conjunction with the Se⋅⋅⋅O chalcogen bonding modes in ebselen and its analogues provides insights into the mechanism of drug action in this class of organoselenium antioxidants. The potential role of the intermolecular Se⋅⋅⋅O chalcogen bonding in forming the intermediate supramolecular assembly that leads to the bond cleavage mechanism has been proposed in terms of electron density topological parameters in a series of molecular complexes of ebselen with reactive oxygen species (ROS). © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

The role of GyrB in the DNA cleavage-religation reaction of DNA gyrase: a proposed two metal-ion mechanism.

PubMed

Noble, Christian G; Maxwell, Anthony

2002-04-26

We have examined the role of the DNA gyrase B protein in cleavage and religation of DNA using site-directed mutagenesis. Three aspartate residues and a glutamate residue: E424, D498, D500 and D502, thought to co-ordinate a magnesium ion, were mutated to alanine; in addition, the glutamate residue and one aspartate residue were mutated to glutamine and asparagine, respectively. We have shown that these residues are important for the cleavage-religation reaction and are likely to be involved in magnesium ion co-ordination. On separate mutation of two of these aspartate residues to cysteine or histidine, the metal ion preference for the DNA relaxation activity of gyrase changed from magnesium to manganese (II). We present evidence to support the idea that cleavage of each DNA strand involves two or more metal ions, and suggest a scheme for the DNA cleavage chemistry of DNA gyrase involving two metal ions. (c) 2002 Elsevier Science Ltd.

Transition metal catalyzed manipulation of non-polar carbon–hydrogen bonds for synthetic purpose

PubMed Central

MURAI, Shinji

2011-01-01

The direct addition of ortho C–H bonds in various aromatic compounds such as ketones, esters, imines, imidates, nitriles, and aldehydes to olefins and acetylenes can be achieved with the aid of transition metal catalysts. The ruthenium catalyzed reaction is usually highly efficient and useful as a general synthetic method. The coordination to the metal center by a heteroatom in a directing group such as carbonyl and imino groups in aromatic compounds is the key step in this process. Mechanistically, the reductive elimination to form a C–C bond is the rate-determining step, while the C–H bond cleavage step is not. PMID:21558759

Crystallization and characterization of human chorionic gonadotropin in chemically deglycosylated and enzymatically desialylated states

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

Lustbader, J.W.; Birken, S.; Pileggi, N.F.

1989-11-28

Crystals suitable for X-ray diffraction studies at moderate resolution have been grown from two forms of human chorionic gonadotropin (hCG): HF-treated hCG and neuraminidase-treated hCG. The enzymatically desialylated form of hCG produced crystals that diffract to 2.8 {angstrom} as compared to the HF-treated hCG crystals that diffract to 3.0 {angstrom}. Although it was assumed that the high and heterogeneous carbohydrate content of the glycoprotein hormones inhibited their crystallization, this report suggests that it is the negatively charged surface sugars and neither the total carbohydrate content nor its heterogeneity which interferes with crystal formation. Chemical deglycosylation resulted in significantly increased proteinmore » degradation during crystal growth. Such peptide bond cleavages were observed to a much lesser extent in the crystals grown from neuraminidase-digested hCG. Sequence analysis of the HF-treated hCG crystals suggested that up to 45% of the molecules within the crystal had an acid-labile peptide bond cleaved. In contrast, the neuraminidase-treated hCG exhibited less than 9% of this type of cleavage. The manner in which hCG was treated prior to crystallization was found to be a very important factor in the extent of peptide bound cleavages occurring during crystal growth. HF treatment of glycoproteins may render glycoproteins more susceptible to peptide bond cleavage during crystal growth.« less

Site-Specific Pyrolysis Induced Cleavage at Aspartic Acid Residue in Peptides and Proteins

PubMed Central

Zhang, Shaofeng; Basile, Franco

2011-01-01

A simple and site-specific non-enzymatic method based on pyrolysis has been developed to cleave peptides and proteins. Pyrolytic cleavage was found to be specific and rapid as it induced a cleavage at the C-terminal side of aspartic acid in the temperature range of 220–250 °C in 10 seconds. Electrospray Ionization (ESI) mass spectrometry (MS) and tandem-MS (MS/MS) were used to characterize and identify pyrolysis cleavage products, confirming that sequence information is conserved after the pyrolysis process in both peptides and protein tested. This suggests that pyrolysis-induced cleavage at aspartyl residues can be used as a rapid protein digestion procedure for the generation of sequence specific protein biomarkers. PMID:17388620

Oxygen vacancies promoted interfacial charge carrier transfer of CdS/ZnO heterostructure for photocatalytic hydrogen generation.

PubMed

Xie, Ying Peng; Yang, Yongqiang; Wang, Guosheng; Liu, Gang

2017-10-01

The solid-state Z-scheme trinary/binary heterostructures show the advantage of utilizing the high-energy photogenerated charge carriers in photocatalysis. However, the key factors controlling such Z-scheme in the binary heterostructures are still unclear. In this paper, we showed that oxygen vacancies could act as an interface electron transfer mediator to promote the direct Z-scheme charge transfer process in binary semiconductor heterostructures of CdS/ZnS. Increasing the concentration of surface oxygen vacancies of ZnO crystal can greatly enhance photocatalytic hydrogen generation of CdS/ZnO heterostructure. This was attributed to the strengthened direct Z-scheme charge transfer process in CdS/ZnO, as evidenced by steady-state/time-resolved photoluminescence spectroscopy and selective photodeposition of metal particles on the heterostructure. Copyright © 2017 Elsevier Inc. All rights reserved.

Deionization and desalination using electrostatic ion pumping

DOEpatents

Bourcier, William L.; Aines, Roger D.; Haslam, Jeffery J.; Schaldach, Charlene M.; O& #x27; Brien, Kevin C.; Cussler, Edward

2013-06-11

The present invention provides a new method and apparatus/system for purifying ionic solutions, such as, for example, desalinating water, using engineered charged surfaces to sorb ions from such solutions. Surface charge is applied externally, and is synchronized with oscillatory fluid movements between substantially parallel charged plates. Ions are held in place during fluid movement in one direction (because they are held in the electrical double layer), and released for transport during fluid movement in the opposite direction by removing the applied electric field. In this way the ions, such as salt, are "ratcheted" across the charged surface from the feed side to the concentrate side. The process itself is very simple and involves only pumps, charged surfaces, and manifolds for fluid collection.

Deionization and desalination using electrostatic ion pumping

DOEpatents

Bourcier, William L [Livermore, CA; Aines, Roger D [Livermore, CA; Haslam, Jeffery J [Livermore, CA; Schaldach, Charlene M [Pleasanton, CA; O'Brien, Kevin C [San Ramon, CA; Cussler, Edward [Edina, MN

2011-07-19

The present invention provides a new method and apparatus/system for purifying ionic solutions, such as, for example, desalinating water, using engineered charged surfaces to sorb ions from such solutions. Surface charge is applied externally, and is synchronized with oscillatory fluid movements between substantially parallel charged plates. Ions are held in place during fluid movement in one direction (because they are held in the electrical double layer), and released for transport during fluid movement in the opposite direction by removing the applied electric field. In this way the ions, such as salt, are "ratcheted" across the charged surface from the feed side to the concentrate side. The process itself is very simple and involves only pumps, charged surfaces, and manifolds for fluid collection.

Essential role of cyclophilin A for hepatitis C virus replication and virus production and possible link to polyprotein cleavage kinetics.

PubMed

Kaul, Artur; Stauffer, Sarah; Berger, Carola; Pertel, Thomas; Schmitt, Jennifer; Kallis, Stephanie; Zayas, Margarita; Lopez, Margarita Zayas; Lohmann, Volker; Luban, Jeremy; Bartenschlager, Ralf

2009-08-01

Viruses are obligate intracellular parasites and therefore their replication completely depends on host cell factors. In case of the hepatitis C virus (HCV), a positive-strand RNA virus that in the majority of infections establishes persistence, cyclophilins are considered to play an important role in RNA replication. Subsequent to the observation that cyclosporines, known to sequester cyclophilins by direct binding, profoundly block HCV replication in cultured human hepatoma cells, conflicting results were obtained as to the particular cyclophilin (Cyp) required for viral RNA replication and the underlying possible mode of action. By using a set of cell lines with stable knock-down of CypA or CypB, we demonstrate in the present work that replication of subgenomic HCV replicons of different genotypes is reduced by CypA depletion up to 1,000-fold whereas knock-down of CypB had no effect. Inhibition of replication was rescued by over-expression of wild type CypA, but not by a mutant lacking isomerase activity. Replication of JFH1-derived full length genomes was even more sensitive to CypA depletion as compared to subgenomic replicons and virus production was completely blocked. These results argue that CypA may target an additional viral factor outside of the minimal replicase contributing to RNA amplification and assembly, presumably nonstructural protein 2. By selecting for resistance against the cyclosporine analogue DEBIO-025 that targets CypA in a dose-dependent manner, we identified two mutations (V2440A and V2440L) close to the cleavage site between nonstructural protein 5A and the RNA-dependent RNA polymerase in nonstructural protein 5B that slow down cleavage kinetics at this site and reduce CypA dependence of viral replication. Further amino acid substitutions at the same cleavage site accelerating processing increase CypA dependence. Our results thus identify an unexpected correlation between HCV polyprotein processing and CypA dependence of HCV replication.

Kinesin motor protein as an electrostatic ratchet machine

NASA Astrophysics Data System (ADS)

Tsironis, George; Ciudad, Aleix; Sancho, Jose Maria

2008-03-01

Kinesin and related motor proteins utilize ATP fuel to propel themselves along the external surface of microtubules in a processive and directional fashion. We show that the observed step-like motion is possible through time varying charge distributions furnished by the ATP hydrolysis circle while the static charge configuration on the microtuble provides the guide for motion. Thus, while the chemical hydrolysis energy induces appropriate local conformational changes, the motor translational energy is fundamentally electrostatic. Numerical simulations of the mechanical equations of motion show that processivity and directionality are direct consequences of the ATP-dependent electrostatic interaction between the different charge distributions of kinesin and microtubule. Treating proterins as continuous dielectric media and using a Green's function formalism we find analytical expressions for the electrostatic energy in the vicinity of the protein surfaces. We calculate the Bjerrum length in the interior of the protein and analyze its dependence on the charge proximity to the protein interface. We apply these results to kinesin and estimate the pure electrostatic ATP-ADP interaction to be larger than 2k T.

Mass spectrometric investigation of buspirone drug in comparison with thermal analyses and MO-calculations

NASA Astrophysics Data System (ADS)

Zayed, M. A.; Fahmey, M. A.; Hawash, M. A.; El-Habeeb, Abeer A.

2007-06-01

The buspirone drug is usually present as hydrochloride form of general formula C 21H 31N 5O 2·HCl, and of molecular weight (MW) = 421.96. It is an analgesic anxiolytic drug, which does not cause sedative or depression of central nervous system. In the present work it is investigated using electron impact mass spectral (EI-MS) fragmentation at 70 eV, in comparison with thermal analyses (TA) measurements (TG/DTG and DTA) and molecular orbital calculation (MOC). Semi-empirical MO calculation, PM3 procedure, has been carried out on buspirone both as neutral molecule (in TA) and the corresponding positively charged species (in MS). The calculated MOC parameters include bond length, bond order, particle charge distribution on different atoms and heats of formation. The fragmentation pathways of buspirone in EI-MS lead to the formation of important primary and secondary fragment ions. The mechanism of formation of some important daughter ions can be illuminated from comparing with that obtained using electrospray ESIMS/MS mode mass spectrometer through the accurate mass measurement determination. The losses of the intermediate aliphatic part (CH 2) 4 due to cleavage of N-C bond from both sides is the primary cleavage in both techniques (MS and TA). The PM3 provides a base for fine distinction among sites of initial bond cleavage and subsequent fragmentation of drug molecule in both TA and MS techniques; consequently the choice of the correct pathway of such fragmentation knowing this structural session of bonds can be used to decide the active sites of this drug responsible for its chemical, biological and medical reactivity.

Selection of hammerhead ribozymes for optimum cleavage of interleukin 6 mRNA.

PubMed Central

Hendrix, C; Anné, J; Joris, B; Van Aerschot, A; Herdewijn, P

1996-01-01

Four GUC triplets in the coding region of the MRNA of interleukin 6 (IL-6) were examined for their suitabilty to serve as a target for hammerhead ribozome-mediated cleavage. This selection procedure was performed with the intention to downregulate IL-6 production as a potential treatment of those diseases in which IL-6 overexpression is involved. Hammerhead ribozymes and their respective short synthetic substrates (19-mers) were synthesized for these four GUC triplets. Notwithstanding the identical catalytic core sequences, the difference in base composition of the helices involved in substrate binding caused substantial variation in cleavage activity. The cleavage reactions on the 1035 nucleotide IL-6 mRNA transcript revealed that two ribozymes were able to cleave this substrate, showing a decrease in catalytic efficiency to 1/30 and 1/300 of the short substrate. This study indicates that the GUC triplet located at nucleotide 510 of the mRNA of IL-6 is the best site for hammerhead ribozyme-mediated cleavage. We suggest that in future targeting of chemically modified hammerhead ribosomes for cleavage of IL-6 RNA should be directed at this location. PMID:8670082

Photoaffinity labeling of the Torpedo californica nicotinic acetylcholine receptor with an aryl azide derivative of phosphatidylserine

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

Blanton, M.P.; Wang, H.H.

1990-02-06

A photoactivatable analogue of phosphatidylserine, {sup 125}I-labeled 4-azidosalicylic acid-phosphatidylserine ({sup 125}I ASA-PS), was used to label both native acetylcholine receptor (AchR)-rich membranes from Torpedo californica and AchR membranes affinity purified from Torpedo reconstituted into asolectin vesicles. The radioiodinated arylazido group attaches directly to the phospholipid head group and thus probes for regions of the AchR structure in contact with the negatively charged head group of phosphatidylserine. All four subunits of the AchR incorporated the label, with the {alpha} subunit incorporating approximately twice as much as each of the other subunits on a per mole basis. The regions of the AchRmore » {alpha} subunit that incorporated {sup 125}I ASA-PS were mapped by Staphylococcus aureus V8 protease digestion. The majority of label incorporated into fragments representing a more complete digestion of the {alpha} subunit was localized to 11.7- and 10.1-kDa V8 cleavage fragments, both beginning at Asn-339 and of sufficient length to contain the hydrophobic region M4. An 18.7-kDa fragment beginning at Ser-173 and of sufficient length to contain the hydrophobic regions M1, M2, and M3 was also significantly labeled. In contrast, V8 cleavage fragments representing roughly a third of the amino-terminal portion of the {alpha} subunit incorporated little or no detectable amount of probe.« less

Biasing genome-editing events toward precise length deletions with an RNA-guided TevCas9 dual nuclease.

PubMed

Wolfs, Jason M; Hamilton, Thomas A; Lant, Jeremy T; Laforet, Marcon; Zhang, Jenny; Salemi, Louisa M; Gloor, Gregory B; Schild-Poulter, Caroline; Edgell, David R

2016-12-27

The CRISPR/Cas9 nuclease is commonly used to make gene knockouts. The blunt DNA ends generated by cleavage can be efficiently ligated by the classical nonhomologous end-joining repair pathway (c-NHEJ), regenerating the target site. This repair creates a cycle of cleavage, ligation, and target site regeneration that persists until sufficient modification of the DNA break by alternative NHEJ prevents further Cas9 cutting, generating a heterogeneous population of insertions and deletions typical of gene knockouts. Here, we develop a strategy to escape this cycle and bias events toward defined length deletions by creating an RNA-guided dual active site nuclease that generates two noncompatible DNA breaks at a target site, effectively deleting the majority of the target site such that it cannot be regenerated. The TevCas9 nuclease, a fusion of the I-TevI nuclease domain to Cas9, functions robustly in HEK293 cells and generates 33- to 36-bp deletions at frequencies up to 40%. Deep sequencing revealed minimal processing of TevCas9 products, consistent with protection of the DNA ends from exonucleolytic degradation and repair by the c-NHEJ pathway. Directed evolution experiments identified I-TevI variants with broadened targeting range, making TevCas9 an easy-to-use reagent. Our results highlight how the sequence-tolerant cleavage properties of the I-TevI homing endonuclease can be harnessed to enhance Cas9 applications, circumventing the cleavage and ligation cycle and biasing genome-editing events toward defined length deletions.

Arm-specific cleavage and mutation during reverse transcription of 2΄,5΄-branched RNA by Moloney murine leukemia virus reverse transcriptase

PubMed Central

Döring, Jessica

2017-01-01

Abstract Branchpoint nucleotides of intron lariats induce pausing of DNA synthesis by reverse transcriptases (RTs), but it is not known yet how they direct RT RNase H activity on branched RNA (bRNA). Here, we report the effects of the two arms of bRNA on branchpoint-directed RNA cleavage and mutation produced by Moloney murine leukemia virus (M-MLV) RT during DNA polymerization. We constructed a long-chained bRNA template by splinted-ligation. The bRNA oligonucleotide is chimeric and contains DNA to identify RNA cleavage products by probe hybridization. Unique sequences surrounding the branchpoint facilitate monitoring of bRNA purification by terminal-restriction fragment length polymorphism analysis. We evaluate the M-MLV RT-generated cleavage and mutational patterns. We find that cleavage of bRNA and misprocessing of the branched nucleotide proceed arm-specifically. Bypass of the branchpoint from the 2΄-arm causes single-mismatch errors, whereas bypass from the 3΄-arm leads to deletion mutations. The non-template arm is cleaved when reverse transcription is primed from the 3΄-arm but not from the 2΄-arm. This suggests that RTs flip ∼180° at branchpoints and RNases H cleave the non-template arm depending on its accessibility. Our observed interplay between M-MLV RT and bRNA would be compatible with a bRNA-mediated control of retroviral and related retrotransposon replication. PMID:28160599

Core protein cleavage by signal peptide peptidase is required for hepatitis C virus-like particle assembly

PubMed Central

Ait-Goughoulte, Malika; Hourioux, Christophe; Patient, Romuald; Trassard, Sylvie; Brand, Denys; Roingeard, Philippe

2006-01-01

SUMMARY Hepatitis C virus (HCV) core protein, expressed with a Semliki forest virus (SFV) replicon, self-assembles into HCV-like particles (HCV-LP) at the endoplasmic reticulum (ER) membrane, providing an opportunity to study HCV assembly and morphogenesis by electron microscopy. We used this model to investigate whether the processing of the HCV core protein by the signal peptide peptidase (SPP) is required for the HCV-LP assembly. We designed several mutants as there are conflicting reports concerning the cleavage of mutant proteins by SPP. Production of the only core mutant protein that escaped SPP processing led to the formation of multiple layers of electron-dense ER membrane, with no evidence of HCV-LP assembly. Our data shed light on the HCV core residues involved in SPP cleavage and suggest that this cleavage is essential for HCV assembly. PMID:16528035

Charged-particle emission tomography

NASA Astrophysics Data System (ADS)

Ding, Yijun

Conventional charged-particle imaging techniques--such as autoradiography-- provide only two-dimensional (2D) images of thin tissue slices. To get volumetric information, images of multiple thin slices are stacked. This process is time consuming and prone to distortions, as registration of 2D images is required. We propose a direct three-dimensional (3D) autoradiography technique, which we call charged-particle emission tomography (CPET). This 3D imaging technique enables imaging of thick sections, thus increasing laboratory throughput and eliminating distortions due to registration. In CPET, molecules or cells of interest are labeled so that they emit charged particles without significant alteration of their biological function. Therefore, by imaging the source of the charged particles, one can gain information about the distribution of the molecules or cells of interest. Two special case of CPET include beta emission tomography (BET) and alpha emission tomography (alphaET), where the charged particles employed are fast electrons and alpha particles, respectively. A crucial component of CPET is the charged-particle detector. Conventional charged-particle detectors are sensitive only to the 2-D positions of the detected particles. We propose a new detector concept, which we call particle-processing detector (PPD). A PPD measures attributes of each detected particle, including location, direction of propagation, and/or the energy deposited in the detector. Reconstruction algorithms for CPET are developed, and reconstruction results from simulated data are presented for both BET and alphaET. The results show that, in addition to position, direction and energy provide valuable information for 3D reconstruction of CPET. Several designs of particle-processing detectors are described. Experimental results for one detector are discussed. With appropriate detector design and careful data analysis, it is possible to measure direction and energy, as well as position of each detected particle. The null functions of CPET with PPDs that measure different combinations of attributes are calculated through singular-value decomposition. In general, the more particle attributes are measured from each detection event, the smaller the null space of CPET is. In other words, the higher dimension the data space is, the more information about an object can be recovered from CPET.

Critical domain interactions for type A RNase P RNA catalysis with and without the specificity domain

PubMed Central

Mao, Guanzhong; Srivastava, Abhishek S.; Wu, Shiying; Kosek, David; Lindell, Magnus

2018-01-01

The natural trans-acting ribozyme RNase P RNA (RPR) is composed of two domains in which the catalytic (C-) domain mediates cleavage of various substrates. The C-domain alone, after removal of the second specificity (S-) domain, catalyzes this reaction as well, albeit with reduced efficiency. Here we provide experimental evidence indicating that efficient cleavage mediated by the Escherichia coli C-domain (Eco CP RPR) with and without the C5 protein likely depends on an interaction referred to as the "P6-mimic". Moreover, the P18 helix connects the C- and S-domains between its loop and the P8 helix in the S-domain (the P8/ P18-interaction). In contrast to the "P6-mimic", the presence of P18 does not contribute to the catalytic performance by the C-domain lacking the S-domain in cleavage of an all ribo model hairpin loop substrate while deletion or disruption of the P8/ P18-interaction in full-size RPR lowers the catalytic efficiency in cleavage of the same model hairpin loop substrate in keeping with previously reported data using precursor tRNAs. Consistent with that P18 is not required for cleavage mediated by the C-domain we show that the archaeal Pyrococcus furiosus RPR C-domain, which lacks the P18 helix, is catalytically active in trans without the S-domain and any protein. Our data also suggest that the S-domain has a larger impact on catalysis for E. coli RPR compared to P. furiosus RPR. Finally, we provide data indicating that the absence of the S-domain and P18, or the P8/ P18-interaction in full-length RPR influences the charge distribution near the cleavage site in the RPR-substrate complex to a small but reproducible extent. PMID:29509761

Proteolytic Processing of Turnip Yellow Mosaic Virus Replication Proteins and Functional Impact on Infectivity▿

PubMed Central

Jakubiec, Anna; Drugeon, Gabrièle; Camborde, Laurent; Jupin, Isabelle

2007-01-01

Turnip yellow mosaic virus (TYMV), a positive-strand RNA virus belonging to the alphavirus-like supergroup, encodes its nonstructural replication proteins as a 206K precursor with domains indicative of methyltransferase (MT), proteinase (PRO), NTPase/helicase (HEL), and polymerase (POL) activities. Subsequent processing of 206K generates a 66K protein encompassing the POL domain and uncharacterized 115K and 85K proteins. Here, we demonstrate that TYMV proteinase mediates an additional cleavage between the PRO and HEL domains of the polyprotein, generating the 115K protein and a 42K protein encompassing the HEL domain that can be detected in plant cells using a specific antiserum. Deletion and substitution mutagenesis experiments and sequence comparisons indicate that the scissile bond is located between residues Ser879 and Gln880. The 85K protein is generated by a host proteinase and is likely to result from nonspecific proteolytic degradation occurring during protein sample extraction or analysis. We also report that TYMV proteinase has the ability to process substrates in trans in vivo. Finally, we examined the processing of the 206K protein containing native, mutated, or shuffled cleavage sites and analyzed the effects of cleavage mutations on viral infectivity and RNA synthesis by performing reverse-genetics experiments. We present evidence that PRO/HEL cleavage is critical for productive virus infection and that the impaired infectivity of PRO/HEL cleavage mutants is due mainly to defective synthesis of positive-strand RNA. PMID:17686855

Observations of cleavage steps, slip traces and dislocation hollow cores on cleaved ?100? faces of ?-arginine phosphate monohydrate single crystals by atomic force microscopy

NASA Astrophysics Data System (ADS)

Sangwal, K.; Torrent-Burgués, J.; Sanz, F.; Servat, J.

1997-03-01

The results of an atomic force microscopy study of the nature of cleavage steps, observation of slip traces and formation of hollow cores at the centres of dislocations on the {100} faces of L-arginine phosphate monohydrate (LAP) single crystals grown from aqueous solutions are described and discussed. It was observed that: (1) most of the cleavage steps and all the slip traces are of elementary height, a = 1.085 nm; (2) the origin of a cleavage step may or may not have a hollow core; and (3) close to its origin, the curvature of a cleavage step may be positive or negative or may change from positive to negative. The results suggest that slip traces observed on the cleaved surfaces of LAP are formed during the cleavage process while the rounding and the rearrangement of elementary cleavage steps take place immediately after the occurrence of cleavage. Analysis of the results also shows that the dislocations responsible for the origin of hollow cores always represent a stress field state corresponding to a trapped solution of different local interface supersaturations.

Stabilization of Clostridium perfringens collagenase mRNA by VR-RNA-dependent cleavage in 5' leader sequence.

PubMed

Obana, Nozomu; Shirahama, Yu; Abe, Kimihiro; Nakamura, Kouji

2010-09-01

The small RNA (sRNA), VR-RNA that is directly regulated by the VirR/VirS two-component system, regulates many genes including toxin genes such as collagenase (colA) and phospholipase C (plc) in Clostridium perfringens. Although the VR-RNA 3' region is sufficient to regulate the colA and plc genes, the molecular mechanism of toxin gene regulation by VR-RNA remains unclear. Here, we found that colA mRNA is cleaved at position -79 and -78 from the A of the first codon (ATG) in the presence of VR-RNA. The processed transcripts were stable compared with longer intact transcripts. On the other hand, colA mRNA was labile in a VR-RNA-deficient strain, and processed transcripts were undetectable. The stability and processing of colA mRNA were restored by transformation of the 3' region of VR-RNA-expression vector. The 3' region of VR-RNA and colA mRNA had significant complementation and interacted in vitro. These results show that VR-RNA base pairs with colA mRNA and induces cleavage in the 5' untranslated region (UTR) of colA mRNA, which leads to the stabilization of colA mRNA and the activation of colA expression. © 2010 Blackwell Publishing Ltd.

Deep Sequencing Insights in Therapeutic shRNA Processing and siRNA Target Cleavage Precision.

PubMed

Denise, Hubert; Moschos, Sterghios A; Sidders, Benjamin; Burden, Frances; Perkins, Hannah; Carter, Nikki; Stroud, Tim; Kennedy, Michael; Fancy, Sally-Ann; Lapthorn, Cris; Lavender, Helen; Kinloch, Ross; Suhy, David; Corbau, Romu

2014-02-04

TT-034 (PF-05095808) is a recombinant adeno-associated virus serotype 8 (AAV8) agent expressing three short hairpin RNA (shRNA) pro-drugs that target the hepatitis C virus (HCV) RNA genome. The cytosolic enzyme Dicer cleaves each shRNA into multiple, potentially active small interfering RNA (siRNA) drugs. Using next-generation sequencing (NGS) to identify and characterize active shRNAs maturation products, we observed that each TT-034-encoded shRNA could be processed into as many as 95 separate siRNA strands. Few of these appeared active as determined by Sanger 5' RNA Ligase-Mediated Rapid Amplification of cDNA Ends (5-RACE) and through synthetic shRNA and siRNA analogue studies. Moreover, NGS scrutiny applied on 5-RACE products (RACE-seq) suggested that synthetic siRNAs could direct cleavage in not one, but up to five separate positions on targeted RNA, in a sequence-dependent manner. These data support an on-target mechanism of action for TT-034 without cytotoxicity and question the accepted precision of substrate processing by the key RNA interference (RNAi) enzymes Dicer and siRNA-induced silencing complex (siRISC).Molecular Therapy-Nucleic Acids (2014) 3, e145; doi:10.1038/mtna.2013.73; published online 4 February 2014.

Mechanistic Insights into Ring Cleavage and Contraction of Benzene over a Titanium Hydride Cluster.

PubMed

Kang, Xiaohui; Luo, Gen; Luo, Lun; Hu, Shaowei; Luo, Yi; Hou, Zhaomin

2016-09-14

Carbon-carbon bond cleavage of benzene by transition metals is of great fundamental interest and practical importance, as this transformation is involved in the production of fuels and other important chemicals in the industrial hydrocracking of naphtha on solid catalysts. Although this transformation is thought to rely on cooperation of multiple metal sites, molecular-level information on the reaction mechanism has remained scarce to date. Here, we report the DFT studies of the ring cleavage and contraction of benzene by a molecular trinuclear titanium hydride cluster. Our studies suggest that the reaction is initiated by benzene coordination, followed by H2 release, C6H6 hydrometalation, repeated C-C and C-H bond cleavage and formation to give a MeC5H4 unit, and insertion of a Ti atom into the MeC5H4 unit with release of H2 to give a metallacycle product. The C-C bond cleavage and ring contraction of toluene can also occur in a similar fashion, though some details are different due to the presence of the methyl substituent. Obviously, the facile release of H2 from the metal hydride cluster to provide electrons and to alter the charge population at the metal centers, in combination with the flexible metal-hydride connections and dynamic redox behavior of the trimetallic framework, has enabled this unusual transformation to occur. This work has not only provided unprecedented insights into the activation and transformation of benzene over a multimetallic framework but it may also offer help in the design of new molecular catalysts for the activation and transformation of inactive aromatics.

Multiphoton near-infrared femtosecond laser pulse-induced DNA damage with and without the photosensitizer proflavine.

PubMed

Shafirovich, V; Dourandin, A; Luneva, N P; Singh, C; Kirigin, F; Geacintov, N E

1999-03-01

The excitation of pBr322 supercoiled plasmid DNA with intense near-IR 810 nm fs laser pulses by a simultaneous multiphoton absorption mechanism results in single-strand breaks after treatment of the irradiated samples with Micrococcus luteus UV endonuclease. This enzyme cleaves DNA strands at sites of cyclobutane dimers that are formed by the simultaneous absorption of three (or more) 810 nm IR photons (pulse width approximately 140 fs, 76 MHz pulse repetition, average power output focused through 10x microscope objective is approximately 1.2 MW/cm2). Direct single-strand breaks (without treatment with M. luteus) were not observed under these conditions. However, in the presence of 6 microM of the intercalator proflavine (PF), both direct single- and double-strand breaks are observed under conditions where substantial fractions of undamaged supercoiled DNA molecules are still present. The fraction of direct double-strand breaks is 30 +/- 5% of all measurable strand cleavage events, is independent of dosage (up to 6.4 GJ/cm2) and is proportional to In, where I is the average power/area of the 810 nm fs laser pulses, and n = 3 +/- 1. The nicking of two DNA strands in the immediate vicinity of the excited PF molecules gives rise to this double-strand cleavage. In contrast, excitation of the same samples under low-power, single-photon absorption conditions (approximately 400-500 nm) gives rise predominantly to single-strand breaks, but some double-strand breaks are observed at the higher dosages. Thus, single-photon excitation with 400-500 nm light and multiphoton activation of PF by near-IR fs laser pulses produces different distributions of single- and double-strand breaks. These results suggest that DNA strand cleavage originates from unrelaxed, higher excited states when PF is excited by simultaneous IR multiphoton absorption processes.

SKI2 mediates degradation of RISC 5′-cleavage fragments and prevents secondary siRNA production from miRNA targets in Arabidopsis

PubMed Central

Branscheid, Anja; Marchais, Antonin; Schott, Gregory; Lange, Heike; Gagliardi, Dominique; Andersen, Stig Uggerhøj; Voinnet, Olivier; Brodersen, Peter

2015-01-01

Small regulatory RNAs are fundamental in eukaryotic and prokaryotic gene regulation. In plants, an important element of post-transcriptional control is effected by 20–24 nt microRNAs (miRNAs) and short interfering RNAs (siRNAs) bound to the ARGONAUTE1 (AGO1) protein in an RNA induced silencing complex (RISC). AGO1 may cleave target mRNAs with small RNA complementarity, but the fate of the resulting cleavage fragments remains incompletely understood. Here, we show that SKI2, SKI3 and SKI8, subunits of a cytoplasmic cofactor of the RNA exosome, are required for degradation of RISC 5′, but not 3′-cleavage fragments in Arabidopsis. In the absence of SKI2 activity, many miRNA targets produce siRNAs via the RNA-dependent RNA polymerase 6 (RDR6) pathway. These siRNAs are low-abundant, and map close to the cleavage site. In most cases, siRNAs were produced 5′ to the cleavage site, but several examples of 3′-spreading were also identified. These observations suggest that siRNAs do not simply derive from RDR6 action on stable 5′-cleavage fragments and hence that SKI2 has a direct role in limiting secondary siRNA production in addition to its function in mediating degradation of 5′-cleavage fragments. PMID:26464441

Crack stability and branching at interfaces

NASA Astrophysics Data System (ADS)

Thomson, Robb

1995-11-01

The various events that occur at a crack on an interface are explored, and described in terms of a simple graphical construction called the crack stability diagram. For simple Griffith cleavage in a homogeneous material, the stability diagram is a sector of a circle in the space of stress intensity factors, KI/KII. The Griffith circle is limited in both positive and negative KII directions by nonblunting dislocation emission on the cleavage plane. For a branching plane inclined at an angle to the original cleavage plane, both cleavage and emission (which blunts the crack) can be described as a balance between an elastic driving force and a lattice resistance for the event. We use an analytic expression obtained by Cotterell and Rice for cleavage, and show that it is an excellent approximation, but show that the lattice resistance includes a cornering resistance, in addition to the standard surface energy in the final cleavage criterion. Our discussion of the lattaice resistance is derived from simulations in two-dimensional hexagonal lattices with UBER force laws with a variety of shapes. Both branching cleavage and blunting emission can be described in terms of a stability diagram in the space of the remote stress intensity factors, and the competition between events on the initial cleavage plane and those on the branching plane can be described by overlays of the two appropriate stability diagrams. The popular criterion that kII=0 on the branching plane is explored for lattices and found to fail significantly, because the lattice stabilizes cleavage by the anisotropy of the surface energy. Also, in the lattice, dislocation emission must must always be considered as an alternative competing event to branching.

Enterovirus 71 Inhibits Pyroptosis through Cleavage of Gasdermin D

PubMed Central

Lei, Xiaobo; Zhang, Zhenzhen; Xiao, Xia; Qi, Jianli

2017-01-01

ABSTRACT Enterovirus 71 (EV71) can cause hand-foot-and-mouth disease (HFMD) in young children. Severe infection with EV71 can lead to neurological complications and even death. However, the molecular basis of viral pathogenesis remains poorly understood. Here, we report that EV71 induces degradation of gasdermin D (GSDMD), an essential component of pyroptosis. Remarkably, the viral protease 3C directly targets GSDMD and induces its cleavage, which is dependent on the protease activity. Further analyses show that the Q193-G194 pair within GSDMD is the cleavage site of 3C. This cleavage produces a shorter N-terminal fragment spanning amino acids 1 to 193 (GSDMD1–193). However, unlike the N-terminal fragment produced by caspase-1 cleavage, this fragment fails to trigger cell death or inhibit EV71 replication. Importantly, a T239D or F240D substitution abrogates the activity of GSDMD consisting of amino acids 1 to 275 (GSDMD1–275). This is correlated with the lack of pyroptosis or inhibition of viral replication. These results reveal a previously unrecognized strategy for EV71 to evade the antiviral response. IMPORTANCE Recently, it has been reported that GSDMD plays a critical role in regulating lipopolysaccharide and NLRP3-mediated interleukin-1β (IL-1β) secretion. In this process, the N-terminal domain of p30 released from GSDMD acts as an effector in cell pyroptosis. We show that EV71 infection downregulates GSDMD. EV71 3C cleaves GSDMD at the Q193-G194 pair, resulting in a truncated N-terminal fragment disrupted for inducing cell pyroptosis. Notably, GSDMD1–275 (p30) inhibits EV71 replication whereas GSDMD1–193 does not. These results reveal a new strategy for EV71 to evade the antiviral response. PMID:28679757

Enterovirus 71 Inhibits Pyroptosis through Cleavage of Gasdermin D.

PubMed

Lei, Xiaobo; Zhang, Zhenzhen; Xiao, Xia; Qi, Jianli; He, Bin; Wang, Jianwei

2017-09-15

Enterovirus 71 (EV71) can cause hand-foot-and-mouth disease (HFMD) in young children. Severe infection with EV71 can lead to neurological complications and even death. However, the molecular basis of viral pathogenesis remains poorly understood. Here, we report that EV71 induces degradation of gasdermin D (GSDMD), an essential component of pyroptosis. Remarkably, the viral protease 3C directly targets GSDMD and induces its cleavage, which is dependent on the protease activity. Further analyses show that the Q193-G194 pair within GSDMD is the cleavage site of 3C. This cleavage produces a shorter N-terminal fragment spanning amino acids 1 to 193 (GSDMD 1-193 ). However, unlike the N-terminal fragment produced by caspase-1 cleavage, this fragment fails to trigger cell death or inhibit EV71 replication. Importantly, a T239D or F240D substitution abrogates the activity of GSDMD consisting of amino acids 1 to 275 (GSDMD 1-275 ). This is correlated with the lack of pyroptosis or inhibition of viral replication. These results reveal a previously unrecognized strategy for EV71 to evade the antiviral response. IMPORTANCE Recently, it has been reported that GSDMD plays a critical role in regulating lipopolysaccharide and NLRP3-mediated interleukin-1β (IL-1β) secretion. In this process, the N-terminal domain of p30 released from GSDMD acts as an effector in cell pyroptosis. We show that EV71 infection downregulates GSDMD. EV71 3C cleaves GSDMD at the Q193-G194 pair, resulting in a truncated N-terminal fragment disrupted for inducing cell pyroptosis. Notably, GSDMD 1-275 (p30) inhibits EV71 replication whereas GSDMD 1-193 does not. These results reveal a new strategy for EV71 to evade the antiviral response. Copyright © 2017 American Society for Microbiology.

Cation Recombination Energy/Coulomb Repulsion Effects in ETD/ECD as Revealed by Variation of Charge per Residue at Fixed Total Charge

PubMed Central

Mentinova, Marija; Crizer, David M.; Baba, Takashi; McGee, William M.; Glish, Gary L.; McLuckey, Scott A.

2013-01-01

Electron capture dissociation (ECD) and electron transfer dissociation (ETD) experiments in electrodynamic ion traps operated in the presence of a bath gas in the 1–10 mTorr range have been conducted on a common set of doubly protonated model peptides of the form X(AG)nX (X = lysine, arginine, or histidine, n=1, 2, or 4). The partitioning of reaction products was measured using thermal electrons, anions of azobenzene, and anions of 1,3-dinitrobenzene as reagents. Variation of n alters the charge per residue of the peptide cation, which affects recombination energy. The ECD experiments showed that H-atom loss is greatest for the n=1 peptides and decreases as n increases. Proton transfer in ETD, on the other hand, is expected to increase as charge per residue decreases (i.e., as n increases). These opposing tendencies were apparent in the data for the K(AG)nK peptides. H-atom loss appeared to be more prevalent in ECD than in ETD and is rationalized on the basis of either internal energy differences, differences in angular momentum transfer associated with the electron capture versus electron transfer processes, or a combination of the two. The histidine peptides showed the greatest extent of charge reduction without dissociation, the arginine peptides showed the greatest extent of side-chain cleavages, and the lysine peptides generally showed the greatest extent of partitioning into the c/z•-product ion channels. The fragmentation patterns for the complementary c- and z•-ions for ETD and ECD were found to be remarkably similar, particularly for the peptides with X = lysine. PMID:23568028

Mechanisms for ribotoxin-induced ribosomal RNA cleavage

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

He, Kaiyu; Center for Integrative Toxicology, Michigan State University, East Lansing, MI 48824; Zhou, Hui-Ren

The Type B trichothecene deoxynivalenol (DON), a ribotoxic mycotoxin known to contaminate cereal-based foods, induces ribosomal RNA (rRNA) cleavage in the macrophage via p38-directed activation of caspases. Here we employed the RAW 264.7 murine macrophage model to test the hypothesis that this rRNA cleavage pathway is similarly induced by other ribotoxins. Capillary electrophoresis confirmed that the antibiotic anisomycin (≥ 25 ng/ml), the macrocylic trichothecene satratoxin G (SG) (≥ 10 ng/ml) and ribosome-inactivating protein ricin (≥ 300 ng/ml) induced 18s and 28s rRNA fragmentation patterns identical to that observed for DON. Also, as found for DON, inhibition of p38, double-stranded RNA-activatedmore » kinase (PKR) and hematopoietic cell kinase (Hck) suppressed MAPK anisomycin-induced rRNA cleavage, while, in contrast, their inhibition did not affect SG- and ricin-induced rRNA fragmentation. The p53 inhibitor pifithrin-μ and pan caspase inhibitor Z-VAD-FMK suppressed rRNA cleavage induced by anisomycin, SG and ricin, indicating that these ribotoxins shared with DON a conserved downstream pathway. Activation of caspases 8, 9 and 3 concurrently with apoptosis further suggested that rRNA cleavage occurred in parallel with both extrinsic and intrinsic pathways of programmed cell death. When specific inhibitors of cathepsins L and B (lysosomal cysteine cathepsins active at cytosolic neutral pH) were tested, only the former impaired anisomycin-, SG-, ricin- and DON-induced rRNA cleavage. Taken together, the data suggest that (1) all four ribotoxins induced p53-dependent rRNA cleavage via activation of cathepsin L and caspase 3, and (2) activation of p53 by DON and anisomycin involved p38 whereas SG and ricin activated p53 by an alternative mechanism. Highlights: ► Deoxynivalenol (DON) anisomycin, satratoxin G (SG) and ricin are ribotoxins. ► Ribotoxins induce 18s and 28s rRNA cleavage in the RAW 264.7 macrophage model. ► Ribotoxins induce rRNA cleavage via activation of p53, caspases and cathepsins. ► DON- and anisomycin-triggered rRNA cleavage is p38-dependent. ► SG- and ricin-induced rRNA cleavage is p38-independent.« less

Photo-catalytic Activities of Plant Hormones on Semiconductor Nanoparticles by Laser-Activated Electron Tunneling and Emitting

PubMed Central

Tang, Xuemei; Huang, Lulu; Zhang, Wenyang; Jiang, Ruowei; Zhong, Hongying

2015-01-01

Understanding of the dynamic process of laser-induced ultrafast electron tunneling is still very limited. It has been thought that the photo-catalytic reaction of adsorbents on the surface is either dependent on the number of resultant electron-hole pairs where excess energy is lost to the lattice through coupling with phonon modes, or dependent on irradiation photon wavelength. We used UV (355 nm) laser pulses to excite electrons from the valence band to the conduction band of titanium dioxide (TiO2), zinc oxide (ZnO) and bismuth cobalt zinc oxide (Bi2O3)0.07(CoO)0.03(ZnO)0.9 semiconductor nanoparticles with different photo catalytic properties. Photoelectrons are extracted, accelerated in a static electric field and eventually captured by charge deficient atoms of adsorbed organic molecules. A time-of-flight mass spectrometer was used to detect negative molecules and fragment ions generated by un-paired electron directed bond cleavages. We show that the probability of electron tunneling is determined by the strength of the static electric field and intrinsic electron mobility of semiconductors. Photo-catalytic dissociation or polymerization reactions of adsorbents are highly dependent on the kinetic energy of tunneling electrons as well as the strength of laser influx. By using this approach, photo-activities of phytohormones have been investigated. PMID:25749635

Photo-catalytic Activities of Plant Hormones on Semiconductor Nanoparticles by Laser-Activated Electron Tunneling and Emitting

NASA Astrophysics Data System (ADS)

Tang, Xuemei; Huang, Lulu; Zhang, Wenyang; Jiang, Ruowei; Zhong, Hongying

2015-03-01

Understanding of the dynamic process of laser-induced ultrafast electron tunneling is still very limited. It has been thought that the photo-catalytic reaction of adsorbents on the surface is either dependent on the number of resultant electron-hole pairs where excess energy is lost to the lattice through coupling with phonon modes, or dependent on irradiation photon wavelength. We used UV (355 nm) laser pulses to excite electrons from the valence band to the conduction band of titanium dioxide (TiO2), zinc oxide (ZnO) and bismuth cobalt zinc oxide (Bi2O3)0.07(CoO)0.03(ZnO)0.9 semiconductor nanoparticles with different photo catalytic properties. Photoelectrons are extracted, accelerated in a static electric field and eventually captured by charge deficient atoms of adsorbed organic molecules. A time-of-flight mass spectrometer was used to detect negative molecules and fragment ions generated by un-paired electron directed bond cleavages. We show that the probability of electron tunneling is determined by the strength of the static electric field and intrinsic electron mobility of semiconductors. Photo-catalytic dissociation or polymerization reactions of adsorbents are highly dependent on the kinetic energy of tunneling electrons as well as the strength of laser influx. By using this approach, photo-activities of phytohormones have been investigated.

Hemoglobin Cleavage Site-Specificity of the Plasmodium falciparum Cysteine Proteases Falcipain-2 and Falcipain-3

PubMed Central

Subramanian, Shoba; Hardt, Markus; Choe, Youngchool; Niles, Richard K.; Johansen, Eric B.; Legac, Jennifer; Gut, Jiri; Kerr, Iain D.; Craik, Charles S.; Rosenthal, Philip J.

2009-01-01

The Plasmodium falciparum cysteine proteases falcipain-2 and falcipain-3 degrade host hemoglobin to provide free amino acids for parasite protein synthesis. Hemoglobin hydrolysis has been described as an ordered process initiated by aspartic proteases, but cysteine protease inhibitors completely block the process, suggesting that cysteine proteases can also initiate hemoglobin hydrolysis. To characterize the specific roles of falcipains, we used three approaches. First, using random P1 – P4 amino acid substrate libraries, falcipain-2 and falcipain-3 demonstrated strong preference for cleavage sites with Leu at the P2 position. Second, with overlapping peptides spanning α and β globin and proteolysis-dependent 18O labeling, hydrolysis was seen at many cleavage sites. Third, with intact hemoglobin, numerous cleavage products were identified. Our results suggest that hemoglobin hydrolysis by malaria parasites is not a highly ordered process, but rather proceeds with rapid cleavage by falcipains at multiple sites. However, falcipain-2 and falcipain-3 show strong specificity for P2 Leu in small peptide substrates, in agreement with the specificity in optimized small molecule inhibitors that was identified previously. These results are consistent with a principal role of falcipain-2 and falcipain-3 in the hydrolysis of hemoglobin by P. falciparum and with the possibility of developing small molecule inhibitors with optimized specificity as antimalarial agents. PMID:19357776

Depletion zones and crystallography on pinched spheres

NASA Astrophysics Data System (ADS)

Chen, Jingyuan; Xing, Xiangjun; Yao, Zhenwei

2018-03-01

Understanding the interplay between ordered structures and substrate curvature is an interesting problem with versatile applications, including functionalization of charged supramolecular surfaces and modern microfluidic technologies. In this work, we investigate the two-dimensional packing structures of charged particles confined on a pinched sphere. By continuously pinching the sphere, we observe cleavage of elongated scars into pleats, proliferation of disclinations, and subsequently, emergence of a depletion zone at the negatively curved waist that is completely void of particles. We systematically study the geometrics and energetics of the depletion zone, and reveal its physical origin as a finite size effect, due to the interplay between Coulomb repulsion and concave geometry of the pinched sphere. These results further our understanding of crystallography on curved surfaces, and have implications in design and manipulation of charged, deformable interfaces in various applications.

The Adam family metalloprotease Kuzbanian regulates the cleavage of the roundabout receptor to control axon repulsion at the midline

PubMed Central

Coleman, Hope A.; Labrador, Juan-Pablo; Chance, Rebecca K.; Bashaw, Greg J.

