Edible carbohydrates from formaldehyde in a spacecraft

NASA Technical Reports Server (NTRS)

Weiss, A. H.

1975-01-01

The autocatalytic nature of the base catalyzed condensation of formaldehyde to formose sugars is eliminated by using as a cocatalyst, an aldose, or ketose having an alpha-hydrogen. This is more strongly complexed by base than is formaldehyde and the cocatalyst and sugar products accumulate as catalyst complexes instead of formaldehyde. Because of the presence of alpha-hydrogen atoms in cocatalysts and formose sugars, their removal by cross Cannizzaro reaction of complexed sugars does not occur, so the formose reaction behaves autocatalytically due to this accumulation. It is believed that a given catalytic formose complex is not a discrete complexed sugar, but rather, a scrambled dynamic mixture of sugars having weakened structures. The sugar complexes derive from a common salt-like formaldehyde complex, which, because of the absence of alpha-hydrogen, has a greater tendency to undergo Cannizzaro reaction, rather than formose condensation. Because of this, the Cannizzaro reaction can proceed without measurable formose condensation. The reverse is not possible.

Processing, stability, and kinetic parameters of C5a peptidase from Streptococcus pyogenes.

PubMed

Anderson, Elizabeth T; Wetherell, Michael G; Winter, Laurie A; Olmsted, Stephen B; Cleary, Patrick P; Matsuka, Yury V

2002-10-01

A recombinant streptococcal C5a peptidase was expressed in Escherichia coli and its catalytic properties and thermal stability were subjected to examination. It was shown that the NH2-terminal region of C5a peptidase (Asn32-Asp79/Lys90) forms the pro-sequence segment. Upon maturation the propeptide is hydrolyzed either via an autocatalytic intramolecular cleavage or by exogenous protease streptopain. At pH 7.4 the enzyme exhibited maximum activity in the narrow range of temperatures between 40 and 43 degrees C. The process of heat denaturation of C5a peptidase investigated by fluorescence and circular dichroism spectroscopy revealed that the protein undergoes biphasic unfolding transition with Tm of 50 and 70 degrees C suggesting melting of different parts of the molecule with different stability. Unfolding of the less stable structures was accompanied by the loss of proteolytic activity. Using synthetic peptides corresponding to the COOH-terminus of human complement C5a we demonstrated that in vitro peptidase catalyzes hydrolysis of two His67-Lys68 and Ala58-Ser59 peptide bonds. The high catalytic efficiency obtained for the SQLRANISHKDMQLGR extended peptide compared to the poor hydrolysis of its derivative Ac-SQLRANISH-pNA that lacks residues at P2'-P7' positions, suggest the importance of C5a peptidase interactions with the P' side of the substrate.

Catalytic Properties of Botulinum Neurotoxins Subtypes A3 and A4

PubMed Central

Henkel, James S.; Jacobson, Mark; Tepp, William; Pier, Christina; Johnson, Eric A.; Barbieri, Joseph T.

2009-01-01

Botulinum toxins (BoNT) are zinc proteases (serotypes A-G) which cause flaccid paralysis through the cleavage of SNARE proteins within motor neurons. BoNT/A was originally organized into two subtypes: BoNT/A1 and BoNT/A2, which are ~ 95 % homologous and possess similar catalytic activities. Subsequently, two additional subtypes were identified; BoNT/A3 (Loch Maree), and BoNT/A4 (657Ba), which have 81 and 88% homology with BoNT/A1, respectively. Alignment studies predicted that BoNT/A3 and BoNT/A4 were sufficiently different to BoNT/A1 to affect SNAP25 binding and cleavage. Recombinant Light Chain (LC) of BoNT/A3 (LC/A3) and BoNT/A4 (LC/A4) were subjected to biochemical analysis. LC/A3 cleaved SNAP25 at 50% the rate of LC/A1, but cleaved SNAPtide® at a faster rate than LC/A1, while LC/A4 cleaved SNAP25 and SNAPtide® at slower rates than LC/A1. LC/A3 and LC/A4 had similar Kms for SNAP25 relative to LC/A1, while the kcat for LC/A4 was 10- fold slower than LC/A1, suggesting a defect in substrate cleavage. Neither LC/A3 nor LC/A4 possessed autocatalytic activity, a property of LC/A1 and LC/A2. Thus, the four subtypes of BoNT/A bind SNAP25 with similar affinity but have different catalytic capacities for SNAP25 cleavage, SNAPtide® cleavage, and autocatalysis. The catalytic properties identified among the subtypes of LC/A may influence strategies for the development of small molecule- or peptide- inhibitors as therapies against botulism. PMID:19256469

Delta-Secretase Phosphorylation by SRPK2 Enhances Its Enzymatic Activity, Provoking Pathogenesis in Alzheimer's Disease.

PubMed

Wang, Zhi-Hao; Liu, Pai; Liu, Xia; Manfredsson, Fredric P; Sandoval, Ivette M; Yu, Shan Ping; Wang, Jian-Zhi; Ye, Keqiang

2017-09-07

Delta-secretase, a lysosomal asparagine endopeptidase (AEP), simultaneously cleaves both APP and tau, controlling the onset of pathogenesis of Alzheimer's disease (AD). However, how this protease is post-translationally regulated remains unclear. Here we report that serine-arginine protein kinase 2 (SRPK2) phosphorylates delta-secretase and enhances its enzymatic activity. SRPK2 phosphorylates serine 226 on delta-secretase and accelerates its autocatalytic cleavage, leading to its cytoplasmic translocation and escalated enzymatic activities. Delta-secretase is highly phosphorylated in human AD brains, tightly correlated with SRPK2 activity. Overexpression of a phosphorylation mimetic (S226D) in young 3xTg mice strongly promotes APP and tau fragmentation and facilitates amyloid plaque deposits and neurofibrillary tangle (NFT) formation, resulting in cognitive impairment. Conversely, viral injection of the non-phosphorylatable mutant (S226A) into 5XFAD mice decreases APP and tau proteolytic cleavage, attenuates AD pathologies, and reverses cognitive defects. Our findings support that delta-secretase phosphorylation by SRPK2 plays a critical role in aggravating AD pathogenesis. Copyright © 2017 Elsevier Inc. All rights reserved.

Single step purification of recombinant proteins using the metal ion-inducible autocleavage (MIIA) domain as linker for tag removal.

PubMed

Ibe, Susan; Schirrmeister, Jana; Zehner, Susanne

2015-08-20

For fast and easy purification, proteins are typically fused with an affinity tag, which often needs to be removed after purification. Here, we present a method for the removal of the affinity tag from the target protein in a single step protocol. The protein VIC_001052 of the coral pathogen Vibrio coralliilyticus ATCC BAA-450 contains a metal ion-inducible autocatalytic cleavage (MIIA) domain. Its coding sequence was inserted into an expression vector for the production of recombinant fusion proteins. Following, the target proteins MalE and mCherry were produced as MIIA-Strep fusion proteins in Escherichia coli. The target proteins could be separated from the MIIA-Strep part simply by the addition of calcium or manganese(II) ions within minutes. The cleavage is not affected in the pH range from 5.0 to 9.0 or at low temperatures (6°C). Autocleavage was also observed with immobilized protein on an affinity column. The protein yield was similar to that achieved with a conventional purification protocol. Copyright © 2015 Elsevier B.V. All rights reserved.

Centrosomes are autocatalytic droplets of pericentriolar material organized by centrioles

NASA Astrophysics Data System (ADS)

Zwicker, David; Decker, Markus; Jaensch, Steffen; Hyman, Anthony A.; Jülicher, Frank

2014-03-01

We propose a physical description of the centrosome, a membrane-less organelle involved in cell division. In our model, centrosome material occurs in a soluble form in the cytosol and a form that tends to undergo phase separation from the cytosol. We find that an autocatalytic chemical transition between these forms accounts for the temporal evolution observed in experiments. Interestingly, the nucleation of centrosomes can be controlled by an enzymatic activity of the centrioles, which are present at the core of all centrosomes. This non-equilibrium feature also allows for multiple stable centrosomes, a situation which is unstable in equilibrium phase separation. Our theory explains the growth dynamics of centrosomes for all cell sizes down to the eight-cell stage of the C. elegans embryo. It also accounts for data acquired in experiments with aberrant numbers of centrosomes and altered cell volumes. Furthermore, our model can describe unequal centrosome sizes observed in cells with disturbed centrioles. Our example suggests a general picture of the organization of membrane-less organelles.

Satellite RNAs of plant viruses: structures and biological effects.

PubMed Central

Roossinck, M J; Sleat, D; Palukaitis, P

1992-01-01

Plant viruses often contain parasites of their own, referred to as satellites. Satellite RNAs are dependent on their associated (helper) virus for both replication and encapsidation. Satellite RNAs vary from 194 to approximately 1,500 nucleotides (nt). The larger satellites (900 to 1,500 nt) contain open reading frames and express proteins in vitro and in vivo, whereas the smaller satellites (194 to 700 nt) do not appear to produce functional proteins. The smaller satellites contain a high degree of secondary structure involving 49 to 73% of their sequences, with the circular satellites containing more base pairing than the linear satellites. Many of the smaller satellites produce multimeric forms during replication. There are various models to account for their formation and role in satellite replication. Some of these smaller satellites encode ribozymes and are able to undergo autocatalytic cleavage. The enzymology of satellite replication is poorly understood, as is the replication of their helper viruses. In many cases the coreplication of satellites suppresses the replication of the helper virus genome. This is usually paralleled by a reduction in the disease induced by the helper virus; however, there are notable exceptions in which the satellite exacerbates the pathogenicity of the helper virus, albeit on only a limited number of hosts. The ameliorative satellites are being assessed as biocontrol agents of virus-induced disease. In greenhouse studies, satellites have been known to "spontaneously" appear in virus cultures. The possible origin of satellites will be briefly considered. PMID:1620065

The Kinetic Rate Law for Autocatalytic Reactions.

ERIC Educational Resources Information Center

Mata-Perez, Fernando; Perez-Benito, Joaquin F.

1987-01-01

Presented is a method of obtaining accurate rate constants for autocatalytic reactions. The autocatalytic oxidation of dimethylamine by permanganate ion in aqueous solution is used as an example. (RH)

Intermittency inhibited by transport: An exactly solvable model

NASA Astrophysics Data System (ADS)

Zanette, Damián H.

1994-04-01

Transport is incorporated in a discrete-time stochastic model of a system undergoing autocatalytic reactions of the type A-->2A and A-->0, whose population field is known to exhibit spatiotemporal intermittency. The temporal evolution is exactly solved, and it is shown that if the transport process is strong enough, intermittency is inhibited. This inhibition is nonuniform, in the sense that, as transport is strengthened, low-order population moments are affected before the high-order ones. Numerical simulations are presented to support the analytical results.

Computational identification of obligatorily autocatalytic replicators embedded in metabolic networks

PubMed Central

Kun, Ádám; Papp, Balázs; Szathmáry, Eörs

2008-01-01

Background If chemical A is necessary for the synthesis of more chemical A, then A has the power of replication (such systems are known as autocatalytic systems). We provide the first systems-level analysis searching for small-molecular autocatalytic components in the metabolisms of diverse organisms, including an inferred minimal metabolism. Results We find that intermediary metabolism is invariably autocatalytic for ATP. Furthermore, we provide evidence for the existence of additional, organism-specific autocatalytic metabolites in the forms of coenzymes (NAD+, coenzyme A, tetrahydrofolate, quinones) and sugars. Although the enzymatic reactions of a number of autocatalytic cycles are present in most of the studied organisms, they display obligatorily autocatalytic behavior in a few networks only, hence demonstrating the need for a systems-level approach to identify metabolic replicators embedded in large networks. Conclusion Metabolic replicators are apparently common and potentially both universal and ancestral: without their presence, kick-starting metabolic networks is impossible, even if all enzymes and genes are present in the same cell. Identification of metabolic replicators is also important for attempts to create synthetic cells, as some of these autocatalytic molecules will presumably be needed to be added to the system as, by definition, the system cannot synthesize them without their initial presence. PMID:18331628

The Sugar Model: Autocatalytic Activity of the Triose-Ammonia Reaction

NASA Technical Reports Server (NTRS)

Weber, Arthur L.

2006-01-01

Reaction of triose sugars with ammonia under anaerobic conditions yielded autocatalytic products. The autocatalytic behavior of the products was examined by measuring the effect of the crude triose-ammonia reaction product on the kinetics of a second identical triose-ammonia reaction. The reaction product showed autocatalytic activity by increasing both the rate of disappearance of triose and the rate formation of pyruvaldehyde, the product of triose dehydration. This synthetic process is considered a reasonable model of origin-of-life chemistry because it uses plausible prebiotic substrates, and resembles modern biosynthesis by employing the energized carbon groups of sugars to drive the synthesis of autocatalytic molecules.

The Sugar Model: Autocatalytic Activity of the Triose Ammonia Reaction

NASA Astrophysics Data System (ADS)

Weber, Arthur L.

2007-04-01

Reaction of triose sugars with ammonia under anaerobic conditions yielded autocatalytic products. The autocatalytic behavior of the products was examined by measuring the effect of the crude triose ammonia reaction product on the kinetics of a second identical triose ammonia reaction. The reaction product showed autocatalytic activity by increasing both the rate of disappearance of triose and the rate of formation of pyruvaldehyde, the product of triose dehydration. This synthetic process is considered a reasonable model of origin-of-life chemistry because it uses plausible prebiotic substrates, and resembles modern biosynthesis by employing the energized carbon groups of sugars to drive the synthesis of autocatalytic molecules.

Structure of the Repulsive Guidance Molecule (RGM)—Neogenin Signaling Hub

PubMed Central

Bell, Christian H.; Bishop, Benjamin; Tang, Chenxiang; Gilbert, Robert J.C.; Aricescu, A. Radu; Pasterkamp, R. Jeroen; Siebold, Christian

2016-01-01

Repulsive guidance molecule family members (RGMs) control fundamental and diverse cellular processes, including motility and adhesion, immune cell regulation, and systemic iron metabolism. However, it is not known how RGMs initiate signaling through their common cell-surface receptor, neogenin (NEO1). Here, we present crystal structures of the NEO1 RGM-binding region and its complex with human RGMB (also called dragon). The RGMB structure reveals a previously unknown protein fold and a functionally important autocatalytic cleavage mechanism and provides a framework to explain numerous disease-linked mutations in RGMs. In the complex, two RGMB ectodomains conformationally stabilize the juxtamembrane regions of two NEO1 receptors in a pH-dependent manner. We demonstrate that all RGM-NEO1 complexes share this architecture, which therefore represents the core of multiple signaling pathways. PMID:23744777

Exonuclease III-assisted cascade signal amplification strategy for label-free and ultrasensitive electrochemical detection of nucleic acids.

PubMed

Xiong, Erhu; Yan, Xiaoxia; Zhang, Xiaohua; Liu, Yunqing; Zhou, Jiawan; Chen, Jinhua

2017-01-15

In this work, a simple, signal-on and label-free electrochemical biosensor for ultrasensitive DNA detection is reported on the basis of an autocatalytic and exonuclease III (Exo III)-assisted cascade signal amplification strategy. In the presence of target DNA (T-DNA), the hybridization between the 3'-protruding DNA fragment of hairpin DNA probe (HP1) and T-DNA triggered the Exo III cleavage process, accompanied by the releasing of T-DNA and autonomous generation of new DNA fragment which was used for the successive hybridization with the another hairpin DNA (HP2) on the electrode. After the Exo III cleavage process, numerous quadruplex-forming oligomers which caged in HP2 were liberated on the electrode surface and folded into G-quadruplex-hemin complexes with the help of K + and hemin to give a remarkable electrochemical response. As a result, a low detection limit of 4.83fM with an excellent selectivity toward T-DNA was achieved. The developed electrochemical biosensor should be further extended for the detection of a wide spectrum of analytes and has great potential for the development of ultrasensitive biosensing platform for early diagnosis in gene-related diseases. Copyright © 2016 Elsevier B.V. All rights reserved.

Centrosomes are autocatalytic droplets of pericentriolar material organized by centrioles.

PubMed

Zwicker, David; Decker, Markus; Jaensch, Steffen; Hyman, Anthony A; Jülicher, Frank

2014-07-01

Centrosomes are highly dynamic, spherical organelles without a membrane. Their physical nature and their assembly are not understood. Using the concept of phase separation, we propose a theoretical description of centrosomes as liquid droplets. In our model, centrosome material occurs in a form soluble in the cytosol and a form that tends to undergo phase separation from the cytosol. We show that an autocatalytic chemical transition between these forms accounts for the temporal evolution observed in experiments. Interestingly, the nucleation of centrosomes can be controlled by an enzymatic activity of the centrioles, which are present at the core of all centrosomes. This nonequilibrium feature also allows for multiple stable centrosomes, a situation that is unstable in equilibrium phase separation. Our theory explains the growth dynamics of centrosomes for all cell sizes down to the eight-cell stage of the Caenorhabditis elegans embryo, and it also accounts for data acquired in experiments with aberrant numbers of centrosomes and altered cell volumes. Furthermore, the model can describe unequal centrosome sizes observed in cells with perturbed centrioles. We also propose an interpretation of the molecular details of the involved proteins in the case of C. elegans. Our example suggests a general picture of the organization of membraneless organelles.

Aqueous phase hydrodeoxygenation of polyols over Pd/WO3-ZrO2: Role of Pd-WO3 interaction and hydrodeoxygenation pathway

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

Liu, Changjun; Sun, Junming; Brown, Heather M.

Aqueous-phase hydrodeoxygenation of sugar and sugar-derived molecules can be used to produce a range of alkanes and oxygenates. In this paper, we have identified the reaction intermediates and reaction chemistry for the aqueous-phase hydrodeoxygenation of sorbitol over a bifunctional catalyst (Pt/SiO2–Al2O3) that contains both metal (Pt) and acid (SiO2–Al2O3) sites. A wide variety of reactions occur in this process including Csingle bondC bond cleavage, Csingle bondO bond cleavage, and hydrogenation reactions. The key Csingle bondC bond cleavage reactions include: retro-aldol condensation and decarbonylation, which both occur on metal catalytic sites. Dehydration is the key Csingle bondO bond cleavage reaction andmore » occurs on acid catalytic sites. Sorbitol initially undergoes dehydration and ring closure to produce cyclic C6 molecules or retro-aldol condensation reactions to produce primarily C3 polyols. Isosorbide is the major final product from sorbitol dehydration. Isosorbide then undergoes ring opening hydrogenation reactions and a dehydration/hydrogenation step to form 1,2,6-hexanetriol. The hexanetriol is then converted into hexanol and hexane by dehydration/hydrogenation. Smaller oxygenates are produced by Csingle bondC bond cleavage. These smaller oxygenates undergo dehydration/hydrogenation reactions to produce alkanes from C1–C5. The results from this paper suggest that hydrodeoxygenation chemistry can be tuned to make a wide variety of products from biomass-derived oxygenates.« less

Genetic and functional characterization of PCSK1.

PubMed

Choquet, Hélène; Stijnen, Pieter; Creemers, John W M

2011-01-01

PC1/3 is a neuroendocrine-specific member of the mammalian subtilisin-like proprotein convertase family. This seven-member family is involved in the endoproteolytic cleavage of a large number of precursor proteins including prohormones, proneuropeptides, zymogens, and proreceptors. PC1/3 is synthesized as a zymogen, proPC1/3, and its propeptide is rapidly and autocatalytically cleaved in the endoplasmic reticulum. The mature protein is sorted and stored in dense-core secretory vesicles, together with its substrates. Compound-inactivating mutations in the PCSK1 gene, which encodes PC1/3, cause monogenic obesity. Furthermore, the contribution of two common nonsynonymous variants in PCSK1 to polygenic obesity risk has recently been established. Additional rare variants have been identified in non-consanguineous extremely obese Europeans but functional characterization has not yet been described. Sequencing efforts of larger cohorts of obese patients might reveal more variants conferring risk of obesity.

Aminoxyl (nitroxyl) radicals in the early decomposition of the nitramine RDX.

PubMed

Irikura, Karl K

2013-03-14

The explosive nitramine RDX (1,3,5-trinitrohexahydro-s-triazine) is thought to decompose largely by homolytic N-N bond cleavage, among other possible initiation reactions. Density-functional theory (DFT) calculations indicate that the resulting secondary aminyl (R2N·) radical can abstract an oxygen atom from NO2 or from a neighboring nitramine molecule, producing an aminoxyl (R2NO·) radical. Persistent aminoxyl radicals have been detected in electron-spin resonance (ESR) experiments and are consistent with autocatalytic "red oils" reported in the experimental literature. When the O-atom donor is a nitramine, a nitrosamine is formed along with the aminoxyl radical. Reactions of aminoxyl radicals can lead readily to the "oxy-s-triazine" product (as the s-triazine N-oxide) observed mass-spectrometrically by Behrens and co-workers. In addition to forming aminoxyl radicals, the initial aminyl radical can catalyze loss of HONO from RDX.

Molecular mechanism of VDE-initiated intein homing in yeast nuclear genome.

PubMed

Fukuda, Tomoyuki; Nagai, Yuri; Ohya, Yoshikazu

2004-01-01

In Saccharomyces cerevisiae, VMA1 intein encodes a homing endonuclease termed VDE which is produced by an autocatalytic protein splicing reaction. VDE introduces a DSB at its recognition sequence on intein-minus allele, resulting in the lateral transfer of VMA1 intein. In this review, we summarize a decade of in vitro study on VDE and describe our recent study on the in vivo behavior of both VDE and host proteins involved in intein mobility. Meiotic DSBs caused by VDE are repaired in the similar pathway to that working in meiotic recombination induced by Spo11p-mediated DSBs. Meiosis-specific DNA cleavage and homing is shown to be guaranteed by the two distinct mechanisms, the subcellular localization of VDE and a requirement of premeiotic DNA replication. Based on these lines of evidence, we present the whole picture of molecular mechanism of VDE-initiated homing in yeast cells.

Molecular mechanism of vde-initiated intein homing in yeast nuclear genome.

PubMed

Fukuda, Tomoyuki; Nagai, Yuri; Ohya, Yoshikazu

2004-01-01

In Saccharomyces cerevisiae, VMAI intein encodes a homing endonuclease termed VDE which is produced by an autocatalytic protein splicing reaction. VDE introduces a DSB at its recognition sequence on intein-minus allele, resulting in the lateral transfer of VMAI intein. In this review, we summarize a decade of in vitro study on VDE and describe our recent study on the in vivo behavior of both VDE and host proteins involved in intein mobility. Meiotic DSBs caused by VDE are repaired in the similar pathway to that working in meiotic recombination induced by Spollp-mediated DSBs. Meiosis-specific DNA cleavage and homing is shown to be guaranteed by the two distinct mechanisms, the subcellular localization of VDE and a requirement of premeiotic DNA replication. Based on these lines of evidence, we present the whole picture of molecular mechanism of VDEinitiated homing in yeast cells.

The identification of disulfides in ricin D using proteolytic cleavage followed by negative-ion nano-electrospray ionization mass spectrometry of the peptide fragments.

PubMed

Tran, T T Nha; Brinkworth, Craig S; Bowie, John H

2015-01-30

To use negative-ion nano-electrospray ionization mass spectrometry of peptides from the tryptic digest of ricin D, to provide sequence information; in particular, to identify disulfide position and connectivity. Negative-ion fragmentations of peptides from the tryptic digest of ricin D was studied using a Waters QTOF2 mass spectrometer operating in MS and MS(2) modes. Twenty-three peptides were obtained following high-performance liquid chromatography and studied by negative-ion mass spectrometry covering 73% of the amino-acid residues of ricin D. Five disulfide-containing peptides were identified, three intermolecular and two intramolecular disulfide-containing peptides. The [M-H](-) anions of the intermolecular disulfides undergo facile cleavage of the disulfide units to produce fragment peptides. In negative-ion collision-induced dissociation (CID) these source-formed anions undergo backbone cleavages, which provide sequencing information. The two intramolecular disulfides were converted proteolytically into intermolecular disulfides, which were identified as outlined above. The positions of the five disulfide groups in ricin D may be determined by characteristic negative-ion cleavage of the disulfide groups, while sequence information may be determined using the standard negative-ion backbone cleavages of the resulting cleaved peptides. Negative-ion mass spectrometry can also be used to provide partial sequencing information for other peptides (i.e. those not containing Cys) using the standard negative-ion backbone cleavages of these peptides. Copyright © 2014 John Wiley & Sons, Ltd.

Granule swelling and cleavage of mitogen-activated protein kinases in human neutrophils undergoing apoptosis

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

Kato, Takayuki, E-mail: tkato@med.osaka-cu.ac.jp; Ikemoto, Masaru; Hato, Fumihiko

2009-04-10

Extracellular signal-regulated kinase and p38 have been shown to be cleaved in human neutrophils undergoing apoptosis induced by tumor necrosis factor-{alpha} and cycloheximide. However, the cleavage products of these molecules were undetected when apoptotic neutrophils were pretreated with phenylmethylsulfonyl fluoride or disrupted by nitrogen cavitation before preparation of cell lysates. The electron microscopy revealed that granules in apoptotic neutrophils were significantly swollen than those in control cells. These findings suggest that granule membrane may become destabilized during neutrophil apoptosis, leading to rapid proteolysis of these molecules by granule-derived serine proteases during preparation of cell lysates with the conventional lysis buffer.

The mechanism by which a propeptide-encoded pH sensor regulates spatiotemporal activation of furin.

PubMed

Williamson, Danielle M; Elferich, Johannes; Ramakrishnan, Parvathy; Thomas, Gary; Shinde, Ujwal

2013-06-28

The proprotein convertase furin requires the pH gradient of the secretory pathway to regulate its multistep, compartment-specific autocatalytic activation. Although His-69 within the furin prodomain serves as the pH sensor that detects transport of the propeptide-enzyme complex to the trans-Golgi network, where it promotes cleavage and release of the inhibitory propeptide, a mechanistic understanding of how His-69 protonation mediates furin activation remains unclear. Here we employ biophysical, biochemical, and computational approaches to elucidate the mechanism underlying the pH-dependent activation of furin. Structural analyses and binding experiments comparing the wild-type furin propeptide with a nonprotonatable His-69 → Leu mutant that blocks furin activation in vivo revealed protonation of His-69 reduces both the thermodynamic stability of the propeptide as well as its affinity for furin at pH 6.0. Structural modeling combined with mathematical modeling and molecular dynamic simulations suggested that His-69 does not directly contribute to the propeptide-enzyme interface but, rather, triggers movement of a loop region in the propeptide that modulates access to the cleavage site and, thus, allows for the tight pH regulation of furin activation. Our work establishes a mechanism by which His-69 functions as a pH sensor that regulates compartment-specific furin activation and provides insights into how other convertases and proteases may regulate their precise spatiotemporal activation.

The Mechanism by Which a Propeptide-encoded pH Sensor Regulates Spatiotemporal Activation of Furin*

PubMed Central

Williamson, Danielle M.; Elferich, Johannes; Ramakrishnan, Parvathy; Thomas, Gary; Shinde, Ujwal

2013-01-01

The proprotein convertase furin requires the pH gradient of the secretory pathway to regulate its multistep, compartment-specific autocatalytic activation. Although His-69 within the furin prodomain serves as the pH sensor that detects transport of the propeptide-enzyme complex to the trans-Golgi network, where it promotes cleavage and release of the inhibitory propeptide, a mechanistic understanding of how His-69 protonation mediates furin activation remains unclear. Here we employ biophysical, biochemical, and computational approaches to elucidate the mechanism underlying the pH-dependent activation of furin. Structural analyses and binding experiments comparing the wild-type furin propeptide with a nonprotonatable His-69 → Leu mutant that blocks furin activation in vivo revealed protonation of His-69 reduces both the thermodynamic stability of the propeptide as well as its affinity for furin at pH 6.0. Structural modeling combined with mathematical modeling and molecular dynamic simulations suggested that His-69 does not directly contribute to the propeptide-enzyme interface but, rather, triggers movement of a loop region in the propeptide that modulates access to the cleavage site and, thus, allows for the tight pH regulation of furin activation. Our work establishes a mechanism by which His-69 functions as a pH sensor that regulates compartment-specific furin activation and provides insights into how other convertases and proteases may regulate their precise spatiotemporal activation. PMID:23653353

Centrosomes are autocatalytic droplets of pericentriolar material organized by centrioles

PubMed Central

Zwicker, David; Decker, Markus; Jaensch, Steffen; Hyman, Anthony A.; Jülicher, Frank

2014-01-01

Centrosomes are highly dynamic, spherical organelles without a membrane. Their physical nature and their assembly are not understood. Using the concept of phase separation, we propose a theoretical description of centrosomes as liquid droplets. In our model, centrosome material occurs in a form soluble in the cytosol and a form that tends to undergo phase separation from the cytosol. We show that an autocatalytic chemical transition between these forms accounts for the temporal evolution observed in experiments. Interestingly, the nucleation of centrosomes can be controlled by an enzymatic activity of the centrioles, which are present at the core of all centrosomes. This nonequilibrium feature also allows for multiple stable centrosomes, a situation that is unstable in equilibrium phase separation. Our theory explains the growth dynamics of centrosomes for all cell sizes down to the eight-cell stage of the Caenorhabditis elegans embryo, and it also accounts for data acquired in experiments with aberrant numbers of centrosomes and altered cell volumes. Furthermore, the model can describe unequal centrosome sizes observed in cells with perturbed centrioles. We also propose an interpretation of the molecular details of the involved proteins in the case of C. elegans. Our example suggests a general picture of the organization of membraneless organelles. PMID:24979791

Biochemical properties of the matrix metalloproteinase NtMMP1 from Nicotiana tabacum cv. BY-2 suspension cells.

PubMed

Mandal, Manoj K; Fischer, Rainer; Schillberg, Stefan; Schiermeyer, Andreas

2010-09-01

A zinc-dependent matrix metalloproteinase (NtMMP1) found in the plasma membrane of Nicotiana tabacum cv. Bright Yellow 2 (BY-2) suspension cells is thought to be responsible for the degradation of recombinant proteins secreted into the culture supernatant. We have characterized the proteolytic activity of NtMMP1 by expressing a recombinant derivative lacking the C-terminal transmembrane domain in yeast. After purifying the protein by affinity chromatography, its autocatalytic activity was analyzed using monoclonal antibodies raised against its N-terminal and C-terminal portions. Both the unprocessed and processed forms of NtMMP1 displayed caseinolytic activity and N-terminal sequencing identified an autocatalytic cleavage site within the sequence motif HFSFFP, which is similar to the corresponding sequences of the human matrix metalloproteinases stromelysin-1 (MMP-3) and stromelysin-2 (MMP-10). Unlike all other matrix metalloproteinases investigated so far, NtMMP1 contains a disulfide bond within its propeptide thus rendering the proenzyme catalytically active. Kinetic analysis of NtMMP1 with a synthetic substrate revealed a K(m) of 10.55 +/- 0.9 microM, a k(cat) of 0.6 +/- 0.01 s(-1) and maximum activity at pH 7.5. We found that NtMMP1 degrades Desmodus rotundus salivary plasminogen activator alpha 1 (DSPAalpha1), a biopharmaceutical protein, that has proven difficult to produce in tobacco BY-2 cells. This provides a likely explanation for the frequent instability of secreted recombinant biopharmaceuticals produced in plant suspension cell cultures. Our data suggest new avenues that can be explored to improve the production of pharmaceutical proteins in plants and plant cells.

Tailoring the degradation rates of thermally responsive hydrogels designed for soft tissue injection by varying the autocatalytic potential.

PubMed

Zhu, Yang; Jiang, Hongbin; Ye, Sang-Ho; Yoshizumi, Tomo; Wagner, William R

2015-01-01

The ability to modulate the degradation properties of biomaterials such as thermally responsive hydrogels is desirable when exploring new therapeutic strategies that rely on the temporary presence of a placed scaffold or gel. Here we report a method of manipulating the absorption rate of a poly(N-isopropylacrylamide) ((poly(NIPAAm)) based hydrogel across a wide range (from 1 d to 5 mo) by small alterations in the composition. Relying upon the autocatalytic effect, the degradation of poly(NIPAAm-co-HEMA-co-MAPLA), (HEMA = 2-hydroxyethyl methacrylate; MAPLA = methacrylate-polylactide) was greatly accelerated by adding a fourth monomer methacrylic acid (MAA) at no more than 2 mol% to obtain poly(NIPAAm-co-HEMA-co-MAPLA-co-MAA) (pNHMMj) where j reflects the MAA molar % in the reactant mixture. MAA residue introduction decreased the pH inside the hydrogels and in surrounding buffered solutions. Accelerated degradation positively correlated with MAA content in pNHMMj polymers, putatively by the accelerated cleavage of MAPLA residues to raise the transition temperature of the polymer above body temperature. Physical properties including thermal transition behavior and initial mechanical strength did not vary significantly with MAA content. A rat hindlimb injection model generally reflected the in vitro observation that higher MAA content resulted in more rapid degradation and cellular infiltration. The strategy of tuning the degradation of thermally responsive hydrogels where degradation or solubilization is determined by their polyester components might be applied to other tissue engineering and regenerative medicine applications where designed biomaterial degradation behavior is needed. Copyright © 2015 Elsevier Ltd. All rights reserved.

A Structural and Functional Comparison Between Infectious and Non-Infectious Autocatalytic Recombinant PrP Conformers

PubMed Central

Noble, Geoffrey P.; Wang, Daphne W.; Walsh, Daniel J.; Barone, Justin R.; Miller, Michael B.; Nishina, Koren A.; Li, Sheng; Supattapone, Surachai

2015-01-01

Infectious prions contain a self-propagating, misfolded conformer of the prion protein termed PrPSc. A critical prediction of the protein-only hypothesis is that autocatalytic PrPSc molecules should be infectious. However, some autocatalytic recombinant PrPSc molecules have low or undetectable levels of specific infectivity in bioassays, and the essential determinants of recombinant prion infectivity remain obscure. To identify structural and functional features specifically associated with infectivity, we compared the properties of two autocatalytic recombinant PrP conformers derived from the same original template, which differ by >105-fold in specific infectivity for wild-type mice. Structurally, hydrogen/deuterium exchange mass spectrometry (DXMS) studies revealed that solvent accessibility profiles of infectious and non-infectious autocatalytic recombinant PrP conformers are remarkably similar throughout their protease-resistant cores, except for two domains encompassing residues 91-115 and 144-163. Raman spectroscopy and immunoprecipitation studies confirm that these domains adopt distinct conformations within infectious versus non-infectious autocatalytic recombinant PrP conformers. Functionally, in vitro prion propagation experiments show that the non-infectious conformer is unable to seed mouse PrPC substrates containing a glycosylphosphatidylinositol (GPI) anchor, including native PrPC. Taken together, these results indicate that having a conformation that can be specifically adopted by post-translationally modified PrPC molecules is an essential determinant of biological infectivity for recombinant prions, and suggest that this ability is associated with discrete features of PrPSc structure. PMID:26125623

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.

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.

Mechanism and Regulation of DNA-Protein Crosslink Repair by the DNA-Dependent Metalloprotease SPRTN

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

Stingele, Julian; Bellelli, Roberto; Alte, Ferdinand

Covalent DNA-protein crosslinks (DPCs) are toxic DNA lesions that interfere with essential chromatin transactions, such as replication and transcription. Little was known about DPC-specific repair mechanisms until the recent identification of a DPC-processing protease in yeast. The existence of a DPC protease in higher eukaryotes is inferred from data in Xenopus laevis egg extracts, but its identity remains elusive. Here we identify the metalloprotease SPRTN as the DPC protease acting in metazoans. Loss of SPRTN results in failure to repair DPCs and hypersensitivity to DPC-inducing agents. SPRTN accomplishes DPC processing through a unique DNA-induced protease activity, which is controlled bymore » several sophisticated regulatory mechanisms. Cellular, biochemical, and structural studies define a DNA switch triggering its protease activity, a ubiquitin switch controlling SPRTN chromatin accessibility, and regulatory autocatalytic cleavage. Our data also provide a molecular explanation on how SPRTN deficiency causes the premature aging and cancer predisposition disorder Ruijs-Aalfs syndrome.« less

Mechanism and Regulation of DNA-Protein Crosslink Repair by the DNA-Dependent Metalloprotease SPRTN

DOE PAGES

Stingele, Julian; Bellelli, Roberto; Alte, Ferdinand; ...

2016-10-27

Covalent DNA-protein crosslinks (DPCs) are toxic DNA lesions that interfere with essential chromatin transactions, such as replication and transcription. Little was known about DPC-specific repair mechanisms until the recent identification of a DPC-processing protease in yeast. The existence of a DPC protease in higher eukaryotes is inferred from data in Xenopus laevis egg extracts, but its identity remains elusive. Here we identify the metalloprotease SPRTN as the DPC protease acting in metazoans. Loss of SPRTN results in failure to repair DPCs and hypersensitivity to DPC-inducing agents. SPRTN accomplishes DPC processing through a unique DNA-induced protease activity, which is controlled bymore » several sophisticated regulatory mechanisms. Cellular, biochemical, and structural studies define a DNA switch triggering its protease activity, a ubiquitin switch controlling SPRTN chromatin accessibility, and regulatory autocatalytic cleavage. Our data also provide a molecular explanation on how SPRTN deficiency causes the premature aging and cancer predisposition disorder Ruijs-Aalfs syndrome.« less

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.

Phenylselenolate Mercury Alkyl Compounds, PhSeHgMe and PhSeHgEt: Molecular Structures, Protolytic Hg–C Bond Cleavage and Phenylselenolate Exchange‡

PubMed Central

Yurkerwich, Kevin; Quinlivan, Patrick J.; Rong, Yi

2015-01-01

The phenylselenolate mercury alkyl compounds, PhSeHgMe and PhSeHgEt, have been structurally characterized by X-ray diffraction, thereby demonstrating that both compounds are monomeric with approximately linear coordination geometries; the mercury centers do, nevertheless, exhibit secondary Hg•••Se intermolecular interactions that serve to increase the coordination number in the solid state. The ethyl derivative, PhSeHgEt, undergoes facile protolytic cleavage of the Hg–C bond to release ethane at room temperature, whereas PhSeHgMe exhibits little reactivity under similar conditions. Interestingly, the cleavage of the Hg–C bond of PhSeHgEt is also more facile than that of the thiolate analogue, PhSHgEt, which demonstrates that coordination by selenium promotes protolytic cleavage of the mercury-carbon bond. The phenylselenolate compounds PhSeHgR (R = Me, Et) also undergo degenerate exchange reactions with, for example, PhSHgR and RHgCl. In each case, the alkyl groups preserve coupling to the 199Hg nuclei, thereby indicating that the exchange process involves metathesis of the Hg–SePh/Hg–X groups rather than metathesis of the Hg–R/Hg–R groups. PMID:26644634

Phenylselenolate Mercury Alkyl Compounds, PhSeHgMe and PhSeHgEt: Molecular Structures, Protolytic Hg-C Bond Cleavage and Phenylselenolate Exchange.

PubMed

Yurkerwich, Kevin; Quinlivan, Patrick J; Rong, Yi; Parkin, Gerard

2016-01-08

The phenylselenolate mercury alkyl compounds, PhSeHgMe and PhSeHgEt, have been structurally characterized by X-ray diffraction, thereby demonstrating that both compounds are monomeric with approximately linear coordination geometries; the mercury centers do, nevertheless, exhibit secondary Hg•••Se intermolecular interactions that serve to increase the coordination number in the solid state. The ethyl derivative, PhSeHgEt, undergoes facile protolytic cleavage of the Hg-C bond to release ethane at room temperature, whereas PhSeHgMe exhibits little reactivity under similar conditions. Interestingly, the cleavage of the Hg-C bond of PhSeHgEt is also more facile than that of the thiolate analogue, PhSHgEt, which demonstrates that coordination by selenium promotes protolytic cleavage of the mercury-carbon bond. The phenylselenolate compounds PhSeHgR (R = Me, Et) also undergo degenerate exchange reactions with, for example, PhSHgR and RHgCl. In each case, the alkyl groups preserve coupling to the 199 Hg nuclei, thereby indicating that the exchange process involves metathesis of the Hg-SePh/Hg-X groups rather than metathesis of the Hg-R/Hg-R groups.

Solid-state stability studies of 13-cis-retinoic acid and all-trans-retinoic acid using microcalorimetry and HPLC analysis.

PubMed

Tan, X; Meltzer, N; Lindebaum, S

1992-09-01

The solid-state stabilities of 13-cis-retinoic acid and all-trans-retinoic acid in the presence and absence of oxygen were investigated. The samples were first evaluated using microcalorimetry. The rate laws of different samples under different conditions were deduced from the shapes of the heat flow curves, and the activation energies of the reactions were determined from Arrhenius plots. Under an air atmosphere, the decomposition of 13-cis-retinoic acid is an autocatalytic reaction, while all-trans-retinoic acid undergoes a zero-order process. The degradation of the two compounds at a selected elevated temperature was also determined utilizing HPLC analysis. This technique confirmed the decomposition kinetics. Hence, their half-lives and shelf lives at room temperature could be calculated. Under a nitrogen atmosphere, the microcalorimetric experiment showed a first-order phenomenon for both samples, but HPLC analysis showed no degradation, suggesting that the two samples, in the absence of oxygen, undergo only a physical change.

Verification of RDX Photolysis Mechanism

DTIC Science & Technology

1999-11-01

which re-addition of HN02 was proposed to yield a hydroxydiazo intermediate that then decomposed to an alcohol . This sequence is shown for...various organic products such as alcohols , or undergo carbon- nitrogen (C-N) bond cleavage (Noller 1965). This reaction is sufficiently quanti...carbon-centered functional group such as the alcohol shown below, or C-N bond cleavage. 42 CERL TR 99/93 N02 N02 No2 ^Nv. N ’ ( ^| H2

Who or What? Self-Replication and Function-Reproduction in the Origin of Life

NASA Technical Reports Server (NTRS)

New, Michael H.; Stassinopoulos, Dimitris; Monaco, Regina; Pohorille, Andrew; DeVincenzi, Donald (Technical Monitor)

2002-01-01

In this presentation, we will present results on the fundamental properties of two classes of replicating systems: autocatalytic replicators that reproduce exact copies of a template molecule, and function reproducers that maintain a set of essential functions without replicating the identities of the functional moieties. We will describe the stability and behavior in-the-large of autocatalytic replicators. Most importantly, we have found no sharp distinction between an autocatalytic and a non-autocatalytic domain. We will also present a new derivation of von Kiedrowski's square-root rate law. Function - reproducers are proposed as an important component of protocells and we will present theoretical results on a simple model system that incorporates known peptide biophysics. For a wide range of parameters, we have shown that this type of system can improve its overall performance, even in the absence of any method for information storage. This type of system improvement is defined to be non-genomic evolution.

Apoptotic cells subjected to cold/warming exposure disorganize apoptotic microtubule network and undergo secondary necrosis.

PubMed

Oropesa-Ávila, Manuel; Fernández-Vega, Alejandro; de la Mata, Mario; Garrido-Maraver, Juan; Cotán, David; Paz, Marina Villanueva; Pavón, Ana Delgado; Cordero, Mario D; Alcocer-Gómez, Elizabet; de Lavera, Isabel; Lema, Rafael; Zaderenko, Ana Paula; Sánchez-Alcázar, José A

2014-09-01

Apoptotic microtubule network (AMN) is organized during apoptosis, forming a cortical structure beneath the plasma membrane which plays a critical role in preserving cell morphology and plasma membrane integrity. The aim of this study was to examine the effect of cold/warming exposure on apoptotic microtubules and plasma membrane integrity during the execution phase of apoptosis. We demonstrated in camptothecin-induced apoptotic H460 cells that cold/warming exposure disorganized apoptotic microtubules and allowed the access of active caspases to the cellular cortex and the cleavage of essential proteins in the preservation of plasma membrane permeability. Cleavage of cellular cortex and plasma membrane proteins, such as α-spectrin, paxilin, focal adhesion kinase and calcium ATPase pump (PMCA-4) involved in cell calcium extrusion resulted in increased plasma permeability and calcium overload leading apoptotic cells to secondary necrosis. The essential role of caspase-mediated cleavage in this process was demonstrated because the addition of the pan-caspase inhibitor z-VAD during cold/warming exposure that induces AMN depolymerization avoided the cleavage of cortical and plasma membrane proteins and prevented apoptotic cells to undergo secondary necrosis. Likewise, apoptotic microtubules stabilization by taxol during cold/warming exposure also prevented cellular cortex and plasma membrane protein cleavage and secondary necrosis. Furthermore, microtubules stabilization or caspase inhibition during cold/warming exposure was also critical for proper phosphatidylserine externalization and apoptotic cell clearance by macrophages. These results indicate that cold/warming exposure of apoptotic cells induces secondary necrosis which can be prevented by both, microtubule stabilization or caspase inhibition.

Characterization of Bleomycin-Mediated Cleavage of a Hairpin DNA Library

PubMed Central

Segerman, Zachary J.; Roy, Basab; Hecht, Sidney M.

2013-01-01

A study of BLM A5 was conducted using a previously isolated library of hairpin DNAs found to bind strongly to metal free BLM. The ability of Fe(II)•BLM to effect cleavage on both the 3' and 5'-arms of the hairpin DNAs was characterized. The strongly bound DNAs were found to be efficient substrates for Fe•BLM A5-mediated hairpin DNA cleavage. Surprisingly, the most prevalent site of BLM-mediated cleavage was found to be the 5′-AT-3′ dinucleotide sequence. This dinucleotide sequence, and other sequences generally not cleaved well by BLM when examined using arbitrarily chosen DNA substrates, were apparent when examining the library of ten hairpin DNAs. In total, 132 sites of DNA cleavage were produced by exposure of the hairpin DNA library to Fe•BLM A5. The existence of multiple sites of cleavage on both the 3′- and 5′-arms of the hairpin DNAs suggested that some of these might be double-strand cleavage events. Accordingly, an assay was developed with which to test the propensity of the hairpin DNAs to undergo double-strand DNA damage. One hairpin DNA was characterized using this method, and gave results consistent with earlier reports of double-strand DNA cleavage, but with a sequence selectivity different from those reported previously. PMID:23834496

C-N bond cleavage of anilines by a (salen)ruthenium(VI) nitrido complex.

PubMed

Man, Wai-Lun; Xie, Jianhui; Pan, Yi; Lam, William W Y; Kwong, Hoi-Ki; Ip, Kwok-Wa; Yiu, Shek-Man; Lau, Kai-Chung; Lau, Tai-Chu

2013-04-17

We report experimental and computational studies of the facile oxidative C-N bond cleavage of anilines by a (salen)ruthenium(VI) nitrido complex. We provide evidence that the initial step involves nucleophilic attack of aniline at the nitrido ligand of the ruthenium complex, which is followed by proton and electron transfer to afford a (salen)ruthenium(II) diazonium intermediate. This intermediate then undergoes unimolecular decomposition to generate benzene and N2.

Men's body mass index in relation to embryo quality and clinical outcomes in couples undergoing in vitro fertilization.

PubMed

Colaci, Daniela S; Afeiche, Myriam; Gaskins, Audrey J; Wright, Diane L; Toth, Thomas L; Tanrikut, Cigdem; Hauser, Russ; Chavarro, Jorge E

2012-11-01

To evaluate the association between men's body mass index (BMI), early embryo quality, and clinical outcomes in couples undergoing in vitro fertilization (IVF). Prospective cohort study. Fertility clinic in an academic medical center. 114 couples who underwent 172 assisted reproduction cycles. None. Fertilization rate, embryo quality, implantation rate, clinical pregnancy rate, and live birth rate. The fertilization rate was higher among obese men than among normal weight men in conventional IVF cycles. No statistically significant associations were found between men's BMI and the proportion of poor-quality embryos on day 3, slow embryo cleavage rate, or accelerated embryo cleavage rate. Men's BMI was unrelated to positive β-human chorionic gonadotropin rate, clinical pregnancy rate, or live-birth rate per embryo transfer. Among couples undergoing intracytoplasmic sperm injection, the odds of live birth in couples with obese male partners was 84% lower than the odds in couples with men with normal BMI. Our data suggest a possible deleterious effect of male obesity on the odds of having a live birth among couples undergoing intracytoplasmic sperm injection. Copyright © 2012 American Society for Reproductive Medicine. Published by Elsevier Inc. All rights reserved.

Production of congopain, the major cysteine protease of Trypanosoma (Nannomonas) congolense, in Pichia pastoris reveals unexpected dimerisation at physiological pH.

PubMed

Boulangé, Alain F; Khamadi, Samoel A; Pillay, Davita; Coetzer, Theresa H T; Authié, Edith

2011-01-01

African animal trypanosomosis (nagana) is arguably the most important parasitic disease affecting livestock in sub-Saharan Africa. Since none of the existing control measures are entirely satisfactory, vaccine development is being actively pursued. However, due to antigenic variation, the quest for a conventional vaccine has proven elusive. As a result, we have sought an alternative 'anti-disease vaccine approach', based on congopain, a cysteine protease of Trypanosoma congolense, which was shown to have pathogenic effects in vivo. Congopain was initially expressed as a recombinant protein in bacterial and baculovirus expression systems, but both the folding and yield obtained proved inadequate. Hence alternative expression systems were investigated, amongst which Pichia pastoris proved to be the most suitable. We report here the expression of full length, and C-terminal domain-truncated congopain in the methylotrophic yeast P. pastoris. Differences in yield were observed between full length and truncated proteins, the full length producing 2-4 mg of protein per litre of culture, while the truncated form produced 20-30 mg/l. The protease was produced as a proenzyme, but underwent spontaneous activation when acidified (pH <5). To investigate whether this activation was due to autolysis, we produced an inactive mutant (active site Cys→Ala) by site-directed mutagenesis. The mutant form was produced at a much higher rate, up to 100mg/l culture, as a proenzyme. It did not undergo spontaneous cleavage of the propeptide when subjected to acidic pH suggesting an autocatalytic process of activation for congopain. These recombinant proteins displayed a very unusual feature for cathepsin L-like proteinases, i.e. complete dimerisation at pH >6, and by reversibly monomerising at acidic pH <5. This attribute is of utmost importance in the context of an anti-disease vaccine, given that the epitopes recognised by the sera of trypanosome-infected trypanotolerant cattle appear dimer-specific. Copyright © 2010 Elsevier Inc. All rights reserved.

Autocatalytic formation of an iron(IV)-oxo complex via scandium ion-promoted radical chain autoxidation of an iron(II) complex with dioxygen and tetraphenylborate.

PubMed

Nishida, Yusuke; Lee, Yong-Min; Nam, Wonwoo; Fukuzumi, Shunichi

2014-06-04

A non-heme iron(IV)-oxo complex, [(TMC)Fe(IV)(O)](2+) (TMC = 1,4,8,11-tetramethyl-1,4,8,11-tetraazacyclotetradecane), was formed by oxidation of an iron(II) complex ([(TMC)Fe(II)](2+)) with dioxygen (O2) and tetraphenylborate (BPh4(-)) in the presence of scandium triflate (Sc(OTf)3) in acetonitrile at 298 K via autocatalytic radical chain reactions rather than by a direct O2 activation pathway. The autocatalytic radical chain reaction is initiated by scandium ion-promoted electron transfer from BPh4(-) to [(TMC)Fe(IV)(O)](2+) to produce phenyl radical (Ph(•)). The chain propagation step is composed of the addition of O2 to Ph(•) and the reduction of the resulting phenylperoxyl radical (PhOO(•)) by scandium ion-promoted electron transfer from BPh4(-) to PhOO(•) to produce phenyl hydroperoxide (PhOOH), accompanied by regeneration of phenyl radical. PhOOH reacts with [(TMC)Fe(II)](2+) to yield phenol (PhOH) and [(TMC)Fe(IV)(O)](2+). Biphenyl (Ph-Ph) was formed via the radical chain autoxidation of BPh3 by O2. The induction period of the autocatalytic radical chain reactions was shortened by addition of a catalytic amount of [(TMC)Fe(IV)(O)](2+), whereas addition of a catalytic amount of ferrocene that can reduce [(TMC)Fe(IV)(O)](2+) resulted in elongation of the induction period. Radical chain autoxidation of BPh4(-) by O2 also occurred in the presence of Sc(OTf)3 without [(TMC)Fe(IV)(O)](2+), initiating the autocatalytic oxidation of [(TMC)Fe(II)](2+) with O2 and BPh4(-) to yield [(TMC)Fe(IV)(O)](2+). Thus, the general view for formation of non-heme iron(IV)-oxo complexes via O2-binding iron species (e.g., Fe(III)(O2(•-))) without contribution of autocatalytic radical chain reactions should be viewed with caution.

A plasmid-based reverse genetics system for influenza A virus.

PubMed Central

Pleschka, S; Jaskunas, R; Engelhardt, O G; Zürcher, T; Palese, P; García-Sastre, A

1996-01-01

A reverse genetics system for negative-strand RNA viruses was first successfully developed for influenza viruses. This technology involved the transfection of in vitro-reconstituted ribonucleoprotein (RNP) complexes into influenza virus-infected cells. We have now developed a method that allows intracellular reconstitution of RNP complexes from plasmid-based expression vectors. Expression of a viral RNA-like transcript is achieved from a plasmid containing a truncated human polymerase I (polI) promoter and a ribozyme sequence that generates the desired 3' end by autocatalytic cleavage. The polI-driven plasmid is cotransfected into human 293 cells with polII-responsive plasmids that express the viral PB1, PB2, PA, and NP proteins. This exclusively plasmid-driven system results in the efficient transcription and replication of the viral RNA-like reporter and allows the study of cis- and trans-acting signals involved in the transcription and replication of influenza virus RNAs. Using this system, we have also been able to rescue a synthetic neuraminidase gene into a recombinant influenza virus. This method represents a convenient alternative to the previously established RNP transfection system. PMID:8648766

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

Reaction fronts of the autocatalytic hydrogenase reaction

NASA Astrophysics Data System (ADS)

Gyevi-Nagy, László; Lantos, Emese; Gehér-Herczegh, Tünde; Tóth, Ágota; Bagyinka, Csaba; Horváth, Dezső

2018-04-01

We have built a model to describe the hydrogenase catalyzed, autocatalytic, reversible hydrogen oxidation reaction where one of the enzyme forms is the autocatalyst. The model not only reproduces the experimentally observed front properties, but also explains the found hydrogen ion dependence. Furthermore, by linear stability analysis, two different front types are found in good agreement with the experiments.

Dynamic graph of an oxy-fuel combustion system using autocatalytic set model

NASA Astrophysics Data System (ADS)

Harish, Noor Ainy; Bakar, Sumarni Abu

2017-08-01

Evaporation process is one of the main processes besides combustion process in an oxy-combustion boiler system. An Autocatalytic Set (ASC) Model has successfully applied in developing graphical representation of the chemical reactions that occurs in the evaporation process in the system. Seventeen variables identified in the process are represented as nodes and the catalytic relationships are represented as edges in the graph. In addition, in this paper graph dynamics of ACS is further investigated. By using Dynamic Autocatalytic Set Graph Algorithm (DAGA), the adjacency matrix for each of the graphs and its relations to Perron-Frobenius Theorem is investigated. The dynamic graph obtained is further investigated where the connection of the graph to fuzzy graph Type 1 is established.

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

The invariant cleavage pattern displayed by ascidian embryos depends on spindle positioning along the cell's longest axis in the apical plane and relies on asynchronous cell divisions

PubMed Central

Dumollard, Rémi; Minc, Nicolas; Salez, Gregory; Aicha, Sameh Ben; Bekkouche, Faisal; Hebras, Céline; Besnardeau, Lydia; McDougall, Alex

2017-01-01

The ascidian embryo is an ideal system to investigate how cell position is determined during embryogenesis. Using 3D timelapse imaging and computational methods we analyzed the planar cell divisions in ascidian early embryos and found that spindles in every cell tend to align at metaphase in the long length of the apical surface except in cells undergoing unequal cleavage. Furthermore, the invariant and conserved cleavage pattern of ascidian embryos was found to consist in alternate planar cell divisions between ectoderm and endomesoderm. In order to test the importance of alternate cell divisions we manipulated zygotic transcription induced by β-catenin or downregulated wee1 activity, both of which abolish this cell cycle asynchrony. Crucially, abolishing cell cycle asynchrony consistently disrupted the spindle orienting mechanism underpinning the invariant cleavage pattern. Our results demonstrate how an evolutionary conserved cell cycle asynchrony maintains the invariant cleavage pattern driving morphogenesis of the ascidian blastula. DOI: http://dx.doi.org/10.7554/eLife.19290.001 PMID:28121291

Directed Laplacians For Fuzzy Autocatalytic Set Of Fuzzy Graph Type-3 Of An Incineration Process

NASA Astrophysics Data System (ADS)

Ahmad, Tahir; Baharun, Sabariah; Bakar, Sumarni Abu

2010-11-01

Fuzzy Autocatalytic Set (FACS) of Fuzzy Graph Type-3 was used in the modeling of a clinical waste incineration process in Malacca. FACS provided more accurate explanations of the incineration process than using crisp graph. In this paper we explore further FACS. Directed and combinatorial Laplacian of FACS are developed and their basic properties are presented.

An autocatalytic network model for stock markets

NASA Astrophysics Data System (ADS)

Caetano, Marco Antonio Leonel; Yoneyama, Takashi

2015-02-01

The stock prices of companies with businesses that are closely related within a specific sector of economy might exhibit movement patterns and correlations in their dynamics. The idea in this work is to use the concept of autocatalytic network to model such correlations and patterns in the trends exhibited by the expected returns. The trends are expressed in terms of positive or negative returns within each fixed time interval. The time series derived from these trends is then used to represent the movement patterns by a probabilistic boolean network with transitions modeled as an autocatalytic network. The proposed method might be of value in short term forecasting and identification of dependencies. The method is illustrated with a case study based on four stocks of companies in the field of natural resource and technology.

Autocatalytic, bistable, oscillatory networks of biologically relevant organic reactions.

PubMed

Semenov, Sergey N; Kraft, Lewis J; Ainla, Alar; Zhao, Mengxia; Baghbanzadeh, Mostafa; Campbell, Victoria E; Kang, Kyungtae; Fox, Jerome M; Whitesides, George M

2016-09-29

Networks of organic chemical reactions are important in life and probably played a central part in its origin. Network dynamics regulate cell division, circadian rhythms, nerve impulses and chemotaxis, and guide the development of organisms. Although out-of-equilibrium networks of chemical reactions have the potential to display emergent network dynamics such as spontaneous pattern formation, bistability and periodic oscillations, the principles that enable networks of organic reactions to develop complex behaviours are incompletely understood. Here we describe a network of biologically relevant organic reactions (amide formation, thiolate-thioester exchange, thiolate-disulfide interchange and conjugate addition) that displays bistability and oscillations in the concentrations of organic thiols and amides. Oscillations arise from the interaction between three subcomponents of the network: an autocatalytic cycle that generates thiols and amides from thioesters and dialkyl disulfides; a trigger that controls autocatalytic growth; and inhibitory processes that remove activating thiol species that are produced during the autocatalytic cycle. In contrast to previous studies that have demonstrated oscillations and bistability using highly evolved biomolecules (enzymes and DNA) or inorganic molecules of questionable biochemical relevance (for example, those used in Belousov-Zhabotinskii-type reactions), the organic molecules we use are relevant to metabolism and similar to those that might have existed on the early Earth. By using small organic molecules to build a network of organic reactions with autocatalytic, bistable and oscillatory behaviour, we identify principles that explain the ways in which dynamic networks relevant to life could have developed. Modifications of this network will clarify the influence of molecular structure on the dynamics of reaction networks, and may enable the design of biomimetic networks and of synthetic self-regulating and evolving chemical systems.

Autocatalytic, bistable, oscillatory networks of biologically relevant organic reactions

NASA Astrophysics Data System (ADS)

Semenov, Sergey N.; Kraft, Lewis J.; Ainla, Alar; Zhao, Mengxia; Baghbanzadeh, Mostafa; Campbell, Victoria E.; Kang, Kyungtae; Fox, Jerome M.; Whitesides, George M.

2016-09-01

Networks of organic chemical reactions are important in life and probably played a central part in its origin. Network dynamics regulate cell division, circadian rhythms, nerve impulses and chemotaxis, and guide the development of organisms. Although out-of-equilibrium networks of chemical reactions have the potential to display emergent network dynamics such as spontaneous pattern formation, bistability and periodic oscillations, the principles that enable networks of organic reactions to develop complex behaviours are incompletely understood. Here we describe a network of biologically relevant organic reactions (amide formation, thiolate-thioester exchange, thiolate-disulfide interchange and conjugate addition) that displays bistability and oscillations in the concentrations of organic thiols and amides. Oscillations arise from the interaction between three subcomponents of the network: an autocatalytic cycle that generates thiols and amides from thioesters and dialkyl disulfides; a trigger that controls autocatalytic growth; and inhibitory processes that remove activating thiol species that are produced during the autocatalytic cycle. In contrast to previous studies that have demonstrated oscillations and bistability using highly evolved biomolecules (enzymes and DNA) or inorganic molecules of questionable biochemical relevance (for example, those used in Belousov-Zhabotinskii-type reactions), the organic molecules we use are relevant to metabolism and similar to those that might have existed on the early Earth. By using small organic molecules to build a network of organic reactions with autocatalytic, bistable and oscillatory behaviour, we identify principles that explain the ways in which dynamic networks relevant to life could have developed. Modifications of this network will clarify the influence of molecular structure on the dynamics of reaction networks, and may enable the design of biomimetic networks and of synthetic self-regulating and evolving chemical systems.

New mechanism for autocatalytic decomposition of H2CO3 in the vapor phase.

PubMed

Ghoshal, Sourav; Hazra, Montu K

2014-04-03

In this article, we present high level ab initio calculations investigating the energetics of a new autocatalytic decomposition mechanism for carbonic acid (H2CO3) in the vapor phase. The calculation have been performed at the MP2 level of theory in conjunction with aug-cc-pVDZ, aug-cc-pVTZ, and 6-311++G(3df,3pd) basis sets as well as at the CCSD(T)/aug-cc-pVTZ level. The present study suggests that this new decomposition mechanism is effectively a near-barrierless process at room temperature and makes vapor phase of H2CO3 unstable even in the absence of water molecules. Our calculation at the MP2/aug-cc-pVTZ level predicts that the effective barrier, defined as the difference between the zero-point vibrational energy (ZPE) corrected energy of the transition state and the total energy of the isolated starting reactants in terms of bimolecular encounters, is nearly zero for the autocatalytic decomposition mechanism. The results at the CCSD(T)/aug-cc-pVTZ level of calculations suggest that the effective barrier, as defined above, is sensitive to some extent to the levels of calculations used, nevertheless, we find that the effective barrier height predicted at the CCSD(T)/aug-cc-pVTZ level is very small or in other words the autocatalytic decomposition mechanism presented in this work is a near-barrierless process as mentioned above. Thus, we suggest that this new autocatalytic decomposition mechanism has to be considered as the primary mechanism for the decomposition of carbonic acid, especially at its source, where the vapor phase concentration of H2CO3 molecules reaches its highest levels.

The Effects of Trivalent Lanthanide Cationization on the Electron Transfer Dissociation of Acidic Fibrinopeptide B and its Analogs

NASA Astrophysics Data System (ADS)

Commodore, Juliette J.; Cassady, Carolyn J.

2016-09-01

Electrospray ionization (ESI) on mixtures of acidic fibrinopeptide B and two peptide analogs with trivalent lanthanide salts generates [M + Met + H]4+, [M + Met]3+, and [M + Met -H]2+, where M = peptide and Met = metal (except radioactive promethium). These ions undergo extensive and highly efficient electron transfer dissociation (ETD) to form metallated and non-metallated c- and z-ions. All metal adducted product ions contain at least two acidic sites, which suggest attachment of the lanthanide cation at the side chains of one or more acidic residues. The three peptides undergo similar fragmentation. ETD on [M + Met + H]4+ leads to cleavage at every residue; the presence of both a metal ion and an extra proton is very effective in promoting sequence-informative fragmentation. Backbone dissociation of [M + Met]3+ is also extensive, although cleavage does not always occur between adjacent glutamic acid residues. For [M + Met - H ]2+, a more limited range of product ions form. All lanthanide metal peptide complexes display similar fragmentation except for europium (Eu). ETD on [M + Eu - H]2+ and [M + Eu]3+ yields a limited amount of peptide backbone cleavage; however, [M + Eu + H]4+ dissociates extensively with cleavage at every residue. With the exception of the results for Eu(III), metallated peptide ion formation by ESI, ETD fragmentation efficiencies, and product ion formation are unaffected by the identity of the lanthanide cation. Adduction with trivalent lanthanide metal ions is a promising tool for sequence analysis of acidic peptides by ETD.

Marginal states in a cubic autocatalytic reaction

NASA Astrophysics Data System (ADS)

Das, Debojyoti; Ghosh, Pushpita; Ray, Deb Shankar

2011-09-01

Marginal steady state belongs to a special class of states in nonlinear dynamics. To realize this state we consider a cubic autocatalytic reaction A + 2B → 3B in a continuous-stirred-tank-reactor, where the flow rate of the reactant A can be controlled to manipulate the dynamical behavior of the open system. We demonstrate that when the flow rate is weakly noisy the autocatalytic reaction admits of a steady state which is marginal in nature and is surrounded by infinite number of periodic trajectories. When the uncatalyzed reaction A → B is included in the reaction scheme, there exists a marginal steady state which is a critical state corresponding to the point of transition between the flow branch and the equilibrium branch, similar to gas-liquid critical point of transition. This state loses its stability in the weak noise limit.

Comparing the Caputo, Caputo-Fabrizio and Atangana-Baleanu derivative with fractional order: Fractional cubic isothermal auto-catalytic chemical system

NASA Astrophysics Data System (ADS)

Saad, K. M.

2018-03-01

In this work we extend the standard model for a cubic isothermal auto-catalytic chemical system (CIACS) to a new model of a fractional cubic isothermal auto-catalytic chemical system (FCIACS) based on Caputo (C), Caputo-Fabrizio (CF) and Atangana-Baleanu in the Liouville-Caputo sense (ABC) fractional time derivatives, respectively. We present approximate solutions for these extended models using the q -homotopy analysis transform method ( q -HATM). We solve the FCIACS with the C derivative and compare our results with those obtained using the CF and ABC derivatives. The ranges of convergence of the solutions are found and the optimal values of h , the auxiliary parameter, are derived. Finally, these solutions are compared with numerical solutions of the various models obtained using finite differences and excellent agreement is found.

Complex Autocatalysis in Simple Chemistries.

PubMed

Virgo, Nathaniel; Ikegami, Takashi; McGregor, Simon

2016-01-01

Life on Earth must originally have arisen from abiotic chemistry. Since the details of this chemistry are unknown, we wish to understand, in general, which types of chemistry can lead to complex, lifelike behavior. Here we show that even very simple chemistries in the thermodynamically reversible regime can self-organize to form complex autocatalytic cycles, with the catalytic effects emerging from the network structure. We demonstrate this with a very simple but thermodynamically reasonable artificial chemistry model. By suppressing the direct reaction from reactants to products, we obtain the simplest kind of autocatalytic cycle, resulting in exponential growth. When these simple first-order cycles are prevented from forming, the system achieves superexponential growth through more complex, higher-order autocatalytic cycles. This leads to nonlinear phenomena such as oscillations and bistability, the latter of which is of particular interest regarding the origins of life.

A Single Glycan at the 99-Loop of Human Kallikrein-related Peptidase 2 Regulates Activation and Enzymatic Activity*

PubMed Central

Guo, Shihui; Skala, Wolfgang; Magdolen, Viktor; Briza, Peter; Biniossek, Martin L.; Schilling, Oliver; Kellermann, Josef; Brandstetter, Hans; Goettig, Peter

2016-01-01

Human kallikrein-related peptidase 2 (KLK2) is a key serine protease in semen liquefaction and prostate cancer together with KLK3/prostate-specific antigen. In order to decipher the function of its potential N-glycosylation site, we produced pro-KLK2 in Leishmania tarentolae cells and compared it with its non-glycosylated counterpart from Escherichia coli expression. Mass spectrometry revealed that Asn-95 carries a core glycan, consisting of two GlcNAc and three hexoses. Autocatalytic activation was retarded in glyco-pro-KLK2, whereas the activated glyco-form exhibited an increased proteolytic resistance. The specificity patterns obtained by the PICS (proteomic identification of protease cleavage sites) method are similar for both KLK2 variants, with a major preference for P1-Arg. However, glycosylation changes the enzymatic activity of KLK2 in a drastically substrate-dependent manner. Although glyco-KLK2 has a considerably lower catalytic efficiency than glycan-free KLK2 toward peptidic substrates with P2-Phe, the situation was reverted toward protein substrates, such as glyco-pro-KLK2 itself. These findings can be rationalized by the glycan-carrying 99-loop that prefers to cover the active site like a lid. By contrast, the non-glycosylated 99-loop seems to favor a wide open conformation, which mostly increases the apparent affinity for the substrates (i.e. by a reduction of Km). Also, the cleavage pattern and kinetics in autolytic inactivation of both KLK2 variants can be explained by a shift of the target sites due to the presence of the glycan. These striking effects of glycosylation pave the way to a deeper understanding of kallikrein-related peptidase biology and pathology. PMID:26582203

Ribosomes are optimized for autocatalytic production

NASA Astrophysics Data System (ADS)

Reuveni, Shlomi; Ehrenberg, Måns; Paulsson, Johan

2017-07-01

Many fine-scale features of ribosomes have been explained in terms of function, revealing a molecular machine that is optimized for error-correction, speed and control. Here we demonstrate mathematically that many less well understood, larger-scale features of ribosomes—such as why a few ribosomal RNA molecules dominate the mass and why the ribosomal protein content is divided into 55-80 small, similarly sized segments—speed up their autocatalytic production.

Dissociation of recombinant prion autocatalysis from infectivity.

PubMed

Noble, Geoffrey P; Supattapone, Surachai

2015-01-01

Within the mammalian prion field, the existence of recombinant prion protein (PrP) conformers with self-replicating (ie. autocatalytic) activity in vitro but little to no infectious activity in vivo challenges a key prediction of the protein-only hypothesis of prion replication--that autocatalytic PrP conformers should be infectious. To understand this dissociation of autocatalysis from infectivity, we recently performed a structural and functional comparison between a highly infectious and non-infectious pair of autocatalytic recombinant PrP conformers derived from the same initial prion strain. (1) We identified restricted, C-terminal structural differences between these 2 conformers and provided evidence that these relatively subtle differences prevent the non-infectious conformer from templating the conversion of native PrP(C) substrates containing a glycosylphosphatidylinositol (GPI) anchor. (1) In this article we discuss a model, consistent with these findings, in which recombinant PrP, lacking post-translational modifications and associated folding constraints, is capable of adopting a wide variety of autocatalytic conformations. Only a subset of these recombinant conformers can be adopted by post-translationally modified native PrP(C), and this subset represents the recombinant conformers with high specific infectivity. We examine this model's implications for the generation of highly infectious recombinant prions and the protein-only hypothesis of prion replication.

Permanganate oxidation of α-amino acids: kinetic correlations for the nonautocatalytic and autocatalytic reaction pathways.

PubMed

Perez-Benito, Joaquin F

2011-09-08

The reactions of permanganate ion with seven α-amino acids in aqueous KH(2)PO(4)/K(2)HPO(4) buffers have been followed spectrophotometrically at two different wavelengths: 526 nm (decay of MnO(4)(-)) and 418 nm (formation of colloidal MnO(2)). All of the reactions studied were autocatalyzed by colloidal MnO(2), with the contribution of the autocatalytic reaction pathway decreasing in the order glycine > l-threonine > l-alanine > l-glutamic acid > l-leucine > l-isoleucine > l-valine. The rate constants corresponding to the nonautocatalytic and autocatalytic pathways were obtained by means of either a differential rate law or an integrated one, the latter requiring the use of an iterative method for its implementation. The activation parameters for the two pathways were determined and analyzed to obtain statistically significant correlations for the series of reactions studied. The activation enthalpy of the nonautocatalytic pathway showed a strong, positive dependence on the standard Gibbs energy for the dissociation of the protonated amino group of the α-amino acid. Linear enthalpy-entropy correlations were found for both pathways, leading to isokinetic temperatures of 370 ± 21 K (nonautocatalytic) and 364 ± 28 K (autocatalytic). Mechanisms in agreement with the experimental data are proposed for the two reaction pathways.

Stepwise Evolution of Nonliving to Living Chemical Systems

NASA Astrophysics Data System (ADS)

Lindahl, Paul A.

2004-08-01

Steps by which a nonliving chemical system could have transformed into a living system are described and discussed, assuming general features of Wächtershäuser's chemo-autotrophic surface theory of the origin of life. Environmental species such as CO2 and H2S are proposed to have reacted to form a quasi-steady state metal-bound intermediate (CH3-M) that slowly decayed into waste (CH4). Unpredictable dispersive reactions expanded the system to include surface-bound forms of the citric acid cycle intermediates (oxaloacetate --> citrate). Further reaction yielded an autocatalytic system in which raw materials are converted into the system at exponential rates. Combinatorial dispersive reactions that improved the performance of this system were automatically selected and incorporated into it. Systems evolved critical features of living systems (proteins, membranes, proteins, nucleic acids, etc.) using two related mechanisms called grafting and waste-conversion. Such living systems were transformed from less recognizable types (characterized by autocatalytic spreading, decentralization, poorly defined boundaries, etc.) into more recognizable ones (encapsulated by membranes, controlled by single-molecule genomes, etc.) that self-replicated by a cell division cycle and could evolve by the standard gene-based Darwinian mechanism. The resulting systems are viewed as having an autocatalytic network composed of three linked autocatalytic subreactions.

A General Diastereoselective Catalytic Vinylogous Aldol Reaction Among Tetramic Acid-Derived Pyrroles

PubMed Central

2015-01-01

A catalytic diastereoselective aldol reaction has been developed for N1-arylated/C2-O-silylated/C3-methylated and brominated/C4-O-methylated pyrroles in its reactions with various aldehydes. Syn adducts emerge with regard to the vicinal nitrogen and oxygen heteroatom substituents. The N1-aryl residue undergoes oxidative cleavage, and the C3-bromine atom undergoes palladium-mediated coupling reactions, both without disturbing the newly created stereocenters. PMID:25119431

Total Synthesis of Plakilactones C, B and des-Hydroxyplakilactone B by the Oxidative Cleavage of Gracilioether Furanylidenes.

PubMed

Norris, Matthew D; Perkins, Michael V

2016-08-05

A chemoselective oxidative cleavage of synthetic gracilioether B, 11-epi-gracilioether C benzoate, and des-hydroxygracilioether C with pyridinium chlorochromate, which proceeds with loss of the furanyl acetate, has enabled total synthesis and stereochemical elucidation of the marine sponge metabolites (4R,6R)-plakilactone C, (4R,6R,9R)-plakilactone B, and (4R,6R)-des-hydroxyplakilactone B. des-Hydroxygracilioether C, the putative biosynthetic precursor to hippolachnin A, was also found to undergo a facile ene cyclization on treatment with SnCl4.

Copper(I)-catalyzed substitution reactions of propargylic amines: importance of C(sp)-C(sp3) bond cleavage in generation of iminium intermediates.

PubMed

Sugiishi, Tsuyuka; Kimura, Akifumi; Nakamura, Hiroyuki

2010-04-21

Substitution reactions of propargylic amines proceed in the presence of copper(I) catalysts. Mechanistic studies showed that C(sp)-C(sp(3)) bond cleavage assisted by nitrogen lone-pair electrons is essential for the reaction, and the resulting iminium intermediates undergo amine exchange, aldehyde exchange, and alkyne addition reactions. Because iminium intermediates are key to aldehyde-alkyne-amine (A(3)) coupling reactions, this transformation is effective not only for reconstruction of propargylic amines but also for chiral induction of racemic compounds in the presence of chiral catalysts.

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

Polycystin-1 Cleavage and the Regulation of Transcriptional Pathways

PubMed Central

Merrick, David; Bertuccio, Claudia A.; Chapin, Hannah C.; Lal, Mark; Chauvet, Veronique; Caplan, Michael J.

2013-01-01

Autosomal dominant polycystic kidney disease (ADPKD) is the most common genetic cause of end stage renal disease, affecting ~1 in 1,000 people. The disease is characterized by the development of numerous large fluid filled renal cysts over the course of decades. These cysts compress the surrounding renal parenchyma and impair its function. Mutations in two genes are responsible for ADPKD. The protein products of both of these genes, polycystin-1 and polycystin-2, localize to the primary cilium and participate in a wide variety of signaling pathways. Polycystin-1 undergoes several proteolytic cleavages that produce fragments that manifest biological activities. Recent results suggest that the production of polycystin-1 cleavage fragments is necessary and sufficient to account for at least some, although certainly not all, of the physiological functions of the parent protein. PMID:23824180

Structure of the Immature Dengue Virus at Low pH Primes Proteolytic Maturation

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

Yu, I-Mei; Zhang, Wei; Holdaway, Heather A.

Intracellular cleavage of immature flaviviruses is a critical step in assembly that generates the membrane fusion potential of the E glycoprotein. With cryo-electron microscopy we show that the immature dengue particles undergo a reversible conformational change at low pH that renders them accessible to furin cleavage. At a pH of 6.0, the E proteins are arranged in a herringbone pattern with the pr peptides docked onto the fusion loops, a configuration similar to that of the mature virion. After cleavage, the dissociation of pr is pH-dependent, suggesting that in the acidic environment of the trans-Golgi network pr is retained onmore » the virion to prevent membrane fusion. These results suggest a mechanism by which flaviviruses are processed and stabilized in the host cell secretory pathway.« less

Autocatalytic polymerization generates persistent random walk of crawling cells.

PubMed

Sambeth, R; Baumgaertner, A

2001-05-28

The autocatalytic polymerization kinetics of the cytoskeletal actin network provides the basic mechanism for a persistent random walk of a crawling cell. It is shown that network remodeling by branching processes near the cell membrane is essential for the bimodal spatial stability of the network which induces a spontaneous breaking of isotropic cell motion. Details of the phenomena are analyzed using a simple polymerization model studied by analytical and simulation methods.

Modeling of autocatalytic hydrolysis of adefovir dipivoxil in solid formulations.

PubMed

Dong, Ying; Zhang, Yan; Xiang, Bingren; Deng, Haishan; Wu, Jingfang

2011-04-01

The stability and hydrolysis kinetics of a phosphate prodrug, adefovir dipivoxil, in solid formulations were studied. The stability relationship between five solid formulations was explored. An autocatalytic mechanism for hydrolysis could be proposed according to the kinetic behavior which fits the Prout-Tompkins model well. For the classical kinetic models could hardly describe and predict the hydrolysis kinetics of adefovir dipivoxil in solid formulations accurately when the temperature is high, a feedforward multilayer perceptron (MLP) neural network was constructed to model the hydrolysis kinetics. The build-in approaches in Weka, such as lazy classifiers and rule-based learners (IBk, KStar, DecisionTable and M5Rules), were used to verify the performance of MLP. The predictability of the models was evaluated by 10-fold cross-validation and an external test set. It reveals that MLP should be of general applicability proposing an alternative efficient way to model and predict autocatalytic hydrolysis kinetics for phosphate prodrugs.

Autocatalytic microtubule nucleation determines the size and mass of Xenopus laevis egg extract spindles

PubMed Central

Decker, Franziska; Oriola, David; Dalton, Benjamin

2018-01-01

Regulation of size and growth is a fundamental problem in biology. A prominent example is the formation of the mitotic spindle, where protein concentration gradients around chromosomes are thought to regulate spindle growth by controlling microtubule nucleation. Previous evidence suggests that microtubules nucleate throughout the spindle structure. However, the mechanisms underlying microtubule nucleation and its spatial regulation are still unclear. Here, we developed an assay based on laser ablation to directly probe microtubule nucleation events in Xenopus laevis egg extracts. Combining this method with theory and quantitative microscopy, we show that the size of a spindle is controlled by autocatalytic growth of microtubules, driven by microtubule-stimulated microtubule nucleation. The autocatalytic activity of this nucleation system is spatially regulated by the limiting amounts of active microtubule nucleators, which decrease with distance from the chromosomes. This mechanism provides an upper limit to spindle size even when resources are not limiting. PMID:29323637

Crystal Structure and Proteomics Analysis of Empty Virus-like Particles of Cowpea Mosaic Virus

PubMed Central

Huynh, Nhung T.; Hesketh, Emma L.; Saxena, Pooja; Meshcheriakova, Yulia; Ku, You-Chan; Hoang, Linh T.; Johnson, John E.; Ranson, Neil A.; Lomonossoff, George P.; Reddy, Vijay S.

2016-01-01

SUMMARY Empty virus-like particles (eVLPs) of Cowpea mosaic virus (CPMV) are currently being utilized as reagents in various biomedical and nanotechnology applications. Here, we report the crystal structure of CPMV eVLPs determined using X-ray crystallography at 2.3 Å resolution and compare it with previously reported cryo-electron microscopy (cryo-EM) of eVLPs and virion crystal structures. Although the X-ray and cryo-EM structures of eVLPs are mostly similar, there exist significant differences at the C terminus of the small (S) subunit. The intact C terminus of the S subunit plays a critical role in enabling the efficient assembly of CPMV virions and eVLPs, but undergoes proteolysis after particle formation. In addition, we report the results of mass spectrometry-based proteomics analysis of coat protein subunits from CPMV eVLPs and virions that identify the C termini of S subunits undergo proteolytic cleavages at multiple sites instead of a single cleavage site as previously observed. PMID:27021160

Prebiological evolution and the metabolic origins of life.

PubMed

Pratt, Andrew J

2011-01-01

The chemoton model of cells posits three subsystems: metabolism, compartmentalization, and information. A specific model for the prebiological evolution of a reproducing system with rudimentary versions of these three interdependent subsystems is presented. This is based on the initial emergence and reproduction of autocatalytic networks in hydrothermal microcompartments containing iron sulfide. The driving force for life was catalysis of the dissipation of the intrinsic redox gradient of the planet. The codependence of life on iron and phosphate provides chemical constraints on the ordering of prebiological evolution. The initial protometabolism was based on positive feedback loops associated with in situ carbon fixation in which the initial protometabolites modified the catalytic capacity and mobility of metal-based catalysts, especially iron-sulfur centers. A number of selection mechanisms, including catalytic efficiency and specificity, hydrolytic stability, and selective solubilization, are proposed as key determinants for autocatalytic reproduction exploited in protometabolic evolution. This evolutionary process led from autocatalytic networks within preexisting compartments to discrete, reproducing, mobile vesicular protocells with the capacity to use soluble sugar phosphates and hence the opportunity to develop nucleic acids. Fidelity of information transfer in the reproduction of these increasingly complex autocatalytic networks is a key selection pressure in prebiological evolution that eventually leads to the selection of nucleic acids as a digital information subsystem and hence the emergence of fully functional chemotons capable of Darwinian evolution.

Identification of a Membrane Targeting and Degradation Signal in the p42 Protein of Influenza C Virus

PubMed Central

Pekosz, Andrew; Lamb, Robert A.

2000-01-01

Two mRNA species are derived from the influenza C virus RNA segment six, (i) a colinear transcript containing a 374-amino-acid residue open reading frame (referred to herein as the seg 6 ORF) which is translated to yield the p42 protein, and (ii) a spliced mRNA which encodes the influenza C virus matrix (CM1) protein consisting of the first 242 amino acids of p42. The p42 protein undergoes proteolytic cleavage at a consensus signal peptidase cleavage site after residue 259, yielding the p31 and CM2 proteins. Translocation of p42 into the endoplasmic reticulum membrane occurs cotranslationally and requires the hydrophobic internal signal peptide (residues 239 to 259), as well as the predicted transmembrane domain of CM2 (residues 285 to 308). The p31 protein was found to undergo rapid degradation after cleavage from p42. Addition of the 26S proteasome inhibitor lactacystin to influenza C virus-infected or seg 6 ORF cDNA-transfected cells drastically reduced p31 degradation. Transfer of the 17-residue C-terminal region of p31 to heterologous proteins resulted in their rapid turnover. The hydrophobic nature, but not the specific amino acid sequence of the 17-amino-acid C terminus of p31 appears to act as the signal for targeting the protein to membranes and for degradation. PMID:11044092

Adenosylcobinamide methyl phosphate as a pseudocoenzyme for diol dehydrase.

PubMed

Ishida, A; Toraya, T

1993-02-16

Adenosylcobinamide methyl phosphate, a novel analog of adenosylcobalamin lacking the nucleotide loop moiety, was synthesized. It did not show detectable coenzymic activity but behaved as a strong competitive inhibitor against AdoCbl with relatively high affinity (Ki = 2.5 microM). When apoenzyme was incubated at 37 degrees C with this analog in the presence of substrate, the Co-C bond of the analog was almost completely and irreversibly cleaved within 10 min, forming an enzyme-bound Co(II)-containing species. The cleavage was not observed in the absence of substrate. The Co-C bond cleavage in the presence of substrate was not catalytic but stoichiometric, implying that the Co-C bond of the analog undergoes activation when the analog binds to the active site of the enzyme. 5'-Deoxyadenosine was the only product derived from the adenosyl group of the analog upon the Co-C bond cleavage. Apoenzyme did not undergo modification during this process. Therefore, it seems likely that adenosylcobinamide methyl phosphate acts as a pseudocoenzyme or a potent suicide coenzyme. Since adenosylcobinamide neither functions as coenzyme nor binds tightly to apoenzyme, it can be concluded that the phosphodiester moiety of the nucleotide loop of adenosylcobalamin is essential for tight binding to apoenzyme and therefore for subsequent activation of the Co-C bond and catalysis. It is also evident that the nucleotide loop is obligatory for the normal progress of catalytic cycle.

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.

Mass Spectra of Some Perfluoroalkyl and Perfluoroalkylether Substituted 1,2,4-Oxadiazoles

NASA Technical Reports Server (NTRS)

Paciorek, Kazimiera J. L.; Nakahara, James H.; Kratzer, Reinhold H.; Rosser, Robert W.

1977-01-01

Electron impact fragmentation patterns were obtained for 1,4-bis[(5-perfluoro-n-heptyl)-1,2,4-oxadiazolyl- benzene, its perfluoroalkylether substituted analogue, 3,5-bis(perfluoroalkyl)-, 3,5-bis(perfluoroalkylether)- and 3-perfluoroalkylether-5-perfluoroalkyl-1,2,4-oxadiazoles. In the compounds containing the phenylene group the molecular ion constituted the base peak; the main process was the breakdown of the oxadiazole ring with concurrent liberation of the perfluoroalkyl or perfluoroalkylether nitrile molecule; cleavage of the fluorinated chain ot to the oxadiazole ring was found to take place to a considerable degree. In the perfluorinated 1,2,4-oxadiazoles cleavage beta to the oxadiazole ring occurred preferentially; fragmentation of the ring itself took place to a limited degree only. The 3-perfluoroalkylether-5-perfluoroalkyl-1,2,4-oxadiazole appeared to undergo the primary beta-cleavage exclusively at the perfluoroalkylether sidechain.

Compositional and stable carbon isotopic fractionation during non-autocatalytic thermochemical sulfate reduction by gaseous hydrocarbons

USGS Publications Warehouse

Xia, Xinyu; Ellis, Geoffrey S.; Ma, Qisheng; Tang, Yongchun

2014-01-01

The possibility of autocatalysis during thermochemical sulfate reduction (TSR) by gaseous hydrocarbons was investigated by examination of previously reported laboratory and field data. This reaction was found to be a kinetically controlled non-autocatalytic process, and the apparent lack of autocatalysis is thought to be due to the absence of the required intermediate species. Kinetic parameters for chemical and carbon isotopic fractionations of gaseous hydrocarbons affected by TSR were calculated and found to be consistent with experimentally derived values for TSR involving long-chain hydrocarbons. Model predictions based on these kinetic values indicate that TSR by gaseous hydrocarbon requires high-temperature conditions. The oxidation of C2–5 hydrocarbons by sulfate reduction is accompanied by carbon isotopic fractionation with the residual C2–5 hydrocarbons becoming more enriched in 13C. Kinetic parameters were calculated for the stable carbon isotopic fractionation of gaseous hydrocarbons that have experienced TSR. Model predictions based on these kinetics indicate that it may be difficult to distinguish the effects of TSR from those of thermal maturation at lower levels of hydrocarbon oxidation; however, unusually heavy δ13C2+ values (>−10‰) can be diagnostic of high levels of conversion (>50%). Stoichiometric and stable carbon isotopic data show that methane is stable under the investigated reaction conditions and is likely a product of TSR by other gaseous hydrocarbons rather than a significant reactant. These results indicate that the overall TSR reaction mechanism for oxidation of organic substrates containing long-chain hydrocarbons involves three distinct phases as follows: (1) an initial slow and non-autocatalytic stage characterized by the reduction of reactive sulfate by long-chain saturated hydrocarbons; (2) a second autocatalytic reaction phase dominated by reactions involving reduced sulfur species and partially oxidized hydrocarbons; (3) and a final, or late-stage, TSR reaction in which hydrocarbon oxidation continues at a slower rate via the non-autocatalytic reduction of sulfate by gaseous hydrocarbons.

Atomic resolution structure of the cytoplasmic domain of Yersinia pestis YscU, a regulatory switch involved in type III secretion

PubMed Central

Lountos, George T; Austin, Brian P; Nallamsetty, Sreedevi; Waugh, David S

2009-01-01

Crystal structures of cleaved and uncleaved forms of the YscU cytoplasmic domain, an essential component of the type III secretion system (T3SS) in Yersinia pestis, have been solved by single-wavelength anomolous dispersion and refined with X-ray diffraction data extending up to atomic resolution (1.13 Å). These crystallographic studies provide structural insights into the conformational changes induced upon auto-cleavage of the cytoplasmic domain of YscU. The structures indicate that the cleaved fragments remain bound to each other. The conserved NPTH sequence that contains the site of the N263-P264 peptide bond cleavage is found on a β-turn which, upon cleavage, undergoes a major reorientation of the loop away from the catalytic N263, resulting in altered electrostatic surface features at the site of cleavage. Additionally, a significant conformational change was observed in the N-terminal linker regions of the cleaved and noncleaved forms of YscU which may correspond to the molecular switch that influences substrate specificity. The YscU structures determined here also are in good agreement with the auto-cleavage mechanism described for the flagellar homolog FlhB and E. coli EscU. PMID:19165725

Convective instabilities in traveling fronts of addition polymerization

NASA Technical Reports Server (NTRS)

Pojman, John A.; Jones, Chris E.; Khan, Akhtar M.

1993-01-01

An autocatalytic reaction in an unstirred vessel can support a constant velocity wavefront resulting from the coupling of diffusion to the chemical reaction. A flare front is a common example in which heat is the autocatalytic species that diffuses into unreacted regions stimulating a reaction that produces more heat. Traveling fronts were studied in synthetic polymerization reactions under high pressure by workers in the former USSR. More recently, propagating fronts of methacrylic acid polymerization were studied under ambient conditions, both with video techniques and by NMR.

Amyloid Beta Mediates Memory Formation

ERIC Educational Resources Information Center

Garcia-Osta, Ana; Alberini, Cristina M.

2009-01-01

The amyloid precursor protein (APP) undergoes sequential cleavages to generate various polypeptides, including the amyloid [beta] (1-42) peptide (A[beta][1-42]), which is believed to play a major role in amyloid plaque formation in Alzheimer's disease (AD). Here we provide evidence that, in contrast with its pathological role when accumulated,…

A review of models of fluctuating protrusion and retraction patterns at the leading edge of motile cells.

PubMed

Ryan, Gillian L; Watanabe, Naoki; Vavylonis, Dimitrios

2012-04-01

A characteristic feature of motile cells as they undergo a change in motile behavior is the development of fluctuating exploratory motions of the leading edge, driven by actin polymerization. We review quantitative models of these protrusion and retraction phenomena. Theoretical studies have been motivated by advances in experimental and computational methods that allow controlled perturbations, single molecule imaging, and analysis of spatiotemporal correlations in microscopic images. To explain oscillations and waves of the leading edge, most theoretical models propose nonlinear interactions and feedback mechanisms among different components of the actin cytoskeleton system. These mechanisms include curvature-sensing membrane proteins, myosin contraction, and autocatalytic biochemical reaction kinetics. We discuss how the combination of experimental studies with modeling promises to quantify the relative importance of these biochemical and biophysical processes at the leading edge and to evaluate their generality across cell types and extracellular environments. Copyright © 2012 Wiley Periodicals, Inc.

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.

Noise-induced symmetry breaking far from equilibrium and the emergence of biological homochirality

NASA Astrophysics Data System (ADS)

Jafarpour, Farshid; Biancalani, Tommaso; Goldenfeld, Nigel

2017-03-01

The origin of homochirality, the observed single-handedness of biological amino acids and sugars, has long been attributed to autocatalysis, a frequently assumed precursor for early life self-replication. However, the stability of homochiral states in deterministic autocatalytic systems relies on cross-inhibition of the two chiral states, an unlikely scenario for early life self-replicators. Here we present a theory for a stochastic individual-level model of autocatalytic prebiotic self-replicators that are maintained out of thermal equilibrium. Without chiral inhibition, the racemic state is the global attractor of the deterministic dynamics, but intrinsic multiplicative noise stabilizes the homochiral states. Moreover, we show that this noise-induced bistability is robust with respect to diffusion of molecules of opposite chirality, and systems of diffusively coupled autocatalytic chemical reactions synchronize their final homochiral states when the self-replication is the dominant production mechanism for the chiral molecules. We conclude that nonequilibrium autocatalysis is a viable mechanism for homochirality, without imposing additional nonlinearities such as chiral inhibition.

Exact solutions of kinetic equations in an autocatalytic growth model.

PubMed

Jędrak, Jakub

2013-02-01

Kinetic equations are introduced for the transition-metal nanocluster nucleation and growth mechanism, as proposed by Watzky and Finke [J. Am. Chem. Soc. 119, 10382 (1997)]. Equations of this type take the form of Smoluchowski coagulation equations supplemented with the terms responsible for the chemical reactions. In the absence of coagulation, we find complete analytical solutions of the model equations for the autocatalytic rate constant both proportional to the cluster mass, and the mass-independent one. In the former case, ξ(k)=s(k)(ξ(1))[proportionality]ξ(1)(k)/k was obtained, while in the latter, the functional form of s(k)(ξ(1)) is more complicated. In both cases, ξ(1)(t)=h(μ)(M(μ)(t)) is a function of the moments of the mass distribution. Both functions, s(k)(ξ(1)) and h(μ)(M(μ)), depend on the assumed mechanism of autocatalytic growth and monomer production, and not on other chemical reactions present in a system.

Design principles of autocatalytic cycles constrain enzyme kinetics and force low substrate saturation at flux branch points

PubMed Central

Barenholz, Uri; Davidi, Dan; Reznik, Ed; Bar-On, Yinon; Antonovsky, Niv; Noor, Elad; Milo, Ron

2017-01-01

A set of chemical reactions that require a metabolite to synthesize more of that metabolite is an autocatalytic cycle. Here, we show that most of the reactions in the core of central carbon metabolism are part of compact autocatalytic cycles. Such metabolic designs must meet specific conditions to support stable fluxes, hence avoiding depletion of intermediate metabolites. As such, they are subjected to constraints that may seem counter-intuitive: the enzymes of branch reactions out of the cycle must be overexpressed and the affinity of these enzymes to their substrates must be relatively weak. We use recent quantitative proteomics and fluxomics measurements to show that the above conditions hold for functioning cycles in central carbon metabolism of E. coli. This work demonstrates that the topology of a metabolic network can shape kinetic parameters of enzymes and lead to seemingly wasteful enzyme usage. DOI: http://dx.doi.org/10.7554/eLife.20667.001 PMID:28169831

Autocatalytic microtubule nucleation determines the size and mass of Xenopus laevis egg extract spindles.

PubMed

Decker, Franziska; Oriola, David; Dalton, Benjamin; Brugués, Jan

2018-01-11

Regulation of size and growth is a fundamental problem in biology. A prominent example is the formation of the mitotic spindle, where protein concentration gradients around chromosomes are thought to regulate spindle growth by controlling microtubule nucleation. Previous evidence suggests that microtubules nucleate throughout the spindle structure. However, the mechanisms underlying microtubule nucleation and its spatial regulation are still unclear. Here, we developed an assay based on laser ablation to directly probe microtubule nucleation events in Xenopus laevis egg extracts. Combining this method with theory and quantitative microscopy, we show that the size of a spindle is controlled by autocatalytic growth of microtubules, driven by microtubule-stimulated microtubule nucleation. The autocatalytic activity of this nucleation system is spatially regulated by the limiting amounts of active microtubule nucleators, which decrease with distance from the chromosomes. This mechanism provides an upper limit to spindle size even when resources are not limiting. © 2018, Decker et al.

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

Cleavage of sp3 C-O bonds via oxidative addition of C-H bonds.

PubMed

Choi, Jongwook; Choliy, Yuriy; Zhang, Xiawei; Emge, Thomas J; Krogh-Jespersen, Karsten; Goldman, Alan S

2009-11-04

(PCP)Ir (PCP = kappa(3)-C(6)H(3)-2,6-[CH(2)P(t-Bu)(2)](2)) is found to undergo oxidative addition of the methyl-oxygen bond of electron-poor methyl aryl ethers, including methoxy-3,5-bis(trifluoromethyl)benzene and methoxypentafluorobenzene, to give the corresponding aryloxide complexes (PCP)Ir(CH(3))(OAr). Although the net reaction is insertion of the Ir center into the C-O bond, density functional theory (DFT) calculations and a significant kinetic isotope effect [k(CH(3))(OAr)/k(CD(3))(OAr) = 4.3(3)] strongly argue against a simple insertion mechanism and in favor of a pathway involving C-H addition and alpha-migration of the OAr group to give a methylene complex followed by hydride-to-methylene migration to give the observed product. Ethoxy aryl ethers, including ethoxybenzene, also undergo C-O bond cleavage by (PCP)Ir, but the net reaction in this case is 1,2-elimination of ArO-H to give (PCP)Ir(H)(OAr) and ethylene. DFT calculations point to a low-barrier pathway for this reaction that proceeds through C-H addition of the ethoxy methyl group followed by beta-aryl oxide elimination and loss of ethylene. Thus, both of these distinct C-O cleavage reactions proceed via initial addition of a C(sp(3))-H bond, despite the fact that such bonds are typically considered inert and are much stronger than C-O bonds.

Phenanthridine synthesis through iron-catalyzed intramolecular N-arylation of O-acetyl oxime.

PubMed

Deb, Indubhusan; Yoshikai, Naohiko

2013-08-16

O-Acetyl oximes derived from 2'-arylacetophenones undergo N-O bond cleavage/intramolecular N-arylation in the presence of a catalytic amount of iron(III) acetylacetonate in acetic acid. In combination with the conventional cross-coupling or directed C-H arylation, the reaction offers a convenient route to substituted phenanthridines.

Crystal Structure and Proteomics Analysis of Empty Virus-like Particles of Cowpea Mosaic Virus.

PubMed

Huynh, Nhung T; Hesketh, Emma L; Saxena, Pooja; Meshcheriakova, Yulia; Ku, You-Chan; Hoang, Linh T; Johnson, John E; Ranson, Neil A; Lomonossoff, George P; Reddy, Vijay S

2016-04-05

Empty virus-like particles (eVLPs) of Cowpea mosaic virus (CPMV) are currently being utilized as reagents in various biomedical and nanotechnology applications. Here, we report the crystal structure of CPMV eVLPs determined using X-ray crystallography at 2.3 Å resolution and compare it with previously reported cryo-electron microscopy (cryo-EM) of eVLPs and virion crystal structures. Although the X-ray and cryo-EM structures of eVLPs are mostly similar, there exist significant differences at the C terminus of the small (S) subunit. The intact C terminus of the S subunit plays a critical role in enabling the efficient assembly of CPMV virions and eVLPs, but undergoes proteolysis after particle formation. In addition, we report the results of mass spectrometry-based proteomics analysis of coat protein subunits from CPMV eVLPs and virions that identify the C termini of S subunits undergo proteolytic cleavages at multiple sites instead of a single cleavage site as previously observed. Copyright © 2016 Elsevier Ltd. All rights reserved.

Stars and Symbiosis: MicroRNA- and MicroRNA*-Mediated Transcript Cleavage Involved in Arbuscular Mycorrhizal Symbiosis1[W][OA

PubMed Central

Devers, Emanuel A.; Branscheid, Anja; May, Patrick; Krajinski, Franziska

2011-01-01

The majority of plants are able to form the arbuscular mycorrhizal (AM) symbiosis in association with AM fungi. During symbiosis development, plant cells undergo a complex reprogramming resulting in profound morphological and physiological changes. MicroRNAs (miRNAs) are important components of the regulatory network of plant cells. To unravel the impact of miRNAs and miRNA-mediated mRNA cleavage on root cell reprogramming during AM symbiosis, we carried out high-throughput (Illumina) sequencing of small RNAs and degradome tags of Medicago truncatula roots. This led to the annotation of 243 novel miRNAs. An increased accumulation of several novel and conserved miRNAs in mycorrhizal roots suggest a role of these miRNAs during AM symbiosis. The degradome analysis led to the identification of 185 root transcripts as mature miRNA and also miRNA*-mediated mRNA cleavage targets. Several of the identified miRNA targets are known to be involved in root symbioses. In summary, the increased accumulation of specific miRNAs and the miRNA-mediated cleavage of symbiosis-relevant genes indicate that miRNAs are an important part of the regulatory network leading to symbiosis development. PMID:21571671

Regulation of blood vessels by prolactin and vasoinhibins.

PubMed

Clapp, Carmen; Thebault, Stéphanie; Macotela, Yazmín; Moreno-Carranza, Bibiana; Triebel, Jakob; Martínez de la Escalera, Gonzalo

2015-01-01

Prolactin (PRL) stimulates the growth of new blood vessels (angiogenesis) either directly through actions on endothelial cells or indirectly by upregulating proangiogenic factors like vascular endothelial growth factor (VEGF). Moreover, PRL acquires antiangiogenic properties after undergoing proteolytic cleavage to vasoinhibins, a family of PRL fragments (including 16 kDa PRL) with potent antiangiogenic, vasoconstrictive, and antivasopermeability effects. In view of the opposing actions of PRL and vasoinhibins, the regulation of the proteases responsible for specific PRL cleavage represents an efficient mechanism for controlling blood vessel growth and function. This review briefly describes the vascular actions of PRL and vasoinhibins, and addresses how their interplay could help drive biological effects of PRL in the context of health and disease.

Solar photochemical oxidation of alcohols using catalytic hydroquinone and copper nanoparticles under oxygen: oxidative cleavage of lignin models.

PubMed

Mitchell, Lorna J; Moody, Christopher J

2014-11-21

Alcohols are converted into to their corresponding carbonyl compounds using catalytic amounts of 1,4-hydroquinone with a copper nanoparticle electron transfer mediator with oxygen as the terminal oxidant in acetone as solvent under visible light irradiation. These conditions employing biorenewable hydroquinone as reagent were developed from initial experiments using stoichiometric amounts of 1,4-benzoquinone as oxidant. A range of benzylic and aliphatic primary and secondary alcohols are oxidized, affording the corresponding aldehydes or ketones in moderate to excellent yields. The methodology is also applicable to the oxidative degradation of lignin model compounds that undergo C-C bond cleavage to give simple aromatic compounds.

Ovarian adipocytokines are associated with early in vitro human embryo development independent of the action of ovarian insulin.

PubMed

Li, Liyun; Ferin, Michel; Sauer, Mark V; Lobo, Roger A

2012-12-01

We aimed to characterize the association between levels of serum and follicular fluid (FF) adipocytokines, reflected by the leptin to adiponectin ratio (L:A ratio), and oocyte quality and in vitro embryo development in women undergoing assisted reproduction. We also aimed to assess whether follicular hormonal pathways mediate this interaction. We prospectively collected FF from up to four individual preovulatory follicles (n = 76) and fasting sera from women (n = 31) without endocrinopathies undergoing in vitro fertilization (IVF) at a university-based center for assisted reproduction. Leptin, total adiponectin, insulin, insulin-like growth factor 1 (IGF-1), and ovarian steriods were measured using enzyme immunoassay. Oocyte maturity, fertilization, and embryo development were assessed. FF leptin was similar to serum levels while FF adiponectin was lower. FF leptin (27.10 ± 4.05 ng/mL) and the L:A ratio (11.48E-3 ± 2.57E-3) were related to FF insulin (R (2) = 0.370 and 0.419, p < 0.001) but not to ovarian steroids or IGF-1, whereas FF adiponectin ( 4.22 ± 0.52 ug/mL) correlated only with leptin (R (2) = -0.138, p = 0.001). Oocytes from a high FF L:A ratio environment were 81 % (RR 1.81 [95%CI 0.97-3.37]) more likely to undergo successful cleavage and 117 % (RR 2.17 [95 % CI 1.06-4.44]) more likely to obtain viable cleavage morphology compared to a low FF L:A ratio environment, even when adjusted for FF insulin, an independent predictor of cleavage. Certain adipocytokines, particularly the L:A ratio in the FF of the preovulatory follicle, are related to successful in vitro embryo development. This action may be independent of FF insulin.

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

Fundamental studies of desulfurization processes: reaction of methanethiol on ZnO and Cs/ZnO

NASA Astrophysics Data System (ADS)

Dvorak, Joseph; Jirsak, Tomas; Rodriguez, José A.

2001-05-01

The reaction of methanethiol on ZnO and Cs promoted ZnO surfaces has been studied with synchrotron based photoemission and thermal desorption spectroscopy. On ZnO, methanethiol undergoes selective reaction to produce carbon monoxide (37-58%), methane (23-38%), formaldehyde (12-15%), ethane (1-11%), and a mixture of ethylene and acetylene (3-13%). At low temperatures (<100 K), methanethiol reacts to yield thiolate intermediate bound to Zn 2+ cations. The thiolate is stable to 500 K. Above this temperature, C-S bond cleavage occurs to yield methyl intermediate and atomic S. Carbon is removed from the surface as gaseous products above 500 K, and atomic sulfur remains bound to the zinc sites of the surface. Submonolayer amounts of cesium do not have a significant promotional effect on C-S bond cleavage, whereas Cs multilayers are found to significantly lower the activation barrier for C-S bond cleavage. This study illustrates the chemistry associated with the desulfurization of thiols on a catalytically relevant oxide surface.

Lithostathine quadruple-helical filaments form proteinase K-resistant deposits in Creutzfeldt-Jakob disease.

PubMed

Laurine, Emmanuelle; Grégoire, Catherine; Fändrich, Marcus; Engemann, Sabine; Marchal, Stéphane; Thion, Laurent; Mohr, Michel; Monsarrat, Bernard; Michel, Bernard; Dobson, Christopher M; Wanker, Erich; Erard, Monique; Verdier, Jean-Michel

2003-12-19

Autocatalytic cleavage of lithostathine leads to the formation of quadruple-helical fibrils (QHF-litho) that are present in Alzheimer's disease. Here we show that such fibrils also occur in Creutzfeldt-Jakob and Gerstmann-Sträussler-Scheinker diseases, where they form protease-K-resistant deposits and co-localize with amyloid plaques formed from prion protein. Lithostathine does not appear to change its native-like, globular structure during fibril formation. However, we obtained evidence that a cluster of six conserved tryptophans, positioned around a surface loop, could act as a mobile structural element that can be swapped between adjacent protein molecules, thereby enabling the formation of higher order fibril bundles. Despite their association with these clinical amyloid deposits, QHF-litho differ from typical amyloid fibrils in several ways, for example they produce a different infrared spectrum and cannot bind Congo Red, suggesting that they may not represent amyloid structures themselves. Instead, we suggest that lithostathine constitutes a novel component decorating disease-associated amyloid fibrils. Interestingly, [6,6']bibenzothiazolyl-2,2'-diamine, an agent found previously to disrupt aggregates of huntingtin associated with Huntington's disease, can dissociate lithostathine bundles into individual protofilaments. Disrupting QHF-litho fibrils could therefore represent a novel therapeutic strategy to combat clinical amyloidoses.

A Dual-Intein Autoprocessing Domain that Directs Synchronized Protein Co-Expression in Both Prokaryotes and Eukaryotes

PubMed Central

Zhang, Bei; Rapolu, Madhusudhan; Liang, Zhibin; Han, Zhenlin; Williams, Philip G.; Su, Wei Wen

2015-01-01

Being able to coordinate co-expression of multiple proteins is necessary for a variety of important applications such as assembly of protein complexes, trait stacking, and metabolic engineering. Currently only few options are available for multiple recombinant protein co-expression, and most of them are not applicable to both prokaryotic and eukaryotic hosts. Here, we report a new polyprotein vector system that is based on a pair of self-excising mini-inteins fused in tandem, termed the dual-intein (DI) domain, to achieve synchronized co-expression of multiple proteins. The DI domain comprises an Ssp DnaE mini-intein N159A mutant and an Ssp DnaB mini-intein C1A mutant connected in tandem by a peptide linker to mediate efficient release of the flanking proteins via autocatalytic cleavage. Essentially complete release of constituent proteins, GFP and RFP (mCherry), from a polyprotein precursor, in bacterial, mammalian, and plant hosts was demonstrated. In addition, successful co-expression of GFP with chloramphenicol acetyltransferase, and thioredoxin with RFP, respectively, further substantiates the general applicability of the DI polyprotein system. Collectively, our results demonstrate the DI-based polyprotein technology as a highly valuable addition to the molecular toolbox for multi-protein co-expression which finds vast applications in biotechnology, biosciences, and biomedicine. PMID:25712612

Design and Analysis of Compact DNA Strand Displacement Circuits for Analog Computation Using Autocatalytic Amplifiers.

PubMed

Song, Tianqi; Garg, Sudhanshu; Mokhtar, Reem; Bui, Hieu; Reif, John

2018-01-19

A main goal in DNA computing is to build DNA circuits to compute designated functions using a minimal number of DNA strands. Here, we propose a novel architecture to build compact DNA strand displacement circuits to compute a broad scope of functions in an analog fashion. A circuit by this architecture is composed of three autocatalytic amplifiers, and the amplifiers interact to perform computation. We show DNA circuits to compute functions sqrt(x), ln(x) and exp(x) for x in tunable ranges with simulation results. A key innovation in our architecture, inspired by Napier's use of logarithm transforms to compute square roots on a slide rule, is to make use of autocatalytic amplifiers to do logarithmic and exponential transforms in concentration and time. In particular, we convert from the input that is encoded by the initial concentration of the input DNA strand, to time, and then back again to the output encoded by the concentration of the output DNA strand at equilibrium. This combined use of strand-concentration and time encoding of computational values may have impact on other forms of molecular computation.

Study on Synthesis of Thoreau-modified 3, 5-Dimethyl-Thioltoluenediamine Used as Epoxy Resin Curing Agent and Its Performance

NASA Astrophysics Data System (ADS)

Peng, Yongli; Xiao, Wenzheng

2017-06-01

A novel curing agent Thoreau modified 3, 5-Dimethyl-thioltoluenediamine was synthesized and its molecular structure was characterized by FTIR and DSC. The curing kinetics of a high toughness and low volume shrinkage ratio epoxy system (modified DMTDA/DGEBA) was studied by differential scanning calorimetry (DSC) under noni so thermal conditions. The data were fitted to an order model and autocatalytic model respectively. The results indicate that in order model deviates significantly from experimental data. Malik’s method was used to prove that the curing kinetics of the system concerned follow single-step autocatalytic model, and a “single-point model-free” approach was employed to calculate meaningful kinetic parameters. The DSC curves derived from autocatalytic model gave satisfactory agreement with that of experiment in the range 5K/min∼25K/min. As the heating rate increased, the predicted DSC curves deviated from experimental curves, and the total exothermic enthalpy declined owing to the transition of competition relationship between kinetics control and diffusion control.

An Adaptor Domain-Mediated Auto-Catalytic Interfacial Kinase Reaction

PubMed Central

Liao, Xiaoli; Su, Jing; Mrksich, Milan

2010-01-01

This paper describes a model system for studying the auto-catalytic phosphorylation of an immobilized substrate by a kinase enzyme. This work uses self-assembled monolayers (SAMs) of alkanethiolates on gold to present the peptide substrate on a planar surface. Treatment of the monolayer with Abl kinase results in phosphorylation of the substrate. The phosphorylated peptide then serves as a ligand for the SH2 adaptor domain of the kinase and thereby directs the kinase activity to nearby peptide substrates. This directed reaction is intramolecular and proceeds with a faster rate than does the initial, intermolecular reaction, making this an auto-catalytic process. The kinetic non-linearity gives rise to properties that have no counterpart in the corresponding homogeneous phase reaction: in one example, the rate for phosphorylation of a mixture of two peptides is faster than the sum of the rates for phosphorylation of each peptide when presented alone. This work highlights the use of an adaptor domain in modulating the activity of a kinase enzyme for an immobilized substrate and offers a new approach for studying biochemical reactions in spatially inhomogeneous settings. PMID:19821459

Parental contributions to early embryo development: influences of urinary phthalate and phthalate alternatives among couples undergoing IVF treatment.