2010-01-01

Slits and their Roundabout (Robo) receptors mediate repulsive axon guidance at the Drosophila ventral midline and in the vertebrate spinal cord. Slit is cleaved to produce fragments with distinct signaling properties. In a screen for genes involved in Slit-Robo repulsion, we have identified the Adam family metalloprotease Kuzbanian (Kuz). Kuz does not regulate midline repulsion through cleavage of Slit, nor is Slit cleavage essential for repulsion. Instead, Kuz acts in neurons to regulate repulsion and Kuz can cleave the Robo extracellular domain in Drosophila cells. Genetic rescue experiments using an uncleavable form of Robo show that this receptor does not maintain normal repellent activity. Finally, Kuz activity is required for Robo to recruit its downstream signaling partner, Son of sevenless (Sos). These observations support the model that Kuz-directed cleavage is important for Robo receptor activation. PMID:20570941

Prenatal Organophosphates Exposure Alternates the Cleavage Plane Orientation of Apical Neural Progenitor in Developing Neocortex

PubMed Central

Chen, Xiao-Ping; Chen, Wei-Feng; Wang, Da-Wei

2014-01-01

Prenatal organophosphate exposure elicits long-term brain cytoarchitecture and cognitive function impairments, but the mechanism underlying the onset and development of neural progenitors remain largely unclear. Using precise positioned brain slices, we observed an alternated cleavage plane bias that emerged in the mitotic neural progenitors of embryonal neocortex with diazinion (DZN) and chlorpyrifos (CPF) pretreatment. In comparison with the control, DZN and CPF treatment induced decrease of vertical orientation, increase of oblique orientation, and increase of horizontal orientation. That is, the cleavage plane orientation bias had been rotated from vertical to horizontal after DZN and CPF treatment. Meanwhile, general morphology and mitotic index of the progenitors were unchanged. Acephate (ACP), another common organophosphate, had no significant effects on the cleavage plane orientation, cell morphology and mitotic index. These results represent direct evidence for the toxicity mechanism in onset multiplication of neural progenitors. PMID:24740262

The Influence of Hydrogen on the Evolving Microstructure During Fatigue Crack Growth in Metastable and Stable Austenitic Stainless Steels

NASA Astrophysics Data System (ADS)

Nygren, Kelly Elizabeth

The effect of high levels of internal hydrogen on the microstructure evolving during stage II fatigue crack growth was investigated through a series of tensile and fatigue studies in metastable (304) and stable (316, 316L) stainless steels. The first, a tensile study in 304 stainless steel, identified the underlying microstructure which resulted in the flat and quasi-cleavage features on the fracture surface of a hydrogen-charged tensile bar. The second study utilized single-edge notched tensile specimens loaded in fatigue, and compared the evolving microstructure ahead of a fatigue crack for cases of an uncharged, 10 wppm hydrogen-charged, and 104 wppm hydrogen-charged 304 and 316L alloy. The final fatigue study, a small fatigue crack growth study in round bars of 304 and 316, provided a contextual comparison of microstructures to previous results in literature. In the metastable 304 stainless steel, hydrogen is found to change the nature of the martensitic transformation and subsequent fracture path. This transformation is attributed to enhanced plasticity and hydrogen-dislocation interactions stabilizing the austenitic matrix and confining slip to particular close-packed planes. The martensite acts as a fast diffusion pathway for hydrogen, leading to final fracture along martensitic laths or cleavage planes. In 316L, the material deforms via slip and twinning and hydrogen does not induce a change in deformation mechanisms. Instead, the enhanced plasticity and hydrogen-dislocation interactions increase the degree of plasticity, leading to smaller dislocation cell sizes with thicker walls before the onset of twinning. The crack interacts with a heavily twinned structure superimposed on dislocation cells, resulting in a curvature of one twin-variant in the direction of crack growth and the formation of a refined region at the fracture surface. These structures are localized to the crack surface and limited in rotation in the presence of hydrogen. The presence of a hydrogen content above the solution-limit led to a strengthening effect over all other samples in the round bar samples for both 304 and 316. In the SENT studies, 304 stainless steel charged to these levels showed the greatest degree of hydrogen embrittlement, while in 316L stainless steel, the samples containing these levels of hydrogen were found to be stronger in fatigue life than the 10 wppm hydrogen-charged sample. Both of the 316L hydrogen-charged samples were weaker than their uncharged counterpart. Differences in the outcomes for the two studies was attributed to the geometry and testing condition employed. When strengthening was observed, the role of hydrogen was resolved to be two-fold: hydrogen hardens the matrix in the lattice, suppressing the process zone at the crack; and hydrogen at the dislocations enhances the plasticity experienced in the region where the material is yielding ahead of crack advance in the strain lobe. Irrespective of material class, increasing stress-state on the crack within the same stage of fatigue life leads to an increase in depth of the refinement layer and level of plasticity. The refinement at the surface can be attributed to the dislocation emission created during crack advance and the deformation induced in the crack wake. The former is the primary contributor to the SENT specimen studies, while the latter is responsible for the differences observed in the round-bar studies. In 316L, when the twin structure was observed to extend to the surface, regions of light and dark contrast were observed between the twinning bundle variants which corresponded to the peaks and troughs of the striations. This observation is counter to the current understanding that striations form during the crack advance process, independently of the evolved microstructure. The resulting microstructures in all hydrogen-induced studies are discussed in terms of the impact of hydrogen-dislocation interactions and enhanced plasticity. The evolving microstructure and unique observation of striations conforming to the microstructure developed ahead of crack advance emphasize the need to revisit how fatigue models are approached. Although a challenge, models must be informed by plasticity based arguments incorporating the deformation mechanisms observed in experiments to accurately reflect fatigue crack growth.

Process of pigment cell specification in the sand dollar, Scaphechinus mirabilis.

PubMed

Kominami, Tetsuya; Takata, Hiromi

2002-04-01

The process of pigment cell specification in the sand dollar Scaphechinus mirabilis was examined by manipulative methods. In half embryos, which were formed by dissociating embryos at the 2-cell stage, the number of pigment cells was significantly greater than half the number of pigment cells observed in control embryos. This relative increase might have been brought about by the change in the arrangement of blastomeres surrounding the micromere progeny. To examine whether such an increase could be induced at a later stage, embryos were bisected with a glass needle. When embryos were bisected before 7 h postfertilization, the sum of pigment cells observed in a pair of embryo fragments was greater than that in control embryos. This relative increase was not seen when embryos were bisected after 7 h postfertilization. From the size of blastomeres, it became clear that the 9th cleavage was completed by 7 h postfertilization. Aphidicolin treatment revealed that 10-15 pigment founder cells were formed. The results obtained suggest that the pigment founder cells were specified through direct cell contact with micromere progeny after the 9th cleavage, and that most of the founder cells had divided three times before they differentiated into pigment cells.

Covalent bond force profile and cleavage in a single polymer chain

NASA Astrophysics Data System (ADS)

Garnier, Lionel; Gauthier-Manuel, Bernard; van der Vegte, Eric W.; Snijders, Jaap; Hadziioannou, Georges

2000-08-01

We present here the measurement of the single-polymer entropic elasticity and the single covalent bond force profile, probed with two types of atomic force microscopes (AFM) on a synthetic polymer molecule: polymethacrylic acid in water. The conventional AFM allowed us to distinguish two types of interactions present in this system when doing force spectroscopic measurements: the first interaction is associated with adsorption sites of the polymer chains onto a bare gold surface, the second interaction is directly correlated to the rupture process of a single covalent bond. All these bridging interactions allowed us to stretch the single polymer chain and to determine the various factors playing a role in the elasticity of these molecules. To obtain a closer insight into the bond rupture process, we moved to a force sensor stable in position when measuring attractive forces. By optimizing the polymer length so as to fulfill the elastic stability conditions, we were able for the first time to map out the entire force profile associated with the cleavage of a single covalent bond. Experimental data coupled with molecular quantum mechanical calculations strongly suggest that the breaking bond is located at one end of the polymer chain.

The p2 domain of human immunodeficiency virus type 1 Gag regulates sequential proteolytic processing and is required to produce fully infectious virions.

PubMed

Pettit, S C; Moody, M D; Wehbie, R S; Kaplan, A H; Nantermet, P V; Klein, C A; Swanstrom, R

1994-12-01

The proteolytic processing sites of the human immunodeficiency virus type 1 (HIV-1) Gag precursor are cleaved in a sequential manner by the viral protease. We investigated the factors that regulate sequential processing. When full-length Gag protein was digested with recombinant HIV-1 protease in vitro, four of the five major processing sites in Gag were cleaved at rates that differ by as much as 400-fold. Three of these four processing sites were cleaved independently of the others. The CA/p2 site, however, was cleaved approximately 20-fold faster when the adjacent downstream p2/NC site was blocked from cleavage or when the p2 domain of Gag was deleted. These results suggest that the presence of a C-terminal p2 tail on processing intermediates slows cleavage at the upstream CA/p2 site. We also found that lower pH selectively accelerated cleavage of the CA/p2 processing site in the full-length precursor and as a peptide primarily by a sequence-based mechanism rather than by a change in protein conformation. Deletion of the p2 domain of Gag results in released virions that are less infectious despite the presence of the processed final products of Gag. These findings suggest that the p2 domain of HIV-1 Gag regulates the rate of cleavage at the CA/p2 processing site during sequential processing in vitro and in infected cells and that p2 may function in the proper assembly of virions.

The contamination of Lake Superior with amphibole gangue minerals.

PubMed

Langer, A M; Maggiore, C M; Nicholson, W J; Rohl, A N; Rubin, I B; Selikoff, I J

1979-01-01

Iron ore called taconite is mined in the Biwabik Iron Formation in the Eastern Mesabi region of the Mesabi Range, in eastern Minnesota. After mining, ore is shipped to Silver Bay, Minnnesota for processing and wet magnetic extraction. Tailings from the process are dumped, as a slurry, into a man-made containment delta constructed in Lake Superior. Submicroscopic amphibole fibers and/or cleavage fragments, a component of the gangue, apparently escape from the delta at Silver Bay, and enter Lake Superior. These particles contaiminate the potable water supplies of municipalities drawing directly from the lake. One of the gangue minerals is the amphibole grunerite, whose asbestiform variety is called amosite. Major emphasis of this study was directed at identification of submicroscopic particle pollutants, based on morphology, structure and chemical composition. Quantitative determination of fibrous amphibole phases, present in a range of water samples, was undertaken. Transmission electron microscopy, selected area electron diffraction, and an electron microprobe technique was used for identification and enumeration and this information was compared with data sets determined from standards. Grunerite fiber and/or acicular cleavage fragments, in some instances indistinguishable from asbestiform grunerite, are present in the tailings, lake water and drinking water of a number of municipalities, a result of contamination of the lake at the Silver Bay milling operation. This amphibole is found in drinking water in concentrations which range from 0.6 to 2.8 X 10(6) fiber/liter. The risk to health, associated with direct ingestion of grunerite fiber is unknown and is extrapolated from the asbestiform grunerite (amosite) data base. The biological activity of other fibrous amphiboles observed, unrelated to any asbestiform silicate variety, is presently unknown and warrants investigation.

Ab initio molecular dynamics simulations of low energy recoil events in MgO

NASA Astrophysics Data System (ADS)

Petersen, B. A.; Liu, B.; Weber, W. J.; Zhang, Y.

2017-04-01

Low-energy recoil events in MgO are studied using ab intio molecular dynamics simulations to reveal the dynamic displacement processes and final defect configurations. Threshold displacement energies, Ed, are obtained for Mg and O along three low-index crystallographic directions, [100], [110], and [111]. The minimum values for Ed are found along the [110] direction consisting of the same element, either Mg or O atoms. Minimum threshold values of 29.5 eV for Mg and 25.5 eV for O, respectively, are suggested from the calculations. For other directions, the threshold energies are considerably higher, 65.5 and 150.0 eV for O along [111] and [100], and 122.5 eV for Mg along both [111] and [100] directions, respectively. These results show that the recoil events in MgO are partial-charge transfer assisted processes where the charge transfer plays an important role. There is a similar trend found in other oxide materials, where the threshold displacement energy correlates linearly with the peak partial-charge transfer, suggesting this behavior might be universal in ceramic oxides.

Higher-order looping and nuclear organization of antigen receptor loci facilitate targeted RAG cleavage and regulated rearrangement in recombination centers

PubMed Central

Chaumeil, Julie; Micsinai, Mariann; Ntziachristos, Panagiotis; Deriano, Ludovic; Wang, Joy M-H; Ji, Yanhong; Nora, Elphege P.; Rodesch, Matthew J.; Jeddeloh, Jeffrey A.; Aifantis, Iannis; Kluger, Yuval; Schatz, David G.; Skok, Jane A.

2013-01-01

SUMMARY V(D)J recombination is essential for generating a diverse array of B and T cell receptors that can recognize and combat foreign antigen. As with any recombination event, tight control is essential to prevent the occurrence of genetic anomalies that drive cellular transformation. One important aspect of regulation is directed targeting of the RAG recombinase. Indeed, RAG accumulates at the 3’ end of individual antigen receptor loci poised for rearrangement, however, it is not known whether focal binding is involved in regulating cleavage, and what mechanisms lead to enrichment of RAG in this region. Here we show that mono-allelic looping out of the 3’ end of Tcra, coupled with transcription and increased chromatin/nuclear accessibility, are linked to focal RAG binding and ATM-mediated regulated mono-allelic cleavage on looped out 3’ regions. Our data identify higher order loop formation as a key determinant of directed RAG targeting and the maintenance of genome stability. PMID:23416051

A low-noise CMOS pixel direct charge sensor, Topmetal-II-

DOE PAGES

An, Mangmang; Chen, Chufeng; Gao, Chaosong; ...

2015-12-12

In this paper, we report the design and characterization of a CMOS pixel direct charge sensor, Topmetal-II-, fabricated in a standard 0.35 μm CMOS Integrated Circuit process. The sensor utilizes exposed metal patches on top of each pixel to directly collect charge. Each pixel contains a low-noise charge-sensitive preamplifier to establish the analog signal and a discriminator with tunable threshold to generate hits. The analog signal from each pixel is accessible through time-shared multiplexing over the entire array. Hits are read out digitally through a column-based priority logic structure. Tests show that the sensor achieved a <15e - analog noisemore » and a 200e - minimum threshold for digital readout per pixel. The sensor is capable of detecting both electrons and ions drifting in gas. Lastly, these characteristics enable its use as the charge readout device in future Time Projection Chambers without gaseous gain mechanism, which has unique advantages in low background and low rate-density experiments.« less

A low-noise CMOS pixel direct charge sensor, Topmetal-II-

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

An, Mangmang; Chen, Chufeng; Gao, Chaosong

In this paper, we report the design and characterization of a CMOS pixel direct charge sensor, Topmetal-II-, fabricated in a standard 0.35 μm CMOS Integrated Circuit process. The sensor utilizes exposed metal patches on top of each pixel to directly collect charge. Each pixel contains a low-noise charge-sensitive preamplifier to establish the analog signal and a discriminator with tunable threshold to generate hits. The analog signal from each pixel is accessible through time-shared multiplexing over the entire array. Hits are read out digitally through a column-based priority logic structure. Tests show that the sensor achieved a <15e - analog noisemore » and a 200e - minimum threshold for digital readout per pixel. The sensor is capable of detecting both electrons and ions drifting in gas. Lastly, these characteristics enable its use as the charge readout device in future Time Projection Chambers without gaseous gain mechanism, which has unique advantages in low background and low rate-density experiments.« less

Characterization of Long-Chain Fatty Acid as N-(4-Aminomethylphenyl) Pyridinium Derivative by MALDI LIFT-TOF/TOF Mass Spectrometry

NASA Astrophysics Data System (ADS)

Frankfater, Cheryl; Jiang, Xuntian; Hsu, Fong-Fu

2018-05-01

Charge remote fragmentation (CRF) elimination of CnH2n+2 residues along the aliphatic tail of long chain fatty acid is hall mark of keV high-energy CID fragmentation process. It is an important fragmentation pathway leading to structural characterization of biomolecules by CID tandem mass spectrometry. In this report, we describe MALDI LIFT TOF-TOF mass spectrometric approach to study a wide variety of fatty acids (FAs), which were derivatized to N-(4-aminomethylphenyl) pyridinium (AMPP) derivative, and desorbed as M+ ions by laser with or without matrix. The high-energy MALDI LIFT TOF-TOF mass spectra of FA-AMPP contain fragment ions mainly deriving from CRF cleavages of CnH2n+2 residues, as expected. These ions together with ions from specific cleavages of the bond(s) involving the functional group within the molecule provide more complete structural identification than those produced by low-energy CID/HCD using a linear ion-trap instrument. However, this LIFT TOF-TOF mass spectrometric approach inherits low sensitivity, a typical feature of high-energy CID tandem mass spectrometry. Because of the lack of unit mass precursor ion selection with sufficient sensitivity of the current LIFT TOF-TOF technology, product ion spectra from same chain length fatty acids with difference in one or two double bonds in a mixture are not distinguishable.

Different residues in the SARS-CoV spike protein determine cleavage and activation by the host cell protease TMPRSS2

PubMed Central

Reinke, Lennart Michel; Hartleib, Anika; Nehlmeier, Inga; Gierer, Stefanie; Hoffmann, Markus; Hofmann-Winkler, Heike; Winkler, Michael

2017-01-01

The spike (S) protein of severe acute respiratory syndrome coronavirus (SARS-CoV) mediates viral entry into target cells. Cleavage and activation of SARS S by a host cell protease is essential for infectious viral entry and the responsible enzymes are potential targets for antiviral intervention. The type II transmembrane serine protease TMPRSS2 cleaves and activates SARS S in cell culture and potentially also in the infected host. Here, we investigated which determinants in SARS S control cleavage and activation by TMPRSS2. We found that SARS S residue R667, a previously identified trypsin cleavage site, is also required for S protein cleavage by TMPRSS2. The cleavage fragments produced by trypsin and TMPRSS2 differed in their decoration with N-glycans, suggesting that these proteases cleave different SARS S glycoforms. Although R667 was required for SARS S cleavage by TMPRSS2, this residue was dispensable for TMPRSS2-mediated S protein activation. Conversely, residue R797, previously reported to be required for SARS S activation by trypsin, was dispensable for S protein cleavage but required for S protein activation by TMPRSS2. Collectively, these results show that different residues in SARS S control cleavage and activation by TMPRSS2, suggesting that these processes are more complex than initially appreciated. PMID:28636671

Different residues in the SARS-CoV spike protein determine cleavage and activation by the host cell protease TMPRSS2.

PubMed

Reinke, Lennart Michel; Spiegel, Martin; Plegge, Teresa; Hartleib, Anika; Nehlmeier, Inga; Gierer, Stefanie; Hoffmann, Markus; Hofmann-Winkler, Heike; Winkler, Michael; Pöhlmann, Stefan

2017-01-01

The spike (S) protein of severe acute respiratory syndrome coronavirus (SARS-CoV) mediates viral entry into target cells. Cleavage and activation of SARS S by a host cell protease is essential for infectious viral entry and the responsible enzymes are potential targets for antiviral intervention. The type II transmembrane serine protease TMPRSS2 cleaves and activates SARS S in cell culture and potentially also in the infected host. Here, we investigated which determinants in SARS S control cleavage and activation by TMPRSS2. We found that SARS S residue R667, a previously identified trypsin cleavage site, is also required for S protein cleavage by TMPRSS2. The cleavage fragments produced by trypsin and TMPRSS2 differed in their decoration with N-glycans, suggesting that these proteases cleave different SARS S glycoforms. Although R667 was required for SARS S cleavage by TMPRSS2, this residue was dispensable for TMPRSS2-mediated S protein activation. Conversely, residue R797, previously reported to be required for SARS S activation by trypsin, was dispensable for S protein cleavage but required for S protein activation by TMPRSS2. Collectively, these results show that different residues in SARS S control cleavage and activation by TMPRSS2, suggesting that these processes are more complex than initially appreciated.

Surface invasive cleavage assay on a maskless light-directed diamond DNA microarray for genome-wide human SNP mapping.

PubMed

Nie, Bei; Yang, Min; Fu, Weiling; Liang, Zhiqing

2015-07-07

The surface invasive cleavage assay, because of its innate accuracy and ability for self-signal amplification, provides a potential route for the mapping of hundreds of thousands of human SNP sites. However, its performance on a high density DNA array has not yet been established, due to the unusual "hairpin" probe design on the microarray and the lack of chemical stability of commercially available substrates. Here we present an applicable method to implement a nanocrystalline diamond thin film as an alternative substrate for fabricating an addressable DNA array using maskless light-directed photochemistry, producing the most chemically stable and biocompatible system for genetic analysis and enzymatic reactions. The surface invasive cleavage reaction, followed by degenerated primer ligation and post-rolling circle amplification is consecutively performed on the addressable diamond DNA array, accurately mapping SNP sites from PCR-amplified human genomic target DNA. Furthermore, a specially-designed DNA array containing dual probes in the same pixel is fabricated by following a reverse light-directed DNA synthesis protocol. This essentially enables us to decipher thousands of SNP alleles in a single-pot reaction by the simple addition of enzyme, target and reaction buffers.

The dehydroalanine effect in the fragmentation of ions derived from polypeptides

PubMed Central

Pilo, Alice L.; Peng, Zhou; McLuckey, Scott A.

2016-01-01

The fragmentation of peptides and proteins upon collision-induced dissociation (CID) is highly dependent on sequence and ion type (e.g. protonated, deprotonated, sodiated, odd electron, etc.). Some amino acids, for example aspartic acid and proline, have been found to enhance certain cleavages along the backbone. Here, we show that peptides and proteins containing dehydroalanine, a non-proteinogenic amino acid with an unsaturated side-chain, undergo enhanced cleavage of the N—Cα bond of the dehydroalanine residue to generate c- and z-ions. Because these fragment ion types are not commonly observed upon activation of positively charged even-electron species, they can be used to identify dehydroalanine residues and localize them within the peptide or protein chain. While dehydroalanine can be generated in solution, it can also be generated in the gas phase upon CID of various species. Oxidized S-alkyl cysteine residues generate dehydroalanine upon activation via highly efficient loss of the alkyl sulfenic acid. Asymmetric cleavage of disulfide bonds upon collisional activation of systems with limited proton mobility also generates dehydroalanine. Furthermore, we show that gas-phase ion/ion reactions can be used to facilitate the generation of dehydroalanine residues via, for example, oxidation of S-alkyl cysteine residues and conversion of multiply-protonated peptides to radical cations. In the latter case, loss of radical side-chains to generate dehydroalanine from some amino acids gives rise to the possibility for residue-specific backbone cleavage of polypeptide ions. PMID:27484024

Direct Observation of Photoinduced Charge Separation in Ruthenium Complex/Ni(OH) 2 Nanoparticle Hybrid

DOE PAGES

Tang, Yu; Pattengale, Brian A.; Ludwig, John M.; ...

2015-12-17

We report that Ni(OH) 2 have emerged as important functional materials for solar fuel conversion because of their potential as cost-effective bifunctional catalysts for both hydrogen and oxygen evolution reactions. However, their roles as photocatalysts in the photoinduced charge separation (CS) reactions remain unexplored. In this paper, we investigate the CS dynamics of a newly designed hybrid catalyst by integrating a Ru complex with Ni(OH) 2 nanoparticles (NPs). Using time resolved X-ray absorption spectroscopy (XTA), we directly observed the formation of the reduced Ni metal site (~60 ps), unambiguously demonstrating CS process in the hybrid through ultrafast electron transfer frommore » Ru complex to Ni(OH) 2 NPs. Compared to the ultrafast CS process, the charge recombination in the hybrid is ultraslow (>>50 ns). These results not only suggest the possibility of developing Ni(OH) 2 as solar fuel catalysts, but also represent the first time direct observation of efficient CS in a hybrid catalyst using XTA.« less

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

PubMed

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

2014-12-01

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

Unusual Fragmentation of Pro-Ser/Thr-Containing Peptides Detected in Collision-Induced Dissociation Spectra

NASA Astrophysics Data System (ADS)

Medzihradszky, Katalin F.; Trinidad, Jonathan C.

2012-04-01

During collision-induced dissociation (CID)-, phosphoserine- and phosphothreonine-containing peptides frequently undergo neutral loss of phosphoric acid. Subsequent amide bond cleavage N-terminal to the site of phosphorylation results in a y ion with a mass 18 Da lower than the corresponding unmodified y fragment. We report here that when the phosphoserine or phosphothreonine is directly preceded by a proline, an unusual fragment with a mass 10 Da higher than the corresponding unmodified y ion is frequently observed. Accurate mass measurements are consistent with elimination of the phosphoric acid followed by fragmentation between the α carbon and the carbonyl group of the proline residue. We propose a cyclic oxazoline structure for this fragment. Our observation may be explained by the charge-directed SN2 neighboring group participation reaction proposed for the phosphoric acid elimination by Palumbo et al. [Palumbo, A. M., Tepe, J. J., Reid, G. E. Mechanistic Insights into the Multistage Gas-Phase Fragmentation Behavior of Phosphoserine- and Phosphothreonine-Containing Peptides. J. Protein Res. 7(2), 771-779 (2008)]. Considering such specific fragment ions for confirmation purposes after regular database searches may boost the confidence of peptide identifications as well as phosphorylation site assignments.

SKI2 mediates degradation of RISC 5'-cleavage fragments and prevents secondary siRNA production from miRNA targets in Arabidopsis.

PubMed

Branscheid, Anja; Marchais, Antonin; Schott, Gregory; Lange, Heike; Gagliardi, Dominique; Andersen, Stig Uggerhøj; Voinnet, Olivier; Brodersen, Peter

2015-12-15

Small regulatory RNAs are fundamental in eukaryotic and prokaryotic gene regulation. In plants, an important element of post-transcriptional control is effected by 20-24 nt microRNAs (miRNAs) and short interfering RNAs (siRNAs) bound to the ARGONAUTE1 (AGO1) protein in an RNA induced silencing complex (RISC). AGO1 may cleave target mRNAs with small RNA complementarity, but the fate of the resulting cleavage fragments remains incompletely understood. Here, we show that SKI2, SKI3 and SKI8, subunits of a cytoplasmic cofactor of the RNA exosome, are required for degradation of RISC 5', but not 3'-cleavage fragments in Arabidopsis. In the absence of SKI2 activity, many miRNA targets produce siRNAs via the RNA-dependent RNA polymerase 6 (RDR6) pathway. These siRNAs are low-abundant, and map close to the cleavage site. In most cases, siRNAs were produced 5' to the cleavage site, but several examples of 3'-spreading were also identified. These observations suggest that siRNAs do not simply derive from RDR6 action on stable 5'-cleavage fragments and hence that SKI2 has a direct role in limiting secondary siRNA production in addition to its function in mediating degradation of 5'-cleavage fragments. © The Author(s) 2015. Published by Oxford University Press on behalf of Nucleic Acids Research.

Novel regulatory mechanisms for generation of the soluble leptin receptor: implications for leptin action.

PubMed

Schaab, Michael; Kausch, Henriette; Klammt, Juergen; Nowicki, Marcin; Anderegg, Ulf; Gebhardt, Rolf; Rose-John, Stefan; Scheller, Juergen; Thiery, Joachim; Kratzsch, Juergen

2012-01-01

The adipokine leptin realizes signal transduction via four different membrane-anchored leptin receptor (Ob-R) isoforms in humans. However, the amount of functionally active Ob-R is affected by constitutive shedding of the extracellular domain via a so far unknown mechanism. The product of the cleavage process the so-called soluble leptin receptor (sOb-R) is the main binding protein for leptin in human blood and modulates its bioavailability. sOb-R levels are differentially regulated in metabolic disorders like type 1 diabetes mellitus or obesity and can, therefore, enhance or reduce leptin sensitivity. To describe mechanisms of Ob-R cleavage and to investigate the functional significance of differential sOb-R levels we established a model of HEK293 cells transiently transfected with different human Ob-R isoforms. Using siRNA knockdown experiments we identified ADAM10 (A Disintegrin And Metalloproteinase 10) as a major protease for constitutive and activated Ob-R cleavage. Additionally, the induction of lipotoxicity and apoptosis led to enhanced shedding shown by increased levels of the soluble leptin receptor (sOb-R) in cell supernatants. Conversely, high leptin concentrations and ER stress reduced sOb-R levels. Decreased amounts of sOb-R due to ER stress were accompanied by impaired leptin signaling and reduced leptin binding. Lipotoxicity and apoptosis increased Ob-R cleavage via ADAM10-dependent mechanisms. In contrast high leptin levels and ER stress led to reduced sOb-R levels. While increased sOb-R concentrations seem to directly block leptin action, reduced amounts of sOb-R may reflect decreased membrane expression of Ob-R. These findings could explain changes of leptin sensitivity which are associated with variations of serum sOb-R levels in metabolic diseases.

Novel Regulatory Mechanisms for Generation of the Soluble Leptin Receptor: Implications for Leptin Action

PubMed Central

Schaab, Michael; Kausch, Henriette; Klammt, Juergen; Nowicki, Marcin; Anderegg, Ulf; Gebhardt, Rolf; Rose-John, Stefan; Scheller, Juergen; Thiery, Joachim; Kratzsch, Juergen

2012-01-01

Background The adipokine leptin realizes signal transduction via four different membrane-anchored leptin receptor (Ob-R) isoforms in humans. However, the amount of functionally active Ob-R is affected by constitutive shedding of the extracellular domain via a so far unknown mechanism. The product of the cleavage process the so-called soluble leptin receptor (sOb-R) is the main binding protein for leptin in human blood and modulates its bioavailability. sOb-R levels are differentially regulated in metabolic disorders like type 1 diabetes mellitus or obesity and can, therefore, enhance or reduce leptin sensitivity. Methodology/Principal Findings To describe mechanisms of Ob-R cleavage and to investigate the functional significance of differential sOb-R levels we established a model of HEK293 cells transiently transfected with different human Ob-R isoforms. Using siRNA knockdown experiments we identified ADAM10 (A Disintegrin And Metalloproteinase 10) as a major protease for constitutive and activated Ob-R cleavage. Additionally, the induction of lipotoxicity and apoptosis led to enhanced shedding shown by increased levels of the soluble leptin receptor (sOb-R) in cell supernatants. Conversely, high leptin concentrations and ER stress reduced sOb-R levels. Decreased amounts of sOb-R due to ER stress were accompanied by impaired leptin signaling and reduced leptin binding. Conclusions Lipotoxicity and apoptosis increased Ob-R cleavage via ADAM10-dependent mechanisms. In contrast high leptin levels and ER stress led to reduced sOb-R levels. While increased sOb-R concentrations seem to directly block leptin action, reduced amounts of sOb-R may reflect decreased membrane expression of Ob-R. These findings could explain changes of leptin sensitivity which are associated with variations of serum sOb-R levels in metabolic diseases. PMID:22545089

Somatostatin increases glucocorticoid binding and signaling in macrophages by blocking the calpain-specific cleavage of Hsp 90.

PubMed

Bellocq, A; Doublier, S; Suberville, S; Perez, J; Escoubet, B; Fouqueray, B; Puyol, D R; Baud, L

1999-12-24

Somatostatin has direct anti-inflammatory actions and participates in the anti-inflammatory actions of glucocorticoids, but the mechanisms underlying this regulation remain poorly understood. The objective of this study was to evaluate whether somatostatin increases glucocorticoid responsiveness by up-regulating glucocorticoid receptor (GR) expression and signaling. Somatostatin promoted a time- and dose-dependent increase in [(3)H]dexamethasone binding to RAW 264.7 macrophages. Cell exposure to 10 nM somatostatin for 18 h promoted a 2-fold increase in the number of GR sites per cell without significant modification of the affinity. Analysis of GR heterocomplex components demonstrated that somatostatin increased the level of heat shock protein (Hsp) 90, whereas the level of GR remained almost unchanged. The increase in Hsp 90 was associated with a decrease in the cleavage of its carboxyl-terminal domain. Evidence for the involvement of calpain inhibition in this process was obtained by the demonstration that 1) somatostatin induced a dose-dependent decrease in calpain activity and 2) calpain inhibitors, calpain inhibitor I and calpeptin, both abolished the cleavage of Hsp 90 and induced a dose-dependent increase in [(3)H]dexamethasone binding. Increases in glucocorticoid binding after somatostatin treatment were associated with similar increases in the ability of GR to transactivate a minimal promoter containing two glucocorticoid response elements (GRE) and to interfere with the activation of nuclear factor-kappaB (NF-kappaB). Thus, the present findings indicate that somatostatin increases glucocorticoid binding and signaling by limiting the calpain-specific cleavage of GR-associated Hsp 90. This mechanism may represent a novel target for intervention to increase glucocorticoid responsiveness.

Donation and back-donation analyzed through a charge transfer model based on density functional theory.

PubMed

Orozco-Valencia, Ulises; Gázquez, José L; Vela, Alberto

2017-07-01

The net charge transfer process that occurs between two species, A and B, interacting with each other, may be decomposed into two processes: one in which A receives charge from B, which can be identified as the electrophilic channel for A or the nucleophilic channel for B, and a second in which A donates charge to B, which can be identified as the nucleophilic channel for A or the electrophilic channel for B. By determining the amount of charge associated with both processes through the minimization of the interaction energy associated with each case, the expressions for the amount of charge involved in each case can be expressed in terms of the directional chemical potentials and the hardnesses of the interacting species. The correlation between the charges obtained for the interaction between phosphine ligands of the type PRR'R'' and Ni, and the A 1 carbonyl stretching frequency provides support for their interpretation as measures of the electrophilicity and nucleophilicity of a chemical species, and, at the same time, allows one to describe the donation and back-donation processes in terms of the density functional theory of chemical reactivity.

Growth and optical properties of Dy doped and undoped n-type InSe single crystal

NASA Astrophysics Data System (ADS)

Gürbulak, B.

1999-02-01

Undoped n-InSe and Dy doped n-InSe (n-InSe : Dy) single crystals were grown by a method which is similar to direct freezing method. Ingots had no cracks and voids on the surface. There were no processes to polish and clean treatment at cleavage faces of these samples because of the natural mirror-like cleavage faces. The absorption measurements were carried out in n-InSe and n-InSe : Dy samples in the temperature range 10-320 K. The first exciton energies for n=1 were calculated as 1.331, 1.248 eV in n-InSe and were 1.326, 1.244 eV in n-InSe : Dy at 10 and 300 K, respectively. The second exciton energies for n=2 in n-InSe were calculated as 1.346, 1.336 eV and in n-InSe : Dy were 1.340, 1.332 eV at 10 and 80 K, respectively. Binding energies of n-InSe and n-InSe : Dy were calculated as 19.47 and 18.87 meV, respectively. The direct bands gap for n-InSe are 1.350, 1.267 eV and for n-InSe : Dy are 1.344, 1.263 eV at 10, 300 K, respectively.

EXPERIMENTAL STUDIES ON EMBRYOGENESIS IN HYDROZOANS (TRACHYLINA AND SIPHONOPHORA) WITH DIRECT DEVELOPMENT.

PubMed

Freeman, Gary

1983-12-01

The normal embryology of the trachymedusa Aglantha digitale and the siphonophores Nanomia cara and Muggiaea atlantica is described. Marking experiments on these embryos indicate that the site of first cleavage initiation corresponds to the oral pole of the oral-aboral axis. In Muggiaea the plane of the first cleavage corresponds to the plane of bilateral symmetry. Experiments in which presumptive aboral and oral regions are isolated from these embryos at different stages of development indicate that there is an early determination of different regions along this axis. Only the oral region of the Muggiaea embryo has the ability to regulate. These eggs have a pronounced centrolecithal organization. As a consequence of cleavage, the outer ectoplasmic layer of the egg ends up in the cells that form the ectoderm, while the inner or endoplasmic region of the egg ends up in the cells that form the endoderm. Experimentally created fragments of fertilized eggs that contain only ectoplasm differentiate to form an unorganized ectodermal cell mass, indicating that endoplasm is necessary in order to differentiate endoderm. The process of embryogenesis in these animals and the developmental mechanisms they use are very different from those used by hydrozoans with indirect development. These embryos use a suite of developmental mechanisms which are very similar to those used by ctenophores. The significance of this similarity is discussed.

F-box only protein 2 (Fbxo2) regulates amyloid precursor protein levels and processing.

PubMed

Atkin, Graham; Hunt, Jack; Minakawa, Eiko; Sharkey, Lisa; Tipper, Nathan; Tennant, William; Paulson, Henry L

2014-03-07

The amyloid precursor protein (APP) is an integral membrane glycoprotein whose cleavage products, particularly amyloid-β, accumulate in Alzheimer disease (AD). APP is present at synapses and is thought to play a role in both the formation and plasticity of these critical neuronal structures. Despite the central role suggested for APP in AD pathogenesis, the mechanisms regulating APP in neurons and its processing into cleavage products remain incompletely understood. F-box only protein 2 (Fbxo2), a neuron-enriched ubiquitin ligase substrate adaptor that preferentially binds high-mannose glycans on glycoproteins, was previously implicated in APP processing by facilitating the degradation of the APP-cleaving β-secretase, β-site APP-cleaving enzyme. Here, we sought to determine whether Fbxo2 plays a similar role for other glycoproteins in the amyloid processing pathway. We present in vitro and in vivo evidence that APP is itself a substrate for Fbxo2. APP levels were decreased in the presence of Fbxo2 in non-neuronal cells, and increased in both cultured hippocampal neurons and brain tissue from Fbxo2 knock-out mice. The processing of APP into its cleavage products was also increased in hippocampi and cultured hippocampal neurons lacking Fbxo2. In hippocampal slices, this increase in cleavage products was accompanied by a significant reduction in APP at the cell surface. Taken together, these results suggest that Fbxo2 regulates APP levels and processing in the brain and may play a role in modulating AD pathogenesis.

Separase is recruited to mitotic chromosomes to dissolve sister chromatid cohesion in a DNA-dependent manner.

PubMed

Sun, Yuxiao; Kucej, Martin; Fan, Heng-Yu; Yu, Hong; Sun, Qing-Yuan; Zou, Hui

2009-04-03

Sister chromatid separation is triggered by the separase-catalyzed cleavage of cohesin. This process is temporally controlled by cell-cycle-dependent factors, but its biochemical mechanism and spatial regulation remain poorly understood. We report that cohesin cleavage by human separase requires DNA in a sequence-nonspecific manner. Separase binds to DNA in vitro, but its proteolytic activity, measured by its autocleavage, is not stimulated by DNA. Instead, biochemical characterizations suggest that DNA mediates cohesin cleavage by bridging the interaction between separase and cohesin. In human cells, a fraction of separase localizes to the mitotic chromosome. The importance of the chromosomal DNA in cohesin cleavage is further demonstrated by the observation that the cleavage of the chromosome-associated cohesins is sensitive to nuclease treatment. Our observations explain why chromosome-associated cohesins are specifically cleaved by separase and the soluble cohesins are left intact in anaphase.

Hawking radiation As tunneling

PubMed

Parikh; Wilczek

2000-12-11

We present a short and direct derivation of Hawking radiation as a tunneling process, based on particles in a dynamical geometry. The imaginary part of the action for the classically forbidden process is related to the Boltzmann factor for emission at the Hawking temperature. Because the derivation respects conservation laws, the exact spectrum is not precisely thermal. We compare and contrast the problem of spontaneous emission of charged particles from a charged conductor.

New Redox Polymers that Exhibit Reversible Cleavage of Sulfur Bonds as Cathode Materials.

PubMed

Baloch, Marya; Ben Youcef, Hicham; Li, Chunmei; Garcia-Calvo, Oihane; Rodriguez, Lide M; Shanmukaraj, Devaraj; Rojo, Teofilo; Armand, Michel

2016-11-23

Two new cathode materials based on redox organosulfur polymers were synthesized and investigated for rechargeable lithium batteries as a proof-of-concept study. These cathodes offered good cycling performance owing to the absence of polysulfide solubility, which plagues Li/S systems. Herein, an aliphatic polyamine or a conjugated polyazomethine was used as the base to tether the redox-active species. The activity comes from the cleavage and formation of S-S or N-S bonds, which is made possible by the rigid conjugated backbone. The synthesized polymers were characterized through FTIR spectroscopy and thermogravimetric analysis (TGA). Galvanostatic measurements were performed to evaluate the discharge/charge cycles and characterize the performance of the lithium-based cells, which displayed initial discharge capacities of approximately 300 mA h g -1 at C/5 over 100 cycles with approximately 98 % Coulombic efficiency. © 2016 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.

A Serendipitous Discover that in situ Proteolysis is Essential for the Crystallization of Yeast CPSF-100 (Ydh1p)

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

Mandel,C.; Gebauer, D.; Zhang, H.

2006-01-01

The cleavage and polyadenylation specificity factor (CPSF) complex is required for the cleavage and polyadenylation of the 3'-end of messenger RNA precursors in eukaryotes. During structural studies of the 100 kDa subunit (CPSF-100, Ydh1p) of the yeast CPSF complex, it was serendipitously discovered that a solution that is infected by a fungus (subsequently identified as Penicillium) is crucial for the crystallization of this protein. Further analyses suggest that the protein has undergone partial proteolysis during crystallization, resulting in the deletion of an internal segment of about 200 highly charged and hydrophilic residues, very likely catalyzed by a protease secreted bymore » the fungus. With the removal of this segment, yeast CPSF-100 (Ydh1p) has greatly reduced solubility and can be crystallized in the presence of a minute amount of precipitant.« less

AB INITIO STUDY OF GRAIN BOUNDARY PROPERTIES OF TUNGSTEN ALLOYS

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

Setyawan, Wahyu; Kurtz, Richard J.

2012-04-17

Density functional theory was employed to investigate the grain boundary (GB) property of W-TM alloys (TM: fifth and sixth row transition metals). GB strengthening was found for Hf, Ta, Nb, Ru, Re, Os and Ir for 27{l_brace}525{r_brace} and to a lesser degree for 11{l_brace}323{r_brace}. Lower valence solutes strengthen the GB at certain substitutional sites, while higher valence elements enforce it at other positions. For 3{l_brace}112{r_brace}, the alloys exhibit reduced cleavage energies. Hence, allowing with TMs increases the GB cohesion more effectively for large-angle GBs whose cleavage energy is, in general, inherently lower than the low-angle ones. Electron density analysis elucidatesmore » the mechanism of charge addition or depletion of the GB bonding region upon TM substitution at various positions leading to stronger or weaker intergranular cohesion, respectively.« less

Charge Separation and Recombination at Polymer-Fullerene Heterojunctions: Delocalization and Hybridization Effects.

PubMed

D'Avino, Gabriele; Muccioli, Luca; Olivier, Yoann; Beljonne, David

2016-02-04

We address charge separation and recombination in polymer/fullerene solar cells with a multiscale modeling built from accurate atomistic inputs and accounting for disorder, interface electrostatics and genuine quantum effects on equal footings. Our results show that bound localized charge transfer states at the interface coexist with a large majority of thermally accessible delocalized space-separated states that can be also reached by direct photoexcitation, thanks to their strong hybridization with singlet polymer excitons. These findings reconcile the recent experimental reports of ultrafast exciton separation ("hot" process) with the evidence that high quantum yields do not require excess electronic or vibrational energy ("cold" process), and show that delocalization, by shifting the density of charge transfer states toward larger effective electron-hole radii, may reduce energy losses through charge recombination.

Hydrogen Embrittlement in 17-4PH Stainless Steel

DTIC Science & Technology

1982-08-01

is observed to exhibit microplastic tearing mixed with some quasi- cleavage. When exposed to longer hydrogen charging times, specimens in the higher...Hours, (a) Central Region Illustrating Dimpled Rupture, (b) and 0-) Shell Region Near Edge Exhibiting Microplastic Tearing. 20 NTAC TP 6 3 43 (a) (b) (c...Shell Region Near Edge Exhibiting Microplastic Tearing. 21 ’JWC TP 6343 FIGURE 15. SEM Fractographv Showing Intergranular Fracture (if 17-4PH- in

12 CFR 1070.22 - Fees for processing requests for CFPB records.

Code of Federal Regulations, 2013 CFR

2013-01-01

... CFPB shall charge the requester for the actual direct cost of the search, including computer search time, runs, and the operator's salary. The fee for computer output will be the actual direct cost. For... and the cost of operating the computer to process a request) equals the equivalent dollar amount of...

A rechargeable hydrogen battery based on Ru catalysis.

PubMed

Hsu, Shih-Fan; Rommel, Susanne; Eversfield, Philipp; Muller, Keven; Klemm, Elias; Thiel, Werner R; Plietker, Bernd

2014-07-01

Apart from energy generation, the storage and liberation of energy are among the major problems in establishing a sustainable energy supply chain. Herein we report the development of a rechargeable H2 battery which is based on the principle of the Ru-catalyzed hydrogenation of CO2 to formic acid (charging process) and the Ru-catalyzed decomposition of formic acid to CO2 and H2 (discharging process). Both processes are driven by the same catalyst at elevated temperature either under pressure (charging process) or pressure-free conditions (discharging process). Up to five charging-discharging cycles were performed without decrease of storage capacity. The resulting CO2/H2 mixture is free of CO and can be employed directly in fuel-cell technology. © 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Blocking of proteolytic processing and deletion of glycosaminoglycan side chain of mouse DMP1 by substituting critical amino acid residues.

PubMed

Peng, Tao; Huang, Bingzhen; Sun, Yao; Lu, Yongbo; Bonewald, Lynda; Chen, Shuo; Butler, William T; Feng, Jerry Q; D'Souza, Rena N; Qin, Chunlin

2009-01-01

Dentin matrix protein 1 (DMP1) is present in the extracellular matrix (ECM) of dentin and bone as processed NH(2)- and COOH-terminal fragments, resulting from proteolytic cleavage at the NH(2) termini of 4 aspartic acid residues during rat DMP1 processing. One cleavage site residue, Asp(181) (corresponding to Asp(197) of mouse DMP1), and its flanking region are highly conserved across species. We speculate that cleavage at the NH(2) terminus of Asp(197) of mouse DMP1 represents an initial, first-step scission in the whole cascade of proteolytic processing. To test if Asp(197) is critical for initiating the proteolytic processing of mouse DMP1, we substituted Asp(197) with Ala(197) by mutating the corresponding nucleotides of mouse cDNA that encode this amino acid residue. This mutant DMP1 cDNA was cloned into a pcDNA3.1 vector. Data from transfection experiments indicated that this single substitution blocked the proteolytic processing of mouse DMP1 in HEK-293 cells, indicating that cleavage at the NH(2) terminus of Asp(197) is essential for exposing other cleavage sites for the conversion of DMP1 to its fragments. The NH(2)-terminal fragment of DMP1 occurs as a proteoglycan form (DMP1-PG) that contains a glycosaminoglycan (GAG) chain. Previously, we showed that a GAG chain is linked to Ser(74) in rat DMP1 (Ser(89) in mouse DMP1). To confirm that mouse DMP1-PG possesses a single GAG chain attached to Ser(89), we substituted Ser(89) by Gly(89). Data from transfection analysis indicated that this substitution completely prevented formation of the GAG-containing form, confirming that DMP1-PG contains a single GAG chain attached to Ser(89) in mouse DMP1. Copyright 2008 S. Karger AG, Basel.

Efficient Cleavage of Ribosome-Associated Poly(A)-Binding Protein by Enterovirus 3C Protease

PubMed Central

Kuyumcu-Martinez, N. Muge; Joachims, Michelle; Lloyd, Richard E.

2002-01-01

Poliovirus (PV) causes a rapid and drastic inhibition of host cell cap-dependent protein synthesis during infection while preferentially allowing cap-independent translation of its own genomic RNA via an internal ribosome entry site element. Inhibition of cap-dependent translation is partly mediated by cleavage of an essential translation initiation factor, eIF4GI, during PV infection. In addition to cleavage of eIF4GI, cleavage of eIF4GII and poly(A)-binding protein (PABP) has been recently proposed to contribute to complete host translation shutoff; however, the relative importance of eIF4GII and PABP cleavage has not been determined. At times when cap-dependent translation is first blocked during infection, only 25 to 35% of the total cellular PABP is cleaved; therefore, we hypothesized that the pool of PABP associated with polysomes may be preferentially targeted by viral proteases. We have investigated what cleavage products of PABP are produced in vivo and the substrate determinants for cleavage of PABP by 2A protease (2Apro) or 3C protease (3Cpro). Our results show that PABP in ribosome-enriched fractions is preferentially cleaved in vitro and in vivo compared to PABP in other fractions. Furthermore, we have identified four N-terminal PABP cleavage products produced during PV infection and have shown that viral 3C protease generates three of the four cleavage products. Also, 3Cpro is more efficient in cleaving PABP in ribosome-enriched fractions than 2Apro in vitro. In addition, binding of PABP to poly(A) RNA stimulates 3Cpro-mediated cleavage and inhibits 2Apro-mediated cleavage. These results suggest that 3Cpro plays a major role in processing PABP during virus infection and that the interaction of PABP with translation initiation factors, ribosomes, or poly(A) RNA may promote its cleavage by viral 2A and 3C proteases. PMID:11836384

Single charging events on colloidal particles in a nonpolar liquid with surfactant

NASA Astrophysics Data System (ADS)

Schreuer, Caspar; Vandewiele, Stijn; Brans, Toon; Strubbe, Filip; Neyts, Kristiaan; Beunis, Filip

2018-01-01

Electrical charging of colloidal particles in nonpolar liquids due to surfactant additives is investigated intensively, motivated by its importance in a variety of applications. Most methods rely on average electrophoretic mobility measurements of many particles, which provide only indirect information on the charging mechanism. In the present work, we present a method that allows us to obtain direct information on the charging mechanism, by measuring the charge fluctuations on individual particles with a precision higher than the elementary charge using optical trapping electrophoresis. We demonstrate the capabilities of the method by studying the influence of added surfactant OLOA 11000 on the charging of single colloidal PMMA particles in dodecane. The particle charge and the frequency of charging events are investigated both below and above the critical micelle concentration (CMC) and with or without applying a DC offset voltage. It is found that at least two separate charging mechanisms are present below the critical micelle concentration. One mechanism is a process where the particle is stripped from negatively charged ionic molecules. An increase in the charging frequency with increased surfactant concentration suggests a second mechanism that involves single surfactant molecules. Above the CMC, neutral inverse micelles can also be involved in the charging process.

4 CFR 28.98 - Individual charges in EEO cases.

Code of Federal Regulations, 2013 CFR

2013-01-01

... complaint process. (d) Special rules for RIF based actions. An individual alleging discrimination issues in connection with a RIF-based separation may follow the procedures outlined above in paragraph (c) of this.... (2) A charging party challenging a RIF action by filing directly with the PAB shall follow the...

4 CFR 28.98 - Individual charges in EEO cases.

Code of Federal Regulations, 2012 CFR

2012-01-01

... complaint process. (d) Special rules for RIF based actions. An individual alleging discrimination issues in connection with a RIF-based separation may follow the procedures outlined above in paragraph (c) of this.... (2) A charging party challenging a RIF action by filing directly with the PAB shall follow the...