PubMed

Wu, Haotian; Ashcraft, Lisa; Whitcomb, Brian W; Rahil, Tayyab; Tougias, Ellen; Sites, Cynthia K; Pilsner, J Richard

2017-01-01

Are preconception urinary concentrations of phthalates and phthalate alternatives associated with diminished early stage embryo quality in couples undergoing IVF? Male, but not female, urinary concentrations of select metabolites of phthalates and phthalate alternatives are associated with diminished blastocyst quality. Although phthalates are endocrine disrupting compounds associated with adverse reproductive health, they are in widespread use across the world. Male and female preconception exposures to select phthalates have been previously associated with adverse reproductive outcomes in both the general population and in those undergoing IVF. This prospective cohort included 50 subfertile couples undergoing IVF in western Massachusetts. This study includes the first 50 couples recruited from the Baystate Medical Center's Fertility Center in Springfield, MA, as part of the Sperm Environmental Epigenetics and Development Study (SEEDS). Relevant data from both partners, including embryo quality at the cleavage (Day 3) and blastocyst (Day 5) stages, were collected by clinic personnel during the normal course of an IVF cycle. A spot urine sample was collected from both male and female partners on the same day as semen sample procurement and oocyte retrieval. Concentrations of 17 urinary metabolite were quantified by liquid chromatography mass spectrometry and normalized via specific gravity. Generalized estimating equations were used to estimate odds ratios (OR) and 95% CI, with urinary phthalates and phthalate alternatives fitted as continuous variables and embryo quality as a binary variable. The 50 couples contributed 761 oocytes, of which 423 progressed to the cleavage stage, 261 were high-quality cleavage stage embryos, 137 were transferrable quality blastocysts and 47 were high-quality blastocysts. At the cleavage stage, male urinary monoethyl phthalate concentrations were positively associated with high-quality cleavage stage embryos (OR = 1.20, 95% CI 1.01-1.43, P = 0.04); no other significant associations were observed at this stage. At the blastocyst stage, male urinary concentrations of monobenzyl phthalate (OR = 0.55, 95% CI 0.36-0.84, P = 0.01), mono-3-hydroxybutyl phthalate (OR = 0.37, 95% CI 0.18-0.76, P = 0.01), mono-n-butyl phthalate (OR = 0.55, 95% CI 0.42-0.73, P < 0.01) and monomethyl phthalate (OR = 0.39, 95% CI 0.26-0.60, P < 0.01) were inversely associated with high-quality blastocysts. A borderline statistically significant relationship was observed for male concentrations of mono(2-ethylhexyl) phthalate (OR = 0.52, 95% CI 0.27-1.00, P = 0.05) and cyclohexane-1,2-dicarboxylic acid-monocarboxy isooctyl ester (OR = 0.21, 95% CI 0.04-1.03, P = 0.05) at the blastocyst stage. Similar inverse associations were observed between male urinary phthalate metabolite concentrations and likelihood of transferrable quality blastocysts. For female partners, select metabolites were positively associated with odds of high or transferrable blastocyst quality, but the observed associations were not consistent across blastocyst quality measures or between sex-specific and couples-level models. All models were adjusted for age of both partners, urinary metabolite concentrations of female partners and male infertility status, while models of blastocysts were additionally adjusted for embryo quality at cleavage stage. Our modest sample included only 50 couples contributing one cycle each. In addition, non-differential misclassification of exposure remains a concern given the single-spot urine collection and the short half-life of phthalates. Our results suggest an inverse association between male preconception concentrations of select phthalate metabolites and blastocyst quality, likely occurring after genomic activation. If corroborated with other studies, such findings will have public health and clinical significance for both the general population and those undergoing IVF. This work was generously supported by grant K22-ES023085 from the National Institute of Environmental Health Sciences. The authors declare no competing interests. N/A. © The Author 2016. Published by Oxford University Press on behalf of the European Society of Human Reproduction and Embryology. All rights reserved. For Permissions, please email: journals.permissions@oup.com.

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.

Halogenated Explosives to Defeat Biological Agents

DTIC Science & Technology

2015-09-01

The synthetic transformation of difluoramination of ketones by difluoramine (HNF2)29 is a specialized, hazardous process that is not likely to become...defluorination in triflic acid; even 4-(trifluoromethyl)propiophenone (ethyl phenyl ketone ) does not undergo C–F cleavage.45 The prospect of this...trifluoroacetaldehyde hydrate to generate trifluoroacet- aldehyde gas, which reacts with liquefied ammonia at low temperature. Upon warming, the hemiaminal

5-Nitrosalicylic Acid as a Novel Matrix for In-Source Decay in Matrix-Assisted Laser Desorption/Ionization Mass Spectrometry

PubMed Central

Asakawa, Daiki

2013-01-01

The matrix-assisted laser desorption/ionization in-source decay (MALDI-ISD) of peptides and glycans was studied using an oxidizing chemical, 5-nitrosalicylic acid (5-NSA) as the matrix. The use of 5-NSA for the MALDI-ISD of peptides and glycans promoted fragmentation pathways involving “hydrogen-deficient” radical precursors. Hydrogen abstraction from peptides resulted in the production of a “hydrogen-deficient” peptide radical that contained a radical site on the amide nitrogen in the peptide backbone with subsequent radical-induced cleavage at the Cα–C bonds. Cleavage at the Cα–C bond leads to the production of an a•/x fragment pair and the radical a• ions then undergo further hydrogen abstraction to form a ions after Cα–C bond cleavage. Since the Pro residue does not contain a nitrogen-centered radical site, Cα–C bond cleavage does not occur at this site. Alternatively, the specific cleavage of CO−N bonds leads to a b•/y fragment pair at Xxx−Pro which occurs via hydrogen abstraction from the Cα−H in the Pro residue. In contrast, “hydrogen-deficient” glycan radicals were generated by hydrogen abstraction from hydroxyl groups in glycans. Both glycosidic and cross-ring cleavages occurred as the result of the degradation of “hydrogen-deficient” glycan radicals. Cross-ring cleavage ions are potentially useful in linkage analysis, one of the most critical steps in the characterization of glycans. Moreover, isobaric glycans could be distinguished by structure specific ISD ions, and the molar ratio of glycan isomers in a mixture can be estimated from their fragment ions abundance ratios. MALDI-ISD with 5-NSA could be a useful method for the sequencing of peptides including the location of post-translational modifications, identification and semi-quantitative analysis of mixtures of glycan isomers. PMID:24860709

Crystallographic Insights into the Autocatalytic Assembly Mechanism of a Bacteriophage Tail Spike

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

Xiang, Ye; Leiman, Petr G.; Li, Long

2010-02-03

The tailed bacteriophage phi29 has 12 'appendages' (gene product 12, gp12) attached to its neck region that participate in host cell recognition and entry. In the cell, monomeric gp12 undergoes proteolytic processing that releases the C-terminal domain during assembly into trimers. We report here crystal structures of the protein before and after catalytic processing and show that the C-terminal domain of gp12 is an 'autochaperone' that aids trimerization. We also show that autocleavage of the C-terminal domain is a posttrimerization event that is followed by a unique ATP-dependent release. The posttranslationally modified N-terminal part has three domains that function tomore » attach the appendages to the phage, digest the cell wall teichoic acids, and bind irreversibly to the host, respectively. Structural and sequence comparisons suggest that some eukaryotic and bacterial viruses as well as bacterial adhesins might have a similar maturation mechanism as is performed by phi29 gp12 for Bacillus subtilis.« less

Degradation mechanisms of bioresorbable polyesters. Part 2. Effects of initial molecular weight and residual monomer.

PubMed

Gleadall, Andrew; Pan, Jingzhe; Kruft, Marc-Anton; Kellomäki, Minna

2014-05-01

This paper presents an understanding of how initial molecular weight and initial monomer fraction affect the degradation of bioresorbable polymers in terms of the underlying hydrolysis mechanisms. A mathematical model was used to analyse the effects of initial molecular weight for various hydrolysis mechanisms including noncatalytic random scission, autocatalytic random scission, noncatalytic end scission or autocatalytic end scission. Different behaviours were identified to relate initial molecular weight to the molecular weight half-life and to the time until the onset of mass loss. The behaviours were validated by fitting the model to experimental data for molecular weight reduction and mass loss of samples with different initial molecular weights. Several publications that consider initial molecular weight were reviewed. The effect of residual monomer on degradation was also analysed, and shown to accelerate the reduction of molecular weight and mass loss. An inverse square root law relationship was found between molecular weight half-life and initial monomer fraction for autocatalytic hydrolysis. The relationship was tested by fitting the model to experimental data with various residual monomer contents. Copyright © 2014 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.

Kinetics of autocatalysis in small systems

NASA Astrophysics Data System (ADS)

Arslan, Erdem; Laurenzi, Ian J.

2008-01-01

Autocatalysis is a ubiquitous chemical process that drives a plethora of biological phenomena, including the self-propagation of prions etiological to the Creutzfeldt-Jakob disease and bovine spongiform encephalopathy. To explain the dynamics of these systems, we have solved the chemical master equation for the irreversible autocatalytic reaction A +B→2A. This solution comprises the first closed form expression describing the probabilistic time evolution of the populations of autocatalytic and noncatalytic molecules from an arbitrary initial state. Grand probability distributions are likewise presented for autocatalysis in the equilibrium limit (A+B⇌2A), allowing for the first mechanistic comparison of this process with chemical isomerization (B⇌A) in small systems. Although the average population of autocatalytic (i.e., prion) molecules largely conforms to the predictions of the classical "rate law" approach in time and the law of mass action at equilibrium, thermodynamic differences between the entropies of isomerization and autocatalysis are revealed, suggesting a "mechanism dependence" of state variables for chemical reaction processes. These results demonstrate the importance of chemical mechanism and molecularity in the development of stochastic processes for chemical systems and the relationship between the stochastic approach to chemical kinetics and nonequilibrium thermodynamics.

Influence of heat losses on nonlinear fingering dynamics of exothermic autocatalytic fronts

NASA Astrophysics Data System (ADS)

D'Hernoncourt, J.; De Wit, A.

2010-06-01

Across traveling exothermic autocatalytic fronts, a density jump can be observed due to changes in composition and temperature. These density changes are prone to induce buoyancy-driven convection around the front when the propagation takes place in absence of gel within the gravity field. Most recent experiments devoted to studying such reaction-diffusion-convection dynamics are performed in Hele-Shaw cells, two glass plates separated by a thin gap width and filled by the chemical solutions. We investigate here the influence of heat losses through the walls of such cells on the nonlinear fingering dynamics of exothermic autocatalytic fronts propagating in vertical Hele-Shaw cells. We show that these heat losses increase tip splittings and modify the properties of the flow field. A comparison of the differences between the dynamics in reactors with respectively insulating and conducting walls is performed as a function of the Lewis number Le, the Newton cooling coefficient α quantifying the amplitude of heat losses and the width of the system. We find that tip splitting is enhanced for intermediate values of α while coarsening towards one single finger dominates for insulated systems or large values of α leading to situations equivalent to isothermal ones.

The PP2A-B56 Phosphatase Opposes Cyclin E Autocatalytic Degradation via Site-Specific Dephosphorylation

PubMed Central

Davis, Ryan J.; Swanger, Jherek; Hughes, Bridget T.

2017-01-01

ABSTRACT Cyclin E, in conjunction with its catalytic partner cyclin-dependent kinase 2 (CDK2), regulates cell cycle progression as cells exit quiescence and enter S-phase. Multiple mechanisms control cyclin E periodicity during the cell cycle, including phosphorylation-dependent cyclin E ubiquitylation by the SCFFbw7 ubiquitin ligase. Serine 384 (S384) is the critical cyclin E phosphorylation site that stimulates Fbw7 binding and cyclin E ubiquitylation and degradation. Because S384 is autophosphorylated by bound CDK2, this presents a paradox as to how cyclin E can evade autocatalytically induced degradation in order to phosphorylate its other substrates. We found that S384 phosphorylation is dynamically regulated in cells and that cyclin E is specifically dephosphorylated at S384 by the PP2A-B56 phosphatase, thereby uncoupling cyclin E degradation from cyclin E-CDK2 activity. Furthermore, the rate of S384 dephosphorylation is high in interphase but low in mitosis. This provides a mechanism whereby interphase cells can oppose autocatalytic cyclin E degradation and maintain cyclin E-CDK2 activity while also enabling cyclin E destruction in mitosis, when inappropriate cyclin E expression is genotoxic. PMID:28137908

Primordial evolvability: Impasses and challenges.

PubMed

Vasas, Vera; Fernando, Chrisantha; Szilágyi, András; Zachár, István; Santos, Mauro; Szathmáry, Eörs

2015-09-21

While it is generally agreed that some kind of replicating non-living compounds were the precursors of life, there is much debate over their possible chemical nature. Metabolism-first approaches propose that mutually catalytic sets of simple organic molecules could be capable of self-replication and rudimentary chemical evolution. In particular, the graded autocatalysis replication domain (GARD) model, depicting assemblies of amphiphilic molecules, has received considerable interest. The system propagates compositional information across generations and is suggested to be a target of natural selection. However, evolutionary simulations indicate that the system lacks selectability (i.e. selection has negligible effect on the equilibrium concentrations). We elaborate on the lessons learnt from the example of the GARD model and, more widely, on the issue of evolvability, and discuss the implications for similar metabolism-first scenarios. We found that simple incorporation-type chemistry based on non-covalent bonds, as assumed in GARD, is unlikely to result in alternative autocatalytic cycles when catalytic interactions are randomly distributed. An even more serious problem stems from the lognormal distribution of catalytic factors, causing inherent kinetic instability of such loops, due to the dominance of efficiently catalyzed components that fail to return catalytic aid. Accordingly, the dynamics of the GARD model is dominated by strongly catalytic, but not auto-catalytic, molecules. Without effective autocatalysis, stable hereditary propagation is not possible. Many repetitions and different scaling of the model come to no rescue. Despite all attempts to show the contrary, the GARD model is not evolvable, in contrast to reflexively autocatalytic networks, complemented by rare uncatalyzed reactions and compartmentation. The latter networks, resting on the creation and breakage of chemical bonds, can generate novel ('mutant') autocatalytic loops from a given set of environmentally available compounds. Real chemical reactions that make or break covalent bonds, rather than mere incorporation of components, are necessary for open-ended evolvability. The issue of whether or not several concrete chemical systems (rather than singular curiosities) could realize reflexively autocatalytic macromolecular networks will ultimately determine the relevance of metabolism-first approaches to the origin of life, as stepping stones towards true open-endedness that requires the combination of rich combinatorial chemistry controlled by information stored in template replicators. Copyright © 2015 Elsevier Ltd. All rights reserved.

Bovine non-competent oocytes (BCB-) negatively impact the capacity of competent (BCB+) oocytes to undergo in vitro maturation, fertilisation and embryonic development.

PubMed

Salviano, M B; Collares, F J F; Becker, B S; Rodrigues, B A; Rodrigues, J L

2016-04-01

Competent oocyte selection remains a bottleneck in the in vitro production (IVP) of mammalian embryos. Among the vital assays described for selecting competent oocytes for IVP, the brilliant cresyl blue (BCB) test has shown consistent results. The aim of the first experiment was to observe if oocytes directly submitted to IVM show similar cleavage and blastocyst rates as those obtained with oocytes maintained under the same in vitro conditions as the oocytes that undergo the BCB test. Bovine cumulus-oocyte complexes (COCs) were recovered from slaughterhouse-derived ovaries and, after morphological evaluation, were randomised grouped into three groups: (1) directly submitted to IVM; (2) oocytes submitted to the BCB test without the addition of BCB stain (BCB control group); and (3) submitted to the BCB test. The results showed that oocytes directly submitted to IVM reached similar cleavage (48/80 - 60%) and embryonic development rates to the blastocyst stage (10/48 - 21%) as the results obtained with the BCB control group oocytes (45/77 - 58% and 08/45 - 18%, respectively). The aim of the second experiment was to determine the cleavage and blastocyst rates obtained from BCB+ oocytes undergoing IVM in the presence of BCB- oocytes at a ratio of 10:1. COCs were recovered from slaughterhouse-derived ovaries and, after morphological evaluation, were randomised into two groups that were submitted to IVM either directly (1: control group) or submitted to the BCB test prior to IVM. After the BCB test, the COCs were classified as either BCB+ (blue cytoplasm) or BCB- (colourless cytoplasm) and then divided into four experimental groups: (2) BCB+; (3) BCB-; and (4) BCB+ matured in same IVM medium drop as (5) BCB- at a ratio of 10:1. After IVM (24 h), oocytes from the different experimental groups were submitted to in vitro fertilisation (IVF) and in vitro culture (IVC) under the same culture conditions until they reached the blastocyst stage (D7). With regards to the cleavage rate (48 h after IVF), only group 3 (102/229 - 44%) differed (P < 0.05) from the other groups [1 (145/241 - 60%); 2 (150/225 - 67%); 4 (201/318 - 63%) and 5 (21/33 - 63%)]. On day 7, the embryos from group 2 (BCB+) achieved the highest blastocyst rate (46/150 - 31%) (P < 0.05) when compared with the embryo development capacity of the other experimental groups (1: 31/145 - 21%; group 3: 17/102 - 17%; group 4: 46/201 - 23%; and group 5: 2/21 - 10%). In conclusion, submitting BCB+ oocytes that were separated from BCB- oocytes to IVM increases the rate of embryonic development to the blastocyst stage when compared to the control group, BCB- oocyte group, BCB+ paracrine group and BCB- paracrine group. The presence of non-competent oocytes during IVM, even in low proportion (1:10), reduces the capacity of competent oocytes to undergo embryo development and achieve blastocyst stage during IVC.

Association of the pr Peptides with Dengue Virus at Acidic pH Blocks Membrane Fusion

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

Yu, I.-M.; Holdaway, H.A.; Chipman, P.R.

Flavivirus assembles into an inert particle that requires proteolytic activation by furin to enable transmission to other hosts. We previously showed that immature virus undergoes a conformational change at low pH that renders it accessible to furin (I. M. Yu, W. Zhang, H. A. Holdaway, L. Li, V. A. Kostyuchenko, P. R. Chipman, R. J. Kuhn, M. G. Rossmann, and J. Chen, Science 319:1834-1837, 2008). Here we show, using cryoelectron microscopy, that the structure of immature dengue virus at pH 6.0 is essentially the same before and after the cleavage of prM. The structure shows that after cleavage, the proteolyticmore » product pr remains associated with the virion at acidic pH, and that furin cleavage by itself does not induce any major conformational changes. We also show by liposome cofloatation experiments that pr retention prevents membrane insertion, suggesting that pr is present on the virion in the trans-Golgi network to protect the progeny virus from fusion within the host cell.« less

Smallpox Antiviral Drug

DTIC Science & Technology

2006-01-01

preparing a Continuation in Part ( CIP ) to add the new I7L cleavage assays recently developed by SIGA. Conclusions By using homology-based... developmental cycle . RNA viruses and retroviruses commonly undergo formative proteolysis in which large polyproteins are cleaved by viral encoded proteinases to...structural model of the vaccinia virus (VV) I7L proteinase was developed at Transtech Pharma. A unique chemical library of ~ 51,000 compounds was

Microtubule organization during human parthenogenesis.

PubMed

Terada, Yukihiro; Hasegawa, Hisataka; Ugajin, Tomohisa; Murakami, Takashi; Yaegashi, Nobuo; Okamura, Kunihiro

2009-04-01

In human fertilization, the sperm centrosome plays a crucial role as a microtubule organizing center (MTOC). We studied microtubule organization during human parthenogenesis, which occurs when a human egg undergoes cleavage without a sperm centrosome. Multiple cytoplasmic asters were organized in the human oocyte after parthenogenetic activation, indicating that multiple MTOC are present in the human oocyte cytoplasm and function like a human sperm centrosome during parthenogenesis.

Overview of reductants utilized in nuclear fuel reprocessing/recycling

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

Patricia Paviet-Hartmann; Catherine Riddle; Keri Campbell

2013-10-01

Most of the aqueous processes developed, or under consideration worldwide for the recycling of used nuclear fuel (UNF) utilize the oxido-reduction properties of actinides to separate them from other radionuclides. Generally, after acid dissolution of the UNF, (essentially in nitric acid solution), actinides are separated from the raffinate by liquid-liquid extraction using specific solvents, associated along the process, with a particular reductant that will allow the separation to occur. For example, the industrial PUREX process utilizes hydroxylamine as a plutonium reductant. Hydroxylamine has numerous advantages: not only does it have the proper attributes to reduce Pu(IV) to Pu(III), but itmore » is also a non-metallic chemical that is readily decomposed to innocuous products by heating. However, it has been observed that the presence of high nitric acid concentrations or impurities (such as metal ions) in hydroxylamine solutions increase the likelihood of the initiation of an autocatalytic reaction. Recently there has been some interest in the application of simple hydrophilic hydroxamic ligands such as acetohydroxamic acid (AHA) for the stripping of tetravalent actinides in the UREX process flowsheet. This approach is based on the high coordinating ability of hydroxamic acids with tetravalent actinides (Np and Pu) compared with hexavalent uranium. Thus, the use of AHA offers a route for controlling neptunium and plutonium in the UREX process by complexant based stripping of Np(IV) and Pu(IV) from the TBP solvent phase, while U(VI) ions are not affected by AHA and remain solvated in the TBP phase. In the European GANEX process, AHA is also used to form hydrophilic complexes with actinides and strip them from the organic phase into nitric acid. However, AHA does not decompose completely when treated with nitric acid and hampers nitric acid recycling. In lieu of using AHA in the UREX + process, formohydroxamic acid (FHA), although not commercially available, hold promises as a replacement for AHA. FHA undergoes hydrolysis to formic acid which is volatile, thus allowing the recycling of nitric acid. Unfortunately, FHA powder was not stable in the experiments we ran in our laboratory. In addition, AHA and FHA also decompose to hydroxylamine which may undergo an autocatalytic reaction. Other reductants are available and could be extremely useful for actinides separation. The review presents the current plutonium reductants used in used nuclear fuel reprocessing and will introduce innovative and novel reductants that could become reducers for future research on UNF separation.« less

The negative influence of sperm cryopreservation on the quality and development of the embryo depends on the morphology of the oocyte.

PubMed

Braga, D P A F; Setti, A S; Figueira, R C S; Iaconelli, A; Borges, E

2015-07-01

The present case-control study aimed to identify the effect of sperm cryopreservation on the quality of the embryo and on the probability of blastocyst formation when oocytes free of dimorphisms are injected and when at least one dymorphism is present. The study included 22 186 zygotes, obtained from 2802 patients undergoing intracytoplasmic sperm injection cycles, in a private assisted reproduction center, using either fresh or cryopreserved sperm. The effect of sperm cryopreservation on the embryo quality on cleavage stage and blastocyst formation chance were evaluated when oocytes free of dimorphisms are injected and when at least one dymorphism is present. The quality of the embryo on cleavage stage as well as the chance for blastocyst formation was not influenced by the origin of the spermatozoa when the quality of the oocyte was not considered. When at least one oocyte defect was present, a negative influence of sperm cryopreservation on cleavage stage embryo quality and the chance for blastocyst formation was noted. In oocytes with extra-cytoplasmic dimorphisms, the injection of cryopreserved sperm did not affect the quality of the embryo during the cleavage stage, but did affect the chance for blastocyst formation. Conversely, in oocytes with intracytoplasmic defects, the quality of the embryos on cleavage stage and the chance of blastocyst formation were negatively influenced by the injection of cryopreserved sperm. The results suggest an oocyte quality-dependent negative effect of sperm cryopreservation on embryo quality and on the probability of blastocyst formation. © 2015 American Society of Andrology and European Academy of Andrology.

Tandem MS Analysis of Selenamide-Derivatized Peptide Ions

NASA Astrophysics Data System (ADS)

Zhang, Yun; Zhang, Hao; Cui, Weidong; Chen, Hao

2011-09-01

Our previous study showed that selenamide reagents such as ebselen and N-(phenylseleno)phthalimide (NPSP) can be used for selective and rapid derivatization of protein/peptide thiols in high conversion yield. This paper reports the systematic investigation of MS/MS dissociation behaviors of selenamide-derivatized peptide ions upon collision induced dissociation (CID) and electron transfer dissociation (ETD). In the positive ion mode, derivatized peptide ions exhibit tag-dependent CID dissociation pathways. For instance, ebselen-derivatized peptide ions preferentially undergo Se-S bond cleavage upon CID to produce a characteristic fragment ion, the protonated ebselen ( m/z 276), which allows selective identification of thiol peptides from protein digest as well as selective detection of thiol proteins from protein mixture using precursor ion scan (PIS). In contrast, NPSP-derivatized peptide ions retain their phenylselenenyl tags during CID, which is useful in sequencing peptides and locating cysteine residues. In the negative ion CID mode, both types of tags are preferentially lost via the Se-S cleavage, analogous to the S-S bond cleavage during CID of disulfide-containing peptide anions. In consideration of the convenience in preparing selenamide-derivatized peptides and the similarity of Se-S of the tag to the S-S bond, we also examined ETD of the derivatized peptide ions to probe the mechanism for electron-based ion dissociation. Interestingly, facile cleavage of Se-S bond occurs to the peptide ions carrying either protons or alkali metal ions, while backbone cleavage to form c/z ions is severely inhibited. These results are in agreement with the Utah-Washington mechanism proposed for depicting electron-based ion dissociation processes.

Splicing fidelity: DEAD/H-box ATPases as molecular clocks.

PubMed

Koodathingal, Prakash; Staley, Jonathan P

2013-07-01

The spliceosome discriminates against suboptimal substrates, both during assembly and catalysis, thereby enhancing specificity during pre-mRNA splicing. Central to such fidelity mechanisms are a conserved subset of the DEAD- and DEAH-box ATPases, which belong to a superfamily of proteins that mediate RNP rearrangements in almost all RNA-dependent processes in the cell. Through an investigation of the mechanisms contributing to the specificity of 5' splice site cleavage, two related reports, one from our lab and the other from the Cheng lab, have provided insights into fidelity mechanisms utilized by the spliceosome. In our work, we found evidence for a kinetic proofreading mechanism in splicing in which the DEAH-box ATPase Prp16 discriminates against substrates undergoing slow 5' splice site cleavage. Additionally, our study revealed that discriminated substrates are discarded through a general spliceosome disassembly pathway, mediated by another DEAH-box ATPase Prp43. In their work, Tseng et al. described the underlying molecular events through which Prp16 discriminates against a splicing substrate during 5' splice site cleavage. Here, we present a synthesis of these two studies and, additionally, provide the first biochemical evidence for discrimination of a suboptimal splicing substrate just prior to 5' splice site cleavage. Together, these findings support a general mechanism for a ubiquitous superfamily of ATPases in enhancing specificity during RNA-dependent processes in the cell.

Growth rates of the buoyancy-driven instability of an autocatalytic reaction front in a narrow cell

PubMed

Bockmann; Muller

2000-09-18

Experimental studies were performed on the buoyancy-driven instability of an autocatalytic reaction front in a quasi-2D cell. The unstable density stratification at an ascending front leads to convection that results in a fingerlike front deformation. The growth rates of the spatial modes of the instability are determined at the initial stage. A stabilization is found at higher wave numbers, while the system is unstable against low wave number perturbations. Whereas comparison with a reported model governed by Hele-Shaw flow fails, a two-dimensional Navier-Stokes model yields more satisfactory results. Still, present deviations suggest the presence of an additional mechanism that suppresses the growth.

The mitochondrial genome of fission yeast: inability of all introns to splice autocatalytically, and construction and characterization of an intronless genome.

PubMed

Schäfer, B; Merlos-Lange, A M; Anderl, C; Welser, F; Zimmer, M; Wolf, K

1991-01-01

In this paper we report the inability of four group I introns in the gene encoding subunit I of cytochrome c oxidase (cox1) and the group II intron in the apocytochrome b gene (cob) to splice autocatalytically. Furthermore we present the characterization of the first cox1 intron in the mutator strain anar-14 and the construction and characterization of strains with intronless mitochondrial genomes. We provide evidence that removal of introns at the DNA level (termed DNA splicing) is dependent on an active RNA maturase. Finally we demonstrate that the absence of introns does not abolish homologous mitochondrial recombination.

Minimal autocatalytic networks.

PubMed

Steel, Mike; Hordijk, Wim; Smith, Joshua

2013-09-07

Self-sustaining autocatalytic chemical networks represent a necessary, though not sufficient condition for the emergence of early living systems. These networks have been formalised and investigated within the framework of RAF theory, which has led to a number of insights and results concerning the likelihood of such networks forming. In this paper, we extend this analysis by focussing on how small autocatalytic networks are likely to be when they first emerge. First we show that simulations are unlikely to settle this question, by establishing that the problem of finding a smallest RAF within a catalytic reaction system is NP-hard. However, irreducible RAFs (irrRAFs) can be constructed in polynomial time, and we show it is possible to determine in polynomial time whether a bounded size set of these irrRAFs contain the smallest RAFs within a system. Moreover, we derive rigorous bounds on the sizes of small RAFs and use simulations to sample irrRAFs under the binary polymer model. We then apply mathematical arguments to prove a new result suggested by those simulations: at the transition catalysis level at which RAFs first form in this model, small RAFs are unlikely to be present. We also investigate further the relationship between RAFs and another formal approach to self-sustaining and closed chemical networks, namely chemical organisation theory (COT). Copyright © 2013 Elsevier Ltd. All rights reserved.

Fluorescence detection of thrombin using autocatalytic strand displacement cycle reaction and a dual-aptamer DNA sandwich assay.

PubMed

Niu, Shuyan; Qu, Lijing; Zhang, Qing; Lin, Jiehua

2012-02-15

A sensitive and specific sandwich assay for the detection of thrombin is described. Two affiliative aptamers were used to increase the assay specificity through sandwich recognition. Recognition DNA loaded on gold nanoparticles (AuNPs) partially hybridized with the initiator DNA, which was displaced by surviving DNA. After the initiator DNA was released into the solution, one hairpin structure was opened, which in turn opened another hairpin structure. The initiator DNA was displaced and released into the solution again by another hairpin structure because of the hybridized reaction. Then the released initiator DNA initiated another autocatalytic strand displacement reaction. A sophisticated network of three such duplex formation cycles was designed to amplify the fluorescence signal. Other proteins, such as bovine serum albumin and lysozyme, did not interfere with the detection of thrombin. This approach enables rapid and specific thrombin detection with reduced costs and minimized material consumption compared with traditional assay processes. The detection limit of thrombin was as low as 4.3 × 10⁻¹³ M based on the AuNP amplification and the autocatalytic strand displacement cycle reaction. This method could be used in biological samples with excellent selectivity. Copyright © 2011 Elsevier Inc. All rights reserved.

Near-IR Light-Cleavable Antibody Conjugates and Conjugate Precursors | NCI Technology Transfer Center | TTC

Cancer.gov

Researchers at the National Cancer Institute (NCI) developed novel groups of cyanine (Cy) based antibody-drug conjugate (ADC) chemical linkers that undergo photolytic cleavage upon irradiation with near-IR light. By using the fluorescent properties of the Cy linker to monitor localization of the ADC, and subsequent near-IR irradiation of cancerous tissue, drug release could be confined to the tumor microenvironment.

A New Replicator: A theoretical framework for analysing replication

PubMed Central

2010-01-01

Background Replicators are the crucial entities in evolution. The notion of a replicator, however, is far less exact than the weight of its importance. Without identifying and classifying multiplying entities exactly, their dynamics cannot be determined appropriately. Therefore, it is importance to decide the nature and characteristics of any multiplying entity, in a detailed and formal way. Results Replication is basically an autocatalytic process which enables us to rest on the notions of formal chemistry. This statement has major implications. Simple autocatalytic cycle intermediates are considered as non-informational replicators. A consequence of which is that any autocatalytically multiplying entity is a replicator, be it simple or overly complex (even nests). A stricter definition refers to entities which can inherit acquired changes (informational replicators). Simple autocatalytic molecules (and nests) are excluded from this group. However, in turn, any entity possessing copiable information is to be named a replicator, even multicellular organisms. In order to deal with the situation, an abstract, formal framework is presented, which allows the proper identification of various types of replicators. This sheds light on the old problem of the units and levels of selection and evolution. A hierarchical classification for the partition of the replicator-continuum is provided where specific replicators are nested within more general ones. The classification should be able to be successfully applied to known replicators and also to future candidates. Conclusion This paper redefines the concept of the replicator from a bottom-up theoretical approach. The formal definition and the abstract models presented can distinguish between among all possible replicator types, based on their quantity of variable and heritable information. This allows for the exact identification of various replicator types and their underlying dynamics. The most important claim is that replication, in general, is basically autocatalysis, with a specific defined environment and selective force. A replicator is not valid unless its working environment, and the selective force to which it is subject, is specified. PMID:20219099

Kinetics of Organic Transformations Under Mild Aqueous Conditions: Implications for the Origin of Life and Its Metabolism

NASA Technical Reports Server (NTRS)

Weber, Arthur L.

2003-01-01

The rates of thermal transformation of organic molecules containing carbon, hydrogen, and oxygen were systematically examined in order to identify the kinetic constraints that governed origin-of-life organic chemistry under mild aqueous conditions. Arrhenius plots of the kinetic data were used to estimate the reaction half-life at 50 C, and to reveal the effect of functional groups on reactivity. This survey showed that hydrocarbons and organic substances containing a single oxygenated group were kinetically the most stable (i. e. acetate decarboxylation half-life was l0(exp 18) years at 50 C); whereas, organic substances containing two oxygenated groups in which one group was a beta-positioned carbonyl group were the most reactive (i. e. acetoacetate decarboxylation half-life was l0(exp-2) years at 50 C). Of all functional groups the beta-positioned carbonyl group (aldehyde or ketone) was the strongest activating group, giving rates of reaction that were up to 10(exp 24)-times faster than rates of similar molecules lacking the beta-carbonyl group. From this knowledge of organic reactivity and the inherent constraints of autocatalytic processes, we concluded that an origins-of-life process based on autocatalytic transformation of C,H,O-substrates was constrained to using the most reactive organic molecules that contain alpha- or beta-carbonyl groups, since small autocatalytic domains of plausible catalytic power that used less reactive substrates could not carry out chemical transformations fast enough to prevent catastrophic efflux (escape) of reaction intermediates. Knowledge of the kinetics of organic transformations is useful, not only in constraining the chemistry of the earliest autocatalytic process related to the origin of life, but also in establishing the relative reactivity of organic molecules on the early Earth and other planets that may or may not be related to the origin of life.

Clinical outcome of fresh and vitrified-warmed blastocyst and cleavage-stage embryo transfers in ethnic Chinese ART patients

PubMed Central

2012-01-01

Objectives This study sought to evaluate the outcome of fresh and vitrified-warmed cleavage-stage and blastocyst-stage embryo transfers in patients undergoing ART treatment within an ethnic Chinese population. Study design We compared the clinical results of embryo transfer on the 3rd (cleavage stage) or 5th (blastocyst stage) day after oocyte retrieval, including clinical pregnancy rates, implantation rates and multiple pregnancy rates. Results Our data showed that blastocyst transfer on day 5 did not significantly increase clinical pregnancy rate (41.07% vs 47.08%, p>0.05) and implantation rate (31.8% vs 31.2%, p>0.05) in patients under 35 years of age, in comparison with day 3 cleavage stage embryo transfer. In patients older than 35 years of age, the clinical pregnancy rate after blastocyst transfer was slightly decreased compared with cleavage stage embryo transfer (33.33% vs 42.31%, p>0.05). Unexpectedly, It was found that vitrified-warmed blastocyst transfer resulted in significantly higher clinical pregnancy rate (56.8%) and implantation rate (47%) compared with fresh blastocyst transfer in controlled stimulation cycles (41.07% and 31.8%, respectively). For patients under 35 years of age, the cumulative clinical pregnancy rate combining fresh and vitrified-warmed blastocyst transfer cycles were significantly higher compared to just cleavage-stage embryo transfer (70.1% versus 51.8%, p<0.05). However, the cumulative multiple pregnancy rates showed no significant difference between the two groups. Conclusions In an ethnic Chinese patient population, fresh blastocyst transfer does not significantly increase clinical pregnancy rate. However, subsequent vitrified-warmed blastocyst transfer in a non-controlled ovarian hyperstimulation cycle dramatically improves clinical outcomes. Therefore, blastocyst culture in tandem with vitrified-warmed blastocyst transfer is recommended as a favourable and promising protocol in human ART treatment, particularly for ethnic Chinese patients. PMID:23039212

Clinical outcome of fresh and vitrified-warmed blastocyst and cleavage-stage embryo transfers in ethnic Chinese ART patients.

PubMed

Tong, Guo Qing; Cao, Shan Ren; Wu, Xun; Zhang, Jun Qiang; Cui, Ji; Heng, Boon Chin; Ling, Xiu Feng

2012-10-05

This study sought to evaluate the outcome of fresh and vitrified-warmed cleavage-stage and blastocyst-stage embryo transfers in patients undergoing ART treatment within an ethnic Chinese population. We compared the clinical results of embryo transfer on the 3rd (cleavage stage) or 5th (blastocyst stage) day after oocyte retrieval, including clinical pregnancy rates, implantation rates and multiple pregnancy rates. Our data showed that blastocyst transfer on day 5 did not significantly increase clinical pregnancy rate (41.07% vs 47.08%, p>0.05) and implantation rate (31.8% vs 31.2%, p>0.05) in patients under 35 years of age, in comparison with day 3 cleavage stage embryo transfer. In patients older than 35 years of age, the clinical pregnancy rate after blastocyst transfer was slightly decreased compared with cleavage stage embryo transfer (33.33% vs 42.31%, p>0.05). Unexpectedly, It was found that vitrified-warmed blastocyst transfer resulted in significantly higher clinical pregnancy rate (56.8%) and implantation rate (47%) compared with fresh blastocyst transfer in controlled stimulation cycles (41.07% and 31.8%, respectively). For patients under 35 years of age, the cumulative clinical pregnancy rate combining fresh and vitrified-warmed blastocyst transfer cycles were significantly higher compared to just cleavage-stage embryo transfer (70.1% versus 51.8%, p<0.05). However, the cumulative multiple pregnancy rates showed no significant difference between the two groups. In an ethnic Chinese patient population, fresh blastocyst transfer does not significantly increase clinical pregnancy rate. However, subsequent vitrified-warmed blastocyst transfer in a non-controlled ovarian hyperstimulation cycle dramatically improves clinical outcomes. Therefore, blastocyst culture in tandem with vitrified-warmed blastocyst transfer is recommended as a favourable and promising protocol in human ART treatment, particularly for ethnic Chinese patients.

Apoptotic microtubules delimit an active caspase free area in the cellular cortex during the execution phase of apoptosis.

PubMed

Oropesa-Ávila, M; Fernández-Vega, A; de la Mata, M; Maraver, J G; Cordero, M D; Cotán, D; de Miguel, M; Calero, C P; Paz, M V; Pavón, A D; Sánchez, M A; Zaderenko, A P; Ybot-González, P; Sánchez-Alcázar, J A

2013-03-07

Apoptotic microtubule network (AMN) is organized during apoptosis, forming a cortical structure beneath plasma membrane, which has an important role in preserving cell morphology and plasma membrane permeability. The aim of this study was to examine the role of AMN in maintaining plasma membrane integrity during the execution phase of apoptosis. We demonstrated in camptothecin-induced apoptosis in H460 cells that AMN delimits an active caspase free area beneath plasma membrane that permits the preservation of cellular cortex and transmembrane proteins. AMN depolymerization in apoptotic cells by a short exposure to colchicine allowed active caspases to reach the cellular cortex and cleave many key proteins involved in plasma membrane structural support, cell adhesion and ionic homeostasis. Cleavage of cellular cortex and plasma membrane proteins, such as α-spectrin, paxilin, focal adhesion kinase (FAK), E-cadherin and integrin subunit β4 was associated with cell collapse and cell detachment. Otherwise, cleavage-mediated inactivation of calcium ATPase pump (PMCA-4) and Na(+)/Ca(2+) exchanger (NCX) involved in cell calcium extrusion resulted in calcium overload. Furthermore, cleavage of Na(+)/K(+) pump subunit β was associated with altered sodium homeostasis. Cleavage of cell cortex and plasma membrane proteins in apoptotic cells after AMN depolymerization increased plasma permeability, ionic imbalance and bioenergetic collapse, leading apoptotic cells to secondary necrosis. The essential role of caspase-mediated cleavage in this process was demonstrated because the concomitant addition of colchicine that induces AMN depolymerization and the pan-caspase inhibitor z-VAD avoided the cleavage of cortical and plasma membrane proteins and prevented apoptotic cells to undergo secondary necrosis. Furthermore, the presence of AMN was also critical for proper phosphatidylserine externalization and apoptotic cell clearance by macrophages. These results indicate that AMN is essential to preserve an active caspase free area in the cellular cortex of apoptotic cells that allows plasma membrane integrity during the execution phase of apoptosis.

Apoptotic microtubules delimit an active caspase free area in the cellular cortex during the execution phase of apoptosis

PubMed Central

Oropesa-Ávila, M; Fernández-Vega, A; de la Mata, M; Maraver, J G; Cordero, M D; Cotán, D; de Miguel, M; Calero, C P; Paz, M V; Pavón, A D; Sánchez, M A; Zaderenko, A P; Ybot-González, P; Sánchez-Alcázar, J A

2013-01-01

Apoptotic microtubule network (AMN) is organized during apoptosis, forming a cortical structure beneath plasma membrane, which has an important role in preserving cell morphology and plasma membrane permeability. The aim of this study was to examine the role of AMN in maintaining plasma membrane integrity during the execution phase of apoptosis. We demonstrated in camptothecin-induced apoptosis in H460 cells that AMN delimits an active caspase free area beneath plasma membrane that permits the preservation of cellular cortex and transmembrane proteins. AMN depolymerization in apoptotic cells by a short exposure to colchicine allowed active caspases to reach the cellular cortex and cleave many key proteins involved in plasma membrane structural support, cell adhesion and ionic homeostasis. Cleavage of cellular cortex and plasma membrane proteins, such as α-spectrin, paxilin, focal adhesion kinase (FAK), E-cadherin and integrin subunit β4 was associated with cell collapse and cell detachment. Otherwise, cleavage-mediated inactivation of calcium ATPase pump (PMCA-4) and Na+/Ca2+ exchanger (NCX) involved in cell calcium extrusion resulted in calcium overload. Furthermore, cleavage of Na+/K+ pump subunit β was associated with altered sodium homeostasis. Cleavage of cell cortex and plasma membrane proteins in apoptotic cells after AMN depolymerization increased plasma permeability, ionic imbalance and bioenergetic collapse, leading apoptotic cells to secondary necrosis. The essential role of caspase-mediated cleavage in this process was demonstrated because the concomitant addition of colchicine that induces AMN depolymerization and the pan-caspase inhibitor z-VAD avoided the cleavage of cortical and plasma membrane proteins and prevented apoptotic cells to undergo secondary necrosis. Furthermore, the presence of AMN was also critical for proper phosphatidylserine externalization and apoptotic cell clearance by macrophages. These results indicate that AMN is essential to preserve an active caspase free area in the cellular cortex of apoptotic cells that allows plasma membrane integrity during the execution phase of apoptosis. PMID:23470534

The transmembrane domain of the p75 neurotrophin receptor stimulates phosphorylation of the TrkB tyrosine kinase receptor.

PubMed

Saadipour, Khalil; MacLean, Michael; Pirkle, Sean; Ali, Solav; Lopez-Redondo, Maria-Luisa; Stokes, David L; Chao, Moses V

2017-10-06

The function of protein products generated from intramembraneous cleavage by the γ-secretase complex is not well defined. The γ-secretase complex is responsible for the cleavage of several transmembrane proteins, most notably the amyloid precursor protein that results in Aβ, a transmembrane (TM) peptide. Another protein that undergoes very similar γ-secretase cleavage is the p75 neurotrophin receptor. However, the fate of the cleaved p75 TM domain is unknown. p75 neurotrophin receptor is highly expressed during early neuronal development and regulates survival and process formation of neurons. Here, we report that the p75 TM can stimulate the phosphorylation of TrkB (tyrosine kinase receptor B). In vitro phosphorylation experiments indicated that a peptide representing p75 TM increases TrkB phosphorylation in a dose- and time-dependent manner. Moreover, mutagenesis analyses revealed that a valine residue at position 264 in the rat p75 neurotrophin receptor is necessary for the ability of p75 TM to induce TrkB phosphorylation. Because this residue is just before the γ-secretase cleavage site, we then investigated whether the p75(αγ) peptide, which is a product of both α- and γ-cleavage events, could also induce TrkB phosphorylation. Experiments using TM domains from other receptors, EGFR and FGFR1, failed to stimulate TrkB phosphorylation. Co-immunoprecipitation and biochemical fractionation data suggested that p75 TM stimulates TrkB phosphorylation at the cell membrane. Altogether, our results suggest that TrkB activation by p75(αγ) peptide may be enhanced in situations where the levels of the p75 receptor are increased, such as during brain injury, Alzheimer's disease, and epilepsy. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

Clomiphene citrate is associated with favorable cycle characteristics but impaired outcomes of obese women with polycystic ovarian syndrome undergoing ovarian stimulation for in vitro fertilization

PubMed Central

Jiang, Shutian; Kuang, Yanping

2017-01-01

Abstract The aim of this study was to explore the effect of clomiphene citrate (CC) on the cycle characteristics and outcomes of obese women with polycystic ovarian syndrome (PCOS) undergoing ovarian stimulation for in vitro fertilization (IVF). This is a retrospective cohort study, and it was conducted at the tertiary-care academic medical center. This study included 174 obese PCOS patients undergoing IVF. In the study group (n = 90), CC and human menopausal gonadotropin (HMG) were administered simultaneously beginning on cycle day 3, while in control group (n = 84) HMG was used only. Both of the 2 groups used medroxyprogesterone acetate (MPA) for preventing premature luteinizing hormone (LH) surges. Ovulation was cotriggered by a GnRH agonist and hCG when dominant follicles matured. The primary outcome measure was the number of oocytes retrieved. Secondary outcomes included the number of top-quality embryos, maturation rate, fertilization rate, cleavage rate, incidence of premature LH surge, and OHSS. The study group received obviously lower total HMG dose [1650 (975–4800) vs 2025 (1350–3300) IU, P = 2.038E–4] but similar HMG duration. While the antral follicle count (AFC) is higher in study group, the number of oocytes retrieved and top-quality embryos are remarkably less [5 (0–30) vs 13 (0–42), P = 6.333E–5; 2 (0–14) vs 3.5 (0–15), P = .003; respectively]. The mature oocyte rate is higher in study group (P = .036). No significant differences were detected in fertilization rate and cleavage rate between 2 groups. CC has a positive influence on cycle characteristics, but might be correlated with the impaired IVF outcomes (less oocytes retrieved and top quality embryos, lower oocyte retrieval rate) in obese PCOS patients undergoing IVF, when HMG and MPA are used simultaneously. PMID:28796038

Implications of Biospheric Energization

NASA Astrophysics Data System (ADS)

Budding, Edd; Demircan, Osman; Gündüz, Güngör; Emin Özel, Mehmet

2016-07-01

Our physical model relating to the origin and development of lifelike processes from very simple beginnings is reviewed. This molecular ('ABC') process is compared with the chemoton model, noting the role of the autocatalytic tuning to the time-dependent source of energy. This substantiates a Darwinian character to evolution. The system evolves from very simple beginnings to a progressively more highly tuned, energized and complex responding biosphere, that grows exponentially; albeit with a very low net growth factor. Rates of growth and complexity in the evolution raise disturbing issues of inherent stability. Autocatalytic processes can include a fractal character to their development allowing recapitulative effects to be observed. This property, in allowing similarities of pattern to be recognized, can be useful in interpreting complex (lifelike) systems.

Resistance of Actin to Cleavage during Apoptosis

NASA Astrophysics Data System (ADS)

Song, Qizhong; Wei, Tie; Lees-Miller, Susan; Alnemri, Emad; Watters, Dianne; Lavin, Martin F.

1997-01-01

A small number of cellular proteins present in the nucleus, cytosol, and membrane fraction are specifically cleaved by the interleukin-1β -converting enzyme (ICE)-like family of proteases during apoptosis. Previous results have demonstrated that one of these, the cytoskeletal protein actin, is degraded in rat PC12 pheochromocytoma cells upon serum withdrawal. Extracts from etoposide-treated U937 cells are also capable of cleaving actin. It was assumed that cleavage of actin represented a general phenomenon, and a mechanism coordinating proteolytic, endonucleolytic, and morphological aspects of apoptosis was proposed. We demonstrate here that actin is resistant to degradation in several different human cells induced to undergo apoptosis in response to a variety of stimuli, including Fas ligation, serum withdrawal, cytotoxic T-cell killing, and DNA damage. On the other hand, cell-free extracts from these cells and the ICE-like protease CPP32 were capable of cleaving actin in vitro. We conclude that while actin contains cleavage sites for ICE-like proteases, it is not degraded in vivo in human cells either because of lack of access of these proteases to actin or due to the presence of other factors that prevent degradation.

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

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

Oxidative Metabolites of Curcumin Poison Human Type II Topoisomerases†

PubMed Central

Ketron, Adam C.; Gordon, Odaine N.; Schneider, Claus; Osheroff, Neil

2013-01-01

The polyphenol curcumin is the principal flavor and color component of the spice turmeric. Beyond its culinary uses, curcumin is believed to positively impact human health and displays antioxidant, anti-inflammatory, antibacterial, and chemopreventive properties. It also is in clinical trials as an anticancer agent. In aqueous solution at physiological pH, curcumin undergoes spontaneous autoxidation that is enhanced by oxidizing agents. The reaction proceeds through a series of quinone methide and other reactive intermediates to form a final dioxygenated bicyclopentadione product. Several naturally occurring polyphenols that can form quinones have been shown to act as topoisomerase II poisons (i.e., increase levels of topoisomerase II-mediated DNA cleavage). Because several of these compounds have chemopreventive properties, we determined the effects of curcumin, its oxidative metabolites, and structurally related degradation products (vanillin, ferulic acid, and feruloylmethane), on the DNA cleavage activities of human topoisomerase IIα and IIβ. Intermediates in the curcumin oxidation pathway increased DNA scission mediated by both enzymes ~4-5–fold. In contrast, curcumin and the bicyclopentadione, as well as vanillin, ferulic acid, and feruloylmethane, had no effect on DNA cleavage. As found for other quinone-based compounds, curcumin oxidation intermediates acted as redox-dependent (as opposed to interfacial) topoisomerase II poisons. Finally, under conditions that promote oxidation, the dietary spice turmeric enhanced topoisomerase II-mediated DNA cleavage. Thus, even within the more complex spice formulation, oxidized curcumin intermediates appear to function as topoisomerase II poisons. PMID:23253398

The Lys-Asp-Tyr Triad within the Mite Allergen Der p 1 Propeptide Is a Critical Structural Element for the pH-Dependent Initiation of the Protease Maturation.

PubMed

Chevigné, Andy; Campizi, Vincenzo; Szpakowska, Martyna; Bourry, David; Dumez, Marie-Eve; Martins, José C; Matagne, André; Galleni, Moreno; Jacquet, Alain

2017-05-20

The major house dust mite allergen, Der p 1, is a papain-like cysteine protease expressed as an inactive precursor, proDer p 1, carrying an N-terminal propeptide with a unique structure. The maturation of the zymogen into an enzymatically-active form of Der p 1 is a multistep autocatalytic process initiated under acidic conditions through conformational changes of the propeptide, leading to the loss of its inhibitory ability and its subsequent gradual cleavage. The aims of this study were to characterize the residues present in the Der p 1 propeptide involved in the initiation of the zymogen maturation process, but also to assess the impact of acidic pH on the propeptide structure, the activity of Der p 1 and the fate of the propeptide. Using various complementary enzymatic and structural approaches, we demonstrated that a structural triad K17p-D51p-Y19p within the N-terminal domain of the propeptide is essential for its stabilization and the sensing of pH changes. Particularly, the protonation of D51p under acidic conditions unfolds the propeptide through disruption of the K17p-D51p salt bridge, reduces its inhibition capacity and unmasks the buried residues K17p and Y19p constituting the first maturation cleavage site of the zymogen. Our results also evidenced that this triad acts in a cooperative manner with other propeptide pH-responsive elements, including residues E56p and E80p, to promote the propeptide unfolding and/or to facilitate its proteolysis. Furthermore, we showed that acidic conditions modify Der p 1 proteolytic specificity and confirmed that the formation of the first intermediate represents the limiting step of the in vitro Der p 1 maturation process. Altogether, our results provide new insights into the early events of the mechanism of proDer p 1 maturation and identify a unique structural triad acting as a stabilizing and a pH-sensing regulatory element.

Description of ground and excited electronic states by ensemble density functional method with extended active space

DOE PAGES

Filatov, Michael; Martínez, Todd J.; Kim, Kwang S.

2017-08-14

An extended variant of the spin-restricted ensemble-referenced Kohn-Sham (REKS) method, the REKS(4,4) method, designed to describe the ground electronic states of strongly multireference systems is modified to enable calculation of excited states within the time-independent variational formalism. The new method, the state-interaction state-averaged REKS(4,4), i.e., SI-SA-REKS(4,4), is capable of describing several excited states of a molecule involving double bond cleavage, polyradical character, or multiple chromophoric units.We demonstrate that the newmethod correctly describes the ground and the lowest singlet excited states of a molecule (ethylene) undergoing double bond cleavage. The applicability of the new method for excitonic states is illustrated withmore » π stacked ethylene and tetracene dimers. We conclude that the new method can describe a wide range of multireference phenomena.« less

Study of a two-stage photobase generator for photolithography in microelectronics.

PubMed

Turro, Nicholas J; Li, Yongjun; Jockusch, Steffen; Hagiwara, Yuji; Okazaki, Masahiro; Mesch, Ryan A; Schuster, David I; Willson, C Grant

2013-03-01

The investigation of the photochemistry of a two-stage photobase generator (PBG) is described. Absorption of a photon by a latent PBG (1) (first step) produces a PBG (2). Irradiation of 2 in the presence of water produces a base (second step). This two-photon sequence (1 + hν → 2 + hν → base) is an important component in the design of photoresists for pitch division technology, a method that doubles the resolution of projection photolithography for the production of microelectronic chips. In the present system, the excitation of 1 results in a Norrish type II intramolecular hydrogen abstraction to generate a 1,4-biradiacal that undergoes cleavage to form 2 and acetophenone (Φ ∼ 0.04). In the second step, excitation of 2 causes cleavage of the oxime ester (Φ = 0.56) followed by base generation after reaction with water.

Description of ground and excited electronic states by ensemble density functional method with extended active space

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

Filatov, Michael; Martínez, Todd J.; Kim, Kwang S.

An extended variant of the spin-restricted ensemble-referenced Kohn-Sham (REKS) method, the REKS(4,4) method, designed to describe the ground electronic states of strongly multireference systems is modified to enable calculation of excited states within the time-independent variational formalism. The new method, the state-interaction state-averaged REKS(4,4), i.e., SI-SA-REKS(4,4), is capable of describing several excited states of a molecule involving double bond cleavage, polyradical character, or multiple chromophoric units.We demonstrate that the newmethod correctly describes the ground and the lowest singlet excited states of a molecule (ethylene) undergoing double bond cleavage. The applicability of the new method for excitonic states is illustrated withmore » π stacked ethylene and tetracene dimers. We conclude that the new method can describe a wide range of multireference phenomena.« less

A kinetic study on the chemical cleavage of nucleoside diphosphate sugars.

PubMed

Huhta, Eija; Parjanen, Atte; Mikkola, Satu

2010-03-30

Nucleoside diphosphate sugars serve in essential roles in metabolic processes. They have, therefore, been used in mechanistic studies on glycosylation reactions, and their analogues have been synthesised as enzyme and receptor inhibitors. Despite extensive biochemical research, little is known about their chemical reactions. In the present work the chemical cleavage of two different types of nucleoside diphosphate sugars has been studied. UDP-Glc is phosphorylated at the anomeric carbon, whereas in ADP-Rib C-1 is unsubstituted, allowing hence the equilibrium between cyclic hemiacetal and acyclic carbonyl forms. Due to the structural difference, these substrates react via different pathways under slightly alkaline conditions: while UDP-Glc reacts exclusively by a nucleophilic attack of a glucose hydroxyl group on the diphosphate moiety, ADP-Rib undergoes a complex reaction sequence that involves isomerisation processes of the acyclic ribose sugar and results in a release of ADP. Copyright 2009 Elsevier Ltd. All rights reserved.

Endogenous peptide profile for elucidating biosynthetic processing of the ghrelin precursor.

PubMed

Tsuchiya, Takashi; Iwakura, Hiroshi; Minamino, Naoto; Kangawa, Kenji; Sasaki, Kazuki

2017-09-02

Ghrelin is an orexigenic peptide primarily produced by gastric endocrine cells. The biosynthetic cleavage site of ghrelin has been well documented, but how its downstream region undergoes proteolytic processing remains poorly explored. Here, we provide the first snapshot of endogenous peptides from the ghrelin precursor by profiling the secretopeptidome of cultured mouse ghrelin-producing cells during exocytosis. Mapping of MS/MS sequenced peptides to the precursor highlighted three atypical monobasic processing sites, including the established C-terminus of ghrelin and the N-terminal cleavage site for obestatin, a putative 23-amino-acid C-terminally amidated peptide. However, we found that mouse obestatin does not occur in the form originally reported, but that a different amidation site is used to generate a shorter peptide. These data can be extended to study and characterize the precursor-derived peptides located downstream of ghrelin in different biological contexts. Copyright © 2017 Elsevier Inc. All rights reserved.

Prebiotic Synthesis of Autocatalytic Products From Formaldehyde-Derived Sugars as the Carbon and Energy Source

NASA Technical Reports Server (NTRS)

Weber, Arthur L.

2003-01-01

Our research objective is to understand and model the chemical processes on the primitive Earth that generated the first autocatalytic molecules and microstructures involved in the origin of life. Our approach involves: (a) investigation of a model origin-of-life process named the Sugar Model that is based on the reaction of formaldehyde- derived sugars (trioses and tetroses) with ammonia, and (b) elucidation of the constraints imposed on the chemistry of the origin of life by the fixed energies and rates of C,H,O-organic reactions under mild aqueous conditions. Recently, we demonstrated that under mild aqueous conditions the Sugar Model process yields autocatalytic products, and generates organic micropherules (2-20 micron dia.) that exhibit budding, size uniformity, and chain formation. We also discovered that the sugar substrates of the Sugar Model are capable of reducing nitrite to ammonia under mild aqueous conditions. In addition studies done in collaboration with Sandra Pizzarrello (Arizona State University) revealed that chiral amino acids (including meteoritic isovaline) catalyze both the synthesis and specific handedness of chiral sugars. Our systematic survey of the energies and rates of reactions of C,H,O-organic substrates under mild aqueous conditions revealed several general principles (rules) that govern the direction and rate of organic reactions. These reactivity principles constrain the structure of chemical pathways used in the origin of life, and in modern and primitive metabolism.

The behaviour of basic autocatalytic signalling modules in isolation and embedded in networks

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

Krishnan, J.; Mois, Kristina; Suwanmajo, Thapanar

2014-11-07

In this paper, we examine the behaviour of basic autocatalytic feedback modules involving a species catalyzing its own production, either directly or indirectly. We first perform a systematic study of the autocatalytic feedback module in isolation, examining the effect of different factors, showing how this module is capable of exhibiting monostable threshold and bistable switch-like behaviour. We then study the behaviour of this module embedded in different kinds of basic networks including (essentially) irreversible cycles, open and closed reversible chains, and networks with additional feedback. We study the behaviour of the networks deterministically and also stochastically, using simulations, analytical work,more » and bifurcation analysis. We find that (i) there are significant differences between the behaviour of this module in isolation and in a network: thresholds may be altered or destroyed and bistability may be destroyed or even induced, even when the ambient network is simple. The global characteristics and topology of this network and the position of the module in the ambient network can play important and unexpected roles. (ii) There can be important differences between the deterministic and stochastic dynamics of the module embedded in networks, which may be accentuated by the ambient network. This provides new insights into the functioning of such enzymatic modules individually and as part of networks, with relevance to other enzymatic signalling modules as well.« less

Microwave Enhancement of Autocatalytic Growth of Nanometals.

PubMed

Ashley, Bridgett; Vakil, Parth N; Lynch, Brian B; Dyer, Christopher M; Tracy, Joseph B; Owens, Jeffery; Strouse, Geoffrey F

2017-10-24

The desire for designing efficient synthetic methods that lead to industrially important nanomaterials has led a desire to more fully understand the mechanism of growth and how modern synthetic techniques can be employed. Microwave (MW) synthesis is one such technique that has attracted attention as a green, sustainable method. The reports of enhancement of formation rates and improved quality for MW driven reactions are intriguing, but the lack of understanding of the reaction mechanism and how coupling to the MW field leads to these observations is concerning. In this manuscript, the growth of a metal nanoparticles (NPs) in a microwave cavity is spectroscopically analyzed and compared with the classical autocatalytic method of NP growth to elucidate the underpinnings for the observed enhanced growth behavior for metal NPs prepared in a MW field. The study illustrates that microwave synthesis of nickel and gold NPs below saturation conditions follows the Finke-Watzky mechanism of nucleation and growth. The enhancement of the reaction arises from the size-dependent increase in MW absorption cross section for the metal NPs. For Ni, the presence of oxides is considered via theoretical computations and compared to dielectric measurements of isolated nickel NPs. The study definitively shows that MW growth can be modeled by an autocatalytic mechanism that directly leads to the observed enhanced rates and improved quality widely reported in the nanomaterial community when MW irradiation is employed.

Rapid autocatalytic activation of the M4 metalloprotease aureolysin is controlled by a conserved N-terminal fungalysin-thermolysin-propeptide domain.

PubMed

Nickerson, Nicholas N; Joag, Vineet; McGavin, Martin J

2008-09-01

The Staphylococcus aureus proteolytic cascade consists of a metalloprotease aureolysin (Aur), which activates a serine protease zymogen proSspA, which in turn activates the SspB cysteine protease. As with other M4 metalloproteases, including elastase of Pseudomonas aeruginosa, the propeptide of proAur contains an N-terminal fungalysin-thermolysin-propeptide (FTP) domain. Autocatalytic activation of proAur was initiated by processing at T85 downward arrowL(86) in the FTP domain. This differed from the mechanism described for proElastase, where the FTP domain has an RY motif in place of TL(86), and processing occurred at the junction of the propeptide and metalloprotease domains, which remained as an inactive complex during passage across the outer membrane. When TL(86) in the FTP domain was replaced with RY, an intact N-terminal propeptide was secreted, but the M4 metalloprotease domain was degraded. Consequently, this segment of the FTP domain promotes intramolecular processing of proAur while bestowing a chaperone function, but discourages processing within the FTP domain of proElastase, where activation must be co-ordinated with passage across a second membrane. We conclude that the FTP domain of proAur is adapted to facilitate a rapid autocatalytic activation mechanism, consistent with the role or proAur as initiator of the staphylococcal proteolytic cascade.

The behaviour of basic autocatalytic signalling modules in isolation and embedded in networks

NASA Astrophysics Data System (ADS)

Krishnan, J.; Mois, Kristina; Suwanmajo, Thapanar

2014-11-01

In this paper, we examine the behaviour of basic autocatalytic feedback modules involving a species catalyzing its own production, either directly or indirectly. We first perform a systematic study of the autocatalytic feedback module in isolation, examining the effect of different factors, showing how this module is capable of exhibiting monostable threshold and bistable switch-like behaviour. We then study the behaviour of this module embedded in different kinds of basic networks including (essentially) irreversible cycles, open and closed reversible chains, and networks with additional feedback. We study the behaviour of the networks deterministically and also stochastically, using simulations, analytical work, and bifurcation analysis. We find that (i) there are significant differences between the behaviour of this module in isolation and in a network: thresholds may be altered or destroyed and bistability may be destroyed or even induced, even when the ambient network is simple. The global characteristics and topology of this network and the position of the module in the ambient network can play important and unexpected roles. (ii) There can be important differences between the deterministic and stochastic dynamics of the module embedded in networks, which may be accentuated by the ambient network. This provides new insights into the functioning of such enzymatic modules individually and as part of networks, with relevance to other enzymatic signalling modules as well.

The Coxsackievirus and Adenovirus Receptor (CAR) Undergoes Ectodomain Shedding and Regulated Intramembrane Proteolysis (RIP)

PubMed Central

Houri, Nadia; Huang, Kuo-Cheng; Nalbantoglu, Josephine

2013-01-01

The Coxsackievirus and Adenovirus Receptor (CAR) is a cell adhesion molecule originally characterized as a virus receptor but subsequently shown to be involved in physiological processes such as neuronal and heart development, epithelial tight junction integrity, and tumour suppression. Proteolysis of cell adhesion molecules and a wide variety of other cell surface proteins serves as a mechanism for protein turnover and, in some cases, cell signaling. Metalloproteases such as A Disintegrin and Metalloprotease (ADAM) family members cleave cell surface receptors to release their substrates’ ectodomains, while the presenilin/ɣ-secretase complex mediates regulated intramembrane proteolysis (RIP), releasing intracellular domain fragments from the plasma membrane. In the case of some substrates such as Notch and amyloid precursor protein (APP), the released intracellular domains enter the nucleus to modulate gene expression. We report that CAR ectodomain is constitutively shed from glioma cells and developing neurons, and is also shed when cells are treated with the phorbol ester phorbol 12-myristate 13-acetate (PMA) and the calcium ionophore ionomycin. We identified ADAM10 as a sheddase of CAR using assays involving shRNA knockdown and rescue, overexpression of wild-type ADAM10 and inhibition of ADAM10 activity by addition of its prodomain. In vitro peptide cleavage, mass spectrometry and mutagenesis revealed the amino acids M224 to L227 of CAR as the site of ADAM10-mediated ectodomain cleavage. CAR also undergoes RIP by the presenilin/γ-secretase complex, and the intracellular domain of CAR enters the nucleus. Ectodomain shedding is a prerequisite for RIP of CAR. Thus, CAR belongs to the increasing list of cell surface molecules that undergo ectodomain shedding and that are substrates for ɣ-secretase-mediated RIP. PMID:24015300

Effect of the Phosphine Steric and Electronic Profile on the Rh-Promoted Dehydrocoupling of Phosphine–Boranes

PubMed Central

2014-01-01

The electronic and steric effects in the stoichiometric dehydrocoupling of secondary and primary phosphine–boranes H3B·PR2H [R = 3,5-(CF3)2C6H3; p-(CF3)C6H4; p-(OMe)C6H4; adamantyl, Ad] and H3B·PCyH2 to form the metal-bound linear diboraphosphines H3B·PR2BH2·PR2H and H3B·PRHBH2·PRH2, respectively, are reported. Reaction of [Rh(L)(η6-FC6H5)][BArF4] [L = Ph2P(CH2)3PPh2, ArF = 3,5-(CF3)2C6H3] with 2 equiv of H3B·PR2H affords [Rh(L)(H)(σ,η-PR2BH3)(η1-H3B·PR2H)][BArF4]. These complexes undergo dehydrocoupling to give the diboraphosphine complexes [Rh(L)(H)(σ,η2-PR2·BH2PR2·BH3)][BArF4]. With electron-withdrawing groups on the phosphine–borane there is the parallel formation of the products of B–P cleavage, [Rh(L)(PR2H)2][BArF4], while with electron-donating groups no parallel product is formed. For the bulky, electron rich, H3B·P(Ad)2H no dehydrocoupling is observed, but an intermediate Rh(I) σ phosphine–borane complex is formed, [Rh(L){η2-H3B·P(Ad)2H}][BArF4], that undergoes B–P bond cleavage to give [Rh(L){η1-H3B·P(Ad)2H}{P(Ad)2H}][BArF4]. The relative rates of dehydrocoupling of H3B·PR2H (R = aryl) show that increasingly electron-withdrawing substituents result in faster dehydrocoupling, but also suffer from the formation of the parallel product resulting from P–B bond cleavage. H3B·PCyH2 undergoes a similar dehydrocoupling process, and gives a mixture of stereoisomers of the resulting metal-bound diboraphosphine that arise from activation of the prochiral P–H bonds, with one stereoisomer favored. This diastereomeric mixture may also be biased by use of a chiral phosphine ligand. The selectivity and efficiencies of resulting catalytic dehydrocoupling processes are also briefly discussed. PMID:24617924

Diastereoselective sp2-sp3 coupling of sugar enol ethers with unactivated cycloalkenes: new entries to C-branched sugars.

PubMed

Hussain, Nazar; Tatina, Madhu Babu; Rasool, Faheem; Mukherjee, Debaraj

2016-10-25

Sugar enol ethers undergo efficient coupling at C-2 with unactivated cycloalkenes under a low Pd loading affording allylic substitution products. High diastereoselectivity was observed at the allylic centre with sterically hindered substrates. Generation of a π-allyl complex by the Pd(ii) catalyst via cleavage of the allylic C-H bond of the cycloalkene may be responsible for the formation of sp 2 -sp 3 coupling products.

Perfluorinated Ligands in Organometallic Chemistry

DTIC Science & Technology

1989-12-12

C49t00ooVER ,or C M’ AD"OV’~mDecember 12) 199IFinal 1/1/86 to 8/31/89C smuS. FUNOING NUMgIERS cJ Perfluorinated Ligands in Organometallic Chemistry 612...compounds, stabilized by tridentate perfluorinated ligands. Dinuclear rhodium complexes of OFCOT undergo a selective C-F bond activation reaction...hexafluorocyclooctatrieneyne ligand. Stereospecific cleavage of a fluorinated C-C bond,#-bond in perfluorocyclopropene by platinum and iridium complexes has been achieved

Regioselective and stereospecific cleavage of a terminal oxirane system: a novel synthetic approach to lipid mediator congeners--1,2(2,3)-diacyl-3(1)-halo-sn-glycerols.

PubMed

Stamatov, Stephan D; Stawinski, Jacek

2006-07-01

Glycidyl esters upon treatment with a mixture of carboxylic acid anhydride (CAA) and trimethylsilyl halide (TMSX) in the presence of tetra-n-butylammonium halide (Bu(4)NX, X=Cl, Br or I) undergo stereospecific and regioselective opening of the oxirane ring to afford mixed-(or mono)-acid 1,2(2,3)-diacyl-3(1)-halo-sn-glycerols in high yields.

40 CFR 413.71 - Specialized definitions.

Code of Federal Regulations, 2012 CFR

2012-07-01

... deposition of conductive material from an autocatalytic plating solution without application of electrical current. (c) The term operation shall mean any step in the electroless plating process in which a metal is...

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

Decay of mRNAs targeted by RISC requires XRN1, the Ski complex, and the exosome

PubMed Central

ORBAN, TAMAS I.; IZAURRALDE, ELISA

2005-01-01

RNA interference (RNAi) is a conserved RNA silencing pathway that leads to sequence-specific mRNA decay in response to the presence of double-stranded RNA (dsRNA). Long dsRNA molecules are first processed by Dicer into 21–22-nucleotide small interfering RNAs (siRNAs). The siRNAs are incorporated into a multimeric RNA-induced silencing complex (RISC) that cleaves mRNAs at a site determined by complementarity with the siRNAs. Following this initial endonucleolytic cleavage, the mRNA is degraded by a mechanism that is not completely understood. We investigated the decay pathway of mRNAs targeted by RISC in Drosophila cells. We show that 5′ mRNA fragments generated by RISC cleavage are rapidly degraded from their 3′ ends by the exosome, whereas the 3′ fragments are degraded from their 5′ ends by XRN1. Exosome-mediated decay of the 5′ fragments requires the Drosophila homologs of yeast Ski2p, Ski3p, and Ski8p, suggesting that their role as regulators of exosome activity is conserved. Our findings indicate that mRNAs targeted by siRNAs are degraded from the ends generated by RISC cleavage, without undergoing decapping or deadenylation. PMID:15703439

Oxidatively Generated Guanine(C8)-Thymine(N3) Intrastrand Cross-links in Double-stranded DNA Are Repaired by Base Excision Repair Pathways.

PubMed

Talhaoui, Ibtissam; Shafirovich, Vladimir; Liu, Zhi; Saint-Pierre, Christine; Akishev, Zhiger; Matkarimov, Bakhyt T; Gasparutto, Didier; Geacintov, Nicholas E; Saparbaev, Murat

2015-06-05

Oxidatively generated guanine radical cations in DNA can undergo various nucleophilic reactions including the formation of C8-guanine cross-links with adjacent or nearby N3-thymines in DNA in the presence of O2. The G*[C8-N3]T* lesions have been identified in the DNA of human cells exposed to oxidative stress, and are most likely genotoxic if not removed by cellular defense mechanisms. It has been shown that the G*[C8-N3]T* lesions are substrates of nucleotide excision repair in human cell extracts. Cleavage at the sites of the lesions was also observed but not further investigated (Ding et al. (2012) Nucleic Acids Res. 40, 2506-2517). Using a panel of eukaryotic and prokaryotic bifunctional DNA glycosylases/lyases (NEIL1, Nei, Fpg, Nth, and NTH1) and apurinic/apyrimidinic (AP) endonucleases (Apn1, APE1, and Nfo), the analysis of cleavage fragments by PAGE and MALDI-TOF/MS show that the G*[C8-N3]T* lesions in 17-mer duplexes are incised on either side of G*, that none of the recovered cleavage fragments contain G*, and that T* is converted to a normal T in the 3'-fragment cleavage products. The abilities of the DNA glycosylases to incise the DNA strand adjacent to G*, while this base is initially cross-linked with T*, is a surprising observation and an indication of the versatility of these base excision repair proteins. © 2015 by The American Society for Biochemistry and Molecular Biology, Inc.

Oxidatively Generated Guanine(C8)-Thymine(N3) Intrastrand Cross-links in Double-stranded DNA Are Repaired by Base Excision Repair Pathways*

PubMed Central

Talhaoui, Ibtissam; Shafirovich, Vladimir; Liu, Zhi; Saint-Pierre, Christine; Akishev, Zhiger; Matkarimov, Bakhyt T.; Gasparutto, Didier; Geacintov, Nicholas E.; Saparbaev, Murat

2015-01-01

Oxidatively generated guanine radical cations in DNA can undergo various nucleophilic reactions including the formation of C8-guanine cross-links with adjacent or nearby N3-thymines in DNA in the presence of O2. The G*[C8-N3]T* lesions have been identified in the DNA of human cells exposed to oxidative stress, and are most likely genotoxic if not removed by cellular defense mechanisms. It has been shown that the G*[C8-N3]T* lesions are substrates of nucleotide excision repair in human cell extracts. Cleavage at the sites of the lesions was also observed but not further investigated (Ding et al. (2012) Nucleic Acids Res. 40, 2506–2517). Using a panel of eukaryotic and prokaryotic bifunctional DNA glycosylases/lyases (NEIL1, Nei, Fpg, Nth, and NTH1) and apurinic/apyrimidinic (AP) endonucleases (Apn1, APE1, and Nfo), the analysis of cleavage fragments by PAGE and MALDI-TOF/MS show that the G*[C8-N3]T* lesions in 17-mer duplexes are incised on either side of G*, that none of the recovered cleavage fragments contain G*, and that T* is converted to a normal T in the 3′-fragment cleavage products. The abilities of the DNA glycosylases to incise the DNA strand adjacent to G*, while this base is initially cross-linked with T*, is a surprising observation and an indication of the versatility of these base excision repair proteins. PMID:25903131

Partial synchronization of relaxation oscillators with repulsive coupling in autocatalytic integrate-and-fire model and electrochemical experiments

NASA Astrophysics Data System (ADS)

Kori, Hiroshi; Kiss, István Z.; Jain, Swati; Hudson, John L.

2018-04-01

Experiments and supporting theoretical analysis are presented to describe the synchronization patterns that can be observed with a population of globally coupled electrochemical oscillators close to a homoclinic, saddle-loop bifurcation, where the coupling is repulsive in the electrode potential. While attractive coupling generates phase clusters and desynchronized states, repulsive coupling results in synchronized oscillations. The experiments are interpreted with a phenomenological model that captures the waveform of the oscillations (exponential increase) followed by a refractory period. The globally coupled autocatalytic integrate-and-fire model predicts the development of partially synchronized states that occur through attracting heteroclinic cycles between out-of-phase two-cluster states. Similar behavior can be expected in many other systems where the oscillations occur close to a saddle-loop bifurcation, e.g., with Morris-Lecar neurons.

A cascade autocatalytic strand displacement amplification and hybridization chain reaction event for label-free and ultrasensitive electrochemical nucleic acid biosensing.

PubMed

Chen, Zhiqiang; Liu, Ying; Xin, Chen; Zhao, Jikuan; Liu, Shufeng

2018-08-15

Herein, an autocatalytic strand displacement amplification (ASDA) strategy was proposed for the first time, which was further ingeniously coupled with hybridization chain reaction (HCR) event for the isothermal, label-free and multiple amplification toward nucleic acid detection. During the ASDA module, the target recognition opens the immobilized hairpin probe (IP) and initiates the annealing of the auxiliary DNA strand (AS) with the opened IP for the successive polymerization and nicking reaction in the presence of DNA polymerase and nicking endonuclease. This induces the target recycling and generation of a large amount of intermediate DNA sequences, which can be used as target analogy to execute the autocatalytic strand displacement amplification. Simultaneously, the introduced AS strand can propagate the HCR between two hairpins (H1 and H2) to form a linear DNA concatamer with cytosine (C)-rich loop region, which can facilitate the in-situ synthesis of silver nanoclusters (AgNCs) as electrochemical tags for further amplification toward target responses. With current cascade ASDA and HCR strategy, the detection of target DNA could be achieved with a low detection limit of about 0.16 fM and a good selectivity. The developed biosensor also exhibits the distinct advantages of flexibility and simplicity in probe design and biosensor fabrication, and label-free electrochemical detection, thus opens a promising avenue for the detection of nucleic acid with low abundance in bioanalysis and clinical biomedicine. Copyright © 2018 Elsevier B.V. All rights reserved.

Competition between Primary Nucleation and Autocatalysis in Amyloid Fibril Self-Assembly

PubMed Central

Eden, Kym; Morris, Ryan; Gillam, Jay; MacPhee, Cait E.; Allen, Rosalind J.

2015-01-01

Kinetic measurements of the self-assembly of proteins into amyloid fibrils are often used to make inferences about molecular mechanisms. In particular, the lag time—the quiescent period before aggregates are detected—is often found to scale with the protein concentration as a power law, whose exponent has been used to infer the presence or absence of autocatalytic growth processes such as fibril fragmentation. Here we show that experimental data for lag time versus protein concentration can show signs of kinks: clear changes in scaling exponent, indicating changes in the dominant molecular mechanism determining the lag time. Classical models for the kinetics of fibril assembly suggest that at least two mechanisms are at play during the lag time: primary nucleation and autocatalytic growth. Using computer simulations and theoretical calculations, we investigate whether the competition between these two processes can account for the kinks which we observe in our and others’ experimental data. We derive theoretical conditions for the crossover between nucleation-dominated and growth-dominated regimes, and analyze their dependence on system volume and autocatalysis mechanism. Comparing these predictions to the data, we find that the experimentally observed kinks cannot be explained by a simple crossover between nucleation-dominated and autocatalytic growth regimes. Our results show that existing kinetic models fail to explain detailed features of lag time versus concentration curves, suggesting that new mechanistic understanding is needed. More broadly, our work demonstrates that care is needed in interpreting lag-time scaling exponents from protein assembly data. PMID:25650930

Investigating an Evolutionarily Conserved Role for the Tousled-like Kinase in Genome Stability and as a Novel Target for the Treatment of Ovarian Cancer

DTIC Science & Technology

2013-10-01

Approved OMB No. 0704-0188 Public reporting burden for this collection of information is estimated to average 1 hour per response, including the...cleavage plane during cytokinesis (15). The anteroposterior (AP) axis of the one- cell embryo is determined at fertilization by the sperm entry point, which...demarcates the posterior pole of the embryo (16). Upon sperm entry, the anteriorly-localized maternal nucleus undergoes two meiotic divisions to

Chemical Principles Exemplified

ERIC Educational Resources Information Center

Plumb, Robert C.

1972-01-01

Collection of two short descriptions of chemical principles seen in life situations: the autocatalytic reaction seen in the bombardier beetle, and molecular potential energy used for quick roasting of beef. Brief reference is also made to methanol lighters. (PS)

TGFbeta regulation of membrane mucin Muc4 via proteosome degradation.

PubMed

Lomako, Wieslawa M; Lomako, Joseph; Soto, Pedro; Carraway, Coralie A Carothers; Carraway, Kermit L

2009-07-01

Muc4 is a heterodimeric membrane mucin implicated in epithelial differentiation and tumor progression. It is expressed from a single gene as a 300 kDa precursor protein which is cleaved in the endoplasmic reticulum to its two subunits. Our previous work has shown that Muc4 is regulated by TGFbeta, which represses the precursor cleavage. Working with Muc4-transfected A375 tumor cells, we now show that Muc4 undergoes proteosomal degradation. Proteosome inhibitors prolong the life of the precursor, shunt the Muc4 into cytoplasmic aggresomes, increase the level of Muc4 associated with the endoplasmic reticulum chaperones calnexin and calreticulin and increase the levels of ubiquitinated Muc4. Most importantly, proteosome inhibitors repress the TGFbeta inhibition of Muc4 expression. These results suggest a model in which TGFbeta inhibits precursor cleavage, shunting the precursor into the proteosomal degradation pathway. Thus, the cells have evolved a mechanism to use the quality control pathway for glycoproteins to control the quantity of the protein produced. 2009 Wiley-Liss, Inc.

Integrin trafficking regulated by Rab21 is necessary for cytokinesis.

PubMed

Pellinen, Teijo; Tuomi, Saara; Arjonen, Antti; Wolf, Maija; Edgren, Henrik; Meyer, Hannelore; Grosse, Robert; Kitzing, Thomas; Rantala, Juha K; Kallioniemi, Olli; Fässler, Reinhard; Kallio, Marko; Ivaska, Johanna

2008-09-01

Adherent cells undergo remarkable changes in shape during cell division. However, the functional interplay between cell adhesion turnover and the mitotic machinery is poorly understood. The endo/exocytic trafficking of integrins is regulated by the small GTPase Rab21, which associates with several integrin alpha subunits. Here, we show that targeted trafficking of integrins to and from the cleavage furrow is required for successful cytokinesis, and that this is regulated by Rab21. Rab21 activity, integrin-Rab21 association, and integrin endocytosis are all necessary for normal cytokinesis, which becomes impaired when integrin-mediated adhesion at the cleavage furrow fails. We also describe a chromosomal deletion and loss of Rab21 gene expression in human cancer, which leads to the accumulation of multinucleate cells. Importantly, reintroduction of Rab21 rescued this phenotype. In conclusion, Rab21-regulated integrin trafficking is essential for normal cell division, and its defects may contribute to multinucleation and genomic instability, which are hallmarks of cancer.

A Stable Bacterial Peroxidase with Novel Halogenating Activity and an Autocatalytically Linked Heme Prosthetic Group*

PubMed Central

Auer, Markus; Gruber, Clemens; Bellei, Marzia; Pirker, Katharina F.; Zamocky, Marcel; Kroiss, Daniela; Teufer, Stefan A.; Hofbauer, Stefan; Soudi, Monika; Battistuzzi, Gianantonio; Furtmüller, Paul G.; Obinger, Christian

2013-01-01

Reconstructing the phylogenetic relationships of the main evolutionary lines of the mammalian peroxidases lactoperoxidase and myeloperoxidase revealed the presence of novel bacterial heme peroxidase subfamilies. Here, for the first time, an ancestral bacterial heme peroxidase is shown to possess a very high bromide oxidation activity (besides conventional peroxidase activity). The recombinant protein allowed monitoring of the autocatalytic peroxide-driven formation of covalent heme to protein bonds. Thereby, the high spin ferric rhombic heme spectrum became similar to lactoperoxidase, the standard reduction potential of the Fe(III)/Fe(II) couple shifted to more positive values (−145 ± 10 mV at pH 7), and the conformational and thermal stability of the protein increased significantly. We discuss structure-function relationships of this new peroxidase in relation to its mammalian counterparts and ask for its putative physiological role. PMID:23918925

Sequence selection by dynamical symmetry breaking in an autocatalytic binary polymer model

NASA Astrophysics Data System (ADS)

Fellermann, Harold; Tanaka, Shinpei; Rasmussen, Steen

2017-12-01

Template-directed replication of nucleic acids is at the essence of all living beings and a major milestone for any origin of life scenario. We present an idealized model of prebiotic sequence replication, where binary polymers act as templates for their autocatalytic replication, thereby serving as each others reactants and products in an intertwined molecular ecology. Our model demonstrates how autocatalysis alters the qualitative and quantitative system dynamics in counterintuitive ways. Most notably, numerical simulations reveal a very strong intrinsic selection mechanism that favors the appearance of a few population structures with highly ordered and repetitive sequence patterns when starting from a pool of monomers. We demonstrate both analytically and through simulation how this "selection of the dullest" is caused by continued symmetry breaking through random fluctuations in the transient dynamics that are amplified by autocatalysis and eventually propagate to the population level. The impact of these observations on related prebiotic mathematical models is discussed.

On the classification of buoyancy-driven chemo-hydrodynamic instabilities of chemical fronts.

PubMed

D'Hernoncourt, J; Zebib, A; De Wit, A

2007-03-01

Exothermic autocatalytic fronts traveling in the gravity field can be deformed by buoyancy-driven convection due to solutal and thermal contributions to changes in the density of the product versus the reactant solutions. We classify the possible instability mechanisms, such as Rayleigh-Benard, Rayleigh-Taylor, and double-diffusive mechanisms known to operate in such conditions in a parameter space spanned by the corresponding solutal and thermal Rayleigh numbers. We also discuss a counterintuitive instability leading to buoyancy-driven deformation of statically stable fronts across which a solute-light and hot solution lies on top of a solute-heavy and colder one. The mechanism of this chemically driven instability lies in the coupling of a localized reaction zone and of differential diffusion of heat and mass. Dispersion curves of the various cases are analyzed. A discussion of the possible candidates of autocatalytic reactions and experimental conditions necessary to observe the various instability scenarios is presented.

Spatiotemporal chaos in the dynamics of buoyantly and diffusively unstable chemical fronts

NASA Astrophysics Data System (ADS)

Baroni, M. P. M. A.; Guéron, E.; De Wit, A.

2012-03-01

Nonlinear dynamics resulting from the interplay between diffusive and buoyancy-driven Rayleigh-Taylor (RT) instabilities of autocatalytic traveling fronts are analyzed numerically for various values of the relevant parameters. These are the Rayleigh numbers of the reactant A and autocatalytic product B solutions as well as the ratio D =DB/DA between the diffusion coefficients of the two key chemical species. The interplay between the coarsening dynamics characteristic of the RT instability and the constant short wavelength modulation of the diffusive instability can lead in some regimes to complex dynamics dominated by irregular succession of birth and death of fingers. By using spectral entropy measurements, we characterize the transition between order and spatial disorder in this system. The analysis of the power spectrum and autocorrelation function, moreover, identifies similarities between the various spatial patterns. The contribution of the diffusive instability to the complex dynamics is discussed.

Thermonuclear plasma with autocatalytic thermomagnetic current amplification by nuclear reactions from fusion neutrons

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

Winterberg, F.

2006-03-15

It is proposed to use the neutrons released from a deuterium-tritium or deuterium-deuterium fusion reaction to drive thermomagnetic currents in a plasma corona surrounding the fusion plasma through the heating of the corona with nuclear reactions by the neutrons released in the fusion reaction. Because the neutron reaction cross sections are larger for slow neutrons, it is proposed to slow them down in a moderator separated from the hot plasma of the corona, giving the configuration a striking similarity to a heterogeneous nuclear fission reactor. While in a fission reactor the separation makes possible a growing neutron chain reaction, itmore » here makes possible the autocatalytic amplification of the thermomagnetic currents by an increase of the fusion reaction rate through a rise of the plasma pressure by the magnetic pressure of the thermomagnetic currents. This is expected to substantially increase the n{tau} product over its Lawson value.« less

Autocatalytic sets and chemical organizations: modeling self-sustaining reaction networks at the origin of life

NASA Astrophysics Data System (ADS)

Hordijk, Wim; Steel, Mike; Dittrich, Peter

2018-01-01

Two related but somewhat different approaches have been proposed to formalize the notion of a self-sustaining chemical reaction network. One is the notion of collectively autocatalytic sets, formalized as RAF theory, and the other is chemical organization theory. Both formalisms have been argued to be relevant to the origin of life. RAF sets and chemical organizations are defined differently, but previously some relationships between the two have been shown. Here, we refine and explore these connections in more detail. In particular, we show that so-called closed RAFs are chemical organizations, but that the converse is not necessarily true. We then introduce and apply a procedure to show how chemical organizations can be used to find all closed RAFs within any chemical reaction system. We end with a discussion of why and how closed RAFs could be important in the context of the origin and early evolution of life.

Steam explosion pretreatment of oil palm empty fruit bunches (EFB) using autocatalytic hydrolysis: A biorefinery approach.

PubMed

Medina, Jesus David Coral; Woiciechowski, Adenise; Filho, Arion Zandona; Nigam, Poonam Singh; Ramos, Luiz Pereira; Soccol, Carlos Ricardo

2016-01-01

The oil palm empty fruit bunches (EFB) are an attractive source of carbon for the production of biochemical products, therefore, the aim of this work is to analyze the effect of the steam explosion (SE) pretreatment under autocatalytic conditions on EFB using a full experimental design. Temperature and reaction time were the operational variables studied. The EFB treated at 195°C for 6 min showed an increase of 34.69% in glycan (mostly cellulose), and a reduction of 68.12% in hemicelluloses, with increased enzymatic digestibility to 33% producing 4.2 g L(-1) of glucose. Scanning electron micrographs of the steam treated EFB exhibited surface erosion and an increased fiber porosity. Fourier transform infrared spectroscopy showed the solubilization of hemicellulose and modification of cellulose in treated EFB. Copyright © 2015 Elsevier Ltd. All rights reserved.

Effects of an inhibitor of the γ-secretase complex on proliferation and apoptotic parameters in a FOXL2-mutated granulosa tumor cell line (KGN).

PubMed

Irusta, Griselda; Pazos, Maria Camila; Maidana, Camila Pazos; Abramovich, Dalhia; De Zúñiga, Ignacio; Parborell, Fernanda; Tesone, Marta

2013-07-01

Ovarian granulosa cell tumors (GCTs) represent 3%-5% of all ovarian malignancies. Treatments have limited proven efficacy and biologically targeted treatment is lacking. The aim of this study was to investigate the role of Notch signaling in the proliferation, steroidogenesis, apoptosis, and phosphatidylinositol 3-kinase (PI3K)/AKT pathway in a FOXL2-mutated granulosa tumor cell line (KGN) representative of the adult form of GCTs. When Notch signaling is initiated, the receptors expose a cleavage site in the extracellular domain to the metalloproteinase TACE and, following this cleavage, Notch undergoes another cleavage mediated by the presenilin-gamma-secretase complex. To achieve our goal, DAPT, an inhibitor of the gamma-secretase complex, was used to investigate the role of the Notch system in parameters associated with cell growth and death, using a human granulosa cell tumor line (KGN) as an experimental model. We observed that JAGGED1, DLL4, NOTCH1, and NOTCH4 were highly expressed in KGN cells as compared to granulosa-lutein cells obtained from assisted reproductive techniques patients. The proliferation and viability of KGN cells, as well as progesterone and estradiol production, decreased in the presence of 20 μM DAPT. Apoptotic parameters like PARP and caspase 8 cleavages, BAX, and BCLXs increased in KGN cells cultured with DAPT, whereas others such as BCL2, BCLXl, FAS, and FAS ligand did not change. AKT phosphorylation decreased and PTEN protein increased when Notch signaling was inhibited in KGN cells. We conclude that the Notch system acts as a survival pathway in KGN cells, and might be interacting with the PI3K/AKT pathway.

The endoproteolytic maturation of progastrin and procholecystokinin.

PubMed

Rehfeld, Jens F

2006-07-01

The homologous brain-gut propeptides, procholecystokinin (proCCK) and progastrin, both undergo extensive posttranslational maturation in specific neuroendocrine cells. The process comprises multiple endoproteolytic cleavages at mono- and dibasic sites, in addition to exoproteolytic trimmings and amino acid derivatizations. Knockout of prohormone convertases (PCs) in mice and studies in cell lines indicate that PC1, PC2 and, to a minor extent, PC5, are responsible for most of the endoproteolytic cleavages of both prohormones. Progastrin in antral G-cells is cleaved by PC1 at two di-Arg sites, R36R37 and R73R74, whereas, PC2 only cleaves at the single di-Lys site, K53K54. Pituitary corticotrophs and intestinal TG-cells, both of which express gastrin, do not cleave K53K54 due to lack of PC2. In proCCK five monobasic (R25, R44, R50, K61 and R75) as well as a single dibasic site (R85R86) can all be cleaved by both PC1 and PC2. But the cleavage differs in a cell-specific manner in that PC1 is responsible for the entire endoproteolytic cleavage in intestinal endocrine I-cells, except for perhaps the K61 site. In contrast PC2 is responsible for most endoproteolysis of proCCK in the cerebral CCK-neurons, which do not express PC1 in significant amounts. Moreover, PC5 appears to contribute to a minor extent to the neuronal proCCK and to the antral progastrin processing. This review emphasizes that prohormone convertases play a decisive but substrate and cell-specific role in the biosynthetic maturation of gastrin and CCK.

Aging pathways for organophosphate-inhibited human butyrylcholinesterase, including novel pathways for isomalathion, resolved by mass spectrometry.

PubMed

Li, He; Schopfer, Lawrence M; Nachon, Florian; Froment, Marie-Thérèse; Masson, Patrick; Lockridge, Oksana

2007-11-01

Some organophosphorus compounds are toxic because they inhibit acetylcholinesterase (AChE) by phosphylation of the active site serine, forming a stable conjugate: Ser-O-P(O)-(Y)-(XR) (where X can be O, N, or S and Y can be methyl, OR, or SR). The inhibited enzyme can undergo an aging process, during which the X-R moiety is dealkylated by breaking either the P-X or the X-R bond depending on the specific compound, leading to a nonreactivatable enzyme. Aging mechanisms have been studied primarily using AChE. However, some recent studies have indicated that organophosphate-inhibited butyrylcholinesterase (BChE) may age through an alternative pathway. Our work utilized matrix-assisted laser desorption/ionization-time-of-flight mass spectrometry to study the aging mechanism of human BChE inhibited by dichlorvos, echothiophate, diisopropylfluorophosphate (DFP), isomalathion, soman, sarin, cyclohexyl sarin, VX, and VR. Inhibited BChE was aged in the presence of H2O18 to allow incorporation of (18)O, if cleavage was at the P-X bond. Tryptic-peptide organophosphate conjugates were identified through peptide mass mapping. Our results showed no aging of VX- and VR-treated BChE at 25 degrees C, pH 7.0. However, BChE inhibited by dichlorvos, echothiophate, DFP, soman, sarin, and cyclohexyl sarin aged exclusively through O-C bond cleavage, i.e., the classical X-R scission pathway. In contrast, isomalathion aged through both X-R and P-X pathways; the main aged product resulted from P-S bond cleavage and a minor product resulted from O-C and/or S-C bond cleavage.

Molecular insight into bacterial cleavage of oceanic dimethylsulfoniopropionate into dimethyl sulfide

PubMed Central

Li, Chun-Yang; Wei, Tian-Di; Zhang, Sheng-Hui; Chen, Xiu-Lan; Gao, Xiang; Wang, Peng; Xie, Bin-Bin; Su, Hai-Nan; Qin, Qi-Long; Zhang, Xi-Ying; Yu, Juan; Zhang, Hong-Hai; Zhou, Bai-Cheng; Yang, Gui-Peng; Zhang, Yu-Zhong

2014-01-01

The microbial cleavage of dimethylsulfoniopropionate (DMSP) generates volatile DMS through the action of DMSP lyases and is important in the global sulfur and carbon cycles. When released into the atmosphere from the oceans, DMS is oxidized, forming cloud condensation nuclei that may influence weather and climate. Six different DMSP lyase genes are found in taxonomically diverse microorganisms, and dddQ is among the most abundant in marine metagenomes. Here, we examine the molecular mechanism of DMSP cleavage by the DMSP lyase, DddQ, from Ruegeria lacuscaerulensis ITI_1157. The structures of DddQ bound to an inhibitory molecule 2-(N-morpholino)ethanesulfonic acid and of DddQ inactivated by a Tyr131Ala mutation and bound to DMSP were solved. DddQ adopts a β-barrel fold structure and contains a Zn2+ ion and six highly conserved hydrophilic residues (Tyr120, His123, His125, Glu129, Tyr131, and His163) in the active site. Mutational and biochemical analyses indicate that these hydrophilic residues are essential to catalysis. In particular, Tyr131 undergoes a conformational change during catalysis, acting as a base to initiate the β-elimination reaction in DMSP lysis. Moreover, structural analyses and molecular dynamics simulations indicate that two loops over the substrate-binding pocket of DddQ can alternate between “open” and “closed” states, serving as a gate for DMSP entry. We also propose a molecular mechanism for DMS production through DMSP cleavage. Our study provides important insight into the mechanism involved in the conversion of DMSP into DMS, which should lead to a better understanding of this globally important biogeochemical reaction. PMID:24395783

Intracellular Domain Fragment of CD44 Alters CD44 Function in Chondrocytes*

PubMed Central

Mellor, Liliana; Knudson, Cheryl B.; Hida, Daisuke; Askew, Emily B.; Knudson, Warren

2013-01-01

The hyaluronan receptor CD44 undergoes sequential proteolytic cleavage at the cell surface. The initial cleavage of the CD44 extracellular domain is followed by a second intramembranous cleavage of the residual CD44 fragment, liberating the C-terminal cytoplasmic tail of CD44. In this study conditions that promote CD44 cleavage resulted in a diminished capacity to assemble and retain pericellular matrices even though sufficient non-degraded full-length CD44 remained. Using stable and transient overexpression of the cytoplasmic domain of CD44, we determined that the intracellular domain interfered with anchoring of the full-length CD44 to the cytoskeleton and disrupted the ability of the cells to bind hyaluronan and assemble a pericellular matrix. Co-immunoprecipitation assays were used to determine whether the mechanism of this interference was due to competition with actin adaptor proteins. CD44 of control chondrocytes was found to interact and co-immunoprecipitate with both the 65- and 130-kDa isoforms of ankyrin-3. Moreover, this interaction with ankyrin-3 proteins was diminished in cells overexpressing the CD44 intracellular domain. Mutating the putative ankyrin binding site of the transiently transfected CD44 intracellular domain diminished the inhibitory effects of this protein on matrix retention. Although CD44 in other cells types has been shown to interact with members of the ezrin/radixin/moesin (ERM) family of adaptor proteins, only modest interactions between CD44 and moesin could be demonstrated in chondrocytes. The data suggest that release of the CD44 intracellular domain into the cytoplasm of cells such as chondrocytes exerts a competitive or dominant-negative effect on the function of full-length CD44. PMID:23884413

Self-catalyzed growth of S layers via an amorphous-to-crystalline transition limited by folding kinetics.

PubMed

Chung, Sungwook; Shin, Seong-Ho; Bertozzi, Carolyn R; De Yoreo, James J

2010-09-21

The importance of nonclassical, multistage crystallization pathways is increasingly evident from theoretical studies on colloidal systems and experimental investigations of proteins and biomineral phases. Although theoretical predictions suggest that proteins follow these pathways as a result of fluctuations that create unstable dense-liquid states, microscopic studies indicate these states are long-lived. Using in situ atomic force microscopy to follow 2D assembly of S-layer proteins on supported lipid bilayers, we have obtained a molecular-scale picture of multistage protein crystallization that reveals the importance of conformational transformations in directing the pathway of assembly. We find that monomers with an extended conformation first form a mobile adsorbed phase, from which they condense into amorphous clusters. These clusters undergo a phase transition through S-layer folding into crystalline clusters composed of compact tetramers. Growth then proceeds by formation of new tetramers exclusively at cluster edges, implying tetramer formation is autocatalytic. Analysis of the growth kinetics leads to a quantitative model in which tetramer creation is rate limiting. However, the estimated barrier is much smaller than expected for folding of isolated S-layer proteins, suggesting an energetic rationale for this multistage pathway.

Nuclear HER4 mediates acquired resistance to trastuzumab and is associated with poor outcome in HER2 positive breast cancer

PubMed Central

Nafi, Siti Norasikin Mohd; Generali, Daniele; Kramer-Marek, Gabriela; Gijsen, Merel; Strina, Carla; Cappelletti, Mariarosa; Andreis, Daniele; Haider, Syed; Li, Ji-Liang; Bridges, Esther; Capala, Jacek; Ioannis, Roxanis; Harris, Adrian L; Kong, Anthony

2014-01-01

The role of HER4 in breast cancer is controversial and its role in relation to trastuzumab resistance remains unclear. We showed that trastuzumab treatment and its acquired resistance induced HER4 upregulation, cleavage and nuclear translocation. However, knockdown of HER4 by specific siRNAs increased trastuzumab sensitivity and reversed its resistance in HER2 positive breast cancer cells. Preventing HER4 cleavage by a γ-secretase inhibitor and inhibiting HER4 tyrosine kinase activity by neratinib decreased trastuzumab-induced HER4 nuclear translocation and enhanced trastuzumab response. There was also increased nuclear HER4 staining in the tumours from BT474 xenograft mice and human patients treated with trastuzumab. Furthermore, nuclear HER4 predicted poor clinical response to trastuzumab monotherapy in patients undergoing a window study and was shown to be an independent poor prognostic factor in HER2 positive breast cancer. Our data suggest that HER4 plays a key role in relation to trastuzumab resistance in HER2 positive breast cancer. Therefore, our study provides novel findings that HER4 activation, cleavage and nuclear translocation influence trastuzumab sensitivity and resistance in HER2 positive breast cancer. Nuclear HER4 could be a potential prognostic and predictive biomarker and understanding the role of HER4 may provide strategies to overcome trastuzumab resistance in HER2 positive breast cancer. PMID:25153719

E. coli derived Von Willebrand Factor-A2 domain FRET proteins that quantify ADAMTS13 activity

PubMed Central

Dayananda, Kannayakanahalli M.; Gogia, Shobhit; Neelamegham, Sriram

2010-01-01

The cleavage of the A2-domain of Von Willebrand Factor (VWF) by the metalloprotease ADAMTS13 regulates VWF size and platelet thrombosis rates. Reduction or inhibition of this enzyme activity leads to thrombotic thrombocytopenic purpura (TTP). We generated a set of novel molecules called VWF-A2 FRET proteins’, where variants of YFP (Venus) and CFP (Cerulean) flank either the entire VWF-A2 domain (175 amino acids) or truncated fragments (141, 113, 77 amino acids) of this domain. These proteins were expressed in E. coli in soluble form, and they exhibited Fluorescence/Förster Resonance Energy Transfer (FRET) properties. Results show that introduction of Venus/Cerulean itself did not alter the ability of VWF-A2 to undergo ADAMTS13 mediated cleavage. The smallest FRET protein, XS-VWF, detected plasma ADAMTS13 activity down to 10% of normal levels. Tests of acquired and inherited TTP could be completed within 30 min. VWF-A2 conformation changed progressively, and not abruptly, upon increasing urea concentration. While proteins with 77 and 113 VWF-A2 residues were cleaved in the absence of denaturant, 4M urea was required for the efficient cleavage of larger constructs. Overall, VWF-A2 FRET proteins can be applied both for the rapid diagnosis of plasma ADAMTS13 activity, and as a tool to study VWF-A2 conformation dynamics. PMID:21146487

An ultraviolet crosslink in the hammerhead ribozyme dependent on 2-thiocytidine or 4-thiouridine substitution.

PubMed Central

Wang, L; Ruffner, D E

1997-01-01

The hammerhead domain is one of the smallest known ribozymes. Like other ribozymes it catalyzes site-specific cleavage of a phosphodiester bond. The hammerhead ribozyme has been the subject of a vast number of biochemical and structural studies aimed at determining the structure and mechanism of cleavage. Recently crystallographic analysis has produced a structure for the hammerhead. As the hammerhead is capable of undergoing cleavage within the crystal, it would appear that the crystal structure is representative of the catalytically active solution structure. However, the crystal structure conflicts with much of the biochemical data and reveals a catalytic metal ion binding site expected to be of very low affinity. Clearly, additional studies are needed to reconcile the discrepancies and provide a clear understanding of the structure and mechanism of the hammerhead ribozyme. Here we demonstrate that a unique crosslink can be induced in the hammerhead with 2-thiocytidine or 4-thiouridine substitution at different locations within the conserved core. Generation of the same crosslink with different modifications at different positions suggests that the structure trapped by the crosslink may be relevant to the catalytically active solution structure of the hammerhead ribozyme. As this crosslink appears to be incompatible with the crystal structure, this provides yet another indication that the active solution and crystal structures may differ significantly. PMID:9336468

Mechanism of endonuclease cleavage by the HigB toxin

PubMed Central

Schureck, Marc A.; Repack, Adrienne; Miles, Stacey J.; Marquez, Jhomar; Dunham, Christine M.

2016-01-01

Bacteria encode multiple type II toxin–antitoxin modules that cleave ribosome-bound mRNAs in response to stress. All ribosome-dependent toxin family members structurally characterized to date adopt similar microbial RNase architectures despite possessing low sequence identities. Therefore, determining which residues are catalytically important in this specialized RNase family has been a challenge in the field. Structural studies of RelE and YoeB toxins bound to the ribosome provided significant insights but biochemical experiments with RelE were required to clearly demonstrate which residues are critical for acid-base catalysis of mRNA cleavage. Here, we solved an X-ray crystal structure of the wild-type, ribosome-dependent toxin HigB bound to the ribosome revealing potential catalytic residues proximal to the mRNA substrate. Using cell-based and biochemical assays, we further determined that HigB residues His54, Asp90, Tyr91 and His92 are critical for activity in vivo, while HigB H54A and Y91A variants have the largest effect on mRNA cleavage in vitro. Comparison of X-ray crystal structures of two catalytically inactive HigB variants with 70S-HigB bound structures reveal that HigB active site residues undergo conformational rearrangements likely required for recognition of its mRNA substrate. These data support the emerging concept that ribosome-dependent toxins have diverse modes of mRNA recognition. PMID:27378776

Hypoxic Switch in Mitochondrial Myeloid Cell Leukemia Factor-1/Mtd Apoptotic Rheostat Contributes to Human Trophoblast Cell Death in Preeclampsia

PubMed Central

Soleymanlou, Nima; Jurisicova, Andrea; Wu, Yuanhong; Chijiiwa, Mari; Ray, Jocelyn E.; Detmar, Jacqui; Todros, Tullia; Zamudio, Stacy; Post, Martin; Caniggia, Isabella

2007-01-01

Preeclampsia, a disorder of pregnancy, is characterized by increased trophoblast cell death and altered trophoblast-mediated remodeling of myometrial spiral arteries resulting in reduced uteroplacental perfusion. Mitochondria-associated Bcl-2 family members are important regulators of programed cell death. The mechanism whereby hypoxia alters the mitochondrial apoptotic rheostat is essential to our understanding of placental disease. Herein, myeloid cell leukemia factor-1 (Mcl-1) isoform expression was examined in physiological/pathological models of placental hypoxia. Preeclamptic placentae were characterized by caspase-dependent cleavage of death-suppressing Mcl-1L and switch toward cell death-inducing Mcl-1S. In vitro, Mcl-1L cleavage was induced by hypoxia-reoxygenation in villous explants, whereas Mcl-1L overexpression under hypoxia-reoxygenation rescued trophoblast cells from undergoing apoptosis. Cleavage was mediated by caspase-3/-7 because pharmacological caspase inhibition prevented this process. Altitude-induced chronic hypoxia was characterized by expression of Mcl-1L; resulting in a reduction of apoptotic markers (cleaved caspase-3/-8 and p85 poly-ADP-ribose polymerase). Moreover, in both physiological (explants and high altitude) and pathological (preeclampsia) placental hypoxia, decreased trophoblast syncytin expression was observed. Hence, although both pathological and physiological placental hypoxia are associated with slowed trophoblast differentiation, trophoblast apoptosis is only up-regulated in preeclampsia, because of a hypoxia-reoxygenation-induced switch in generation of proapoptotic Mcl-1 isoforms. PMID:17600131

Structure and photochemistry of a saccharyl thiotetrazole.

PubMed

Ismael, A; Borba, A; Henriques, M S C; Paixão, J A; Fausto, R; Cristiano, M L S

2015-01-02

The molecular structure and photochemistry of 5-thiosaccharyl-1-methyltetrazole (TSMT) were studied by means of matrix-isolation FTIR spectroscopy, X-ray crystallography, and theoretical calculations. The calculations predicted two conformers of TSMT that differ in energy by more than 15 kJ mol(-1). The infrared spectrum of TSMT isolated in solid argon was fully assigned on the basis of the spectrum calculated (O3LYP/6-311++G(3df,3pd)) for the most stable conformer. In the crystal, TSMT molecules were found to assume the same conformation as for the isolated molecule, with each molecule forming four hydrogen bonds with three neighboring molecules, leading to a network of TSMT oligomers. Upon UV (λ = 265 nm) irradiation of the matrix-isolated TSMT, two photodegradation pathways were observed, both arising from cleavage of the tetrazolyl ring. Pathway a involves cleavage of the N1-N2 and N3-N4 bonds with extrusion of N2, leading to photostable diazirine and thiocarbodiimide derivatives. The photostability of the photoproduced diazirine under the conditions used precluded its rearrangement to the nitrile imine, as reported for 5-phenyltetrazole by Bégué et al. ( J. Am. Chem. Soc. 2012 , 134 , 5339 ). Pathway b involves cleavage of the C5-N1 and N4-N3 bonds, leading to a thiocyanate and methyl azide, the latter undergoing subsequent fragmentation to give CNH.

The influence of differential processing of procathepsin H on its aminopeptidase activity, secretion and subcellular localization in human cell lines.

PubMed

Rojnik, Matija; Jevnikar, Zala R; Doljak, Bojan; Turk, Samo; Zidar, Nace; Kos, Janko

2012-10-01

Cathepsin H is a unique member of the cysteine cathepsins that acts primarily as an aminopeptidase. Like other cysteine cathepsins, it is synthesized as an inactive precursor and activated by proteolytic removal of its propeptide. Here we demonstrate that, in human cells, the processing of the propeptide is an autocatalytic, multistep process proceeding from an inactive 41kDa pro-form, through a 30kDa intermediate form, to the 28kDa mature form. Tyr87P and Gly90P were identified as the two major endopeptidase cleavage sites, converting the 30kDa form into the mature 28kDa form. The level of processing differs significantly in different human cell lines. In monocyte-derived macrophages U937 and prostate cancer cells PC-3, the 28kDa form is predominant, whereas in osteoblasts HOS the processing from the 30kDa form to the 28kDa form is significantly lower. The aminopeptidase activity of the enzyme and its subcellular localization are independent of the product, however the 30kDa form was not secreted in HOS cells. The activity of the resulting cathepsin H in U937 cells was significantly lower than that in HOS cells, presumably due to the high levels of endogenous cysteine protease inhibitor cystatin F present specifically in this cell line. These results provide an insight into the dependence of human cathepsin H processing and regulation on cell type. Copyright © 2012 Elsevier GmbH. All rights reserved.

Kissing nevus of the penis: a case report and dermatoscopic findings*

PubMed Central

Godinho, Nivea; Nai, Gisele Alborghetti; Schaefer, Arnaldo Luiz Flávio; Schaefer, Luiza Vasconcelos

2017-01-01

Divided nevus, also known as kissing nevus, is a rare variant of congenital melanocytic nevi in which there are two adjacent nevi in areas of the body that undergo embryonic cleavage. The original description of this type of lesion was on the eyelid. The location on the penis is even rarer, with only 17 case reports in the literature so far, and only one of them described the dermoscopic findings. We report the case of a patient with divided nevus of the penis and its clinical, dermoscopic and histopathological features. PMID:29267459

An Inexpensive, Point-of-Care Urine Test for Bladder Cancer in Patients Undergoing Hematuria Evaluation.