4 CFR 28.98 - Individual charges in EEO cases.

Code of Federal Regulations, 2014 CFR

2014-01-01

... complaint process. (d) Special rules for RIF based actions. An individual alleging discrimination issues in connection with a RIF-based separation may follow the procedures outlined above in paragraph (c) of this.... (2) A charging party challenging a RIF action by filing directly with the PAB shall follow the...

Time-resolved electric force microscopy of charge trapping in polycrystalline pentacene.

PubMed

Jaquith, Michael; Muller, Erik M; Marohn, John A

2007-07-12

Here we introduce time-resolved electric force microscopy measurements to directly and locally probe the kinetics of charge trap formation in a polycrystalline pentacene thin-film transistor. We find that the trapping rate depends strongly on the initial concentration of free holes and that trapped charge is highly localized. The observed dependence of trapping rate on the hole chemical potential suggests that the trapping process should not be viewed as a filling of midgap energy levels, but instead as a process in which the very creation of trapped states requires the presence of free holes.

Dose control in electron beam processing: Comparison of results from a graphite charge collector, routine dosimeters and the ISS alanine-based dosimeter

NASA Astrophysics Data System (ADS)

Fuochi, P. G.; Onori, S.; Casali, F.; Chirco, P.

1993-10-01

A 12 MeV linear accelerator is currently used for electron beam processing of power semiconductor devices for lifetime control and, on an experimental basis, for food irradiation, sludge treatment etc. In order to control the irradiation process a simple, quick and reliable method for a direct evaluation of dose and fluence in a broad electron beam has been developed. This paper presents the results obtained using a "charge collector" which measures the charge absorbed in a graphite target exposed in air. Calibration of the system with super-Fricke dosimeter and comparison of absorbed dose results obtained with plastic dosimeters and alanine pellets are discussed.

Mo-Mo Quintuple Bond is Highly Reactive in H-H, C-H, and O-H σ-Bond Cleavages Because of the Polarized Electronic Structure in Transition State.

PubMed

Chen, Yue; Sakaki, Shigeyoshi

2017-04-03

The recently reported high reactivity of the Mo-Mo quintuple bond of Mo 2 (N ∧ N) 2 (1) {N ∧ N = μ-κ 2 -CH[N(2,6-iPr 2 C 6 H 3 )] 2 } in the H-H σ-bond cleavage was investigated. DFT calculations disclosed that the H-H σ-bond cleavage by 1 occurs with nearly no barrier to afford the cis-dihydride species followed by cis-trans isomerization to form the trans-dihydride product, which is consistent with the experimental result. The O-H and C-H bond cleavages by 1 were computationally predicted to occur with moderate (ΔG° ⧧ = 9.0 kcal/mol) and acceptable activation energies (ΔG° ⧧ = 22.5 kcal/mol), respectively, suggesting that the Mo-Mo quintuple bond can be applied to various σ-bond cleavages. In these σ-bond cleavage reactions, the charge-transfer (CT Mo→XH ) from the Mo-Mo quintuple bond to the X-H (X = H, C, or O) bond and that (CT XH→Mo ) from the X-H bond to the Mo-Mo bond play crucial roles. Though the HOMO (dδ-MO) of 1 is at lower energy and the LUMO + 2 (dδ*-MO) of 1 is at higher energy than those of RhCl(PMe 3 ) 2 (LUMO and LUMO + 1 of 1 are not frontier MO), the H-H σ-bond cleavage by 1 more easily occurs than that by the Rh complex. Hence, the frontier MO energies are not the reason for the high reactivity of 1. The high reactivity of 1 arises from the polarization of dδ-type MOs of the Mo-Mo quintuple bond in the transition state. Such a polarized electronic structure enhances the bonding overlap between the dδ-MO of the Mo-Mo bond and the σ*-antibonding MO of the X-H bond to facilitate the CT Mo→XH and reduce the exchange repulsion between the Mo-Mo bond and the X-H bond. This polarized electronic structure of the transition state is similar to that of a frustrated Lewis pair. The easy polarization of the dδ-type MOs is one of the advantages of the metal-metal multiple bond, because such polarization is impossible in the mononuclear metal complex.

Identification of succinimide sites in proteins by N-terminal sequence analysis after alkaline hydroxylamine cleavage.

PubMed Central

Kwong, M. Y.; Harris, R. J.

1994-01-01

Under favorable conditions, Asp or Asn residues can undergo rearrangement to a succinimide (cyclic imide), which may also serve as an intermediate for deamidation and/or isoaspartate formation. Direct identification of such succinimides by peptide mapping is hampered by their lability at neutral and alkaline pH. We determined that incubation in 2 M hydroxylamine, 0.2 M Tris buffer, pH 9, for 2 h at 45 degrees C will specifically cleave on the C-terminal side of succinimides without cleavage at Asn-Gly bonds; yields are typically approximately 50%. N-terminal sequence analysis can then be used to identify an internal sequence generated by cleavage of the succinimide, hence identifying the succinimide site. PMID:8142891

A new class of antibacterials, the imidazopyrazinones, reveal structural transitions involved in DNA gyrase poisoning and mechanisms of resistance.

PubMed

Germe, Thomas; Vörös, Judit; Jeannot, Frederic; Taillier, Thomas; Stavenger, Robert A; Bacqué, Eric; Maxwell, Anthony; Bax, Benjamin D

2018-05-04

Imidazopyrazinones (IPYs) are a new class of compounds that target bacterial topoisomerases as a basis for their antibacterial activity. We have characterized the mechanism of these compounds through structural/mechanistic studies showing they bind and stabilize a cleavage complex between DNA gyrase and DNA ('poisoning') in an analogous fashion to fluoroquinolones, but without the requirement for the water-metal-ion bridge. Biochemical experiments and structural studies of cleavage complexes of IPYs compared with an uncleaved gyrase-DNA complex, reveal conformational transitions coupled to DNA cleavage at the DNA gate. These involve movement at the GyrA interface and tilting of the TOPRIM domains toward the scissile phosphate coupled to capture of the catalytic metal ion. Our experiments show that these structural transitions are involved generally in poisoning of gyrase by therapeutic compounds and resemble those undergone by the enzyme during its adenosine triphosphate-coupled strand-passage cycle. In addition to resistance mutations affecting residues that directly interact with the compounds, we characterized a mutant (D82N) that inhibits formation of the cleavage complex by the unpoisoned enzyme. The D82N mutant appears to act by stabilizing the binary conformation of DNA gyrase with uncleaved DNA without direct interaction with the compounds. This provides general insight into the resistance mechanisms to antibiotics targeting bacterial type II topoisomerases.

A new class of antibacterials, the imidazopyrazinones, reveal structural transitions involved in DNA gyrase poisoning and mechanisms of resistance

PubMed Central

Germe, Thomas; Vörös, Judit; Jeannot, Frederic; Taillier, Thomas; Stavenger, Robert A; Bacqué, Eric; Bax, Benjamin D

2018-01-01

Abstract Imidazopyrazinones (IPYs) are a new class of compounds that target bacterial topoisomerases as a basis for their antibacterial activity. We have characterized the mechanism of these compounds through structural/mechanistic studies showing they bind and stabilize a cleavage complex between DNA gyrase and DNA (‘poisoning’) in an analogous fashion to fluoroquinolones, but without the requirement for the water–metal–ion bridge. Biochemical experiments and structural studies of cleavage complexes of IPYs compared with an uncleaved gyrase–DNA complex, reveal conformational transitions coupled to DNA cleavage at the DNA gate. These involve movement at the GyrA interface and tilting of the TOPRIM domains toward the scissile phosphate coupled to capture of the catalytic metal ion. Our experiments show that these structural transitions are involved generally in poisoning of gyrase by therapeutic compounds and resemble those undergone by the enzyme during its adenosine triphosphate-coupled strand-passage cycle. In addition to resistance mutations affecting residues that directly interact with the compounds, we characterized a mutant (D82N) that inhibits formation of the cleavage complex by the unpoisoned enzyme. The D82N mutant appears to act by stabilizing the binary conformation of DNA gyrase with uncleaved DNA without direct interaction with the compounds. This provides general insight into the resistance mechanisms to antibiotics targeting bacterial type II topoisomerases. PMID:29538767

Progress in Genome Editing Technology and Its Application in Plants

PubMed Central

Zhang, Kai; Raboanatahiry, Nadia; Zhu, Bin; Li, Maoteng

2017-01-01

Genome editing technology (GET) is a versatile approach that has progressed rapidly as a mechanism to alter the genotype and phenotype of organisms. However, conventional genome modification using GET cannot satisfy current demand for high-efficiency and site-directed mutagenesis, retrofitting of artificial nucleases has developed into a new avenue within this field. Based on mechanisms to recognize target genes, newly-developed GETs can generally be subdivided into three cleavage systems, protein-dependent DNA cleavage systems (i.e., zinc-finger nucleases, ZFN, and transcription activator-like effector nucleases, TALEN), RNA-dependent DNA cleavage systems (i.e., clustered regularly interspaced short palindromic repeats-CRISPR associated proteins, CRISPR-Cas9, CRISPR-Cpf1, and CRISPR-C2c1), and RNA-dependent RNA cleavage systems (i.e., RNA interference, RNAi, and CRISPR-C2c2). All these techniques can lead to double-stranded (DSB) or single-stranded breaks (SSB), and result in either random mutations via non-homologous end-joining (NHEJ) or targeted mutation via homologous recombination (HR). Thus, site-directed mutagenesis can be induced via targeted gene knock-out, knock-in, or replacement to modify specific characteristics including morphology-modification, resistance-enhancement, and physiological mechanism-improvement along with plant growth and development. In this paper, an non-comprehensive review on the development of different GETs as applied to plants is presented. PMID:28261237

Kallikrein-8 Proteolytically Processes Human Papillomaviruses in the Extracellular Space To Facilitate Entry into Host Cells

PubMed Central

Cerqueira, Carla; Samperio Ventayol, Pilar; Vogeley, Christian

2015-01-01

ABSTRACT The entry of human papillomaviruses into host cells is a complex process. It involves conformational changes at the cell surface, receptor switching, internalization by a novel endocytic mechanism, uncoating in endosomes, trafficking of a subviral complex to the Golgi complex, and nuclear entry during mitosis. Here, we addressed how the stabilizing contacts in the capsid of human papillomavirus 16 (HPV16) may be reversed to allow uncoating of the viral genome. Using biochemical and cell-biological analyses, we determined that the major capsid protein L1 underwent proteolytic cleavage during entry. In addition to a dispensable cathepsin-mediated proteolysis that occurred likely after removal of capsomers from the subviral complex in endosomes, at least two further proteolytic cleavages of L1 were observed, one of which was independent of the low-pH environment of endosomes. This cleavage occurred extracellularly. Further analysis showed that the responsible protease was the secreted trypsin-like serine protease kallikrein-8 (KLK8) involved in epidermal homeostasis and wound healing. Required for infection, the cleavage was facilitated by prior interaction of viral particles with heparan sulfate proteoglycans. KLK8-mediated cleavage was crucial for further conformational changes exposing an important epitope of the minor capsid protein L2. Occurring independently of cyclophilins and of furin that mediate L2 exposure, KLK8-mediated cleavage of L1 likely facilitated access to L2, located in the capsid lumen, and potentially uncoating. Since HPV6 and HPV18 also required KLK8 for entry, we propose that the KLK8-dependent entry step is conserved. IMPORTANCE Our analysis of the proteolytic processing of incoming HPV16, an etiological agent of cervical cancer, demonstrated that the capsid is cleaved extracellularly by a serine protease active during wound healing and that this cleavage was crucial for infection. The cleavage of L1 is one of at least four structural alterations that prime the virus extracellularly for receptor switching, internalization, and possibly uncoating. This step was also important for HPV6 and HPV18, which may suggest that it is conserved among the papillomaviruses. This study advances the understanding of how HPV16 initially infects cells, strengthens the notion that wounding facilitates infection of epidermal tissue, and may help the development of antiviral measures. PMID:25926655

Enzymatic cleavage specificity of the proalpha1(V) chain processing analysed by site-directed mutagenesis.

PubMed

Bonod-Bidaud, Christelle; Beraud, Mickaël; Vaganay, Elisabeth; Delacoux, Frédéric; Font, Bernard; Hulmes, David J S; Ruggiero, Florence

2007-07-15

The proteolytic processing of procollagen V is complex and depends on the activity of several enzymes among which the BMP-1 (bone morphogenetic protein-1)/tolloid metalloproteinase and the furin-like proprotein convertases. Few of these processing interactions could have been predicted by analysing the presence of conserved consensus sequences in the proalpha1(V) chain. In the present study we opted for a cell approach that allows a straightforward identification of processing interactions. A construct encompassing the complete N-terminal end of the proalpha1(V) chain, referred to as Nalpha1, was recombinantly expressed to be used for enzymatic assays and for antibody production. Structural analysis showed that Nalpha1 is a monomer composed of a compact globule and an extended tail, which correspond respectively to the non-collagenous Nalpha1 subdomains, TSPN-1 (thrombospondin-1 N-terminal domain-like) and the variable region. Nalpha1 was efficiently cleaved by BMP-1 indicating that the triple helix is not required for enzyme activity. By mutating residues flanking the cleavage site, we showed that the aspartate residue at position P2' is essential for BMP-1 activity. BMP-1 activity at the C-terminal end of the procollagen V was assessed by generating a furin double mutant (R1584A/R1585A). We showed that, in absence of furin activity, BMP-1 is capable of processing the C-propeptide even though less efficiently than furin. Altogether, our results provide new relevant information on this complex and poorly understood mechanism of enzymatic processing in procollagen V function.

RNase L targets distinct sites in influenza A virus RNAs.

PubMed

Cooper, Daphne A; Banerjee, Shuvojit; Chakrabarti, Arindam; García-Sastre, Adolfo; Hesselberth, Jay R; Silverman, Robert H; Barton, David J

2015-03-01

Influenza A virus (IAV) infections are influenced by type 1 interferon-mediated antiviral defenses and by viral countermeasures to these defenses. When IAV NS1 protein is disabled, RNase L restricts virus replication; however, the RNAs targeted for cleavage by RNase L under these conditions have not been defined. In this study, we used deep-sequencing methods to identify RNase L cleavage sites within host and viral RNAs from IAV PR8ΔNS1-infected A549 cells. Short hairpin RNA knockdown of RNase L allowed us to distinguish between RNase L-dependent and RNase L-independent cleavage sites. RNase L-dependent cleavage sites were evident at discrete locations in IAV RNA segments (both positive and negative strands). Cleavage in PB2, PB1, and PA genomic RNAs suggests that viral RNPs are susceptible to cleavage by RNase L. Prominent amounts of cleavage mapped to specific regions within IAV RNAs, including some areas of increased synonymous-site conservation. Among cellular RNAs, RNase L-dependent cleavage was most frequent at precise locations in rRNAs. Our data show that RNase L targets specific sites in both host and viral RNAs to restrict influenza virus replication when NS1 protein is disabled. RNase L is a critical component of interferon-regulated and double-stranded-RNA-activated antiviral host responses. We sought to determine how RNase L exerts its antiviral activity during influenza virus infection. We enhanced the antiviral activity of RNase L by disabling a viral protein, NS1, that inhibits the activation of RNase L. Then, using deep-sequencing methods, we identified the host and viral RNAs targeted by RNase L. We found that RNase L cleaved viral RNAs and rRNAs at very precise locations. The direct cleavage of IAV RNAs by RNase L highlights an intimate battle between viral RNAs and an antiviral endonuclease. Copyright © 2015, American Society for Microbiology. All Rights Reserved.

Extended Hall-Petch Relationships for Yield, Cleavage and Intergranular Fracture Strengths of bcc Steel and Its Deformation and Fracture Behaviors

NASA Astrophysics Data System (ADS)

Heo, N. H.; Heo, Y.-U.; Kwon, S. K.; Kim, N. J.; Kim, S.-J.; Lee, H.-C.

2018-03-01

Extended Hall-Petch relationships for yield ( σy ), cleavage ( σ_{cl} ) and intergranular fracture ( σ_{ig} ) strengths of pure iron have been established through the direct calculation of the proportional constant (k) and the estimation of the friction stress (σ0 ) . The magnitude orders of k and σ0 are generally ky < k_{cl} < k_{ig} and σ_{y0} < σ_{cl0} < σ_{ig0} , respectively. Based on the Hall-Petch relationships, micro-yielding in a bcc steel occurs at the instance that the pile-up dislocations within a specific grain showing the Schmid factor of 0.5 propagate into the neighboring grain. The initial brittle crack is formed at the instance that the flow strength exceeds the brittle fracture strength. Once the brittle crack is formed, it grows catastrophically. Due to the smallest and ky and σ_{y0} , the cleavage and the intergranular fracture occur always after micro-yielding. The {100} cleavage fracture of the steel is due to the lowest theoretical {100} cleavage strength. Due to the thermal components included in cleavage and intergranular fracture strengths, they show also the temperature and strain rate dependence observed in yield strength. The increase in susceptibility to brittle fracture with decreasing temperature and increasing strain rate is due to the increase in dislocation density which causes the high work hardening rate.

APASdb: a database describing alternative poly(A) sites and selection of heterogeneous cleavage sites downstream of poly(A) signals

PubMed Central

You, Leiming; Wu, Jiexin; Feng, Yuchao; Fu, Yonggui; Guo, Yanan; Long, Liyuan; Zhang, Hui; Luan, Yijie; Tian, Peng; Chen, Liangfu; Huang, Guangrui; Huang, Shengfeng; Li, Yuxin; Li, Jie; Chen, Chengyong; Zhang, Yaqing; Chen, Shangwu; Xu, Anlong

2015-01-01

Increasing amounts of genes have been shown to utilize alternative polyadenylation (APA) 3′-processing sites depending on the cell and tissue type and/or physiological and pathological conditions at the time of processing, and the construction of genome-wide database regarding APA is urgently needed for better understanding poly(A) site selection and APA-directed gene expression regulation for a given biology. Here we present a web-accessible database, named APASdb (http://mosas.sysu.edu.cn/utr), which can visualize the precise map and usage quantification of different APA isoforms for all genes. The datasets are deeply profiled by the sequencing alternative polyadenylation sites (SAPAS) method capable of high-throughput sequencing 3′-ends of polyadenylated transcripts. Thus, APASdb details all the heterogeneous cleavage sites downstream of poly(A) signals, and maintains near complete coverage for APA sites, much better than the previous databases using conventional methods. Furthermore, APASdb provides the quantification of a given APA variant among transcripts with different APA sites by computing their corresponding normalized-reads, making our database more useful. In addition, APASdb supports URL-based retrieval, browsing and display of exon-intron structure, poly(A) signals, poly(A) sites location and usage reads, and 3′-untranslated regions (3′-UTRs). Currently, APASdb involves APA in various biological processes and diseases in human, mouse and zebrafish. PMID:25378337

Pulsed radiolysis of model aromatic polymers and epoxy based matrix materials

NASA Technical Reports Server (NTRS)

Gupta, A.; Moacanin, J.; Liang, R.; Coulter, D.

1982-01-01

Models of primary processes leading to deactivation of energy deposited by a pulse of high energy electrons were derived for epoxy matrix materials and polyl-vinyl naphthalene. The basic conclusion is that recombination of initially formed charged states is complete within 1 nanosecond, and subsequent degradation chemistry is controlled by the reactivity of these excited states. Excited states in both systems form complexes with ground state molecules. These excimers or exciplexes have their characteristics emissive and absorptive properties and may decay to form separated pairs of ground state molecules, cross over to the triplet manifold or emit fluorescence. ESR studies and chemical analyses subsequent to pulse radiolysis were performed in order to estimate bond cleavage probabilities and net reaction rates. The energy deactivation models which were proposed to interpret these data have led to the development of radiation stabilization criteria for these systems.

Unconjugated Bilirubin Inhibits Proteolytic Cleavage of von Willebrand Factor by ADAMTS13 Protease

PubMed Central

Lu, Rui-Nan; Yang, Shangbin; Wu, Haifeng M.; Zheng, X. Long

2015-01-01

Summary Background Bilirubin is a yellow breakdown product of heme catabolism. Increased serum levels of unconjugated bilirubin are conditions commonly seen in premature neonates and adults with acute hemolysis including thrombotic microangiopathy. Previous studies have shown that unconjugated bilirubin lowers plasma ADAMTS13 activity, but the mechanism is not fully understood. Objectives The study is to determine whether unconjugated bilirubin directly inhibits the cleavage of von Willebrand factor (VWF) and its analogs by ADAMTS13. Methods Fluorogenic, SELDI-TOF mass spectrometric assay, and Western blotting analyses were employed to address this question. Results Unconjugated bilirubin inhibits the cleavage of F485-rVWF73-H, D633-rVWF73-H, and GST-rVWF71-11K by ADAMTS13 in a concentration-dependent manner with a half-maximal inhibitory concentration (IC50) of ~13 μM, ~70 μM, and ~17 μM, respectively. Unconjugated bilirubin also dose-dependently inhibits the cleavage of multimeric VWF by ADAMTS13 under denaturing conditions. The inhibitory activity of bilirubin on the cleavage of D633-rVWF73-H and multimeric VWF, but not F485-rVWF73-H, was eliminated after incubation with bilirubin oxidase that converts bilirubin to biliverdin. Furthermore, plasma ADAMTS13 activity in patients with hyperbilirubinemia is lower prior to than after treatment with bilirubin oxidase. Conclusions unconjugated bilirubin directly inhibits ADAMTS13’s ability to cleave both peptidyl and native VWF substrates in addition to its interference with certain fluorogenic assays. Our findings may help proper interpretation of ADAMTS13 results under pathological conditions. Whether elevated serum unconjugated bilirubin has an adverse effect in vivo remains to be determined in our future study. PMID:25782102

Are cleavage anomalies, multinucleation, or specific cell cycle kinetics observed with time-lapse imaging predictive of embryo developmental capacity or ploidy?

PubMed

Desai, Nina; Goldberg, Jeffrey M; Austin, Cynthia; Falcone, Tommaso

2018-04-01

To determine whether cleavage anomalies, multinucleation, and specific cellular kinetic parameters available from time-lapse imaging are predictive of developmental capacity or blastocyst chromosomal status. Retrospective analysis of prospectively collected data. Single academic center. A total of 1,478 zygotes from patients with blastocysts biopsied for preimplantation genetic screening were cultured in the EmbryoScope. Trophectoderm biopsy. Embryo dysmorphisms, developmental kinetics, and euploidy. Of the 767 biopsied blastocysts, 41.6% (95% confidence interval [CI], 38%-45%) were diagnosed as euploid. Individual dysmorphisms such as multinucleation, reverse cleavage, irregular chaotic division, or direct uneven cleavage were not associated with aneuploidy. Direct uneven cleavage and irregular chaotic division embryos did, however, exhibit lower developmental potential. The presence of two or more dysmorphisms was associated with an overall lower euploidy rate, 27.6% (95% CI 19%-39%). Early embryo kinetics were predictive of blastocyst development but not ploidy status. In contrast, chromosomal status correlated significantly with start time of blastulation (tSB), expansion (tEB), and the tEB-tSB interval. A lower euploidy rate, 36.6% (95% CI 33%-42%) was observed with tSB ≥ 96.2 hours, compared with 48.2% with tSB < 96.2 (95% CI 42%-54%). A drop in euploidy rate to 30% (95% CI 25%-37%) was observed in blastocysts with delayed expansion (tEB > 116). The proportion of euploid blastocysts was increased with tEB-tSB intervals of ≤13 hours. A logistic regression model to enhance the probability of selecting a euploid blastocyst was constructed. Morphokinetics may aid in selection of euploid embryos from a cohort of day 5/6 blastocysts. Copyright © 2018 The Authors. Published by Elsevier Inc. All rights reserved.

Direct detection of a transport-blocking trap in a nanoscaled silicon single-electron transistor by radio-frequency reflectometry

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

Villis, B. J.; Sanquer, M.; Jehl, X.

2014-06-09

The continuous downscaling of transistors results in nanoscale devices which require fewer and fewer charged carriers for their operation. The ultimate charge controlled device, the single-electron transistor (SET), controls the transfer of individual electrons. It is also the most sensitive electrometer, and as a result the electron transport through it can be dramatically affected by nearby charges. Standard direct-current characterization techniques, however, are often unable to unambiguously detect and resolve the origin of the observed changes in SET behavior arising from changes in the charge state of a capacitively coupled trap. Using a radio-frequency (RF) reflectometry technique, we are ablemore » to unequivocally detect this process, in very close agreement with modeling of the trap's occupation probability.« less

Charge transfer in model peptides: obtaining Marcus parameters from molecular simulation.

PubMed

Heck, Alexander; Woiczikowski, P Benjamin; Kubař, Tomáš; Giese, Bernd; Elstner, Marcus; Steinbrecher, Thomas B

2012-02-23

Charge transfer within and between biomolecules remains a highly active field of biophysics. Due to the complexities of real systems, model compounds are a useful alternative to study the mechanistic fundamentals of charge transfer. In recent years, such model experiments have been underpinned by molecular simulation methods as well. In this work, we study electron hole transfer in helical model peptides by means of molecular dynamics simulations. A theoretical framework to extract Marcus parameters of charge transfer from simulations is presented. We find that the peptides form stable helical structures with sequence dependent small deviations from ideal PPII helices. We identify direct exposure of charged side chains to solvent as a cause of high reorganization energies, significantly larger than typical for electron transfer in proteins. This, together with small direct couplings, makes long-range superexchange electron transport in this system very slow. In good agreement with experiment, direct transfer between the terminal amino acid side chains can be dicounted in favor of a two-step hopping process if appropriate bridging groups exist. © 2012 American Chemical Society

Ab initio molecular dynamics simulations of low energy recoil events in MgO

DOE PAGES

Petersen, B. A.; Liu, B.; Weber, W. J.; ...

2017-01-11

In this paper, low-energy recoil events in MgO are studied using ab initio molecular dynamics simulations to reveal the dynamic displacement processes and final defect configurations. Threshold displacement energies, E d, are obtained for Mg and O along three low-index crystallographic directions, [100], [110], and [111]. The minimum values for E d are found along the [110] direction consisting of the same element, either Mg or O atoms. Minimum threshold values of 29.5 eV for Mg and 25.5 eV for O, respectively, are suggested from the calculations. For other directions, the threshold energies are considerably higher, 65.5 and 150.0 eVmore » for O along [111] and [100], and 122.5 eV for Mg along both [111] and [100] directions, respectively. These results show that the recoil events in MgO are partial-charge transfer assisted processes where the charge transfer plays an important role. Finally, there is a similar trend found in other oxide materials, where the threshold displacement energy correlates linearly with the peak partial-charge transfer, suggesting this behavior might be universal in ceramic oxides.« less

Porcine Epidemic Diarrhea Virus 3C-Like Protease-Mediated Nucleocapsid Processing: Possible Link to Viral Cell Culture Adaptability.

PubMed

Jaru-Ampornpan, Peera; Jengarn, Juggragarn; Wanitchang, Asawin; Jongkaewwattana, Anan

2017-01-15

Porcine epidemic diarrhea virus (PEDV) causes severe diarrhea and high mortality rates in newborn piglets, leading to massive losses to the swine industry worldwide during recent epidemics. Intense research efforts are now focusing on defining viral characteristics that confer a growth advantage, pathogenicity, or cell adaptability in order to better understand the PEDV life cycle and identify suitable targets for antiviral or vaccine development. Here, we report a unique phenomenon of PEDV nucleocapsid (N) cleavage by the PEDV-encoded 3C-like protease (3Cpro) during infection. The identification of the 3Cpro cleavage site at the C terminus of N supported previous observations that PEDV 3Cpro showed a substrate requirement slightly different from that of severe acute respiratory syndrome coronavirus (SARS-CoV) 3Cpro and revealed a greater flexibility in its substrate recognition site. This cleavage motif is present in the majority of cell culture-adapted PEDV strains but is missing in emerging field isolates. Remarkably, reverse-genetics-derived cell culture-adapted PEDV AVCT12 harboring uncleavable N displayed growth retardation in Vero E6-APN cells compared to the wild-type virus. These observations altogether shed new light on the investigation and characterization of the PEDV nucleocapsid protein and its possible link to cell culture adaptation. Recurrent PEDV outbreaks have resulted in enormous economic losses to swine industries worldwide. To gain the upper hand in combating this disease, it is necessary to understand how this virus replicates and evades host immunity. Characterization of viral proteins provides important clues to mechanisms by which viruses survive and spread. Here, we characterized an intriguing phenomenon in which the nucleocapsids of some PEDV strains are proteolytically processed by the virally encoded main protease. Growth retardation in recombinant PEDV carrying uncleavable N suggests a replication advantage provided by the cleavage event, at least in the cell culture system. These findings may direct us to a more complete understanding of PEDV replication and pathogenicity. Copyright © 2017 American Society for Microbiology.

A novel proteolytic processing of prolysyl oxidase

PubMed Central

Atsawasuwan, Phimon; Mochida, Yoshiyuki; Katafuchi, Michitsuna; Tokutomi, Kentaro; Mocanu, Viorel; Parker, Carol E.; Yamauchi, Mitsuo

2012-01-01

Lysyl oxidase (LOX) is an amine oxidase that is critical for the stability of connective tissues. The secreted proLOX is enzymatically quiescent and is activated through proteolytic cleavage between residue Gly162 and Asp163 (residue numbers according to the mouse LOX) by bone morphogenetic protein (BMP)-1 gene products. Here we report a novel processing of proLOX identified in vitro and in vivo. Two forms of mature LOX were identified and characterized by their immunoreactivity to specific antibodies, amine oxidase activity and mass spectrometry. One form was identified as a well characterized BMP-1 processed LOX protein. Another was found to be a truncated form of LOX (tLOX) resulting from the cleavage at the carboxy terminus of Arg192. The tLOX still appeared to retain amine oxidase activity. The results from the proLOX gene deletion and mutation experiments indicated that the processing occurs independent of the cleavage of proLOX by BMP-1 gene products and likely requires the presence of LOX propeptide. These results indicate that proLOX could be processed by two different mechanisms producing two forms of active LOX. PMID:21591931

A novel proteolytic processing of prolysyl oxidase.

PubMed

Atsawasuwan, Phimon; Mochida, Yoshiyuki; Katafuchi, Michitsuna; Tokutomi, Kentaro; Mocanu, Viorel; Parker, Carol E; Yamauchi, Mitsuo

2011-01-01

Lysyl oxidase (LOX) is an amine oxidase that is critical for the stability of connective tissues. The secreted proLOX is enzymatically quiescent and is activated through proteolytic cleavage between residues Gly(162) and Asp(163) (residue numbers according to the mouse LOX) by bone morphogenetic protein (BMP)-1 gene products. Here we report a novel processing of proLOX identified in vitro and in vivo. Two forms of mature LOX were identified and characterized by their immunoreactivity to specific antibodies, amine oxidase activity, and mass spectrometry. One form was identified as a well-characterized BMP-1 processed LOX protein. Another was found to be a truncated form of LOX resulting from the cleavage at the carboxy terminus of Arg(192). The truncated form of LOX still appeared to retain amine oxidase activity. The results from the proLOX gene deletion and mutation experiments indicated that the processing occurs independent of the cleavage of proLOX by BMP-1 gene products and likely requires the presence of LOX propeptide. These results indicate that proLOX could be processed by two different mechanisms producing two forms of active LOX.

Structure investigation of codeine drug using mass spectrometry, thermal analyses and semi-emperical molecular orbital (MO) calculations

NASA Astrophysics Data System (ADS)

Zayed, M. A.; Hawash, M. F.; Fahmey, M. A.

2006-05-01

Codeine is an analgesic with uses similar to morphine, but it has a mild sedative effect. It is preferable used as phosphate form and it is often administrated by mouth with aspirin or paracetamol. Therefore, it is important to investigate its structure to know the active groups and weak bonds responsible for its medical activity. Consequently in the present work, codeine was investigated by mass spectrometry and thermal analyses (TG, DTG and DTA) and confirming by semi-empirical MO-calculation (PM3 method) in the neutral and positively charged forms of the drug. Some results of studying the d-block element complexes of codeine were used to declare the relationship between drug structure and its chemical reactivity in vitro system. The mass spectra and thermal analyses fragmentation pathways were proposed and compared to each other to select the most suitable scheme representing the correct fragmentation of this drug. From EI mass spectra, the main primary cleavage site of the charged drug molecule is that due to β-cleavage to nitrogen atom in its skeleton. It occurs in two parallel mechanisms with the same possibility, i.e. no difference in appearance activation energy between them. In the neutral drug form the primary site cleavage is that occurs in the ether ring. Thermal analyses of the neutral form of the drug revealed the high response of the drug to the temperature variation with very fast rate. It decomposed in several sequential steps in the temperature range 200-600 °C. The initial thermal fragments are very similar to that obtained by mass spectrometric fragmentation. Therefore, comparison between mass and thermal helps in selection of the proper pathway representing the fragmentation of this drug. This comparison successfully confirmed by MOC. These calculations give the bond order, charge distribution, heat of formation and possible hybridization of some atoms in different position of the drug skeleton. This helps the successful choice of the weakest bond at which both mass and thermal fragmentation occurs. Therefore, the best fragmentation pathway of this drug is correctly selected. The effect of such fragmentation on the drug behavior in the human body can be expected as a result of comparing these data with that obtained on studying codeine metal complexes using mass and thermal fragmentation techniques.

piRNA-directed cleavage of meiotic transcripts regulates spermatogenesis.

PubMed

Goh, Wee Siong Sho; Falciatori, Ilaria; Tam, Oliver H; Burgess, Ralph; Meikar, Oliver; Kotaja, Noora; Hammell, Molly; Hannon, Gregory J

2015-05-15

MIWI catalytic activity is required for spermatogenesis, indicating that piRNA-guided cleavage is critical for germ cell development. To identify meiotic piRNA targets, we augmented the mouse piRNA repertoire by introducing a human meiotic piRNA cluster. This triggered a spermatogenesis defect by inappropriately targeting the piRNA machinery to mouse mRNAs essential for germ cell development. Analysis of such de novo targets revealed a signature for pachytene piRNA target recognition. This enabled identification of both transposable elements and meiotically expressed protein-coding genes as targets of native piRNAs. Cleavage of genic targets began at the pachytene stage and resulted in progressive repression through meiosis, driven at least in part via the ping-pong cycle. Our data support the idea that meiotic piRNA populations must be strongly selected to enable successful spermatogenesis, both driving the response away from essential genes and directing the pathway toward mRNA targets that are regulated by small RNAs in meiotic cells. © 2015 Goh et al.; Published by Cold Spring Harbor Laboratory Press.

Characterization of CMPO and its radiolysis products by Direct Infusion ESI-MS

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

G. S. Groenewold; G. Elias; B. J. Mincher

2012-09-01

Direct infusion electrospray ionization-mass spectrometry (ESI-MS) approaches were developed for rapid identification of octyl,phenyl,(N,N-(diisobutyl)carbamoylmethyl) phosphine oxide (CMPO) and impurity compounds formed during alpha and gamma irradiation experiments. CMPO is an aggressive Lewis base, and produces extremely abundant metal complex ions in the ESI-MS analysis that make identification of low abundance compounds that are less nucleophilic challenging. Radiolysis products were identified using several approaches including restricting ion trapping so as to exclude the abundant natiated CMPO ions, extraction of acidic products using aqueous NaOH, and extraction of basic products using HNO3. These approaches generated protonated, natiated and deprotonated species derived frommore » CMPO degradation products formed via radiolytic cleavages of several different bonds. Cleavages of the amide and methylene-phosphoryl bonds appear to be favored by both forms of irradiation, while alpha irradiation also appears to induce cleavage of the methylene-carbonyl bond. The degradation products observed are formed from recombination of the initially formed radicals with hydrogen, methyl, isopropyl and hydroxyl radicals that are derived either from CMPO, or the dodecane solvent.« less

Inverse Thio Effects in the Hepatitis Delta Virus Ribozyme Reveal that the Reaction Pathway Is Controlled by Metal Ion Charge Density

PubMed Central

2015-01-01

The hepatitis delta virus (HDV) ribozyme self-cleaves in the presence of a wide range of monovalent and divalent ions. Prior theoretical studies provided evidence that self-cleavage proceeds via a concerted or stepwise pathway, with the outcome dictated by the valency of the metal ion. In the present study, we measure stereospecific thio effects at the nonbridging oxygens of the scissile phosphate under a wide range of experimental conditions, including varying concentrations of diverse monovalent and divalent ions, and combine these with quantum mechanical/molecular mechanical (QM/MM) free energy simulations on the stereospecific thio substrates. The RP substrate gives large normal thio effects in the presence of all monovalent ions. The SP substrate also gives normal or no thio effects, but only for smaller monovalent and divalent cations, such as Li+, Mg2+, Ca2+, and Sr2+; in contrast, sizable inverse thio effects are found for larger monovalent and divalent cations, including Na+, K+, NH4+, and Ba2+. Proton inventories are found to be unity in the presence of the larger monovalent and divalent ions, but two in the presence of Mg2+. Additionally, rate–pH profiles are inverted for the low charge density ions, and only imidazole plus ammonium ions rescue an inactive C75Δ variant in the absence of Mg2+. Results from the thio effect experiments, rate–pH profiles, proton inventories, and ammonium/imidazole rescue experiments, combined with QM/MM free energy simulations, support a change in the mechanism of HDV ribozyme self-cleavage from concerted and metal ion-stabilized to stepwise and proton transfer-stabilized as the charge density of the metal ion decreases. PMID:25799319

Cleavage and polyadenylation: Ending the message expands gene regulation

PubMed Central

Neve, Jonathan

2017-01-01

ABSTRACT Cleavage and polyadenylation (pA) is a fundamental step that is required for the maturation of primary protein encoding transcripts into functional mRNAs that can be exported from the nucleus and translated in the cytoplasm. 3′end processing is dependent on the assembly of a multiprotein processing complex on the pA signals that reside in the pre-mRNAs. Most eukaryotic genes have multiple pA signals, resulting in alternative cleavage and polyadenylation (APA), a widespread phenomenon that is important to establish cell state and cell type specific transcriptomes. Here, we review how pA sites are recognized and comprehensively summarize how APA is regulated and creates mRNA isoform profiles that are characteristic for cell types, tissues, cellular states and disease. PMID:28453393

Structural analysis and implicit 3D modelling of Jwaneng Mine: Insights into deformation of the Transvaal Supergroup in SE Botswana

NASA Astrophysics Data System (ADS)

Creus, P. K.; Basson, I. J.; Stoch, B.; Mogorosi, O.; Gabanakgosi, K.; Ramsden, F.; Gaegopolwe, P.

2018-01-01

Country rock at Jwaneng Diamond Mine provides a rare insight into the deformational history of the Transvaal Supergroup in southern Botswana. The ca. 235 Ma kimberlite diatremes intruded into late Archaean to Early Proterozoic, mixed, siliciclastic-carbonate sediments, that were subjected to at least three deformational events. The first deformational event (D1), caused by NW-SE directed compression, is responsible for NE-trending, open folds (F1) with associated diverging, fanning, axial planar cleavage. The second deformational event (D2) is probably progressive, involving a clockwise rotation of the principal stress to NE-SW trends. Early D2, which was N-S directed, involved left-lateral, oblique shearing along cleavage planes that developed around F1 folds, along with the development of antithetic structures. Progressive clockwise rotation of far-field forces saw the development of NW-trending folds (F2) and its associated, weak, axial planar cleavage. D3 is an extensional event in which normal faulting, along pre-existing cleavage planes, created a series of rhomboid-shaped, fault-bounded blocks. Normal faults, which bound these blocks, are the dominant structures at Jwaneng Mine. Combined with block rotation and NW-dipping bedding, a horst-like structure on the northwestern limb of a broad, gentle, NE-trending anticline is indicated. The early compressional and subsequent extensional events are consistent throughout the Jwaneng-Ramotswa-Lobatse-Thabazimbi area, suggesting that a large area records the same fault geometry and, consequently, deformational history. It is proposed that Jwaneng Mine is at or near the northernmost limit of the initial, northwards-directed compressional event.

The catalytic chain of human complement subcomponent C1r. Purification and N-terminal amino acid sequences of the major cyanogen bromide-cleavage fragments.

PubMed

Arlaud, G J; Gagnon, J; Porter, R R

1982-01-01

1. The a- and b-chains of reduced and alkylated human complement subcomponent C1r were separated by high-pressure gel-permeation chromatography and isolated in good yield and in pure form. 2. CNBr cleavage of C1r b-chain yielded eight major peptides, which were purified by gel filtration and high-pressure reversed-phase chromatography. As determined from the sum of their amino acid compositions, these peptides accounted for a minimum molecular weight of 28 000, close to the value 29 100 calculated from the whole b-chain. 3. N-Terminal sequence determinations of C1r b-chain and its CNBr-cleavage peptides allowed the identification of about two-thirds of the amino acids of C1r b-chain. From our results, and on the basis of homology with other serine proteinases, an alignment of the eight CNBr-cleavage peptides from C1r b-chain is proposed. 4. The residues forming the 'charge-relay' system of the active site of serine proteinases (His-57, Asp-102 and Ser-195 in the chymotrypsinogen numbering) are found in the corresponding regions of C1r b-chain, and the amino acid sequence around these residues has been determined. 5. The N-terminal sequence of C1r b-chain has been extended to residue 60 and reveals that C1r b-chain lacks the 'histidine loop', a disulphide bond that is present in all other known serine proteinases.

Simulation study of spheroidal dust gains charging: Applicable to dust grain alignment

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

Zahed, H.; Sobhanian, S.; Mahmoodi, J.

2006-09-15

The charging process of nonspherical dust grains in an unmagnetized plasma as well as in the presence of a magnetic field is studied. It is shown that unlike the spherical dust grain, due to nonhomogeneity of charge distribution on the spheroidal dust surface, the resultant electric forces on electrons and ions are different. This process produces some surface charge density gradient on the nonspherical grain surface. Effects of a magnetic field and other plasma parameters on the properties of the dust particulate are studied. It has been shown that the alignment direction could be changed or even reversed with themore » magnetic field and plasma parameters. Finally, the charge distribution on the spheroidal grain surface is studied for different ambient parameters including plasma temperature, neutral collision frequency, and the magnitude of the magnetic field.« less

Specificity and kinetics of haloalkane dehalogenase.

PubMed

Schanstra, J P; Kingma, J; Janssen, D B

1996-06-21

Haloalkane dehalogenase converts halogenated alkanes to their corresponding alcohols. The active site is buried inside the protein and lined with hydrophobic residues. The reaction proceeds via a covalent substrate-enzyme complex. This paper describes a steady-state and pre-steady-state kinetic analysis of the conversion of a number of substrates of the dehalogenase. The kinetic mechanism for the "natural" substrate 1,2-dichloroethane and for the brominated analog and nematocide 1,2-dibromoethane are given. In general, brominated substrates had a lower Km, but a similar kcat than the chlorinated analogs. The rate of C-Br bond cleavage was higher than the rate of C-Cl bond cleavage, which is in agreement with the leaving group abilities of these halogens. The lower Km for brominated compounds therefore originates both from the higher rate of C-Br bond cleavage and from a lower Ks for bromo-compounds. However, the rate-determining step in the conversion (kcat) of 1, 2-dibromoethane and 1,2-dichloroethane was found to be release of the charged halide ion out of the active site cavity, explaining the different Km but similar kcat values for these compounds. The study provides a basis for the analysis of rate-determining steps in the hydrolysis of various environmentally important substrates.

RNase MRP is required for entry of 35S precursor rRNA into the canonical processing pathway.

PubMed

Lindahl, Lasse; Bommankanti, Ananth; Li, Xing; Hayden, Lauren; Jones, Adrienne; Khan, Miriam; Oni, Tolulope; Zengel, Janice M

2009-07-01

RNase MRP is a nucleolar RNA-protein enzyme that participates in the processing of rRNA during ribosome biogenesis. Previous experiments suggested that RNase MRP makes a nonessential cleavage in the first internal transcribed spacer. Here we report experiments with new temperature-sensitive RNase MRP mutants in Saccharomyces cerevisiae that show that the abundance of all early intermediates in the processing pathway is severely reduced upon inactivation of RNase MRP. Transcription of rRNA continues unabated as determined by RNA polymerase run-on transcription, but the precursor rRNA transcript does not accumulate, and appears to be unstable. Taken together, these observations suggest that inactivation of RNase MRP blocks cleavage at sites A0, A1, A2, and A3, which in turn, prevents precursor rRNA from entering the canonical processing pathway (35S > 20S + 27S > 18S + 25S + 5.8S rRNA). Nevertheless, at least some cleavage at the processing site in the second internal transcribed spacer takes place to form an unusual 24S intermediate, suggesting that cleavage at C2 is not blocked. Furthermore, the long form of 5.8S rRNA is made in the absence of RNase MRP activity, but only in the presence of Xrn1p (exonuclease 1), an enzyme not required for the canonical pathway. We conclude that RNase MRP is a key enzyme for initiating the canonical processing of precursor rRNA transcripts, but alternative pathway(s) might provide a backup for production of small amounts of rRNA.

MicroRNA superfamilies descended from miR390 and their roles in secondary small interfering RNA biogenesis in eudicots

USDA-ARS?s Scientific Manuscript database

MiRNAs have been demonstrated to regulate diverse biological processes through cleavage of gene transcripts. Some of miRNAs acquire additional function and their cleavage can incite production of secondary small RNAs which possibly provoke a novel regulatory cascade. In this study, we investigated...

The Role of Magnesium for Geometry and Charge in GTP Hydrolysis, Revealed by Quantum Mechanics/Molecular Mechanics Simulations

PubMed Central

Rudack, Till; Xia, Fei; Schlitter, Jürgen; Kötting, Carsten; Gerwert, Klaus

2012-01-01

The coordination of the magnesium ion in proteins by triphosphates plays an important role in catalytic hydrolysis of GTP or ATP, either in signal transduction or energy conversion. For example, in Ras the magnesium ion contributes to the catalysis of GTP hydrolysis. The cleavage of GTP to GDP and Pi in Ras switches off cellular signaling. We analyzed GTP hydrolysis in water, Ras, and Ras·Ras-GTPase-activating protein using quantum mechanics/molecular mechanics simulations. By comparison of the theoretical IR-difference spectra for magnesium ion coordinated triphosphate to experimental ones, the simulations are validated. We elucidated thereby how the magnesium ion contributes to catalysis. It provides a temporary storage for the electrons taken from the triphosphate and it returns them after bond cleavage and Pi release back to the diphosphate. Furthermore, the Ras·Mg2+ complex forces the triphosphate into a stretched conformation in which the β- and γ-phosphates are coordinated in a bidentate manner. In this conformation, the triphosphate elongates the bond, which has to be cleaved during hydrolysis. Furthermore, the γ-phosphate adopts a more planar structure, driving the conformation of the molecule closer to the hydrolysis transition state. GTPase-activating protein enhances these changes in GTP conformation and charge distribution via the intruding arginine finger. PMID:22853907

Prediction of proprotein convertase cleavage sites.

PubMed

Duckert, Peter; Brunak, Søren; Blom, Nikolaj

2004-01-01

Many secretory proteins and peptides are synthesized as inactive precursors that in addition to signal peptide cleavage undergo post-translational processing to become biologically active polypeptides. Precursors are usually cleaved at sites composed of single or paired basic amino acid residues by members of the subtilisin/kexin-like proprotein convertase (PC) family. In mammals, seven members have been identified, with furin being the one first discovered and best characterized. Recently, the involvement of furin in diseases ranging from Alzheimer's disease and cancer to anthrax and Ebola fever has created additional focus on proprotein processing. We have developed a method for prediction of cleavage sites for PCs based on artificial neural networks. Two different types of neural networks have been constructed: a furin-specific network based on experimental results derived from the literature, and a general PC-specific network trained on data from the Swiss-Prot protein database. The method predicts cleavage sites in independent sequences with a sensitivity of 95% for the furin neural network and 62% for the general PC network. The ProP method is made publicly available at http://www.cbs.dtu.dk/services/ProP.

Biochemical analyses indicate that binding and cleavage specificities define the ordered processing of human Okazaki fragments by Dna2 and FEN1.

PubMed

Gloor, Jason W; Balakrishnan, Lata; Campbell, Judith L; Bambara, Robert A

2012-08-01

In eukaryotic Okazaki fragment processing, the RNA primer is displaced into a single-stranded flap prior to removal. Evidence suggests that some flaps become long before they are cleaved, and that this cleavage involves the sequential action of two nucleases. Strand displacement characteristics of the polymerase show that a short gap precedes the flap during synthesis. Using biochemical techniques, binding and cleavage assays presented here indicate that when the flap is ∼ 30 nt long the nuclease Dna2 can bind with high affinity to the flap and downstream double strand and begin cleavage. When the polymerase idles or dissociates the Dna2 can reorient for additional contacts with the upstream primer region, allowing the nuclease to remain stably bound as the flap is further shortened. The DNA can then equilibrate to a double flap that can bind Dna2 and flap endonuclease (FEN1) simultaneously. When Dna2 shortens the flap even more, FEN1 can displace the Dna2 and cleave at the flap base to make a nick for ligation.

Molecular orbital (SCF-Xα-SW) theory of metal-metal charge transfer processes in minerals

USGS Publications Warehouse

Sherman, David M.