PubMed

Acharya, Abhinav P; Theisen, Kathryn M; Correa, Andres; Meyyappan, Thiagarajan; Apfel, Abraham; Sun, Tao; Tarin, Tatum V; Little, Steven R

2017-11-01

Although hematuria (blood in urine) is the most common symptom of bladder cancer, 70-98% of hematuria cases are benign. These hematuria patients unnecessarily undergo costly, invasive, and expensive evaluation for bladder cancer. Therefore, there remains a need for noninvasive office-based tests that can rapidly and reliably rule out bladder cancer in patients undergoing hematuria evaluation. Herein, a clinical assay for matrix metalloproteinases ("Ammps") is presented, which generates a visual signal based on the collagenase activity (in urine of patients) on the Ammps substrates. Ammps substrates are generated by crosslinking gelatin with Fe(II) chelated alginate nanoparticles, which precipitate in urine samples. The cleavage of gelatin-conjugated alginate (Fe(II)) nanoparticles by collagenases generates free-floating alginate (Fe(II)) nanoparticles that participate in Fenton's reaction to generate a visual signal. In a pilot study of 88 patients, Ammps had 100% sensitivity, 85% specificity, and a negative predictive value (NPV) of 100% for diagnosing bladder cancer. This high NPV can be useful in ruling out bladder cancer in patients referred for hematuria evaluation. © 2017 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

A Simple Demonstration of Convective Effects on Reaction-Diffusion Systems: A Burning Cigarette.

ERIC Educational Resources Information Center

Pojman, John A.

1990-01-01

Described is a demonstration that provides an introduction to nonequilibrium reaction-diffusion systems and the coupling of hydrodynamics to chemical reactions. Experiments that demonstrate autocatalytic behavior that are effected by gravity and convection are included. (KR)

Analysis of positional isotope exchange in ATP by cleavage of the beta P-O gamma P bond. Demonstration of negligible positional isotope exchange by myosin.

PubMed

Dale, M P; Hackney, D D

1987-12-15

A method for analysis of positional isotope exchange (PIX) during ATP in equilibrium with HOH oxygen exchange is presented that uses a two-step degradation of ATP resulting in cleavage of the beta P-O gamma P bond. This cleavage yields Pi derived from the gamma-phosphoryl of ATP that contains all four of the gamma oxygens. Both PIX between the beta,gamma-bridge and beta-nonbridge positions and washout of the gamma-nonbridge oxygens can be simultaneously followed by using ATP labeled with 17O at the beta-nonbridge positions and 18O at the beta,gamma-bridge and gamma-nonbridge positions. Application of this method to ATP in equilibrium with HOH exchange during single turnovers of myosin indicates that the bulk of the ATP undergoes rapid washout of gamma-nonbridge oxygens in the virtual absence of PIX. At 25 degrees C with subfragment 1 the scrambling rate is at the limit of detectability of approximately 0.001 s-1, which is 50-fold slower than the steady-state rate. This corresponds to a probability of scrambling for the beta-oxygens of bound ADP of 1 in 10,000 for each cycle of reversible hydrolysis of bound ATP. A fraction of the ATP, however, does not undergo rapid washout. With myosin and stoichiometric ATP at 0 degrees C, this fraction corresponds to 10% of the ATP remaining at 36 s, or 2% of the initial ATP, and an equivalent level of ATP is found that does not bind irreversibly to myosin in a cold chase experiment. A significant level of apparent PIX is observed with subfragment 1 in the fraction that resists washout, and this apparent PIX is shown to be due to contaminant adenylate kinase activity. This apparent PIX due to adenylate kinase provides a possible explanation for the PIX observed by Geeves et al. [Geeves, M. A., Webb, M. R., Midelfort, C. F., & Trentham, D. R. (1980) Biochemistry 19, 4748-4754] with subfragment 1.

PrPC Undergoes Basal to Apical Transcytosis in Polarized Epithelial MDCK Cells

PubMed Central

Arkhipenko, Alexander; Syan, Sylvie; Victoria, Guiliana Soraya

2016-01-01

The Prion Protein (PrP) is an ubiquitously expressed glycosylated membrane protein attached to the external leaflet of the plasma membrane via a glycosylphosphatidylinositol anchor (GPI). While the misfolded PrPSc scrapie isoform is the infectious agent of prion disease, the cellular isoform (PrPC) is an enigmatic protein with unclear function. Of interest, PrP localization in polarized MDCK cells is controversial and its mechanism of trafficking is not clear. Here we investigated PrP traffic in MDCK cells polarized on filters and in three-dimensional MDCK cysts, a more physiological model of polarized epithelia. We found that, unlike other GPI-anchored proteins (GPI-APs), PrP undergoes basolateral-to-apical transcytosis in fully polarized MDCK cells. Following this event full-length PrP and its cleavage fragments are segregated in different domains of the plasma membrane in polarized cells in both 2D and 3D cultures. PMID:27389581

Autoproteolysis coupled to protein folding in the SEA domain of the membrane-bound MUC1 mucin.

PubMed

Macao, Bertil; Johansson, Denny G A; Hansson, Gunnar C; Härd, Torleif

2006-01-01

The single cell layer of the lungs and the gastrointestinal tract is protected by the mucus formed by large glycoproteins called mucins. Transmembrane mucins typically contain 110-residue SEA domains located next to the membrane. These domains undergo post-translational cleavage between glycine and serine in a characteristic GSVVV sequence, but the two peptides remain tightly associated. We show that the SEA domain of the human MUC1 transmembrane mucin undergoes a novel type of autoproteolysis, which is catalyzed by conformational stress and the conserved serine hydroxyl. We propose that self-cleaving SEA domains have evolved to dissociate as a result of mechanical rather than chemical stress at the apical cell membrane and that this protects epithelial cells from rupture. We further suggest that the cell can register mechanical shear at the mucosal surface if the dissociation is signaled via loss of a SEA-binding protein.

[The efficacy of autocatalytic casapse-3 driven by human telomerase reverse transcriptase promoter on human ovarian carcinoma].

PubMed

Song, Yue; Shen, Keng; Yu, Jing-rong

2007-11-06

To construct recombinant adenoviral vector expressing autocatalysis caspase-3 driven by human telomerase reverse transcriptase promoter (hTERTp), and investigate its antitumor effect on ovarian cancer in vitro and in vivo. Recombinant adenovirus expressing autocatalytic caspase-3 (rev-csapase-3) driven by hTERTp, AdHT-rev-casp3, was constructed. Ad-rev-casp3 expressing rev-caspase-3 driven by cytomegalovirus promoter (CMVp) was used as a positive control. hTERT positive human ovarian cancer cells of the line AO and hTERT-negative human umbilical venous endothelial cells (HUVECs) were cultured and transfected with AdHT-rev-casp3, Ad-rev-casp3, or Ad-EGFG expressing enhanced green fluorescent protein as control group. Western blotting, Cell Counting Kit (CCK-8), flow cytometry, and TUNEL were used to detect the expression of p17, active subunit of caspase-3, and p85, a poly ADP-ribose polymerase (PARP) cleavage fragment, and they were also used to measure the cell survival rate and apoptotic rate. Western blotting was used to detect the expression of active caspase-3 and its substrate PARP in the AO cells and HUVECs. Twenty nude BALB/c mice were inoculated subcutaneously with AO cells to establish subcutaneous tumor models, when the tumor grew to the volume of 150 mm3 the rats were divided into 4 equal groups to undergo intra-tumor injection of AdHT-rev-casp3, Ad-rev-casp3, Ad-EGFG, and phosphate-buffered saline (PBS) respectively, the survival rate tumor inhibition rate was observed, 72 days later the mice were killed with their livers and tumors taken out, and Western blotting was used to detect the expression of active caspase-3. Another 40 mice underwent intraperitoneal injection of AO cells to establish intraperitoneal transplanted tumor models, 21 days later the rats were divided into 4 equal groups to be injected intraperitoneally with AdHT-rev-casp3, Ad-rev-casp3, Ad-EGFG, or PBS, the survival rate was observed, and the blood levels of alanine transaminase (ALT) and aspartate transaminase (AST) were detected. Following the administration of AdHT-rev-casp3, active caspase-3 protein was significantly expressed, and the levels of p17 and p85 expressions were significantly elevated in AO cells, while no expressions of p17 and p85 was observed in HUVEC. In contrast, both AO and HUVEC expressed high levels of p17 and p85 protein after administrations of Ad-rev-casp3. AdHT-rev-casp3 dose-dependently killed the hTERT positive AO cells, however, showed no killing effect on the hTERT-negative HUVEC cells; whereas Ad-rev-casp3 was cytotoxic independent of the hTERT status of the cells. The killing effect of Ad-rev-casp3 was stronger than that of AdHT-rev-casp3. Treated with AdHT-rev-cap3 the expression levels of the caspase-3 fragment p17 and PARP cleavage fragment p85 of the AO cells were significantly higher than those before the treatment, however, the expression levels of p17 and p85 were both weaker than those of the AO cells treated with Ad-rev-casp-3. Though treated with AdHT-rev-casp-3, there was still no remarkable expression of p17 and p85 in the HUVECs, however, rather high protein expression levels of p17 and p85 was shown. After treatment with AdHT-rev-casp3 remarkable expression of active caspase-3 was seen in the tumor collected from the mouse body, but not in the liver; however, high caspase-3 expression level was shown in both the liver and tumor after the treatment of Ad-rev-casp-3. 53 days after treatment the tumor suppression rate of the AdHT-rev-casp3 and ad-rev-casp-3 groups were 60% and 70% respectively, both significantly higher than that of the control group. The survival rates of the mice treated with AdHT-rev-casp3 and Ad-rev-casp-3 were both significantly longer than that of the PBS group; however the survival rate of the Ad-rev-casp-3 group was longer than that of the AdHT-rev-casp3 group. The serum ALT and AST levels were not significantly elevated in the AdHT-rev-casp3-treated mice, whereas 7-9-times that before treatment in the Ad-rev-casp3-treated mice. Recombinant adenovirus AdHT-rev-casp3 expressing rev-caspase-3 driven by hTERTp effectively causes cell apoptosis targeting tumor, significantly suppresses tumor growth and prolongs the mouse survival duration, with mild liver toxicity.

The effect of chromosomal polymorphisms on the outcomes of fresh IVF/ICSI-ET cycles in a Chinese population.

PubMed

Xu, Xiaojuan; Zhang, Rui; Wang, Wei; Liu, Hongfang; Liu, Lin; Mao, Bin; Zeng, Xiangwu; Zhang, Xuehong

2016-11-01

Chromosomal polymorphisms (CPs) have been reported to be associated with infertility; however, their effects on the outcomes of in vitro fertilization/intracytoplasmic sperm injection-embryo transfer (IVF/ICSI-ET) are still controversial. In this retrospective study, we aimed to evaluate the effect of CPs on IVF/ICSI-ET outcomes. To investigate whether CPs affected the outcomes of fresh IVF/ICSI-ET cycles in a Chinese population, we evaluated infertile couples with male carriers of CPs (n = 348), infertile couples with female carriers (n = 99), and unaffected couples (n = 400) who had received their first treatment cycles in our hospital between January 2013 and March 2015. CPs in either male or female carriers seemed to have adverse effects on IVF/ICSI-ET outcomes. CPs in male carriers affected outcomes mainly by decreasing the rates of fertilization, embryo cleavage, good quality embryos, clinical pregnancies, ongoing pregnancies, and deliveries as well as increasing the biochemical pregnancy rate (P < 0.05); CPs in female carriers affected outcomes only by lowering the embryo cleavage rate (P < 0.05). The mean fertilization rate of couples with male CP carriers undergoing IVF was significantly lower than that in those undergoing ICSI (61.1 versus 66.5 %, respectively; P = 0.0004). Our data provide evidence for the involvement of CPs in the poor outcomes of fresh IVF/ICSI-ET cycles in a Chinese population. The use of ICSI might improve outcomes by increasing the fertilization rate for men with CPs.

Study of dynamics of glucose-glucose oxidase-ferricyanide reaction

NASA Astrophysics Data System (ADS)

Nováková, A.; Schreiberová, L.; Schreiber, I.

2011-12-01

This work is focused on dynamics of the glucose-glucose oxidase-ferricyanide enzymatic reaction with or without sodium hydroxide in a continuous-flow stirred tank reactor (CSTR) and in a batch reactor. This reaction exhibits pH-variations having autocatalytic character and is reported to provide nonlinear dynamic behavior (bistability, excitability). The dynamical behavior of the reaction was examined within a wide range of inlet parameters. The main inlet parameters were the ratio of concentrations of sodium hydroxide and ferricyanide and the flow rate. In a batch reactor we observed an autocatalytic drop of pH from slightly basic to medium acidic values. In a CSTR our aim was to find bistability in the presence of sodium hydroxide. However, only a basic steady state was found. In order to reach an acidic steady state, we investigated the system in the absence of sodium hydroxide. Under these conditions the transition from the basic to the acidic steady state was observed when inlet glucose concentration was increased.

Local pH oscillations witness autocatalytic self-organization of biomorphic nanostructures

NASA Astrophysics Data System (ADS)

Montalti, M.; Zhang, G.; Genovese, D.; Morales, J.; Kellermeier, M.; García-Ruiz, J. M.

2017-02-01

Bottom-up self-assembly of simple molecular compounds is a prime pathway to complex materials with interesting structures and functions. Coupled reaction systems are known to spontaneously produce highly ordered patterns, so far observed in soft matter. Here we show that similar phenomena can occur during silica-carbonate crystallization, the emerging order being preserved. The resulting materials, called silica biomorphs, exhibit non-crystallographic curved morphologies and hierarchical textures, much reminiscent of structural principles found in natural biominerals. We have used a fluorescent chemosensor to probe local conditions during the growth of such self-organized nanostructures. We demonstrate that the pH oscillates in the local microenvironment near the growth front due to chemical coupling, which becomes manifest in the final mineralized architectures as intrinsic banding patterns with the same periodicity. A better understanding of dynamic autocatalytic crystallization processes in such simple model systems is key to the rational development of advanced materials and to unravel the mechanisms of biomineralization.

The mechanisms of hydrothermal deconstruction of lignocellulose: New insights from thermal–analytical and complementary studies

PubMed Central

Ibbett, Roger; Gaddipati, Sanyasi; Davies, Scott; Hill, Sandra; Tucker, Greg

2011-01-01

Differential Scanning Calorimetry, Dynamic Mechanical Thermal Analysis, gravimetric and chemical techniques have been used to study hydrothermal reactions of straw biomass. Exothermic degradation initiates above 195 °C, due to breakdown of the xylose ring from hemicellulose, which may be similar to reactions occurring during the early stage pyrolysis of dry biomass, though activated at lower temperature through water mediation. The temperature and magnitude of the exotherm reduce with increasing acid concentration, suggesting a reduction in activation energy and a change in the balance of reaction pathways. The presence of xylan oligomers in auto-catalytic hydrolysates is believed to be due to a low rate constant rather than a specific reaction mechanism. The loss of the lignin glass transition indicates that the lignin phase is reorganised under high temperature auto-catalytic conditions, but remains partially intact under lower temperature acid-catalytic conditions. This shows that lignin degradation reactions are activated thermally but are not effectively catalysed by aqueous acid. PMID:21763128

A model for the emergence of cooperation, interdependence, and structure in evolving networks.

PubMed

Jain, S; Krishna, S

2001-01-16

Evolution produces complex and structured networks of interacting components in chemical, biological, and social systems. We describe a simple mathematical model for the evolution of an idealized chemical system to study how a network of cooperative molecular species arises and evolves to become more complex and structured. The network is modeled by a directed weighted graph whose positive and negative links represent "catalytic" and "inhibitory" interactions among the molecular species, and which evolves as the least populated species (typically those that go extinct) are replaced by new ones. A small autocatalytic set, appearing by chance, provides the seed for the spontaneous growth of connectivity and cooperation in the graph. A highly structured chemical organization arises inevitably as the autocatalytic set enlarges and percolates through the network in a short analytically determined timescale. This self organization does not require the presence of self-replicating species. The network also exhibits catastrophes over long timescales triggered by the chance elimination of "keystone" species, followed by recoveries.

Intracellular signal propagation in a two-dimensional autocatalytic reaction model.

PubMed

Castiglione, F; Bernaschi, M; Succi, S; Heinrich, R; Kirschner, M W

2002-09-01

We study a simple reaction scheme in a two-dimensional lattice of particles or molecules with a refractory state. We analyze the dynamics of the propagating front as a function of physical-chemical properties of the host medium. The anisotropy of the medium significantly affects the smoothness of the wave front. Similarly, if particles or molecules may diffuse slowly to neighboring sites, then the front wave is more likely to be irregular. Both situations affect the ability of the whole system to relax to the original state, which is a required feature in the biological cells. Attempts to map this simple reaction scheme to reactions involved in the intracellular pathways suggest that, in some cases, signal transduction might take both connotation of a random walk and a propagating wave, depending on the local density of the medium. In particular, a sufficient condition for the appearance of waves in high-density regions of the media, is the existence of at least one autocatalytic reaction in the chain of reactions characterizing the pathway.

Temporal Control of Gelation and Polymerization Fronts Driven by an Autocatalytic Enzyme Reaction.

PubMed

Jee, Elizabeth; Bánsági, Tamás; Taylor, Annette F; Pojman, John A

2016-02-05

Chemical systems that remain kinetically dormant until activated have numerous applications in materials science. Herein we present a method for the control of gelation that exploits an inbuilt switch: the increase in pH after an induction period in the urease-catalyzed hydrolysis of urea was used to trigger the base-catalyzed Michael addition of a water-soluble trithiol to a polyethylene glycol diacrylate. The time to gelation (minutes to hours) was either preset through the initial concentrations or the reaction was initiated locally by a base, thus resulting in polymerization fronts that converted the mixture from a liquid into a gel (ca. 0.1 mm min -1 ). The rate of hydrolytic degradation of the hydrogel depended on the initial concentrations, thus resulting in a gel lifetime of hours to months. In this way, temporal programming of gelation was possible under mild conditions by using the output of an autocatalytic enzyme reaction to drive both the polymerization and subsequent degradation of a hydrogel.

A model for the emergence of cooperation, interdependence, and structure in evolving networks

NASA Astrophysics Data System (ADS)

Jain, Sanjay; Krishna, Sandeep

2001-01-01

Evolution produces complex and structured networks of interacting components in chemical, biological, and social systems. We describe a simple mathematical model for the evolution of an idealized chemical system to study how a network of cooperative molecular species arises and evolves to become more complex and structured. The network is modeled by a directed weighted graph whose positive and negative links represent "catalytic" and "inhibitory" interactions among the molecular species, and which evolves as the least populated species (typically those that go extinct) are replaced by new ones. A small autocatalytic set, appearing by chance, provides the seed for the spontaneous growth of connectivity and cooperation in the graph. A highly structured chemical organization arises inevitably as the autocatalytic set enlarges and percolates through the network in a short analytically determined timescale. This self organization does not require the presence of self-replicating species. The network also exhibits catastrophes over long timescales triggered by the chance elimination of "keystone" species, followed by recoveries.

Kinetic study of isothermal crystallization process of Gd2Ti2O7 precursor's powder prepared through the Pechini synthetic approach

NASA Astrophysics Data System (ADS)

Janković, Bojan; Marinović-Cincović, Milena; Dramićanin, Miroslav

2015-10-01

Crystallization process of Gd2Ti2O7 precursor's powder prepared by Pechini-type polymerized complex route has been studied under isothermal experimental conditions in an air atmosphere. It was found that the crystallization proceeds through two-parameter Šesták-Berggren (SB) autocatalytic model, in the operating temperature range of 550 °C≤T≤750 °C. Based on the behavior of SB parameters (M, N), it was found that in the lower operating temperature range, the crystallites with relatively low compactness exist, which probably disclosed low dimensionality of crystal growth from numerous nucleation sites, where the amorphous solid is produced. In the higher operating temperature region (above 750 °C), it was established that a morphological well-defined and high-dimensional particles of the formed pyrochlore phase can be expected. It was found that at T=850 °C, there is a change in the rate-determining reaction step, from autocatalytic into the contracting volume mechanism.

Kinetics and Mechanism of the Hydrogenation of CpCr(CO)3•/[CpCr(CO)3]2 Equilibrium to CpCr(CO)3H

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

Norton, Jack R.; Spataru, Tudor; Camaioni, Donald M.

2014-05-26

The kinetics of the hydrogenation of 2 CpCr(CO)3•/[CpCr(CO)3]2 to CpCr(CO)3H has been investigated. The reaction is second-order in Cr and first-order in H2, with a rate constant of 45 M 2s 1 at 25 °C in benzene. DFT calculations rule out an H2 complex as an intermediate, and suggest (a) end-on approach of H2 to one Cr of [CpCr(CO)3]2 as the Cr-Cr bond undergoes heterolytic cleavage, (b) heterolytic cleavage of the coordinated H2 between O and Cr, and (c) isomerization of the resulting O-protonated CpCr(CO)2(COH) to CpCr(CO)3H. The work at Pacific Northwest National Laboratory (PNNL) was supported by the U.S.more » Department of Energy, Office of Science, Office of Basic Energy Sciences, Division of Chemical Sciences, Geosciences and Biosciences; Battelle operates PNNL for DOE.« less

Design of Enzymatically Cleavable Prodrugs of a Potent Platinum-Containing Anticancer Agent

PubMed Central

Ding, Song; Pickard, Amanda J.; Kucera, Gregory L.

2014-01-01

Using a versatile synthetic approach, a new class of potential ester prodrugs of highly potent, but systemically too toxic, platinum–acridine anticancer agents was generated. The new hybrids contain a hydroxyl group, which has been masked with a cleavable lipophilic acyl moiety. Both butanoic (butyric) and bulkier 2-propanepentanoic (valproic) esters were introduced. The goals of this design were to improve the drug-like properties (e.g., logD) and to reduce the systemic toxicity of the pharmacophore. Two distinct pathways by which the target compounds undergo effective ester hydrolysis, the proposed activating step, have been confirmed: platinum-assisted, self-immolative ester cleavage in a low-chloride environment (LC-ESMS, NMR spectroscopy) and enzymatic cleavage by human carboxylesterase-2 (hCES-2) (LC-ESMS). The valproic acid ester derivatives are the first example of a metal-containing agent cleavable by the pro-drug-converting enzyme. They show excellent chemical stability and reduced systemic toxicity. Preliminary results from screening in lung adenocarcinoma cell lines (A549, NCI-H1435) suggest that the mechanism of the valproic esters may involve intracellular deesterification. PMID:25303639

Catalytic routes and oxidation mechanisms in photoreforming of polyols

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

Sanwald, Kai E.; Berto, Tobias F.; Eisenreich, Wolfgang

2016-12-01

Photocatalytic reforming of biomass-derived oxygenates leads to H 2 generation and evolution of CO 2 via parallel formation of organic intermediates through anodic oxidations on a Rh/TiO 2 photocatalyst. The reaction pathways and kinetics in the photoreforming of C 3–C 6 polyols were explored. Polyols are converted via direct and indirect hole transfer pathways resulting in (i) oxidative rupture of C–C bonds, (ii) oxidation to a-oxygen functionalized aldoses and ketoses (carbonyl group formation) and (iii) light-driven dehydration. Direct hole transfer to chemisorbed oxygenates on terminal Ti(IV)-OH groups, generating alkoxy-radicals that undergo ß-C–C-cleavage, is proposed for the oxidative C–C rupture. Carbonylmore » group formation and dehydration are attributed to indirect hole transfer at surface lattice oxygen sites [Ti_ _ _O_ _ _Ti] followed by the generation of carbon-centered radicals. Polyol chain length impacts the contribution of the oxidation mechanisms favoring the C–C bond cleavage (internal preferred over terminal) as the dominant pathway with higher polyol carbon number.« less

Rational design of a split-Cas9 enzyme complex

DOE PAGES

Wright, Addison V.; Sternberg, Samuel H.; Taylor, David W.; ...

2015-02-23

Cas9, an RNA-guided DNA endonuclease found in clustered regularly interspaced short palindromic repeats (CRISPR) bacterial immune systems, is a versatile tool for genome editing, transcriptional regulation, and cellular imaging applications. Structures of Streptococcus pyogenes Cas9 alone or bound to single-guide RNA (sgRNA) and target DNA revealed a bilobed protein architecture that undergoes major conformational changes upon guide RNA and DNA binding. To investigate the molecular determinants and relevance of the interlobe rearrangement for target recognition and cleavage, we designed a split-Cas9 enzyme in which the nuclease lobe and α-helical lobe are expressed as separate polypeptides. The lobes do not interactmore » on their own, the sgRNA recruits them into a ternary complex that recapitulates the activity of full-length Cas9 and catalyzes site-specific DNA cleavage. The use of a modified sgRNA abrogates split-Cas9 activity by preventing dimerization, allowing for the development of an inducible dimerization system. We propose that split-Cas9 can act as a highly regulatable platform for genome-engineering applications.« less

Rational design of a split-Cas9 enzyme complex

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

Wright, Addison V.; Sternberg, Samuel H.; Taylor, David W.

Cas9, an RNA-guided DNA endonuclease found in clustered regularly interspaced short palindromic repeats (CRISPR) bacterial immune systems, is a versatile tool for genome editing, transcriptional regulation, and cellular imaging applications. Structures of Streptococcus pyogenes Cas9 alone or bound to single-guide RNA (sgRNA) and target DNA revealed a bilobed protein architecture that undergoes major conformational changes upon guide RNA and DNA binding. To investigate the molecular determinants and relevance of the interlobe rearrangement for target recognition and cleavage, we designed a split-Cas9 enzyme in which the nuclease lobe and α-helical lobe are expressed as separate polypeptides. The lobes do not interactmore » on their own, the sgRNA recruits them into a ternary complex that recapitulates the activity of full-length Cas9 and catalyzes site-specific DNA cleavage. The use of a modified sgRNA abrogates split-Cas9 activity by preventing dimerization, allowing for the development of an inducible dimerization system. We propose that split-Cas9 can act as a highly regulatable platform for genome-engineering applications.« less

The α-Secretase-derived N-terminal Product of Cellular Prion, N1, Displays Neuroprotective Function in Vitro and in Vivo*

PubMed Central

Guillot-Sestier, Marie-Victoire; Sunyach, Claire; Druon, Charlotte; Scarzello, Sabine; Checler, Frédéric

2009-01-01

Cellular prion protein (PrPc) undergoes a disintegrin-mediated physiological cleavage, generating a soluble amino-terminal fragment (N1), the function of which remained unknown. Recombinant N1 inhibits staurosporine-induced caspase-3 activation by modulating p53 transcription and activity, whereas the PrPc-derived pathological fragment (N2) remains biologically inert. Furthermore, N1 protects retinal ganglion cells from hypoxia-induced apoptosis, reduces the number of terminal deoxynucleotidyltransferase-mediated biotinylated UTP nick end labeling-positive and p53-immunoreactive neurons in a pressure-induced ischemia model of the rat retina and triggers a partial recovery of b-waves but not a-waves of rat electroretinograms. Our work is the first demonstration that the α-secretase-derived PrPc fragment N1, but not N2, displays in vivo and in vitro neuroprotective function by modulating p53 pathway. It further demonstrates that distinct N-terminal cleavage products of PrPc harbor different biological activities underlying the various phenotypes linking PrPc to cell survival. PMID:19850936

Tau truncation is a productive posttranslational modification of neurofibrillary degeneration in Alzheimer's disease.

PubMed

Kovacech, B; Novak, M

2010-12-01

Deposits of the misfolded neuronal protein tau are major hallmarks of neurodegeneration in Alzheimer's disease (AD) and other tauopathies. The etiology of the transformation process of the intrinsically disordered soluble protein tau into the insoluble misordered aggregate has attracted much attention. Tau undergoes multiple modifications in AD, most notably hyperphosphorylation and truncation. Hyperphosphorylation is widely regarded as the hottest candidate for the inducer of the neurofibrillary pathology. However, the true nature of the impetus that initiates the whole process in the human brains remains unknown. In AD, several site-specific tau cleavages were identified and became connected to the progression of the disease. In addition, western blot analyses of tau species in AD brains reveal multitudes of various truncated forms. In this review we summarize evidence showing that tau truncation alone is sufficient to induce the complete cascade of neurofibrillary pathology, including hyperphosphorylation and accumulation of misfolded insoluble forms of tau. Therefore, proteolytical abnormalities in the stressed neurons and production of aberrant tau cleavage products deserve closer attention and should be considered as early therapeutic targets for Alzheimer's disease.

X-ray topographic fractography of single crystals of molybdenum and niobium

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

Hmelo, A.B.

1982-12-01

The semi-brittle (001) cleavage fracture at 77/sup 0/K of Mo and outgassed Nb single crystals was related to their fracture toughness. Berg-Barrett x-ray reflection topography and White Beam Synchrotron Fractography were used. Calibrated electropolishing in layers followed by Synchrotron Fractography revealed that under the conditions which existed at the tip of the running crack, Mo underwent general yielding which resulted in a plastically curved lattice. This result correlates with the substantial fracture toughness for Mo measured previously. On the other hand, double (112) slip was observed for Nb. The plastic zone associated with the crack-tip is shown to have amore » structure typicla of shock-loaded materials, consisting of arrays of screw dislocations, micro- and macrotwins. This result correlates with the low fracture toughness of Nb at 77/sup 0/K. It is proposed that these extremes in cleavage behavior for Mo vs Nb can be rationalized in terms of the capacity of a bcc structure to undergo a slip to twinning transition at high strain rates. 32 figures, 5 tables.« less

Rational design of a split-Cas9 enzyme complex.

PubMed

Wright, Addison V; Sternberg, Samuel H; Taylor, David W; Staahl, Brett T; Bardales, Jorge A; Kornfeld, Jack E; Doudna, Jennifer A

2015-03-10

Cas9, an RNA-guided DNA endonuclease found in clustered regularly interspaced short palindromic repeats (CRISPR) bacterial immune systems, is a versatile tool for genome editing, transcriptional regulation, and cellular imaging applications. Structures of Streptococcus pyogenes Cas9 alone or bound to single-guide RNA (sgRNA) and target DNA revealed a bilobed protein architecture that undergoes major conformational changes upon guide RNA and DNA binding. To investigate the molecular determinants and relevance of the interlobe rearrangement for target recognition and cleavage, we designed a split-Cas9 enzyme in which the nuclease lobe and α-helical lobe are expressed as separate polypeptides. Although the lobes do not interact on their own, the sgRNA recruits them into a ternary complex that recapitulates the activity of full-length Cas9 and catalyzes site-specific DNA cleavage. The use of a modified sgRNA abrogates split-Cas9 activity by preventing dimerization, allowing for the development of an inducible dimerization system. We propose that split-Cas9 can act as a highly regulatable platform for genome-engineering applications.

Intra- and intermolecular nonenzymatic ligations occur within transcripts derived from the peach latent mosaic viroid.

PubMed

Lafontaine, D; Beaudry, D; Marquis, P; Perreault, J P

1995-10-01

We report here the nonenzymatic self-ligation of transcripts corresponding to the peach latent mosaic viroid (PLMVd). This is the first description of this process with viroid sequences, although it has been reported to occur with human hepatitis delta virus RNA. Self-ligation occurs when the 5'-hydroxyl and the 2',3'-cyclic phosphate termini produced by the hammerhead self-cleavage of the viroid RNA are juxtaposed by the viroid rod-like structure, and a phosphodiester bond is formed between the two following hydrolysis of the cyclic phosphate. Unit-length transcripts undergo intramolecular folding, and their subsequent self-ligation produces circular molecules. The self-ligation observed in vitro may contribute to PLMVd circularization during rolling circle replication; however, this does not exclude the possibility that a host RNA ligase catalyzes the ligation steps in vivo. Like self-cleavage, self-ligation is probably an ancestral reaction, and the enzyme-catalyzed ligation most likely evolved from this primitive mechanism. Furthermore, the intermolecular self-ligation of annealed transcripts derived from PLMVd is demonstrated, suggesting a possible mechanism for sequence reassortment in viroids.

Residues in the Hendra Virus Fusion Protein Transmembrane Domain Are Critical for Endocytic Recycling

PubMed Central

Popa, Andreea; Carter, James R.; Smith, Stacy E.; Hellman, Lance; Fried, Michael G.

2012-01-01

Hendra virus is a highly pathogenic paramyxovirus classified as a biosafety level four agent. The fusion (F) protein of Hendra virus is critical for promoting viral entry and cell-to-cell fusion. To be fusogenically active, Hendra virus F must undergo endocytic recycling and cleavage by the endosomal/lysosomal protease cathepsin L, but the route of Hendra virus F following internalization and the recycling signals involved are poorly understood. We examined the intracellular distribution of Hendra virus F following endocytosis and showed that it is primarily present in Rab5- and Rab4-positive endosomal compartments, suggesting that cathepsin L cleavage occurs in early endosomes. Hendra virus F transmembrane domain (TMD) residues S490 and Y498 were found to be important for correct Hendra virus F recycling, with the hydroxyl group of S490 and the aromatic ring of Y498 important for this process. In addition, changes in association of isolated Hendra virus F TMDs correlated with alterations to Hendra virus F recycling, suggesting that appropriate TMD interactions play an important role in endocytic trafficking. PMID:22238299

Oxidative metabolism of phenanthrene and anthracene by soil pseudomonads. The ring-fission mechanism

PubMed Central

Evans, W. C.; Fernley, H. N.; Griffiths, E.

1965-01-01

1. Phenanthrene is oxidatively metabolized by soil pseudomonads through trans-3,4-dihydro-3,4-dihydroxyphenanthrene to 3,4-dihydroxyphenanthrene, which then undergoes cleavage. 2. Some properties of the ring-fission product, cis-4-(1-hydroxynaphth-2-yl)-2-oxobut-3-enoic acid, are described. The Fe2+-dependent oxygenase therefore disrupts the bond between C-4 and the angular C of the phenanthrene nucleus. 3. An enzyme of the aldolase type converts the fission product into 1-hydroxy-2-naphthaldehyde (2-formyl-1-hydroxynaphthalene). An NAD-specific dehydrogenase is also present in the cell-free extract, which oxidizes the aldehyde to 1-hydroxy-2-naphthoic acid. This is then oxidatively decarboxylated to 1,2-dihydroxynaphthalene, thus allowing continuation of metabolism via the naphthalene pathway. 4. Anthracene is similarly metabolized, through 1,2-dihydro-1,2-dihydroxyanthracene to 1,2-dihydroxyanthracene, in which ring-fission occurs to give cis-4-(2-hydroxynaphth-3-yl)-2-oxobut-3-enoic acid. The position of cleavage is again at the bond between the angular C and C-1 of the anthracene nucleus. 5. Enzymes that convert the fission product through 2-hydroxy-3-naphthaldehyde into 2-hydroxy-3-naphthoic acid were demonstrated. The further metabolism of this acid is discussed. 6. The Fe2+-dependent oxygenase responsible for cleavage of all the o-dihydroxyphenol derivatives appears to be catechol 2,3-oxygenase, and is a constitutive enzyme in the Pseudomonas strains used. PMID:14342521

Electrosynthesis of vanillin from isoeugenol using platinum electrode

NASA Astrophysics Data System (ADS)

Mubarok, H.; Hilyatudini; Saepudin, E.; Ivandini, T. A.

2017-04-01

Vanillin was synthesized from isoeugenol through electrochemical method in one compartment cell using platinum electrode. Cyclic voltammetry in 0.1 M TBAP in methanol and acetonitrile indicated the first oxidation potential at +0.21 and +0.16 V (vs. Ag/AgCl), respectively. Isoeugenolis was proposed to undergo the oxidation accompanied by oxidative cleavage of alkene bond into aldehyde. Accordingly, the synthesis of vanillin was conducted using chronoamperometry technique. The electrosynthesis result was analyzed by HPLC and GC/MS. The optimum condition of the oxidation potential, solvent ratio, time of electrolysis and amount of water was investigated.

Thermal Decomposition of the Solid Phase of Nitromethane: Ab Initio Molecular Dynamics Simulations

NASA Astrophysics Data System (ADS)

Chang, Jing; Lian, Peng; Wei, Dong-Qing; Chen, Xiang-Rong; Zhang, Qing-Ming; Gong, Zi-Zheng

2010-10-01

The Car-Parrinello molecular dynamics simulations were employed to investigate thermal decomposition of the solid nitromethane. It is found that it undergoes chemical decomposition at about 2200 K under ambient pressure. The initiation of reactions involves both proton transfer and commonly known C-N bond cleavage. About 75 species and 100 elementary reactions were observed with the final products being H2O, CO2, N2, and CNCNC. It represents the first complete simulation of solid-phase explosive reactions reported to date, which is of far-reaching implication for design and development of new energetic materials.

A new and efficient synthetic method for 15N3-labeled cytosine nucleosides: Dimroth rearrangement of cytidine N3-oxides.

PubMed

Sako, Magoichi; Kawada, Hiroyoshi

2004-11-12

The treatment of (15)N(4)-labeled cytidine N(3)-oxide and (15)N(4)-labeled 2'-deoxycytidine N(3)-oxide, prepared from the appropriate unprotected uridines in three reaction steps, with benzyl bromide in the presence of excess lithium methoxide allowed the smooth occurrence of their Dimroth rearrangement even under mild conditions leading to the corresponding (15)N(3)-labeled uridine 4-O-benzyloximes which can easily undergo the reductive N-O bond cleavage to give the desirable (15)N(3)-labeled cytosine nucleosides in high total yields.

Sperm chromatin maturity and integrity correlated to zygote development in ICSI program.

PubMed

Asmarinah; Syauqy, Ahmad; Umar, Liya Agustin; Lestari, Silvia Werdhy; Mansyur, Eliza; Hestiantoro, Andon; Paradowszka-Dogan, Agnieszka

2016-10-01

This study aimed to evaluate sperm chromatin maturity and integrity of that injected into good-quality oocytes in an in vitro fertilization-intra cytoplasmic sperm injection (IVF-ICSI) program. A cut-off value of sperm chromatin maturity and integrity was developed as a function of their correlation to the zygote development, i.e., embryo formation and cleavage rate. The study assessed sperm chromatin maturity using aniline blue (AB) staining, whereas toluidine blue (TB) staining was used to assess sperm chromatin integrity. Ejaculates from 59 patients undergoing ICSI and 46 fertile normozoospermic donors for determination of normal values of sperm chromatin status were used in this study. Embryo formation and cleavage rates were observed for the period of 3 days after ICSI. There was a significant difference in the percentage of sperm with mature chromatin between ejaculate from ICSI patients and fertile donor (p=0.020); while there was no significant difference in sperm chromatin integrity of both samples (p=0.120). There was no significant correlation between sperm chromatin maturity and either embryo formation or cleavage rate; as well as sperm chromatin integrity to both parameters of zygote development (p>0.05). Furthermore, we found that the cut-off value of sperm chromatin maturity and integrity of the fertile normozoospermic ejaculates were 87.2% and 80.2%, respectively. Using the cut-offs, we found that low sperm chromatin maturity at the level of <87% correlated significantly with the cleavage rate of the zygote (p=0.022; r=0.371); whereas poor sperm chromatin integrity at the level of <80% correlated with embryo formation (p=0.048; r=0,485). In conclusion, this study showed that poor maturity and integrity of sperm chromatin (AB<87% and TB<80%, respectively), could affect zygote development following ICSI. AB: aniline blue; CMA3: chromomycin A3; ICSI: intra cytoplasmic sperm injection; IVF: in vitro fertilization; PBS: phosphate buffer saline; SPSS: Statistical Package for Social Science; TB: toluidine blue; WHO: World Health Organization.

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

PubMed Central

2016-01-01

The obligate human pathogen Neisseria gonorrhoeae is the sole aetiologic agent of the sexually transmitted infection, gonorrhea. Required for gonococcal infection, Type IV pili (Tfp) mediate many functions including adherence, twitching motility, defense against neutrophil killing, and natural transformation. Critical for immune escape, the gonococcal Tfp undergoes antigenic variation, a recombination event at the pilE locus that varies the surface exposed residues of the major pilus subunit PilE (pilin) in the pilus fiber. This programmed recombination system has the potential to produce thousands of pilin variants and can produce strains with unproductive pilin molecules that are completely unable to form Tfp. Saturating mutagenesis of the 3’ third of the pilE gene identified 68 unique single nucleotide mutations that each resulted in an underpiliated colony morphology. Notably, all isolates, including those with undetectable levels of pilin protein and no observable surface-exposed pili, retained an intermediate level of transformation competence not exhibited in ΔpilE strains. Site-directed, nonsense mutations revealed that only the first 38 amino acids of the mature pilin N-terminus (the N-terminal domain or Ntd) are required for transformation competence, and microscopy, ELISAs and pilus purification demonstrate that extended Tfp are not required for competence. Transformation in strains producing only the pilin Ntd has the same genetic determinants as wild-type transformation. The Ntd corresponds to the alternative product of S-pilin cleavage, a specific proteolysis unique to pathogenic Neisseria. Mutation of the S-pilin cleavage site demonstrated that S-pilin cleavage mediated release of the Ntd is required for competence when a strain produces unproductive pilin molecules that cannot assemble into a Tfp through mutation or antigenic variation. We conclude that S-pilin cleavage evolved as a mechanism to maintain competence in nonpiliated antigenic variants and suggest there are alternate forms of the Tfp assembly apparatus that mediate various functions including transformation. PMID:27213957

Cryopreservation of feline oocytes by vitrification using commercial kits and slush nitrogen technique.

PubMed

Fernandez-Gonzalez, L; Jewgenow, K

2017-04-01

Assisted reproductive techniques are a valuable tool for conservation breeding of endangered species. Cryopreservation methods are the basis of gamete banks, supporting genetic diversity preservation. Unfortunately, cryopreservation of feline oocytes is still considered an experimental technique. The aim of this study was to compare two commercial kits, with our protocol for vitrification of cat oocytes (IZW), which comprises a three-step method with ethylene glycol, DMSO, fetal calf serum, trehalose and Ficoll PM-70. Furthermore, we applied slush nitrogen (SN 2 ) for ultra-rapid freezing to improve survival rates. Cumulus-oocyte complexes were collected from domestic cat ovaries by slicing and vitrified at immature stage using Cryotop as storage device. Vit Kit ® Freeze/Thaw (n = 89) showed the lowest maturation percentage obtained after warming (10.1%). A significant difference in maturation percentage of oocytes was found between Kitazato ® kit (38.7%, n = 137) and IZW protocol (24.5%, n = 143). The cleavage after ICSI of warmed and matured oocytes (20.7% and 28.6%, respectively) and the morula percentage (18. 2% and 22.5%, respectively), however, did not reveal any significant difference between the two methods. Application of SN 2 did not result in any improvement of oocytes' cryopreservation. Maturation percentage of the oocytes vitrified by IZW method with SN 2 (n = 144) decreased until 6.1%, without any cleavage after fertilization. For Kitazato ® (n = 62), only 17.7% were able to undergo maturation and cleavage percentage dropped to 18.2%, not reaching morula stage. These data demonstrate that feline oocytes can be vitrified either by our IZW method or by commercial Kitazato ® kit, but the use of SN 2 is improving neither maturation nor cleavage percentages when combined with these procedures. © 2016 Blackwell Verlag GmbH.

C-terminal cleavage of DeltaNp63alpha is associated with TSA-induced apoptosis in immortalized corneal epithelial cells.

PubMed

Robertson, Danielle M; Ho, Su-Inn; Cavanagh, H Dwight

2010-08-01

In the central human corneal epithelium, loss of DeltaNp63 occurs in all surface epithelial cells preparing to undergo desquamation, suggesting a potential role for DeltaNp63 isoforms in mediating surface cell apoptotic shedding. In this study, the authors investigated a role for DeltaNp63 isoforms in caspase-mediated apoptosis in a telomerase-immortalized corneal epithelial cell line. For in vitro studies, hTCEpi cells were cultured in KGM-2 serum-free culture media containing 0.15 mM calcium. To assess dynamic protein interactions among individual DeltaNp63 isoforms, DeltaNp63-EGFP expression plasmids were transiently expressed in hTCEpi cells and evaluated by FRAP. Trichostatin-A (TSA; 3.31 muM) was used to induce cell death as measured by caspase activity. Cleavage and loss of endogenous DeltaNp63alpha, DeltaNp63-EGFP expression plasmids, and p53 were assessed after treatment with TSA and siRNA. Transient expression of DeltaNp63-EGFP alpha and beta isoforms resulted in the formation of a smaller isoform similar in size to DeltaNp63gamma-EGFP. FRAP demonstrated that DeltaNp63alpha-EGFP has greater immobile fraction than beta or gamma. TSA induced caspase-mediated apoptotic pathways; caspase induction was accompanied by a decrease in endogenous DeltaNp63alpha and p53. TSA upregulated DeltaNp63-EGFP plasmid expression; this was accompanied by a selective increase in cleavage of DeltaNp63alpha-EGFP. siRNA knockdown of DeltaNp63alpha correlated with a reduction in p53 independently of TSA. DeltaNp63alpha is the dominant active isoform in corneal epithelial cell nuclei. Loss of DeltaNp63alpha occurs during apoptotic signaling by cleavage at the C terminus. The corresponding loss of p53 suggests that a significant relationship appears to exist between these two regulatory proteins.

Deciphering the chemoselectivity of nickel-dependent quercetin 2,4-dioxygenase.

PubMed

Wang, Wen-Juan; Wei, Wen-Jie; Liao, Rong-Zhen

2018-06-13

The reaction mechanism and chemoselectivity of nickel-dependent quercetin 2,4-dioxygenase (2,4-QueD) have been investigated using the QM/MM approach. The protonation state of the Glu74 residue, a first-shell ligand of Ni, has been considered to be either neutral or deprotonated. QM/MM calculations predict that Glu74 must be deprotonated to rationalize the chemoselectivity and steer the 2,4-dioxygenolytic cleavage of quercetin, which harvests the experimentally-observed product, 2-protocatechuoylphloroglucinol carboxylic acid, coupled with the release of carbon monoxide. If the enzyme has a neutral Glu74 residue, the undesired 2,3-dioxygenolytic cleavage of quercetin becomes the dominant pathway, leading to the formation of α-keto acid. The calculations suggest that the reaction takes place via three major steps: (1) attack of the superoxide on the C2 of the substrate pyrone ring to generate a NiII-peroxide intermediate; (2) formation of the second C-O bond between C4 and the peroxide to produce a peroxide bridge; (3) simultaneous cleavage of the C2-C3, C3-C4, and O1-O2 bonds with the formation of 2-protocatechuoylphloroglucinol carboxylic acid and carbon monoxide. The third step was found to be rate-limiting, with a barrier of 17.4 kcal mol-1, which is in very good agreement with the experimental kinetic data. For the second C-O bond formation, an alternative pathway is that the peroxide attacks the C3 of the substrate pyrone ring, leading to the formation of a four-membered ring intermediate, which then undergoes concerted C2-C3 and O1-O2 bond cleavages to produce an α-keto acid. This pathway is associated with a barrier of 30.6 kcal mol-1, which is much higher than the major pathway. When Glu74 is protonated, the 2,3-dioxygenolytic pathway, however, has a lower barrier (21.8 kcal mol-1) than the 2,4-dioxygenolytic pathway.

Structure Predictions of Two Bauhinia variegata Lectins Reveal Patterns of C-Terminal Properties in Single Chain Legume Lectins

PubMed Central

Moreira, Gustavo M. S. G.; Conceição, Fabricio R.; McBride, Alan J. A.; Pinto, Luciano da S.

2013-01-01

Bauhinia variegata lectins (BVL-I and BVL-II) are single chain lectins isolated from the plant Bauhinia variegata. Single chain lectins undergo post-translational processing on its N-terminal and C-terminal regions, which determines their physiological targeting, carbohydrate binding activity and pattern of quaternary association. These two lectins are isoforms, BVL-I being highly glycosylated, and thus far, it has not been possible to determine their structures. The present study used prediction and validation algorithms to elucidate the likely structures of BVL-I and -II. The program Bhageerath-H was chosen from among three different structure prediction programs due to its better overall reliability. In order to predict the C-terminal region cleavage sites, other lectins known to have this modification were analysed and three rules were created: (1) the first amino acid of the excised peptide is small or hydrophobic; (2) the cleavage occurs after an acid, polar, or hydrophobic residue, but not after a basic one; and (3) the cleavage spot is located 5-8 residues after a conserved Leu amino acid. These rules predicted that BVL-I and –II would have fifteen C-terminal residues cleaved, and this was confirmed experimentally by Edman degradation sequencing of BVL-I. Furthermore, the C-terminal analyses predicted that only BVL-II underwent α-helical folding in this region, similar to that seen in SBA and DBL. Conversely, BVL-I and -II contained four conserved regions of a GS-I association, providing evidence of a previously undescribed X4+unusual oligomerisation between the truncated BVL-I and the intact BVL-II. This is the first report on the structural analysis of lectins from Bauhinia spp. and therefore is important for the characterisation C-terminal cleavage and patterns of quaternary association of single chain lectins. PMID:24260572

Structure predictions of two Bauhinia variegata lectins reveal patterns of C-terminal properties in single chain legume lectins.

PubMed

Moreira, Gustavo M S G; Conceição, Fabricio R; McBride, Alan J A; Pinto, Luciano da S

2013-01-01

Bauhinia variegata lectins (BVL-I and BVL-II) are single chain lectins isolated from the plant Bauhinia variegata. Single chain lectins undergo post-translational processing on its N-terminal and C-terminal regions, which determines their physiological targeting, carbohydrate binding activity and pattern of quaternary association. These two lectins are isoforms, BVL-I being highly glycosylated, and thus far, it has not been possible to determine their structures. The present study used prediction and validation algorithms to elucidate the likely structures of BVL-I and -II. The program Bhageerath-H was chosen from among three different structure prediction programs due to its better overall reliability. In order to predict the C-terminal region cleavage sites, other lectins known to have this modification were analysed and three rules were created: (1) the first amino acid of the excised peptide is small or hydrophobic; (2) the cleavage occurs after an acid, polar, or hydrophobic residue, but not after a basic one; and (3) the cleavage spot is located 5-8 residues after a conserved Leu amino acid. These rules predicted that BVL-I and -II would have fifteen C-terminal residues cleaved, and this was confirmed experimentally by Edman degradation sequencing of BVL-I. Furthermore, the C-terminal analyses predicted that only BVL-II underwent α-helical folding in this region, similar to that seen in SBA and DBL. Conversely, BVL-I and -II contained four conserved regions of a GS-I association, providing evidence of a previously undescribed X4+unusual oligomerisation between the truncated BVL-I and the intact BVL-II. This is the first report on the structural analysis of lectins from Bauhinia spp. and therefore is important for the characterisation C-terminal cleavage and patterns of quaternary association of single chain lectins.

Loss-of-function PCSK9 mutants evade the unfolded protein response sensor GRP78 and fail to induce endoplasmic reticulum stress when retained.

PubMed

Lebeau, Paul; Platko, Khrystyna; Al-Hashimi, Ali A; Byun, Jae Hyun; Lhoták, Šárka; Holzapfel, Nicholas; Gyulay, Gabriel; Igdoura, Suleiman A; Cool, David R; Trigatti, Bernardo; Seidah, Nabil G; Austin, Richard C

2018-05-11

The proprotein convertase subtilisin/kexin type-9 (PCSK9) plays a central role in cardiovascular disease (CVD) by degrading hepatic low-density lipoprotein receptor (LDLR). As such, loss-of-function (LOF) PCSK9 variants that fail to exit the endoplasmic reticulum (ER) increase hepatic LDLR levels and lower the risk of developing CVD. The retention of misfolded protein in the ER can cause ER stress and activate the unfolded protein response (UPR). In this study, we investigated whether a variety of LOF PCSK9 variants that are retained in the ER can cause ER stress and hepatic cytotoxicity. Although overexpression of these PCSK9 variants caused an accumulation in the ER of hepatocytes, UPR activation or apoptosis was not observed. Furthermore, ER retention of endogenous PCSK9 via splice switching also failed to induce the UPR. Consistent with these in vitro studies, overexpression of PCSK9 in the livers of mice had no impact on UPR activation. To elucidate the cellular mechanism to explain these surprising findings, we observed that the 94-kDa glucose-regulated protein (GRP94) sequesters PCSK9 away from the 78-kDa glucose-regulated protein (GRP78), the major activator of the UPR. As a result, GRP94 knockdown increased the stability of GRP78-PCSK9 complex and resulted in UPR activation following overexpression of ER-retained PCSK9 variants relative to WT secreted controls. Given that overexpression of these LOF PCSK9 variants does not cause UPR activation under normal homeostatic conditions, therapeutic strategies aimed at blocking the autocatalytic cleavage of PCSK9 in the ER represent a viable strategy for reducing circulating PCSK9. © 2018 by The American Society for Biochemistry and Molecular Biology, Inc.

Structure-function analyses of human kallikrein-related peptidase 2 establish the 99-loop as master regulator of activity.

PubMed

Skala, Wolfgang; Utzschneider, Daniel T; Magdolen, Viktor; Debela, Mekdes; Guo, Shihui; Craik, Charles S; Brandstetter, Hans; Goettig, Peter

2014-12-05

Human kallikrein-related peptidase 2 (KLK2) is a tryptic serine protease predominantly expressed in prostatic tissue and secreted into prostatic fluid, a major component of seminal fluid. Most likely it activates and complements chymotryptic KLK3 (prostate-specific antigen) in cleaving seminal clotting proteins, resulting in sperm liquefaction. KLK2 belongs to the "classical" KLKs 1-3, which share an extended 99- or kallikrein loop near their non-primed substrate binding site. Here, we report the 1.9 Å crystal structures of two KLK2-small molecule inhibitor complexes. In both structures discontinuous electron density for the 99-loop indicates that this loop is largely disordered. We provide evidence that the 99-loop is responsible for two biochemical peculiarities of KLK2, i.e. reversible inhibition by micromolar Zn(2+) concentrations and permanent inactivation by autocatalytic cleavage. Indeed, several 99-loop mutants of KLK2 displayed an altered susceptibility to Zn(2+), which located the Zn(2+) binding site at the 99-loop/active site interface. In addition, we identified an autolysis site between residues 95e and 95f in the 99-loop, whose elimination prevented the mature enzyme from limited autolysis and irreversible inactivation. An exhaustive comparison of KLK2 with related structures revealed that in the KLK family the 99-, 148-, and 220-loop exist in open and closed conformations, allowing or preventing substrate access, which extends the concept of conformational selection in trypsin-related proteases. Taken together, our novel biochemical and structural data on KLK2 identify its 99-loop as a key player in activity regulation. © 2014 by The American Society for Biochemistry and Molecular Biology, Inc.

Structure-Function Analyses of Human Kallikrein-related Peptidase 2 Establish the 99-Loop as Master Regulator of Activity*

PubMed Central

Skala, Wolfgang; Utzschneider, Daniel T.; Magdolen, Viktor; Debela, Mekdes; Guo, Shihui; Craik, Charles S.; Brandstetter, Hans; Goettig, Peter

2014-01-01

Human kallikrein-related peptidase 2 (KLK2) is a tryptic serine protease predominantly expressed in prostatic tissue and secreted into prostatic fluid, a major component of seminal fluid. Most likely it activates and complements chymotryptic KLK3 (prostate-specific antigen) in cleaving seminal clotting proteins, resulting in sperm liquefaction. KLK2 belongs to the “classical” KLKs 1–3, which share an extended 99- or kallikrein loop near their non-primed substrate binding site. Here, we report the 1.9 Å crystal structures of two KLK2-small molecule inhibitor complexes. In both structures discontinuous electron density for the 99-loop indicates that this loop is largely disordered. We provide evidence that the 99-loop is responsible for two biochemical peculiarities of KLK2, i.e. reversible inhibition by micromolar Zn2+ concentrations and permanent inactivation by autocatalytic cleavage. Indeed, several 99-loop mutants of KLK2 displayed an altered susceptibility to Zn2+, which located the Zn2+ binding site at the 99-loop/active site interface. In addition, we identified an autolysis site between residues 95e and 95f in the 99-loop, whose elimination prevented the mature enzyme from limited autolysis and irreversible inactivation. An exhaustive comparison of KLK2 with related structures revealed that in the KLK family the 99-, 148-, and 220-loop exist in open and closed conformations, allowing or preventing substrate access, which extends the concept of conformational selection in trypsin-related proteases. Taken together, our novel biochemical and structural data on KLK2 identify its 99-loop as a key player in activity regulation. PMID:25326387

Role of water in aging of human butyrylcholinesterase inhibited by echothiophate: the crystal structure suggests two alternative mechanisms of aging.

PubMed

Nachon, Florian; Asojo, Oluwatoyin A; Borgstahl, Gloria E O; Masson, Patrick; Lockridge, Oksana

2005-02-01

Organophosphorus poisons (OP) bind covalently to the active-site serine of cholinesterases. The inhibited enzyme can usually be reactivated with powerful nucleophiles such as oximes. However, the covalently bound OP can undergo a suicide reaction (termed aging) yielding nonreactivatable enzyme. In human butyrylcholinesterase (hBChE), aging involves the residues His438 and Glu197 that are proximal to the active-site serine (Ser198). The mechanism of aging is known in detail for the nerve gases soman, sarin, and tabun as well as the pesticide metabolite isomalathion. Aging of soman- and sarin-inhibited acetylcholinesterase occurs by C-O bond cleavage, whereas that of tabun- and isomalathion-inhibited acetylcholinesterase occurs by P-N and P-S bond cleavage, respectively. In this work, the crystal structures of hBChE inhibited by the ophthalmic reagents echothiophate (nonaged and aged) and diisopropylfluorophosphate (aged) were solved and refined to 2.1, 2.25, and 2.2 A resolution, respectively. No appreciable shift in the position of the catalytic triad histidine was observed between the aged and nonaged conjugates of hBChE. This absence of shift contrasts with the aged and nonaged crystal structures of Torpedo californica acetylcholinesterase inhibited by the nerve agent VX. The nonaged hBChE structure shows one water molecule interacting with Glu197 and the catalytic triad histidine (His438). Interestingly, this water molecule is ideally positioned to promote aging by two mechanisms: breaking either a C-O bond or a P-O bond. Pesticides and certain stereoisomers of nerve agents are expected to undergo aging by breaking the P-O bond.

Neurotensin and neuromedin N undergo distinct catabolic processes in murine astrocytes and primary cultured neurons.

PubMed

Vincent, B; Vincent, J P; Checler, F

1994-04-01

We examined the occurrence of various endopeptidases and exopeptidases and their subcellular partition within soluble and membrane-associated compartments of 15-day-old astrocytes and 4-day-old primary cultured neurons. Peptidases were monitored with chromogenic or fluorimetric substrates and identified by means of specific inhibitors. We assessed the contribution of these peptidases in the catabolism of two related neuropeptides, neurotensin and neuromedin N. Metabolites were separated by HPLC and the identity of the proteolytic activities involved in their formation was established using specific inhibitors. Neuromedin N and neurotensin undergo both quantitative and qualitative differential proteolysis. Initial maximal rates of neuromedin N degradation were higher than those of neurotensin in both cell types. Furthermore, the two peptides were inactivated much more rapidly by the soluble than by the membrane-associated fractions prepared from both cell cultures. Neuromedin N was rapidly broken down by an aminopeptidase M/leucine aminopeptidase attack, leading to the functionally silent Des-Lys1-neuromedin N metabolite. In the astrocytic membrane-associated fraction, neuromedin N underwent an additional minor endoproteolytic cleavage at the Pro3-Tyr4 bond elicited by endopeptidase 24.11, as suggested by the protective effect of its blocking agent phosphoramidon. Unlike neuromedin N, neurotensin totally resisted hydrolysis by aminopeptidases. Primary inactivating cleavages detected in both cell types appeared mainly located at the Arg8-Arg9 and Pro10-Tyr11 bonds, leading to the formations of neurotensin-(1-8) and neurotensin-(1-10) as the major biologically inactive neurotensin catabolites. Endopeptidase 24.15 appeared mainly responsible for neurotensin-(1-8) formation by the soluble fraction of neurons and astrocytes. In contrast, endopeptidase 24.16 was involved in neurotensin-(1-10) formation by both soluble and membrane-associated fractions of the two cell types. An additional cleavage leading to neurotensin-(1-11) formation and ascribed to endopeptidase 24.11 was detected mainly in the membrane-associated fraction from astrocytes. Finally, the secondary processing of neurotensin degradation products indicated that: (a) neurotensin-(1-11) was converted into neurotensin-(1-8) in the membrane fraction prepared from astrocytes; (b) neurotensin-(1-10) was transformed into neurotensin-(1-8) by an unidentified peptidase belonging to the class of metalloenzymes. The significance of distinct quantitative and qualitative catabolic fates of neuromedin N and neurotensin in cultured astrocytes and neurons is discussed.

MicroRNA: Biogenesis, Function and Role in Cancer

PubMed Central

MacFarlane, Leigh-Ann; Murphy, Paul R.

2010-01-01

MicroRNAs are small, highly conserved non-coding RNA molecules involved in the regulation of gene expression. MicroRNAs are transcribed by RNA polymerases II and III, generating precursors that undergo a series of cleavage events to form mature microRNA. The conventional biogenesis pathway consists of two cleavage events, one nuclear and one cytoplasmic. However, alternative biogenesis pathways exist that differ in the number of cleavage events and enzymes responsible. How microRNA precursors are sorted to the different pathways is unclear but appears to be determined by the site of origin of the microRNA, its sequence and thermodynamic stability. The regulatory functions of microRNAs are accomplished through the RNA-induced silencing complex (RISC). MicroRNA assembles into RISC, activating the complex to target messenger RNA (mRNA) specified by the microRNA. Various RISC assembly models have been proposed and research continues to explore the mechanism(s) of RISC loading and activation. The degree and nature of the complementarity between the microRNA and target determine the gene silencing mechanism, slicer-dependent mRNA degradation or slicer-independent translation inhibition. Recent evidence indicates that P-bodies are essential for microRNA-mediated gene silencing and that RISC assembly and silencing occurs primarily within P-bodies. The P-body model outlines microRNA sorting and shuttling between specialized P-body compartments that house enzymes required for slicer –dependent and –independent silencing, addressing the reversibility of these silencing mechanisms. Detailed knowledge of the microRNA pathways is essential for understanding their physiological role and the implications associated with dysfunction and dysregulation. PMID:21532838

Amyloid-β Peptide Exacerbates the Memory Deficit Caused by Amyloid Precursor Protein Loss-of-Function in Drosophila

PubMed Central

Bourdet, Isabelle; Lampin-Saint-Amaux, Aurélie; Preat, Thomas; Goguel, Valérie

2015-01-01

The amyloid precursor protein (APP) plays a central role in Alzheimer’s disease (AD). APP can undergo two exclusive proteolytic pathways: cleavage by the α-secretase initiates the non-amyloidogenic pathway while cleavage by the β-secretase initiates the amyloidogenic pathway that leads, after a second cleavage by the γ-secretase, to amyloid-β (Aβ) peptides that can form toxic extracellular deposits, a hallmark of AD. The initial events leading to AD are still unknown. Importantly, aside from Aβ toxicity whose molecular mechanisms remain elusive, several studies have shown that APP plays a positive role in memory, raising the possibility that APP loss-of-function may participate to AD. We previously showed that APPL, the Drosophila APP ortholog, is required for associative memory in young flies. In the present report, we provide the first analysis of the amyloidogenic pathway’s influence on memory in the adult. We show that transient overexpression of the β-secretase in the mushroom bodies, the center for olfactory memory, did not alter memory. In sharp contrast, β-secretase overexpression affected memory when associated with APPL partial loss-of-function. Interestingly, similar results were observed with Drosophila Aβ peptide. Because Aβ overexpression impaired memory only when combined to APPL partial loss-of-function, the data suggest that Aβ affects memory through the APPL pathway. Thus, memory is altered by two connected mechanisms—APPL loss-of-function and amyloid peptide toxicity—revealing in Drosophila a functional interaction between APPL and amyloid peptide. PMID:26274614

Effects of inhibition of ubiquitin-proteasome pathway on human primary leukemic cells.

PubMed

Lan, Yu; Zhang, Xuemin; Yang, Pingdi; Hu, Meiru; Yu, Ming; Yang, Yi; Shen, Beifen

2002-12-01

Though there were a lot of reports about the totally different responses to the inhibition of ubiquitin-proteasome pathway in different kinds of cell lines, much less has been known about the responses in primary human leukemic cells. In this study, the effects of inhibition of ubiquitin-proteasome pathway on human bone marrow (BM) mononuclear cells (MNCs) obtained from 10 normal persons and 8 leukemia patients were examined. The results showed that the responses obviously varied individually. Among them, BM MNCs in 3 cases of leukemic patients were extremely sensitive, demonstrated by that > 90% cells were induced to undergo apoptosis within 24 h, but MNCs in 10 cases of normal persons showed resistance to the inhibition and no apoptosis was observed. Furthermore, Western blots revealed that the Bcl-2 expression was relatively high in the sensitive primary leukemia cells, and especially the cleavage of 26 ku Bcl-2 into a 22 ku fragment occurred during the induction of apoptosis. In contrast, the Bcl-2 expression was either undetectable or detectable but no cleavage of that above was observed in the cells insensitive to the inhibition of the pathway (including BM MNCs in normal persons). Together with the observations on the leukemic cell lines, these findings suggested the correlation of the specific cleavage of Bcl-2 into a shortened fragment with the sensitivity of cells to the inhibition of ubiquitin-proteasome pathway, which provides clues to the further understanding of the mechanisms of that dramatically different responses existing in different kinds of cells to the inhibition of ubiquitin-proteasome pathway.

Solvent isotope-induced equilibrium perturbation for isocitrate lyase.

PubMed

Quartararo, Christine E; Hadi, Timin; Cahill, Sean M; Blanchard, John S

2013-12-23

Isocitrate lyase (ICL) catalyzes the reversible retro-aldol cleavage of isocitrate to generate glyoxylate and succinate. ICL is the first enzyme of the glyoxylate shunt, which allows for the anaplerosis of citric acid cycle intermediates under nutrient limiting conditions. In Mycobacterium tuberculosis, the source of ICL for these studies, ICL is vital for the persistence phase of the bacterium's life cycle. Solvent kinetic isotope effects (KIEs) in the direction of isocitrate cleavage ((D₂O)V = 2.0 ± 0.1, and (D₂O)[V/K(isocitrate)] = 2.2 ± 0.3) arise from the initial deprotonation of the C2 hydroxyl group of isocitrate or the protonation of the aci-acid of the succinate product of the isocitrate aldol cleavage by a solvent-derived proton. This KIE suggested that an equilibrium mixture of all protiated isocitrate, glyoxylate, and succinate prepared in D₂O would undergo transient changes in equilibrium concentrations as a result of the solvent KIE and solvent-derived deuterium incorporation into both succinate and isocitrate. No change in the isotopic composition of glyoxylate was expected or observed. We have directly monitored the changing concentrations of all isotopic species of all reactants and products using a combination of nuclear magnetic resonance (NMR) spectroscopy and mass spectrometry. Continuous monitoring of glyoxylate by ¹H NMR spectroscopy shows a clear equilibrium perturbation in D₂O. The final equilibrium isotopic composition of reactants in D₂O revealed dideuterated succinate, protiated glyoxylate, and monodeuterated isocitrate, with the transient appearance and disappearance of monodeuterated succinate. A model for the equilibrium perturbation of substrate species and their time-dependent isotopic composition is presented.

Specificity determinants for autoproteolysis of LexA, a key regulator of bacterial SOS mutagenesis.

PubMed

Mo, Charlie Y; Birdwell, L Dillon; Kohli, Rahul M

2014-05-20

Bacteria utilize the tightly regulated stress response (SOS) pathway to respond to a variety of genotoxic agents, including antimicrobials. Activation of the SOS response is regulated by a key repressor-protease, LexA, which undergoes autoproteolysis in the setting of stress, resulting in derepression of SOS genes. Remarkably, genetic inactivation of LexA's self-cleavage activity significantly decreases acquired antibiotic resistance in infection models and renders bacteria hypersensitive to traditional antibiotics, suggesting that a mechanistic study of LexA could help inform its viability as a novel target for combating acquired drug resistance. Despite structural insights into LexA, a detailed knowledge of the enzyme's protease specificity is lacking. Here, we employ saturation and positional scanning mutagenesis on LexA's internal cleavage region to analyze >140 mutants and generate a comprehensive specificity profile of LexA from the human pathogen Pseudomonas aeruginosa (LexAPa). We find that the LexAPa active site possesses a unique mode of substrate recognition. Positions P1-P3 prefer small hydrophobic residues that suggest specific contacts with the active site, while positions P5 and P1' show a preference for flexible glycine residues that may facilitate the conformational change that permits autoproteolysis. We further show that stabilizing the β-turn within the cleavage region enhances LexA autoproteolytic activity. Finally, we identify permissive positions flanking the scissile bond (P4 and P2') that are tolerant to extensive mutagenesis. Our studies shed light on the active site architecture of the LexA autoprotease and provide insights that may inform the design of probes of the SOS pathway.

Two different types of double-strand breaks in Saccharomyces cerevisiae are repaired by similar RAD52-independent, nonhomologous recombination events.

PubMed Central

Kramer, K M; Brock, J A; Bloom, K; Moore, J K; Haber, J E

1994-01-01

In haploid rad52 Saccharomyces cerevisiae strains unable to undergo homologous recombination, a chromosomal double-strand break (DSB) can be repaired by imprecise rejoining of the broken chromosome ends. We have used two different strategies to generate broken chromosomes: (i) a site-specific DSB generated at the MAT locus by HO endonuclease cutting or (ii) a random DSB generated by mechanical rupture during mitotic segregation of a conditionally dicentric chromosome. Broken chromosomes were repaired by deletions that were highly variable in size, all of which removed more sequences than was required either to prevent subsequent HO cleavage or to eliminate a functional centromere, respectively. The junction of the deletions frequently occurred where complementary strands from the flanking DNA could anneal to form 1 to 5 bp, although 12% (4 of 34) of the events appear to have occurred by blunt-end ligation. These types of deletions are very similar to the junctions observed in the repair of DSBs by mammalian cells (D. B. Roth and J. H. Wilson, Mol. Cell. Biol. 6:4295-4304, 1986). When a high level of HO endonuclease, expressed in all phases of the cell cycle, was used to create DSBs, we also recovered a large class of very small (2- or 3-bp) insertions in the HO cleavage site. These insertions appear to represent still another mechanism of DSB repair, apparently by annealing and filling in the overhanging 3' ends of the cleavage site. These types of events have also been well documented for vertebrate cells. PMID:8289808

Determination of Kinetic and Thermodynamic Parameters that Describe Isothermal Seed Germination: A Student Research Project.

ERIC Educational Resources Information Center

Hageseth, Gaylord T.

1982-01-01

Describes a project for students to collect and fit data to a theoretical mathematical model that describes the rate of isothermal seed germination, including activation energy for substrate and produce and the autocatalytic reaction, and changes in enthalpy, entropy, and the Gibb's free energy. (Author/SK)

Foldamer hypothesis for the growth and sequence differentiation of prebiotic polymers

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

Guseva, Elizaveta; Zuckermann, Ronald N.; Dill, Ken A.

It is not known how life originated. It is thought that prebiotic processes were able to synthesize short random polymers. However, then, how do short-chain molecules spontaneously grow longer? Also, how would random chains grow more informational and become autocatalytic (i.e., increasing their own concentrations)? We study the folding and binding of random sequences of hydrophobic ( H) and polar ( P) monomers in a computational model. We find that even short hydrophobic polar ( HP) chains can collapse into relatively compact structures, exposing hydrophobic surfaces. In this way, they act as primitive versions of today’s protein catalysts, elongating othermore » such HP polymers as ribosomes would now do. Such foldamer catalysts are shown to form an autocatalytic set, through which short chains grow into longer chains that have particular sequences. An attractive feature of this model is that it does not overconverge to a single solution; it gives ensembles that could further evolve under selection. This mechanism describes how specific sequences and conformations could contribute to the chemistry-to-biology (CTB) transition.« less

Necessary conditions for the emergence of homochirality via autocatalytic self-replication

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

Stich, Michael; Ribó, Josep M.; Blackmond, Donna G., E-mail: blackmond@scripps.edu

We analyze a recent proposal for spontaneous mirror symmetry breaking based on the coupling of first-order enantioselective autocatalysis and direct production of the enantiomers that invokes a critical role for intrinsic reaction noise. For isolated systems, the racemic state is the unique stable outcome for both stochastic and deterministic dynamics when the system is in compliance with the constraints dictated by the thermodynamics of chemical reaction processes. In open systems, the racemic outcome also results for both stochastic and deterministic dynamics when driving the autocatalysis unidirectionally by external reagents. Nonracemic states can result in the latter only if the reversemore » reactions are strictly zero: these are kinetically controlled outcomes for small populations and volumes, and can be simulated by stochastic dynamics. However, the stability of the thermodynamic limit proves that the racemic outcome is the unique stable state for strictly irreversible externally driven autocatalysis. These findings contradict the suggestion that the inhibition requirement of the Frank autocatalytic model for the emergence of homochirality may be relaxed in a noise-induced mechanism.« less

Dynamics of intracellular information decoding.

PubMed

Kobayashi, Tetsuya J; Kamimura, Atsushi

2011-10-01

A variety of cellular functions are robust even to substantial intrinsic and extrinsic noise in intracellular reactions and the environment that could be strong enough to impair or limit them. In particular, of substantial importance is cellular decision-making in which a cell chooses a fate or behavior on the basis of information conveyed in noisy external signals. For robust decoding, the crucial step is filtering out the noise inevitably added during information transmission. As a minimal and optimal implementation of such an information decoding process, the autocatalytic phosphorylation and autocatalytic dephosphorylation (aPadP) cycle was recently proposed. Here, we analyze the dynamical properties of the aPadP cycle in detail. We describe the dynamical roles of the stationary and short-term responses in determining the efficiency of information decoding and clarify the optimality of the threshold value of the stationary response and its information-theoretical meaning. Furthermore, we investigate the robustness of the aPadP cycle against the receptor inactivation time and intrinsic noise. Finally, we discuss the relationship among information decoding with information-dependent actions, bet-hedging and network modularity.

Influence of thermal effects on buoyancy-driven convection around autocatalytic chemical fronts propagating horizontally.

PubMed

Rongy, L; Schuszter, G; Sinkó, Z; Tóth, T; Horváth, D; Tóth, A; De Wit, A

2009-06-01

The spatiotemporal dynamics of vertical autocatalytic fronts traveling horizontally in thin solution layers closed to the air can be influenced by buoyancy-driven convection induced by density gradients across the front. We perform here a combined experimental and theoretical study of the competition between solutal and thermal effects on such convection. Experimentally, we focus on the antagonistic chlorite-tetrathionate reaction for which solutal and thermal contributions to the density jump across the front have opposite signs. We show that in isothermal conditions the heavier products sink below the lighter reactants, providing an asymptotic constant finger shape deformation of the front by convection. When thermal effects are present, the hotter products, on the contrary, climb above the reactants for strongly exothermic conditions. These various observations as well as the influence of the relative weight of the solutal and thermal effects and of the thickness of the solution layer on the dynamics are discussed in terms of a two-dimensional reaction-diffusion-convection model parametrized by a solutal R(C) and a thermal R(T) Rayleigh number.

Highly efficient and autocatalytic H2₂ dissociation for CO₂ reduction into formic acid with zinc.

PubMed

Jin, Fangming; Zeng, Xu; Liu, Jianke; Jin, Yujia; Wang, Lunying; Zhong, Heng; Yao, Guodong; Huo, Zhibao

2014-03-28

Artificial photosynthesis, specifically H2O dissociation for CO2 reduction with solar energy, is regarded as one of the most promising methods for sustainable energy and utilisation of environmental resources. However, a highly efficient conversion still remains extremely challenging. The hydrogenation of CO2 is regarded as the most commercially feasible method, but this method requires either exotic catalysts or high-purity hydrogen and hydrogen storage, which are regarded as an energy-intensive process. Here we report a highly efficient method of H2O dissociation for reducing CO2 into chemicals with Zn powder that produces formic acid with a high yield of approximately 80%, and this reaction is revealed for the first time as an autocatalytic process in which an active intermediate, ZnH(-) complex, serves as the active hydrogen. The proposed process can assist in developing a new concept for improving artificial photosynthetic efficiency by coupling geochemistry, specifically the metal-based reduction of H2O and CO2, with solar-driven thermochemistry for reducing metal oxide into metal.

Highly efficient and autocatalytic H2O dissociation for CO2 reduction into formic acid with zinc

PubMed Central

Jin, Fangming; Zeng, Xu; Liu, Jianke; Jin, Yujia; Wang, Lunying; Zhong, Heng; Yao, Guodong; Huo, Zhibao

2014-01-01

Artificial photosynthesis, specifically H2O dissociation for CO2 reduction with solar energy, is regarded as one of the most promising methods for sustainable energy and utilisation of environmental resources. However, a highly efficient conversion still remains extremely challenging. The hydrogenation of CO2 is regarded as the most commercially feasible method, but this method requires either exotic catalysts or high-purity hydrogen and hydrogen storage, which are regarded as an energy-intensive process. Here we report a highly efficient method of H2O dissociation for reducing CO2 into chemicals with Zn powder that produces formic acid with a high yield of approximately 80%, and this reaction is revealed for the first time as an autocatalytic process in which an active intermediate, ZnH− complex, serves as the active hydrogen. The proposed process can assist in developing a new concept for improving artificial photosynthetic efficiency by coupling geochemistry, specifically the metal-based reduction of H2O and CO2, with solar-driven thermochemistry for reducing metal oxide into metal. PMID:24675820

Temporal Control of Gelation and Polymerization Fronts Driven by an Autocatalytic Enzyme Reaction.

PubMed

Jee, Elizabeth; Bánsági, Tamás; Taylor, Annette F; Pojman, John A

2016-02-05

Chemical systems that remain kinetically dormant until activated have numerous applications in materials science. Herein we present a method for the control of gelation that exploits an inbuilt switch: the increase in pH after an induction period in the urease-catalyzed hydrolysis of urea was used to trigger the base-catalyzed Michael addition of a water-soluble trithiol to a polyethylene glycol diacrylate. The time to gelation (minutes to hours) was either preset through the initial concentrations or the reaction was initiated locally by a base, thus resulting in polymerization fronts that converted the mixture from a liquid into a gel (ca. 0.1 mm min(-1)). The rate of hydrolytic degradation of the hydrogel depended on the initial concentrations, thus resulting in a gel lifetime of hours to months. In this way, temporal programming of gelation was possible under mild conditions by using the output of an autocatalytic enzyme reaction to drive both the polymerization and subsequent degradation of a hydrogel. © 2015 The Authors. Published by Wiley-VCH Verlag GmbH & Co. KGaA.

EGF-dependent re-routing of vesicular recycling switches spontaneous phosphorylation suppression to EGFR signaling

PubMed Central

Baumdick, Martin; Brüggemann, Yannick; Schmick, Malte; Xouri, Georgia; Sabet, Ola; Davis, Lloyd; Chin, Jason W; Bastiaens, Philippe IH

2015-01-01

Autocatalytic activation of epidermal growth factor receptor (EGFR) coupled to dephosphorylating activity of protein tyrosine phosphatases (PTPs) ensures robust yet diverse responses to extracellular stimuli. The inevitable tradeoff of this plasticity is spontaneous receptor activation and spurious signaling. We show that a ligand-mediated switch in EGFR trafficking enables suppression of spontaneous activation while maintaining EGFR’s capacity to transduce extracellular signals. Autocatalytic phosphorylation of tyrosine 845 on unliganded EGFR monomers is suppressed by vesicular recycling through perinuclear areas with high PTP1B activity. Ligand-binding results in phosphorylation of the c-Cbl docking tyrosine and ubiquitination of the receptor. This secondary signal relies on EGF-induced EGFR self-association and switches suppressive recycling to directional trafficking. The re-routing regulates EGFR signaling response by the transit-time to late endosomes where it is switched-off by high PTP1B activity. This ubiquitin-mediated switch in EGFR trafficking is a uniquely suited solution to suppress spontaneous activation while maintaining responsiveness to EGF. DOI: http://dx.doi.org/10.7554/eLife.12223.001 PMID:26609808

Physical basis of large microtubule aster growth

PubMed Central

Ishihara, Keisuke; Korolev, Kirill S; Mitchison, Timothy J

2016-01-01

Microtubule asters - radial arrays of microtubules organized by centrosomes - play a fundamental role in the spatial coordination of animal cells. The standard model of aster growth assumes a fixed number of microtubules originating from the centrosomes. However, aster morphology in this model does not scale with cell size, and we recently found evidence for non-centrosomal microtubule nucleation. Here, we combine autocatalytic nucleation and polymerization dynamics to develop a biophysical model of aster growth. Our model predicts that asters expand as traveling waves and recapitulates all major aspects of aster growth. With increasing nucleation rate, the model predicts an explosive transition from stationary to growing asters with a discontinuous jump of the aster velocity to a nonzero value. Experiments in frog egg extract confirm the main theoretical predictions. Our results suggest that asters observed in large fish and amphibian eggs are a meshwork of short, unstable microtubules maintained by autocatalytic nucleation and provide a paradigm for the assembly of robust and evolvable polymer networks. DOI: http://dx.doi.org/10.7554/eLife.19145.001 PMID:27892852

Foldamer hypothesis for the growth and sequence differentiation of prebiotic polymers

DOE PAGES

Guseva, Elizaveta; Zuckermann, Ronald N.; Dill, Ken A.

2017-08-22

It is not known how life originated. It is thought that prebiotic processes were able to synthesize short random polymers. However, then, how do short-chain molecules spontaneously grow longer? Also, how would random chains grow more informational and become autocatalytic (i.e., increasing their own concentrations)? We study the folding and binding of random sequences of hydrophobic ( H) and polar ( P) monomers in a computational model. We find that even short hydrophobic polar ( HP) chains can collapse into relatively compact structures, exposing hydrophobic surfaces. In this way, they act as primitive versions of today’s protein catalysts, elongating othermore » such HP polymers as ribosomes would now do. Such foldamer catalysts are shown to form an autocatalytic set, through which short chains grow into longer chains that have particular sequences. An attractive feature of this model is that it does not overconverge to a single solution; it gives ensembles that could further evolve under selection. This mechanism describes how specific sequences and conformations could contribute to the chemistry-to-biology (CTB) transition.« less

Foldamer hypothesis for the growth and sequence differentiation of prebiotic polymers

PubMed Central

Guseva, Elizaveta; Zuckermann, Ronald N.; Dill, Ken A.

2017-01-01

It is not known how life originated. It is thought that prebiotic processes were able to synthesize short random polymers. However, then, how do short-chain molecules spontaneously grow longer? Also, how would random chains grow more informational and become autocatalytic (i.e., increasing their own concentrations)? We study the folding and binding of random sequences of hydrophobic (H) and polar (P) monomers in a computational model. We find that even short hydrophobic polar (HP) chains can collapse into relatively compact structures, exposing hydrophobic surfaces. In this way, they act as primitive versions of today’s protein catalysts, elongating other such HP polymers as ribosomes would now do. Such foldamer catalysts are shown to form an autocatalytic set, through which short chains grow into longer chains that have particular sequences. An attractive feature of this model is that it does not overconverge to a single solution; it gives ensembles that could further evolve under selection. This mechanism describes how specific sequences and conformations could contribute to the chemistry-to-biology (CTB) transition. PMID:28831002

Thermal decomposition of the solid phase of nitromethane: ab initio molecular dynamics simulations.

PubMed

Chang, Jing; Lian, Peng; Wei, Dong-Qing; Chen, Xiang-Rong; Zhang, Qing-Ming; Gong, Zi-Zheng

2010-10-29

The Car-Parrinello molecular dynamics simulations were employed to investigate thermal decomposition of the solid nitromethane. It is found that it undergoes chemical decomposition at about 2200 K under ambient pressure. The initiation of reactions involves both proton transfer and commonly known C-N bond cleavage. About 75 species and 100 elementary reactions were observed with the final products being H2O, CO2, N2, and CNCNC. It represents the first complete simulation of solid-phase explosive reactions reported to date, which is of far-reaching implication for design and development of new energetic materials.

Role of heme in intracellular trafficking of thyroperoxidase and involvement of H2O2 generated at the apical surface of thyroid cells in autocatalytic covalent heme binding.

PubMed

Fayadat, L; Niccoli-Sire, P; Lanet, J; Franc, J L

1999-04-09

Thyroperoxidase (TPO) is a glycosylated hemoprotein that plays a key role in thyroid hormone synthesis. We previously showed that in CHO cells expressing human TPO (hTPO) only 2% of synthesized hTPO reaches the cell surface. Herein, we investigated the role of heme moiety insertion in the exit of hTPO from the endoplasmic reticulum. Peroxidase activity at the cell surface and cell surface expression of hTPO were decreased by approximately 30 and approximately 80%, respectively, with succinyl acetone, an inhibitor of heme biosynthesis, and were increased by 20% with holotransferrin and aminolevulinic acid, precursors of heme biosynthesis. Results were similar with holotransferrin plus aminolevulinic acid or hemin, but hemin increased cell surface activity more efficiently (+120%) relative to the control. It had been suggested (DePillis, G., Ozaki, S., Kuo, J. M., Maltby, D. A., and Ortiz de Montellano, P. R. (1997) J. Biol. Chem. 272, 8857-8960) that covalent attachment of heme to mammalian peroxidases could be an H2O2-dependent autocatalytic processing. In our study, heme associated intracellularly with hTPO, and we hypothesized that there was insufficient exposure to H2O2 in Chinese hamster ovary cells before hTPO reached the cell surface. After a 10-min incubation, 10 microM H2O2 led to a 65% increase in cell surface activity. In contrast, in thyroid cells, H2O2 was synthesized at the apical cell surface and allowed covalent attachment of heme. Two-day incubation of primocultures of thyroid cells with catalase led to a 30% decrease in TPO activity at the cell surface. In conclusion, we provide compelling evidence for an essential role of 1) heme incorporation in the intracellular trafficking of hTPO and of 2) H2O2 generated at the apical pole of thyroid cells in the autocatalytic covalent heme binding to the TPO molecule.

Oxidation and gum formation in diesel fuels. Interim report No. 2, 10 September 1984-30 April 1985

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

Mayo, F.R.

1985-05-03

Rates of oxygen absorption (R sub O) and gum formation (R sub g) are now related by the R sub g/ R/sub 0/ ratio, the reciprocal of the ratio previously used. The change was made because when the oxygen content of the gum is known, the R sub g/ R/sub 0/ ratio is proportional to the fraction of oxygen absorbed that appears in the gum. Oxidations of tetralin (TET) and 2-ethyl-naphthalene (EtN) at 130 C are initially fast but the rates decrease regularly. The oxidation of n-dodecane (DOD) is clearly autocatalytic; it requires the most oxygen to produce a milligrammore » of gum while EtN, among the pure hydrocarbons, requires the least. Rates of oxygen absorption for DOD at 100 C appear to be erratic. Part of the problem is autocatalysis; part is near exhaustion of oxygen. The best results are those for K92A and K92C. R/sub 0/ without t-Bu2O2 is autocatalytic. Trends in oxidations of Fuels 14 and 14A, which are different at 130 C, are not yet clear at 100, and 60 C. The important points in the above discussion are: The oxidation of DOD is autocatalytic; oxidations of TET, EtN, and Fuel 14 are self-retarding. In our oxidations of Fuel 14 with shaking, all the deposits at 100 and 130 C appear as films on glass; no deposits have yet been obtained at lower temperatures. The ratio R sub g/ R/sub 0/, still appears to be essentially constant for any fuel at a single temperature, even with large differences in R/sub 0/ from addition of t-Bu202. Thus, gum can be accumulated relatively rapidly for experimental purposes. We are accumulating new data at 43 and 60 C. These findings should assist materially in our efforts to understand and devise a test for fuel stability.« less

Put a tiger in your tank: the polyclad flatworm Maritigrella crozieri as a proposed model for evo-devo.

PubMed

Lapraz, François; Rawlinson, Kate A; Girstmair, Johannes; Tomiczek, Bartłomiej; Berger, Jürgen; Jékely, Gáspár; Telford, Maximilian J; Egger, Bernhard

2013-10-09

Polyclad flatworms are an early branching clade within the rhabditophoran Platyhelminthes. They provide an interesting system with which to explore the evolution of development within Platyhelminthes and amongst Spiralia (Lophotrochozoa). Unlike most other flatworms, polyclads undergo spiral cleavage (similar to that seen in some other spiralian taxa), they are the only free-living flatworms where development via a larval stage occurs, and they are the only flatworms in which embryos can be reared outside of their protective egg case, enabling embryonic manipulations. Past work has focused on comparing early cleavage patterns and larval anatomy between polyclads and other spiralians. We have selected Maritigrella crozieri, the tiger flatworm, as a suitable polyclad species for developmental studies, because it is abundant and large in size compared to other species. These characteristics have facilitated the generation of a transcriptome from embryonic and larval material and are enabling us to develop methods for gene expression analysis and immunofluorescence techniques. Here we give an overview of M. crozieri and its development, we highlight the advantages and current limitations of this animal as a potential evo-devo model and discuss current lines of research.

Methods to Increase the Metabolic Stability of (18)F-Radiotracers.

PubMed

Kuchar, Manuela; Mamat, Constantin

2015-09-03

The majority of pharmaceuticals and other organic compounds incorporating radiotracers that are considered foreign to the body undergo metabolic changes in vivo. Metabolic degradation of these drugs is commonly caused by a system of enzymes of low substrate specificity requirement, which is present mainly in the liver, but drug metabolism may also take place in the kidneys or other organs. Thus, radiotracers and all other pharmaceuticals are faced with enormous challenges to maintain their stability in vivo highlighting the importance of their structure. Often in practice, such biologically active molecules exhibit these properties in vitro, but fail during in vivo studies due to obtaining an increased metabolism within minutes. Many pharmacologically and biologically interesting compounds never see application due to their lack of stability. One of the most important issues of radiotracers development based on fluorine-18 is the stability in vitro and in vivo. Sometimes, the metabolism of (18)F-radiotracers goes along with the cleavage of the C-F bond and with the rejection of [(18)F]fluoride mostly combined with high background and accumulation in the skeleton. This review deals with the impact of radiodefluorination and with approaches to stabilize the C-F bond to avoid the cleavage between fluorine and carbon.

Put a tiger in your tank: the polyclad flatworm Maritigrella crozieri as a proposed model for evo-devo

PubMed Central

2013-01-01

Polyclad flatworms are an early branching clade within the rhabditophoran Platyhelminthes. They provide an interesting system with which to explore the evolution of development within Platyhelminthes and amongst Spiralia (Lophotrochozoa). Unlike most other flatworms, polyclads undergo spiral cleavage (similar to that seen in some other spiralian taxa), they are the only free-living flatworms where development via a larval stage occurs, and they are the only flatworms in which embryos can be reared outside of their protective egg case, enabling embryonic manipulations. Past work has focused on comparing early cleavage patterns and larval anatomy between polyclads and other spiralians. We have selected Maritigrella crozieri, the tiger flatworm, as a suitable polyclad species for developmental studies, because it is abundant and large in size compared to other species. These characteristics have facilitated the generation of a transcriptome from embryonic and larval material and are enabling us to develop methods for gene expression analysis and immunofluorescence techniques. Here we give an overview of M. crozieri and its development, we highlight the advantages and current limitations of this animal as a potential evo-devo model and discuss current lines of research. PMID:24107307

Degradation of nitrobenzene by a Pseudomonas pseudoalcaligenes.

PubMed Central

Nishino, S F; Spain, J C

1993-01-01

A Pseudomonas pseudoalcaligenes able to use nitrobenzene as the sole source of carbon, nitrogen, and energy was isolated from soil and groundwater contaminated with nitrobenzene. The range of aromatic substrates able to support growth was limited to nitrobenzene, hydroxylaminobenzene, and 2-aminophenol. Washed suspensions of nitrobenzene-grown cells removed nitrobenzene from culture fluids with the concomitant release of ammonia. Nitrobenzene, nitrosobenzene, hydroxylaminobenzene, and 2-aminophenol stimulated oxygen uptake in resting cells and in extracts of nitrobenzene-grown cells. Under aerobic and anaerobic conditions, crude extracts converted nitrobenzene to 2-aminophenol with oxidation of 2 mol of NADPH. Ring cleavage, which required ferrous iron, produced a transient yellow product with a maximum A380. In the presence of NAD, the product disappeared and NADH was produced. In the absence of NAD, the ring fission product was spontaneously converted to picolinic acid, which was not further metabolized. These results indicate that the catabolic pathway involves the reduction of nitrobenzene to nitrosobenzene and then to hydroxylaminobenzene; each of these steps requires 1 mol of NADPH. An enzyme-mediated Bamberger-like rearrangement converts hydroxylaminobenzene to 2-aminophenol, which then undergoes meta ring cleavage to 2-aminomuconic semialdehyde. The mechanism for release of ammonia and subsequent metabolism are under investigation. PMID:8368838

The effect of glucocorticoids on ERK-1/2 phosphorylation during maturation of lamb oocytes and their subsequent fertilization and cleavage ability in vitro.

PubMed

González, Raquel; Ruiz-León, Yolanda; Gomendio, Montserrat; Roldan, Eduardo R S

2010-04-01

High levels of glucocorticoids may alter reproduction, but little is known about their direct actions on oocyte maturation, fertilization and subsequent development. Earlier work suggested negative effects of cortisol or dexamethasone on oocyte maturation but differences were noted between animal models. Both glucocorticoids reduce the p34(cdc2)-cyclin B1 complex but it is unknown if other signaling pathways important for meiosis progression are affected. In this study, using sheep oocytes as a model system, we assessed in vitro the effects of increasing concentration of glucocorticoids (0-250 microM) on oocyte maturation and underlying changes in the MAP kinase pathway, and the ability of oocytes to undergo fertilization and embryo development. Cortisol decreased oocyte maturation but only at the highest concentration, whereas dexamethasone had no effect. Fertilization and cleavage were not affected. On the other hand, both cortisol and dexamethasone inhibited ERK-1/2 activation in a concentration-dependent manner. It thus seems that oocytes can overcome deleterious effects of glucocorticoids during maturation despite the decrease in ERK-1/2 activity, but repercussions in vivo should be further explored. Copyright 2009 Elsevier Inc. All rights reserved.

Temperature-independent energy expenditure in early development of the African clawed frog Xenopus laevis.

PubMed

Nagano, Yatsuhisa; Ode, Koji L

2014-08-01

The thermal dissipation of activated eggs and embryos undergoing development from cleavage to the tailbud stage of the African clawed frog Xenopus laevis was measured as a function of incubation time at temperatures ranging from T = 288.2 K to 295.2 K, using a high-precision isothermal calorimeter. A23187-mediated activation of mature eggs induced stable periodic thermal oscillations lasting for 8-34 h. The frequency agreed well with the cell cycle frequency of initial cleavages at the identical temperature. In the developing embryo, energy metabolism switches from embryonic to adult features during gastrulation. The thermal dissipation after gastrulation fit well with a single modified Avrami equation, which has been used for modeling crystal-growth. Both the oscillation frequency of the activated egg and the growth rate of the embryo strongly depend on temperature with the same apparent activation energy of approximately 87 kJ mole(-1). This result suggests that early development proceeds as a single biological time, attributable to a single metabolic rate. A temperature-independent growth curve was derived by scaling the thermogram to the biological time, indicating that the amount of energy expenditure during each developmental stage is constant over the optimal temperature range.

Structural Aberrations in T-Even Bacteriophage V. Effects of Canavanine on the Maturation and Utilization of Specific Gene Products

PubMed Central

Bolin, Rex W.; Cummings, Donald J.

1974-01-01

Previous results have shown that when a T-even bacteriophage-infected cell was exposed to l-canavanine followed by an exposure to l-arginine, a monster phage particle, termed a lollipop, was formed. l-Canavanine was necessary for the induction event but l-arginine was required for the maturation of the particle. We now describe the effects of canavanine on the maturation of certain T4 proteins and their role in the induction of lollipops. The cleavage reactions of the head proteins P22, P23, P24, and IPIII are prevented by l-canavanine as shown by the accumulation of the precursor proteins and the failure of the cleaved products to appear. l-Canavanine also prevents the appearance of P12 (tailplate protein) and P20 (head protein) indicating that these proteins may undergo a proteolytic cleavage during normal assembly. The formation of P10 (tailplate protein) and P18 (tail sheath protein) is also affected by l-canavanine. The data suggest that P23 in conjunction with P20 plays a major role in the determination of the length of the phage head. Images PMID:4833614

Purification and characterization of VDE, a site-specific endonuclease from the yeast Saccharomyces cerevisiae.

PubMed

Gimble, F S; Thorner, J

1993-10-15

The 119-kDa primary translation product of the VMA1 gene of Saccharomyces cerevisiae undergoes a self-catalyzed rearrangement ("protein splicing") that excises an internal 50-kDa segment of the polypeptide and joins the amino-terminal and carboxyl-terminal segments to generate the 69-kDa subunit of the vacuolar membrane-associated H(+)-ATPase. We have shown previously that the internal segment is a site-specific endonuclease (Gimble, F. S., and Thorner, J. (1992) Nature 357, 301-306). Here we describe methods for the high level expression and purification to near homogeneity of both the authentic VMA1-derived endonuclease (or VDE) from yeast (yield 18%) and a recombinant form of VDE made in bacteria (yield 29%). Detailed characterization of these preparations demonstrated that the yeast-derived and bacterially produced enzymes were indistinguishable, as judged by: (a) behavior during purification; (b) apparent native molecular mass (50 kDa); (c) immunological reactivity; and (d) catalytic properties (specific activity; cleavage site recognition; and optima for pH, temperature, divalent cation and ionic strength). The minimal site required for VDE cleavage was delimited to a 30-base pair sequence within its specific substrate (the VMA1 delta vde allele).

Analysis of protein prenylation and S-acylation using gas chromatography-coupled mass spectrometry.

PubMed

Sorek, Nadav; Akerman, Amir; Yalovsky, Shaul

2013-01-01

Lipid modifications play a key role in protein targeting and function. The two Arabidopsis Gγ subunits, AGG1 and AGG2, have been shown to undergo prenylation (AGG1) and S-acylation (AGG2). Prenylation involves covalent nonreversible attachment of either farnesyl (15 carbons) or geranylgeranyl (20 carbons) isoprenoids to conserved cysteine residues at or near the C-terminus of proteins. S-acylation, frequently referred to as palmitoylation, involves the attachment of acyl fatty acids to thiol groups of cysteine residues through a reversible thioester bond. The procedures described below allow direct analysis of the prenyl and acyl moieties using gas chromatography-coupled mass spectrometry (GC-MS). These methods are based on (1) cleavage of prenyl groups with the Raney nickel catalyst and (2) analysis of protein S-acylation following cleavage of the acyl fatty acids from proteins by hydrogenation with platinum (IV) oxide. The hydrogenation under these conditions causes an acid transesterification of the acyl moieties, adding an ethyl group to the carboxyl head of the fatty acid. The addition of the ethyl group reduces the polarity of the fatty acids, allowing their efficient separation by gas chromatography.

Polysorbates 20 and 80 used in the formulation of protein biotherapeutics: structure and degradation pathways.

PubMed

Kerwin, Bruce A

2008-08-01

Polysorbates 20 and 80 (Tween 20 and Tween 80) are used in the formulation of biotherapeutic products for both preventing surface adsorption and as stabilizers against protein aggregation. The polysorbates are amphipathic, nonionic surfactants composed of fatty acid esters of polyoxyethylene sorbitan being polyoxyethylene sorbitan monolaurate for polysorbate 20 and polyoxyethylene sorbitan monooleate for polysorbate 80. The polysorbates used in the formulation of biopharmaceuticals are mixtures of different fatty acid esters with the monolaurate fraction of polysorbate 20 making up only 40-60% of the mixture and the monooleate fraction of polysorbate 80 making up >58% of the mixture. The polysorbates undergo autooxidation, cleavage at the ethylene oxide subunits and hydrolysis of the fatty acid ester bond. Autooxidation results in hydroperoxide formation, side-chain cleavage and eventually formation of short chain acids such as formic acid all of which could influence the stability of a biopharmaceutical product. Oxidation of the fatty acid moiety while well described in the literature has not been specifically investigated for polysorbate. This review focuses on the chemical structure of the polysorbates, factors influencing micelle formation and factors and excipients influencing stability and degradation of the polyoxyethylene and fatty acid ester linkages.

Poly(ethyl glyoxylate)-Poly(ethylene oxide) Nanoparticles: Stimuli-Responsive Drug Release via End-to-End Polyglyoxylate Depolymerization.

PubMed

Fan, Bo; Gillies, Elizabeth R

2017-08-07

The ability to disrupt polymer assemblies in response to specific stimuli provides the potential to release drugs selectively at certain sites or conditions in vivo. However, most stimuli-responsive delivery systems require many stimuli-initiated events to release drugs. "Self-immolative polymers" offer the potential to provide amplified responses to stimuli as they undergo complete end-to-end depolymerization following the cleavage of a single end-cap. Herein, linker end-caps were developed to conjugate self-immolative poly(ethyl glyoxylate) (PEtG) with poly(ethylene oxide) (PEO) to form amphiphilic block copolymers. These copolymers were self-assembled to form nanoparticles in aqueous solution. Cleavage of the linker end-caps were triggered by a thiol reducing agent, UV light, H 2 O 2 , and combinations of these stimuli, resulting in nanoparticle disintegration. Low stimuli concentrations were effective in rapidly disrupting the nanoparticles. Nile red, doxorubin, and curcumin were encapsulated into the nanoparticles and were selectively released upon application of the appropriate stimulus. The ability to tune the stimuli-responsiveness simply by changing the linker end-cap makes this new platform highly attractive for applications in drug delivery.

Studies of Premixed Laminar and Turbulent Flames at Microgravity

NASA Technical Reports Server (NTRS)

Abid, M.; Aung, K.; Ronney, P. D.; Sharif, J. A.; Wu, M.-S.

1999-01-01

Several topics relating to combustion limits in premixed flames at reduced gravity have been studied. These topics include: (1) flame balls; (2) numerical simulation of flame ball and planar flame structure and stability; (3) experimental simulation of buoyancy effects in premixed flames using aqueous autocatalytic reactions; and (4) premixed flame propagation in Hele-Shaw cells.

Autocatalytic Patterning with Silver Using Tin(II) Chloride Sensitizer

ERIC Educational Resources Information Center

Mbindyo, Jeremiah K. N.; Anna, Laura J.; Fell, B. Andrew; Patton, David A.

2011-01-01

A silver mirror can be deposited on many types of surfaces from the reduction of the silver-diammine complex by a reducing sugar as proposed by Kemp in this "Journal". Three extensions of Kemp's demonstration that highlight the role of SnCl[subscript 2] sensitizer in the deposition of a silver mirror on surfaces are presented. The demonstration…

Posttranslational processing of the prohormone-cleaving Kex2 protease in the Saccharomyces cerevisiae secretory pathway.

PubMed

Wilcox, C A; Fuller, R S

1991-10-01

The Kex2 protease of the yeast Saccharomyces cerevisiae is a prototypical eukaryotic prohormone-processing enzyme that cleaves precursors of secreted peptides at pairs of basic residues. Here we have established the pathway of posttranslational modification of Kex2 protein using immunoprecipitation of the biosynthetically pulse-labeled protein from a variety of wild-type and mutant yeast strains as the principal methodology. Kex2 protein is initially synthesized as a prepro-enzyme that undergoes cotranslational signal peptide cleavage and addition of Asn-linked core oligosaccharide and Ser/Thr-linked mannose in the ER. The earliest detectable species, I1 (approximately 129 kD), undergoes rapid amino-terminal proteolytic removal of a approximately 9-kD pro-segment yielding species I2 (approximately 120 kD) before arrival at the Golgi complex. Transport to the Golgi complex is marked by extensive elaboration of Ser/Thr-linked chains and minor modification of Asn-linked oligosaccharide. During the latter phase of its lifetime, Kex2 protein undergoes a gradual increase in apparent molecular weight. This final modification serves as a marker for association of Kex2 protease with a late compartment of the yeast Golgi complex in which it is concentrated about 27-fold relative to other secretory proteins.

Investigating inner-sphere reorganization via secondary kinetic isotope effects in the C-H cleavage reaction catalyzed by soybean lipoxygenase: tunneling in the substrate backbone as well as the transferred hydrogen.

PubMed

Meyer, Matthew P; Klinman, Judith P

2011-01-26

This work describes the application of NMR to the measurement of secondary deuterium (2° (2)H) and carbon-13 ((13)C) kinetic isotope effects (KIEs) at positions 9-13 within the substrate linoleic acid (LA) of soybean lipoxygenase-1. The KIEs have been measured using LA labeled with either protium (11,11-h2-LA) or deuterium (11,11-d2-LA) at the reactive C11 position, which has been previously shown to yield a primary deuterium isotope effect of ca. 80. The conditions of measurement yield the intrinsic 2° (2)H and (13)C KIEs on k(cat)/K(m) directly for 11,11-d2-LA, whereas the values for the 2° (2)H KIEs for 11,11-h2-LA are obtained after correction for a kinetic commitment. The pattern of the resulting 2° (2)H and (13)C isotope effects reveals values that lie far above those predicted from changes in local force constants. Additionally, many of the experimental values cannot be modeled by electronic effects, torsional strain, or the simple inclusion of a tunneling correction to the rate. Although previous studies have shown the importance of extensive tunneling for cleavage of the primary hydrogen at C11 of LA, the present findings can only be interpreted by extending the conclusion of nonclassical behavior to the secondary hydrogens and carbons that flank the position undergoing C-H bond cleavage. A quantum mechanical method introduced by Buhks et al. [J. Phys. Chem. 1981, 85, 3763] to model the inner-sphere reorganization that accompanies electron transfer has been shown to be able to reproduce the scale of the 2° (2)H KIEs.

Solvent Isotope-induced Equilibrium Perturbation for Isocitrate Lyase

PubMed Central

Quartararo, Christine E.; Hadi, Timin; Cahill, Sean M.; Blanchard, John S.

2014-01-01

Isocitrate lyase (ICL) catalyzes the reversible retro-aldol cleavage of isocitrate to generate glyoxylate and succinate. ICL is the first enzyme of the glyoxylate shunt, which allows for the anaplerosis of citric acid cycle intermediates under nutrient limiting conditions. In Mycobacterium tuberculosis, the source of ICL for these studies, ICL is vital for the persistence phase of the bacteria’s life cycle. Solvent kinetic isotope effects (KIEs) in the direction of isocitrate cleavage of D2OV = 2.0 ± 0.1 and D2O[V/Kisocitrate] = 2.2 ± 0.3 arise from the initial deprotonation of the C2 hydroxyl group of isocitrate or the protonation of the aci-acid of succinate product of the isocitrate aldol cleavage by a solvent-derived proton. This KIE suggested that an equilibrium mixture of all protiated isocitrate, glyoxylate and succinate prepared in D2O, would undergo transient changes in equilibrium concentrations as a result of the solvent KIE and solvent-derived deuterium incorporation into both succinate and isocitrate. No change in the isotopic composition of glyoxylate was expected or observed. We have directly monitored the changing concentrations of all isotopic species of all reactants and products using a combination of NMR spectroscopy and mass spectrometry. Continuous monitoring of glyoxylate by 1H NMR spectroscopy shows a clear equilibrium perturbation in D2O. The final equilibrium isotopic composition of reactants in D2O revealed di-deuterated succinate, protiated glyoxylate, and mono-deuterated isocitrate, with the transient appearance and disappearance of mono-deuterated succinate. A model for the equilibrium perturbation of substrate species, and their time-dependent isotopic composition is presented. PMID:24261638

Does dietary fat intake influence oocyte competence and embryo quality by inducing oxidative stress in follicular fluid?

PubMed

Kazemi, Ashraf; Ramezanzadeh, Fatemeh; Nasr-Esfahani, Mohammad Hosein; Saboor Yaraghi, Ali Akbar; Ahmadi, Mehdi

2013-12-01

Fat-rich diet may alter oocyte development and maturation and embryonic development by inducing oxidative stress (OS) in follicular environment. To investigate the relationship between fat intake and oxidative stress with oocyte competence and embryo quality. In observational study follicular fluid was collected from 236 women undergoing assisted reproduction program. Malon-di-aldehyde (MDA) levels and total antioxidant capacity (TAC) levels of follicular fluid were assessed as oxidative stress biomarkers. In assisted reproduction treatment cycle fat consumption and its component were assessed. A percentage of metaphase ΙΙ stage oocytes, fertilization rate were considered as markers of oocyte competence and non-fragmented embryo rate, mean of blastomer and good cleavage (embryos with more than 5 cells on 3 days post insemination) rate were considered as markers of embryo quality. The MDA level in follicular fluid was positively related to polyunsaturated fatty acids intake level (p=0.02) and negatively associated with good cleavage rate (p=0.045). Also good cleavage rate (p=0.005) and mean of blastomer (p=0.006) was negatively associated with polyunsaturated fatty acids intake levels. The percentage of metaphase ΙΙ stage oocyte was positively related to the TAC levels in follicular fluid (p=0.046). The relationship between the OS biomarkers in FF and the fertilization rate was not significant. These findings revealed that fat rich diet may induce the OS in oocyte environment and negatively influence embryonic development. This effect can partially be accounted by polyunsaturated fatty acids uptake while oocyte maturation is related to TAC and oocytes with low total antioxidant capacity have lower chance for fertilization and further development.

Decomposition of amino diazeniumdiolates (NONOates): molecular mechanisms.

PubMed

Shaikh, Nizamuddin; Valiev, Marat; Lymar, Sergei V

2014-12-01

Although diazeniumdiolates (X[N(O)NO](-)) are extensively used in biochemical, physiological, and pharmacological studies due to their ability to release NO and/or its congeneric nitroxyl, the mechanisms of these processes remain obscure. In this work, we used a combination of spectroscopic, kinetic, and computational techniques to arrive at a quantitatively consistent molecular mechanism for decomposition of amino diazeniumdiolates (amino NONOates: R2N[N(O)NO](-), where R=N(C2H5)2 (1), N(C3H4NH2)2 (2), or N(C2H4NH2)2 (3)). Decomposition of these NONOates is triggered by protonation of their [NN(O)NO](-) group with the apparent pKa and decomposition rate constants of 4.6 and 1 s(-1) for 1; 3.5 and 0.083 s(-1) for 2; and 3.8 and 0.0033 s(-1) for 3. Although protonation occurs mainly on the O atoms of the functional group, only the minor R2N(H)N(O)NO tautomer (population ~10(-7), for 1) undergoes the NN heterolytic bond cleavage (kd~10(7) s(-1) for 1) leading to amine and NO. Decompositions of protonated amino NONOates are strongly temperature-dependent; activation enthalpies are 20.4 and 19.4 kcal/mol for 1 and 2, respectively, which includes contributions from both the tautomerization and bond cleavage. The bond cleavage rates exhibit exceptional sensitivity to the nature of R substituents which strongly modulate activation entropy. At pH<2, decompositions of all three NONOates that have been investigated are subject to additional acid catalysis that occurs through di-protonation of the [NN(O)NO](-) group. Copyright © 2014 Elsevier Inc. All rights reserved.

Decomposition of amino diazeniumdiolates (NONOates): Molecular mechanisms

DOE PAGES

Shaikh, Nizamuddin; Valiev, Marat; Lymar, Sergei V.

2014-08-23

Although diazeniumdiolates (X[N(O)NO] -) are extensively used in biochemical, physiological, and pharmacological studies due to their ability to release NO and/or its congeneric nitroxyl, the mechanisms of these processes remain obscure. In this work, we used a combination of spectroscopic, kinetic, and computational techniques to arrive at a quantitatively consistent molecular mechanism for decomposition of amino diazeniumdiolates (amino NONOates: R 2N[N(O)NO] -, where R = —N(C 2H 5) 2(1), —N(C 3H 4NH 2) 2(2), or —N(C 2H 4NH 2) 2(3)). Decomposition of these NONOates is triggered by protonation of their [NN(O)NO] - group with the apparent pKa and decomposition ratemore » constants of 4.6 and 1 s -1 for 1; 3.5 and 0.083 s -1 for 2; and 3.8 and 0.0033 s -1 for 3. Although protonation occurs mainly on the O atoms of the functional group, only the minor R 2N(H)N(O)NO tautomer (population ~ 10 -7, for 1) undergoes the N—N heterolytic bond cleavage (k d ~ 107 s -1 for 1) leading to amine and NO. Decompositions of protonated amino NONOates are strongly temperature-dependent; activation enthalpies are 20.4 and 19.4 kcal/mol for 1 and 2, respectively, which includes contributions from both the tautomerization and bond cleavage. Thus, the bond cleavage rates exhibit exceptional sensitivity to the nature of R substituents which strongly modulate activation entropy. At pH < 2, decompositions of all three NONOates that have been investigated are subject to additional acid catalysis that occurs through di-protonation of the [NN(O)NO] - group.« less

Synthesis, DNA Cleavage Activity, Cytotoxicity, Acetylcholinesterase Inhibition, and Acute Murine Toxicity of Redox-Active Ruthenium(II) Polypyridyl Complexes.