1987-01-01

Electronic transitions between the Fe-Fe bonding and Fe-Fe antibonding orbitals results in the optically-induced intervalence charge transfer bands observed in the electronic spectra of mixed valence minerals. Such transitions are predicted to be polarized along the metal-metal bond direction, in agreement with experimental observations.

Evidence and implications of direct charge excitation as the dominant mechanism in plasmon-mediated photocatalysis

DOE PAGES

Boerigter, Calvin; Campana, Robert; Morabito, Matthew; ...

2016-01-28

Plasmonic metal nanoparticles enhance chemical reactions on their surface when illuminated with light of particular frequencies. It has been shown that these processes are driven by excitation of localized surface plasmon resonance (LSPR). The interaction of LSPR with adsorbate orbitals can lead to the injection of energized charge carriers into the adsorbate, which can result in chemical transformations. The mechanism of the charge injection process (and role of LSPR) is not well understood. Here we shed light on the specifics of this mechanism by coupling optical characterization methods, mainly wavelength-dependent Stokes and anti-Stokes SERS, with kinetic analysis of photocatalytic reactionsmore » in an Ag nanocube–methylene blue plasmonic system. We propose that localized LSPR-induced electric fields result in a direct charge transfer within the molecule–adsorbate system. Lastly, these observations provide a foundation for the development of plasmonic catalysts that can selectively activate targeted chemical bonds, since the mechanism allows for tuning plasmonic nanomaterials in such a way that illumination can selectively enhance desired chemical pathways.« less

Podocyte-associated talin1 is critical for glomerular filtration barrier maintenance

PubMed Central

Tian, Xuefei; Kim, Jin Ju; Monkley, Susan M.; Gotoh, Nanami; Nandez, Ramiro; Soda, Keita; Inoue, Kazunori; Balkin, Daniel M.; Hassan, Hossam; Son, Sung Hyun; Lee, Yashang; Moeckel, Gilbert; Calderwood, David A.; Holzman, Lawrence B.; Critchley, David R.; Zent, Roy; Reiser, Jochen; Ishibe, Shuta

2014-01-01

Podocytes are specialized actin-rich epithelial cells that line the kidney glomerular filtration barrier. The interface between the podocyte and the glomerular basement membrane requires integrins, and defects in either α3 or β1 integrin, or the α3β1 ligand laminin result in nephrotic syndrome in murine models. The large cytoskeletal protein talin1 is not only pivotal for integrin activation, but also directly links integrins to the actin cytoskeleton. Here, we found that mice lacking talin1 specifically in podocytes display severe proteinuria, foot process effacement, and kidney failure. Loss of talin1 in podocytes caused only a modest reduction in β1 integrin activation, podocyte cell adhesion, and cell spreading; however, the actin cytoskeleton of podocytes was profoundly altered by the loss of talin1. Evaluation of murine models of glomerular injury and patients with nephrotic syndrome revealed that calpain-induced talin1 cleavage in podocytes might promote pathogenesis of nephrotic syndrome. Furthermore, pharmacologic inhibition of calpain activity following glomerular injury substantially reduced talin1 cleavage, albuminuria, and foot process effacement. Collectively, these findings indicate that podocyte talin1 is critical for maintaining the integrity of the glomerular filtration barrier and provide insight into the pathogenesis of nephrotic syndrome. PMID:24531545

Product release mechanism and the complete enzyme catalysis cycle in yeast cytosine deaminase (yCD): A computational study.

PubMed

Zhao, Yuan; She, Nai; Zhang, Xin; Wang, Chaojie; Mo, Yirong

2017-08-01

Yeast cytosine deaminase (yCD) is critical in gene-directed enzyme prodrug therapy as it catalyzes the hydrolytic deamination of cytosine. The product (uracil) release process is considered as rate-limiting in the whole enzymatic catalysis and includes the cleavage of the uracil-metal bond and the delivery of free uracil out of the reactive site. Herein extensive combined random acceleration molecular dynamics (RAMD) and molecular dynamics (MD) simulations coupled with the umbrella sampling technique have been performed to study the product transport mechanism. Five channels have been identified, and the thermodynamic and dynamic characterizations for the two most favorable channels have been determined and analyzed. The free energy barrier for the most beneficial pathway is about 13kcal/mol and mainly results from the cleavage of hydrogen bonds between the ligand uracil and surrounding residues Asn51, Glu64, and Asp155. The conjugated rings of Phe114 and Trp152 play gating and guiding roles in the product delivery via π⋯π van der Waals interactions with the product. Finally, the full cycle of the enzymatic catalysis has been determined, making the whole process computationally more precise. Copyright © 2017 Elsevier B.V. All rights reserved.

β- but not γ-secretase proteolysis of APP causes synaptic and memory deficits in a mouse model of dementia.

PubMed

Tamayev, Robert; Matsuda, Shuji; Arancio, Ottavio; D'Adamio, Luciano

2012-03-01

A mutation in the BRI2/ITM2b gene causes loss of BRI2 protein leading to familial Danish dementia (FDD). BRI2 deficiency of FDD provokes an increase in amyloid-β precursor protein (APP) processing since BRI2 is an inhibitor of APP proteolysis, and APP mediates the synaptic/memory deficits in FDD. APP processing is linked to Alzheimer disease (AD) pathogenesis, which is consistent with a common mechanism involving toxic APP metabolites in both dementias. We show that inhibition of APP cleavage by β-secretase rescues synaptic/memory deficits in a mouse model of FDD. β-cleavage of APP yields amino-terminal-soluble APPβ (sAPPβ) and β-carboxyl-terminal fragments (β-CTF). Processing of β-CTF by γ-secretase releases amyloid-β (Aβ), which is assumed to cause AD. However, inhibition of γ-secretase did not ameliorate synaptic/memory deficits of FDD mice. These results suggest that sAPPβ and/or β-CTF, rather than Aβ, are the toxic species causing dementia, and indicate that reducing β-cleavage of APP is an appropriate therapeutic approach to treating human dementias. Our data and the failures of anti-Aβ therapies in humans advise against targeting γ-secretase cleavage of APP and/or Aβ. Copyright © 2012 EMBO Molecular Medicine.

β- but not γ-secretase proteolysis of APP causes synaptic and memory deficits in a mouse model of dementia

PubMed Central

Tamayev, Robert; Matsuda, Shuji; Arancio, Ottavio; D'Adamio, Luciano

2012-01-01

A mutation in the BRI2/ITM2b gene causes loss of BRI2 protein leading to familial Danish dementia (FDD). BRI2 deficiency of FDD provokes an increase in amyloid-β precursor protein (APP) processing since BRI2 is an inhibitor of APP proteolysis, and APP mediates the synaptic/memory deficits in FDD. APP processing is linked to Alzheimer disease (AD) pathogenesis, which is consistent with a common mechanism involving toxic APP metabolites in both dementias. We show that inhibition of APP cleavage by β-secretase rescues synaptic/memory deficits in a mouse model of FDD. β-cleavage of APP yields amino-terminal-soluble APPβ (sAPPβ) and β-carboxyl-terminal fragments (β-CTF). Processing of β-CTF by γ-secretase releases amyloid-β (Aβ), which is assumed to cause AD. However, inhibition of γ-secretase did not ameliorate synaptic/memory deficits of FDD mice. These results suggest that sAPPβ and/or β-CTF, rather than Aβ, are the toxic species causing dementia, and indicate that reducing β-cleavage of APP is an appropriate therapeutic approach to treating human dementias. Our data and the failures of anti-Aβ therapies in humans advise against targeting γ-secretase cleavage of APP and/or Aβ. PMID:22170863

Helium diffusion in carbonates

NASA Astrophysics Data System (ADS)

Amidon, W. H.; Cherniak, D. J.; Watson, E. B.; Hobbs, D.

2013-12-01

The abundance and large grain size of carbonate minerals make them a potentially attractive target for 4He thermochronology and 3He cosmogenic dating, although the diffusive properties of helium in carbonates remain poorly understood. This work characterizes helium diffusion in calcite and dolomite to better understand the crystal-chemical factors controlling He transport and retentivity. Slabs of cleaved natural calcite and dolomite, and polished sections of calcite cut parallel or normal to c, were implanted with 3He at 3 MeV with a dose of 5x1015/cm2. Implanted carbonates were heated in 1-atm furnaces, and 3He distributions following diffusion anneals were profiled with Nuclear Reaction Analysis using the reaction 3He(d,p)4He. For 3He transport normal to cleavage surfaces in calcite, we obtain the following Arrhenius relation over the temperature range 78-300°C: Dcalcite = 9.0x10-9exp(-55 × 6 kJ mol-1/RT) m2sec-1. Diffusion in calcite exhibits marked anisotropy, with diffusion parallel to c about two orders of magnitude slower than diffusion normal to cleavage faces. He diffusivities for transport normal to the c-axis are similar in value to those normal to cleavage surfaces. Our findings are broadly consistent with helium diffusivities from step-heating measurements of calcite by Copeland et al. (2007); these bulk degassing data may reflect varying effects of diffusional anisotropy. Helium diffusion normal to cleavage surfaces in dolomite is significantly slower than diffusion in calcite, and has a much higher activation energy for diffusion. For dolomite, we obtain the following Arrhenius relation for He diffusion over the temperature range 150-400°C: Ddolomite = 9.0x10-8exp(-92 × 9 kJ mol-1/RT) m2sec-1. The role of crystallographic structure in influencing these differences among diffusivities was evaluated using the maximum aperture approach of Cherniak and Watson (2011), in which crystallographic structures are sectioned along possible diffusion directions and the maximum interstitial apertures in each 'slice' in the structure are identified. Preliminary results show that observed differences in diffusivities are consistent with the size of the smallest maximum aperture along each diffusion direction. In calcite, the smallest maximum apertures are ~0.92 and ~0.66 angstroms for cleavage-normal and c-axis parallel directions respectively. In dolomite, the smallest maximum aperture is ~0.78 angstroms for the cleavage normal direction. Work is in progress on characterizing helium diffusion for other orientations in dolomite, and in other carbonates, including aragonite and magnesite, and in implementing these diffusion findings in the interpretation and modeling of bulk volume diffusion in heterogeneous calcite crystals common in many geologic applications. Copeland et al. (2007) GCA 71, 4488-4511 Cherniak and Watson, (2011) Chem. Geo. 288, 149-161

Copper-Catalyzed Oxidative Reaction of β-Keto Sulfones with Alcohols via C-S Bond Cleavage: Reaction Development and Mechanism Study.

PubMed

Du, Bingnan; Wang, Wenmin; Wang, Yang; Qi, Zhenghang; Tian, Jiaqi; Zhou, Jie; Wang, Xiaochen; Han, Jianlin; Ma, Jing; Pan, Yi

2018-02-16

A Cu-catalyzed cascade oxidative radical process of β-keto sulfones with alcohols has been achieved by using oxygen as an oxidant. In this reaction, β-keto sulfones were converted into sulfinate esters under the oxidative conditions via cleavage of C-S bond. Experimental and computational studies demonstrate that a new pathway is involved in this reaction, which proceeds through the formation of the key four-coordinated Cu II intermediate, O-O bond homolysis induced C-S bond cleavage and Cu-catalyzed esterification to form the final products. This reaction provides a new strategy to sulfonate esters and enriches the research content of C-S bond cleavage and transformations. © 2018 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.

Direct probing of electron and hole trapping into nano-floating-gate in organic field-effect transistor nonvolatile memories

NASA Astrophysics Data System (ADS)

Cui, Ze-Qun; Wang, Shun; Chen, Jian-Mei; Gao, Xu; Dong, Bin; Chi, Li-Feng; Wang, Sui-Dong

2015-03-01

Electron and hole trapping into the nano-floating-gate of a pentacene-based organic field-effect transistor nonvolatile memory is directly probed by Kelvin probe force microscopy. The probing is straightforward and non-destructive. The measured surface potential change can quantitatively profile the charge trapping, and the surface characterization results are in good accord with the corresponding device behavior. Both electrons and holes can be trapped into the nano-floating-gate, with a preference of electron trapping than hole trapping. The trapped charge quantity has an approximately linear relation with the programming/erasing gate bias, indicating that the charge trapping in the device is a field-controlled process.

In-vivo quantification of primary microRNA processing by Drosha with a luciferase based system

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

Allegra, Danilo; Cooperation Unit 'Mechanisms of Leukemogenesis', B061, DKFZ, Im Neuenheimer Feld 280, 69120 Heidelberg; Mertens, Daniel, E-mail: daniel.mertens@uniklinik-ulm.de

2011-03-25

Research highlights: {yields} Posttranscriptional regulation of miRNA processing is difficult to quantify. {yields} Our in-vivo processing assay can quantify Drosha cleavage in live cells. {yields} It is based on luciferase reporters fused with pri-miRNAs. {yields} The assay validates the processing defect caused by a mutation in pri-16-1. {yields} It is a sensitive method to quantify pri-miRNA cleavage by Drosha in live cells. -- Abstract: The RNAse III Drosha is responsible for the first step of microRNA maturation, the cleavage of primary miRNA to produce the precursor miRNA. Processing by Drosha is finely regulated and influences the amount of mature microRNAmore » in a cell. We describe in the present work a method to quantify Drosha processing activity in-vivo, which is applicable to any microRNA. With respect to other methods for measuring Drosha activity, our system is faster and scalable, can be used with any cellular system and does not require cell sorting or use of radioactive isotopes. This system is useful to study regulation of Drosha activity in physiological and pathological conditions.« less

[A microstructural approach to fatigue crack processes in poly crystalline BCC materials]. Progress report

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

Gerberich, W.W.

1992-12-31

Objective was to study fatigue where a combination of low temperature and cyclic loading produced cyclic cleavage in bcc Fe-base systems. Both dislocation dynamics and quasi-statics of crack growth were probed. This document reviews progress over the past 6 years: hydrogen embrittlement and cleavage, computations (stress near crack tip), dislocation emission from grain boundaries, fracture process zones, and understanding brittle fracture at the atomistic/dislocation scales and at the microscopic/macroscopic scale.

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

Nabavi, Sadeq; Nazar, Ross N., E-mail: rnnazar@uoguelph.ca

The maturation of many small nuclear RNAs is dependent on RNase III-like endonuclease mediated cleavage, which generates a loading site for the exosome complex that trims the precursor at its 3' end. Using a temperature sensitive Pac1 nuclease, here we show that the endonuclease cleavage is equally important in terminating the transcription of the U2 snRNA in Schizosaccharomyces pombe. Using a temperature sensitive Dhp1p 5' {yields} 3' exonuclease, we demonstrate that it also is an essential component of the termination pathway. Taken together the results support a 'reversed torpedoes' model for the termination and maturation of the U2 snRNA; themore » Pac1 endonuclease cleavage provides entry sites for the 3' and 5' exonuclease activities, leading to RNA maturation in one direction and transcript termination in the other.« less

Recent Advances in Ring-Opening Functionalization of Cycloalkanols by C-C σ-Bond Cleavage.

PubMed

Wu, Xinxin; Zhu, Chen

2018-06-01

Cycloalkanols prove to be privileged precursors for the synthesis of distally substituted alkyl ketones and polycyclic aromatic hydrocarbons (PAHs) by virtue of cleavage of their cyclic C-C bonds. Direct functionalization of cyclobutanols to build up other chemical bonds (e. g., C-F, C-Cl, C-Br, C-N, C-S, C-Se, C-C, etc.) has been achieved by using the ring-opening strategy. Mechanistically, the C-C cleavage of cyclobutanols can be involved in two pathways: (a) transition-metal catalyzed β-carbon elimination; (b) radical-mediated 'radical clock'-type ring opening. The recent advances of our group for the ring-opening functionalization of tertiary cycloalkanols are described in this account. © 2018 The Chemical Society of Japan & Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.

Experimental exploration of the Mulliken-Hush relationship for intramolecular electron transfer reactions.

PubMed

Mukherjee, Tamal; Ito, Naoki; Gould, Ian R

2011-03-17

The Mulliken-Hush (M-H) relationship provides the critical link between optical and thermal electron transfer processes, and yet very little direct experimental support for its applicability has been provided. Dicyanovinylazaadamantane (DCVA) represents a simple two-state (neutral/charge-transfer) intramolecular electron transfer system that exhibits charge-transfer absorption and emission spectra that are readily measurable in solvents with a wide range of polarities. In this regard it represents an ideal model system for studying the factors that control both optical charge separation (absorption) and recombination (emission) processes in solution. Here we explore the applicability of the M-H relation to quantitative descriptions of the optical charge-transfer processes in DCVA. For DCVA, the measured radiative rate constants exhibit a linear dependence on transition energy, and transition dipole moments exhibit an inverse dependence on transition energy, consistent with the M-H relationship.

Where does the electron go? The nature of ortho/para and meta group directing in electrophilic aromatic substitution

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

Liu, Shubin, E-mail: shubin@email.unc.edu

Electrophilic aromatic substitution as one of the most fundamental chemical processes is affected by atoms or groups already attached to the aromatic ring. The groups that promote substitution at the ortho/para or meta positions are, respectively, called ortho/para and meta directing groups, which are often characterized by their capability to donate electrons to or withdraw electrons from the ring. Though resonance and inductive effects have been employed in textbooks to explain this phenomenon, no satisfactory quantitative interpretation is available in the literature. Here, based on the theoretical framework we recently established in density functional reactivity theory (DFRT), where electrophilicity andmore » nucleophilicity are simultaneously quantified by the Hirshfeld charge, the nature of ortho/para and meta group directing is systematically investigated for a total of 85 systems. We find that regioselectivity of electrophilic attacks is determined by the Hirshfeld charge distribution on the aromatic ring. Ortho/para directing groups have most negative charges on the ortho/para positions, while meta directing groups often possess the largest negative charge on the meta position. Our results do not support that ortho/para directing groups are electron donors and meta directing groups are electron acceptors. Most neutral species we studied here are electron withdrawal in nature. Anionic systems are always electron donors. There are also electron donors serving as meta directing groups. We predicted ortho/para and meta group directing behaviors for a list of groups whose regioselectivity is previously unknown. In addition, strong linear correlations between the Hirshfeld charge and the highest occupied molecular orbital have been observed, providing the first link between the frontier molecular orbital theory and DFRT.« less

Characterization of geothermal paleosystem in the Lesser Antilles volcanic arc: structural, petrographic, thermodynamic and petrophysics analysis of Terre-de-Haut (Les Saintes archipelago, Lesser Antilles)

NASA Astrophysics Data System (ADS)

Favier, Alexiane; Navelot, Vivien; Verati, Chrystèle; Lardeaux, Jean-Marc; Corsini, Michel; Diraison, Marc; Géraud, Yves; Mercier de Lépinay, Jeanne; Munschy, Marc

2017-04-01

This survey takes part in the GEOTREF project (high enthalpy geothermal energy in fractured reservoirs), supported by the French government program "Investments for the future". The program focuses on the exploration of geothermal resource in the Lesser Antilles volcanic arc. An exclusive license has been issued in the Vieux-Habitants area (Basse-Terre, Guadeloupe) to carry on the development of high-temperature geothermal energy in this active volcanic region. The deep geothermal reservoir on the Basse-Terre island could be characterized in exhumed paleosystems. The reference paleosystem in the Guadeloupe archipelago is located in Terre-de-Haut. Four major fault directions have been highlighted N000-N020, N050-N070, N090-N110 and N130-N140. Field observations emphasize three major cleavage directions overlaying the fault systems: N035-N060, N080-N110, N145-N165. Volcanic rocks affected by cleavage display several metamorphic transformation grades. The more transformed calc-alkaline rocks are located at the intersection of several cleavage directions. Mineralogical transformations due to metamorphism and surimposed fractures are also responsible for strong changes of petrophysical properties. In comparison with the reference protolith of andesitic lava flows outcropping in Vieux-Habitants, which have porosity and permeability lower than 5 % and 10-15 m2, andesites of Terre-de-Haut have better reservoir properties with connected porosity and permeability higher than 15 % and 10-14-10-15 m2 respectively. Thermodynamic modelling based on petrography and chemical composition of the most transformed rocks highlights a steady state mineral assemblage between 0.25 - 1.5 kbar and 350 - 450 ˚ C. It corresponds to a geothermal gradient higher than 120 to 150˚ C/km. This is consistent with temperatures measured in Bouillante wells. However, this geothermal gradient is notably higher to a usual volcanic arc conductive gradient estimated to 70-100˚ C/km. It can be explained by the addition of a convective processes caused by hydrothermal fluid flows.

Special raster scanning for reduction of charging effects in scanning electron microscopy.

PubMed

Suzuki, Kazuhiko; Oho, Eisaku

2014-01-01

A special raster scanning (SRS) method for reduction of charging effects is developed for the field of SEM. Both a conventional fast scan (horizontal direction) and an unusual scan (vertical direction) are adopted for acquiring raw data consisting of many sub-images. These data are converted to a proper SEM image using digital image processing techniques. About sharpness of the image and reduction of charging effects, the SRS is compared with the conventional fast scan (with frame-averaging) and the conventional slow scan. Experimental results show the effectiveness of SRS images. By a successful combination of the proposed scanning method and low accelerating voltage (LV)-SEMs, it is expected that higher-quality SEM images can be more easily acquired by the considerable reduction of charging effects, while maintaining the resolution. © 2013 Wiley Periodicals, Inc.

Proteomic Analysis of Tendon Extracellular Matrix Reveals Disease Stage-specific Fragmentation and Differential Cleavage of COMP (Cartilage Oligomeric Matrix Protein)*

PubMed Central

Dakin, Stephanie Georgina; Smith, Roger Kenneth Whealands; Heinegård, Dick; Önnerfjord, Patrik; Khabut, Areej; Dudhia, Jayesh

2014-01-01

During inflammatory processes the extracellular matrix (ECM) is extensively remodeled, and many of the constituent components are released as proteolytically cleaved fragments. These degradative processes are better documented for inflammatory joint diseases than tendinopathy even though the pathogenesis has many similarities. The aims of this study were to investigate the proteomic composition of injured tendons during early and late disease stages to identify disease-specific cleavage patterns of the ECM protein cartilage oligomeric matrix protein (COMP). In addition to characterizing fragments released in naturally occurring disease, we hypothesized that stimulation of tendon explants with proinflammatory mediators in vitro would induce fragments of COMP analogous to natural disease. Therefore, normal tendon explants were stimulated with IL-1β and prostaglandin E2, and their effects on the release of COMP and its cleavage patterns were characterized. Analyses of injured tendons identified an altered proteomic composition of the ECM at all stages post injury, showing protein fragments that were specific to disease stage. IL-1β enhanced the proteolytic cleavage and release of COMP from tendon explants, whereas PGE2 had no catabolic effect. Of the cleavage fragments identified in early stage tendon disease, two fragments were generated by an IL-1-mediated mechanism. These fragments provide a platform for the development of neo-epitope assays specific to injury stage for tendon disease. PMID:24398684

Crystal Structure of the 25 kDa Subunit of Human Cleavage Factor I{m}

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

Coseno,M.; Martin, G.; Berger, C.

Cleavage factor Im is an essential component of the pre-messenger RNA 3'-end processing machinery in higher eukaryotes, participating in both the polyadenylation and cleavage steps. Cleavage factor Im is an oligomer composed of a small 25 kDa subunit (CF Im25) and a variable larger subunit of either 59, 68 or 72 kDa. The small subunit also interacts with RNA, poly(A) polymerase, and the nuclear poly(A)-binding protein. These protein-protein interactions are thought to be facilitated by the Nudix domain of CF Im25, a hydrolase motif with a characteristic {alpha}/{beta}/{alpha} fold and a conserved catalytic sequence or Nudix box. We present heremore » the crystal structures of human CF Im25 in its free and diadenosine tetraphosphate (Ap4A) bound forms at 1.85 and 1.80 Angstroms, respectively. CF Im25 crystallizes as a dimer and presents the classical Nudix fold. Results from crystallographic and biochemical experiments suggest that CF Im25 makes use of its Nudix fold to bind but not hydrolyze ATP and Ap4A. The complex and apo protein structures provide insight into the active oligomeric state of CF Im and suggest a possible role of nucleotide binding in either the polyadenylation and/or cleavage steps of pre-messenger RNA 3'-end processing.« less

Crystal structure of the 25 kDa subunit of human cleavage factor Im

PubMed Central

Coseno, Molly; Martin, Georges; Berger, Christopher; Gilmartin, Gregory; Keller, Walter; Doublié, Sylvie

2008-01-01

Cleavage factor Im is an essential component of the pre-messenger RNA 3′-end processing machinery in higher eukaryotes, participating in both the polyadenylation and cleavage steps. Cleavage factor Im is an oligomer composed of a small 25 kDa subunit (CF Im25) and a variable larger subunit of either 59, 68 or 72 kDa. The small subunit also interacts with RNA, poly(A) polymerase, and the nuclear poly(A)-binding protein. These protein–protein interactions are thought to be facilitated by the Nudix domain of CF Im25, a hydrolase motif with a characteristic α/β/α fold and a conserved catalytic sequence or Nudix box. We present here the crystal structures of human CF Im25 in its free and diadenosine tetraphosphate (Ap4A) bound forms at 1.85 and 1.80 Å, respectively. CF Im25 crystallizes as a dimer and presents the classical Nudix fold. Results from crystallographic and biochemical experiments suggest that CF Im25 makes use of its Nudix fold to bind but not hydrolyze ATP and Ap4A. The complex and apo protein structures provide insight into the active oligomeric state of CF Im and suggest a possible role of nucleotide binding in either the polyadenylation and/or cleavage steps of pre-messenger RNA 3′-end processing. PMID:18445629

Individual breathing reactions measured in hemoglobin by hydrogen exchange methods.

PubMed Central

Englander, S W; Calhoun, D B; Englander, J J; Kallenbach, N R; Liem, R K; Malin, E L; Mandal, C; Rogero, J R

1980-01-01

Protein hydrogen exchange is generally believed to register some aspects of internal protein dynamics, but the kind of motion at work is not clear. Experiments are being done to identify the determinants of protein hydrogen exchange and to distinguish between local unfolding and accessibility-penetration mechanisms. Results with small molecules, polynucleotides, and proteins demonstrate that solvent accessibility is by no means sufficient for fast exchange. H-exchange slowing is quite generally connected with intramolecular H-bonding, and the exchange process depends pivotally on transient H-bond cleavage. At least in alpha-helical structures, the cooperative aspect of H-bond cleavage must be expressed in local unfolding reactions. Results obtained by use of a difference hydrogen exchange method appear to provide a direct measurement of transient, cooperative, local unfolding reactions in hemoglobin. The reality of these supposed coherent breathing units is being tested by using the difference H-exchange approach to tritium label the units one at a time and then attempting to locate the tritium by fragmenting the protein, separating the fragments, and testing them for label. Early results demonstrate the feasibility of this approach. PMID:7248462

Intra-domain phage display (ID-PhD) of peptides and protein mini-domains censored from canonical pIII phage display.

PubMed

Tjhung, Katrina F; Deiss, Frédérique; Tran, Jessica; Chou, Ying; Derda, Ratmir

2015-01-01

In this paper, we describe multivalent display of peptide and protein sequences typically censored from traditional N-terminal display on protein pIII of filamentous bacteriophage M13. Using site-directed mutagenesis of commercially available M13KE phage cloning vector, we introduced sites that permit efficient cloning using restriction enzymes between domains N1 and N2 of the pIII protein. As infectivity of phage is directly linked to the integrity of the connection between N1 and N2 domains, intra-domain phage display (ID-PhD) allows for simple quality control of the display and the natural variations in the displayed sequences. Additionally, direct linkage to phage propagation allows efficient monitoring of sequence cleavage, providing a convenient system for selection and evolution of protease-susceptible or protease-resistant sequences. As an example of the benefits of such an ID-PhD system, we displayed a negatively charged FLAG sequence, which is known to be post-translationally excised from pIII when displayed on the N-terminus, as well as positively charged sequences which suppress production of phage when displayed on the N-terminus. ID-PhD of FLAG exhibited sub-nanomolar apparent Kd suggesting multivalent nature of the display. A TEV-protease recognition sequence (TEVrs) co-expressed in tandem with FLAG, allowed us to demonstrate that 99.9997% of the phage displayed the FLAG-TEVrs tandem and can be recognized and cleaved by TEV-protease. The residual 0.0003% consisted of phage clones that have excised the insert from their genome. ID-PhD is also amenable to display of protein mini-domains, such as the 33-residue minimized Z-domain of protein A. We show that it is thus possible to use ID-PhD for multivalent display and selection of mini-domain proteins (Affibodies, scFv, etc.).

Energetics and dynamics of the fragmentation reactions of protonated peptides containing methionine sulfoxide or aspartic acid via energy- and time-resolved surface induced dissociation.

PubMed

Lioe, Hadi; Laskin, Julia; Reid, Gavin E; O'Hair, Richard A J

2007-10-25

The surface-induced dissociation (SID) of six model peptides containing either methionine sulfoxide or aspartic acid (GAILM(O)GAILR, GAILM(O)GAILK, GAILM(O)GAILA, GAILDGAILR, GAILDGAILK, and GAILDGAILA) have been studied using a specially configured Fourier transform ion-cyclotron resonance mass spectrometer (FT-ICR MS). In particular, we have investigated the energetics and dynamics associated with (i) preferential cleavage of the methionine sulfoxide side chain via the loss of CH3SOH (64 Da), and (ii) preferential cleavage of the amide bond C-terminal to aspartic acid. The role of proton mobility in these selective bond cleavage reactions was examined by changing the C-terminal residue of the peptide from arginine (nonmobile proton conditions) to lysine (partially mobile proton conditions) to alanine (mobile proton conditions). Time- and energy-resolved fragmentation efficiency curves (TFECs) reveal that selective cleavages due to the methionine sulfoxide and aspartic acid residues are characterized by slow fragmentation kinetics. RRKM modeling of the experimental data suggests that the slow kinetics is associated with large negative entropy effects and these may be due to the presence of rearrangements prior to fragmentation. It was found that the Arrhenius pre-exponential factor (A) for peptide fragmentations occurring via selective bond cleavages are 1-2 orders of magnitude lower than nonselective peptide fragmentation reactions, while the dissociation threshold (E0) is relatively invariant. This means that selective bond cleavage is kinetically disfavored compared to nonselective amide bond cleavage. It was also found that the energetics and dynamics for the preferential loss of CH3SOH from peptide ions containing methionine sulfoxide are very similar to selective C-terminal amide bond cleavage at the aspartic acid residue. These results suggest that while preferential cleavage can compete with amide bond cleavage energetically, dynamically, these processes are much slower compared to amide bond cleavage, explaining why these selective bond cleavages are not observed if fragmentation is performed under mobile proton conditions. This study further affirms that fragmentation of peptide ions in the gas phase are predominantly governed by entropic effects.

Thermal decomposition pathways of ethane

NASA Astrophysics Data System (ADS)

Gordon, Mark S.; Truong, Thanh N.; Pople, John A.

1986-10-01

The alternate thermal decomposition pathways for ethane in its ground state have been investigated, using ab initio electronic structure calculations. Single-point energies were obtained at the full MP4/6-311 G ∗∗ level, using 6-31 G ∗ geometries for reactant, products, and transition states. The thermodynamically favored products are ethylene and molecular hydrogen, but a very large barrier (130 kcal/mol) is found for the direct 1,2-elimination of hydrogen. When calculated barriers are taken into account, the lowest-energy process is the homolytic cleavage of the C-C bond to form two methyl radicals.

Initial decomposition of the condensed-phase β-HMX under shock waves: molecular dynamics simulations.

PubMed

Ge, Ni-Na; Wei, Yong-Kai; Ji, Guang-Fu; Chen, Xiang-Rong; Zhao, Feng; Wei, Dong-Qing

2012-11-26

We have performed quantum-based multiscale simulations to study the initial chemical processes of condensed-phase octahydro-1,3,5,7-tetranitro-1,3,5,7-tetrazocine (HMX) under shock wave loading. A self-consistent charge density-functional tight-binding (SCC-DFTB) method was employed. The results show that the initial decomposition of shocked HMX is triggered by the N-NO(2) bond breaking under the low velocity impact (8 km/s). As the shock velocity increases (11 km/s), the homolytic cleavage of the N-NO(2) bond is suppressed under high pressure, the C-H bond dissociation becomes the primary pathway for HMX decomposition in its early stages. It is accompanied by a five-membered ring formation and hydrogen transfer from the CH(2) group to the -NO(2) group. Our simulations suggest that the initial chemical processes of shocked HMX are dependent on the impact velocity, which gain new insights into the initial decomposition mechanism of HMX upon shock loading at the atomistic level, and have important implications for understanding and development of energetic materials.

Dengue and Zika viruses subvert reticulophagy by NS2B3-mediated cleavage of FAM134B.

PubMed

Lennemann, Nicholas J; Coyne, Carolyn B

2017-02-01

The endoplasmic reticulum (ER) is exploited by several diverse viruses during their infectious life cycles. Flaviviruses, including dengue virus (DENV) and Zika virus (ZIKV), utilize the ER as a source of membranes to establish their replication organelles and to facilitate their assembly and eventual maturation along the secretory pathway. To maintain normal homeostasis, host cells have evolved highly efficient processes to dynamically regulate the ER, such as through reticulophagy, a selective form of autophagy that leads to ER degradation. Here, we identify the ER-localized reticulophagy receptor FAM134B as a host cell restriction factor for both DENV and ZIKV. We show that RNAi-mediated depletion of FAM134B significantly enhances both DENV and ZIKV replication at an early stage of the viral life cycle. Consistent with its role as an antiviral host factor, we found that several flaviviruses including DENV, ZIKV, and West Nile virus (WNV), utilize their NS3 virally-encoded proteases to directly cleave FAM134B at a single site within its reticulon homology domain (RHD). Mechanistically, we show that NS3-mediated cleavage of FAM134B blocks the formation of ER and viral protein-enriched autophagosomes, suggesting that the cleavage of FAM134B serves to specifically suppress the reticulophagy pathway. These findings thus point to an important role for FAM134B and reticulophagy in the regulation of flavivirus infection and suggest that these viruses specifically target these pathways to promote viral replication.

Effects of flexibility of the α2 chain of type I collagen on collagenase cleavage.

PubMed

Mekkat, Arya; Poppleton, Erik; An, Bo; Visse, Robert; Nagase, Hideaki; Kaplan, David L; Brodsky, Barbara; Lin, Yu-Shan

2018-05-12

Cleavage of collagen by collagenases such as matrix metalloproteinase 1 (MMP-1) is a key step in development, tissue remodeling, and tumor proliferation. The abundant heterotrimeric type I collagen composed of two α1(I) chains and one α2(I) chain is efficiently cleaved by MMP-1 at a unique site in the triple helix, a process which may be initiated by local unfolding within the peptide chains. Atypical homotrimers of the α1(I) chain, found in embryonic and cancer tissues, are very resistant to MMP cleavage. To investigate MMP-1 cleavage, recombinant homotrimers were constructed with sequences from the MMP cleavage regions of human collagen chains inserted into a host bacterial collagen protein system. All triple-helical constructs were cleaved by MMP-1, with α2(I) homotrimers cleaved efficiently at a rate similar to that seen for α1(II) and α1(III) homotrimers, while α1(I) homotrimers were cleaved at a much slower rate. The introduction of destabilizing Gly to Ser mutations within the human collagenase susceptible region of the α2(I) chain did not interfere with MMP-1 cleavage. Molecular dynamics simulations indicated a greater degree of transient hydrogen bond breaking in α2(I) homotrimers compared with α1(I) homotrimers at the MMP-1 cleavage site, and showed an extensive disruption of hydrogen bonding in the presence of a Gly to Ser mutation, consistent with chymotrypsin digestion results. This study indicates that α2(I) homotrimers are susceptible to MMP-1, proves that the presence of an α1(I) chain is not a requirement for α2(I) cleavage, and supports the importance of local unfolding of α2(I) in collagenase cleavage. Copyright © 2018. Published by Elsevier Inc.

A peptide-based approach to evaluate the adaptability of influenza A virus to humans based on its hemagglutinin proteolytic cleavage site

PubMed Central

Straus, Marco R.; Whittaker, Gary R.

2017-01-01

Cleavage activation of the hemagglutinin (HA) protein by host proteases is a crucial step in the infection process of influenza A viruses (IAV). However, IAV exists in eighteen different HA subtypes in nature and their cleavage sites vary considerably. There is uncertainty regarding which specific proteases activate a given HA in the human respiratory tract. Understanding the relationship between different HA subtypes and human-specific proteases will be valuable in assessing the pandemic potential of circulating viruses. Here we utilized fluorogenic peptides mimicking the HA cleavage motif of representative IAV strains causing disease in humans or of zoonotic/pandemic potential and tested them with a range of proteases known to be present in the human respiratory tract. Our results show that peptides from the H1, H2 and H3 subtypes are cleaved efficiently by a wide range of proteases including trypsin, matriptase, human airway tryptase (HAT), kallikrein-related peptidases 5 (KLK5) and 12 (KLK12) and plasmin. Regarding IAVs currently of concern for human adaptation, cleavage site peptides from H10 viruses showed very limited cleavage by respiratory tract proteases. Peptide mimics from H6 viruses showed broader cleavage by respiratory tract proteases, while H5, H7 and H9 subtypes showed variable cleavage; particularly matriptase appeared to be a key protease capable of activating IAVs. We also tested HA substrate specificity of Factor Xa, a protease required for HA cleavage in chicken embryos and relevant for influenza virus production in eggs. Overall our data provide novel tool allowing the assessment of human adaptation of IAV HA subtypes. PMID:28358853

Electron Transfer Dissociation of iTRAQ Labeled Peptide Ions

PubMed Central

Han, Hongling; Pappin, Darryl J.; Ross, Philip L; McLuckey, Scott A.

2009-01-01

Triply and doubly charged iTRAQ (isobaric tagging for relative and absolute quantitation) labeled peptide cations from a tryptic peptide mixture of bovine carbonic anhydrase II were subjected to electron transfer ion/ion reactions to investigate the effect of charge bearing modifications associated with iTRAQ on the fragmentation pattern. It was noted that electron transfer dissociation (ETD) of triply charged or activated ETD (ETD + supplemental collisional activation of intact electron transfer species) of doubly charged iTRAQ tagged peptide ions yielded extensive sequence information, in analogy with ETD of unmodified peptide ions. That is, addition of the fixed charge iTRAQ tag showed relatively little deleterious effect on the ETD performance of the modified peptides. ETD of the triply charged iTRAQ labeled peptide ions followed by collision-induced dissociation (CID) of the product ion at m/z 162 yielded the reporter ion at m/z 116, which is the reporter ion used for quantitation via CID of the same precursor ions. The reporter ion formed via the two-step activation process is expected to provide quantitative information similar to that directly produced from CID. A 103 Da neutral loss species observed in the ETD spectra of all the triply and doubly charged iTRAQ labeled peptide ions is unique to the 116 Da iTRAQ reagent, which implies that this process also has potential for quantitation of peptides/proteins. Therefore, ETD with or without supplemental collisional activation, depending on the precursor ion charge state, has the potential to directly identify and quantify the peptides/proteins simultaneously using existing iTRAQ reagents. PMID:18646790

Stochastic resetting in backtrack recovery by RNA polymerases

NASA Astrophysics Data System (ADS)

Roldán, Édgar; Lisica, Ana; Sánchez-Taltavull, Daniel; Grill, Stephan W.

2016-06-01

Transcription is a key process in gene expression, in which RNA polymerases produce a complementary RNA copy from a DNA template. RNA polymerization is frequently interrupted by backtracking, a process in which polymerases perform a random walk along the DNA template. Recovery of polymerases from the transcriptionally inactive backtracked state is determined by a kinetic competition between one-dimensional diffusion and RNA cleavage. Here we describe backtrack recovery as a continuous-time random walk, where the time for a polymerase to recover from a backtrack of a given depth is described as a first-passage time of a random walker to reach an absorbing state. We represent RNA cleavage as a stochastic resetting process and derive exact expressions for the recovery time distributions and mean recovery times from a given initial backtrack depth for both continuous and discrete-lattice descriptions of the random walk. We show that recovery time statistics do not depend on the discreteness of the DNA lattice when the rate of one-dimensional diffusion is large compared to the rate of cleavage.

Progressive deformation and superposed fabrics related to Cretaceous crustal underthrusting in western Arizona, U.S.A.

USGS Publications Warehouse

Laubach, S.E.; Reynolds, S.J.; Spencer, J.E.; Marshak, S.

1989-01-01

In the Maria fold and thrust belt, a newly recognized E-trending Cretaceous orogenic belt in the southwestern United States, ductile thrusts, large folds and superposed cleavages record discordant emplacement of crystalline thrust sheets across previously tilted sections of crust. Style of deformation and direction of thrusting are in sharp contrast to those of the foreland fold-thrust belt in adjacent segments of the Cordillera. The net effect of polyphase deformation in the Maria belt was underthrusting of Paleozoic and Mesozoic metasedimentary rocks under the Proterozoic crystalline basement of North America. The structure of the Maria belt is illustrated by the Granite Wash Mountains in west-central Arizona, where at least four non-coaxial deformation events (D1-D4) occurred during the Cretaceous. SSE-facing D1 folds are associated with S-directed thrusts and a low-grade slaty cleavage. D1 structures are truncated by the gently-dipping Hercules thrust zone (D2), a regional SW-vergent shear zone that placed Proterozoic and Jurassic crystalline rocks over upturned Paleozoic and Mesozoic supracrustal rocks. Exposures across the footwall margin of the Hercules thrust zone show the progressive development of folds, cleavage and metamorphism related to thrusting. D3 and D4 structures include open folds and spaced cleavages that refold or transect D1 and D2 folds. The D2 Hercules thrust zone and a D3 shear zone are discordantly crosscut by late Cretaceous plutons. ?? 1989.

Cleavage strain in the Variscan fold belt, County Cork, Ireland, estimated from stretched arsenopyrite rosettes

USGS Publications Warehouse

Ford, M.; Ferguson, C.C.

1985-01-01

In south-west Ireland, hydrothermally formed arsenopyrite crystals in a Devonian mudstone have responded to Variscan deformation by brittle extension fracture and fragment separation. The interfragment gaps and terminal extension zones of each crystal are infilled with fibrous quartz. Stretches within the cleavage plane have been calculated by the various methods available, most of which can be modified to incorporate terminal extension zones. The Strain Reversal Method is the most accurate currently available but still gives a minimum estimate of the overall strain. The more direct Hossain method, which gives only slightly lower estimates with this data, is more practical for field use. A strain ellipse can be estimated from each crystal rosette composed of three laths (assuming the original interlimb angles were all 60??) and, because actual rather than relative stretches are estimated, this provides a lower bound to the area increase in the plane of cleavage. Based on the average of our calculated strain ellipses this area increase is at least 114% and implies an average shortening across the cleavage of at least 53%. However, several lines of evidence suggest that the cleavage deformation was more intense and more oblate than that calculated, and we argue that a 300% area increase in the cleavage plane and 75% shortening across the cleavage are more realistic estimates of the true strain. Furthermore, the along-strike elongation indicated is at least 80%, which may be regionally significant. Estimates of orogenic contraction derived from balanced section construction should therefore take into account the possibility of a substantial strike elongation, and tectonic models that can accommodate such elongations need to be developed. ?? 1985.

Structural and Functional Characterization of Cleavage and Inactivation of Human Serine Protease Inhibitors by the Bacterial SPATE Protease EspPα from Enterohemorrhagic E. coli

PubMed Central

Weiss, André; Joerss, Hanna; Brockmeyer, Jens

2014-01-01

EspPα and EspI are serine protease autotransporters found in enterohemorrhagic Escherichia coli. They both belong to the SPATE autotransporter family and are believed to contribute to pathogenicity via proteolytic cleavage and inactivation of different key host proteins during infection. Here, we describe the specific cleavage and functional inactivation of serine protease inhibitors (serpins) by EspPα and compare this activity with the related SPATE EspI. Serpins are structurally related proteins that regulate vital protease cascades, such as blood coagulation and inflammatory host response. For the rapid determination of serpin cleavage sites, we applied direct MALDI-TOF-MS or ESI-FTMS analysis of coincubations of serpins and SPATE proteases and confirmed observed cleavage positions using in-gel-digest of SDS-PAGE-separated degradation products. Activities of both serpin and SPATE protease were assessed in a newly developed photometrical assay using chromogenic peptide substrates. EspPα cleaved the serpins α1-protease inhibitor (α1-PI), α1-antichymotrypsin, angiotensinogen, and α2-antiplasmin. Serpin cleavage led to loss of inhibitory function as demonstrated for α1-PI while EspPα activity was not affected. Notably, EspPα showed pronounced specificity and cleaved procoagulatory serpins such as α2-antiplasmin while the anticoagulatory antithrombin III was not affected. Together with recently published research, this underlines the interference of EspPα with hemostasis or inflammatory responses during infection, while the observed interaction of EspI with serpins is likely to be not physiologically relevant. EspPα-mediated serpin cleavage occurred always in flexible loops, indicating that this structural motif might be required for substrate recognition. PMID:25347319

Sequence features associated with the cleavage efficiency of CRISPR/Cas9 system.

PubMed

Liu, Xiaoxi; Homma, Ayaka; Sayadi, Jamasb; Yang, Shu; Ohashi, Jun; Takumi, Toru

2016-01-27

The CRISPR-Cas9 system has recently emerged as a versatile tool for biological and medical research. In this system, a single guide RNA (sgRNA) directs the endonuclease Cas9 to a targeted DNA sequence for site-specific manipulation. In addition to this targeting function, the sgRNA has also been shown to play a role in activating the endonuclease activity of Cas9. This dual function of the sgRNA likely underlies observations that different sgRNAs have varying on-target activities. Currently, our understanding of the relationship between sequence features of sgRNAs and their on-target cleavage efficiencies remains limited, largely due to difficulties in assessing the cleavage capacity of a large number of sgRNAs. In this study, we evaluated the cleavage activities of 218 sgRNAs using in vitro Surveyor assays. We found that nucleotides at both PAM-distal and PAM-proximal regions of the sgRNA are significantly correlated with on-target efficiency. Furthermore, we also demonstrated that the genomic context of the targeted DNA, the GC percentage, and the secondary structure of sgRNA are critical factors contributing to cleavage efficiency. In summary, our study reveals important parameters for the design of sgRNAs with high on-target efficiencies, especially in the context of high throughput applications.

Novel meiotic miRNAs and indications for a role of phasiRNAs in meiosis

USDA-ARS?s Scientific Manuscript database

Small RNAs (sRNA) add additional layers to the regulation of gene expression, with siRNAs directing gene silencing at the DNA level by RdDM (RNA-directed DNA methylation), and miRNAs directing post-transcriptional regulation of specific target genes, mostly by mRNA cleavage. We used manually isolate...

What Hinders Electron Transfer Dissociation (ETD) of DNA Cations?

NASA Astrophysics Data System (ADS)

Hari, Yvonne; Leumann, Christian J.; Schürch, Stefan

2017-12-01

Radical activation methods, such as electron transfer dissociation (ETD), produce structural information complementary to collision-induced dissociation. Herein, electron transfer dissociation of 3-fold protonated DNA hexamers was studied to gain insight into the fragmentation mechanism. The fragmentation patterns of a large set of DNA hexamers confirm cytosine as the primary target of electron transfer. The reported data reveal backbone cleavage by internal electron transfer from the nucleobase to the phosphate linker leading either to a•/ w or d/ z• ion pairs. This reaction pathway contrasts with previous findings on the dissociation processes after electron capture by DNA cations, suggesting multiple, parallel dissociation channels. However, all these channels merely result in partial fragmentation of the precursor ion because the charge-reduced DNA radical cations are quite stable. Two hypotheses are put forward to explain the low dissociation yield of DNA radical cations: it is either attributed to non-covalent interactions between complementary fragments or to the stabilization of the unpaired electron in stacked nucleobases. MS3 experiments suggest that the charge-reduced species is the intact oligonucleotide. Moreover, introducing abasic sites significantly increases the dissociation yield of DNA cations. Consequently, the stabilization of the unpaired electron by π-π-stacking provides an appropriate rationale for the high intensity of DNA radical cations after electron transfer. [Figure not available: see fulltext.

Low-energy collision-induced fragmentation of negative ions derived from ortho-, meta-, and para-hydroxyphenyl carbaldehydes, ketones, and related compounds.

PubMed

Attygalle, Athula B; Ruzicka, Josef; Varughese, Deepu; Bialecki, Jason B; Jafri, Sayed

2007-09-01

Collision-induced dissociation (CID) mass spectra of anions derived from several hydroxyphenyl carbaldehydes and ketones were recorded and mechanistically rationalized. For example, the spectrum of m/z 121 ion of deprotonated ortho-hydroxybenzaldehyde shows an intense peak at m/z 93 for a loss of carbon monoxide attributable to an ortho-effect mediated by a charge-directed heterolytic fragmentation mechanism. In contrast, the m/z 121 ion derived from meta and para isomers undergoes a charge-remote homolytic cleavage to eliminate an *H and form a distonic anion radical, which eventually loses CO to produce a peak at m/z 92. In fact, for the para isomer, this two-step homolytic mechanism is the most dominant fragmentation pathway. The spectrum of the meta isomer on the other hand, shows two predominant peaks at m/z 92 and 93 representing both homolytic and heterolytic fragmentations, respectively. (18)O-isotope-labeling studies confirmed that the oxygen in the CO molecule that is eliminated from the anion of meta-hydroxybenzaldehyde originates from either the aldehydic or the phenolic group. In contrast, anions of ortho-hydroxybenzaldehyde and 2-hydroxy-1-naphthaldehyde, both of which show two consecutive CO eliminations, specifically lose the carbonyl oxygen first, followed by that of the phenolic group. Anions from 2-hydroxyphenyl alkyl ketones lose a ketene by a hydrogen transfer predominantly from the alpha position. Interestingly, a very significant charge-remote 1,4-elimination of a H(2) molecule was observed from the anion derived from 2,4-dihydroxybenzaldehyde. For this mechanism to operate, a labile hydrogen atom should be available on the hydroxyl group adjacent to the carbaldehyde functionality.