PubMed

Alatrash, Nagham; Narh, Eugenia S; Yadav, Abhishek; Kim, Mahn-Jong; Janaratne, Thamara; Gabriel, James; MacDonnell, Frederick M

2017-07-06

Four mononuclear [(L-L) 2 Ru(tatpp)] 2+ and two dinuclear [(L-L) 2 Ru(tatpp)Ru(L-L) 2 ] 4+ ruthenium(II) polypyridyl complexes (RPCs) containing the 9,11,20,22-tetraazatetrapyrido[3,2-a:2',3'-c:3'',2''-l:2''',3'''-n]pentacene (tatpp) ligand were synthesized, in which L-L is a chelating diamine ligand such as 2,2'-bipyridine (bpy), 1,10-phenanthroline (phen), 3,4,7,8-tetramethyl-1,10-phenanthroline (Me 4 phen) or 4,7-diphenyl-1,10-phenanthroline (Ph 2 phen). These Ru-tatpp analogues all undergo reduction reactions with modest reducing agents, such as glutathione (GSH), at pH 7. These, plus several structurally related but non-redox-active RPCs, were screened for DNA cleavage activity, cytotoxicity, acetylcholinesterase (AChE) inhibition, and acute mouse toxicity, and their activities were examined with respect to redox activity and lipophilicity. All of the redox-active RPCs show single-strand DNA cleavage in the presence of GSH, whereas none of the non-redox-active RPCs do. Low-micromolar cytotoxicity (IC 50 ) against malignant H358, CCL228, and MCF7 cultured cell lines was mainly restricted to the redox-active RPCs; however, they were substantially less toxic toward nonmalignant MCF10 cells. The IC 50 values for AChE inhibition in cell-free assays and the acute toxicity of RPCs in mice revealed that whereas most RPCs show potent inhibitory action against AChE (IC 50 values <15 μm), Ru-tatpp complexes as a class are surprisingly well tolerated in animals relative to other RPCs. © 2017 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.

Site-specific O-Glycosylation by Polypeptide N-Acetylgalactosaminyltransferase 2 (GalNAc-transferase T2) Co-regulates β1-Adrenergic Receptor N-terminal Cleavage*

PubMed Central

Goth, Christoffer K.; Tuhkanen, Hanna E.; Khan, Hamayun; Lackman, Jarkko J.; Wang, Shengjun; Narimatsu, Yoshiki; Hansen, Lasse H.; Overall, Christopher M.; Clausen, Henrik; Schjoldager, Katrine T.; Petäjä-Repo, Ulla E.

2017-01-01

The β1-adrenergic receptor (β1AR) is a G protein-coupled receptor (GPCR) and the predominant adrenergic receptor subtype in the heart, where it mediates cardiac contractility and the force of contraction. Although it is the most important target for β-adrenergic antagonists, such as β-blockers, relatively little is yet known about its regulation. We have shown previously that β1AR undergoes constitutive and regulated N-terminal cleavage participating in receptor down-regulation and, moreover, that the receptor is modified by O-glycosylation. Here we demonstrate that the polypeptide GalNAc-transferase 2 (GalNAc-T2) specifically O-glycosylates β1AR at five residues in the extracellular N terminus, including the Ser-49 residue at the location of the common S49G single-nucleotide polymorphism. Using in vitro O-glycosylation and proteolytic cleavage assays, a cell line deficient in O-glycosylation, GalNAc-T-edited cell line model systems, and a GalNAc-T2 knock-out rat model, we show that GalNAc-T2 co-regulates the metalloproteinase-mediated limited proteolysis of β1AR. Furthermore, we demonstrate that impaired O-glycosylation and enhanced proteolysis lead to attenuated receptor signaling, because the maximal response elicited by the βAR agonist isoproterenol and its potency in a cAMP accumulation assay were decreased in HEK293 cells lacking GalNAc-T2. Our findings reveal, for the first time, a GPCR as a target for co-regulatory functions of site-specific O-glycosylation mediated by a unique GalNAc-T isoform. The results provide a new level of β1AR regulation that may open up possibilities for new therapeutic strategies for cardiovascular diseases. PMID:28167537

A homogeneous 2D deformation of geological interest: Rotation shear

NASA Astrophysics Data System (ADS)

Bastida, Fernando; Bobillo-Ares, Nilo C.; Aller, Jesús; Lisle, Richard J.

2018-07-01

We define a simple two-dimensional deformation called "rotation shear". It has one line of no finite longitudinal strain with invariant direction and another one that rotates with the deformation. An analysis of this deformation is carried out. Rotation shear superficially resembles simple shear but the analysis reveals that the two deformations have very different properties. In general, lines deformed by simple shear show a more complex deformation history and undergo greater longitudinal strain, i.e. are more extended, than lines deformed by rotation shear. Rotation shear is used to explain the development of geological structures such as kink bands, ideal similar folds, crenulation and crenulation cleavage and shear zones.

Early development and acquisition of Zooxanthellae in the temperate symbiotic sea anemone Anthopleura ballii (Cocks).

PubMed

Davy, Simon K; Turner, John R

2003-08-01

The ova of Anthopleura ballii become infected with zooxanthellae (endosymbiotic dinoflagellates) of maternal origin just prior to spawning. After fertilization, the zygotes undergo radial, holoblastic cleavage, and then gastrulate by invagination to form ciliated planulae. Because the zooxanthellae are localized on one side of the ovum-and later, within the blastomeres at one end of the embryo-invagination leads to the zooxanthellae being restricted to the planular endoderm and hence to the gastrodermal cells of the adult anemone. We propose that maternal inheritance of zooxanthellae plays an important part in the success of these temperate sea anemones, which live in regions where potential sources of zooxanthellae are scarce.

Improvements upon the "Colorful Cobalt Catalysis" Demonstration and Evidence for the Presence of an Autocatalytic Mechanism

ERIC Educational Resources Information Center

Wright, Stephen W.

2010-01-01

The oxidation of potassium sodium tartrate by hydrogen peroxide catalyzed by cobalt(II) chloride is a favorite lecture demonstration. I present conditions under which this experiment may be performed without need for 30% hydrogen peroxide and without need for controlled heating or any heating of the reaction mixture. I further show that this…

The DTIC Review: Volume 3, Number 3. Microtechnologies and Microelectronics

DTIC Science & Technology

1997-09-01

high resolution rnicroanalytical experiments in the martensitic alloys address control of autocatalytic coherent precipitation to achieve efficient...the dependence of hardness at completion of precipitation on alloy carbon content for various particle diameters, including the critical size dc for the...control of microvoid nucleating particle dispersions governing plastic shear localization [22-25], and (b) dispersed- phase transformation toughening by

Ethylene regulates Apple (Malus x domestica) fruit softening through a dose x time-dependent mechanism and through differential sensitivities and dependencies of cell wall-modifying genes.

PubMed

Ireland, Hilary S; Gunaseelan, Kularajathevan; Muddumage, Ratnasiri; Tacken, Emma J; Putterill, Jo; Johnston, Jason W; Schaffer, Robert J

2014-05-01

In fleshy fruit species that have a strong requirement for ethylene to ripen, ethylene is synthesized autocatalytically, producing increasing concentrations as the fruits ripen. Apple fruit with the ACC OXIDASE 1 (ACO1) gene suppressed cannot produce ethylene autocatalytically at ripening. Using these apple lines, an ethylene sensitivity dependency model was previously proposed, with traits such as softening showing a high dependency for ethylene as well as low sensitivity. In this study, it is shown that the molecular control of fruit softening is a complex process, with different cell wall-related genes being independently regulated and exhibiting differential sensitivities to and dependencies on ethylene at the transcriptional level. This regulation is controlled through a dose × time mechanism, which results in a temporal transcriptional response that would allow for progressive cell wall disassembly and thus softening. This research builds on the sensitivity dependency model and shows that ethylene-dependent traits can progress over time to the same degree with lower levels of ethylene. This suggests that a developmental clock measuring cumulative ethylene controls the fruit ripening process.

THREE-STAGE ANALYSIS OF BLOOD COAGULATION

PubMed Central

Milstone, J. H.

1948-01-01

1. Blood-clotting mechanism has been analyzed by a procedure which devotes a separate experimental step to each of the three primary reactions: 1. Prothrombokinase → thrombokinase 2. Prothrombin → thrombin 3. Fibrinogen → fibrin 2. Activation of prothrombin by thrombokinase followed the course of a unimolecular reaction, and the concentration of thrombokinase determined the initial rate. By this relation thrombokinase was measured, and the activation of its precursor was charted. 3. When the activation of prothrombokinase was plotted against time, the experimental points fell close to the theoretical curve for a simple autocatalytic reaction. Moreover, the process was accelerated by seeding with a small amount of crude thrombokinase. It was concluded that the activation of prothrombokinase involves an autocatalytic or chain reaction. 4. The three-stage procedure made possible the separate estimation of the power to activate prothrombin, on one hand, and the capacity to accelerate the transformation of prothrombokinase on the other. Drastic losses of both activities occurred when crude thrombokinase solutions were heated at 60°C., or adsorbed with barium sulfate. 5. The concentration of calcium was important for the normal progress of prothrombin activation, and also for the transformation of prothrombokinase. PMID:18904755

Chlorine dioxide-induced and Congo red-inhibited Marangoni effect on the chlorite-trithionate reaction front

NASA Astrophysics Data System (ADS)

Liu, Yang; Ren, Xingfeng; Pan, Changwei; Zheng, Ting; Yuan, Ling; Zheng, Juhua; Gao, Qingyu

2017-10-01

Hydrodynamic flows can exert multiple effects on an exothermal autocatalytic reaction, such as buoyancy and the Marangoni convection, which can change the structure and velocity of chemical waves. Here we report that in the chlorite-trithionate reaction, the production and consumption of chlorine dioxide can induce and inhibit Marangoni flow, respectively, leading to different chemo-hydrodynamic patterns. The horizontal propagation of a reaction-diffusion-convection front was investigated with the upper surface open to the air. The Marangoni convection, induced by gaseous chlorine dioxide on the surface, produced from chlorite disproportionation after the proton autocatalysis, has the same effect as the heat convection. When the Marangoni effect is removed by the reaction of chlorine dioxide with the Congo red (CR) indicator, an oscillatory propagation of the front tip is observed under suitable conditions. Replacing CR with bromophenol blue (BPB) distinctly enhanced the floating, resulting in multiple vortexes, owing to the coexistence between BPB and chlorine dioxide. Using the incompressible Navier-Stokes equations coupled with reaction-diffusion and heat conduction equations, we numerically obtain various experimental scenarios of front instability for the exothermic autocatalytic reaction coupled with buoyancy-driven convection and Marangoni convection.

Phase field simulations of autocatalytic formation of alpha lamellar colonies in Ti-6Al-4V

DOE PAGES

Radhakrishnan, Bala; Gorti, Sarma; Babu, Suresh Sudharsanam

2016-09-13

Here, we present phase field simulations incorporating energy contributions due to thermodynamics, and anisotropic interfacial and strain energies, to demonstrate the nucleation and growth of multiple variants of alpha from beta in Ti-6Al-4V under isothermal conditions. The simulations focused on the effect of thermodynamic driving force and nucleation rate on the morphology of the transformed alpha assuming that the partitioning of V between beta and alpha is negligible for short isothermal holds. The results indicate that a high nucleation rate favors the formation of the basket-weave structure. However, at a lower nucleation rate the simulations show the intragranular nucleation ofmore » a colony structure by an autocatalytic nucleation mechanism adjacent to a pre-existing alpha variant. New side-plates of the same variant appear to nucleate progressively and grow to form the colony. The isothermal simulation results are used to offer a possible explanation for the transition from a largely basket weave structure to a colony structure inside narrow layer bands occurring during continuous heating and cooling conditions encountered during laser additive manufacturing of Ti-6Al-4V.« less

Discontinuous non-equilibrium phase transition in a threshold Schloegl model for autocatalysis: Generic two-phase coexistence and metastability

DOE PAGES

Wang, Chi -Jen; Liu, Da -Jiang; Evans, James W.

2015-04-28

Threshold versions of Schloegl’s model on a lattice, which involve autocatalytic creation and spontaneous annihilation of particles, can provide a simple prototype for discontinuous non-equilibrium phase transitions. These models are equivalent to so-called threshold contact processes. A discontinuous transition between populated and vacuum states can occur selecting a threshold of N ≥ 2 for the minimum number, N, of neighboring particles enabling autocatalytic creation at an empty site. Fundamental open questions remain given the lack of a thermodynamic framework for analysis. For a square lattice with N = 2, we show that phase coexistence occurs not at a unique valuemore » but for a finite range of particle annihilation rate (the natural control parameter). This generic two-phase coexistence also persists when perturbing the model to allow spontaneous particle creation. Such behavior contrasts both the Gibbs phase rule for thermodynamic systems and also previous analysis for this model. We find metastability near the transition corresponding to a non-zero effective line tension, also contrasting previously suggested critical behavior. As a result, mean-field type analysis, extended to treat spatially heterogeneous states, further elucidates model behavior.« less

Selective autocatalytic reduction of NO from sintering flue gas by the hot sintered ore in the presence of NH3.

PubMed

Chen, Wangsheng; Luo, Jing; Qin, Linbo; Han, Jun

2015-12-01

In this paper, the selective autocatalytic reduction of NO by NH3 combined with multi-metal oxides in the hot sintered ore was studied, and the catalytic activity of the hot sintered ore was investigated as a function of temperature, NH3/NO ratio, O2 content, H2O and SO2. The experimental results indicated that the hot sintered ore, when combined with NH3, had a maximum denitration efficiency of 37.67% at 450 °C, 3000 h(-1) gas hourly space velocity (GHSV) and a NH3/NO ratio of 0.4/1. Additionally, it was found that O2 played an important role in removing NOx. However, high O2 content had a negative effect on NO reduction. H2O was found to promote the denitration efficiency in the absence of SO2, while SO2 inhibited the catalytic activity of the sintered ore. In the presence of H2O and SO2, the catalytic activity of the sintered ore was dramatically suppressed. Copyright © 2015 Elsevier Ltd. All rights reserved.

On initial steps of chemical prebiotic evolution: Triggering autocatalytic reaction of oligomerization

NASA Astrophysics Data System (ADS)

Bartsev, S. I.; Mezhevikin, V. V.

2008-12-01

Searching for extraterrestrial life attracts more and more attention. However this searching hardly can be effective without sufficiently universal concept of life origin, which incidentally tackles a problem of origin of life on the Earth. A concept of initial stages of life origin is stated in the paper. The concept eliminates key difficulties in the problem of life origin, and allows experimental verification of it. According to the concept the predecessor of living beings has to be sufficiently simple to provide non-zero probability of self-assembling during short (in geological or cosmic scale) time. In addition the predecessor has to be capable of autocatalysis, and further complication (evolution). A possible scenario of initial stage of life origin, which can be realized both on other planets, and inside experimental facility is considered. In the scope of the scenario a theoretical model of multivariate oligomeric autocatalyst is presented. Results of computer simulation of two versions of oligomeric autocatalytic reactions are presented. It is shown that the contribution of monomer activation reaction is essential, and in some cases autocatalysis in polymerizing reaction can be achieved without catalyzing proper monomer binding reaction.

Discontinuous non-equilibrium phase transition in a threshold Schloegl model for autocatalysis: Generic two-phase coexistence and metastability

NASA Astrophysics Data System (ADS)

Wang, Chi-Jen; Liu, Da-Jiang; Evans, James W.

2015-04-01

Threshold versions of Schloegl's model on a lattice, which involve autocatalytic creation and spontaneous annihilation of particles, can provide a simple prototype for discontinuous non-equilibrium phase transitions. These models are equivalent to so-called threshold contact processes. A discontinuous transition between populated and vacuum states can occur selecting a threshold of N ≥ 2 for the minimum number, N, of neighboring particles enabling autocatalytic creation at an empty site. Fundamental open questions remain given the lack of a thermodynamic framework for analysis. For a square lattice with N = 2, we show that phase coexistence occurs not at a unique value but for a finite range of particle annihilation rate (the natural control parameter). This generic two-phase coexistence also persists when perturbing the model to allow spontaneous particle creation. Such behavior contrasts both the Gibbs phase rule for thermodynamic systems and also previous analysis for this model. We find metastability near the transition corresponding to a non-zero effective line tension, also contrasting previously suggested critical behavior. Mean-field type analysis, extended to treat spatially heterogeneous states, further elucidates model behavior.

To Be or Not To Be T4: Evidence of a Complex Evolutionary Pathway of Head Structure and Assembly in Giant Salmonella Virus SPN3US

PubMed Central

Ali, Bazla; Desmond, Maxim I.; Mallory, Sara A.; Benítez, Andrea D.; Buckley, Larry J.; Weintraub, Susan T.; Osier, Michael V.; Black, Lindsay W.; Thomas, Julie A.

2017-01-01

Giant Salmonella phage SPN3US has a 240-kb dsDNA genome and a large complex virion composed of many proteins for which the functions of most are undefined. We recently determined that SPN3US shares a core set of genes with related giant phages and sequenced and characterized 18 amber mutants to facilitate its use as a genetic model system. Notably, SPN3US and related giant phages contain a bolus of ejection proteins within their heads, including a multi-subunit virion RNA polymerase (vRNAP), that enter the host cell with the DNA during infection. In this study, we characterized the SPN3US virion using mass spectrometry to gain insight into its head composition and the features that its head shares with those of related giant phages and with T4 phage. SPN3US has only homologs to the T4 proteins critical for prohead shell formation, the portal and major capsid proteins, as well as to the major enzymes essential for head maturation, the prohead protease and large terminase subunit. Eight of ~50 SPN3US head proteins were found to undergo proteolytic processing at a cleavage motif by the prohead protease gp245. Gp245 undergoes auto-cleavage of its C-terminus, suggesting this is a conserved activation and/or maturation feature of related phage proteases. Analyses of essential head gene mutants showed that the five subunits of the vRNAP must be assembled for any subunit to be incorporated into the prohead, although the assembled vRNAP must then undergo subsequent major conformational rearrangements in the DNA packed capsid to allow ejection through the ~30 Å diameter tail tube for transcription from the injected DNA. In addition, ejection protein candidate gp243 was found to play a critical role in head assembly. Our analyses of the vRNAP and gp243 mutants highlighted an unexpected dichotomy in giant phage head maturation: while all analyzed giant phages have a homologous protease that processes major capsid and portal proteins, processing of ejection proteins is not always a stable/defining feature. Our identification in SPN3US, and related phages, of a diverged paralog to the prohead protease further hints toward a complicated evolutionary pathway for giant phage head structure and assembly. PMID:29187846

Developmental expression of Hsp90, Hsp70 and HSF during morphogenesis in the vetigastropod Haliotis asinina.

PubMed

Gunter, Helen M; Degnan, Bernard M

2007-08-01

Heat shock proteins (Hsps) have dual functions, participating in both the stress response and a broad range of developmental processes. At physiological temperatures, it has been demonstrated in deuterostomes (vertebrates) and ecdysozoans (insects) that Hsps are expressed in tissues that are undergoing differentiation and morphogenesis. Here we investigate the developmental expression of Hsp70, Hsp90 and their regulatory transcription factor heat shock transcription factor (HSF) in the marine gastropod Haliotis asinina, a representative of the 3rd major lineage of bilaterian animals, the Lophotrochozoa. HasHsp70, HasHsp90 and HasHSF are maternally expressed in H. asinina and are progressively restricted to the micromere lineage during cleavage. During larval morphogenesis, they are expressed in unique and overlapping patterns in the prototroch, foot, and mantle. Hsp expression peaked in these tissues during periods of cell differentiation and morphogenesis, returning to lower levels after morphogenesis was complete. These patterns of Hsp and HSF expression in H. asinina are akin to those observed in ecdysozoans and deuterostomes, with Hsps being activated in cells and tissues undergoing morphogenesis.

AKT delays the early-activated apoptotic pathway in UVB-irradiated keratinocytes via BAD translocation.

PubMed

Claerhout, Sofie; Decraene, David; Van Laethem, An; Van Kelst, Sofie; Agostinis, Patrizia; Garmyn, Marjan

2007-02-01

Upon irradiation with a high dose of UVB, keratinocytes undergo apoptosis as a protective mechanism. In previous work, we demonstrated the existence of an early-activated UVB-induced apoptotic pathway in growth factor-depleted human keratinocytes, which can be substantially delayed by the exclusive supplementation of IGF-1. We now show that in human keratinocytes, IGF-1 inhibits the onset of UVB-triggered apoptosis through a transcriptional independent, AKT-mediated mechanism, involving BAD serine 136 phosphorylation. Our results show that the early UVB-induced apoptosis in growth factor-depleted human keratinocytes is exclusively triggered through the mitochondrial pathway. It is accompanied by BAX translocation, cytochrome c release, and procaspase-9 cleavage, but not by procaspase-8 or BID cleavage. In human keratinocytes, IGF-1 supplementation inhibits these events in a transcription-independent manner. Both IGF-1 supplementation and the transduction of a membrane-targeted form of AKT result in a shift of the BH3-only protein BAD from the mitochondria to the cytoplasm, paralleled by an increase of AKT-specific Ser136 phospho-BAD bound to 14-3-3zeta protein. These data indicate that AKT-induced BAD phosphorylation and its subsequent cytoplasmic sequestration by 14-3-3zeta is a major mechanism responsible for the postponement of UVB-induced apoptosis in human keratinocytes.

Interstrand cross-links arising from strand breaks at true abasic sites in duplex DNA

PubMed Central

Yang, Zhiyu; Price, Nathan E.; Johnson, Kevin M.

2017-01-01

Abstract Interstrand cross-links are exceptionally bioactive DNA lesions. Endogenous generation of interstrand cross-links in genomic DNA may contribute to aging, neurodegeneration, and cancer. Abasic (Ap) sites are common lesions in genomic DNA that readily undergo spontaneous and amine-catalyzed strand cleavage reactions that generate a 2,3-didehydro-2,3-dideoxyribose sugar remnant (3’ddR5p) at the 3’-terminus of the strand break. Interestingly, this strand scission process leaves an electrophilic α,β-unsaturated aldehyde residue embedded within the resulting nicked duplex. Here we present evidence that 3’ddR5p derivatives generated by spermine-catalyzed strand cleavage at Ap sites in duplex DNA can react with adenine residues on the opposing strand to generate a complex lesion consisting of an interstrand cross-link adjacent to a strand break. The cross-link blocks DNA replication by ϕ29 DNA polymerase, a highly processive polymerase enzyme that couples synthesis with strand displacement. This suggests that 3’ddR5p-derived cross-links have the potential to block critical cellular DNA transactions that require strand separation. LC-MS/MS methods developed herein provide powerful tools for studying the occurrence and properties of these cross-links in biochemical and biological systems. PMID:28531327

Complexes of a dianionic bis(diphosphinomethanide) with lithium, sodium, potassium and zirconium. Effect of substitution on the Schlenk dimerisation of vinylidene phosphines.

PubMed

Izod, Keith; McFarlane, William; Tyson, Brent V; Clegg, William; Harrington, Ross W

2004-12-07

The vinylidene phosphine (Pr(n)(2)P)(2)C=CH(2) (1) undergoes Schlenk dimerisation on treatment with an excess of any of the alkali metals Li, Na or K to give the butane-1,4-diide complexes [(L)M{(Pr(n)(2)P)(2)CCH(2)}](2)[(L)M =(THF)(2)Li (6), (THF)(3)Na (7b), (DME)(2)K (8b)], after recrystallisation. Whereas the reaction between the analogous phenyl derivative (Ph(2)P)(2)C=CH(2) and K results in cleavage of a P-C bond, 1 reacts smoothly with K to give 8, with no evidence for P-C cleavage. Compound 6 is an excellent ligand transfer reagent: metathesis reactions between either 6 or its phenyl analogue [(THF)(2)Li{(Ph(2)P)(2)CCH(2)}](2) (2) and two equivalents of Cp(2)ZrCl(2) in THF give the corresponding dinuclear zirconocene derivatives [Cp(2)Zr(Cl){(R(2)P)(2)CCH(2)}](2) in good yields [R = Ph (11), Pr(n)(12)]. Compounds 6, 7b, 8b, 11 and 12 have been characterised by multi-element NMR spectroscopy and, where possible, by elemental analysis; compounds 6, 7b, 11 and 12 have additionally been characterised by X-ray crystallography.

Bcl2-low-expressing MCF7 cells undergo necrosis rather than apoptosis upon staurosporine treatment.

PubMed Central

Poliseno, Laura; Bianchi, Laura; Citti, Lorenzo; Liberatori, Sabrina; Mariani, Laura; Salvetti, Alessandra; Evangelista, Monica; Bini, Luca; Pallini, Vitaliano; Rainaldi, Giuseppe

2004-01-01

We present a ribozyme-based strategy for studying the effects of Bcl2 down-regulation. The anti-bcl2 hammerhead ribozyme Rz-bcl2 was stably transfected into MCF7 cancer cells and the cleavage of Bcl2 mRNA was demonstrated using a new assay for cleavage product detection, while Western blot analysis showed a concomitant depletion of Bcl2 protein. Rz-bcl2-expressing cells were more sensitive to staurosporine than control cells. Moreover, both molecular and cellular read-outs indicated that staurosporine-induced cell death was necrosis rather than apoptosis in these cells. The study of the effects of Bcl2 down-regulation was extended to the global MCF7 protein expression profile, exploiting a proteomic approach. Two reference electro-pherograms of Rz-bcl2-transfected cells, one with the ribozyme in a catalytically active form and the other with the ribozyme in a catalytically inactive form, were obtained. When comparing the two-dimensional maps, 53 differentially expressed spots were found, four of which were identified by MALDI-TOF (matrix-assisted laser-desorption ionization-time-of-flight) MS as calreticulin, nucleophosmin, phosphoglycerate kinase and pyruvate kinase. How the up-regulation of these proteins might help to explain the modification of Bcl2 activity is discussed. PMID:14748742

Imidazoline ring cleavage in 1,3,6,10-tetraazatetracyclo-(7. 3. 1. 0/sup 2,7/. 0/sup 6,13/)trideca-4,11-dienes, leading to the formation of diquinoxalino(1,2-. cap alpha. :2',3'-d)pyrrole derivatives

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

Charushin, V.N.; Petrova, G.M.; Aleksandrov, G.G.

1987-10-01

Dibenzo(d,k)-1,3,6,10-tetraazatetracyclo(7.3.1.0/sup 2,7/.0/sup 6,13/) trideca-4,11-dienes undergo addition reactions at the C/sub (2)/ carbon atom with alcohols and thiols, accompanied by cleavage of the C-N bond of the imidazoline ring, to generate diquinoxalino(1,2-..cap alpha..:2',3'-d)pyrrole derivatives. /sup 1/H NMR spectra were recorded on Perkin-Elmer R 12B (60 MHz) and Bruker WH-90 spectrometer for CDCl/sub 3/ solutions at 40/sup 0/C and with TMS as internal standard. /sup 13/C NMR spectra were obtained on a Bruker WH-90 (22.62 MHz) spectrometer. /sup 13/C chemical shifts were measured relative to solvent signals (deltaCDCl/sub 3/ 77.0 ppm). /sup 13/C NMR spectra of compounds IIa and g were takenmore » using full spin-spin carbon-proton decoupling. In order to measure SSCC the spectrum was recorded both with proton coupling and also with selective decoupling of individual protons and their attached /sup 13/C carbon nuclei.« less

Acid/base-regulated reversible electron transfer disproportionation of N–N linked bicarbazole and biacridine derivatives† †Electronic supplementary information (ESI) available: Experimental information, synthesis and characterization data, NMR spectra, solid state NMR data, X-ray data, ESR spectra, UV-Vis-NIR spectra, fluorescence spectra, kinetic experiments, theoretical calculations, Tables S1–S8, Scheme S1, Fig. S1–12, References. CCDC 1025063, 1038914, 1049677 and 1040722. For ESI and crystallographic data in CIF or other electronic format see DOI: 10.1039/c5sc00946d

PubMed Central

Pandit, Palash; Yamamoto, Koji; Nakamura, Toshikazu; Nishimura, Katsuyuki; Kurashige, Yuki; Yanai, Takeshi; Nakamura, Go; Masaoka, Shigeyuki; Furukawa, Ko; Yakiyama, Yumi; Kawano, Masaki

2015-01-01

Regulation of electron transfer on organic substances by external stimuli is a fundamental issue in science and technology, which affects organic materials, chemical synthesis, and biological metabolism. Nevertheless, acid/base-responsive organic materials that exhibit reversible electron transfer have not been well studied and developed, owing to the difficulty in inventing a mechanism to associate acid/base stimuli and electron transfer. We discovered a new phenomenon in which N–N linked bicarbazole (BC) and tetramethylbiacridine (TBA) derivatives undergo electron transfer disproportionation by acid stimulus, forming their stable radical cations and reduced species. The reaction occurs through a biradical intermediate generated by the acid-triggered N–N bond cleavage reaction of BC or TBA, which acts as a two electron acceptor to undergo electron transfer reactions with two equivalents of BC or TBA. In addition, in the case of TBA the disproportionation reaction is highly reversible through neutralization with NEt3, which recovers TBA through back electron transfer and N–N bond formation reactions. This highly reversible electron transfer reaction is possible due to the association between the acid stimulus and electron transfer via the acid-regulated N–N bond cleavage/formation reactions which provide an efficient switching mechanism, the ability of the organic molecules to act as multi-electron donors and acceptors, the extraordinary stability of the radical species, the highly selective reactivity, and the balance of the redox potentials. This discovery provides new design concepts for acid/base-regulated organic electron transfer systems, chemical reagents, or organic materials. PMID:29218181

Arabidopsis Type II Phosphatidylinositol 4-Kinase PI4Kγ5 Regulates Auxin Biosynthesis and Leaf Margin Development through Interacting with Membrane-Bound Transcription Factor ANAC078

PubMed Central

Tan, Shu-Tang; Xue, Hong-Wei

2016-01-01

Normal leaf margin development is important for leaf morphogenesis and contributes to diverse leaf shapes in higher plants. We here show the crucial roles of an atypical type II phosphatidylinositol 4-kinase, PI4Kγ5, in Arabidopsis leaf margin development. PI4Kγ5 presents a dynamics expression pattern along with leaf development and a T-DNA mutant lacking PI4Kγ5, pi4kγ5–1, presents serrated leaves, which is resulted from the accelerated cell division and increased auxin concentration at serration tips. Studies revealed that PI4Kγ5 interacts with and phosphorylates a membrane-bound NAC transcription factor, ANAC078. Previous studies demonstrated that membrane-bound transcription factors regulate gene transcription by undergoing proteolytic process to translocate into nucleus, and ANAC078 undergoes proteolysis by cleaving off the transmembrane region and carboxyl terminal. Western blot analysis indeed showed that ANAC078 deleting of carboxyl terminal is significantly reduced in pi4kγ5–1, indicating that PI4Kγ5 is important for the cleavage of ANAC078. This is consistent with the subcellular localization observation showing that fluorescence by GFP-ANAC078 is detected at plasma membrane but not nucleus in pi4kγ5–1 mutant and that expression of ANAC078 deleting of carboxyl terminal, driven by PI4Kγ5 promoter, could rescue the leaf serration defects of pi4kγ5–1. Further analysis showed that ANAC078 suppresses the auxin synthesis by directly binding and regulating the expression of auxin synthesis-related genes. These results indicate that PI4Kγ5 interacts with ANAC078 to negatively regulate auxin synthesis and hence influences cell proliferation and leaf development, providing informative clues for the regulation of in situ auxin synthesis and cell division, as well as the cleavage and functional mechanism of membrane-bound transcription factors. PMID:27529511

Sensitive Detection Using Microfluidics and Nonlinear Amplification

DTIC Science & Technology

2011-07-22

Quantification of Nucleic Acids via Simultaneous Chemical Initiation of Recombinase Polymerase Amplification Reactions on SlipChip" 2011, 83, 3533... Amplification 5a. CONTRACT NUMBER 5b. GRANT NUMBER N00014-08-1-0936 5c. PROGRAM ELEMENT NUMBER 6. AUTHOR(S) Rustem F. Ismagilov 5d. PROJECT NUMBER 5e...concentrations by combining controlled chemical autocatalytic amplification and stochastic confinement of small particles with the microfluidic

Zinc-dependent cleavage in the catalytic core of the hammerhead ribozyme: evidence for a pH-dependent conformational change

PubMed Central

Borda, Emily J.; Markley, John C.; Sigurdsson, Snorri Th.

2003-01-01

We have characterized a novel Zn2+-catalyzed cleavage site between nucleotides C3 and U4 in the catalytic core of the hammerhead ribozyme. In contrast to previously described divalent metal-ion-dependent cleavage of RNA, U4 cleavage is only observed in the presence of Zn2+. This new cleavage site has an unusual pH dependence, in that U4 cleavage products are only observed above pH 7.9 and reach a maximum yield at about pH 8.5. These data, together with the fact that no metal ion-binding site is observed in proximity to the U4 cleavage site in either of the crystal structures, point toward a pH-dependent conformational change in the hammerhead ribozyme. We have described previously Zn2+-dependent cleavage between G8 and A9 in the hammerhead ribozyme and have discovered that U4 cleavage occurs only after A9 cleavage. To our knowledge, this is the first example of sequential cleavage events as a possible regulatory mechanism in ribozymes. PMID:12736309

Crenulation cleavage development by partitioning of deformation into zones of progressive shearing (combined shearing, shortening and volume loss) and progressive shortening (no volume loss): quantification of solution shortening and intermicrolithon-movement

NASA Astrophysics Data System (ADS)

Stewart, L. K.

1997-11-01

An analytical method for determining amounts of cleavage-normal dissolution and cleavage-parallel shear movement that occurred between adjacent microlithons during crenulation cleavage seam formation within a deformed slate is developed for the progressive bulk inhomogeneous shortening (PBIS) mechanism of crenulation cleavage formation. The method utilises structural information obtained from samples where a diverging bed and vein are offset by a crenulation cleavage seam. Several samples analysed using this method produced ratios of relative, cleavage-parallel movement of microlithons to the material thickness removed by dissolution typically in the range of 1.1-3.4:1. The mean amount of solution shortening attributed to the formation of the cleavage seams examined is 24%. The results indicate that a relationship may exist between the width of microlithons and the amount of cleavage-parallel intermicrolithon-movement. The method presented here has the potential to help determine whether crenulation cleavage seams formed by the progressive bulk inhomogeneous shortening mechanism or by that involving cleavage-normal pressure solution alone.

Synthesis of 3-iodoindoles by the Pd/Cu-catalyzed coupling of N,N-dialkyl-2-iodoanilines and terminal acetylenes, followed by electrophilic cyclization.

PubMed

Yue, Dawei; Yao, Tuanli; Larock, Richard C

2006-01-06

[reaction: see text] 3-Iodoindoles have been prepared in excellent yields by coupling terminal acetylenes with N,N-dialkyl-o-iodoanilines in the presence of a Pd/Cu catalyst, followed by an electrophilic cyclization of the resulting N,N-dialkyl-o-(1-alkynyl)anilines using I2 in CH2Cl2. Aryl-, vinylic-, alkyl-, and silyl-substituted terminal acetylenes undergo this process to produce excellent yields of 3-iodoindoles. The reactivity of the carbon-nitrogen bond cleavage during cyclization follows the following order: Me > n-Bu, Me > Ph, and cyclohexyl > Me. Subsequent palladium-catalyzed Sonogashira, Suzuki, and Heck reactions of the resulting 3-iodoindoles proceed smoothly in good yields.

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.

Trypanosoma cruzi has not lost its S-adenosylmethionine decarboxylase: characterization of the gene and the encoded enzyme.

PubMed Central

Persson, K; Aslund, L; Grahn, B; Hanke, J; Heby, O

1998-01-01

All attempts to identify ornithine decarboxylase in the human pathogen Trypanosoma cruzi have failed. The parasites have instead been assumed to depend on putrescine uptake and S-adenosylmethionine decarboxylase (AdoMetDC) for their synthesis of the polyamines spermidine and spermine. We have now identified the gene encoding AdoMetDC in T. cruzi by PCR cloning, with degenerate primers corresponding to conserved amino acid sequences in AdoMetDC proteins of other trypanosomatids. The amplified DNA fragment was used as a probe to isolate the complete AdoMetDC gene from a T. cruzi genomic library. The AdoMetDC gene was located on chromosomes with a size of approx. 1.4 Mbp, and contained a coding region of 1110 bp, specifying a sequence of 370 amino acid residues. The protein showed a sequence identity of only 25% with human AdoMetDC, the major differences being additional amino acids present in the terminal regions of the T. cruzi enzyme. As expected, a higher sequence identity (68-72%) was found in comparison with trypanosomatid AdoMetDCs. When the coding region was expressed in Escherichia coli, the recombinant protein underwent autocatalytic cleavage, generating a 33-34 kDa alpha subunit and a 9 kDa beta subunit. The encoded protein catalysed the decarboxylation of AdoMet (Km 0.21 mM) and was stimulated by putrescine but inhibited by the polyamines, weakly by spermidine and strongly by spermine. Methylglyoxal-bis(guanylhydrazone) (MGBG), a potent inhibitor of human AdoMetDC, was a poor inhibitor of the T. cruzi enzyme. This differential sensitivity to MGBG suggests that the two enzymes are sufficiently different to warrant the search for compounds that might interfere with the progression of Chagas' disease by selectively inhibiting T. cruzi AdoMetDC. PMID:9677309

Capsule Depolymerase Overexpression Reduces Bacillus anthracis Virulence

DTIC Science & Technology

2010-01-01

protein that autocatalytically forms a heterodimer consisting of 35 kDa and 15 kDa subunits. CapD shares 32 % identity with the Bacillus subtilis GGT and 35...Immun 49, 291–297. Kimura, K., Tran, L. S., Uchida, I. & Itoh, Y. (2004). Characterization of Bacillus subtilis gamma-glutamyltransferase and its...Capsule depolymerase overexpression reduces Bacillus anthracis virulence Angelo Scorpio,3 Donald J. Chabot, William A. Day,4 Timothy A. Hoover and

Studies of Premixed Laminar and Turbulent Flames at Microgravity

NASA Technical Reports Server (NTRS)

Ronney, Paul D.

1993-01-01

The work of the Principal Investigator (PI) has encompassed four topics related to the experimental and theoretical study of combustion limits in premixed flames at microgravity, as discussed in the following sections. These topics include: (1) radiation effects on premixed gas flames; (2) flame structure and stability at low Lewis number; (3) flame propagation and extinction is cylindrical tubes; and (4) experimental simulation of combustion processes using autocatalytic chemical reactions.

Cellular self-organization by autocatalytic alignment feedback

PubMed Central

Junkin, Michael; Leung, Siu Ling; Whitman, Samantha; Gregorio, Carol C.; Wong, Pak Kin

2011-01-01

Myoblasts aggregate, differentiate and fuse to form skeletal muscle during both embryogenesis and tissue regeneration. For proper muscle function, long-range self-organization of myoblasts is required to create organized muscle architecture globally aligned to neighboring tissue. However, how the cells process geometric information over distances considerably longer than individual cells to self-organize into well-ordered, aligned and multinucleated myofibers remains a central question in developmental biology and regenerative medicine. Using plasma lithography micropatterning to create spatial cues for cell guidance, we show a physical mechanism by which orientation information can propagate for a long distance from a geometric boundary to guide development of muscle tissue. This long-range alignment occurs only in differentiating myoblasts, but not in non-fusing myoblasts perturbed by microfluidic disturbances or other non-fusing cell types. Computational cellular automata analysis of the spatiotemporal evolution of the self-organization process reveals that myogenic fusion in conjunction with rotational inertia functions in a self-reinforcing manner to enhance long-range propagation of alignment information. With this autocatalytic alignment feedback, well-ordered alignment of muscle could reinforce existing orientations and help promote proper arrangement with neighboring tissue and overall organization. Such physical self-enhancement might represent a fundamental mechanism for long-range pattern formation during tissue morphogenesis. PMID:22193956

Cure Kinetics of Epoxy Nanocomposites Affected by MWCNTs Functionalization: A Review

PubMed Central

Saeb, Mohammad Reza; Bakhshandeh, Ehsan; Khonakdar, Hossein Ali; Mäder, Edith; Scheffler, Christina; Heinrich, Gert

2013-01-01

The current paper provides an overview to emphasize the role of functionalization of multiwalled carbon nanotubes (MWCNTs) in manipulating cure kinetics of epoxy nanocomposites, which itself determines ultimate properties of the resulting compound. In this regard, the most commonly used functionalization schemes, that is, carboxylation and amidation, are thoroughly surveyed to highlight the role of functionalized nanotubes in controlling the rate of autocatalytic and vitrification kinetics. The current literature elucidates that the mechanism of curing in epoxy/MWCNTs nanocomposites remains almost unaffected by the functionalization of carbon nanotubes. On the other hand, early stage facilitation of autocatalytic reactions in the presence of MWCNTs bearing amine groups has been addressed by several researchers. When carboxylated nanotubes were used to modify MWCNTs, the rate of such reactions diminished as a consequence of heterogeneous dispersion within the epoxy matrix. At later stages of curing, however, the prolonged vitrification was seen to be dominant. Thus, the type of functional groups covalently located on the surface of MWCNTs directly affects the degree of polymer-nanotube interaction followed by enhancement of curing reaction. Our survey demonstrated that most widespread efforts ever made to represent multifarious surface-treated MWCNTs have not been directed towards preparation of epoxy nanocomposites, but they could result in property synergism. PMID:24348181

Pre-biotic stage of life origin under non-photosynthetic conditions

NASA Technical Reports Server (NTRS)

Bartsev, S. I.; Mezhevikin, V. V.

2005-01-01

Spontaneous assembling of a simplest bacterial cell even if all necessary molecules are present in a solution seems to be extremely rare event and from the scientific standpoint has to be considered as impossible. Therefore, a predecessor of a living cell has to be very simple for providing its self-assembling and at the same time it should be able of progressive increase in complexity. Now phase-separated particles, first of all micelles, are put forward as possible predecessors of living cell. According to the offered working concept only phase-separated particles possessing autocatalytic properties can be considered as predecessors of living cells. The first stage of evolution of these phase-separated autocatalytic systems is the appearance of pre-biotic metabolism providing synthesis of amphiphiles for formation of capsules of these systems. This synthesis is maintained by the energy of a base reaction being a component of a planet-chemical cycle. Catalytic system providing functioning of pre-biotic metabolism is based on multivariate oligomeric autocatalyst, which reproduces itself from monomers, penetrating the particles from the outside. Since the autocatalyst realizes random polymerization then a collection of other oligomers possessing different catalytic functions is produced. In the paper the functioning of multivariate oligomeric autocatalyst in flow reactor is analyzed. c2005 Published by Elsevier Ltd on behalf of COSPAR.

Three Rate-Constant Kinetic Model for Permanganate Reactions Autocatalyzed by Colloidal Manganese Dioxide: The Oxidation of L-Phenylalanine.

PubMed

Perez-Benito, Joaquin F; Ferrando, Jordi

2014-12-26

The reduction of permanganate ion to MnO(2)-Mn(2)O(3) soluble colloidal mixed oxide by l-phenylalanine in aqueous phosphate-buffered neutral solutions has been followed by a spectrophotometric method, monitoring the decay of permanganate ion at 525 nm and the formation of the colloidal oxide at 420 nm. The reaction is autocatalyzed by the manganese product, and three rate constants have been required to fit the experimental absorbance-time kinetic data. The reaction shows base catalysis, and the values of the activation parameters at different pHs have been determined. A mechanism including both the nonautocatalytic and the autocatalytic reaction pathways, and in agreement with the available experimental data, has been proposed. Some key features of this mechanism are the following: (i) of the two predominant forms of the amino acid, the anionic form exhibits a stronger reducing power than the zwitterionic form; (ii) the nonautocatalytic reaction pathway starts with the transfer of the hydrogen atom in the α position of the amino acid to permanganate ion; and (iii) the autocatalytic reaction pathway involves the reduction of Mn(IV) to Mn(II) by the amino acid and the posterior reoxidation of Mn(II) to Mn(IV) by permanganate ion.

Recycling Frank: Spontaneous emergence of homochirality in noncatalytic systems

PubMed Central

Plasson, Raphaël; Bersini, Hugues; Commeyras, Auguste

2004-01-01

In this work, we introduce a prebiotically relevant protometabolic pattern corresponding to an engine of deracemization by using an external energy source. The spontaneous formation of a nonracemic mixture of chiral compounds can be observed in out-of-equilibrium systems via a symmetry-breaking phenomenon. This observation is possible thanks to chirally selective autocatalytic reactions (Frank's model) [Frank, F. C. (1953) Biochim. Biophys. Acta 11, 459–463]. We show that the use of a Frank-like model in a recycled system composed of reversible chemical reactions, rather than the classical irreversible system, allows for the emergence of a synergetic autoinduction from simple reactions, without any autocatalytic or even catalytic reaction. This model is described as a theoretical framework, based on the stereoselective reactivity of preexisting chiral monomeric building blocks (polymerization, epimerization, and depolymerization) maintained out of equilibrium by a continuous energy income, via an activation reaction. It permits the self-conversion of all monomeric subunits into a single chiral configuration. Real prebiotic systems of amino acid derivatives can be described on this basis. They are shown to be able to spontaneously reach a stable nonracemic state in a few centuries. In such systems, the presence of epimerization reactions is no more destructive, but in contrast is the central driving force of the unstabilization of the racemic state. PMID:15548617

Structure-based cleavage mechanism of Thermus thermophilus Argonaute DNA guide strand-mediated DNA target cleavage

PubMed Central

Sheng, Gang; Zhao, Hongtu; Wang, Jiuyu; Rao, Yu; Tian, Wenwen; Swarts, Daan C.; van der Oost, John; Patel, Dinshaw J.; Wang, Yanli

2014-01-01

We report on crystal structures of ternary Thermus thermophilus Argonaute (TtAgo) complexes with 5′-phosphorylated guide DNA and a series of DNA targets. These ternary complex structures of cleavage-incompatible, cleavage-compatible, and postcleavage states solved at improved resolution up to 2.2 Å have provided molecular insights into the orchestrated positioning of catalytic residues, a pair of Mg2+ cations, and the putative water nucleophile positioned for in-line attack on the cleavable phosphate for TtAgo-mediated target cleavage by a RNase H-type mechanism. In addition, these ternary complex structures have provided insights into protein and DNA conformational changes that facilitate transition between cleavage-incompatible and cleavage-compatible states, including the role of a Glu finger in generating a cleavage-competent catalytic Asp-Glu-Asp-Asp tetrad. Following cleavage, the seed segment forms a stable duplex with the complementary segment of the target strand. PMID:24374628

Characteristics of eyes with inner retinal cleavage.

PubMed

Hwang, Young Hoon; Kim, Yong Yeon; Kim, Hwang Ki; Sohn, Yong Ho

2015-02-01

Inner retinal cleavage can be misdiagnosed as a glaucomatous retinal nerve fiber layer (RNFL) defect. This study was performed to characterize eyes with inner retinal cleavage. Inner retinal cleavage is defined as the appearance of a dark spindle-shaped space between the nerve fibers. Patients who presented at our institution with inner retinal cleavage were enrolled in the study. All participants were evaluated by fundus examination, visual field testing with standard automated perimetry, and optical coherence tomography (OCT) imaging. A total of 15 eyes of 11 subjects with inner retinal cleavage were included in the study. The median age of the subjects was 57 years (age range, 30-67 years). In each case, inner retinal cleavage was located adjacent to retinal blood vessels. Tissue bridging the cleavage area was observed in ten eyes. Six eyes had epiretinal membranes (ERMs), two eyes had glaucoma, and one eye had ERM in addition to glaucoma. Six eyes with inner retinal cleavage without combined ocular abnormalities had highly myopic refractive error (-6.50 to -8.50 diopters). Cross-sectional OCT images of the areas of inner retinal cleavage demonstrated defects with irregular margins and empty spaces in the inner layers of the retina. During the follow-up period, no eye showed changes in inner retinal layer cleavage or visual field sensitivity. Inner retinal cleavage was found in eyes with high myopia or ERMs. Inner retinal cleavage was associated with structural changes distinct from those associated with glaucomatous RNFL defects.

Intrauterine administration of human chorionic gonadotropin (hCG) for subfertile women undergoing assisted reproduction.

PubMed

Craciunas, Laurentiu; Tsampras, Nikolaos; Coomarasamy, Arri; Raine-Fenning, Nick

2016-05-20

Subfertility affects 15% of couples and represents the inability to conceive naturally following 12 months of regular unprotected sexual intercourse. Assisted reproduction refers to procedures involving the in vitro handling of both human gametes and represents a key option for many subfertile couples. Most women undergoing assisted reproduction treatment will reach the stage of embryo transfer (ET) but the proportion of embryos that successfully implant following ET has remained small since the mid-1990s. Human chorionic gonadotropin (hCG) is a hormone synthesised and released by the syncytiotrophoblast and has a fundamental role in embryo implantation and the early stages of pregnancy. Intrauterine administration of synthetic or natural hCG via an ET catheter during a mock procedure around the time of ET is a novel approach that has recently been suggested to improve the outcomes of assisted reproduction. To investigate whether the intrauterine administration of hCG around the time of ET improves the clinical outcomes in subfertile women undergoing assisted reproduction. We performed a comprehensive literature search of the Cochrane Gynaecology and Fertility Group Specialised Register, Cochrane Central Register of Controlled Trials (CENTRAL), MEDLINE, EMBASE, CINAHL, PsycINFO, registers of ongoing trials andreference lists of all included studies and relevant reviews (from inception to 10 November 2015), in consultation with the Cochrane Gynaecology and Fertility Group Trials Search Co-ordinator. We included all randomised controlled trials (RCTs) evaluating intrauterine administration of hCG around the time of ET in this review irrespective of language and country of origin. Two authors independently selected studies, assessed risk of bias, extracted data from studies and attempted to contact the authors where data were missing. We performed statistical analysis using Review Manager 5 in accordance with the Cochrane Handbook for Systematic Reviews of Interventions. We assessed evidence quality using GRADE methods. Twelve RCTs investigated the effect of intrauterine administration of hCG for 4038 subfertile women undergoing assisted reproduction. The intra-cavity hCG (IC-hCG) was administered in variable doses at different timings before the ET. The source of hCG was from the urine of pregnant women or from cell cultures using recombinant DNA technology.Most of the studies (9/12) were at high risk of bias in at least one of the seven domains assessed. Common problems were unclear reporting of study methods and lack of blinding. The main limitations in the overall quality of the evidence were high risk of bias and serious imprecision.For the analyses of live birth and clinical pregnancy, there was considerable heterogeneity (I(2) greater than 75%) and we did not undertake a meta-analysis. Exploration for the sources of heterogeneity identified two key pre-specified variables as important determinants: stage of ET (cleavage versus blastocyst stage) and dose of IC-hCG (less than 500 international units (IU) versus 500 IU or greater). We then performed meta-analysis for these analyses within the subgroups defined by stage of embryo and dose of IC-hCG.There was an increase in live birth rate in the subgroup of women having cleavage-stage ETs with an IC-hCG dose of 500 IU or greater compared to women having cleavage-stage ETs with no IC-hCG (risk ratio (RR) 1.57, 95% confidence interval (CI) 1.32 to 1.87, three RCTs, n = 914, I(2) = 0%, moderate quality evidence). In a clinic with a live birth rate of 25% per cycle then the use of IC-hCG -500 IU or greater would be associated with a live birth rate that varies from 33% to 46%. We did not observe a significant effect on live birth in any of the other subgroups.The was an increase in clinical pregnancy rate in the subgroup of women having cleavage-stage ETs with an IC-hCG dose of 500 IU or greater compared to women having cleavage-stage ETs with no IC-hCG (RR 1.41, 95% CI 1.25 to 1.58, seven RCTs, n = 1414, I(2) = 0%, moderate quality evidence). We did not observe a significant effect on clinical pregnancy in either of the other subgroups.There was no evidence that miscarriage was influenced by intrauterine hCG administration (RR 1.09, 95% CI 0.83 to 1.43, seven RCTs, n = 3395, I(2) = 0%, very low quality evidence).Other complications reported in the included studies were ectopic pregnancy (three RCTs, n = 915, three events overall), heterotopic pregnancy (one RCT, n = 495, one event), intrauterine death (two RCTs, n = 978, 21 events) and triplets (one RCT, n = 48, three events). There was no evidence of a difference between the groups, but there were too few events to allow any conclusions to be drawn and the evidence was very low quality. The pregnancy outcome for cleavage-stage ETs using an IC-hCG dose of 500 IU or greater is promising. However, given the small size and the variable quality of the trials and the fact that the positive finding was from a subgroup analysis, the current evidence for IC-hCG treatment does not support its use in assisted reproduction cycles. A definitive large clinical trial with live birth as the primary outcome is recommended. There was no evidence that miscarriage was influenced by intrauterine hCG administration, irrespective of embryo stage at transfer or dose of IC-hCG. There were too few events to allow any conclusions to be drawn with regard to other complications.

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

Deletion mapping of the Aequorea victoria green fluorescent protein.

PubMed

Dopf, J; Horiagon, T M

1996-01-01

Aequorea victoria green fluorescent protein (GFP) is a promising fluorescent marker which is active in a diverse array of prokaryotic and eukaryotic organisms. A key feature underlying the versatility of GFP is its capacity to undergo heterocyclic chromophore formation by cyclization of a tripeptide present in its primary sequence and thereby acquiring fluorescent activity in a variety of intracellular environments. In order to define further the primary structure requirements for chromophore formation and fluorescence in GFP, a series of N- and C-terminal GFP deletion variant expression vectors were created using the polymerase chain reaction. Scanning spectrofluorometric analyses of crude soluble protein extracts derived from eleven GFP expression constructs revealed that amino acid (aa) residues 2-232, of a total of 238 aa in the native protein, were required for the characteristic emission and absorption spectra of native GFP. Heterocyclic chromophore formation was assayed by comparing the absorption spectrum of GFP deletion variants over the 300-500-nm range to the absorption spectra of full-length GFP and GFP deletion variants missing the chromophore substrate domain from the primary sequence. GFP deletion variants lacking fluorescent activity showed no evidence of heterocyclic ring structure formation when the soluble extracts of their bacterial expression hosts were studied at pH 7.9. These observations suggest that the primary structure requirements for the fluorescent activity of GFP are relatively extensive and are compatible with the view that much of the primary structure serves an autocatalytic function.

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.

The use of morphokinetics as a predictor of embryo implantation.

PubMed

Meseguer, Marcos; Herrero, Javier; Tejera, Alberto; Hilligsøe, Karen Marie; Ramsing, Niels Birger; Remohí, Jose

2011-10-01

Time-lapse observation presents an opportunity for optimizing embryo selection based on morphological grading as well as providing novel kinetic parameters, which may further improve accurate selection of viable embryos. The objective of this retrospective study was to identify the morphokinetic parameters specific to embryos that were capable of implanting. In order to compare a large number of embryos, with minimal variation in culture conditions, we have used an automatic embryo monitoring system. Using a tri-gas IVF incubator with a built-in camera designed to automatically acquire images at defined time points, we have simultaneously monitored up to 72 individual embryos without removing the embryos from the controlled environment. Images were acquired every 15 min in five different focal planes for at least 64 h for each embryo. We have monitored the development of transferred embryos from 285 couples undergoing their first ICSI cycle. The total number of transferred embryos was 522, of which 247 either failed to implant or fully implanted, with full implantation meaning that all transferred embryos in a treatment implanted. A detailed retrospective analysis of cleavage times, blastomere size and multinucleation was made for the 247 transferred embryos with either failed or full implantation. We found that several parameters were significantly correlated with subsequent implantation (e.g. time of first and subsequent cleavages as well as the time between cleavages). The most predictive parameters were: (i) time of division to 5 cells, t5 (48.8-56.6 h after ICSI); (ii) time between division to 3 cells and subsequent division to 4 cells, s2 (≤ 0.76 h) and (iii) duration of cell cycle two, i.e. time between division to 2 cells and division to 3 cells, cc2 (≤ 11.9 h). We also observed aberrant behavior such as multinucleation at the 4 cell stage, uneven blastomere size at the 2 cell stage and abrupt cell division to three or more cells, which appeared to largely preclude implantation. The image acquisition and time-lapse analysis system makes it possible to determine exact timing of embryo cleavages in a clinical setting. We propose a multivariable model based on our findings to classify embryos according to their probability of implantation. The efficacy of this classification will be evaluated in a prospective randomized study that ultimately will determine if implantation rates can be improved by time-lapse analysis.

Complex Networks

DTIC Science & Technology

2006-11-01

The Internet architecture was designed w/o a “theory” • Many academic theorists said it would never work • Recent “emergent” theories are wildly...architecture was designed w/o a “theory” • Many academic theorists said it would never work • Recent “emergent” theories are wildly wrong (there have...Co-factors Fatty acids Sug ars Nucleotides Amino A cids Catabolism Polymerization and complex assembly Proteins P r e c u r s o r s Autocatalytic

Interstrand cross-links arising from strand breaks at true abasic sites in duplex DNA.