[Causal mechanisms of nuclear movement and division during early cleavage stages in the egg of a gall midge,Wachtliella persicariae L.

PubMed

Wolf, Rainer

1973-03-01

Between each mitotic cycle, the cleavage nuclei ofWachtliella move over long distances, thus populating the ooplasm within a short time. Besides being shifted passively by flowing pulses of the ooplasm, thenuclei are also migrating actively. The active movements are accompanied by such oscillations of yolk particles as are known from the eggs of other insects, too. For a closer analysis of these "quivering movements" the inseminated eggs werepressed, either totally or partially, reducing their smaller diameters to ooplasmic layers of 30 μm or between 12 and 4 μm, respectively. Along with the experimental reduction of the radius of the curvature at the egg surface, there is anincreased tendency of cell membrane formation, resulting in anearly total cleavage already at the 2-nuclei-stage. Furthermore, theinitial region of cleavage (= initial region of quivering movements)may be shifted to a site free from nuclei; the initial region even may becomesplit up into two, one near each of the egg's poles. Yet, in flattened eggs, division and migration activities of the nuclei are not prevented. Untreated as well as flattened eggs have been analysed by means of time-lapse motion pictures taken either by the phase contrast or by the differential interference contrast method, using apochromatic objectives of maximum resolution, combined with an inverted microscope.According to the rhythm of the cleavage divisions,waves of irregular quivering movements spread from the initial region(s) of cleavage throughout the whole egg space. They are composed of irregular oscillations of yolk particles, probably caused by the effect of actively shortening, dynamic elements irregularly spread within the ooplasm. The presence ofcleavage nuclei obviously exerts a kind ofregulative effect: Shortly before such a wave of quivering movements reaches a metaphasic cleavage energide, regular oscillations and approximations of yolk particles are visible in the surroundings of the nucleus. The movements in question are radially adjusted towards the spindle poles, starting at the one which is reached first by the wave of quivering movements. These "radial quivering movements" are caused by abig cytaster, each originating from its spindle pole and distally reaching far into the ooplasm. Synchronous with the beginning of the shortening process of the astral rays, the cleavage nucleus passes through anaphase and telophase, and the spindle poles arepulled apart. During the then following migration of each daughter nucleus, its spindle pole-the kinetocentre of the previous spindle-is preserved and becomes the centre of a "migration cytaster". Its longest rays measure up to at least 80 μm. Their distal ends temporarily insert either in motile, or in elastically suspended, or in rigid egg components.By the recurrent short-time insertions and irregular shortening processes of the astral rays,the nucleus, displaying a strong affinity to its own kinetocentre,is pulled foreward. This movement always occurs in the direction of the biggest ooplasmic region still free from nuclei and therefore permitting the greatest spacial extension of the migration cytasters. This could explain the so-called mutual "repulsion" of the energides, leading to their even dispersion all over the egg space.In some of the eggs it has been possible toseparate the cleavage nuclei from their cytaster systems experimentally. Deprived of their nuclei such migration cytasters behave autonomously, i. e. they are actively moving within the ooplasm, possibly even retaining their division power. On the other hand, thenuclei without their cytasters have lost their mobility and therefore at first remain in their sites. But they seem to be capable ofinducing new spindle poles and migration cytasters of their own and to carry out further cleavage divisions.The migration cytasters of all cleavage energides develop by division from the very cytaster whose formation had been induced by the sperm nucleus after entering the egg. On the other hand thefemale pronucleus, remainingwithout a migration cytaster and therefore lacking migration activity, ismoved towards the male nucleus, pulled by the probably permanently inserted astral rays of the latter. Thus the final act of fertilization, i.e. nuclear fusion, comes about by the affinity between the (female pro-)nucleus and the (alien) migration cytaster (of the male nucleus).Judged by their derivation from the "polar rays" of the spindle apparatus, the astral rays with high probability are built up oftubuli, the evidence being left to electron microscopical investigations. Functional structures related to the causal mechanism of the migration cytaster are suggested and their supposed derivation from the mitotic apparatus is discussed. The existence of migration cytasters might not only represent an adaptation to the specific conditions of cleavage within spacious eggs, but also could be essential for the stretching of the spindle and the separation of the daughter nuclei during the division process of many other animal cells.

Prothrombin activation on the activated platelet surface optimizes expression of procoagulant activity

PubMed Central

Wood, Jeremy P.; Silveira, Jay R.; Maille, Nicole M.; Haynes, Laura M.

2011-01-01

Effective hemostasis relies on the timely formation of α-thrombin via prothrombinase, a Ca2+-dependent complex of factors Va and Xa assembled on the activated platelet surface, which cleaves prothrombin at Arg271 and Arg320. Whereas initial cleavage at Arg271 generates the inactive intermediate prethrombin-2, initial cleavage at Arg320 generates the enzymatically active intermediate meizothrombin. To determine which of these intermediates is formed when prothrombin is processed on the activated platelet surface, the cleavage of prothrombin, and prothrombin mutants lacking either one of the cleavage sites, was monitored on the surface of either thrombin- or collagen-activated platelets. Regardless of the agonist used, prothrombin was initially cleaved at Arg271 generating prethrombin-2, with α-thrombin formation quickly after via cleavage at Arg320. The pathway used was independent of the source of factor Va (plasma- or platelet-derived) and was unaffected by soluble components of the platelet releasate. When both cleavage sites are presented within the same substrate molecule, Arg271 effectively competes against Arg320 (with an apparent IC50 = 0.3μM), such that more than 90% to 95% of the initial cleavage occurs at Arg271. We hypothesize that use of the prethrombin-2 pathway serves to optimize the procoagulant activity expressed by activated platelets, by limiting the anticoagulant functions of the alternate intermediate, meizothrombin. PMID:21131592

Prothrombin activation on the activated platelet surface optimizes expression of procoagulant activity.

PubMed

Wood, Jeremy P; Silveira, Jay R; Maille, Nicole M; Haynes, Laura M; Tracy, Paula B

2011-02-03

Effective hemostasis relies on the timely formation of α-thrombin via prothrombinase, a Ca(2+)-dependent complex of factors Va and Xa assembled on the activated platelet surface, which cleaves prothrombin at Arg271 and Arg320. Whereas initial cleavage at Arg271 generates the inactive intermediate prethrombin-2, initial cleavage at Arg320 generates the enzymatically active intermediate meizothrombin. To determine which of these intermediates is formed when prothrombin is processed on the activated platelet surface, the cleavage of prothrombin, and prothrombin mutants lacking either one of the cleavage sites, was monitored on the surface of either thrombin- or collagen-activated platelets. Regardless of the agonist used, prothrombin was initially cleaved at Arg271 generating prethrombin-2, with α-thrombin formation quickly after via cleavage at Arg320. The pathway used was independent of the source of factor Va (plasma- or platelet-derived) and was unaffected by soluble components of the platelet releasate. When both cleavage sites are presented within the same substrate molecule, Arg271 effectively competes against Arg320 (with an apparent IC(50) = 0.3μM), such that more than 90% to 95% of the initial cleavage occurs at Arg271. We hypothesize that use of the prethrombin-2 pathway serves to optimize the procoagulant activity expressed by activated platelets, by limiting the anticoagulant functions of the alternate intermediate, meizothrombin.

On the temperature independence of statistical model parameters for cleavage fracture in ferritic steels

NASA Astrophysics Data System (ADS)

Qian, Guian; Lei, Wei-Sheng; Niffenegger, M.; González-Albuixech, V. F.

2018-04-01

The work relates to the effect of temperature on the model parameters in local approaches (LAs) to cleavage fracture. According to a recently developed LA model, the physical consensus of plastic deformation being a prerequisite to cleavage fracture enforces any LA model of cleavage fracture to observe initial yielding of a volume element as its threshold stress state to incur cleavage fracture in addition to the conventional practice of confining the fracture process zone within the plastic deformation zone. The physical consistency of the new LA model to the basic LA methodology and the differences between the new LA model and other existing models are interpreted. Then this new LA model is adopted to investigate the temperature dependence of LA model parameters using circumferentially notched round tensile specimens. With the published strength data as input, finite element (FE) calculation is conducted for elastic-perfectly plastic deformation and the realistic elastic-plastic hardening, respectively, to provide stress distributions for model calibration. The calibration results in temperature independent model parameters. This leads to the establishment of a 'master curve' characteristic to synchronise the correlation between the nominal strength and the corresponding cleavage fracture probability at different temperatures. This 'master curve' behaviour is verified by strength data from three different steels, providing a new path to calculate cleavage fracture probability with significantly reduced FE efforts.

Neutron Detection Utilizing Gadolinium Doped Hafnium Oxide Films

DTIC Science & Technology

2008-03-01

2.2. Charge Carriers ................................................................................................ 2-2 2.3. Dopants and Impurities...the movement of the charge carries can be assumed to be at this drift velocity and in the direction of the electric field. 2.3. Dopants and...present even with the best purification processes. However, a material, or dopant , can be intentionally added to vary the electrical

Electrokinetic Supercapacitor for Simultaneous Harvesting and Storage of Mechanical Energy.

PubMed

Yang, Peihua; Qu, Xiaopeng; Liu, Kang; Duan, Jiangjiang; Li, Jia; Chen, Qian; Xue, Guobin; Xie, Wenke; Xu, Zhimou; Zhou, Jun

2018-03-07

Energy harvesting and storage are two distinct processes that are generally achieved using two separated parts based on different physical and chemical principles. Here we report a self-charging electrokinetic supercapacitor that directly couples the energy harvesting and storage processes into one device. The device consists of two identical carbon nanotube/titanium electrodes, separated by a piece of anodic aluminum oxide nanochannels membrane. Pressure-driven electrolyte flow through the nanochannels generates streaming potential, which can be used to charge the capacitive electrodes, accomplishing simultaneous energy generation and storage. The device stores electric charge density of 0.4 mC cm -2 after fully charging under pressure of 2.5 bar. This work may offer a train of thought for the development of a new type of energy unit for self-powered systems.

Characterization of the Trans Watson-Crick GU Base Pair Located in the Catalytic Core of the Antigenomic HDV Ribozyme

PubMed Central

Lévesque, Dominique; Reymond, Cédric; Perreault, Jean-Pierre

2012-01-01

The HDV ribozyme’s folding pathway is, by far, the most complex folding pathway elucidated to date for a small ribozyme. It includes 6 different steps that have been shown to occur before the chemical cleavage. It is likely that other steps remain to be discovered. One of the most critical of these unknown steps is the formation of the trans Watson-Crick GU base pair within loop III. The U23 and G28 nucleotides that form this base pair are perfectly conserved in all natural variants of the HDV ribozyme, and therefore are considered as being part of the signature of HDV-like ribozymes. Both the formation and the transformation of this base pair have been studied mainly by crystal structure and by molecular dynamic simulations. In order to obtain physical support for the formation of this base pair in solution, a set of experiments, including direct mutagenesis, the site-specific substitution of chemical groups, kinetic studies, chemical probing and magnesium-induced cleavage, were performed with the specific goal of characterizing this trans Watson-Crick GU base pair in an antigenomic HDV ribozyme. Both U23 and G28 can be substituted for nucleotides that likely preserve some of the H-bond interactions present before and after the cleavage step. The formation of the more stable trans Watson-Crick base pair is shown to be a post-cleavage event, while a possibly weaker trans Watson-Crick/Hoogsteen interaction seems to form before the cleavage step. The formation of this unusually stable post-cleavage base pair may act as a driving force on the chemical cleavage by favouring the formation of a more stable ground state of the product-ribozyme complex. To our knowledge, this represents the first demonstration of a potential stabilising role of a post-cleavage conformational switch event in a ribozyme-catalyzed reaction. PMID:22768274

Ab Initio energetics of SiO bond cleavage.

PubMed

Hühn, Carolin; Erlebach, Andreas; Mey, Dorothea; Wondraczek, Lothar; Sierka, Marek

2017-10-15

A multilevel approach that combines high-level ab initio quantum chemical methods applied to a molecular model of a single, strain-free SiOSi bridge has been used to derive accurate energetics for SiO bond cleavage. The calculated SiO bond dissociation energy and the activation energy for water-assisted SiO bond cleavage of 624 and 163 kJ mol -1 , respectively, are in excellent agreement with values derived recently from experimental data. In addition, the activation energy for H 2 O-assisted SiO bond cleavage is found virtually independent of the amount of water molecules in the vicinity of the reaction site. The estimated reaction energy for this process including zero-point vibrational contribution is in the range of -5 to 19 kJ mol -1 . © 2017 Wiley Periodicals, Inc. © 2017 Wiley Periodicals, Inc.

Tectono-metamorphic evolution of the Kibuye-Gitarama-Gatumba area (Rwanda): using petrochronology to unravel the geodynamic framework of the Karagwe-Ankole Belt

NASA Astrophysics Data System (ADS)

Van Daele, Johanna; Jacques, Dominique; Hulsbosch, Niels; Dewaele, Stijn; Muchez, Philippe

2017-04-01

The Mesoproterozoic Karagwe-Ankole Belt (KAB) extends from Burundi over Rwanda and NW-Tanzania to S-Uganda (Central Africa). The integration of the metamorphic and magmatic evolution of this orogenic belt in a consistent geodynamic framework is still controversial. Additionally, geochronological information on the deformation phases is limited. This tectono-metamorphic model is, however, a crucial component in the understanding of the Meso- to Early Neoproterozoic mineralization processes. A detailed structural mapping of road and river transects was performed in the Kibuye-Gitarama-Gatumba area (West Rwanda) to determine the deformation history of the KAB. Structural analyses and petrographic studies identified two main compressive deformation phases. A locally observed foliation with a N45W-N50W orientation is interpreted as the consequence of a first compressional phase (D1, shortening direction N40E-N45E). Additionally, a well-developed crenulation cleavage and a regionally pervasive foliation were found. The cleavage and foliation have an orientation of N20W-N30W (exceptionally N20E) and are indicative of a second compressional phase (D2) with an EW shortening direction. Final extension (D3) along a N30W-N10E direction resulted in boudinage and joint development. Fieldwork observations combined with known ages of the granites in the KAB indicate that D1 and D2 took place prior to 986 Ma while D3 is younger than 986 Ma. Based on thin section petrography, a petrochronological strategy was outlined to fill in the gaps of the currently broadly defined timeframe. The regional metamorphic grade of the study area is upper greenschist, with the formation of muscovite, biotite, chlorite, garnet, staurolite and cordierite. The main penetrative tectonic foliations (D1 and D2) are expressed by the preferential orientation of muscovite or biotite. In some cases, muscovite growing along the crenulation cleavage (syn-D2) was observed. Furthermore, pre- and syn-D2 garnets were identified. Different generations of quartz veins (post-dating D3) contain muscovite, garnet and/or biotite. These minerals provide excellent dating possibilities (Ar-Ar, in-situ Rb-Sr and Sm-Nd). The obtained results will be used to refine the chronological aspects of the deformation history of the KAB. Furthermore, these data will be combined with regional structural data, petrographic and geothermobarometric analyses to reconstruct the tectono-metamorphic evolution of the Karagwe-Ankole Belt, which will allow to obtain a better insight in the geodynamic evolution and the ore-forming processes.

Solid state optical microscope

DOEpatents

Young, I.T.

1983-08-09

A solid state optical microscope wherein wide-field and high-resolution images of an object are produced at a rapid rate by utilizing conventional optics with a charge-coupled photodiode array. A galvanometer scanning mirror, for scanning in one of two orthogonal directions is provided, while the charge-coupled photodiode array scans in the other orthogonal direction. Illumination light from the object is incident upon the photodiodes, creating packets of electrons (signals) which are representative of the illuminated object. The signals are then processed, stored in a memory, and finally displayed as a video signal. 2 figs.

Solid-state optical microscope

DOEpatents

Young, I.T.

1981-01-07

A solid state optical microscope is described wherein wide-field and high-resolution images of an object are produced at a rapid rate by utilizing conventional optics with a charge-coupled photodiode array. Means for scanning in one of two orthogonal directions are provided, while the charge-coupled photodiode array scans in the other orthogonal direction. Illumination light from the object is incident upon the photodiodes, creating packets of electrons (signals) which are representative of the illuminated object. The signals are then processed, stored in a memory, and finally displayed as a video signal.

Solid state optical microscope

DOEpatents

Young, Ian T.

1983-01-01

A solid state optical microscope wherein wide-field and high-resolution images of an object are produced at a rapid rate by utilizing conventional optics with a charge-coupled photodiode array. A galvanometer scanning mirror, for scanning in one of two orthogonal directions is provided, while the charge-coupled photodiode array scans in the other orthogonal direction. Illumination light from the object is incident upon the photodiodes, creating packets of electrons (signals) which are representative of the illuminated object. The signals are then processed, stored in a memory, and finally displayed as a video signal.

Molecular-dynamics Simulation-based Cohesive Zone Representation of Intergranular Fracture Processes in Aluminum

NASA Technical Reports Server (NTRS)

Yamakov, Vesselin I.; Saether, Erik; Phillips, Dawn R.; Glaessgen, Edward H.

2006-01-01

A traction-displacement relationship that may be embedded into a cohesive zone model for microscale problems of intergranular fracture is extracted from atomistic molecular-dynamics simulations. A molecular-dynamics model for crack propagation under steady-state conditions is developed to analyze intergranular fracture along a flat 99 [1 1 0] symmetric tilt grain boundary in aluminum. Under hydrostatic tensile load, the simulation reveals asymmetric crack propagation in the two opposite directions along the grain boundary. In one direction, the crack propagates in a brittle manner by cleavage with very little or no dislocation emission, and in the other direction, the propagation is ductile through the mechanism of deformation twinning. This behavior is consistent with the Rice criterion for cleavage vs. dislocation blunting transition at the crack tip. The preference for twinning to dislocation slip is in agreement with the predictions of the Tadmor and Hai criterion. A comparison with finite element calculations shows that while the stress field around the brittle crack tip follows the expected elastic solution for the given boundary conditions of the model, the stress field around the twinning crack tip has a strong plastic contribution. Through the definition of a Cohesive-Zone-Volume-Element an atomistic analog to a continuum cohesive zone model element - the results from the molecular-dynamics simulation are recast to obtain an average continuum traction-displacement relationship to represent cohesive zone interaction along a characteristic length of the grain boundary interface for the cases of ductile and brittle decohesion. Keywords: Crack-tip plasticity; Cohesive zone model; Grain boundary decohesion; Intergranular fracture; Molecular-dynamics simulation

Cast-to-shape electrokinetic trapping medium

DOEpatents

Shepodd, Timothy J.; Franklin, Elizabeth; Prickett, Zane T.; Artau, Alexander

2004-08-03

A three-dimensional microporous polymer network material, or monolith, cast-to-shape in a microchannel. The polymer monolith, produced by a phase separation process, is capable of trapping and retaining charged protein species from a mixture of charged and uncharged species under the influence of an applied electric field. The retained charged protein species are released from the porous polymer monolith by a pressure driven flow in the substantial absence of the electric field. The pressure driven flow is independent of direction and thus neither means to reverse fluid flow nor a multi-directional flow field is required, a single flow through the porous polymer monolith can be employed, in contrast to prior art systems. The monolithic polymer material produced by the invention can function as a chromatographic medium. Moreover, by virtue of its ability to retain charged protein species and quantitatively release the retained species the porous polymer monolith can serve as a means for concentrating charged protein species from, for example, a dilute solution.

Cast-to-shape electrokinetic trapping medium

DOEpatents

Shepodd, Timothy J [Livermore, CA; Franklin, Elizabeth [Rolla, MO; Prickett, Zane T [Golden, CO; Artau, Alexander [Pleasanton, CA

2006-05-30

A three-dimensional microporous polymer network material, or monolith, cast-to-shape in a microchannel. The polymer monolith, produced by a phase separation process, is capable of trapping and retaining charged protein species from a mixture of charged and uncharged species under the influence of an applied electric field. The retained charged protein species are released from the porous polymer monolith by a pressure driven flow in the substantial absence of the electric field. The pressure driven flow is independent of direction and thus neither means to reverse fluid flow nor a multi-directional flow field is required, a single flow through the porous polymer monolith can be employed, in contrast to prior art systems. The monolithic polymer material produced by the invention can function as a chromatographic medium. Moreover, by virtue of its ability to retain charged protein species and quantitatively release the retained species the porous polymer monolith can serve as a means for concentrating charged protein species from, for example, a dilute solution.

Directional rolling of positively charged nanoparticles along a flexibility gradient on long DNA molecules.

PubMed

Park, Suehyun; Joo, Heesun; Kim, Jun Soo

2018-01-31

Directing the motion of molecules/colloids in any specific direction is of great interest in many applications of chemistry, physics, and biological sciences, where regulated positioning or transportation of materials is highly desired. Using Brownian dynamics simulations of coarse-grained models of a long, double-stranded DNA molecule and positively charged nanoparticles, we observed that the motion of a single nanoparticle bound to and wrapped by the DNA molecule can be directed along a gradient of DNA local flexibility. The flexibility gradient is constructed along a 0.8 kilobase-pair DNA molecule such that local persistence length decreases gradually from 50 nm to 40 nm, mimicking a gradual change in sequence-dependent flexibility. Nanoparticles roll over a long DNA molecule from less flexible regions towards more flexible ones as a result of the decreasing energetic cost of DNA bending and wrapping. In addition, the rolling becomes slightly accelerated as the positive charge of nanoparticles decreases due to a lower free energy barrier of DNA detachment from charged nanoparticle for processive rolling. This study suggests that the variation in DNA local flexibility can be utilized in constructing and manipulating supramolecular assemblies of DNA molecules and nanoparticles in structural DNA nanotechnology.

Charge and transverse momentum correlations in deep inelastic muon-proton scattering

NASA Astrophysics Data System (ADS)

Arneodo, M.; Arvidson, A.; Aubert, J. J.; Badelek, B.; Beaufays, J.; Bee, C.; Benchouk, C.; Berghoff, G.; Bird, I.; Blurn, D.; Bohm, E.; de Bouard, X.; Brasse, F. W.; Braun, H.; Broll, C.; Brown, S.; Hruck, H.; Calen, H.; Chima, J. S.; Ciborowski, J.; Clifft, R.; Coignet, G.; Combley, F.; Coughlan, J.; Agostini, G. D'; Dahlgren, S.; Dengler, F.; Derado, I.; Dreyer, T.; Drees, J.; Düren, M.; Eckardt, V.; Edwards, A.; Adwards, M.; Ernst, T.; Eszes, G.; Favier, J.; Ferrero, M. I.; Figiel, J.; Flauger, W.; Foster, J.; Gabathuler, E.; Gajewski, J.; Gamet, R.; Gayler, J.; Geddes, N.; Grafström, P.; Grard, F.; Haas, J.; Hagberg, E.; Hasert, F. J.; Hayman, P.; Heusse, P.; Jaffré, M.; Jacholkowska, A.; Janata, F.; Jancso, G.; Johnson, A. S.; Kabuss, E. M.; Kellner, G.; Korbel, V.; Krüger, J.; Kullander, S.; Landgraf, U.; Lanske, D.; Loken, J.; Long, K.; Maire, M.; Malecki, P.; Manz, A.; Maselli, S.; Mohr, W.; Montanet, F.; Montgomery, H. E.; Nagy, E.; Nassalski, J.; Norton, P. R.; Oakham, F. G.; Osborne, A. M.; Pascaud, C.; Pawlik, B.; Payre, P.; Peroni, C.; Pessard, H.; Pettingale, J.; Pietrzyk, B.; Poensgen, B.; Pötsch, M.; Renton, P.; Ribarics, P.; Rith, K.; Rondio, E.; Scheer, M.; Schlagböhmer, A.; Schiemann, H.; Schmitz, N.; Schneegans, M.; Scholz, M.; Schröder, T.; Schouten, M.; Schultze, K.; Sloan, T.; Stier, H. E.; Studt, M.; Taylor, G. N.; Thénard, J. M.; Thompson, J. C.; de La Torre, A.; Toth, J.; Urban, L.; Wallucks, W.; Whalley, M.; Wheeler, S.; Williams, W. S. C.; Wimpenny, S.; Windmolders, R.; Wolf, G.

1986-09-01

Correlations between charged hadrons are investigated in a 280 GeV muon-proton scattering experiment. Although most of the observed particles are decay products it is shown that the correlations found originate in the fragmentation process and are not due simply to resonance production. Correlations are demonstrated between hadrons close in rapidity with respect to their charges and to the directions of their momentum components perpendicular to the virtual photon axis. Such short range correlations are predicted by the standard hadronization models.

Structural characterization by both positive and negative electrospray ion trap mass spectrometry of oligogalacturonates purified by high-performance anion-exchange chromatography.

PubMed

Quéméner, Bernard; Désiré, Cédric; Debrauwer, Laurent; Rathahao, Estelle

2003-01-17

The off-line coupling of high-performance anion-exchange chromatography to electrospray ion trap mass spectrometry (ESI-IT-MS) is described. Two sets of isocratic conditions were optimised for the semi-preparative purification of oligogalacturonates of degree of polymerisation from 4 to 6 by monitoring eluates with either pulsed amperometric detection or evaporative light scattering detection in the presence of an online Dionex Carbohydrate Membrane Desalter (CMD). In these conditions, purified oligogalacturonate solutions were suitable, without further desalting steps, for infusion ESI-IT-MS experiments. This paper provides some interesting features of positive and negative ESI-IT-multiple MS (MSn) of these acidic oligosaccharides. The spectra acquired in both ion modes show characteristic fragments resulting from glycosidic bond and cross-ring cleavages. Under negative ionization conditions, the fragmentation of the singly-charged [M-H]- ions, as well as the Ci-, and Zi-, fragment ions through sequential MSn experiments, was always dominated by product ions from C- and Z-type glycosidic cleavages. All spectra also displayed 0.2 A-type cross-ring cleavage ions which carry linkage information. Collision-induced dissociation (CID) spectra of sodium-cationized species obtained under positive ionization conditions were more complex. Successive MSn experiments also led to the 0.2 A-type cross-ring cleavage ions observed together with B- and Y-type ions. The presence of the 0.2 A ion series was related to Mr 60 (C2H4O2) losses. Combined with the absence of the Mr 30 (CH2O) and the Mr 90 (C3H6O3) ions, these ions were indicative of 1-4 type glycosidic linkage.

Process-independent strong running coupling

DOE PAGES

Binosi, Daniele; Mezrag, Cedric; Papavassiliou, Joannis; ...

2017-09-25

Here, we unify two widely different approaches to understanding the infrared behavior of quantum chromodynamics (QCD), one essentially phenomenological, based on data, and the other computational, realized via quantum field equations in the continuum theory. Using the latter, we explain and calculate a process-independent running-coupling for QCD, a new type of effective charge that is an analogue of the Gell-Mann–Low effective coupling in quantum electrodynamics. The result is almost identical to the process-dependent effective charge defined via the Bjorken sum rule, which provides one of the most basic constraints on our knowledge of nucleon spin structure. As a result, thismore » reveals the Bjorken sum to be a near direct means by which to gain empirical insight into QCD's Gell-Mann–Low effective charge.« less

Process-independent strong running coupling

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

Binosi, Daniele; Mezrag, Cedric; Papavassiliou, Joannis

Here, we unify two widely different approaches to understanding the infrared behavior of quantum chromodynamics (QCD), one essentially phenomenological, based on data, and the other computational, realized via quantum field equations in the continuum theory. Using the latter, we explain and calculate a process-independent running-coupling for QCD, a new type of effective charge that is an analogue of the Gell-Mann–Low effective coupling in quantum electrodynamics. The result is almost identical to the process-dependent effective charge defined via the Bjorken sum rule, which provides one of the most basic constraints on our knowledge of nucleon spin structure. As a result, thismore » reveals the Bjorken sum to be a near direct means by which to gain empirical insight into QCD's Gell-Mann–Low effective charge.« less

Thermolysin damages animal life through degradation of plasma proteins enhanced by rapid cleavage of serpins and activation of proteases.

PubMed

Kong, Lulu; Lu, Anrui; Guan, Jingmin; Yang, Bing; Li, Muwang; Hillyer, Julián F; Ramarao, Nalini; Söderhäll, Kenneth; Liu, Chaoliang; Ling, Erjun

2015-01-01

Thermolysin, a metallopeptidase secreted by pathogenic microbes, is concluded as an important virulence factor due to cleaving purified host proteins in vitro. Using the silkworm Bombyx mori as a model system, we found that thermolysin injection into larvae induces the destruction of the coagulation response and the activation of hemolymph melanization, which results in larval death. Thermolysin triggers the rapid degradation of insect and mammalian plasma proteins at a level that is considerably greater than expected in vitro and/or in vivo. To more specifically explore the mechanism, thermolysin-induced changes to key proteins belonging to the insect melanization pathway were assessed as a window for observing plasma protein cleavage. The application of thermolysin induced the rapid cleavage of the melanization negative regulator serpin-3, but did not directly activate the melanization rate-limiting enzyme prophenoloxidase (PPO) or the terminal serine proteases responsible for PPO activation. Terminal serine proteases of melanization are activated indirectly after thermolysin exposure. We hypothesize that thermolysin induces the rapid degradation of serpins and the activation of proteases directly or indirectly, boosting uncontrolled plasma protein degradation in insects and mammalians. © 2014 Wiley Periodicals, Inc.

Cu(II)-catalyzed esterification reaction via aerobic oxidative cleavage of C(CO)-C(alkyl) bonds.

PubMed

Ma, Ran; He, Liang-Nian; Liu, An-Hua; Song, Qing-Wen

2016-02-04

A novel Cu(II)-catalyzed aerobic oxidative esterification of simple ketones for the synthesis of esters has been developed with wide functional group tolerance. This process is assumed to go through a tandem sequence consisting of α-oxygenation/esterification/nucleophilic addition/C-C bond cleavage and carbon dioxide is released as the only byproduct.

Urokinase links plasminogen activation and cell adhesion by cleavage of the RGD motif in vitronectin.

PubMed

De Lorenzi, Valentina; Sarra Ferraris, Gian Maria; Madsen, Jeppe B; Lupia, Michela; Andreasen, Peter A; Sidenius, Nicolai

2016-07-01

Components of the plasminogen activation system including urokinase (uPA), its inhibitor (PAI-1) and its cell surface receptor (uPAR) have been implicated in a wide variety of biological processes related to tissue homoeostasis. Firstly, the binding of uPA to uPAR favours extracellular proteolysis by enhancing cell surface plasminogen activation. Secondly, it promotes cell adhesion and signalling through binding of the provisional matrix protein vitronectin. We now report that uPA and plasmin induces a potent negative feedback on cell adhesion through specific cleavage of the RGD motif in vitronectin. Cleavage of vitronectin by uPA displays a remarkable receptor dependence and requires concomitant binding of both uPA and vitronectin to uPAR Moreover, we show that PAI-1 counteracts the negative feedback and behaves as a proteolysis-triggered stabilizer of uPAR-mediated cell adhesion to vitronectin. These findings identify a novel and highly specific function for the plasminogen activation system in the regulation of cell adhesion to vitronectin. The cleavage of vitronectin by uPA and plasmin results in the release of N-terminal vitronectin fragments that can be detected in vivo, underscoring the potential physiological relevance of the process. © 2016 The Authors.

A serendipitous discovery that in situ proteolysis is essential for the crystallization of yeast CPSF-100 (Ydh1p)

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

Mandel, Corey R.; Gebauer, Damara; Zhang, Hailong

2006-10-01

Proteolysis in situ by a protease secreted by a contaminating fungus is essential for the crystallization of yeast CPSF-100. The cleavage and polyadenylation specificity factor (CPSF) complex is required for the cleavage and polyadenylation of the 3′-end of messenger RNA precursors in eukaryotes. During structural studies of the 100 kDa subunit (CPSF-100, Ydh1p) of the yeast CPSF complex, it was serendipitously discovered that a solution that is infected by a fungus (subsequently identified as Penicillium) is crucial for the crystallization of this protein. Further analyses suggest that the protein has undergone partial proteolysis during crystallization, resulting in the deletion ofmore » an internal segment of about 200 highly charged and hydrophilic residues, very likely catalyzed by a protease secreted by the fungus. With the removal of this segment, yeast CPSF-100 (Ydh1p) has greatly reduced solubility and can be crystallized in the presence of a minute amount of precipitant.« less

Direct evidence for radiative charge transfer after inner-shell excitation and ionization of large clusters

NASA Astrophysics Data System (ADS)

Hans, Andreas; Stumpf, Vasili; Holzapfel, Xaver; Wiegandt, Florian; Schmidt, Philipp; Ozga, Christian; Reiß, Philipp; Ben Ltaief, Ltaief; Küstner-Wetekam, Catmarna; Jahnke, Till; Ehresmann, Arno; Demekhin, Philipp V.; Gokhberg, Kirill; Knie, André

2018-01-01

We directly observe radiative charge transfer (RCT) in Ne clusters by dispersed vacuum-ultraviolet photon detection. The doubly ionized Ne2+-{{{N}}{{e}}}n-1 initial states of RCT are populated after resonant 1s-3p photoexcitation or 1s photoionization of Ne n clusters with < n> ≈ 2800. These states relax further producing Ne+-Ne+-{{{N}}{{e}}}n-2 final states, and the RCT photon is emitted. Ab initio calculations assign the observed RCT signal to the{}{{{N}}{{e}}}2+(2{{{p}}}-2{[}1{{D}}]){--}{{{N}}{{e}}}n-1 initial state, while transitions from other possible initial states are proposed to be quenched by competing relaxation processes. The present results are in agreement with the commonly discussed scenario, where the doubly ionized atom in a noble gas cluster forms a dimer which dissipates its vibrational energy on a picosecond timescale. Our study complements the picture of the RCT process in weakly bound clusters, providing information which is inaccessible by charged particle detection techniques.

Impact of local electrostatic field rearrangement on field ionization

NASA Astrophysics Data System (ADS)

Katnagallu, Shyam; Dagan, Michal; Parviainen, Stefan; Nematollahi, Ali; Grabowski, Blazej; Bagot, Paul A. J.; Rolland, Nicolas; Neugebauer, Jörg; Raabe, Dierk; Vurpillot, François; Moody, Michael P.; Gault, Baptiste

2018-03-01

Field ion microscopy allows for direct imaging of surfaces with true atomic resolution. The high charge density distribution on the surface generates an intense electric field that can induce ionization of gas atoms. We investigate the dynamic nature of the charge and the consequent electrostatic field redistribution following the departure of atoms initially constituting the surface in the form of an ion, a process known as field evaporation. We report on a new algorithm for image processing and tracking of individual atoms on the specimen surface enabling quantitative assessment of shifts in the imaged atomic positions. By combining experimental investigations with molecular dynamics simulations, which include the full electric charge, we confirm that change is directly associated with the rearrangement of the electrostatic field that modifies the imaging gas ionization zone. We derive important considerations for future developments of data reconstruction in 3D field ion microscopy, in particular for precise quantification of lattice strains and characterization of crystalline defects at the atomic scale.

Photodetachment process for beam neutralization

DOEpatents

Fink, Joel H. [Livermore, CA; Frank, Alan M. [Livermore, CA

1979-02-20

A process for neutralization of accelerated ions employing photo-induced charge detachment. The process involves directing a laser beam across the path of a negative ion beam such as to effect photodetachment of electrons from the beam ions. The frequency of the laser beam employed is selected to provide the maximum cross-section for the photodetachment process.

2D Raman band splitting in graphene: Charge screening and lifting of the K-point Kohn anomaly.

PubMed

Wang, Xuanye; Christopher, Jason W; Swan, Anna K

2017-10-19

Pristine graphene encapsulated in hexagonal boron nitride has transport properties rivalling suspended graphene, while being protected from contamination and mechanical damage. For high quality devices, it is important to avoid and monitor accidental doping and charge fluctuations. The 2D Raman double peak in intrinsic graphene can be used to optically determine charge density, with decreasing peak split corresponding to increasing charge density. We find strong correlations between the 2D 1 and 2D 2 split vs 2D line widths, intensities, and peak positions. Charge density fluctuations can be measured with orders of magnitude higher precision than previously accomplished using the G-band shift with charge. The two 2D intrinsic peaks can be associated with the "inner" and "outer" Raman scattering processes, with the counterintuitive assignment of the phonon closer to the K point in the KM direction (outer process) as the higher energy peak. Even low charge screening lifts the phonon Kohn anomaly near the K point for graphene encapsulated in hBN, and shifts the dominant intensity from the lower to the higher energy peak.

Catalyst system and process for benzyl ether fragmentation and coal liquefaction

DOEpatents

Zoeller, Joseph Robert

1998-04-28

Dibenzyl ether can be readily cleaved to form primarily benzaldehyde and toluene as products, along with minor amounts of bibenzyl and benzyl benzoate, in the presence of a catalyst system comprising a Group 6 metal, preferably molybdenum, a salt, and an organic halide. Although useful synthetically for the cleavage of benzyl ethers, this cleavage also represents a key model reaction for the liquefaction of coal; thus this catalyst system and process should be useful in coal liquefaction with the advantage of operating at significantly lower temperatures and pressures.

Probing the radio emission from air showers with polarization measurements

NASA Astrophysics Data System (ADS)

Aab, A.; Abreu, P.; Aglietta, M.; Ahlers, M.; Ahn, E. J.; Albuquerque, I. F. M.; Allekotte, I.; Allen, J.; Allison, P.; Almela, A.; Alvarez Castillo, J.; Alvarez-Muñiz, J.; Alves Batista, R.; Ambrosio, M.; Aminaei, A.; Anchordoqui, L.; Andringa, S.; Antičić, T.; Aramo, C.; Arqueros, F.; Asorey, H.; Assis, P.; Aublin, J.; Ave, M.; Avenier, M.; Avila, G.; Badescu, A. M.; Barber, K. B.; Bardenet, R.; Bäuml, J.; Baus, C.; Beatty, J. J.; Becker, K. H.; Bellido, J. A.; BenZvi, S.; Berat, C.; Bertou, X.; Biermann, P. L.; Billoir, P.; Blanco, F.; Blanco, M.; Bleve, C.; Blümer, H.; Boháčová, M.; Boncioli, D.; Bonifazi, C.; Bonino, R.; Borodai, N.; Brack, J.; Brancus, I.; Brogueira, P.; Brown, W. C.; Buchholz, P.; Bueno, A.; Buscemi, M.; Caballero-Mora, K. S.; Caccianiga, B.; Caccianiga, L.; Candusso, M.; Caramete, L.; Caruso, R.; Castellina, A.; Cataldi, G.; Cazon, L.; Cester, R.; Cheng, S. H.; Chiavassa, A.; Chinellato, J. A.; Chudoba, J.; Cilmo, M.; Clay, R. W.; Cocciolo, G.; Colalillo, R.; Collica, L.; Coluccia, M. R.; Conceição, R.; Contreras, F.; Cooper, M. J.; Coutu, S.; Covault, C. E.; Criss, A.; Cronin, J.; Curutiu, A.; Dallier, R.; Daniel, B.; Dasso, S.; Daumiller, K.; Dawson, B. R.; de Almeida, R. M.; De Domenico, M.; de Jong, S. J.; De La Vega, G.; de Mello Junior, W. J. M.; de Mello Neto, J. R. T.; De Mitri, I.; de Souza, V.; de Vries, K. D.; del Peral, L.; Deligny, O.; Dembinski, H.; Dhital, N.; Di Giulio, C.; Di Matteo, A.; Diaz, J. C.; Díaz Castro, M. L.; Diep, P. N.; Diogo, F.; Dobrigkeit, C.; Docters, W.; D'Olivo, J. C.; Dong, P. N.; Dorofeev, A.; dos Anjos, J. C.; Dova, M. T.; Ebr, J.; Engel, R.; Erdmann, M.; Escobar, C. O.; Espadanal, J.; Etchegoyen, A.; Facal San Luis, P.; Falcke, H.; Fang, K.; Farrar, G.; Fauth, A. C.; Fazzini, N.; Ferguson, A. P.; Fick, B.; Figueira, J. M.; Filevich, A.; Filipčič, A.; Foerster, N.; Fox, B. D.; Fracchiolla, C. E.; Fraenkel, E. D.; Fratu, O.; Fröhlich, U.; Fuchs, B.; Gaior, R.; Gamarra, R. F.; Gambetta, S.; García, B.; Garcia Roca, S. T.; Garcia-Gamez, D.; Garcia-Pinto, D.; Garilli, G.; Gascon Bravo, A.; Gemmeke, H.; Ghia, P. L.; Giammarchi, M.; Giller, M.; Gitto, J.; Glaser, C.; Glass, H.; Gomez Albarracin, F.; Gómez Berisso, M.; Gómez Vitale, P. F.; Gonçalves, P.; Gonzalez, J. G.; Gookin, B.; Gorgi, A.; Gorham, P.; Gouffon, P.; Grebe, S.; Griffith, N.; Grillo, A. F.; Grubb, T. D.; Guardincerri, Y.; Guarino, F.; Guedes, G. P.; Hansen, P.; Harari, D.; Harrison, T. A.; Harton, J. L.; Haungs, A.; Hebbeker, T.; Heck, D.; Herve, A. E.; Hill, G. C.; Hojvat, C.; Hollon, N.; Holt, E.; Homola, P.; Hörandel, J. R.; Horvath, P.; Hrabovský, M.; Huber, D.; Huege, T.; Insolia, A.; Isar, P. G.; Jansen, S.; Jarne, C.; Josebachuili, M.; Kadija, K.; Kambeitz, O.; Kampert, K. H.; Karhan, P.; Kasper, P.; Katkov, I.; Kégl, B.; Keilhauer, B.; Keivani, A.; Kemp, E.; Kieckhafer, R. M.; Klages, H. O.; Kleifges, M.; Kleinfeller, J.; Knapp, J.; Krause, R.; Krohm, N.; Krömer, O.; Kruppke-Hansen, D.; Kuempel, D.; Kunka, N.; La Rosa, G.; LaHurd, D.; Latronico, L.; Lauer, R.; Lauscher, M.; Lautridou, P.; Le Coz, S.; Leão, M. S. A. B.; Lebrun, D.; Lebrun, P.; Leigui de Oliveira, M. A.; Letessier-Selvon, A.; Lhenry-Yvon, I.; Link, K.; López, R.; Lopez Agüera, A.; Louedec, K.; Lozano Bahilo, J.; Lu, L.; Lucero, A.; Ludwig, M.; Lyberis, H.; Maccarone, M. C.; Malacari, M.; Maldera, S.; Maller, J.; Mandat, D.; Mantsch, P.; Mariazzi, A. G.; Marin, V.; Mariş, I. C.; Marquez Falcon, H. R.; Marsella, G.; Martello, D.; Martin, L.; Martinez, H.; Martínez Bravo, O.; Martraire, D.; Masías Meza, J. J.; Mathes, H. J.; Matthews, J.; Matthews, J. A. J.; Matthiae, G.; Maurel, D.; Maurizio, D.; Mayotte, E.; Mazur, P. O.; Medina, C.; Medina-Tanco, G.; Melissas, M.; Melo, D.; Menichetti, E.; Menshikov, A.; Messina, S.; Meyhandan, R.; Mićanović, S.; Micheletti, M. I.; Middendorf, L.; Minaya, I. A.; Miramonti, L.; Mitrica, B.; Molina-Bueno, L.; Mollerach, S.; Monasor, M.; Monnier Ragaigne, D.; Montanet, F.; Morales, B.; Morello, C.; Moreno, J. C.; Mostafá, M.; Moura, C. A.; Muller, M. A.; Müller, G.; Münchmeyer, M.; Mussa, R.; Navarra, G.; Navarro, J. L.; Navas, S.; Necesal, P.; Nellen, L.; Nelles, A.; Neuser, J.; Nhung, P. T.; Niechciol, M.; Niemietz, L.; Niggemann, T.; Nitz, D.; Nosek, D.; Nožka, L.; Oehlschläger, J.; Olinto, A.; Oliveira, M.; Ortiz, M.; Pacheco, N.; Pakk Selmi-Dei, D.; Palatka, M.; Pallotta, J.; Palmieri, N.; Parente, G.; Parra, A.; Pastor, S.; Paul, T.; Pech, M.; PeÂķala, J.; Pelayo, R.; Pepe, I. M.; Perrone, L.; Pesce, R.; Petermann, E.; Petrera, S.; Petrolini, A.; Petrov, Y.; Piegaia, R.; Pierog, T.; Pieroni, P.; Pimenta, M.; Pirronello, V.; Platino, M.; Plum, M.; Pontz, M.; Porcelli, A.; Preda, T.; Privitera, P.; Prouza, M.; Quel, E. J.; Querchfeld, S.; Quinn, S.; Rautenberg, J.; Ravel, O.; Ravignani, D.; Revenu, B.; Ridky, J.; Riggi, S.; Risse, M.; Ristori, P.; Rivera, H.; Rizi, V.; Roberts, J.; Rodrigues de Carvalho, W.; Rodriguez Cabo, I.; Rodriguez Fernandez, G.; Rodriguez Martino, J.; Rodriguez Rojo, J.; Rodríguez-Frías, M. D.; Ros, G.; Rosado, J.; Rossler, T.; Roth, M.; Rouillé-d'Orfeuil, B.; Roulet, E.; Rovero, A. C.; Rühle, C.; Saffi, S. J.; Saftoiu, A.; Salamida, F.; Salazar, H.; Salesa Greus, F.; Salina, G.; Sánchez, F.; Sanchez-Lucas, P.; Santo, C. E.; Santos, E.; Santos, E. M.; Sarazin, F.; Sarkar, B.; Sarmento, R.; Sato, R.; Scharf, N.; Scherini, V.; Schieler, H.; Schiffer, P.; Schmidt, A.; Scholten, O.; Schoorlemmer, H.; Schovánek, P.; Schröder, F. G.; Schulz, A.; Schulz, J.; Sciutto, S. J.; Scuderi, M.; Segreto, A.; Settimo, M.; Shadkam, A.; Shellard, R. C.; Sidelnik, I.; Sigl, G.; Sima, O.; Śmiałkowski, A.; Šmída, R.; Snow, G. R.; Sommers, P.; Sorokin, J.; Spinka, H.; Squartini, R.; Srivastava, Y. N.; Stanič, S.; Stapleton, J.; Stasielak, J.; Stephan, M.; Straub, M.; Stutz, A.; Suarez, F.; Suomijärvi, T.; Supanitsky, A. D.; Šuša, T.; Sutherland, M. S.; Swain, J.; Szadkowski, Z.; Szuba, M.; Tapia, A.; Tartare, M.; Taşcǎu, O.; Thao, N. T.; Tiffenberg, J.; Timmermans, C.; Tkaczyk, W.; Todero Peixoto, C. J.; Toma, G.; Tomankova, L.; Tomé, B.; Tonachini, A.; Torralba Elipe, G.; Torres Machado, D.; Travnicek, P.; Tridapalli, D. B.; Trovato, E.; Tueros, M.; Ulrich, R.; Unger, M.; Valdés Galicia, J. F.; Valiño, I.; Valore, L.; van Aar, G.; van den Berg, A. M.; van Velzen, S.; van Vliet, A.; Varela, E.; Vargas Cárdenas, B.; Varner, G.; Vázquez, J. R.; Vázquez, R. A.; Veberič, D.; Verzi, V.; Vicha, J.; Videla, M.; Villaseñor, L.; Wahlberg, H.; Wahrlich, P.; Wainberg, O.; Walz, D.; Watson, A. A.; Weber, M.; Weidenhaupt, K.; Weindl, A.; Werner, F.; Westerhoff, S.; Whelan, B. J.; Widom, A.; Wieczorek, G.; Wiencke, L.; Wilczyńska, B.; Wilczyński, H.; Will, M.; Williams, C.; Winchen, T.; Wundheiler, B.; Wykes, S.; Yamamoto, T.; Yapici, T.; Younk, P.; Yuan, G.; Yushkov, A.; Zamorano, B.; Zas, E.; Zavrtanik, D.; Zavrtanik, M.; Zaw, I.; Zepeda, A.; Zhou, J.; Zhu, Y.; Zimbres Silva, M.; Ziolkowski, M.; Pierre Auger Collaboration

2014-03-01

The emission of radio waves from air showers has been attributed to the so-called geomagnetic emission process. At frequencies around 50 MHz this process leads to coherent radiation which can be observed with rather simple setups. The direction of the electric field induced by this emission process depends only on the local magnetic field vector and on the incoming direction of the air shower. We report on measurements of the electric field vector where, in addition to this geomagnetic component, another component has been observed that cannot be described by the geomagnetic emission process. The data provide strong evidence that the other electric field component is polarized radially with respect to the shower axis, in agreement with predictions made by Askaryan who described radio emission from particle showers due to a negative charge excess in the front of the shower. Our results are compared to calculations which include the radiation mechanism induced by this charge-excess process.

Quantum design of photosynthesis for bio-inspired solar-energy conversion.

PubMed

Romero, Elisabet; Novoderezhkin, Vladimir I; van Grondelle, Rienk

2017-03-15

Photosynthesis is the natural process that converts solar photons into energy-rich products that are needed to drive the biochemistry of life. Two ultrafast processes form the basis of photosynthesis: excitation energy transfer and charge separation. Under optimal conditions, every photon that is absorbed is used by the photosynthetic organism. Fundamental quantum mechanics phenomena, including delocalization, underlie the speed, efficiency and directionality of the charge-separation process. At least four design principles are active in natural photosynthesis, and these can be applied practically to stimulate the development of bio-inspired, human-made energy conversion systems.

The TORC1-Regulated CPA Complex Rewires an RNA Processing Network to Drive Autophagy and Metabolic Reprogramming.

PubMed

Tang, Hong-Wen; Hu, Yanhui; Chen, Chiao-Lin; Xia, Baolong; Zirin, Jonathan; Yuan, Min; Asara, John M; Rabinow, Leonard; Perrimon, Norbert

2018-05-01

Nutrient deprivation induces autophagy through inhibiting TORC1 activity. We describe a novel mechanism in Drosophila by which TORC1 regulates RNA processing of Atg transcripts and alters ATG protein levels and activities via the cleavage and polyadenylation (CPA) complex. We show that TORC1 signaling inhibits CDK8 and DOA kinases, which directly phosphorylate CPSF6, a component of the CPA complex. These phosphorylation events regulate CPSF6 localization, RNA binding, and starvation-induced alternative RNA processing of transcripts involved in autophagy, nutrient, and energy metabolism, thereby controlling autophagosome formation and metabolism. Similarly, we find that mammalian CDK8 and CLK2, a DOA ortholog, phosphorylate CPSF6 to regulate autophagy and metabolic changes upon starvation, revealing an evolutionarily conserved mechanism linking TORC1 signaling with RNA processing, autophagy, and metabolism. Copyright © 2018 Elsevier Inc. All rights reserved.

Charged-particle spectroscopy in organic semiconducting single crystals

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

Ciavatti, A.; Basiricò, L.; Fraboni, B.

2016-04-11

The use of organic materials as radiation detectors has grown, due to the easy processability in liquid phase at room temperature and the possibility to cover large areas by means of low cost deposition techniques. Direct charged-particle detectors based on solution-grown Organic Semiconducting Single Crystals (OSSCs) are shown to be capable to detect charged particles in pulse mode, with very good peak discrimination. The direct charged-particle detection in OSSCs has been assessed both in the planar and in the vertical axes, and a digital pulse processing algorithm has been used to perform pulse height spectroscopy and to study the chargemore » collection efficiency as a function of the applied bias voltage. Taking advantage of the charge spectroscopy and the good peak discrimination of pulse height spectra, an Hecht-like behavior of OSSCs radiation detectors is demonstrated. It has been possible to estimate the mobility-lifetime value in organic materials, a fundamental parameter for the characterization of radiation detectors, whose results are equal to μτ{sub coplanar} = (5 .5 ± 0.6 ) × 10{sup −6} cm{sup 2}/V and μτ{sub sandwich} = (1 .9 ± 0.2 ) × 10{sup −6} cm{sup 2}/V, values comparable to those of polycrystalline inorganic detectors. Moreover, alpha particles Time-of-Flight experiments have been carried out to estimate the drift mobility value. The results reported here indicate how charged-particle detectors based on OSSCs possess a great potential as low-cost, large area, solid-state direct detectors operating at room temperature. More interestingly, the good detection efficiency and peak discrimination observed for charged-particle detection in organic materials (hydrogen-rich molecules) are encouraging for their further exploitation in the detection of thermal and high-energy neutrons.« less

Polarized electrons, trions, and nuclei in charged quantum dots

NASA Astrophysics Data System (ADS)

Bracker, A. S.; Tischler, J. G.; Korenev, V. L.; Gammon, D.

2003-07-01

We have investigated spin polarization in GaAs quantum dots. Excitons and trions are polarized directly by optical excitation and studied through polarization of photoluminescence. Electrons and nuclei are polarized indirectly through subsequent relaxation processes. Polarized electrons are identified by the Hanle effect for exciton and trion photoluminescence, while polarized nuclei are identified through the Overhauser effect in individual charged quantum dots.

Regulation of Gene Editing Activity Directed by Single-Stranded Oligonucleotides and CRISPR/Cas9 Systems

PubMed Central

Bialk, Pawel; Rivera-Torres, Natalia; Strouse, Bryan; Kmiec, Eric B.

2015-01-01

Single-stranded DNA oligonucleotides (ssODNs) can direct the repair of a single base mutation in human genes. While the regulation of this gene editing reaction has been partially elucidated, the low frequency with which repair occurs has hampered development toward clinical application. In this work a CRISPR/Cas9 complex is employed to induce double strand DNA breakage at specific sites surrounding the nucleotide designated for exchange. The result is a significant elevation in ssODN-directed gene repair, validated by a phenotypic readout. By analysing reaction parameters, we have uncovered restrictions on gene editing activity involving CRISPR/Cas9 complexes. First, ssODNs that hybridize to the non-transcribed strand direct a higher level of gene repair than those that hybridize to the transcribed strand. Second, cleavage must be proximal to the targeted mutant base to enable higher levels of gene editing. Third, DNA cleavage enables a higher level of gene editing activity as compared to single-stranded DNA nicks, created by modified Cas9 (Nickases). Fourth, we calculated the hybridization potential and free energy levels of ssODNs that are complementary to the guide RNA sequences of CRISPRs used in this study. We find a correlation between free energy potential and the capacity of single-stranded oligonucleotides to inhibit specific DNA cleavage activity, thereby indirectly reducing gene editing activity. Our data provide novel information that might be taken into consideration in the design and usage of CRISPR/Cas9 systems with ssODNs for gene editing. PMID:26053390

Regulation of Gene Editing Activity Directed by Single-Stranded Oligonucleotides and CRISPR/Cas9 Systems.

PubMed

Bialk, Pawel; Rivera-Torres, Natalia; Strouse, Bryan; Kmiec, Eric B

2015-01-01

Single-stranded DNA oligonucleotides (ssODNs) can direct the repair of a single base mutation in human genes. While the regulation of this gene editing reaction has been partially elucidated, the low frequency with which repair occurs has hampered development toward clinical application. In this work a CRISPR/Cas9 complex is employed to induce double strand DNA breakage at specific sites surrounding the nucleotide designated for exchange. The result is a significant elevation in ssODN-directed gene repair, validated by a phenotypic readout. By analysing reaction parameters, we have uncovered restrictions on gene editing activity involving CRISPR/Cas9 complexes. First, ssODNs that hybridize to the non-transcribed strand direct a higher level of gene repair than those that hybridize to the transcribed strand. Second, cleavage must be proximal to the targeted mutant base to enable higher levels of gene editing. Third, DNA cleavage enables a higher level of gene editing activity as compared to single-stranded DNA nicks, created by modified Cas9 (Nickases). Fourth, we calculated the hybridization potential and free energy levels of ssODNs that are complementary to the guide RNA sequences of CRISPRs used in this study. We find a correlation between free energy potential and the capacity of single-stranded oligonucleotides to inhibit specific DNA cleavage activity, thereby indirectly reducing gene editing activity. Our data provide novel information that might be taken into consideration in the design and usage of CRISPR/Cas9 systems with ssODNs for gene editing.

Truncation of the TAR DNA-binding protein 43 is not a prerequisite for cytoplasmic relocalization, and is suppressed by caspase inhibition and by introduction of the A90V sequence variant

PubMed Central

Brandon, Nicholas J.; Moss, Stephen J.

2017-01-01

The RNA-binding and -processing protein TAR DNA-binding protein 43 (TDP-43) is heavily linked to the underlying causes and pathology of neurodegenerative diseases such as amyotrophic lateral sclerosis and frontotemporal lobar degeneration. In these diseases, TDP-43 is mislocalized, hyperphosphorylated, ubiquitinated, aggregated and cleaved. The importance of TDP-43 cleavage in the disease pathogenesis is still poorly understood. Here we detail the use of D-sorbitol as an exogenous stressor that causes TDP-43 cleavage in HeLa cells, resulting in a 35 kDa truncated product that accumulates in the cytoplasm within one hour of treatment. We confirm that the formation of this 35 kDa cleavage product is mediated by the activation of caspases. Inhibition of caspases blocks the cleavage of TDP-43, but does not prevent the accumulation of full-length protein in the cytoplasm. Using D-sorbitol as a stressor and caspase activator, we also demonstrate that the A90V variant of TDP-43, which lies adjacent to the caspase cleavage site within the nuclear localization sequence of TDP-43, confers partial resistance against caspase-mediated generation of the 35 kDa cleavage product. PMID:28510586

Determination of the protease cleavage site repertoire—The RNase H but not the RT domain is essential for foamy viral protease activity

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

Spannaus, Ralf; Bodem, Jochen, E-mail: Jochen.Bodem@vim.uni-wuerzburg.de

2014-04-15

In contrast to orthoretroviruses, the foamy virus protease is only active as a protease-reverse transcriptase fusion protein and requires viral RNA for activation. Maturation of foamy viral proteins seems to be restricted to a single cleavage site in Gag and Pol. We provide evidence that unprocessed Gag is required for optimal infectivity, which is unique among retroviruses. Analyses of the cleavage site sequences of the Gag and Pol cleavage sites revealed a high similarity compared to those of Lentiviruses. We show that positions P2' and P2 are invariant and that Gag and Pol cleavage sites are processed with similar efficiencies.more » The RNase H domain is essential for protease activity, but can functionally be substituted by RNase H domains of other retroviruses. Thus, the RNase H domain might be involved in the stabilization of the protease dimer, while the RT domain is essential for RNA dependent protease activation. - Highlights: • Unprocessed Gag is required for optimal infectivity of foamy viruses. • Positions P2 and P2' are invariant in the foamy viral cleavage sites. • The RNaseH domain is essential for protease activity. • The RNaseH domains of other retroviruses support foamy viral protease activity.« less

Multifaceted regulation of V(D)J recombination

NASA Astrophysics Data System (ADS)

Wang, Guannan

V(D)J recombination is responsible for generating an enormous repertoire of immunoglobulins and T cell receptors, therefore it is a centerpiece to the formation of the adaptive immune system. The V(D)J recombination process proceeds through two steps, site-specific cleavage at RSS (Recombination Signal Sequence) site mediated by the RAG recombinase (RAG1/2) and the subsequent imprecise resolution of the DNA ends, which is carried out by the ubiquitous non-homologous end joining pathway (NHEJ). The V(D)J recombination reaction is obliged to be tightly controlled under all circumstances, as it involves generations of DNA double strand breaks, which are considered the most dangerous lesion to a cell. Multifaceted regulatory mechanisms have been evolved to create great diversity of the antigen receptor repertoire while ensuring genome stability. The RAG-mediated cleavage reaction is stringently regulated at both the pre-cleavage stage and the post-cleavage stage. Specifically, RAG1/2 first forms a pre-cleavage complex assembled at the boarder of RSS and coding flank, which ensures the appropriate DNA targeting. Subsequently, this complex initiates site-specific cleavage, generating two types of double stranded DNA breaks, hairpin-ended coding ends (HP-CEs) and blunt signal ends (SEs). After the cleavage, RAG1/2 proteins bind and retain the recombination ends to form post-cleavage complexes (PCC), which collaborates with the NHEJ machinery for appropriate transfer of recombination ends to NHEJ for proper end resolution. However, little is known about the molecular basis of this collaboration, partly attributed to the lack of sensitive assays to reveal the interaction of PCC with HP-CEs. Here, for the first time, by using two complementary fluorescence-based techniques, fluorescence anisotropy and fluorescence resonance energy transfer (FRET), I managed to monitor the RAG1/2-catalyzed cleavage reaction in real time, from the pre-cleavage to the post-cleavage stages. By examining the dynamic fluorescence changes during the RAG-mediated cleavage reactions, and by manipulating the reaction conditions, I was able to characterize some fundamental properties of RAG-DNA interactions before and after cleavage. Firstly, Mg 2+, known as a physiological cofactor at the excision step, also promotes the HP-CEs retention in the RAG complex after cleavage. Secondly, the structure of pre-cleavage complex may affect the subsequent collaborations with NHEJ for end resolution. Thirdly, the non-core region of RAG2 may have differential influences on the PCC retention of HP-CEs and SEs. Furthermore, I also provide the first evidence of RAG1-mediated regulation of RAG2. Our study provides important insights into the multilayered regulatory mechanisms, in modulating recombination events in developing lymphocytes and paves the way for possible development of detection and diagnotic markers for defective recombination events that are often associated immunodeficiency and/or lymphoid malignancy.

Optimization of microelectrophoresis to select highly negatively charged sperm.

PubMed

Simon, Luke; Murphy, Kristin; Aston, Kenneth I; Emery, Benjamin R; Hotaling, James M; Carrell, Douglas T

2016-06-01

The sperm membrane undergoes extensive surface remodeling as it matures in the epididymis. During this process, the sperm is encapsulated in an extensive glycocalyx layer, which provides the membrane with its characteristic negative electrostatic charge. In this study, we develop a method of microelectrophoresis and standardize the protocol to isolate sperm with high negative membrane charge. Under an electric field, the percentage of positively charged sperm (PCS), negatively charged sperm (NCS), and neutrally charged sperm was determined for each ejaculate prior to and following density gradient centrifugation (DGC), and evaluated for sperm DNA damage, and histone retention. Subsequently, PCS, NCS, and neutrally charged sperm were selected using an ICSI needle and directly analyzed for DNA damage. When raw semen was analyzed using microelectrophoresis, 94 % were NCS. In contrast, DGC completely or partially stripped the negative membrane charge from sperm resulting PCS and neutrally charged sperm, while the charged sperm populations are increased with an increase in electrophoretic current. Following DGC, high sperm DNA damage and abnormal histone retention were inversely correlated with percentage NCS and directly correlated with percentage PCS. NCS exhibited significantly lower DNA damage when compared with control (P < 0.05) and PCS (P < 0.05). When the charged sperm population was corrected for neutrally charged sperm, sperm DNA damage was strongly associated with NCS at a lower electrophoretic current. The results suggest that selection of NCS at lower current may be an important biomarker to select healthy sperm for assisted reproductive treatment.

Modifications to the Foot-and-Mouth Disease Virus 2A Peptide: Influence on Polyprotein Processing and Virus Replication.

PubMed

Kjær, Jonas; Belsham, Graham J

2018-04-15

Foot-and-mouth disease virus (FMDV) has a positive-sense single-stranded RNA (ssRNA) genome that includes a single, large open reading frame encoding a polyprotein. The cotranslational "cleavage" of this polyprotein at the 2A/2B junction is mediated by the 2A peptide (18 residues in length) using a nonproteolytic mechanism termed "ribosome skipping" or "StopGo." Multiple variants of the 2A polypeptide with this property among the picornaviruses share a conserved C-terminal motif [D(V/I)E(S/T)NPG↓P]. The impact of 2A modifications within this motif on FMDV protein synthesis, polyprotein processing, and virus viability were investigated. Amino acid substitutions are tolerated at residues E 14 , S 15 , and N 16 within the 2A sequences of infectious FMDVs despite their reported "cleavage" efficiencies at the 2A/2B junction of only ca. 30 to 50% compared to that of the wild type (wt). In contrast, no viruses containing substitutions at residue P 17 , G 18 , or P 19 , which displayed little or no "cleavage" activity in vitro , were rescued, but wt revertants were obtained. The 2A substitutions impaired the replication of an FMDV replicon. Using transient-expression assays, it was shown that certain amino acid substitutions at residues E 14 , S 15 , N 16 , and P 19 resulted in partial "cleavage" of a protease-free polyprotein, indicating that these specific residues are not essential for cotranslational "cleavage." Immunofluorescence studies, using full-length FMDV RNA transcripts encoding mutant 2A peptides, indicated that the 2A peptide remained attached to adjacent proteins, presumably 2B. These results show that efficient "cleavage" at the 2A/2B junction is required for optimal virus replication. However, maximal StopGo activity does not appear to be essential for the viability of FMDV. IMPORTANCE Foot-and-mouth disease virus (FMDV) causes one of the most economically important diseases of farm animals. Cotranslational "cleavage" of the FMDV polyprotein precursor at the 2A/2B junction, termed StopGo, is mediated by the short 2A peptide through a nonproteolytic mechanism which leads to release of the nascent protein and continued translation of the downstream sequence. Improved understanding of this process will not only give a better insight into how this peptide influences the FMDV replication cycle but may also assist the application of this sequence in biotechnology for the production of multiple proteins from a single mRNA. Our data show that single amino acid substitutions in the 2A peptide can have a major influence on viral protein synthesis, virus viability, and polyprotein processing. They also indicate that efficient "cleavage" at the 2A/2B junction is required for optimal virus replication. However, maximal StopGo activity is not essential for the viability of FMDV. Copyright © 2018 American Society for Microbiology.

A Power-Efficient Wireless Capacitor Charging System Through an Inductive Link

PubMed Central

Lee, Hyung-Min; Ghovanloo, Maysam

2014-01-01

A power-efficient wireless capacitor charging system for inductively powered applications has been presented. A bank of capacitors can be directly charged from an ac source by generating a current through a series charge injection capacitor and a capacitor charger circuit. The fixed charging current reduces energy loss in switches, while maximizing the charging efficiency. An adaptive capacitor tuner compensates for the resonant capacitance variations during charging to keep the amplitude of the ac input voltage at its peak. We have fabricated the capacitor charging system prototype in a 0.35-μm 4-metal 2-poly standard CMOS process in 2.1 mm2 of chip area. It can charge four pairs of capacitors sequentially. While receiving 2.7-V peak ac input through a 2-MHz inductive link, the capacitor charging system can charge each pair of 1 μF capacitors up to ±2 V in 420 μs, achieving a high measured charging efficiency of 82%. PMID:24678284

A Power-Efficient Wireless Capacitor Charging System Through an Inductive Link.

PubMed

Lee, Hyung-Min; Ghovanloo, Maysam

2013-10-01

A power-efficient wireless capacitor charging system for inductively powered applications has been presented. A bank of capacitors can be directly charged from an ac source by generating a current through a series charge injection capacitor and a capacitor charger circuit. The fixed charging current reduces energy loss in switches, while maximizing the charging efficiency. An adaptive capacitor tuner compensates for the resonant capacitance variations during charging to keep the amplitude of the ac input voltage at its peak. We have fabricated the capacitor charging system prototype in a 0.35- μ m 4-metal 2-poly standard CMOS process in 2.1 mm 2 of chip area. It can charge four pairs of capacitors sequentially. While receiving 2.7-V peak ac input through a 2-MHz inductive link, the capacitor charging system can charge each pair of 1 μ F capacitors up to ±2 V in 420 μ s, achieving a high measured charging efficiency of 82%.

Packaging of DNA by shell crosslinked nanoparticles.

PubMed

Thurmond, K B; Remsen, E E; Kowalewski, T; Wooley, K L

1999-07-15

We demonstrate compaction of DNA with nanoscale biomimetic constructs which are robust synthetic analogs of globular proteins. These constructs are approximately 15 nm in diameter, shell crosslinked knedel-like (SCKs) nanoparticles, which are prepared by covalent stabilization of amphiphilic di-block co-polymer micelles, self-assembled in an aqueous solution. This synthetic approach yields size-controlled nanoparticles of persistent shape and containing positively charged functional groups at and near the particle surface. Such properties allow SCKs to bind with DNA through electrostatic interactions and facilitate reduction of the DNA hydrodynamic diameter through reversible compaction. Compaction of DNA by SCKs was evident in dynamic light scattering experiments and was directly observed by in situ atomic force microscopy. Moreover, enzymatic digestion of the DNA plasmid (pBR322, 4361 bp) by Eco RI was inhibited at low SCK:DNA ratios and prevented when [le]60 DNA bp were bound per SCK. Digestion by Msp I in the presence of SCKs resulted in longer DNA fragments, indicating that not all enzyme cleavage sites were accessible within the DNA/SCK aggregates. These results have implications for the development of vehicles for successful gene therapy applications.

Asparatic acid 221 is critical in the calcium-induced modulation of the enzymatic activity of human aminopeptidase A.

PubMed

Goto, Yoshikuni; Hattori, Akira; Mizutani, Shigehiko; Tsujimoto, Masafumi

2007-12-21

Aminopeptidase A (APA) plays an important role in the regulation of blood pressure by mediating angiotensin II degradation in the renin-angiotensin system. The Ca2+-induced modulation of enzymatic activity is the most characteristic feature of APA among the M1 family of aminopeptidases. In this study, we used site-directed mutagenesis for any residues responsible for the Ca2+ modulation of human APA. Alignment of sequences of the M1 family members led to the identification of Asp-221 as a significant residue of APA among the family members. Replacement of Asp-221 with Asn or Gln resulted in a loss of Ca2+ responsiveness toward synthetic substrates. These enzymes were also unresponsive to Ca2+ when peptide hormones, such as angiotensin II, cholecystokinin-8, neurokinin B, and kallidin, were employed as substrates. These results suggest that the negative charge of Asp-221 is essential for Ca2+ modulation of the enzymatic activity of APA and causes preferential cleavage of acidic amino acid at the N-terminal end of substrate peptides.

Pressure modulates the self-cleavage step of the hairpin ribozyme

NASA Astrophysics Data System (ADS)

Schuabb, Caroline; Kumar, Narendra; Pataraia, Salome; Marx, Dominik; Winter, Roland

2017-03-01

The ability of certain RNAs, denoted as ribozymes, to not only store genetic information but also catalyse chemical reactions gave support to the RNA world hypothesis as a putative step in the development of early life on Earth. This, however, might have evolved under extreme environmental conditions, including the deep sea with pressures in the kbar regime. Here we study pressure-induced effects on the self-cleavage of hairpin ribozyme by following structural changes in real-time. Our results suggest that compression of the ribozyme leads to an accelerated transesterification reaction, being the self-cleavage step, although the overall process is retarded in the high-pressure regime. The results reveal that favourable interactions between the reaction site and neighbouring nucleobases are strengthened under pressure, resulting therefore in an accelerated self-cleavage step upon compression. These results suggest that properly engineered ribozymes may also act as piezophilic biocatalysts in addition to their hitherto known properties.

Single-Chain Soluble BG505.SOSIP gp140 Trimers as Structural and Antigenic Mimics of Mature Closed HIV-1 Env.

PubMed

Georgiev, Ivelin S; Joyce, M Gordon; Yang, Yongping; Sastry, Mallika; Zhang, Baoshan; Baxa, Ulrich; Chen, Rita E; Druz, Aliaksandr; Lees, Christopher R; Narpala, Sandeep; Schön, Arne; Van Galen, Joseph; Chuang, Gwo-Yu; Gorman, Jason; Harned, Adam; Pancera, Marie; Stewart-Jones, Guillaume B E; Cheng, Cheng; Freire, Ernesto; McDermott, Adrian B; Mascola, John R; Kwong, Peter D

2015-05-01

Similar to other type I fusion machines, the HIV-1 envelope glycoprotein (Env) requires proteolytic activation; specifically, cleavage of a gp160 precursor into gp120 and gp41 subunits creates an N-terminal gp41 fusion peptide and permits folding from an immature uncleaved state to a mature closed state. While the atomic-level consequences of cleavage for HIV-1 Env are still being determined, the uncleaved state is antigenically distinct from the mature closed state, and cleavage has been reported to be essential for mimicry of the mature viral spike by soluble versions of Env. Here we report the redesign of a current state-of-the-art soluble Env mimic, BG505.SOSIP, to make it cleavage independent. Specifically, we replaced the furin cleavage site between gp120 and gp41 with Gly-Ser linkers of various lengths. The resultant linked gp120-gp41 constructs, termed single-chain gp140 (sc-gp140), exhibited different levels of structural and antigenic mimicry of the parent cleaved BG505.SOSIP. When constructs were subjected to negative selection to remove subspecies recognized by poorly neutralizing antibodies, trimers of high antigenic mimicry of BG505.SOSIP could be obtained; negative-stain electron microscopy indicated these to resemble the mature closed state. Higher proportions of BG505.SOSIP-trimer mimicry were observed in sc-gp140s with linkers of 6 or more residues, with a linker length of 15 residues exhibiting especially promising traits. Overall, flexible linkages between gp120 and gp41 in BG505.SOSIP can thus substitute for cleavage, and sc-gp140s that closely mimicked the vaccine-preferred mature closed state of Env could be obtained. The trimeric HIV-1 envelope glycoprotein (Env) is the sole target of virus-directed neutralizing antibody responses and a primary focus of vaccine design. Soluble mimics of Env have proven challenging to obtain and have been thought to require proteolytic cleavage into two-component subunits, gp120 and gp41, to achieve structural and antigenic mimicry of mature Env spikes on virions. Here we show that replacement of the cleavage site between gp120 and gp41 in a lead soluble gp140 construct, BG505.SOSIP, with flexible linkers can result in molecules that do not require cleavage to fold efficiently into the mature closed state. Our results provide insights into the impact of cleavage on HIV-1 Env folding. In some contexts such as genetic immunization, optimized cleavage-independent soluble gp140 constructs may have utility over the parental BG505.SOSIP, as they would not require furin cleavage to achieve mimicry of mature Env spikes on virions. Copyright © 2015, American Society for Microbiology. All Rights Reserved.

Characterization of the ScAlMgO4 cleaving layer by X-ray crystal truncation rod scattering

NASA Astrophysics Data System (ADS)

Hanada, Takashi; Tajiri, Hiroo; Sakata, Osami; Fukuda, Tsuguo; Matsuoka, Takashi

2018-05-01

ScAlMgO4—easily cleaved in c-plane—forms a natural superlattice structure of a ScO2 layer and two Al0.5Mg0.5O layers stacking along c-axis. ScAlMgO4 is one of the RAMO4-type layered multicomponent oxides and a promising lattice-matching substrate material for InGaN and ZnO. Identification of the topmost layer and the surface atomic structure of the cleaved ScAlMgO4 (0001) are investigated by the X-ray crystal truncation rod scattering method. It is confirmed that ScAlMgO4 is cleaved between the two Al0.5Mg0.5O layers. The two parts separated at this interlayer are inversion symmetric to each other and without surface charge. This prevents parallel-plate-capacitor-like electrostatic force during the cleavage. Two different mechanisms are proposed for the two types of cleavage caused by the impact of a wedge and by the in-plane stress due to an overgrown thick GaN film. It is also revealed that about 10%-20% of the topmost O atoms are desorbed during a surface cleaning at 600 °C in ultra-high vacuum. Surface observations using reflection high-energy electron diffraction are possible only after the high-temperature cleaning because the electrical conduction caused by the oxygen deficiency prevents the charge-up of the insulating sample.

Anisotropic surface chemistry properties and adsorption behavior of silicate mineral crystals.

PubMed

Xu, Longhua; Tian, Jia; Wu, Houqin; Fang, Shuai; Lu, Zhongyuan; Ma, Caifeng; Sun, Wei; Hu, Yuehua

2018-03-07

Anisotropic surface properties of minerals play an important role in a variety of fields. With a focus on the two most intensively investigated silicate minerals (i.e., phyllosilicate minerals and pegmatite aluminosilicate minerals), this review highlights the research on their anisotropic surface properties based on their crystal structures. Four surface features comprise the anisotropic surface chemistry of minerals: broken bonds, energy, wettability, and charge. Analysis of surface broken bond and energy anisotropy helps to explain the cleavage and growth properties of mineral crystals, and understanding surface wettability and charge anisotropy is critical to the analysis of minerals' solution behavior, such as their flotation performance and rheological properties. In a specific reaction, the anisotropic surface properties of minerals are reflected in the adsorption strengths of reagents on different mineral surfaces. Combined with the knowledge of mineral crushing and grinding, a thorough understanding of the anisotropic surface chemistry properties and the anisotropic adsorption behavior of minerals will lead to the development of effective relational models comprising their crystal structure, surface chemistry properties, and targeted reagent adsorption. Overall, such a comprehensive approach is expected to firmly establish the connection between selective cleavage of mineral crystals for desired surfaces and designing novel reagents selectively adsorbed on the mineral surfaces. As tools to characterize the anisotropic surface chemistry properties of minerals, DLVO theory, atomic force microscopy (AFM), and molecular dynamics (MD) simulations are also reviewed. Copyright © 2017 Elsevier B.V. All rights reserved.

Proteolytic cleavage by the inner membrane peptidase (IMP) complex or Oct1 peptidase controls the localization of the yeast peroxiredoxin Prx1 to distinct mitochondrial compartments.

PubMed

Gomes, Fernando; Palma, Flávio Romero; Barros, Mario H; Tsuchida, Eduardo T; Turano, Helena G; Alegria, Thiago G P; Demasi, Marilene; Netto, Luis E S

2017-10-13

Yeast Prx1 is a mitochondrial 1-Cys peroxiredoxin that catalyzes the reduction of endogenously generated H 2 O 2 Prx1 is synthesized on cytosolic ribosomes as a preprotein with a cleavable N-terminal presequence that is the mitochondrial targeting signal, but the mechanisms underlying Prx1 distribution to distinct mitochondrial subcompartments are unknown. Here, we provide direct evidence of the following dual mitochondrial localization of Prx1: a soluble form in the intermembrane space and a form in the matrix weakly associated with the inner mitochondrial membrane. We show that Prx1 sorting into the intermembrane space likely involves the release of the protein precursor within the lipid bilayer of the inner membrane, followed by cleavage by the inner membrane peptidase. We also found that during its import into the matrix compartment, Prx1 is sequentially cleaved by mitochondrial processing peptidase and then by octapeptidyl aminopeptidase 1 (Oct1). Oct1 cleaved eight amino acid residues from the N-terminal region of Prx1 inside the matrix, without interfering with its peroxidase activity in vitro Remarkably, the processing of peroxiredoxin (Prx) proteins by Oct1 appears to be an evolutionarily conserved process because yeast Oct1 could cleave the human mitochondrial peroxiredoxin Prx3 when expressed in Saccharomyces cerevisiae Altogether, the processing of peroxiredoxins by Imp2 or Oct1 likely represents systems that control the localization of Prxs into distinct compartments and thereby contribute to various mitochondrial redox processes. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

Selected classes of minimised hammerhead ribozyme have very high cleavage rates at low Mg2+ concentration.

PubMed Central

Conaty, J; Hendry, P; Lockett, T

1999-01-01

In vitro selection was used to enrich for highly efficient RNA phosphodiesterases within a size-constrained (18 nt) ribonucleotide domain. The starting population (g0) was directed in trans against an RNA oligonucleotide substrate immobilised to an avidin-magnetic phase. Four rounds of selection were conducted using 20 mM Mg2+to fractionate the population on the basis of divalent metal ion-dependent phosphodiesterase activity. The resulting generation 4 (g4) RNA was then directed through a further two rounds of selection using low concentrations of Mg2+. Generation 6 (g6) was composed of sets of active, trans cleaving minimised ribozymes, containing recognised hammerhead motifs in the conserved nucleotides, but with highly variable linker domains (loop II-L.1-L.4). Cleavage rate constants in the g6 population ranged from 0.004 to 1.3 min-1at 1 mM Mg2+(pH 8.0, 37 degrees C). Selection was further used to define conserved positions between G(10.1) and C(11.1) required for high cleavage activity at low Mg2+concentration. At 10 mM MgCl2the kinetic phenotype of these molecules was comparable to a hammerhead ribozyme with 4 bp in helix II. At low Mg2+concentration, the disparity in cleavage rate constants increases in favour of the minimised ribozymes. Favourable kinetic traits appeared to be a general property for specific selected linker sequences, as the high rates of catalysis were transferable to a different substrate system. PMID:10325431

Cnidarian microRNAs frequently regulate targets by cleavage.

PubMed

Moran, Yehu; Fredman, David; Praher, Daniela; Li, Xin Z; Wee, Liang Meng; Rentzsch, Fabian; Zamore, Phillip D; Technau, Ulrich; Seitz, Hervé

2014-04-01

In bilaterians, which comprise most of extant animals, microRNAs (miRNAs) regulate the majority of messenger RNAs (mRNAs) via base-pairing of a short sequence (the miRNA "seed") to the target, subsequently promoting translational inhibition and transcript instability. In plants, many miRNAs guide endonucleolytic cleavage of highly complementary targets. Because little is known about miRNA function in nonbilaterian animals, we investigated the repertoire and biological activity of miRNAs in the sea anemone Nematostella vectensis, a representative of Cnidaria, the sister phylum of Bilateria. Our work uncovers scores of novel miRNAs in Nematostella, increasing the total miRNA gene count to 87. Yet only a handful are conserved in corals and hydras, suggesting that microRNA gene turnover in Cnidaria greatly exceeds that of other metazoan groups. We further show that Nematostella miRNAs frequently direct the cleavage of their mRNA targets via nearly perfect complementarity. This mode of action resembles that of small interfering RNAs (siRNAs) and plant miRNAs. It appears to be common in Cnidaria, as several of the miRNA target sites are conserved among distantly related anemone species, and we also detected miRNA-directed cleavage in Hydra. Unlike in bilaterians, Nematostella miRNAs are commonly coexpressed with their target transcripts. In light of these findings, we propose that post-transcriptional regulation by miRNAs functions differently in Cnidaria and Bilateria. The similar, siRNA-like mode of action of miRNAs in Cnidaria and plants suggests that this may be an ancestral state.

Modulation of procaspase-7 self-activation by PEST amino acid residues of the N-terminal prodomain and intersubunit linker.

PubMed

Alves, Juliano; Garay-Malpartida, Miguel; Occhiucci, João M; Belizário, José E

2017-12-01

Procaspase-7 zymogen polypeptide is composed of a short prodomain, a large subunit (p20), and a small subunit (p10) connected to an intersubunit linker. Caspase-7 is activated by an initiator caspase-8 and -9, or by autocatalysis after specific cleavage at IQAD 198 ↓S located at the intersubunit linker. Previously, we identified that PEST regions made of amino acid residues Pro (P), Glu (E), Asp (D), Ser (S), Thr (T), Asn (N), and Gln (Q) are conserved flanking amino acid residues in the cleavage sites within a prodomain and intersubunit linker of all caspase family members. Here we tested the impact of alanine substitution of PEST amino acid residues on procaspase-7 proteolytic self-activation directly in Escherichia coli. The p20 and p10 subunit cleavage were significantly delayed in double caspase-7 mutants in the prodomain (N18A/P26A) and intersubunit linker (S199A/P201A), compared with the wild-type caspase-7. The S199A/P201A mutants effectively inhibited the p10 small subunit cleavage. However, the mutations did not change the kinetic parameters (k cat /K M ) and optimal tetrapeptide specificity (DEVD) of the purified mutant enzymes. The results suggest a role of PEST-amino acid residues in the molecular mechanism for prodomain and intersubunit cleavage and caspase-7 self-activation.

Structural Determinants of Autoproteolysis of the Haemophilus influenzae Hap Autotransporter▿

PubMed Central

Kenjale, Roma; Meng, Guoyu; Fink, Doran L.; Juehne, Twyla; Ohashi, Tomoo; Erickson, Harold P.; Waksman, Gabriel; St. Geme, Joseph W.

2009-01-01

Haemophilus influenzae is a gram-negative bacterium that initiates infection by colonizing the upper respiratory tract. The H. influenzae Hap autotransporter protein mediates adherence, invasion, and microcolony formation in assays with respiratory epithelial cells and presumably facilitates colonization. The serine protease activity of Hap is associated with autoproteolytic cleavage and extracellular release of the HapS passenger domain, leaving the Hapβ C-terminal domain embedded in the outer membrane. Cleavage occurs most efficiently at the LN1036-37 peptide bond and to a lesser extent at three other sites. In this study, we utilized site-directed mutagenesis, homology modeling, and assays with a peptide library to characterize the structural determinants of Hap proteolytic activity and cleavage specificity. In addition, we used homology modeling to predict the S1, S2, and S4 subsite residues of the Hap substrate groove. Our results indicate that the P1 and P2 positions at the Hap cleavage sites are critical for cleavage, with leucine preferred over larger hydrophobic residues or other amino acids in these positions. The substrate groove is formed by L263 and N274 at the S1 subsite, R264 at the S2 subsite, and E265 at the S4 subsite. This information may facilitate design of approaches to block Hap activity and interfere with H. influenzae colonization. PMID:19687208

Dual CRISPR-Cas9 Cleavage Mediated Gene Excision and Targeted Integration in Yarrowia lipolytica.

PubMed

Gao, Difeng; Smith, Spencer; Spagnuolo, Michael; Rodriguez, Gabriel; Blenner, Mark

2018-05-29

CRISPR-Cas9 technology has been successfully applied in Yarrowia lipolytica for targeted genomic editing including gene disruption and integration; however, disruptions by existing methods typically result from small frameshift mutations caused by indels within the coding region, which usually resulted in unnatural protein. In this study, a dual cleavage strategy directed by paired sgRNAs is developed for gene knockout. This method allows fast and robust gene excision, demonstrated on six genes of interest. The targeted regions for excision vary in length from 0.3 kb up to 3.5 kb and contain both non-coding and coding regions. The majority of the gene excisions are repaired by perfect nonhomologous end-joining without indel. Based on this dual cleavage system, two targeted markerless integration methods are developed by providing repair templates. While both strategies are effective, homology mediated end joining (HMEJ) based method are twice as efficient as homology recombination (HR) based method. In both cases, dual cleavage leads to similar or improved gene integration efficiencies compared to gene excision without integration. This dual cleavage strategy will be useful for not only generating more predictable and robust gene knockout, but also for efficient targeted markerless integration, and simultaneous knockout and integration in Y. lipolytica. © 2018 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Proteolytic Cleavage of ProBDNF into Mature BDNF in the Basolateral Amygdala Is Necessary for Defeat-Induced Social Avoidance

ERIC Educational Resources Information Center

Dulka, Brooke N.; Ford, Ellen C.; Lee, Melissa A.; Donnell, Nathaniel J.; Goode, Travis D.; Prosser, Rebecca; Cooper, Matthew A.

2016-01-01

Brain-derived neurotrophic factor (BDNF) is essential for memory processes. The present study tested whether proteolytic cleavage of proBDNF into mature BDNF (mBDNF) within the basolateral amygdala (BLA) regulates the consolidation of defeat-related memories. We found that acute social defeat increases the expression of mBDNF, but not proBDNF, in…

Thiazolylidene-catalyzed cleavage of methyl oleate-derived α-hydroxy ketone to the corresponding free aldehydes.

PubMed

Deruer, Elsa; Duguet, Nicolas; Lemaire, Marc

2015-08-10

The thiazolylidene-catalyzed cleavage of the α-hydroxy ketone derived from methyl oleate gave the corresponding aldehydes under nonoxidative conditions through a retro-benzoin process. The aldehydes produced are in equilibrium with their corresponding acyloins. To illustrate the synthetic utility of this protocol, the aldehydes were recovered by distillation. © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

A novel quantitative electrochemical method to monitor DNA double-strand breaks caused by a DNA cleavage agent at a DNA sensor.

PubMed

Banasiak, Anna; Cassidy, John; Colleran, John

2018-06-01

To date, DNA cleavage, caused by cleavage agents, has been monitored mainly by gel and capillary electrophoresis. However, these techniques are time-consuming, non-quantitative and require gel stains. In this work, a novel, simple and, importantly, a quantitative method for monitoring the DNA nuclease activity of potential anti-cancer drugs, at a DNA electrochemical sensor, is presented. The DNA sensors were prepared using thiol-modified oligonucleotides that self-assembled to create a DNA monolayer at gold electrode surfaces. The quantification of DNA double-strand breaks is based on calculating the DNA surface coverage, before and after exposure to a DNA cleavage agent. The nuclease properties of a model DNA cleavage agent, copper bis-phenanthroline ([Cu II (phen) 2 ] 2+ ), that can cleave DNA in a Fenton-type reaction, were quantified electrochemically. The DNA surface coverage decreased on average by 21% after subjecting the DNA sensor to a nuclease assay containing [Cu II (phen) 2 ] 2+ , a reductant and an oxidant. This percentage indicates that 6 base pairs were cleaved in the nuclease assay from the immobilised 30 base pair strands. The DNA cleavage can be also induced electrochemically in the absence of a chemical reductant. [Cu II (phen) 2 ] 2+ intercalates between DNA base pairs and, on application of a suitable potential, can be reduced to [Cu I (phen) 2 ] + , with dissolved oxygen acting as the required oxidant. This reduction process is facilitated through DNA strands via long-range electron transfer, resulting in DNA cleavage of 23%. The control measurements for both chemically and electrochemically induced cleavage revealed that DNA strand breaks did not occur under experimental conditions in the absence of [Cu II (phen) 2 ] 2+ . Copyright © 2018 Elsevier B.V. All rights reserved.

Destruction of microcystins (cyanotoxins) by UV-254 nm-based direct photolysis and advanced oxidation processes (AOPs): influence of variable amino acids on the degradation kinetics and reaction mechanisms.

PubMed

He, Xuexiang; de la Cruz, Armah A; Hiskia, Anastasia; Kaloudis, Triantafyllos; O'Shea, Kevin; Dionysiou, Dionysios D

2015-05-01

Hepatotoxic microcystins (MCs) are the most frequently detected group of cyanobacterial toxins. This study investigated the degradation of common MC variants in water, MC-LR, MC-RR, MC-YR and MC-LA, by UV-254 nm-based processes, UV only, UV/H2O2, UV/S2O8(2-) and UV/HSO5(-). Limited direct photolysis of MCs was observed, while the addition of an oxidant significantly improved the degradation efficiency with an order of UV/S2O8(2-) > UV/HSO5(-) > UV/H2O2 at the same initial molar concentration of the oxidant. The removal of MC-LR by UV/H2O2 appeared to be faster than another cyanotoxin, cylindrospermopsin, at either the same initial molar concentration or the same initial organic carbon concentration of the toxin. It suggested a faster reaction of MC-LR with hydroxyl radical, which was further supported by the determined second-order rate constant of MCs with hydroxyl radical. Both isomerization and photohydration byproducts were observed in UV only process for all four MCs; while in UV/H2O2, hydroxylation and diene-Adda double bond cleavage byproducts were detected. The presence of a tyrosine in the structure of MC-YR significantly promoted the formation of monohydroxylation byproduct m/z 1061; while the presence of a second arginine in MC-RR led to the elimination of a guanidine group and the absence of double bond cleavage byproducts. It was therefore demonstrated in this study that the variable amino acids in the structure of MCs influenced not only the degradation kinetics but also the preferable reaction mechanisms. Copyright © 2015 Elsevier Ltd. All rights reserved.

Theoretical Study of the Mechanism of Oxoiron(IV) Formation from H2O2 and a Nonheme Iron(II) Complex: O–O Cleavage Involving Proton-Coupled Electron Transfer

PubMed Central

Hirao, Hajime; Li, Feifei; Que, Lawrence; Morokuma, Keiji

2011-01-01

It has recently been shown that the nonheme oxoiron(IV) species supported by the 1,4,8,11-tetramethyl-1,4,8,11-tetraazacyclotetradecane ligand (TMC) can be generated in near-quantitative yield by reacting [FeII(TMC)(OTf)2] with a stoichiometric amount of H2O2 in CH3CN in the presence of 2,6-lutidine (Li, F.; England, J.; Que L., Jr. J. Am. Chem. Soc. 2010, 132, 2134–2135). This finding has major implications for O–O bond cleavage events in both Fenton chemistry and nonheme iron enzymes. To understand the mechanism of this process, especially the intimate details of the O–O bond cleavage step, a series of density functional theory (DFT) calculations and analyses have been carried out. Two distinct reaction paths (A and B) were identified. Path A consists of two principal steps: (1) coordination of H2O2 to Fe(II) and (2) a combination of partial homolytic O–O bond cleavage and proton-coupled electron transfer (PCET). The latter combination renders the rate-limiting O–O cleavage effectively a heterolytic process. Path B proceeds via a simultaneous homolytic O–O bond cleavage of H2O2 and Fe–O bond formation. This is followed by H-abstraction from the resultant Fe(III)–OH species by an •OH radical. Calculations suggest that path B is plausible in the absence of base. However, once 2,6-lutidine is added to the reacting system, the reaction barrier is lowered and more importantly the mechanistic path switches to path A, where 2,6-lutidine plays an essential role as an acid-base catalyst in a manner similar to how the distal histidine or glutamate residue assists in Compound I formation in heme peroxidases. The reaction was found to proceed predominantly on the quintet spin state surface, and a transition to the triplet state, the experimentally known ground state for the TMC-oxoiron(IV) species, occurs in the last stage of the oxoiron(IV) formation process. PMID:21678930

Injection-modulated polarity conversion by charge carrier density control via a self-assembled monolayer for all-solution-processed organic field-effect transistors

NASA Astrophysics Data System (ADS)

Roh, Jeongkyun; Lee, Taesoo; Kang, Chan-Mo; Kwak, Jeonghun; Lang, Philippe; Horowitz, Gilles; Kim, Hyeok; Lee, Changhee

2017-04-01

We demonstrated modulation of charge carrier densities in all-solution-processed organic field-effect transistors (OFETs) by modifying the injection properties with self-assembled monolayers (SAMs). The all-solution-processed OFETs based on an n-type polymer with inkjet-printed Ag electrodes were fabricated as a test platform, and the injection properties were modified by the SAMs. Two types of SAMs with different dipole direction, thiophenol (TP) and pentafluorobenzene thiol (PFBT) were employed, modifying the work function of the inkjet-printed Ag (4.9 eV) to 4.66 eV and 5.24 eV with TP and PFBT treatments, respectively. The charge carrier densities were controlled by the SAM treatment in both dominant and non-dominant carrier-channel regimes. This work demonstrates that control of the charge carrier densities can be efficiently achieved by modifying the injection property with SAM treatment; thus, this approach can achieve polarity conversion of the OFETs.

Construction of a Direct Water-Injected Two-Stroke Engine for Phased Direct Fuel Injection-High Pressure Charging Investigations

NASA Technical Reports Server (NTRS)

Somsel, James P.

1998-01-01

The development of a water injected Orbital Combustion Process (OCP) engine was conducted to assess the viability of using the powerplant for high altitude NASA aircraft and General Aviation (GA) applications. An OCP direct fuel injected, 1.2 liter, three cylinder, two-stroke engine has been enhanced to independently inject water directly into the combustion chamber. The engine currently demonstrates low brake specific fuel consumption capability and an excellent power to weight ratio. With direct water injection, significant improvements can be made to engine power, to knock limits/ignition advance timing, and to engine NO(x) emissions. The principal aim of the testing was to validate a cyclic model developed by the Systems Analysis Branch at NASA Ames Research Center. The work is a continuation of Ames' investigations into a Phased Direct Fuel Injection Engine with High Pressure Charging (PDFI-ITPC).

Photodetachment process for beam neutralization

DOEpatents

Fink, J.H.; Frank, A.M.

1979-02-20

A process for neutralization of accelerated ions employing photo-induced charge detachment is disclosed. The process involves directing a laser beam across the path of a negative ion beam such as to effect photodetachment of electrons from the beam ions. The frequency of the laser beam employed is selected to provide the maximum cross-section for the photodetachment process. 2 figs.

Reactions in 1,1,1-trifluoroacetone triggered by low energy electrons (0-10 eV): from simple bond cleavages to complex unimolecular reactions.

PubMed

Illenberger, Eugen; Meinke, Martina C

2014-08-21

The impact of low energy electrons (0-10 eV) to 1,1,1-trifluoroacetone yields a variety of fragment anions which are formed via dissociative electron attachment (DEA) through three pronounced resonances located at 0.8 eV, near 4 eV, and in the energy range 8-9 eV. The fragment ions arise from different reactions ranging from the direct cleavage of one single or double bond (formation of F(-), CF3(-), O(-), (M-H)(-), and M-F)(-)) to remarkably complex unimolecular reactions associated with substantial geometric and electronic rearrangement in the transitory intermediate (formation of OH(-), FHF(-), (M-HF)(-), CCH(-), and HCCO(-). The ion CCH(-), for example, is formed by an excision of unit from the target molecule through the concerted cleavage of four bonds and recombination to H2O within the neutral component of the reaction.

Rational design of micro-RNA-like bifunctional siRNAs targeting HIV and the HIV coreceptor CCR5.

PubMed

Ehsani, Ali; Saetrom, Pål; Zhang, Jane; Alluin, Jessica; Li, Haitang; Snøve, Ola; Aagaard, Lars; Rossi, John J

2010-04-01

Small-interfering RNAs (siRNAs) and micro-RNAs (miRNAs) are distinguished by their modes of action. SiRNAs serve as guides for sequence-specific cleavage of complementary mRNAs and the targets can be in coding or noncoding regions of the target transcripts. MiRNAs inhibit translation via partially complementary base-pairing to 3' untranslated regions (UTRs) and are generally ineffective when targeting coding regions of a transcript. In this study, we deliberately designed siRNAs that simultaneously direct cleavage and translational suppression of HIV RNAs, or cleavage of the mRNA encoding the HIV coreceptor CCR5 and suppression of translation of HIV. These bifunctional siRNAs trigger inhibition of HIV infection and replication in cell culture. The design principles have wide applications throughout the genome, as about 90% of genes harbor sites that make the design of bifunctional siRNAs possible.

Analysis of Turbulent Combustion in Simplified Stratified Charge Conditions

NASA Astrophysics Data System (ADS)

Moriyoshi, Yasuo; Morikawa, Hideaki; Komatsu, Eiji

The stratified charge combustion system has been widely studied due to the significant potentials for low fuel consumption rate and low exhaust gas emissions. The fuel-air mixture formation process in a direct-injection stratified charge engine is influenced by various parameters, such as atomization, evaporation, and in-cylinder gas motion at high temperature and high pressure conditions. It is difficult to observe the in-cylinder phenomena in such conditions and also challenging to analyze the following stratified charge combustion. Therefore, the combustion phenomena in simplified stratified charge conditions aiming to analyze the fundamental stratified charge combustion are examined. That is, an experimental apparatus which can control the mixture distribution and the gas motion at ignition timing was developed, and the effects of turbulence intensity, mixture concentration distribution, and mixture composition on stratified charge combustion were examined. As a result, the effects of fuel, charge stratification, and turbulence on combustion characteristics were clarified.

Scales of equilibrium and disequilibrium during cleavage formation in chlorite and biotite-grade phyllites, SE Vermont

USGS Publications Warehouse

McWilliams, C.K.; Wintsch, R.P.; Kunk, Michael J.

2007-01-01

Detailed electron microprobe analyses of phyllosilicates in crenulated phyllites from south-eastern Vermont show that grain-scale zoning is common, and sympathetic zoning in adjacent minerals is nearly universal. We interpret this to reflect a pressure-solution mechanism for cleavage development, where precipitation from a very small fluid reservoir fractionated that fluid. Multiple analyses along single muscovite, biotite and chlorite grains (30–200 μm in length) show zoning patterns indicating Tschermakitic substitutions in muscovite and both Tschermakitic and di/trioctahedral substitutions in biotite and chlorite. Using cross-cutting relationships and mineral chemistry it is shown that these patterns persist in cleavages produced at metamorphic conditions of chlorite-grade, chlorite-grade overprinted by biotite-grade and biotite-grade. Zoning patterns are comparable in all three settings, requiring a similar cleavage-forming mechanism independent of metamorphic grade. Moreover, the use of 40Ar/39Ar geochronology demonstrates this is true regardless of age. Furthermore, samples with chlorite-grade cleavages overprinted by biotite porphyroblasts suggest the closure temperatures for the diffusion of Al, Si, Mg and Fe ions are greater than the temperature of the biotite isograd (>∼400 °C). Parallel and smoothly fanning tie lines produced by coexisting muscovite–chlorite, and muscovite–biotite pairs on compositional diagrams demonstrate effectively instantaneous chemical equilibrium and probably indicate simultaneous crystallization.These results do not support theories suggesting cleavages form in fluid-dominated systems. If crenulation cleavages formed in systems in which the chemical potentials of all major components are fixed by an external reservoir, then the compositions of individual grains defining these cleavages would be uniform. On the contrary, the fine-scale chemical zoning observed probably reflects a grain-scale process consistent with a pressure-solution mechanism in which the aqueous activities of major components are defined by local dissolution and precipitation. Thus the role of fluids was probably limited to one of catalysing pressure-solution and fluids apparently did not drive cleavage development.

Performance of 20:1 multiplexer for large area charge readouts in directional dark matter TPC detectors

NASA Astrophysics Data System (ADS)

Ezeribe, A. C.; Robinson, M.; Robinson, N.; Scarff, A.; Spooner, N. J. C.; Yuriev, L.

2018-02-01

More target mass is required in current TPC based directional dark matter detectors for improved detector sensitivity. This can be achieved by scaling up the detector volumes, but this results in the need for more analogue signal channels. A possible solution to reducing the overall cost of the charge readout electronics is to multiplex the signal readout channels. Here, we present a multiplexer system in expanded mode based on LMH6574 chips produced by Texas Instruments, originally designed for video processing. The setup has a capability of reducing the number of readouts in such TPC detectors by a factor of 20. Results indicate that the important charge distribution asymmetry along an ionization track is retained after multiplexed signals are demultiplexed.

Relativistic direct Urca processes in cooling neutron stars

NASA Astrophysics Data System (ADS)

Leinson, L. B.; Pérez, A.

2001-10-01

We derive a relativistic expression for neutrino energy losses caused by the direct Urca processes in degenerate baryon matter of neutron stars. We use two different ways to calculate the emissivity caused by the reactions to our interest. First we perform a standard calculation by Fermi's ``golden'' rule. The second calculation, resulting in the same expression, is performed with the aid of polarization functions of the medium. Our result for neutrino energy losses strongly differs from previous nonrelativistic results. We also discuss nonconservation of the baryon vector current in reactions through weak charged currents in the medium, when the asymmetry between protons and neutrons is considered. The above effects, not discussed in the literature before, substantially modify the polarization functions responsible for the induced weak charged currents in baryon matter.

Caspases and Kinases in a Death Grip

PubMed Central

Kurokawa, Manabu; Kornbluth, Sally

2011-01-01

The complex process of apoptosis is orchestrated by caspases, a family of cysteine proteases with unique substrate specificities. Accumulating evidence suggests that cell death pathways are finely tuned by multiple signaling events, including direct phosphorylation of caspases, whereas kinases are often substrates of active caspases. Importantly, caspase-mediated cleavage of kinases can terminate prosurvival signaling or generate proapoptotic peptide fragments that help to execute the death program and facilitate packaging of the dying cells. Here, we review caspases as kinase substrates and kinases as caspase substrates and discuss how the balance between cell survival and cell death can be shifted through crosstalk between these two enzyme families. PMID:19737514

A Microplate Format Assay for Real-Time Screening for New Aldolases that Accept Aryl-Substituted Acceptor Substrates.

PubMed

Ma, Huan; Enugala, Thilak Reddy; Widersten, Mikael

2015-12-01

Aldolases are potentially important biocatalysts for asymmetric synthesis of polyhydroxylated compounds. Fructose 6-phosphate aldolase (FSA) is of particular interest by virtue of its unusually relaxed dependency on phosphorylated substrates. FSA has been reported to be a promising catalyst of aldol addition involving aryl-substituted acceptors such as phenylacetaldehyde that can react with donor ketones such as hydroxyacetone. Improvement of the low intrinsic activity with bulky acceptor substrates of this type is of great interest but has been hampered by the lack of powerful screening protocols applicable in directed evolution strategies. Here we present a new screen allowing for direct spectrophotometric recording of retro-aldol cleavage. The assay utilizes an aldehyde reductase produced in vitro by directed evolution; it reduces the aldehyde product formed after cleavage of the aldol by FSA. The assay is suitable both for steady-state enzyme kinetics and for real-time activity screening in a 96-well format. © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Network Analyses Reveal Pervasive Functional Regulation Between Proteases in the Human Protease Web

PubMed Central

Fortelny, Nikolaus; Cox, Jennifer H.; Kappelhoff, Reinhild; Starr, Amanda E.; Lange, Philipp F.; Pavlidis, Paul; Overall, Christopher M.

2014-01-01

Proteolytic processing is an irreversible posttranslational modification affecting a large portion of the proteome. Protease-cleaved mediators frequently exhibit altered activity, and biological pathways are often regulated by proteolytic processing. Many of these mechanisms have not been appreciated as being protease-dependent, and the potential in unraveling a complex new dimension of biological control is increasingly recognized. Proteases are currently believed to act individually or in isolated cascades. However, conclusive but scattered biochemical evidence indicates broader regulation of proteases by protease and inhibitor interactions. Therefore, to systematically study such interactions, we assembled curated protease cleavage and inhibition data into a global, computational representation, termed the protease web. This revealed that proteases pervasively influence the activity of other proteases directly or by cleaving intermediate proteases or protease inhibitors. The protease web spans four classes of proteases and inhibitors and so links both recently and classically described protease groups and cascades, which can no longer be viewed as operating in isolation in vivo. We demonstrated that this observation, termed reachability, is robust to alterations in the data and will only increase in the future as additional data are added. We further show how subnetworks of the web are operational in 23 different tissues reflecting different phenotypes. We applied our network to develop novel insights into biologically relevant protease interactions using cell-specific proteases of the polymorphonuclear leukocyte as a system. Predictions from the protease web on the activity of matrix metalloproteinase 8 (MMP8) and neutrophil elastase being linked by an inactivating cleavage of serpinA1 by MMP8 were validated and explain perplexing Mmp8 −/− versus wild-type polymorphonuclear chemokine cleavages in vivo. Our findings supply systematically derived and validated evidence for the existence of the protease web, a network that affects the activity of most proteases and thereby influences the functional state of the proteome and cell activity. PMID:24865846

Manduca sexta proprophenoloxidase activating proteinase-3 (PAP3) stimulates melanization by activating proPAP3, proSPHs, and proPOs

PubMed Central

Wang, Yang; Lu, Zhiqiang; Jiang, Haobo

2014-01-01

Melanization participates in various insect physiological processes including antimicrobial immune responses. Phenoloxidase (PO), a critical component of the enzyme system catalyzing melanin formation, is produced as an inactive precursor prophenoloxidase (proPO) and becomes active via specific proteolytic cleavage by proPO activating proteinase (PAP). In Manduca sexta, three PAPs can activate proPOs in the presence of two serine proteinase homologs (SPH1 and SPH2). While the hemolymph proteinases (HPs) that generate the active PAPs are known, it is unclear how the proSPHs (especially proSPH1) are activated. In this study, we isolated from plasma of bar-stage M. sexta larvae an Ile-Glu-Ala-Arg-p-nitroanilide hydrolyzing enzyme that cleaved the proSPHs. This proteinase, PAP3, generated active SPH1 and SPH2, which function as cofactors for PAP3 in proPO activation. Cleavage of the purified recombinant proSPHs by PAP3 yielded 38 kDa bands similar in mobility to the SPHs formed in vivo. Surprisingly, PAP3 also can activate proPAP3 to stimulate melanization in a direct positive feedback loop. The enhanced proPO activation concurred with the cleavage activation of proHP6, proHP8, proPAP1, proPAP3, proSPH1, proSPH2, proPOs, but not proHP14 or proHP21. These results indicate that PAP3, like PAP1, is a key factor of the self-reinforcing mechanism in the proPO activation system, which is linked to other immune responses in M. sexta. PMID:24768974

Infectious rotavirus enters cells by direct cell membrane penetration, not by endocytosis

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

Kaljot, K.T.; Shaw, R.D.; Greenberg, H.B.

1988-04-01

Rotaviruses are icosahedral viruses with a segmented, double-stranded RNA genome. They are the major cause of severe infantile infectious diarrhea. Rotavirus growth in tissue culture is markedly enhanced by pretreatment of virus with trypsin. Trypsin activation is associated with cleavage of the viral hemagglutinin (viral protein 3 (VP3); 88 kilodaltons) into two fragments (60 and 28 kilodaltons). The mechanism by which proteolytic cleavage leads to enhanced growth is unknown. To determine whether trypsin treatment affected rotavirus internalization, the authors studied the kinetics of entry of infectious rhesus rotavirus (RRV) into MA104 cells. Trypsin-activated RRV was internalized with a half-time ofmore » 3 to 5 min, while nonactivated virus disappeared from the cell surface with a half-time of 30 to 50 min. In contrast to trypsin-activated RRV, loss of nonactivated RRV from the cell surface did not result in the appearance of infection, as measured by plaque formation. Purified trypsin-activated RRV added to cell monolayers at pH 7.4 mediated {sup 51}Cr, ({sup 14}C)choline, and ({sup 3}H)inositol released from prelabeled MA104 cells. This release could be specifically blocked by neutralizing antibodies to VP3. These results suggest that MA104 cell infection follows the rapid entry of trypsin-activated RRV by direct cell membrane penetration. Cell membrane penetration of infectious RRV is initiated by trypsin cleavage of VP3. Neutralizing antibodies can inhibit this direct membrane penetration.« less

Dark States in the Light-Harvesting complex 2 Revealed by Two-dimensional Electronic Spectroscopy

PubMed Central

Ferretti, Marco; Hendrikx, Ruud; Romero, Elisabet; Southall, June; Cogdell, Richard J.; Novoderezhkin, Vladimir I.; Scholes, Gregory D.; van Grondelle, Rienk

2016-01-01

Energy transfer and trapping in the light harvesting antennae of purple photosynthetic bacteria is an ultrafast process, which occurs with a quantum efficiency close to unity. However the mechanisms behind this process have not yet been fully understood. Recently it was proposed that low-lying energy dark states, such as charge transfer states and polaron pairs, play an important role in the dynamics and directionality of energy transfer. However, it is difficult to directly detect those states because of their small transition dipole moment and overlap with the B850/B870 exciton bands. Here we present a new experimental approach, which combines the selectivity of two-dimensional electronic spectroscopy with the availability of genetically modified light harvesting complexes, to reveal the presence of those dark states in both the genetically modified and the wild-type light harvesting 2 complexes of Rhodopseudomonas palustris. We suggest that Nature has used the unavoidable charge transfer processes that occur when LH pigments are concentrated to enhance and direct the flow of energy. PMID:26857477

Dark States in the Light-Harvesting complex 2 Revealed by Two-dimensional Electronic Spectroscopy

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

Ferretti, Marco; Hendrikx, Ruud; Romero, Elisabet

Energy transfer and trapping in the light harvesting antennae of purple photosynthetic bacteria is an ultrafast process, which occurs with a quantum efficiency close to unity. However the mechanisms behind this process have not yet been fully understood. Recently it was proposed that low-lying energy dark states, such as charge transfer states and polaron pairs, play an important role in the dynamics and directionality of energy transfer. However, it is difficult to directly detect those states because of their small transition dipole moment and overlap with the B850/B870 exciton bands. Here we present a new experimental approach, which combines themore » selectivity of two-dimensional electronic spectroscopy with the availability of genetically modified light harvesting complexes, to reveal the presence of those dark states in both the genetically modified and the wild-type light harvesting 2 complexes of Rhodopseudomonas palustris. In conclusion, we suggest that Nature has used the unavoidable charge transfer processes that occur when LH pigments are concentrated to enhance and direct the flow of energy.« less

Dark States in the Light-Harvesting complex 2 Revealed by Two-dimensional Electronic Spectroscopy

DOE PAGES

Ferretti, Marco; Hendrikx, Ruud; Romero, Elisabet; ...

2016-02-09

Energy transfer and trapping in the light harvesting antennae of purple photosynthetic bacteria is an ultrafast process, which occurs with a quantum efficiency close to unity. However the mechanisms behind this process have not yet been fully understood. Recently it was proposed that low-lying energy dark states, such as charge transfer states and polaron pairs, play an important role in the dynamics and directionality of energy transfer. However, it is difficult to directly detect those states because of their small transition dipole moment and overlap with the B850/B870 exciton bands. Here we present a new experimental approach, which combines themore » selectivity of two-dimensional electronic spectroscopy with the availability of genetically modified light harvesting complexes, to reveal the presence of those dark states in both the genetically modified and the wild-type light harvesting 2 complexes of Rhodopseudomonas palustris. In conclusion, we suggest that Nature has used the unavoidable charge transfer processes that occur when LH pigments are concentrated to enhance and direct the flow of energy.« less

Light, Molecules, Action: Using Ultrafast Uv-Visible and X-Ray Spectroscopy to Probe Excited State Dynamics in Photoactive Molecules

NASA Astrophysics Data System (ADS)

Sension, R. J.

2017-06-01

Light provides a versatile energy source capable of precise manipulation of material systems on size scales ranging from molecular to macroscopic. Photochemistry provides the means for transforming light energy from photon to process via movement of charge, a change in shape, a change in size, or the cleavage of a bond. Photochemistry produces action. In the work to be presented here ultrafast UV-Visible pump-probe, and pump-repump-probe methods have been used to probe the excited state dynamics of stilbene-based molecular motors, cyclohexadiene-based switches, and polyene-based photoacids. Both ultrafast UV-Visible and X-ray absorption spectroscopies have been applied to the study of cobalamin (vitamin B_{12}) based compounds. Optical measurements provide precise characterization of spectroscopic signatures of the intermediate species on the S_{1} surface, while time-resolved XANES spectra at the Co K-edge probe the structural changes that accompany these transformations.

Membrane-bound transcription factors: regulated release by RIP or RUP.

PubMed

Hoppe, T; Rape, M; Jentsch, S

2001-06-01

Regulated nuclear transport of transcription factors from cytoplasmic pools is a major route by which eukaryotes control gene expression. Exquisite examples are transcription factors that are kept in a dormant state in the cytosol by membrane anchors; such proteins are released from membranes by proteolytic cleavage, which enables these transcription factors to enter the nucleus. Cleavage can be mediated either by regulated intramembrane proteolysis (RIP) catalysed by specific membrane-bound proteases or by regulated ubiquitin/proteasome-dependent processing (RUP). In both cases processing can be controlled by cues that originate at or in the vicinity of the membrane.

First-principles modeling of hardness in transition-metal diborides

NASA Astrophysics Data System (ADS)

Lazar, Petr; Chen, Xing-Qiu; Podloucky, Raimund

2009-07-01

Based on recent experiments, the diborides OsB2 and ReB2 were proposed to be ultraincompressible and superhard materials. By application of an ab initio density-functional theory approach we investigate the elastic and cleavage fracture properties of the borides MB2 ( M=Hf , Ta, W, Re, Os, and Ir). We derive a direct correlation between the lowest calculated critical cleavage stress and the experimental (micro)hardness. By calculating the critical shear stress and estimating the possibility of dislocation emission we can justify the prediction that ReB2 is indeed a superhard material.

Two Antagonistic MALT1 Auto-Cleavage Mechanisms Reveal a Role for TRAF6 to Unleash MALT1 Activation

PubMed Central

Renner, Florian; Lam, Stephen; Freuler, Felix; Gerrits, Bertran; Voshol, Johannes; Calzascia, Thomas; Régnier, Catherine H.; Renatus, Martin; Nikolay, Rainer; Israël, Laura; Bornancin, Frédéric

2017-01-01

The paracaspase MALT1 has arginine-directed proteolytic activity triggered by engagement of immune receptors. Recruitment of MALT1 into activation complexes is required for MALT1 proteolytic function. Here, co-expression of MALT1 in HEK293 cells, either with activated CARD11 and BCL10 or with TRAF6, was used to explore the mechanism of MALT1 activation at the molecular level. This work identified a prominent self-cleavage site of MALT1 isoform A (MALT1A) at R781 (R770 in MALT1B) and revealed that TRAF6 can activate MALT1 independently of the CBM. Intramolecular cleavage at R781/R770 removes a C-terminal TRAF6-binding site in both MALT1 isoforms, leaving MALT1B devoid of the two key interaction sites with TRAF6. A previously identified auto-proteolysis site of MALT1 at R149 leads to deletion of the death-domain, thereby abolishing interaction with BCL10. By using MALT1 isoforms and cleaved fragments thereof, as well as TRAF6 WT and mutant forms, this work shows that TRAF6 induces N-terminal auto-proteolytic cleavage of MALT1 at R149 and accelerates MALT1 protein turnover. The MALT1 fragment generated by N-terminal self-cleavage at R149 was labile and displayed enhanced signaling properties that required an intact K644 residue, previously shown to be a site for mono-ubiquitination of MALT1. Conversely, C-terminal self-cleavage at R781/R770 hampered the ability for self-cleavage at R149 and stabilized MALT1 by hindering interaction with TRAF6. C-terminal self-cleavage had limited impact on MALT1A but severely reduced MALT1B proteolytic and signaling functions. It also abrogated NF-κB activation by N-terminally cleaved MALT1A. Altogether, this study provides further insights into mechanisms that regulate the scaffolding and activation cycle of MALT1. It also emphasizes the reduced functional capacity of MALT1B as compared to MALT1A. PMID:28052131

Homoharringtonine combined with aclarubicin and cytarabine synergistically induces apoptosis in t(8;21) leukemia cells and triggers caspase-3-mediated cleavage of the AML1-ETO oncoprotein.

PubMed

Cao, Jiang; Feng, Hao; Ding, Ning-Ning; Wu, Qing-Yun; Chen, Chong; Niu, Ming-Shan; Chen, Wei; Qiu, Ting-Ting; Zhu, Hong-Hu; Xu, Kai-Lin

2016-11-01

Homoharringtonine combined with aclarubicin and cytarabine (HAA) is a highly effective treatment for acute myeloid leukemia (AML), especially for t(8;21) AML. However, the underlying mechanisms by which HAA kills t(8;21) AML cells remain unclear. In this study, SKNO-1 and Kasumi-1 cells with t(8;21) were used. Compared with individual or pairwise administration of homoharringtonine, aclarubicin, or cytarabine, HAA showed the strongest inhibition of growth and induction of apoptosis in SKNO-1 and Kasumi-1 cells. HAA caused cleavage of the AML1-ETO (AE) oncoprotein to form truncated AE (ΔAE). Pretreatment with the caspase-3 inhibitor caspase-3 inhibitor Q-DEVD-OPh (QDO) not only suppressed HAA-induced apoptosis but also abrogated the cleavage of AE and generation of ΔAE. These results suggest that HAA synergistically induces apoptosis in t(8;21) leukemia cells and triggers caspase-3-mediated cleavage of the AML1-ETO oncoprotein, thus providing direct evidence for the strong activity of HAA toward t(8;21) AML. © 2016 The Authors. Cancer Medicine published by John Wiley & Sons Ltd.

SEM stereo-section fractography (SSF) observations

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

Zhang, X.J.; Tregoning, R.L.; Armstrong, R.W.

1997-12-31

Cleavage initiation in engineering materials is governed by local microstructural inhomogeneities. These features are often the principal reason for the large scatter evident in fracture toughness measurements which, in extreme cases, can mask the fundamental relationship between cracking resistance and global material properties. The SEM stereo-section fractography (SSF) technique can be used to carefully evaluate these local inhomogeneities through simultaneous observation of both the fracture surface and the underlying microstructure. By sectioning the fracture surface close to the cleavage initiation site (within 10 {micro}m), and perpendicular to both the fracture surface and the precrack front, a direct correspondence between initiationmore » and the local microstructure can be established. Information obtained from this technique can provide quantitative input about important, local microstructural features which can then be used to calibrate or create realistic micromechanical models. A compendium of SSF results is presented herein for cleavage cracking in disparate materials (A533B steel plates, MIL-70S multi-pass weldments, and Ti6A14V forgings), under various testing conditions. In each case, the SSF technique was able to unambiguously identify the dominant, local features which triggered cleavage initiation.« less

SEM stereo-section fractography observations. Final report

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

Zhang, X.J.; Tregoning, R.L.; Armstrong, R.W.

1998-05-01

Cleavage initiation in engineering materials is governed by local microstructural inhomogeneities. These features are often the principal reason for the large scatter evident in fracture toughness measurements which, in extreme cases, can mask the fundamental relationship between cracking resistance and global material properties. The SEM stereo-section fractography (SSF) technique can be used to carefully evaluate these local inhomogeneities through simultaneous observation of both the fracture surface and the underlying microstructure. By sectioning the fracture surface close to the cleavage initiation site (within 10 microns), and perpendicular to both the fracture surface and the pre crack front, a direct correspondence betweenmore » initiation and the local microstructure can be established. Information obtained from this technique can provide quantitative input about important, local microstructural features which can then be used to calibrate or create realistic micromechanical models. A compendium of SSF results is presented herein for cleavage cracking in disparate materials (A533B steel plates, MIL-70S multi-pass weldments, and Ti6A14V forgings), under various testing conditions. In each case, the SSF technique was able to unambiguously identify the dominant, local features which triggered cleavage initiation.« less

Time and voltage dependences of nanoscale dielectric constant modulation on indium tin oxide films

NASA Astrophysics Data System (ADS)

Li, Liang; Hao, Haoyue; Zhao, Hua

2017-01-01

The modulation of indium tin oxide (ITO) films through surface charge accumulation plays an important role in many different applications. In order to elaborately study the modulation, we measured the dielectric constant of the modulated layer through examining the excitation of surface plasmon polaritons. Charges were pumped on the surfaces of ITO films through applying high voltage in appropriate directions. Experiments unveiled that the dielectric constant of the modulated layer had large variation along with the nanoscale charge accumulation. Corresponding numerical results were worked out through combining Drude model and Mayadas-Shatzkes model. Based on the above results, we deduced the time and voltage dependences of accumulated charge density, which revealed a long-time charge accumulation process.

Quantifying reactivity for electrophilic aromatic substitution reactions with Hirshfeld charge.

PubMed

Liu, Shubin

2015-03-26

An electrophilic aromatic substitution is a process where one atom or group on an aromatic ring is replaced by an incoming electrophile. The reactivity and regioselectivity of this category of reactions is significantly impacted by the group that is already attached to the aromatic ring. Groups promoting substitution at the ortho/para and meta position are called ortho/para and meta directing groups, respectively. Earlier, we have shown that regioselectivity of the electrophilic aromatic substitution is dictated by the nucleophilicity of the substituted aromatic ring, which is proportional to the Hirshfeld charge on the regioselective site. Ortho/para directing groups have the largest negative charge values at the ortho/para positions, whereas meta directing groups often have the largest negative charge value at the meta position. The electron donation or acceptance feature of a substitution group is irrelevant to the regioselectivity. In this contribution, we extend our previous study by quantifying the reactivity for this kind of reactions. To that end, we examine the transition-state structure and activation energy of an identity reaction for a series of monosubstituted-benzene molecules reacting with hydrogen fluoride using BF3 as the catalyst in the gas phase. A total of 18 substitution groups will be considered, nine of which are ortho/para directing and the other nine groups meta directing. From this study, we found that the barrier height of these reactions strongly correlates with the Hirshfeld charge on the regioselective site for both ortho/para and meta directing groups, with the correlation coefficient R(2) both better than 0.96. We also discovered a less accurate correlation between the barrier height and HOMO energy. These results reconfirm the validity and effectiveness of employing the Hirshfeld charge as a reliable descriptor of both reactivity and regioselectivity for this vastly important category of chemical transformations.

Cationized Carbohydrate Gas-Phase Fragmentation Chemistry

NASA Astrophysics Data System (ADS)

Bythell, Benjamin J.; Abutokaikah, Maha T.; Wagoner, Ashley R.; Guan, Shanshan; Rabus, Jordan M.

2017-04-01

We investigate the fragmentation chemistry of cationized carbohydrates using a combination of tandem mass spectrometry, regioselective labeling, and computational methods. Our model system is D-lactose. Barriers to the fundamental glyosidic bond cleavage reactions, neutral loss pathways, and structurally informative cross-ring cleavages are investigated. The most energetically favorable conformations of cationized D-lactose were found to be similar. In agreement with the literature, larger group I cations result in structures with increased cation coordination number which require greater collision energy to dissociate. In contrast with earlier proposals, the B n -Y m fragmentation pathways of both protonated and sodium-cationized analytes proceed via protonation of the glycosidic oxygen with concerted glycosidic bond cleavage. Additionally, for the sodiated congeners our calculations support sodiated 1,6-anhydrogalactose B n ion structures, unlike the preceding literature. This affects the subsequent propensity of formation and prediction of B n /Y m branching ratio. The nature of the anomeric center (α/β) affects the relative energies of these processes, but not the overall ranking. Low-energy cross-ring cleavages are observed for the metal-cationized analytes with a retro-aldol mechanism producing the 0,2 A 2 ion from the sodiated forms . Theory and experiment support the importance of consecutive fragmentation processes, particularly for the protonated congeners at higher collision energies.

Transition from direct to inverted charge transport Marcus regions in molecular junctions via molecular orbital gating

NASA Astrophysics Data System (ADS)

Yuan, Li; Wang, Lejia; Garrigues, Alvar R.; Jiang, Li; Annadata, Harshini Venkata; Anguera Antonana, Marta; Barco, Enrique; Nijhuis, Christian A.

2018-04-01

Solid-state molecular tunnel junctions are often assumed to operate in the Landauer regime, which describes essentially activationless coherent tunnelling processes. In solution, on the other hand, charge transfer is described by Marcus theory, which accounts for thermally activated processes. In practice, however, thermally activated transport phenomena are frequently observed also in solid-state molecular junctions but remain poorly understood. Here, we show experimentally the transition from the Marcus to the inverted Marcus region in a solid-state molecular tunnel junction by means of intra-molecular orbital gating that can be tuned via the chemical structure of the molecule and applied bias. In the inverted Marcus region, charge transport is incoherent, yet virtually independent of temperature. Our experimental results fit well to a theoretical model that combines Landauer and Marcus theories and may have implications for the interpretation of temperature-dependent charge transport measurements in molecular junctions.

Superposition of \\sqrt{13}\\times \\sqrt{13} and 3 × 3 supermodulations in TaS2 probed by scanning tunneling microscopy

NASA Astrophysics Data System (ADS)

Fujisawa, Y.; Iwasaki, T.; Fujii, D.; Ohta, S.; Iwashita, J.; Fujita, T.; Nakata, M.; Kishimoto, K.; Demura, S.; Sakata, H.

2018-03-01

We report on a scanning tunnelling microscopy study of TaS2 at 4.2 K. A surface prepared by cleavage showed a superimposed pattern of two types of charge density waves with 3a 0 × 3a 0 and \\sqrt{13}{a}0× \\sqrt{13}{a}0 periodicity, which had never been observed previously. We attribute the superposition to regular stacking of 4H b polytypes or irregular stacking of 2H and 4H b layers.

The in vitro cleavage of the hAtg proteins by cell death proteases.

PubMed

Norman, Joanna M; Cohen, Gerald M; Bampton, Edward T W

2010-11-01

It is becoming increasingly clear that there is crosstalk between the apoptotic and autophagic pathways, with autophagy helping to contribute to cell death by providing energy to allow the energy-requiring programmed cell death process to complete, as well as degrading cellular material in its own right. Recent evidence has suggested that Atg proteins can themselves be targets of caspases, providing potential regulation of autophagy as well as uncovering novel functions for fragments derived from Atg proteins. However, to date there has not been a detailed examination of which Atg proteins may be the targets of which death proteases. We show that the majority of human Atg (hAtg) proteins can be cleaved by calpain 1, which is activated in some apoptotic paradigms, as well as other forms of death. We also show that hAtg3 is cleaved by caspases-3, -6 and -8, hAtg6 (Beclin 1) is cleaved by caspase-3 and -6, while hAtg9, hAtg7 and the hAtg4 homologues can be cleaved by caspase-3. Cleavage of Beclin 1 was also seen in apoptosis of HeLa cells induced by staurosporine and TRAIL, along with cleavage of Atg3 and Atg4C. There were subtle effects of caspase inhibition on GFP-LC3 lipidation but more marked effects on the formation of GFP-LC3 puncta (a marker of autophagosome formation) and p62 degradation, indicating that caspase cleavage of autophagy-related proteins can affect the autophagic process. Notably we show that p62 is a target for caspase-6 and -8 cleavage.

The preparation and application of white graphene

NASA Astrophysics Data System (ADS)

Zhou, Chenghong

2014-12-01

In this article, another thin film named white graphene is introduced, containing its properties, preparation and potential applications. White graphene, which has the same structure with graphene but quite different electrical properties, can be exfoliated from its layered crystal, hexagonal boron nitride. Here two preparation methods of white graphene including supersonic cleavage and supercritical cleavage are presented. Inspired by the cleavage of graphene oxide, supersonic is applied to BN and few-layered films are obtained. Compared with supersonic cleavage, supercritical cleavage proves to be more successful. As supercritical fluid can diffuse into interlayer space of the layered hexagonal boron nitride easily, once reduce the pressure of the supercritical system fast, supercritical fluid among layers expands and escapes form interlayer, consequently exfoliating the hexagonal boron nitride into few layered structure. A series of characterization demonstrate that the monolayer white graphene prepared in the process matches its theoretical thickness 0.333nm and has lateral sizes at the order of 10μm. Supercritical cleavage proves to be successful and shows many advantages, such as good production quality and fast production cycle. Furthermore, the band energy of white graphene, which shows quite different from graphene, is simulated via tight-bonding in theory. The excellent properties will lead to extensive applications of white graphene. As white graphene has not received enough concern and exploration, it's potential to play a significant role in the fields of industry and science.

Targeted Catalytic Inactivation of Angiotensin Converting Enzyme by Lisinopril-Coupled Transition Metal Chelates

PubMed Central

Joyner, Jeff C.; Hocharoen, Lalintip; Cowan, J. A.

2012-01-01

A series of compounds that target reactive transition metal chelates to somatic Angiotensin Converting Enzyme (sACE-1) have been synthesized. Half maximal inhibitory concentrations (IC50) and rate constants for both inactivation and cleavage of full length sACE-1 have been determined and evaluated in terms of metal-chelate size, charge, reduction potential, coordination unsaturation, and coreactant selectivity. Ethylenediamine-tetraacetic acid (EDTA), nitrilotriacetic acid (NTA), 1,4,7,10-tetraazacyclo-dodecane-1,4,7,10-tetraacetic acid (DOTA), and tripeptide GGH were linked to the lysine sidechain of lisinopril by EDC/NHS coupling. The resulting amide-linked chelate-lisinopril (EDTA-lisinopril, NTA-lisinopril, DOTA-lisinopril, and GGH-lisinopril) conjugates were used to form coordination complexes with iron, cobalt, nickel and copper, such that lisinopril could mediate localization of the reactive metal chelates to sACE-1. ACE activity was assayed by monitoring cleavage of the fluorogenic substrate Mca-RPPGFSAFK(Dnp)-OH, a derivative of bradykinin, following pre-incubation with metal-chelate-lisinopril compounds. Concentration-dependent inhibition of sACE-1 by metal-chelate-lisinopril complexes revealed IC50 values ranging from 44 nM to 4,500 nM for Ni-NTA-lisinopril and Ni-DOTA-lisinopril, respectively, versus 1.9 nM for lisinopril. Stronger inhibition was correlated with smaller size and lower negative charge of the attached metal chelates. Time-dependent inactivation of sACE-1 by metal-chelate-lisinopril complexes revealed a remarkable range of catalytic activities, with second order rate constants as high as 150,000 M−1min−1 (Cu-GGH-lisinopril), while catalyst-mediated cleavage of sACE-1 typically occurred at much lower rates, indicating that inactivation arose primary from sidechain modification. Optimal inactivation of sACE-1 was observed when the reduction potential for the metal center was poised near 1000 mV, reflecting the difficulty of protein oxidation. This class of metal-chelate-lisinopril complexes possesses a range of high-affinity binding to ACE, introduces the advantage of irreversible catalytic turnover, and marks an important step toward the development of multiple-turnover drugs for selective inactivation of sACE-1. PMID:22200082

Targeted catalytic inactivation of angiotensin converting enzyme by lisinopril-coupled transition-metal chelates.

PubMed

Joyner, Jeff C; Hocharoen, Lalintip; Cowan, J A

2012-02-22

A series of compounds that target reactive transition-metal chelates to somatic angiotensin converting enzyme (sACE-1) have been synthesized. Half-maximal inhibitory concentrations (IC(50)) and rate constants for both inactivation and cleavage of full-length sACE-1 have been determined and evaluated in terms of metal chelate size, charge, reduction potential, coordination unsaturation, and coreactant selectivity. Ethylenediaminetetraacetic acid (EDTA), nitrilotriacetic acid (NTA), 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA), and tripeptide GGH were linked to the lysine side chain of lisinopril by 1-ethyl-3-[3-(dimethylamino)propyl]carbodiimide hydrochloride/N-hydroxysuccinimide coupling. The resulting amide-linked chelate-lisinopril (EDTA-lisinopril, NTA-lisinopril, DOTA-lisinopril, and GGH-lisinopril) conjugates were used to form coordination complexes with iron, cobalt, nickel, and copper, such that lisinopril could mediate localization of the reactive metal chelates to sACE-1. ACE activity was assayed by monitoring cleavage of the fluorogenic substrate Mca-RPPGFSAFK(Dnp)-OH, a derivative of bradykinin, following preincubation with metal chelate-lisinopril compounds. Concentration-dependent inhibition of sACE-1 by metal chelate-lisinopril complexes revealed IC(50) values ranging from 44 to 4500 nM for Ni-NTA-lisinopril and Ni-DOTA-lisinopril, respectively, versus 1.9 nM for lisinopril. Stronger inhibition was correlated with smaller size and lower negative charge of the attached metal chelates. Time-dependent inactivation of sACE-1 by metal chelate-lisinopril complexes revealed a remarkable range of catalytic activities, with second-order rate constants as high as 150,000 M(-1) min(-1) (Cu-GGH-lisinopril), while catalyst-mediated cleavage of sACE-1 typically occurred at much lower rates, indicating that inactivation arose primarily from side chain modification. Optimal inactivation of sACE-1 was observed when the reduction potential for the metal center was poised near 1000 mV, reflecting the difficulty of protein oxidation. This class of metal chelate-lisinopril complexes possesses a range of high-affinity binding to ACE, introduces the advantage of irreversible catalytic turnover, and marks an important step toward the development of multiple-turnover drugs for selective inactivation of sACE-1.

LBSizeCleav: improved support vector machine (SVM)-based prediction of Dicer cleavage sites using loop/bulge length.

PubMed

Bao, Yu; Hayashida, Morihiro; Akutsu, Tatsuya

2016-11-25

Dicer is necessary for the process of mature microRNA (miRNA) formation because the Dicer enzyme cleaves pre-miRNA correctly to generate miRNA with correct seed regions. Nonetheless, the mechanism underlying the selection of a Dicer cleavage site is still not fully understood. To date, several studies have been conducted to solve this problem, for example, a recent discovery indicates that the loop/bulge structure plays a central role in the selection of Dicer cleavage sites. In accordance with this breakthrough, a support vector machine (SVM)-based method called PHDCleav was developed to predict Dicer cleavage sites which outperforms other methods based on random forest and naive Bayes. PHDCleav, however, tests only whether a position in the shift window belongs to a loop/bulge structure. In this paper, we used the length of loop/bulge structures (in addition to their presence or absence) to develop an improved method, LBSizeCleav, for predicting Dicer cleavage sites. To evaluate our method, we used 810 empirically validated sequences of human pre-miRNAs and performed fivefold cross-validation. In both 5p and 3p arms of pre-miRNAs, LBSizeCleav showed greater prediction accuracy than PHDCleav did. This result suggests that the length of loop/bulge structures is useful for prediction of Dicer cleavage sites. We developed a novel algorithm for feature space mapping based on the length of a loop/bulge for predicting Dicer cleavage sites. The better performance of our method indicates the usefulness of the length of loop/bulge structures for such predictions.

Direct Production of Propene from the Thermolysis of Poly(β-hydroxybutyrate) (PHB). An Experimental and DFT Investigation.

PubMed

Clark, Jared M; Pilath, Heidi M; Mittal, Ashutosh; Michener, William E; Robichaud, David J; Johnson, David K

2016-01-28

We demonstrate a synthetic route toward the production of propene directly from poly(β-hydroxybutyrate) (PHB), the most common of a wide range of high-molecular-mass microbial polyhydroxyalkanoates. Propene, a major commercial hydrocarbon, was obtained from the depolymerization of PHB and subsequent decarboxylation of the crotonic acid monomer in good yields (up to 75 mol %). The energetics of PHB depolymerization and the gas-phase decarboxylation of crotonic acid were also studied using density functional theory (DFT). The average activation energy for the cleavage of the R'C(O)O-R linkage is calculated to be 163.9 ± 7.0 kJ mol(-1). Intramolecular, autoacceleration effects regarding the depolymerization of PHB, as suggested in some literature accounts, arising from the formation of crotonyl and carboxyl functional groups in the products could not be confirmed by the results of DFT and microkinetic modeling. DFT results, however, suggest that intermolecular catalysis involving terminal carboxyl groups may accelerate PHB depolymerization. Activation energies for this process were estimated to be about 20 kJ mol(-1) lower than that for the noncatalyzed ester cleavage, 144.3 ± 6.4 kJ mol(-1). DFT calculations predict the decarboxylation of crotonic acid to follow second-order kinetics with an activation energy of 147.5 ± 6.3 kJ mol(-1), consistent with that measured experimentally, 146.9 kJ mol(-1). Microkinetic modeling of the PHB to propene overall reaction predicts decarboxylation of crotonic acid to be the rate-limiting step, consistent with experimental observations. The results also indicate that improvements made to enhance the isomerization of crotonic acid to vinylacetic acid will improve the direct conversion of PHB to propene.

Amyloid-β Production Via Cleavage of Amyloid-β Protein Precursor is Modulated by Cell Density

PubMed Central

Zhang, Can; Browne, Andrew; DiVito, Jason R.; Stevenson, Jesse A.; Romano, Donna; Dong, Yuanlin; Xie, Zhongcong; Tanzi, Rudolph E.

2012-01-01

Mounting evidence suggests that Alzheimer disease (AD) is caused by the accumulation of the small peptide, Aβ, a proteolytic cleavage product of amyloid-β protein precursor (AβPP; or APP). Aβ is generated through a serial cleavage of APP by β- and γ-secretase. Aβ40 and Aβ42 are the two main components of amyloid plaques in AD brains, with Aβ42 being more prone to aggregation. APP can also be processed by α-secretase, which cleaves APP within the Aβ sequence, thereby preventing the generation of Aβ. Little is currently known regarding the effects of cell density on APP processing and Aβ generation. Here we assessed the effects of cell density on APP processing in neuronal and non-neuronal cell lines, as well as mouse primary cortical neurons. We found that decreased cell density significantly increases levels of Aβ40, Aβ42, total Aβ, and the ratio of Aβ42:Aβ40. These results also indicate that cell density is a significant modulator of APP processing. Overall, these findings carry profound implications for both previous and forthcoming studies aiming to assess the effects of various conditions and genetic/chemical factors, e.g. novel drugs on APP processing and Aβ generation in cell-based systems. Moreover, it is interesting to speculate whether cell density changes in vivo may also affect APP processing and Aβ levels in the AD brain. PMID:20847415

Interplay of catalysis, fidelity, threading, and processivity in the exo- and endonucleolytic reactions of human exonuclease I

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

Shi, Yuqian; Hellinga, Homme W.; Beese, Lorena S.

Human exonuclease 1 (hExo1) is a member of the RAD2/XPG structure-specific 5'-nuclease superfamily. Its dominant, processive 5'–3' exonuclease and secondary 5'-flap endonuclease activities participate in various DNA repair, recombination, and replication processes. A single active site processes both recessed ends and 5'-flap substrates. By initiating enzyme reactions in crystals, we have trapped hExo1 reaction intermediates that reveal structures of these substrates before and after their exo- and endonucleolytic cleavage, as well as structures of uncleaved, unthreaded, and partially threaded 5' flaps. Their distinctive 5' ends are accommodated by a small, mobile arch in the active site that binds recessed endsmore » at its base and threads 5' flaps through a narrow aperture within its interior. A sequence of successive, interlocking conformational changes guides the two substrate types into a shared reaction mechanism that catalyzes their cleavage by an elaborated variant of the two-metal, in-line hydrolysis mechanism. Coupling of substrate-dependent arch motions to transition-state stabilization suppresses inappropriate or premature cleavage, enhancing processing fidelity. The striking reduction in flap conformational entropy is catalyzed, in part, by arch motions and transient binding interactions between the flap and unprocessed DNA strand. At the end of the observed reaction sequence, hExo1 resets without relinquishing DNA binding, suggesting a structural basis for its processivity.« less

Interplay of catalysis, fidelity, threading, and processivity in the exo- and endonucleolytic reactions of human exonuclease I.

PubMed

Shi, Yuqian; Hellinga, Homme W; Beese, Lorena S

2017-06-06

Human exonuclease 1 (hExo1) is a member of the RAD2/XPG structure-specific 5'-nuclease superfamily. Its dominant, processive 5'-3' exonuclease and secondary 5'-flap endonuclease activities participate in various DNA repair, recombination, and replication processes. A single active site processes both recessed ends and 5'-flap substrates. By initiating enzyme reactions in crystals, we have trapped hExo1 reaction intermediates that reveal structures of these substrates before and after their exo- and endonucleolytic cleavage, as well as structures of uncleaved, unthreaded, and partially threaded 5' flaps. Their distinctive 5' ends are accommodated by a small, mobile arch in the active site that binds recessed ends at its base and threads 5' flaps through a narrow aperture within its interior. A sequence of successive, interlocking conformational changes guides the two substrate types into a shared reaction mechanism that catalyzes their cleavage by an elaborated variant of the two-metal, in-line hydrolysis mechanism. Coupling of substrate-dependent arch motions to transition-state stabilization suppresses inappropriate or premature cleavage, enhancing processing fidelity. The striking reduction in flap conformational entropy is catalyzed, in part, by arch motions and transient binding interactions between the flap and unprocessed DNA strand. At the end of the observed reaction sequence, hExo1 resets without relinquishing DNA binding, suggesting a structural basis for its processivity.

Characterization of clade 2.3.4.4 H5N8 highly pathogenic avian influenza viruses from wild birds possessing atypical hemagglutinin polybasic cleavage sites.

PubMed

Usui, Tatsufumi; Soda, Kosuke; Tomioka, Yukiko; Ito, Hiroshi; Yabuta, Toshiyo; Takakuwa, Hiroki; Otsuki, Koichi; Ito, Toshihiro; Yamaguchi, Tsuyoshi

2017-02-01

Since 2014, clade 2.3.4.4 H5 subtype highly pathogenic avian influenza viruses (HPAIVs) have been distributed worldwide. These viruses, which were reported to be highly virulent in chickens by intravenous inoculation, have a consensus HPAI motif PLRERRRKR at the HA cleavage site. However, two-clade 2.3.4.4 H5N8 viruses which we isolated from wild migratory birds in late 2014 in Japan possessed atypical HA cleavage sequences. A swan isolate, Tottori/C6, had a novel polybasic cleavage sequence, PLGERRRKR, and another isolate from a dead mandarin duck, Gifu/01, had a heterogeneous mixture of consensus PLRERRRKR and variant PLRERRRRKR sequences. The polybasic HA cleavage site is the prime virulence determinant of AIVs. Therefore, in the present study, we examined the pathogenicity of these H5N8 isolates in chickens by intravenous inoculation. When 10 6 EID 50 of these viruses were intravenously inoculated into chickens, the mean death time associated with Tottori/C6 was substantially longer (>6.1 days) than that associated with Gifu/01 (2.5 days). These viruses had comparable abilities to replicate in tissue culture cells in the presence and absence of exogenous trypsin, but the growth of Tottori/C6 was hampered. These results indicate that the novel cleavage motif of Tottori/C6 did not directly affect the infectivity of the virus, but Tottori/C6 caused attenuated pathogenicity in chickens because of hampered replication efficiency. It is important to test for the emergence of diversified HPAIVs, because introduction of HPAIVs with a lower virulence like Tottori/C6 might hinder early detection of affected birds in poultry farms.

Familial Alzheimer’s mutations within APPTM increase Aβ42 production by enhancing accessibility of ɛ-cleavage site

NASA Astrophysics Data System (ADS)

Chen, Wen; Gamache, Eric; Rosenman, David J.; Xie, Jian; Lopez, Maria M.; Li, Yue-Ming; Wang, Chunyu

2014-01-01

The high Aβ42/Aβ40 production ratio is a hallmark of familial Alzheimer’s disease, which can be caused by mutations in the amyloid precursor protein (APP). The C-terminus of Aβ is generated by γ-secretase cleavage within the transmembrane domain of APP (APPTM), a process that is primed by an initial ɛ-cleavage at either T48 or L49, resulting in subsequent production of Aβ42 or Aβ40, respectively. Here we solve the dimer structures of wild-type APPTM (AAPTM WT) and mutant APPTM (FAD mutants V44M) with solution NMR. The right-handed APPTM helical dimer is mediated by GXXXA motif. From the NMR structural and dynamic data, we show that the V44M and V44A mutations can selectively expose the T48 site by weakening helical hydrogen bonds and increasing hydrogen-deuterium exchange rate (kex). We propose a structural model in which FAD mutations (V44M and V44A) can open the T48 site γ-secretase for the initial ɛ-cleavage, and consequently shift cleavage preference towards Aβ42.

Activation and reactivation of the RNA polymerase II trigger loop for intrinsic RNA cleavage and catalysis

PubMed Central

Čabart, Pavel; Jin, Huiyan; Li, Liangtao; Kaplan, Craig D

2014-01-01

In addition to RNA synthesis, multisubunit RNA polymerases (msRNAPs) support enzymatic reactions such as intrinsic transcript cleavage. msRNAP active sites from different species appear to exhibit differential intrinsic transcript cleavage efficiency and have likely evolved to allow fine-tuning of the transcription process. Here we show that a single amino-acid substitution in the trigger loop (TL) of Saccharomyces RNAP II, Rpb1 H1085Y, engenders a gain of intrinsic cleavage activity where the substituted tyrosine appears to participate in acid-base chemistry at alkaline pH for both intrinsic cleavage and nucleotidyl transfer. We extensively characterize this TL substitution for each of these reactions by examining the responses RNAP II enzymes to catalytic metals, altered pH, and factor inputs. We demonstrate that TFIIF stimulation of the first phosphodiester bond formation by RNAP II requires wild type TL function and that H1085Y substitution within the TL compromises or alters RNAP II responsiveness to both TFIIB and TFIIF. Finally, Mn2+ stimulation of H1085Y RNAP II reveals possible allosteric effects of TFIIB on the active center and cooperation between TFIIB and TFIIF. PMID:25764335

The substrate degradome of meprin metalloproteases reveals an unexpected proteolytic link between meprin β and ADAM10.

PubMed

Jefferson, Tamara; Auf dem Keller, Ulrich; Bellac, Caroline; Metz, Verena V; Broder, Claudia; Hedrich, Jana; Ohler, Anke; Maier, Wladislaw; Magdolen, Viktor; Sterchi, Erwin; Bond, Judith S; Jayakumar, Arumugam; Traupe, Heiko; Chalaris, Athena; Rose-John, Stefan; Pietrzik, Claus U; Postina, Rolf; Overall, Christopher M; Becker-Pauly, Christoph

2013-01-01

The in vivo roles of meprin metalloproteases in pathophysiological conditions remain elusive. Substrates define protease roles. Therefore, to identify natural substrates for human meprin α and β we employed TAILS (terminal amine isotopic labeling of substrates), a proteomics approach that enriches for N-terminal peptides of proteins and cleavage fragments. Of the 151 new extracellular substrates we identified, it was notable that ADAM10 (a disintegrin and metalloprotease domain-containing protein 10)-the constitutive α-secretase-is activated by meprin β through cleavage of the propeptide. To validate this cleavage event, we expressed recombinant proADAM10 and after preincubation with meprin β, this resulted in significantly elevated ADAM10 activity. Cellular expression in murine primary fibroblasts confirmed activation. Other novel substrates including extracellular matrix proteins, growth factors and inhibitors were validated by western analyses and enzyme activity assays with Edman sequencing confirming the exact cleavage sites identified by TAILS. Cleavages in vivo were confirmed by comparing wild-type and meprin(-/-) mice. Our finding of cystatin C, elafin and fetuin-A as substrates and natural inhibitors for meprins reveal new mechanisms in the regulation of protease activity important for understanding pathophysiological processes.

Effect of Local Crystallographic Texture on the Fissure Formation During Charpy Impact Testing of Low-Carbon Steel

NASA Astrophysics Data System (ADS)

Ghosh, Abhijit; Patra, Sudipta; Chatterjee, Arya; Chakrabarti, Debalay

2016-06-01

The severity of the formation of fissures (also known as splitting or delamination) on the fracture surface of Charpy impact-tested samples of a low-carbon steel has been found to increase with the decrease in finish rolling temperature [1093 K to 923 K (820 °C to 650 °C)]. Combined scanning electron microscopy and electron back-scattered diffraction study revealed that crystallographic texture was the prime factor responsible for the fissure formation. Through-thickness texture band composed of cube [Normal Direction (ND)║<001>] and gamma [ND║<111>] orientations developed during the inter-critical rolling treatment. Strain incompatibility between these two texture bands causes fissure cracking on the main fracture plane. A new approach based on the angle between {001} planes of neighboring crystals has been employed in order to estimate the `effective grain size,' which is used to determine the cleavage fracture stress on different planes of a sample. The severity of fissure formation was found to be directly related to the difference in cleavage fracture stress between the `main fracture plane' and `fissure plane.' Clustering of ferrite grains having cube texture promoted the fissure crack propagation along the transverse `fissure plane,' by increasing the `effective grain size' and decreasing the cleavage fracture stress on that plane.

Combined application of electron backscatter diffraction and stereo-photogrammetry in fractography studies.

PubMed

Davies, P A; Randle, V

2001-10-01

The main aim of this paper is to report on recent experimental developments that have succeeded in combining electron back-scatter diffraction (EBSD) with stereo-photogrammetry, compared with two other methods for study of fracture surfaces, namely visual fractography analysis in the scanning electron microscope (SEM) and EBSD directly from facets. These approaches will be illustrated with data relating to the cleavage plane orientation analysis in a ferritic and C-Mn steel. It is demonstrated that the combined use of EBSD and stereo-photogrammetry represents a significant advance in the methodology for facet crystallography analysis. The results of point counting from fractograph characterization determined that the proportions of intergranular fracture in C-Mn and ferritic steels were 10.4% and 9.4%, respectively. The crystallographic orientation was determined directly from the fracture surface of a ferritic steel sample and produced an orientation distribution with a clear trend towards the [001] plane. A stereo-photogrammetry technique was validated using the known geometry of a Vickers hardness indent. The technique was then successfully employed to measure the macroscopic orientation of individual cleavage facets in the same reference frame as the EBSD measurements. Correlating the results of these measurements indicated that the actual crystallographic orientation of every cleavage facet identified in the steel specimens is [001].

Key roles of Arf small G proteins and biosynthetic trafficking for animal development.

PubMed

Rodrigues, Francisco F; Harris, Tony J C

2017-04-14

Although biosynthetic trafficking can function constitutively, it also functions specifically for certain developmental processes. These processes require either a large increase to biosynthesis or the biosynthesis and targeted trafficking of specific players. We review the conserved molecular mechanisms that direct biosynthetic trafficking, and discuss how their genetic disruption affects animal development. Specifically, we consider Arf small G proteins, such as Arf1 and Sar1, and their coat effectors, COPI and COPII, and how these proteins promote biosynthetic trafficking for cleavage of the Drosophila embryo, the growth of neuronal dendrites and synapses, extracellular matrix secretion for bone development, lumen development in epithelial tubes, notochord and neural tube development, and ciliogenesis. Specific need for the biosynthetic trafficking system is also evident from conserved CrebA/Creb3-like transcription factors increasing the expression of secretory machinery during several of these developmental processes. Moreover, dysfunctional trafficking leads to a range of developmental syndromes.

TiO2-Photoanode-Assisted Direct-Solar-Energy Harvesting and Storage in a Solar-Powered Redox Cell Using Halides as Active Materials.

PubMed

Zhang, Shun; Chen, Chen; Zhou, Yangen; Qian, Yumin; Ye, Jing; Xiong, Shiyun; Zhao, Yu; Zhang, Xiaohong

2018-06-27

The rapid deployment of renewable energy is resulting in significant energy security, climate change mitigation, and economic benefits. We demonstrate here the direct solar-energy harvesting and storage in a rechargeable solar-powered redox cell, which can be charged solely by solar irradiation. The cell follows a conventional redox-flow cell design with one integrated TiO 2 photoanode in the cathode side. Direct charging of the cell by solar irradiation results in the conversion of solar energy in to chemical energy. Whereas discharging the cell leads to the release of chemical energy in the form of electricity. The cell integrates energy conversion and storage processes in a single device, making the solar energy directly and efficiently dispatchable. When using redox couples of Br 2 /Br - and I 3 - /I - in the cathode side and anode side, respectively, the cell can be directly charged upon solar irradiation, yielding a discharge potential of 0.5 V with good round-trip efficiencies. This design is expected to be a potential alternative toward the development of affordable, inexhaustible, and clean solar-energy technologies.

Control of Bacteriophage T4 Tail Lysozyme Activity During the Infection Process

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

Kanamaru, Shuji; Ishiwata, Yasutaka; Suzuki, Toshiharu

2010-07-19

Bacteriophage T4 has an efficient mechanism for injecting the host Escherichia coli cell with genomic DNA. Its gene product 5 (gp5) has a needle-like structure attached to the end of a tube through which the DNA passes on its way out of the head and into the host. The gp5 needle punctures the outer cell membrane and then digests the peptidoglycan cell wall in the periplasmic space. gp5 is normally post-translationally cleaved between residues 351 and 352. The function of this process in controlling the lysozyme activity of gp5 has now been investigated. When gp5 is over-expressed in E. coli,more » two mutants (S351H and S351A) showed a reduction of cleavage products and five other mutants (S351L, S351K, S351Y, S351Q, and S351T) showed no cleavage. Furthermore, in a complementation assay at 20 C, the mutants that had no cleavage of gp5 produced a reduced number of plaques compared to wild-type T4. The crystal structure of the non-cleavage phenotype mutant of gp5, S351L, complexed with gene product 27, showed that the 18 residues in the vicinity of the potential cleavage site (disordered in the wild-type structure) had visible electron density. The polypeptide around the potential cleavage site is exposed, thus allowing access for an E. coli protease. The lysozyme activity is inhibited in the wild-type structure by a loop from the adjacent gp5 monomer that binds into the substrate-binding site. The same inhibition is apparent in the mutant structure, showing that the lysozyme is inhibited before gp5 is cleaved and, presumably, the lysozyme is activated only after gp5 has penetrated the outer membrane.« less

Base Pairing between U3 Small Nucleolar RNA and the 5′ End of 18S rRNA Is Required for Pre-rRNA Processing

PubMed Central

Sharma, Kishor; Tollervey, David

1999-01-01

The loop of a stem structure close to the 5′ end of the 18S rRNA is complementary to the box A region of the U3 small nucleolar RNA (snoRNA). Substitution of the 18S loop nucleotides inhibited pre-rRNA cleavage at site A1, the 5′ end of the 18S rRNA, and at site A2, located 1.9 kb away in internal transcribed spacer 1. This inhibition was largely suppressed by a compensatory mutation in U3, demonstrating functional base pairing. The U3–pre-rRNA base pairing is incompatible with the structure that forms in the mature 18S rRNA and may prevent premature folding of the pre-rRNA. In the Escherichia coli pre-rRNA the homologous region of the 16S rRNA is also sequestered, in that case by base pairing to the 5′ external transcribed spacer (5′ ETS). Cleavage at site A0 in the yeast 5′ ETS strictly requires base pairing between U3 and a sequence within the 5′ ETS. In contrast, the U3-18S interaction is not required for A0 cleavage. U3 therefore carries out at least two functionally distinct base pair interactions with the pre-rRNA. The nucleotide at the site of A1 cleavage was shown to be specified by two distinct signals; one of these is the stem-loop structure within the 18S rRNA. However, in contrast to the efficiency of cleavage, the position of A1 cleavage is not dependent on the U3-loop interaction. We conclude that the 18S stem-loop structure is recognized at least twice during pre-rRNA processing. PMID:10454548

Hydroxynitrile Lyases with α/β-Hydrolase Fold: Two Enzymes with Almost Identical 3D Structures but Opposite Enantioselectivities and Different Reaction Mechanisms

PubMed Central

Andexer, Jennifer N; Staunig, Nicole; Eggert, Thorsten; Kratky, Christoph; Pohl, Martina; Gruber, Karl

2012-01-01

Hydroxynitrile lyases (HNLs) catalyze the cleavage of cyanohydrins to yield hydrocyanic acid (HCN) and the respective carbonyl compound and are key enzymes in the process of cyanogenesis in plants. In organic syntheses, HNLs are used as biocatalysts for the formation of enantiopure cyanohydrins. We determined the structure of the recently identified, R-selective HNL from Arabidopsis thaliana (AtHNL) at a crystallographic resolution of 2.5 Å. The structure exhibits an α/β-hydrolase fold, very similar to the homologous, but S-selective, HNL from Hevea brasiliensis (HbHNL). The similarities also extend to the active sites of these enzymes, with a Ser-His-Asp catalytic triad present in all three cases. In order to elucidate the mode of substrate binding and to understand the unexpected opposite enantioselectivity of AtHNL, complexes of the enzyme with both (R)- and (S)-mandelonitrile were modeled using molecular docking simulations. Compared to the complex of HbHNL with (S)-mandelonitrile, the calculations produced an approximate mirror image binding mode of the substrate with the phenyl rings located at very similar positions, but with the cyano groups pointing in opposite directions. A catalytic mechanism for AtHNL is proposed, in which His236 from the catalytic triad acts as a general base and the emerging negative charge on the cyano group is stabilized by main-chain amide groups and an α-helix dipole very similar to α/β-hydrolases. This mechanistic proposal is additionally supported by mutagenesis studies. PMID:22851196

Transmission of low-energy negative ions through insulating nanocapillaries

NASA Astrophysics Data System (ADS)

Zhang, Qi; Liu, Zhonglin; Li, Pengfei; Jin, Bo; Song, Guangyin; Jin, Dingkun; Niu, Ben; Wei, Long; Ha, Shuai; Xie, Yiming; Ma, Yue; Wan, Chengliang; Cui, Ying; Zhou, Peng; Zhang, Hongqiang; Chen, Ximeng

2018-04-01

A simulation is performed to study the transmission of low-energy C l- ions through A l2O3 nanocapillaries. For the trajectory simulations, there are several processes involved: the image forces induced by the projectile; the electrostatic force from the deposited charges; the scattering from the inner surface and charge exchange. The simulation reproduces the main features of the experiments; i.e., the double peak structure in the transmitted angular distribution and the transmitted fractions of C l- , C l+ , and C l0 were found in the charge state distribution. The transmitted C l- ions are centered around the beam direction while the transmitted fractions of C l0 and C l+ are centered around the tilt angles. The role of the deposited charge is also studied by simulations. With the deposited charge, it is found that C l- is dominant in the transmission and the majority of the ions, centered around the tilt angle, are mainly from the single deflection by the negative charge patches on the inner surfaces of the capillaries, and only a few directly transmitted C l- ions are centered around the incident direction. There are also a few transmitted fractions of C l0 and C l+ from close surface scatterings. In the case that there are no negative charge patches, the simulation agrees with the experiment in detail: The majority of the directly transmitted C l- ions are centered around the incident direction while only a few scattered C l- ions are centered around the tilt angle from the single close collisions with the inner surfaces of the capillaries. There is a portion, comparable to the transmitted fraction of C l- , of the transmitted fractions of C l0 and C l+ , centered around the tilt angle, from the single scatterings with the inner surfaces of the capillaries. This confirms that at the present experimental conditions there are most probably no negative charge patches formed to guide the negative ions through insulating A l2O3 nanocapillaries.

The role of inserted polymers in polymeric insulation materials: insights from QM/MD simulations.

PubMed

Li, Chunyang; Zhao, Hong; Zhang, Hui; Wang, Ying; Wu, Zhijian; Han, Baozhong

2018-02-28

In this study, we performed a quantum chemical molecular dynamics (QM/MD) simulation to investigate the space charge accumulation process in copolymers of polyethylene (PE) with ethylene acrylic acid (EAA), ethylene vinyl acetate (EVA), styrene-ethylene-butadiene-styrene (SEBS), and black carbon (BC). We predicted that BC, especially branched BC, would possess the highest electron affinity and is identified as the most promising filler in power cable insulation. Following incorporations of 0-4 high-energy electrons into the composites, branched BC exhibited the highest stability and almost all electrons were trapped by it. Therefore, PE was protected efficiently and BC can be considered as an efficient filler for high voltage cables and an inhibitor of tree formation. On the contrary, although EAA, EVA, and SEBS can trap high-energy electrons, the latter can be supersaturated in composites of EAA, EVA, and SEBS with PE. The inserted polymers was unavoidably destroyed following C-H and C-O bond cleavage, which results from the interactions and charge transfer between PE and inserted polymers. The content effects of -COOH, benzene, and -OCOCH 3 groups on the electron trapping, mobility and stability of PE were also investigated systematically. We hope this knowledge gained from this work will be helpful in understanding the role of inserted polymers and the growth mechanisms of electrical treeing in high voltage cable insulation.

In situ monitoring magnetism and resistance of nanophase platinum upon electrochemical oxidation.

PubMed

Steyskal, Eva-Maria; Topolovec, Stefan; Landgraf, Stephan; Krenn, Heinz; Würschum, Roland

2013-01-01

Controlled tuning of material properties by external stimuli represents one of the major topics of current research in the field of functional materials. Electrochemically induced property tuning has recently emerged as a promising pathway in this direction making use of nanophase materials with a high fraction of electrode-electrolyte interfaces. The present letter reports on electrochemical property tuning of porous nanocrystalline Pt. Deeper insight into the underlying processes could be gained by means of a direct comparison of the charge-induced response of two different properties, namely electrical resistance and magnetic moment. For this purpose, four-point resistance measurements and SQUID magnetometry were performed under identical in situ electrochemical control focussing on the regime of electrooxidation. Fully reversible variations of the electrical resistance and the magnetic moment of 6% and 1% were observed upon the formation or dissolution of a subatomic chemisorbed oxygen surface layer, respectively. The increase of the resistance, which is directly correlated to the amount of deposited oxygen, is considered to be primarily caused by charge-carrier scattering processes at the metal-electrolyte interfaces. In comparison, the decrease of the magnetic moment upon positive charging appears to be governed by the electric field at the nanocrystallite-electrolyte interfaces due to spin-orbit coupling.

Cell cycle-dependent regulation of Aurora kinase B mRNA by the Microprocessor complex.

PubMed

Jung, Eunsun; Seong, Youngmo; Seo, Jae Hong; Kwon, Young-Soo; Song, Hoseok

2014-03-28

Aurora kinase B regulates the segregation of chromosomes and the spindle checkpoint during mitosis. In this study, we showed that the Microprocessor complex, which is responsible for the processing of the primary transcripts during the generation of microRNAs, destabilizes the mRNA of Aurora kinase B in human cells. The Microprocessor-mediated cleavage kept Aurora kinase B at a low level and prevented premature entrance into mitosis. The cleavage was reduced during mitosis leading to the accumulation of Aurora kinase B mRNA and protein. In addition to Aurora kinase B mRNA, the processing of other primary transcripts of miRNAs were also decreased during mitosis. We found that the cleavage was dependent on an RNA helicase, DDX5, and the association of DDX5 and DDX17 with the Microprocessor was reduced during mitosis. Thus, we propose a novel mechanism by which the Microprocessor complex regulates stability of Aurora kinase B mRNA and cell cycle progression. Copyright © 2014 Elsevier Inc. All rights reserved.

Specific RNA self-cleavage in coconut cadang cadang viroid: potential for a role in rolling circle replication.

PubMed Central

Liu, Y H; Symons, R H

1998-01-01

The rolling circle replication of the small, single-stranded viroid RNAs requires a specific processing reaction to produce monomeric RNAs that are ligated into the final circular form. For avocado sunblotch viroid, peach latent mosaic viroid, and chrysanthemum chlorotic mottle viroid, the hammerhead self-cleavage reaction is considered to provide this processing reaction. We have searched for a similar type of reaction in the 246-nt coconut cadang cadang viroid, the smallest viroid of the 24-member potato spindle tuber viroid (PSTV) group. RNA transcripts prepared from the cloned central or C domain of this viroid self-cleaved specifically after denaturation with methylmercuric hydroxide followed by incubation in the presence of spermidine but in the absence of added magnesium ions. The unique cleavage site was located in the bottom strand of the C domain within a potential hairpin structure that is conserved within members of all three subgroups of the PSTV group of viroids. PMID:9630248

Femtosecond Fluorescence Upconversion Study of a Naphthalimide-Bithiophene-Triphenylamine Push-Pull Dye in Solution.

PubMed

Maffeis, Valentin; Brisse, Romain; Labet, Vanessa; Jousselme, Bruno; Gustavsson, Thomas

2018-06-13

There is a high interest in the development of new push-pull dyes for the use in dye sensitized solar cells. The pronounced charge transfer character of the directly photoexcited state is in principle favorable for a charge injection. Here, we report a time-resolved fluorescence study of a triphenylamine-bithiophene-naphthalimide dye in four solvents of varying polarity using fluorescence upconversion. The recording of femtosecond time-resolved fluorescence spectra corrected for the group velocity dispersion allows for a detailed analysis discriminating between spectral shifts and total intensity decays. After photoexcitation, the directly populated state (S 1 /FC) evolves toward a relaxed charge transfer state (S 1 /CT). This S 1 /CT state is characterized by a lower radiative transition moment and a higher nonradiative quenching. The fast dynamic shift of the fluorescence band is well described by solvation dynamics in polar solvents, but less so in nonpolar solvents, hinting that the excited-state relaxation process occurs on a free energy surface whose topology is strongly governed by the solvent polarity. This study underlines the influence of the environment on the intramolecular charge transfer (ICT) process, and the necessity to analyze time-resolved data in detail when solvation and ICT occur simultaneously.

Measurement of the direct C P -violating parameter A C P in the decay D + → K - π + π +

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

Abazov, V. M.; Abbott, B.; Acharya, B. S.

2014-12-01

We measure the direct C P -violating parameter A C P for the decay of the charged charm meson, D + → K - π + π + (and charge conjugate), using the full 10.4 fb - 1more » sample of p p ¯ collisions at s = 1.96 TeV collected by the D0 detector at the Fermilab Tevatron collider. We extract the raw reconstructed charge asymmetry by fitting the invariant mass distributions for the sum and difference of charge-specific samples. This quantity is then corrected for detector-related asymmetries using data-driven methods and for possible physics asymmetries (from B → D processes) using input from Monte Carlo simulation. We measure A C P = [ - 0.16 ± 0.15 ( stat ) ± 0.09 ( syst ) ] % , which is consistent with zero, as expected from the standard model prediction of C P conservation, and is the most precise measurement of this quantity to date.« less

Opposite Roles of Furin and PC5A in N-Cadherin Processing12

PubMed Central

Maret, Deborah; Sadr, Mohamad Seyed; Sadr, Emad Seyed; Colman, David R; Del Maestro, Rolando F; Seidah, Nabil G

2012-01-01

We recently demonstrated that lack of Furin-processing of the N-cadherin precursor (proNCAD) in highly invasive melanoma and brain tumor cells results in the cell-surface expression of a nonadhesive protein favoring cell migration and invasion in vitro. Quantitative polymerase chain reaction analysis of malignant human brain tumor cells revealed that of all proprotein convertases (PCs) only the levels of Furin and PC5A are modulated, being inversely (Furin) or directly (PC5A) correlated with brain tumor invasive capacity. Intriguingly, the N-terminal sequence following the Furin-activated NCAD site (RQKR↓DW161, mouse nomenclature) reveals a second putative PC-processing site (RIRSDR↓DK189) located in the first extracellular domain. Cleavage at this site would abolish the adhesive functions of NCAD because of the loss of the critical Trp161. This was confirmed upon analysis of the fate of the endogenous prosegment of proNCAD in human malignant glioma cells expressing high levels of Furin and low levels of PC5A (U343) or high levels of PC5A and negligible Furin levels (U251). Cellular analyses revealed that Furin is the best activating convertase releasing an ∼17-kDa prosegment, whereas PC5A is the major inactivating enzyme resulting in the secretion of an ∼20-kDa product. Like expression of proNCAD at the cell surface, cleavage of the NCAD molecule at RIRSDR↓DK189 renders the U251 cancer cells less adhesive to one another and more migratory. Our work modifies the present view on posttranslational processing and surface expression of classic cadherins and clarifies how NCAD possesses a range of adhesive potentials and plays a critical role in tumor progression. PMID:23097623

Membrane Composition Tunes the Outer Hair Cell Motor

NASA Astrophysics Data System (ADS)

Rajagopalan, L.; Sfondouris, J.; Oghalai, J. S.; Pereira, F. A.; Brownell, W. E.

2009-02-01

Cholesterol and docosahexaenoic acid (DHA), an ω-3 fatty acid, affect membrane mechanical properties in different ways and modulate the function of membrane proteins. We have probed the functional consequence of altering cholesterol and DHA levels in the membranes of OHCs and prestin expressing HEK cells. Large, dynamic and reversible changes in prestin-associated charge movement and OHC motor activity result from altering the concentration of membrane cholesterol. Increasing membrane cholesterol shifts the q/V function ~ 50 mV in the hyperpolarizing direction, possibly a response related to increases in membrane stiffness. The voltage shift is linearly related to total membrane cholesterol. Increasing cholesterol also decreases the total charge moved in a linear fashion. Decreasing membrane cholesterol shifts the q/V function ~ 50 mV in the depolarizing direction with little or no effect on the amount of charge moved. In vivo increases in membrane cholesterol transiently increase but ultimately lead to decreases in DPOAE. Docosahexaenoic acid shifts the q/V function in the hyperpolarizing direction < 15 mV and increases total charge moved. Tuning of cochlear function by membrane cholesterol contributes to the exquisite temporal and frequency processing of mammalian hearing by optimizing the cochlear amplifier.

Quantitative separation of murine leukemia virus proteins by reversed-phase high-pressure liquid chromatography reveals newly described gag and env cleavage products.

PubMed Central

Henderson, L E; Sowder, R; Copeland, T D; Smythers, G; Oroszlan, S

1984-01-01

The structural proteins of murine type C retroviruses are proteolytic cleavage products of two different precursor polyproteins coded by the viral gag and env genes. To further investigate the nature and number of proteolytic cleavages involved in virus maturation, we quantitatively isolated the structural proteins of the Rauscher and Moloney strains of type C murine leukemia virus (R-MuLV and M-MuLV, respectively) by reversed-phase high-pressure liquid chromatography. Proteins and polypeptides isolated from R-MuLV included p10, p12, p15, p30, p15(E), gp69, and gp71 and three previously undescribed virus components designated here as p10', p2(E), and p2(E). Homologous proteins and polypeptides were isolated from M-MuLV. Complete or partial amino acid sequences of all the proteins listed above were either determined in this study or were available in previous reports from this laboratory. These data were compared with those from the translation of the M-MuLV proviral DNA sequence (Shinnick et al., Nature [London] 293:543-548, 1981) to determine the exact nature of proteolytic cleavages for all the structural proteins described above and to determine the origin of p10' and p2(E)s. The results showed that, during proteolytic processing of gp80env from M-MuLV (M-gp 80env), a single Arg residue was excised between gp70 and p15(E) and a single peptide bond was cleaved between p15(E) and p2(E). The structure of M-gPr80env is gp70-(Arg)-p15(E)-p2(E). The data suggest that proteolytic cleavage sites in R-gp85env are identical to corresponding cleavage sites in M-gp80env. The p2(E)s are shown to be different genetic variants of p2(E) present in the uncloned-virus preparations. The data for R- and M-p10's shows that they are cleavage products of the gag precursor with the structure p10-Thr-Leu-Asp-Asp-OH. The complete structure of Pr65gag is p15-p12-p30-p10'. Stoichiometries of the gag and env cleavage products in mature R- and M-MuLV were determined. In each virus, gag cleavage products (p15, p12, p30, and p10 plus p10') were found in equimolar amounts and p15(E)s were equimolar with p2(E)s. The stoichiometry of gag to env cleavage products was 4:1. These data are consistent with the proposal that proteolytic processing of precursor polyproteins occurs after virus assembly and that the C-terminal portion of Pr15(E) [i.e., p15(E)-p2(E)] is located on the inner side of the lipid bilayer of the virus. Images PMID:6333515

Post-translational processing of progastrin: inhibition of cleavage, phosphorylation and sulphation by brefeldin A.

PubMed Central

Varro, A; Dockray, G J

1993-01-01

The precursor for the acid-stimulating hormone gastrin provides a useful model for studies of post-translational processing because defined sites of cleavage, amidation, sulphation and phosphorylation occur within a dodecapeptide sequence. The factors determining these post-translational processing events are still poorly understood. We have used brefeldin A, which disrupts transport from rough endoplasmic reticulum to the Golgi complex, to examine the mechanisms of cleavage, phosphorylation and sulphation of rat progastrin-derived peptides. Biosynthetic products were detected after immunoprecipitation using antibodies specific for the extreme C-terminus of progastrin, followed by reversed-phase and ion-exchange h.p.l.c. Gastrin cells incorporated [3H]tyrosine, [32P]phosphate and [35S]sulphate into both progastrin and its extreme C-terminal tryptic (nona-) peptide. Ion-exchange chromatography resolved four forms of the C-terminal tryptic fragment of progastrin which differed in whether they were phosphorylated at Ser96, sulphated at Tyr103, both or neither. The specific activity of [3H]tyrosine in the peak that was both phosphorylated and sulphated was higher than in the others. Brefeldin A inhibited the appearance of [3H]tyrosine-labelled C-terminal tryptic fragment but there was an accumulation of labelled progastrin and a peptide corresponding to the C-terminal 46 residues of progastrin. Brefeldin A also inhibited incorporation of 32P and 35S into both progastrin and its C-terminal fragment. Thus phosphorylation of Ser96, sulphation of Tyr103 and cleavage at Arg94-Arg95 depend on passage of newly synthesized progastrin along the secretory pathway; as brefeldin A is thought to act proximal to the trans-Golgi, these processing steps would appear to occur distal to this point. The data also indicate that the stores of unphosphorylated C-terminal tryptic fragment are not available for phosphorylation, implying that this modification occurs proximal to the secretory granule; cleavage is known to occur in the secretory granule which suggests that it occurs after phosphorylation. Images Figure 1 PMID:8240296

Real-time observation of DNA target interrogation and product release by the RNA-guided endonuclease CRISPR Cpf1 (Cas12a).

PubMed

Singh, Digvijay; Mallon, John; Poddar, Anustup; Wang, Yanbo; Tippana, Ramreddy; Yang, Olivia; Bailey, Scott; Ha, Taekjip

2018-05-22

CRISPR-Cas9, which imparts adaptive immunity against foreign genomic invaders in certain prokaryotes, has been repurposed for genome-engineering applications. More recently, another RNA-guided CRISPR endonuclease called Cpf1 (also known as Cas12a) was identified and is also being repurposed. Little is known about the kinetics and mechanism of Cpf1 DNA interaction and how sequence mismatches between the DNA target and guide-RNA influence this interaction. We used single-molecule fluorescence analysis and biochemical assays to characterize DNA interrogation, cleavage, and product release by three Cpf1 orthologs. Our Cpf1 data are consistent with the DNA interrogation mechanism proposed for Cas9. They both bind any DNA in search of protospacer-adjacent motif (PAM) sequences, verify the target sequence directionally from the PAM-proximal end, and rapidly reject any targets that lack a PAM or that are poorly matched with the guide-RNA. Unlike Cas9, which requires 9 bp for stable binding and ∼16 bp for cleavage, Cpf1 requires an ∼17-bp sequence match for both stable binding and cleavage. Unlike Cas9, which does not release the DNA cleavage products, Cpf1 rapidly releases the PAM-distal cleavage product, but not the PAM-proximal product. Solution pH, reducing conditions, and 5' guanine in guide-RNA differentially affected different Cpf1 orthologs. Our findings have important implications on Cpf1-based genome engineering and manipulation applications.

Birth of normal infants after transfer of embryos that were twice vitrified/warmed at cleavage stages: report of two cases.

PubMed

Valle, Marcello; Guimarães, Fernando; Cavagnoli, Melissa; Sampaio, Marcos; Geber, Selmo

2012-12-01

The role of cryopreservation in assisted reproductive technology programs has increased within the last years allowing the transfer of a limited number of embryos and the storage of the remaining for future use. The reduction in the number of transferred embryos decreases the frequency of multiple pregnancy rates and of ovarian hyperstimulation syndrome while the cumulative pregnancy rate can be maximized. Moreover, as not all embryos will survive the warming process more cleavage stage embryos are warmed to improve selection for transfer. Therefore, surplus good quality cleavage stage embryos and/or blastocysts must be re-vitrified for further transfer to achieve pregnancy. To our knowledge, there have been no reports demonstrating that human embryos can be successfully vitrified/warmed twice at the cleavage stage. Thus we report two successful pregnancies and deliveries of healthy babies after transfer of embryos that were twice vitrified/warmed at 2-4 cells stage. Copyright © 2012 Elsevier Inc. All rights reserved.

Laboratory Studies of Thermal Energy Charge Transfer of Silicon and Iron Ions in Astrophysical Plasmas

NASA Technical Reports Server (NTRS)

Kwong, Victor H. S.

1997-01-01

The laser ablation/ion storage facility at the UNLV Physics Department is dedicated to the study of atomic processes in low temperature plasmas. Our current program is directed to the study of charge transfer of multiply charged ions and neutrals that are of importance to astrophysics at energies less than 1 eV (about 10(exp 4) K). Specifically, we measure the charge transfer rate coefficient of ions such as N(2+), Si(3+), Si(3+), with helium and Fe(2+) with molecular and atomic hydrogen. All these ions are found in a variety of astrophysical plasmas. Their electron transfer reactions with neutral atoms can affect the ionization equilibrium of the plasma.

Structural and biochemical characterization of the protease domain of the mosaic botulinum neurotoxin type HA.

PubMed

Lam, Kwok-Ho; Sikorra, Stefan; Weisemann, Jasmin; Maatsch, Hannah; Perry, Kay; Rummel, Andreas; Binz, Thomas; Jin, Rongsheng

2018-04-23

The extreme toxicity of botulinum neurotoxins (BoNTs) relies on their specific cleavage of SNARE proteins, which eventually leads to muscle paralysis. One newly identified mosaic toxin, BoNT/HA (aka H or FA), cleaves VAMP-2 at a unique position between residues L54 and E55, but the molecular basis underlying VAMP-2-recognition of BoNT/HA remains poorly characterized. Here, we report a ∼2.09 Å resolution crystal structure of the light chain protease domain of BoNT/HA (LC/HA). Structural comparison between LC/HA and LC of BoNT/F1 (LC/F1) reveals distinctive hydrophobic and electrostatic features near the active sites, which may explain their different VAMP-2 cleavage sites. When compared to BoNT/F5 that cleaves VAMP-2 at the same site as BoNT/HA, LC/HA displays higher affinity for VAMP-2, which could be caused by their different surface charge properties surrounding a VAMP-2 exosite-binding cleft. Furthermore, systematic mutagenesis studies on VAMP-2 and structural modeling demonstrate that residues R47 to K59 spanning the cleavage site in VAMP-2 may adopt a novel extended conformation when interacting with LC/HA and LC/F5. Taken together, our structure provides new insights into substrate-recognition of BoNT/HA and paves the way for rational design of small molecule or peptide inhibitors against LC/HA.

On the binding of indeno[1,2-c]isoquinolines in the DNA-topoisomerase I cleavage complex.

PubMed

Xiao, Xiangshu; Antony, Smitha; Pommier, Yves; Cushman, Mark

2005-05-05

An ab initio quantum mechanics calculation is reported which predicts the orientation of indenoisoquinoline 4 in the ternary cleavage complex formed from DNA and topoisomerase I (top1). The results of this calculation are consistent with the hypothetical structures previously proposed for the indenoisoquinoline-DNA-top1 ternary complexes based on molecular modeling, the crystal structure of a recently reported ternary complex, and the biological results obtained with a pair of diaminoalkyl-substituted indenoisoquinoline enantiomers. The results of these studies indicate that the pi-pi stacking interactions between the indenoisoquinolines and the neighboring DNA base pairs play a major role in determining binding orientation. The calculation of the electrostatic potential surface maps of the indenoisoquinolines and the adjacent DNA base pairs shows electrostatic complementarity in the observed binding orientation, leading to the conclusion that electrostatic attraction between the intercalators and the base pairs in the cleavage complex plays a major stabilizing role. On the other hand, the calculation of LUMO and HOMO energies of indenoisoquinoline 13b and neighboring DNA base pairs in conjunction with NBO analysis indicates that charge transfer complex formation plays a relatively minor role in stabilizing the ternary complexes derived from indenoisoquinolines, DNA, and top1. The results of these studies are important in understanding the existing structure-activity relationships for the indenoisoquinolines as top1 inhibitors and as anticancer agents, and they will be important in the future design of indenoisoquinoline-based top1 inhibitors.

Multi-phase-field modeling of anisotropic crack propagation for polycrystalline materials

NASA Astrophysics Data System (ADS)

Nguyen, Thanh-Tung; Réthoré, Julien; Yvonnet, Julien; Baietto, Marie-Christine

2017-08-01

A new multi-phase-field method is developed for modeling the fracture of polycrystals at the microstructural level. Inter and transgranular cracking, as well as anisotropic effects of both elasticity and preferential cleavage directions within each randomly oriented crystal are taken into account. For this purpose, the proposed phase field formulation includes: (a) a smeared description of grain boundaries as cohesive zones avoiding defining an additional phase for grains; (b) an anisotropic phase field model; (c) a multi-phase field formulation where each preferential cleavage direction is associated with a damage (phase field) variable. The obtained framework allows modeling interactions and competition between grains and grain boundary cracks, as well as their effects on the effective response of the material. The proposed model is illustrated through several numerical examples involving a full description of complex crack initiation and propagation within 2D and 3D models of polycrystals.

Method and system for aligning fibers during electrospinning

NASA Technical Reports Server (NTRS)

Scott-Carnell, Lisa A. (Inventor); Stephens, Ralph M (Inventor); Holloway, Nancy M. (Inventor); Rhim, Caroline (Inventor); Niklason, Laura (Inventor); Clark, Robert L. (Inventor); Siochi, Emilie J. (Inventor)

2011-01-01

A method and system are provided for aligning fibers in an electrospinning process. A jet of a fiberizable material is directed towards an uncharged collector from a dispensing location that is spaced apart from the collector. While the fiberizable material is directed towards the collector, an elliptical electric field is generated via the electrically charged dispenser and an oppositely-charged control location. The field spans between the dispensing location and the control location that is within line-of-sight of the dispensing location, and impinges upon at least a portion of the collector. Various combinations of numbers and geometries of dispensers, collectors, and electrodes can be used.

A numerical investigation of a thermodielectric power generation system

NASA Astrophysics Data System (ADS)

Sklar, Akiva A.

The performance of a novel micro-thermodielectric power generation system was investigated in order to determine if thermodielectric power generation can be practically employed and if its performance can compete with current portable power generation technologies. Thermodielectric power generation is a direct energy conversion technology that converts heat directly into high voltage direct current. It requires dielectric (i.e., capacitive) materials whose charge storing capabilities are a function of temperature. This property can be exploited by heating these materials after they are charged; as their temperature increases, their charge storage capability decreases, forcing them to eject a portion of their surface charge. This ejected charge can then be supplied to an appropriate electronic storage device. There are several advantages associated with thermodielectric energy conversion; first, it requires heat addition at relatively low conventional power generation temperatures, i.e., less than 600 °K, and second, devices that utilize it have the potential for excellent power density and device reliability. The predominant disadvantage of using this power generation technique is that the device must operate in an unsteady manner; this can lead to substantial heat transfer losses that limit the device's thermal efficiency. The studied power generation system was designed so that the power generating components of the system (i.e., the thermodielectric materials) are integrated within a micro-scale heat exchange apparatus designed specifically to provide the thermodielectric materials with the unsteady heating and cooling necessary for efficient power generation. This apparatus is designed to utilize a liquid as a working fluid in order to maximize its heat transfer capabilities, minimize the size of the heat exchanger, and maximize the power density of the power generation system. The thermodielectric materials are operated through a power generation cycle that consists of four processes; the first process is a charging process, during which an electric field is applied to a thermodielectric material, causing it to acquire electrical charge on its surface (this process is analogous to the isentropic compression process of a Brayton cycle). The second process is a heating process in which the temperature of the dielectric material is increased via heat transfer from an external source. During this process, the thermodielectric material is forced to eject a portion of its surface charge because its charge storing capability decreases as the temperature increases; the ejected charge is intended for capture by external circuitry connected to the thermodielectric material, where it can be routed to an electrochemical storage device or an electromechanical device requiring high voltage direct current. The third process is a discharging process, during which the applied electric field is reduced to its initial strength (analogous to the isentropic expansion process of a Brayton cycle). The final process is a cooling process in which the temperature of the dielectric material is decreased via heat transfer from an external source, returning it to its initial temperature. Previously, predicting the performance of a thermodielectric power generator was hindered by a poor understanding of the material's thermodynamic properties and the effect unsteady heat transfer losses have on system performance. In order to improve predictive capabilities in this study, a thermodielectric equation of state was developed that relates the strength of the applied electric field, the amount of surface charge stored by the thermodielectric material, and its temperature. This state equation was then used to derive expressions for the material's thermodynamic states (internal energy, entropy), which were subsequently used to determine the optimum material properties for power generation. Next, a numerical simulation code was developed to determine the heat transfer capabilities of a micro-scale parallel plate heat recuperator (MPPHR), a device designed specifically to (a) provide the unsteady heating and cooling necessary for thermodielectric power generation and (b) minimize the unsteady heat transfer losses of the system. The simulation code was used to find the optimum heat transfer and heat recuperation regimes of the MPPHR. The previously derived thermodynamic equations that describe the behavior of the thermodielectric materials were then incorporated into the model for the walls of the parallel plate channel in the numerical simulation code, creating a tool capable of determining the thermodynamic performance of an MTDPG, in terms of the thermal efficiency, percent Carnot efficiency, and energy/power density. A detailed parameterization of the MTDPG with the simulation code yielded the critical non-dimensional numbers that determine the relationship between the heat exchange/recuperation abilities of the flow and the power generation capabilities of the thermodielectric materials. These relationships were subsequently used to optimize the performance of an MTDPG with an operating temperature range of 300--500 °K. The optimization predicted that the MTDPG could provide a thermal efficiency of 29.7 percent with the potential to reach 34 percent. These thermal efficiencies correspond to 74.2 and 85 percent of the Carnot efficiency, respectively. The power density of this MTDPG depends on the operating frequency and can exceed 1,000,000 W/m3.

Monitoring state-of-charge of Ni-MH and Ni-Cd batteries using impedance spectroscopy

NASA Astrophysics Data System (ADS)

Hammouche, Abderrezak; Karden, Eckhard; De Doncker, Rik W.

This paper reports on laboratory studies into the ac impedance spectra of nickel-metal hydride and nickel-cadmium batteries, aiming at finding out possible correlation between electrical parameters, extracted directly from the high frequency region, and the battery state-of-charge (SoC). Impedance diagrams were recorded immediately after interrupting the dc charge, or discharge, current. The study revealed that the series resonance frequency, at which the dynamic cell behavior switches from an inductive character ( Z″>0) to a capacitive one ( Z″<0), varied monotonously as a function of state-of-charge. This behavior was reproducible after intermittent charge and discharge. Half-cell measurements were also conducted to associate the cell impedance with either processes occurring at the positive or negative plates.

Measurements of Charging of Apollo 17 Lunar Dust Grains by Electron Impact

NASA Technical Reports Server (NTRS)

Abbas, Mian M.; Tankosic, Dragana; Spann, James F.; Dube, Michael J.

2008-01-01

It is well known since the Apollo missions that the lunar surface is covered with a thick layer of micron size dust grains with unusually high adhesive characteristics. The dust grains observed to be levitated and transported on the lunar surface are believed to have a hazardous impact on the robotic and human missions to the Moon. The observed dust phenomena are attributed to the lunar dust being charged positively during the day by UV photoelectric emissions, and negatively during the night by the solar wind electrons. The current dust charging and the levitation models, however, do not fully explain the observed phenomena, with the uncertainty of dust charging processes and the equilibrium potentials of the individual dust grains. It is well recognized that the charging properties of individual dust grains are substantially different from those determined from measurements made on bulk materials that are currently available. An experimental facility has been developed in the Dusty Plasma Laboratory at MSFC for investigating the charging and optical properties of individual micron/sub-micron size positively or negatively charged dust grains by levitating them in an electrodynamic balance in simulated space environments. In this paper, we present the laboratory measurements on charging of Apollo 17 individual lunar dust grains by a low energy electron beam. The charging rates and the equilibrium potentials produced by direct electron impact and by secondary electron emission process are discussed.

Laboratory Measurements of Charging of Apollo 17 Lunar Dust Grains by Low Energy Electrons

NASA Technical Reports Server (NTRS)

Abbas, Mian M.; Tankosic, Dragana; Spann, James F.; Dube, Michael J.; Gaskin, Jessica

2007-01-01

It is well recognized that the charging properties of individual micron/sub-micron size dust grains by various processes are expected to be substantially different from the currently available measurements made on bulk materials. Solar UV radiation and the solar wind plasma charge micron size dust grains on the lunar surface with virtually no atmosphere. The electrostatically charged dust grains are believed to be levitated and transported long distances over the lunar terminator from the day to the night side. The current models do not fully explain the lunar dust phenomena and laboratory measurements are needed to experimentally determine the charging properties of lunar dust grains. An experimental facility has been developed in the Dusty Plasma Laboratory at NASA Marshall Space Flight Center MSFC for investigating the charging properties of individual micron/sub-micron size positively or negatively charged dust grains by levitating them in an electrodynamic balance in simulated space environments. In this paper, we present laboratory measurements on charging of Apollo 17 individual lunar dust grains by low energy electron beams in the 5-100 eV energy range. The measurements are made by levitating Apollo 17 dust grains of 0.2 to 10 micrometer diameters, in an electrodynamic balance and exposing them to mono-energetic electron beams. The charging rates and the equilibrium potentials produced by direct electron impact and by secondary electron emission processes are discussed.

Specific Inhibition of β-Secretase Processing of the Alzheimer Disease Amyloid Precursor Protein.

PubMed

Ben Halima, Saoussen; Mishra, Sabyashachi; Raja, K Muruga Poopathi; Willem, Michael; Baici, Antonio; Simons, Kai; Brüstle, Oliver; Koch, Philipp; Haass, Christian; Caflisch, Amedeo; Rajendran, Lawrence

2016-03-08

Development of disease-modifying therapeutics is urgently needed for treating Alzheimer disease (AD). AD is characterized by toxic β-amyloid (Aβ) peptides produced by β- and γ-secretase-mediated cleavage of the amyloid precursor protein (APP). β-secretase inhibitors reduce Aβ levels, but mechanism-based side effects arise because they also inhibit β-cleavage of non-amyloid substrates like Neuregulin. We report that β-secretase has a higher affinity for Neuregulin than it does for APP. Kinetic studies demonstrate that the affinities and catalytic efficiencies of β-secretase are higher toward non-amyloid substrates than toward APP. We show that non-amyloid substrates are processed by β-secretase in an endocytosis-independent manner. Exploiting this compartmentalization of substrates, we specifically target the endosomal β-secretase by an endosomally targeted β-secretase inhibitor, which blocked cleavage of APP but not non-amyloid substrates in many cell systems, including induced pluripotent stem cell (iPSC)-derived neurons. β-secretase inhibitors can be designed to specifically inhibit the Alzheimer process, enhancing their potential as AD therapeutics without undesired side effects. Copyright © 2016 The Authors. Published by Elsevier Inc. All rights reserved.

Degradation of tropoelastin and skin elastin by neprilysin.

PubMed

Mora Huertas, Angela C; Schmelzer, Christian E H; Luise, Chiara; Sippl, Wolfgang; Pietzsch, Markus; Hoehenwarter, Wolfgang; Heinz, Andrea

2018-03-01

Neprilysin is also known as skin fibroblast-derived elastase, and its up-regulation during aging is associated with impairments of the elastic fiber network, loss of skin elasticity and wrinkle formation. However, information on its elastase activity is still limited. The aim of this study was to investigate the degradation of fibrillar skin elastin by neprilysin and the influence of the donor's age on the degradation process using mass spectrometry and bioinformatics approaches. The results showed that cleavage by neprilysin is dependent on previous damage of elastin. While neprilysin does not cleave young and intact skin elastin well, it degrades elastin fibers from older donors, which may further promote aging processes. With regards to the cleavage behavior of neprilysin, a strong preference for Gly at P1 was found, while Gly, Ala and Val were well accepted at P1' upon cleavage of tropoelastin and skin elastin. The results of the study indicate that the progressive release of bioactive elastin peptides by neprilysin upon skin aging may enhance local tissue damage and accelerate extracellular matrix aging processes. Copyright © 2017 Elsevier B.V. and Société Française de Biochimie et Biologie Moléculaire (SFBBM). All rights reserved.

Poly(A)-tag deep sequencing data processing to extract poly(A) sites.

PubMed

Wu, Xiaohui; Ji, Guoli; Li, Qingshun Quinn

2015-01-01

Polyadenylation [poly(A)] is an essential posttranscriptional processing step in the maturation of eukaryotic mRNA. The advent of next-generation sequencing (NGS) technology has offered feasible means to generate large-scale data and new opportunities for intensive study of polyadenylation, particularly deep sequencing of the transcriptome targeting the junction of 3'-UTR and the poly(A) tail of the transcript. To take advantage of this unprecedented amount of data, we present an automated workflow to identify polyadenylation sites by integrating NGS data cleaning, processing, mapping, normalizing, and clustering. In this pipeline, a series of Perl scripts are seamlessly integrated to iteratively map the single- or paired-end sequences to the reference genome. After mapping, the poly(A) tags (PATs) at the same genome coordinate are grouped into one cleavage site, and the internal priming artifacts removed. Then the ambiguous region is introduced to parse the genome annotation for cleavage site clustering. Finally, cleavage sites within a close range of 24 nucleotides and from different samples can be clustered into poly(A) clusters. This procedure could be used to identify thousands of reliable poly(A) clusters from millions of NGS sequences in different tissues or treatments.

Quantitative proteomics reveals the kinetics of trypsin-catalyzed protein digestion.

PubMed

Pan, Yanbo; Cheng, Kai; Mao, Jiawei; Liu, Fangjie; Liu, Jing; Ye, Mingliang; Zou, Hanfa

2014-10-01

Trypsin is the popular protease to digest proteins into peptides in shotgun proteomics, but few studies have attempted to systematically investigate the kinetics of trypsin-catalyzed protein digestion in proteome samples. In this study, we applied quantitative proteomics via triplex stable isotope dimethyl labeling to investigate the kinetics of trypsin-catalyzed cleavage. It was found that trypsin cleaves the C-terminal to lysine (K) and arginine (R) residues with higher rates for R. And the cleavage sites surrounded by neutral residues could be quickly cut, while those with neighboring charged residues (D/E/K/R) or proline residue (P) could be slowly cut. In a proteome sample, a huge number of proteins with different physical chemical properties coexists. If any type of protein could be preferably digested, then limited digestion could be applied to reduce the sample complexity. However, we found that protein abundance and other physicochemical properties, such as molecular weight (Mw), grand average of hydropathicity (GRAVY), aliphatic index, and isoelectric point (pI) have no notable correlation with digestion priority of proteins.