PubMed

Yang, Zhiyu; Price, Nathan E; Johnson, Kevin M; Wang, Yinsheng; Gates, Kent S

2017-06-20

Interstrand cross-links are exceptionally bioactive DNA lesions. Endogenous generation of interstrand cross-links in genomic DNA may contribute to aging, neurodegeneration, and cancer. Abasic (Ap) sites are common lesions in genomic DNA that readily undergo spontaneous and amine-catalyzed strand cleavage reactions that generate a 2,3-didehydro-2,3-dideoxyribose sugar remnant (3'ddR5p) at the 3'-terminus of the strand break. Interestingly, this strand scission process leaves an electrophilic α,β-unsaturated aldehyde residue embedded within the resulting nicked duplex. Here we present evidence that 3'ddR5p derivatives generated by spermine-catalyzed strand cleavage at Ap sites in duplex DNA can react with adenine residues on the opposing strand to generate a complex lesion consisting of an interstrand cross-link adjacent to a strand break. The cross-link blocks DNA replication by ϕ29 DNA polymerase, a highly processive polymerase enzyme that couples synthesis with strand displacement. This suggests that 3'ddR5p-derived cross-links have the potential to block critical cellular DNA transactions that require strand separation. LC-MS/MS methods developed herein provide powerful tools for studying the occurrence and properties of these cross-links in biochemical and biological systems. © The Author(s) 2017. Published by Oxford University Press on behalf of Nucleic Acids Research.

Single-cell analysis of differences in transcriptomic profiles of oocytes and cumulus cells at GV, MI, MII stages from PCOS patients

PubMed Central

Liu, Qiwei; Li, Yumei; Feng, Yun; Liu, Chaojie; Ma, Jieliang; Li, Yifei; Xiang, Huifen; Ji, Yazhong; Cao, Yunxia; Tong, Xiaowen; Xue, Zhigang

2016-01-01

Polycystic ovary syndrome (PCOS) is a common frequent endocrine disorder among women of reproductive age. Although assisted reproductive techniques (ARTs) are used to address subfertility in PCOS women, their effectiveness is not clear. Our aim was to compare transcriptomic profiles of oocytes and cumulus cells (CCs) between women with and without PCOS, and assess the effectiveness of ARTs in treating PCOS patients. We collected oocytes and CCs from 16 patients with and without PCOS patients to categorize them into 6 groups according to oocyte nuclear maturation. Transcriptional gene expression of oocyte and CCs was determined via single-cell RNA sequencing. The ratio of fertilization and cleavage was higher in PCOS patients than in non-PCOS patients undergoing ARTs, and there was no difference in the number of high-quality embryos between the groups. Differentially expressed genes including PPP2R1A, PDGFRA, EGFR, GJA1, PTGS2, TNFAIP6, TGF-β1, CAV1, INHBB et al. were investigated as potential causes of PCOS oocytes and CCs disorder at early stages, but their expression returned to the normal level at the metaphase II (MII) stage via ARTs. In conclusion, ARTs can improve the quality of cumulus-oocyte complex (COC) and increase the ratio of fertilization and cleavage in PCOS women. PMID:28004769

Time-lapse monitoring of zona pellucida-free embryos obtained through in vitro fertilization: a retrospective case series.

PubMed

Bodri, Daniel; Kato, Ryutaro; Kondo, Masae; Hosomi, Naoko; Katsumata, Yoshinari; Kawachiya, Satoshi; Matsumoto, Tsunekazu

2015-05-01

To report time-lapse monitoring of human oocytes in which the damaged zona pellucida was removed, producing zona-free (ZF) oocytes that were cultured until the blastocyst stage in time-lapse incubators. Retrospective case series. Private infertility clinic. Infertile patients (n = 32) undergoing minimal ovarian stimulation or natural cycle IVF treatment between October 2012 and June 2014. Intracytoplasmic sperm injection (ICSI) fertilization of ZF oocytes, prolonged embryo culture in time-lapse incubators, elective vitrification, and subsequent single vitrified-thawed blastocyst transfer (SVBT). Rate of fertilization, cleavage and blastocyst development, live-birth rate per SVBT cycle. In spite of advanced maternal age (39 ± 4.2; range, 30-46 years), good fertilization (94%), cleavage (94%), and blastocyst development rates (38%) were reached after fertilization and culturing of ZF oocytes/embryos. All thawed ZF blastocysts survived, and up to this date seven SVBT transfers were performed, yielding three (43%) term live births with healthy newborns. Time-lapse imagery gives a unique insight into the dynamics of embryo development in ZF embryos. Moreover, our case series demonstrate that an oocyte with a damaged zona pellucida that has been removed could be successfully fertilized with ICSI, cultured until blastocyst stage in a time-lapse incubator and vitrified electively for subsequent use. Copyright © 2015 American Society for Reproductive Medicine. Published by Elsevier Inc. All rights reserved.

Effects of reactive oxygen species levels in prepared culture media on embryo development: a comparison of two media.

PubMed

Shih, Ying-Fu; Lee, Tsung-Hsien; Liu, Chung-Hsien; Tsao, Hui-Mei; Huang, Chun-Chia; Lee, Maw-Sheng

2014-12-01

This study determined the correlation between the levels of reactive oxygen species (ROS) in prepared culture media and the early development of human embryos. This was an autocontrolled comparison study. A total of 159 patients undergoing in vitro fertilization/intracytoplasmic sperm injection treatment were recruited in this study. The pH values, osmolarity pressures, and ROS levels of 15 batches of two culture media were measured. Sibling oocytes or embryos from individual patients were randomly assigned to two culture groups with Quinn's Advantage Cleavage and Blastocyst media (QAC/QAB) or GIII series cleavage and blastocyst media (G1.3/G2.3). The difference between the two culture groups was analyzed using one-sample t test. The QAC/QAB and G1.3/G2.3 media exhibited similar pH values and osmolarity pressures. However, the prepared QAC/QAB media were characterized to contain lower amounts of ROS than the G1.3/G2.3 media. Furthermore, the blastocysts that developed under the QAC/QAB media were morphologically superior to those that developed under the G1.3/G2.3 media. The elevated ROS levels in culture media were associated with poor development of blastocyst-stage embryos. Measurement of ROS levels may be a valuable process for medium selection or modification. Copyright © 2014. Published by Elsevier B.V.

Effects of dimethyl sulfoxide on asymmetric division and cytokinesis in mouse oocytes.

PubMed

Zhou, Dongjie; Shen, Xinghui; Gu, Yanli; Zhang, Na; Li, Tong; Wu, Xi; Lei, Lei

2014-06-21

Dimethyl sulfoxide (DMSO) is used extensively as a permeable cryoprotectant and is a common solvent utilized for several water-insoluble substances. DMSO has various biological and pharmacological activities; however, the effect of DMSO on mouse oocyte meiotic maturation remains unknown. In DMSO-treated oocytes, we observed abnormal MII oocytes that contained large polar bodies, including 2-cell-like MII oocytes, during in vitro maturation. Oocyte polarization did not occur, due to the absence of actin cap formation and spindle migration. These features are among the primary causes of abnormal symmetric division; however, analysis of the mRNA expression levels of genes related to asymmetric division revealed no significant difference in the expression of these factors between the 3% DMSO-treated group and the control group. After each "blastomere" of the 2-cell-like MII stage oocytes was injected by one sperm head respectively, the oocytes still possessed the ability to extrude the second polar body from each "blastomere" and to begin cleavage. However, MII oocytes with large polar bodies developed to the blastocyst stage after intracytoplasmic sperm injection (ICSI). Furthermore, other permeable cryoprotectants, such as ethylene glycol and glycerol, also caused asymmetric division failure. Permeable cryoprotectants, such as DMSO, ethylene glycol, and glycerol, affect asymmetric division. DMSO disrupts cytokinesis completion by inhibiting cortical reorganization and polarization. Oocytes that undergo symmetric division maintain the ability to begin cleavage after ICSI.

Single-cell analysis of differences in transcriptomic profiles of oocytes and cumulus cells at GV, MI, MII stages from PCOS patients.

PubMed

Liu, Qiwei; Li, Yumei; Feng, Yun; Liu, Chaojie; Ma, Jieliang; Li, Yifei; Xiang, Huifen; Ji, Yazhong; Cao, Yunxia; Tong, Xiaowen; Xue, Zhigang

2016-12-22

Polycystic ovary syndrome (PCOS) is a common frequent endocrine disorder among women of reproductive age. Although assisted reproductive techniques (ARTs) are used to address subfertility in PCOS women, their effectiveness is not clear. Our aim was to compare transcriptomic profiles of oocytes and cumulus cells (CCs) between women with and without PCOS, and assess the effectiveness of ARTs in treating PCOS patients. We collected oocytes and CCs from 16 patients with and without PCOS patients to categorize them into 6 groups according to oocyte nuclear maturation. Transcriptional gene expression of oocyte and CCs was determined via single-cell RNA sequencing. The ratio of fertilization and cleavage was higher in PCOS patients than in non-PCOS patients undergoing ARTs, and there was no difference in the number of high-quality embryos between the groups. Differentially expressed genes including PPP2R1A, PDGFRA, EGFR, GJA1, PTGS2, TNFAIP6, TGF-β1, CAV1, INHBB et al. were investigated as potential causes of PCOS oocytes and CCs disorder at early stages, but their expression returned to the normal level at the metaphase II (MII) stage via ARTs. In conclusion, ARTs can improve the quality of cumulus-oocyte complex (COC) and increase the ratio of fertilization and cleavage in PCOS women.

Dissociation reactions of protonated anthracycline antibiotics following electrospray ionization-tandem mass spectrometry

NASA Astrophysics Data System (ADS)

Sleno, Lekha; Campagna-Slater, Valerie; Volmer, Dietrich A.

2006-09-01

Fragmentation pathways of doxorubicin, a common cancer therapy agent, and three closely related analogs (epirubicin, daunorubicin, idarubicin) were compared using electrospray ionization with tandem mass spectrometry. This class of antibiotics with anti-tumour activity has important structural features, with a tetracyclic aromatic, polyketide portion, which is glycosylated with an amino sugar in order to exhibit its biological activity. Collision-induced dissociation spectra revealed very similar product ions for each analog, however, important differences were seen in the relative abundances and the ease at which certain fragments were formed. Fragment ions observed included those from cleavage of the glycosidic bond, loss of the side chain from the aglycone moiety, water losses and loss of a methyl radical. Following cleavage of the glycosidic bond, the charge can either reside on the aglycone portion or the sugar moiety, and each of these primary fragments undergoes several secondary dissociation pathways, depending on the collision energy. By ramping the collision voltage, we were able to correlate the changes in fragmentation behavior with small alterations in the structure of the precursor ion. The detailed study of the fragmentation behavior of doxorubicin was supported by accurate mass measurements, using an electrospray-time of flight instrument, as well as MS3 data from a quadrupole-linear ion trap mass spectrometer. Computational studies were also performed to help explain the role of certain functional groups in the fragmentation reactions.

Photochemical transformation of five novel brominated flame retardants: Kinetics and photoproducts.

PubMed

Zhang, Ya-Nan; Chen, Jingwen; Xie, Qing; Li, Yingjie; Zhou, Chengzhi

2016-05-01

Many novel brominated flame retardants (NBFRs) are used as substitutes of polybrominated diphenyl ethers (PBDEs) in recent years. However, little is known about their phototransformation behavior, which may influence the environmental fate of these chemicals. In this study, photochemical behavior of five NBFRs, allyl-2,4,6-tribromophenyl ether (ATE), 2-bromoallyl-2,4,6-tribromophenyl ether (BATE), 2,3-dibromopropyl-2,4,6-tribromophenyl ether (DPTE), 1,2-bis(2,4,6-tribromophenoxy)ethane (BTBPE), and 2,4,6-tris(2,4,6-tribromophenoxy)-1,3,5-triazine (TTBP-TAZ) was investigated. Results show all the five NBFRs can undergo photochemical transformation under simulated sunlight irradiation. Quantum yields (Φ) of the five NBFRs varied from 0.012 of TTBP-TAZ in hexane to 0.091 of BTBPE in methanol. Half-lives (t1/2) relevant with solar irradiation of these NBFRs were estimated using the determined Φ, and the values are 1.5-12.0 d in summer and 17.1-165.0 d in winter. Debrominated and ether bond cleavage products were identified in the phototransformation of DPTE and BTBPE. Debromination on the phenyl is a main phototransformation pathway for DPTE, and both debromination and ether bond cleavage are main phototransformation pathways for BTBPE. This study is helpful to better understand the phototransformation behavior of the NBFRs. Copyright © 2015 Elsevier Ltd. All rights reserved.

The impact of food intake and social habits on embryo quality and the likelihood of blastocyst formation.

PubMed

Braga, Daniela Paes Almeida Ferreira; Halpern, Gabriela; Setti, Amanda S; Figueira, Rita Cássia S; Iaconelli, Assumpto; Borges, Edson

2015-07-01

The aim of this study was to evaluate the influence of patients' lifestyle factors and eating habits on embryo development. A total of 2659 embryos recovered from 269 patients undergoing intracytoplasmic sperm injection cycles were included. The frequency of intake of food items and social habits were registered and its influences on embryo development evaluated. The consumption of cereals, vegetables and fruits positively influenced the embryo quality at the cleavage stage. The quality of the embryo at the cleavage stage was also negatively correlated with the consumption of alcoholic drinks and smoking habits. The consumption of fruits influenced the likelihood of blastocyst formation, which was also positively affected by the consumption of fish. Being on a weight-loss diet and consumption of red meat had a negative influence on the likelihood of blastocyst formation. The likelihood of blastocyst formation was also negatively influenced by the consumption of alcoholic drinks and by smoking habits. The consumption of red meat and body mass index had a negative effect on the implantation rate and the likelihood of pregnancy. In addition, being on a weight-loss diet had a negative influence on implantation rate. Our evidence suggests a possible relationship between environmental factors and ovary biology. Copyright © 2015 Reproductive Healthcare Ltd. Published by Elsevier Ltd. All rights reserved.

APC/C-Cdc20 mediates deprotection of centromeric cohesin at meiosis II in yeast.

PubMed

Jonak, Katarzyna; Zagoriy, Ievgeniia; Oz, Tugce; Graf, Peter; Rojas, Julie; Mengoli, Valentina; Zachariae, Wolfgang

2017-06-18

Cells undergoing meiosis produce haploid gametes through one round of DNA replication followed by 2 rounds of chromosome segregation. This requires that cohesin complexes, which establish sister chromatid cohesion during S phase, are removed in a stepwise manner. At meiosis I, the separase protease triggers the segregation of homologous chromosomes by cleaving cohesin's Rec8 subunit on chromosome arms. Cohesin persists at centromeres because the PP2A phosphatase, recruited by the shugoshin protein, dephosphorylates Rec8 and thereby protects it from cleavage. While chromatids disjoin upon cleavage of centromeric Rec8 at meiosis II, it was unclear how and when centromeric Rec8 is liberated from its protector PP2A. One proposal is that bipolar spindle forces separate PP2A from Rec8 as cells enter metaphase II. We show here that sister centromere biorientation is not sufficient to "deprotect" Rec8 at meiosis II in yeast. Instead, our data suggest that the ubiquitin-ligase APC/C Cdc20 removes PP2A from centromeres by targeting for degradation the shugoshin Sgo1 and the kinase Mps1. This implies that Rec8 remains protected until entry into anaphase II when it is phosphorylated concurrently with the activation of separase. Here, we provide further support for this model and speculate on its relevance to mammalian oocytes.

APC/C-Cdc20 mediates deprotection of centromeric cohesin at meiosis II in yeast

PubMed Central

Jonak, Katarzyna; Oz, Tugce; Graf, Peter; Rojas, Julie; Mengoli, Valentina; Zachariae, Wolfgang

2017-01-01

ABSTRACT Cells undergoing meiosis produce haploid gametes through one round of DNA replication followed by 2 rounds of chromosome segregation. This requires that cohesin complexes, which establish sister chromatid cohesion during S phase, are removed in a stepwise manner. At meiosis I, the separase protease triggers the segregation of homologous chromosomes by cleaving cohesin's Rec8 subunit on chromosome arms. Cohesin persists at centromeres because the PP2A phosphatase, recruited by the shugoshin protein, dephosphorylates Rec8 and thereby protects it from cleavage. While chromatids disjoin upon cleavage of centromeric Rec8 at meiosis II, it was unclear how and when centromeric Rec8 is liberated from its protector PP2A. One proposal is that bipolar spindle forces separate PP2A from Rec8 as cells enter metaphase II. We show here that sister centromere biorientation is not sufficient to “deprotect” Rec8 at meiosis II in yeast. Instead, our data suggest that the ubiquitin-ligase APC/CCdc20 removes PP2A from centromeres by targeting for degradation the shugoshin Sgo1 and the kinase Mps1. This implies that Rec8 remains protected until entry into anaphase II when it is phosphorylated concurrently with the activation of separase. Here, we provide further support for this model and speculate on its relevance to mammalian oocytes. PMID:28514186

The cathelicidin LL-37 activates human mast cells and is degraded by mast cell tryptase: counter-regulation by CXCL4.

PubMed

Schiemann, Florian; Brandt, Ernst; Gross, Roland; Lindner, Buko; Mittelstädt, Jessica; Sommerhoff, Christian P; Schulmistrat, Jan; Petersen, Frank

2009-08-15

The cathelicidin LL-37 represents a potent antimicrobial and cell-stimulating agent, most abundantly expressed in peripheral organs such as lung and skin during inflammation. Because mast cells (MC) overtake prominent immunomodulatory roles in these organs, we wondered whether interactions exist between MC and LL-37. In this study, we show for the first time to our knowledge that physiological concentrations of LL-37 induce degranulation in purified human lung MC. Intriguingly, as a consequence LL-37 rapidly undergoes limited cleavage by a released protease. The enzyme was identified as beta-tryptase by inhibitor studies and by comparison to the recombinant protease. Examining the resulting LL-37 fragments for their functional activity, we found that none of the typical capacities of intact LL-37, i.e., MC degranulation, bactericidal activity, and neutralization of LPS, were retained. Conversely, we found that another inflammatory protein, the platelet-derived chemokine CXCL4, protects LL-37 from cleavage by beta-tryptase. Interestingly, CXCL4 did not act as a direct enzyme inhibitor, but destabilized active tetrameric beta-tryptase by antagonizing the heparin component required for the integrity of the tetramer. Altogether our results suggest that interaction of LL-37 and MC initiates an effective feedback loop to limit cathelicidin activity during inflammation, whereas CXCL4 may represent a physiological counter-regulator of beta-tryptase activity.

Pyroptosis is driven by non-selective gasdermin-D pore and its morphology is different from MLKL channel-mediated necroptosis

PubMed Central

Chen, Xin; He, Wan-ting; Hu, Lichen; Li, Jingxian; Fang, Yuan; Wang, Xin; Xu, Xiaozheng; Wang, Zhuo; Huang, Kai; Han, Jiahuai

2016-01-01

Necroptosis and pyroptosis are two forms of programmed cell death with a common feature of plasma membrane rupture. Here we studied the morphology and mechanism of pyroptosis in comparison with necroptosis. Different from necroptosis, pyroptosis undergoes membrane blebbing and produces apoptotic body-like cell protrusions (termed pyroptotic bodies) prior to plasma membrane rupture. The rupture in necroptosis is explosion-like, whereas in pyroptosis it leads to flattening of cells. It is known that the execution of necroptosis is mediated by mixed lineage kinase domain-like (MLKL) oligomers in the plasma membrane, whereas gasdermin-D (GSDMD) mediates pyroptosis after its cleavage by caspase-1 or caspase-11. We show that N-terminal fragment of GSDMD (GSDMD-N) generated by caspase cleavage also forms oligomer and migrates to the plasma membrane to kill cells. Both MLKL and GSDMD-N are lipophilic and the N-terminal sequences of both proteins are important for their oligomerization and plasma membrane translocation. Unlike MLKL which forms channels on the plasma membrane that induces influx of selected ions which osmotically swell the cells to burst, GSDMD-N forms non-selective pores and does not rely on increased osmolarity to disrupt cells. Our study reveals the pore-forming activity of GSDMD and channel-forming activity of MLKL determine different ways of plasma membrane rupture in pyroptosis and necroptosis. PMID:27573174

Pyroptosis is driven by non-selective gasdermin-D pore and its morphology is different from MLKL channel-mediated necroptosis.

PubMed

Chen, Xin; He, Wan-Ting; Hu, Lichen; Li, Jingxian; Fang, Yuan; Wang, Xin; Xu, Xiaozheng; Wang, Zhuo; Huang, Kai; Han, Jiahuai

2016-09-01

Necroptosis and pyroptosis are two forms of programmed cell death with a common feature of plasma membrane rupture. Here we studied the morphology and mechanism of pyroptosis in comparison with necroptosis. Different from necroptosis, pyroptosis undergoes membrane blebbing and produces apoptotic body-like cell protrusions (termed pyroptotic bodies) prior to plasma membrane rupture. The rupture in necroptosis is explosion-like, whereas in pyroptosis it leads to flattening of cells. It is known that the execution of necroptosis is mediated by mixed lineage kinase domain-like (MLKL) oligomers in the plasma membrane, whereas gasdermin-D (GSDMD) mediates pyroptosis after its cleavage by caspase-1 or caspase-11. We show that N-terminal fragment of GSDMD (GSDMD-N) generated by caspase cleavage also forms oligomer and migrates to the plasma membrane to kill cells. Both MLKL and GSDMD-N are lipophilic and the N-terminal sequences of both proteins are important for their oligomerization and plasma membrane translocation. Unlike MLKL which forms channels on the plasma membrane that induces influx of selected ions which osmotically swell the cells to burst, GSDMD-N forms non-selective pores and does not rely on increased osmolarity to disrupt cells. Our study reveals the pore-forming activity of GSDMD and channel-forming activity of MLKL determine different ways of plasma membrane rupture in pyroptosis and necroptosis.

Phototransformation of azoxystrobin fungicide in organic solvents. Photoisomerization vs. photodegradation.

PubMed

Chastain, Jeoffrey; ter Halle, Alexandra; de Sainte Claire, Pascal; Voyard, Guillaume; Traikïa, Mounir; Richard, Claire

2013-12-01

Azoxystrobin is a systemic fungicide that has a tendency to accumulate at the surface of crop leaves or inside their cuticle where it undergoes photodegradation. Its photochemistry was investigated in n-heptane and isopropanol to mimic the polarity of wax leaves. Using analytical and kinetic data, we demonstrate that azoxystrobin (isomer E) undergoes efficient photoisomerization into the isomer Z with a quantum yield of 0.75 ± 0.08. This value is 30-fold higher than that reported in aqueous solution. The photoisomerization of isomer Z into azoxystrobin is more efficient with a chemical yield of 0.95 ± 0.1. In addition, a pseudo photostationary equilibrium is reached when the ratio [azoxystrobin]/[isomer Z] is 2.0 ± 0.1. Photodegradation also takes place from azoxystrobin (quantum yield = 0.073 ± 0.008). Photoproducts mainly arise from bond cleavage between rings and from demethylation of the ether with or without saturation of the acrylate double bond. Theoretical calculations were undertaken to investigate the photoisomerization mechanism and the solvent effect. These data show that the photochemical reactivity of azoxystrobin is enhanced when the solvent polarity decreases and thus should be significant in leaf waxes.

Successful ongoing pregnancies after vitrification of oocytes.

PubMed

Lucena, Elkin; Bernal, Diana Patricia; Lucena, Carolina; Rojas, Alejandro; Moran, Abby; Lucena, Andrés

2006-01-01

To demonstrate the efficiency of vitrifying mature human oocytes for different clinical indications. Descriptive case series. Cryobiology laboratory, Centro Colombiano de Fertilidad y Esterilidad-CECOLFES LTDA. (Bogotá, Colombia). Oocyte vitrification was offered as an alternative management for patients undergoing infertility treatment because of ovarian hyperstimulation syndrome, premature ovarian failure, natural ovarian failure, male factor, poor response, or oocyte donation. Mature oocytes were obtained from 33 donor women and 40 patients undergoing infertility treatment. Oocytes were retrieved by ultrasound-guided transvaginal aspiration and vitrified with the Cryotops method, with 30% ethylene glycol, 30% dimethyl sulfoxide, and 0.5 mol/L sucrose. Viability was assessed 3 hours after thawing. The surviving oocytes were inseminated by intracytoplasmic sperm injection. Fertilization was evaluated after 24 hours. The zygotes were further cultured in vitro for up to 72 hours until time of embryo transfer. Recovery, viability, fertilization, and pregnancy rates. Oocyte vitrification with the Cryotop method resulted in high rates of recovery, viability, fertilization, cleavage, and ongoing pregnancy. Vitrification with the Cryotop method is an efficient, fast, and economical method for oocyte cryopreservation that offers high rates of survival, fertilization, embryo development, and ongoing normal pregnancies, providing a new alternative for the management of female infertility.

Pertussis toxin-catalyzed ADP-ribosylation of a G protein in mouse oocytes, eggs, and preimplantation embryos: Developmental changes and possible functional roles

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

Jones, J.; Schultz, R.M.

1990-06-01

G proteins, which in many somatic cells serve as mediators of signal transduction, were identified in preimplantation mouse embryos by their capacity to undergo pertussis toxin-catalyzed ADP-ribosylation. Two pertussis toxin (PT) substrates with Mr = 38,000 and 39,000 (alpha 38 and alpha 39) are present in approximately equal amounts. Relative to the amount in freshly isolated germinal vesicle (GV)-intact oocytes, the amount of PT-catalyzed ADP-ribosylation of alpha 38-39 falls during oocyte maturation, rises between the one- and two-cell stages, falls by the eight-cell and morula stages, and increases again by the blastocyst stage. The decrease in PT-catalyzed ADP-ribosylation of alphamore » 38-39 that occurs during oocyte maturation, however, does not require germinal vesicle breakdown (GVBD), since inhibiting GVBD with 3-isobutyl-1-methyl xanthine (IBMX) does not prevent the decrease in the extent of PT-catalyzed ADP-ribosylation. A biologically active phorbol diester (12-O-tetradecanoyl phorbol 13-acetate), but not an inactive one (4 alpha-phorbol 12,13-didecanoate, 4 alpha-PDD), totally inhibits the increase in PT-catalyzed ADP-ribosylation of alpha 38-39 that occurs between the one- and two-cell stage; TPA inhibits cleavage, but not transcriptional activation, which occurs in the two-cell embryo. In contrast, cytochalasin D, genistein, or aphidicolin, each of which inhibits cleavage of one-cell embryos, or alpha-amanitin or H8, each of which inhibits transcriptional activation but not cleavage of one-cell embryos, have little or inhibitory effects on the increase in PT-catalyzed ADP-ribosylation of alpha 38-39. Results of immunoblotting experiments using an antibody that is highly specific for alpha il-3 reveal the presence of a cross-reactive species of Mr = 38,000 (alpha 38) in the GV-intact oocyte, metaphase II-arrested egg, and one-, two-cell embryos.« less

Molecular Basis for the Recognition and Cleavages of IGF-II, TGF-[alpha], and Amylin by Human Insulin-Degrading Enzyme

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

Guo, Qing; Manolopoulou, Marika; Bian, Yao

2010-02-11

Insulin-degrading enzyme (IDE) is involved in the clearance of many bioactive peptide substrates, including insulin and amyloid-{beta}, peptides vital to the development of diabetes and Alzheimer's disease, respectively. IDE can also rapidly degrade hormones that are held together by intramolecular disulfide bond(s) without their reduction. Furthermore, IDE exhibits a remarkable ability to preferentially degrade structurally similar peptides such as the selective degradation of insulin-like growth factor (IGF)-II and transforming growth factor-{alpha} (TGF-{alpha}) over IGF-I and epidermal growth factor, respectively. Here, we used high-accuracy mass spectrometry to identify the cleavage sites of human IGF-II, TGF-{alpha}, amylin, reduced amylin, and amyloid-{beta} bymore » human IDE. We also determined the structures of human IDE-IGF-II and IDE-TGF-{alpha} at 2.3 {angstrom} and IDE-amylin at 2.9 {angstrom}. We found that IDE cleaves its substrates at multiple sites in a biased stochastic manner. Furthermore, the presence of a disulfide bond in amylin allows IDE to cut at an additional site in the middle of the peptide (amino acids 18-19). Our amylin-bound IDE structure offers insight into how the structural constraint from a disulfide bond in amylin can alter IDE cleavage sites. Together with NMR structures of amylin and the IGF and epidermal growth factor families, our work also reveals the structural basis of how the high dipole moment of substrates complements the charge distribution of the IDE catalytic chamber for the substrate selectivity. In addition, we show how the ability of substrates to properly anchor their N-terminus to the exosite of IDE and undergo a conformational switch upon binding to the catalytic chamber of IDE can also contribute to the selective degradation of structurally related growth factors.« less

Site-specific O-Glycosylation by Polypeptide N-Acetylgalactosaminyltransferase 2 (GalNAc-transferase T2) Co-regulates β1-Adrenergic Receptor N-terminal Cleavage.

PubMed

Goth, Christoffer K; Tuhkanen, Hanna E; Khan, Hamayun; Lackman, Jarkko J; Wang, Shengjun; Narimatsu, Yoshiki; Hansen, Lasse H; Overall, Christopher M; Clausen, Henrik; Schjoldager, Katrine T; Petäjä-Repo, Ulla E

2017-03-17

The β 1 -adrenergic receptor (β 1 AR) is a G protein-coupled receptor (GPCR) and the predominant adrenergic receptor subtype in the heart, where it mediates cardiac contractility and the force of contraction. Although it is the most important target for β-adrenergic antagonists, such as β-blockers, relatively little is yet known about its regulation. We have shown previously that β 1 AR undergoes constitutive and regulated N-terminal cleavage participating in receptor down-regulation and, moreover, that the receptor is modified by O -glycosylation. Here we demonstrate that the polypeptide GalNAc-transferase 2 (GalNAc-T2) specifically O -glycosylates β 1 AR at five residues in the extracellular N terminus, including the Ser-49 residue at the location of the common S49G single-nucleotide polymorphism. Using in vitro O -glycosylation and proteolytic cleavage assays, a cell line deficient in O -glycosylation, GalNAc-T-edited cell line model systems, and a GalNAc-T2 knock-out rat model, we show that GalNAc-T2 co-regulates the metalloproteinase-mediated limited proteolysis of β 1 AR. Furthermore, we demonstrate that impaired O -glycosylation and enhanced proteolysis lead to attenuated receptor signaling, because the maximal response elicited by the βAR agonist isoproterenol and its potency in a cAMP accumulation assay were decreased in HEK293 cells lacking GalNAc-T2. Our findings reveal, for the first time, a GPCR as a target for co-regulatory functions of site-specific O -glycosylation mediated by a unique GalNAc-T isoform. The results provide a new level of β 1 AR regulation that may open up possibilities for new therapeutic strategies for cardiovascular diseases. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

Diagnostic cyclisation reactions which follow phosphate transfer to carboxylate anion centres for energised [M-H]- anions of pTyr-containing peptides.

PubMed

Tran, T T Nha; Wang, Tianfang; Hack, Sandra; Bowie, John H

2011-09-15

The low-energy negative ion phosphoTyr to C-terminal -CO(2)PO(3)H(2) rearrangement occurs for energised peptide [M-H](-) anions even when there are seven amino acid residues between the pTyr and C-terminal amino acid residues. The rearranged C-terminal -CO(2)PO(2)H(O(-)) group effects characteristic S(N)i cyclisation/cleavage reactions. The most pronounced of these involves the electrophilic central backbone carbon of the penultimate amino acid residue. This reaction is aided by the intermediacy of an H-bonded intermediate in which the nucleophilic and electrophilic reaction centres are held in proximity in order for the S(N)i cyclisation/cleavage to proceed. The ΔG(reaction) is +184 kJ mol(-1) with the barrier to the S(N)i transition state being +240 kJ mol(-1) at the HF/6-31 + G(d)//AM1 level of theory. A similar phosphate rearrangement from pTyr to side chain CO(2)(-) (of Asp or Glu) may also occur for energised peptide [M-H](-) anions. The reaction is favourable: ΔG(reaction) is -44 kJ mol(-1) with a maximum barrier of +21 kJ mol(-1) (to the initial transition state) when Asp and Tyr are adjacent. The rearranged species R(1)-Tyr-NHCH(CH(2)CO(2)PO(3)H(-))COR(2) (R(1)  = CHO; R(2)  = OCH(3)) may undergo an S(N)i six-centred cyclisation/cleavage reaction to form the product anion R(1)-Tyr(NH(-)). This process has a high energy requirement [ΔG(reaction)  = +224 kJ mol(-1), with the barrier to the S(N)i transition state being +299 kJ mol(-1)]. Copyright © 2011 John Wiley & Sons, Ltd.

Accurate and rapid modeling of iron-bleomycin-induced DNA damage using tethered duplex oligonucleotides and electrospray ionization ion trap mass spectrometric analysis.

PubMed

Harsch, A; Marzilli, L A; Bunt, R C; Stubbe, J; Vouros, P

2000-05-01

Bleomycin B(2)(BLM) in the presence of iron [Fe(II)] and O(2)catalyzes single-stranded (ss) and double-stranded (ds) cleavage of DNA. Electrospray ionization ion trap mass spectrometry was used to monitor these cleavage processes. Two duplex oligonucleotides containing an ethylene oxide tether between both strands were used in this investigation, allowing facile monitoring of all ss and ds cleavage events. A sequence for site-specific binding and cleavage by Fe-BLM was incorporated into each analyte. One of these core sequences, GTAC, is a known hot-spot for ds cleavage, while the other sequence, GGCC, is a hot-spot for ss cleavage. Incubation of each oligo-nucleotide under anaerobic conditions with Fe(II)-BLM allowed detection of the non-covalent ternary Fe-BLM/oligonucleotide complex in the gas phase. Cleavage studies were then performed utilizing O(2)-activated Fe(II)-BLM. No work-up or separation steps were required and direct MS and MS/MS analyses of the crude reaction mixtures confirmed sequence-specific Fe-BLM-induced cleavage. Comparison of the cleavage patterns for both oligonucleotides revealed sequence-dependent preferences for ss and ds cleavages in accordance with previously established gel electrophoresis analysis of hairpin oligonucleotides. This novel methodology allowed direct, rapid and accurate determination of cleavage profiles of model duplex oligonucleotides after exposure to activated Fe-BLM.

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.

Solvent-induced controllable synthesis, single-crystal to single-crystal transformation and encapsulation of Alq3 for modulated luminescence in (4,8)-connected metal-organic frameworks.

PubMed

Lan, Ya-Qian; Jiang, Hai-Long; Li, Shun-Li; Xu, Qiang

2012-07-16

In this work, for the first time, we have systematically demonstrated that solvent plays crucial roles in both controllable synthesis of metal-organic frameworks (MOFs) and their structural transformation process. With solvent as the only variable, five new MOFs with different structures have been constructed, in which one MOF undergoes solvent-induced single-crystal to single-crystal (SCSC) transformation that involves not only solvent exchange but also the cleavage and formation of coordination bonds. Particularly, a significant crystallographic change has been realized through an unprecedented three-step SCSC transformation process. Furthermore, we have demonstrated that the obtained MOF could be an excellent host for chromophores such as Alq3 for modulated luminescent properties.

Fast Fmoc synthesis of hAmylin1-37 with pseudoproline assisted on-resin disulfide formation.

PubMed

Page, Karen; Hood, Christina A; Patel, Hirendra; Fuentes, German; Menakuru, Mahendra; Park, Jae H

2007-12-01

Human amylin (1-37) and the (1-13) fragment were synthesized with and without pseudoproline dipeptides. Thallium (III) trifluoroacetate, a mild oxidant, was used to cyclize the peptides by forming a disulfide bridge from C(2) to C(7). On the basis of our model studies, incorporation of a pseudoproline dipeptide decreases the amount of time necessary for the crude linear amylin (1-13) to cyclize on the resin. Without pseudoproline dipeptides, the 1-37 crude linear amylin was not pure enough to undergo the cyclization reaction. Following the cyclization studies, the synthesis time of the linear human amylin (1-37) was systematically reduced from 58 h to 8.5 h by shortening the reaction times. Cyclization and cleavage times were also reduced to 1.5 h.

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.

The origin of life is a spatially localized stochastic transition

PubMed Central

2012-01-01

Background Life depends on biopolymer sequences as catalysts and as genetic material. A key step in the Origin of Life is the emergence of an autocatalytic system of biopolymers. Here we study computational models that address the way a living autocatalytic system could have emerged from a non-living chemical system, as envisaged in the RNA World hypothesis. Results We consider (i) a chemical reaction system describing RNA polymerization, and (ii) a simple model of catalytic replicators that we call the Two’s Company model. Both systems have two stable states: a non-living state, characterized by a slow spontaneous rate of RNA synthesis, and a living state, characterized by rapid autocatalytic RNA synthesis. The origin of life is a transition between these two stable states. The transition is driven by stochastic concentration fluctuations involving relatively small numbers of molecules in a localized region of space. These models are simulated on a two-dimensional lattice in which reactions occur locally on single sites and diffusion occurs by hopping of molecules to neighbouring sites. Conclusions If diffusion is very rapid, the system is well-mixed. The transition to life becomes increasingly difficult as the lattice size is increased because the concentration fluctuations that drive the transition become relatively smaller when larger numbers of molecules are involved. In contrast, when diffusion occurs at a finite rate, concentration fluctuations are local. The transition to life occurs in one local region and then spreads across the rest of the surface. The transition becomes easier with larger lattice sizes because there are more independent regions in which it could occur. The key observations that apply to our models and to the real world are that the origin of life is a rare stochastic event that is localized in one region of space due to the limited rate of diffusion of the molecules involved and that the subsequent spread across the surface is deterministic. It is likely that the time required for the deterministic spread is much shorter than the waiting time for the origin, in which case life evolves only once on a planet, and then rapidly occupies the whole surface. Reviewers Reviewed by Omer Markovitch (nominated by Doron Lancet), Claus Wilke, and Nobuto Takeuchi (nominated by Eugene Koonin). PMID:23176307

DNA cleavage site selection by Type III restriction enzymes provides evidence for head-on protein collisions following 1D bidirectional motion

PubMed Central

Schwarz, Friedrich W.; van Aelst, Kara; Tóth, Júlia; Seidel, Ralf; Szczelkun, Mark D.

2011-01-01

DNA cleavage by the Type III Restriction–Modification enzymes requires communication in 1D between two distant indirectly-repeated recognitions sites, yet results in non-specific dsDNA cleavage close to only one of the two sites. To test a recently proposed ATP-triggered DNA sliding model, we addressed why one site is selected over another during cleavage. We examined the relative cleavage of a pair of identical sites on DNA substrates with different distances to a free or protein blocked end, and on a DNA substrate using different relative concentrations of protein. Under these conditions a bias can be induced in the cleavage of one site over the other. Monte-Carlo simulations based on the sliding model reproduce the experimentally observed behaviour. This suggests that cleavage site selection simply reflects the dynamics of the preceding stochastic enzyme events that are consistent with bidirectional motion in 1D and DNA cleavage following head-on protein collision. PMID:21724613

Adiabatic Compression Sensitivity of AF-M315E

DTIC Science & Technology

2015-07-01

the current work is to expand the knowledge base from previous experiments completed at AFRL for AF-M315E in stainless steel U-tubes at room...addressed, to some degree, with the use of clamps and a large stainless steel plate to dissipate any major vibrations. A large preheated bath of 50:50 v/v...autocatalytic chain decomposition in the propellant. This exothermic decomposition decreases the fume -off initiation temperature of the propellant and its

Studies of Premixed Laminar and Turbulent Flames at Microgravity

NASA Technical Reports Server (NTRS)

Kwon, O. C.; Abid, M.; Porres, J.; Liu, J. B.; Ronney, P. D.; Struk, P. M.; Weiland, K. J.

2003-01-01

Several topics relating to premixed flame behavior at reduced gravity have been studied. These topics include: (1) flame balls; (2) flame structure and stability at low Lewis number; (3) experimental simulation of buoyancy effects in premixed flames using aqueous autocatalytic reactions; and (4) premixed flame propagation in Hele-Shaw cells. Because of space limitations, only topic (1) is discussed here, emphasizing results from experiments on the recent STS-107 Space Shuttle mission, along with numerical modeling efforts.

Experiment on large scale plume interaction with a stratified gas environment resembling the thermal activity of a autocatalytic recombiner

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

Mignot, G.; Kapulla, R.; Paladino, D.

Computational Fluid Dynamics codes (CFD) are increasingly being used to simulate containment conditions after various transient accident scenarios. Consequently, the reliability of such codes must be tested against experimental data. Such validation experiments related to gas mixing and hydrogen transport within containment compartments addressing the effect of heat source are presented in this paper. The experiments were conducted in the large-scale thermal-hydraulics PANDA facility located at the Paul-Scherrer-Inst. (PSI) in Switzerland, in the frame of the OECD/SETH-2 project. A 10 kW electric heater simulating the thermal activity of the autocatalytic recombiner was activated at full power in a containment vesselmore » at the top of which a thick helium layer is initially present. The hot plume interacts with the bottom of the helium layer which is slowly eroded until complete break up at 1350 s. After final erosion of the layer a strong temperature and concentration gradient is maintained in the vessel below the heater inlet as well as in the adjacent vessel below the interconnecting pipe. A detailed characterization of the operating heater suggests the presence of cold gas ingress at the outlet that affects the flow in the chimney. This can be of concern if present in a real PAR unit. (authors)« less

Modelling the degradation and elastic properties of poly(lactic-co-glycolic acid) films and regular open-cell tissue engineering scaffolds.

PubMed

Shirazi, Reyhaneh Neghabat; Ronan, William; Rochev, Yury; McHugh, Peter

2016-02-01

Scaffolding plays a critical rule in tissue engineering and an appropriate degradation rate and sufficient mechanical integrity are required during degradation and healing of tissue. This paper presents a computational investigation of the molecular weight degradation and the mechanical performance of poly(lactic-co-glycolic acid) (PLGA) films and tissue engineering scaffolds. A reaction-diffusion model which predicts the degradation behaviour is coupled with an entropy-based mechanical model which relates Young׳s modulus and the molecular weight. The model parameters are determined based on experimental data for in-vitro degradation of a PLGA film. Microstructural models of three different scaffold architectures are used to investigate the degradation and mechanical behaviour of each scaffold. Although the architecture of the scaffold does not have a significant influence on the degradation rate, it determines the initial stiffness of the scaffold. It is revealed that the size of the scaffold strut controls the degradation rate and the mechanical collapse. A critical length scale due to competition between diffusion of degradation products and autocatalytic degradation is determined to be in the range 2-100μm. Below this range, slower homogenous degradation occurs; however, for larger samples monomers are trapped inside the sample and faster autocatalytic degradation occurs. Copyright © 2015 Elsevier Ltd. All rights reserved.

Crystallization processes in Ge{sub 2}Sb{sub 2}Se{sub 4}Te glass

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

Svoboda, Roman, E-mail: roman.svoboda@upce.cz; Bezdička, Petr; Gutwirth, Jan

2015-01-15

Highlights: • Crystallization kinetics of Ge{sub 2}Sb{sub 2}Se{sub 4}Te glass was studied in dependence on particle size by DSC. • All studied fractions were described in terms of the SB autocatalytic model. • Relatively high amount of Te enhances manifestation of bulk crystallization mechanisms. • XRD analysis of samples crystallized under different conditions showed correlation with DSC data. • XRD analysis revealed a new crystallization mechanism indistinguishable by DSC. - Abstract: Differential scanning calorimetry (DSC) and X-ray diffraction (XRD) analysis were used to study crystallization in Ge{sub 2}Sb{sub 2}Se{sub 4}Te glass under non-isothermal conditions as a function of the particlemore » size. The crystallization kinetics was described in terms of the autocatalytic Šesták–Berggren model. An extensive discussion of all aspects of a full-scale kinetic study of a crystallization process was undertaken. Dominance of the crystallization process originating from mechanically induced strains and heterogeneities was confirmed. Substitution of Se by Te was found to enhance the manifestation of the bulk crystallization mechanisms (at the expense of surface crystallization). The XRD analysis showed significant dependence of the crystalline structural parameters on the crystallization conditions (initial particle size of the glassy grains and applied heating rate). Based on this information, a new microstructural crystallization mechanism, indistinguishable by DSC, was proposed.« less

Can the analyte-triggered asymmetric autocatalytic Soai reaction serve as a universal analytical tool for measuring enantiopurity and assigning absolute configuration?

PubMed

Welch, Christopher J; Zawatzky, Kerstin; Makarov, Alexey A; Fujiwara, Satoshi; Matsumoto, Arimasa; Soai, Kenso

2016-12-20

An investigation is reported on the use of the autocatalytic enantioselective Soai reaction, known to be influenced by the presence of a wide variety of chiral materials, as a generic tool for measuring the enantiopurity and absolute configuration of any substance. Good generality for the reaction across a small group of test analytes was observed, consistent with literature reports suggesting a diversity of compound types that can influence the stereochemical outcome of this reaction. Some trends in the absolute sense of stereochemical enrichment were noted, suggesting the possible utility of the approach for assigning absolute configuration to unknown compounds, by analogy to closely related species with known outcomes. Considerable variation was observed in the triggering strength of different enantiopure materials, an undesirable characteristic when dealing with mixtures containing minor impurities with strong triggering strength in the presence of major components with weak triggering strength. A strong tendency of the reaction toward an 'all or none' type of behavior makes the reaction most sensitive for detecting enantioenrichment close to zero. Consequently, the ability to discern modest from excellent enantioselectivity was relatively poor. While these properties limit the ability to obtain precise enantiopurity measurements in a simple single addition experiment, prospects may exist for more complex experimental setups that may potentially offer improved performance.

Multicolor fluorescent biosensor for multiplexed detection of DNA.

PubMed

Hu, Rong; Liu, Tao; Zhang, Xiao-Bing; Huan, Shuang-Yan; Wu, Cuichen; Fu, Ting; Tan, Weihong

2014-05-20

Development of efficient methods for highly sensitive and rapid screening of specific oligonucleotide sequences is essential to the early diagnosis of serious diseases. In this work, an aggregated cationic perylene diimide (PDI) derivative was found to efficiently quench the fluorescence emission of a variety of anionic oligonucleotide-labeled fluorophores that emit at wavelengths from the visible to NIR region. This broad-spectrum quencher was then adopted to develop a multicolor biosensor via a label-free approach for multiplexed fluorescent detection of DNA. The aggregated perylene derivative exhibits a very high quenching efficiency on all ssDNA-labeled dyes associated with biosensor detection, having efficiency values of 98.3 ± 0.9%, 97 ± 1.1%, and 98.2 ± 0.6% for FAM, TAMRA, and Cy5, respectively. An exonuclease-assisted autocatalytic target recycling amplification was also integrated into the sensing system. High quenching efficiency combined with autocatalytic target recycling amplification afforded the biosensor with high sensitivity toward target DNA, resulting in a detection limit of 20 pM, which is about 50-fold lower than that of traditional unamplified homogeneous fluorescent assay methods. The quencher did not interfere with the catalytic activity of nuclease, and the biosensor could be manipulated in either preaddition or postaddition manner with similar sensitivity. Moreover, the proposed sensing system allows for simultaneous and multicolor analysis of several oligonucleotides in homogeneous solution, demonstrating its potential application in the rapid screening of multiple biotargets.

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

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

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.

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

Chemical Amplification with Encapsulated Reagents

NASA Technical Reports Server (NTRS)

Chen, Jian; Koemer, Steffi; Craig, Stephen; Lin, Shirley; Rudkevich, Dmitry M.; Rebek, Julius, Jr.

2002-01-01

Autocatalysis and chemical amplification are characteristic properties of living systems, and they give rise to behaviors such as increased sensitivity, responsiveness, and self-replication. Here we report a synthetic system in which a unique form of compartmentalization leads to nonlinear, autocatalytic behavior. The compartment is a reversibly formed capsule in which a reagent is sequestered. Reaction products displace the reagent from the capsule into solution and the reaction rate is accelerated. The resulting self-regulation is sensitive to the highly selective molecular recognition properties of the capsule.

Discreteness effects in a reacting system of particles with finite interaction radius.

PubMed

Berti, S; López, C; Vergni, D; Vulpiani, A

2007-09-01

An autocatalytic reacting system with particles interacting at a finite distance is studied. We investigate the effects of the discrete-particle character of the model on properties like reaction rate, quenching phenomenon, and front propagation, focusing on differences with respect to the continuous case. We introduce a renormalized reaction rate depending both on the interaction radius and the particle density, and we relate it to macroscopic observables (e.g., front speed and front thickness) of the system.

Enhanced Detection of Orthogonal Radar Waveforms Using Time-Frequency and Bi-Frequency Signal Processing Techniques

DTIC Science & Technology

2008-09-01

using the principle of pheromones . The termite senses the pheromone , which tells him to gather up dirt in its mouth and deposit it where the scent of...the pheromone is the strongest. The termite gathers dirt and moves to the location of the strongest scent and deposits the mud there. As this...process is repeated by a number of termites all leaving their own contribution of pheromone at the scene, the autocatalytic effect takes over. This

Stable power laws in variable economies; Lotka-Volterra implies Pareto-Zipf

NASA Astrophysics Data System (ADS)

Solomon, S.; Richmond, P.

2002-05-01

In recent years we have found that logistic systems of the Generalized Lotka-Volterra type (GLV) describing statistical systems of auto-catalytic elements posses power law distributions of the Pareto-Zipf type. In particular, when applied to economic systems, GLV leads to power laws in the relative individual wealth distribution and in market returns. These power laws and their exponent α are invariant to arbitrary variations in the total wealth of the system and to other endogenously and exogenously induced variations.

Identification, Characterization, and Three-Dimensional Structure of the Novel Circular Bacteriocin, Enterocin NKR-5-3B, from Enterococcus faecium.

PubMed

Himeno, Kohei; Rosengren, K Johan; Inoue, Tomoko; Perez, Rodney H; Colgrave, Michelle L; Lee, Han Siean; Chan, Lai Y; Henriques, Sónia Troeira; Fujita, Koji; Ishibashi, Naoki; Zendo, Takeshi; Wilaipun, Pongtep; Nakayama, Jiro; Leelawatcharamas, Vichien; Jikuya, Hiroyuki; Craik, David J; Sonomoto, Kenji

2015-08-11

Enterocin NKR-5-3B, one of the multiple bacteriocins produced by Enterococcus faecium NKR-5-3, is a 64-amino acid novel circular bacteriocin that displays broad-spectrum antimicrobial activity. Here we report the identification, characterization, and three-dimensional nuclear magnetic resonance solution structure determination of enterocin NKR-5-3B. Enterocin NKR-5-3B is characterized by four helical segments that enclose a compact hydrophobic core, which together with its circular backbone impart high stability and structural integrity. We also report the corresponding structural gene, enkB, that encodes an 87-amino acid precursor peptide that undergoes a yet to be described enzymatic processing that involves adjacent cleavage and ligation of Leu(24) and Trp(87) to yield the mature (circular) enterocin NKR-5-3B.

Photochemical Kinetics of a Phosphine Oxide Free Radical Initiator from Femtosecond UV-Pump/Mid-IR-Probe Spectroscopy.

PubMed

Straub, Steffen; Lindner, Jörg; Vöhringer, Peter

2017-07-06

Femtosecond UV-pump/mid-infrared-probe spectroscopy was used to explore in detail the primary photochemical events of the free radical initiator, (2,4,6-trimethylbenzoyl)diphenylphosphine oxide, in liquid dichloromethane solution at room temperature. Following electronic excitation of its lowest excited singlet state, S 1 , the radical initiator undergoes an intersystem crossing to the triplet ground state, T 1 , with a time constant of 135 ps. A subsequent α-cleavage occurs from the triplet state with a time constant of 15 ps and yields a trimethylbenzoyl radical together with a diphenylphosphinoyl radical. Transient absorptions from the S 1 and T 1 states are observed that can be assigned to the P═O stretching mode and the symmetric in-plane deformation mode of the trimethylphenyl moiety of the radical initiator.

Single embryo transfer by Day 3 time-lapse selection versus Day 5 conventional morphological selection: a randomized, open-label, non-inferiority trial.

PubMed

Yang, Lanlin; Cai, Sufen; Zhang, Shuoping; Kong, Xiangyi; Gu, Yifan; Lu, Changfu; Dai, Jing; Gong, Fei; Lu, Guangxiu; Lin, Ge

2018-05-01

Does single cleavage-stage (Day 3) embryo transfer using a time-lapse (TL) hierarchical classification model achieve comparable ongoing pregnancy rates (OPR) to single blastocyst (Day 5) transfer by conventional morphological (CM) selection? Day 3 single embryo transfer (SET) with a hierarchical classification model had a significantly lower OPR compared with Day 5 SET with CM selection. Cleavage-stage SET is an alternative to blastocyst SET. Time-lapse imaging assists better embryo selection, based on studies of pregnancy outcomes when adding time-lapse imaging to CM selection at the cleavage or blastocyst stage. This single-centre, randomized, open-label, active-controlled, non-inferiority study included 600 women between October 2015 and April 2017. Eligible patients were Chinese females, aged ≤36 years, who were undergoing their first or second fresh IVF cycle using their own oocytes, and who had FSH levels ≤12 IU/mL on Day 3 of the cycle and 10 or more oocytes retrieved. Patients who had underlying uterine conditions, oocyte donation, recurrent pregnancy loss, abnormal oocytes or <6 normally fertilized embryos (2PN) were excluded from the study participation. Patients were randomized 1:1 to either the cleavage-stage SET with a time-lapse hierarchical classification model for selection (D3 + TL) or blastocyst SET with CM selection (D5 + CM). All normally fertilized zygotes were cultured in Primo Vision. The study was conducted at a tertiary IVF centre (CITIC-Xiangya) and OPR was the primary outcome. A total of 600 patients were randomized to the two groups, among which 585 (D3 + TL = 290, D5 + CM = 295) were included in the Modified-intention-to-treat (mITT) population and 517 (D3 + TL = 261, D5 + CM = 256) were included in the PP population. In the per protocol (PP) population, OPR was significantly lower in the D3 group (59.4%, 155/261) than in the D5 group (68.4%, 175/256) (difference: -9.0%, 95% CI: -17.1%, -0.7%, P = 0.03). Analysis in mITT population showed a marginally significant difference in the OPR between the D3 + TL and D5 + CM groups (56.6 versus 64.1%, difference: -7.5%, 95% CI: -15.4%, 0.4%, P = 0.06). The D3 + TL group resulted in a markedly lower implantation rate than the D5 + CM group (64.4 versus 77.0%; P = 0.002) in the PP analysis, however, the early miscarriage rate did not significantly differ between the two groups. The study lacked a direct comparison between time-lapse and CM selections at cleavage-stage SET and was statistically underpowered to detect non-inferiority. The subject's eligibility criteria favouring women with a good prognosis for IVF weakened the generalizability of the results. The OPR from Day 3 cleavage-stage SET using hierarchical classification time-lapse selection was significantly lower compared with that from Day 5 blastocyst SET using conventional morphology, yet it appeared to be clinically acceptable in women underwent IVF. This study is supported by grants from Ferring Pharmaceuticals and the Program for New Century Excellent Talents in University, China. ChiCTR-ICR-15006600. 16 June 2015. 1 October 2015.

Corticosteroid-Binding Globulin: Structure-Function Implications from Species Differences

PubMed Central

Gardill, Bernd R.; Vogl, Michael R.; Lin, Hai-Yan; Hammond, Geoffrey L.; Muller, Yves A.

2012-01-01

Corticosteroid-binding globulin (CBG) transports glucocorticoids and progesterone in the blood and thereby modulates the tissue availability of these hormones. As a member of the serine protease inhibitor (SERPIN) family, CBG displays a reactive center loop (RCL) that is targeted by proteinases. Cleavage of the RCL is thought to trigger a SERPIN-typical stressed-to-relaxed (S-to-R) transition that leads to marked structural rearrangements and a reduced steroid-binding affinity. To characterize structure-function relationships in CBG we studied various conformational states of E. coli-produced rat and human CBG. In the 2.5 Å crystal structure of human CBG in complex with progesterone, the RCL is cleaved at a novel site that differs from the known human neutrophil elastase recognition site. Although the cleaved RCL segment is five residues longer than anticipated, it becomes an integral part of β-sheet A as a result of the S-to-R transition. The atomic interactions observed between progesterone and CBG explain the lower affinity of progesterone in comparison to corticosteroids. Surprisingly, CD measurements in combination with thermal unfolding experiments show that rat CBG fails to undergo an S-to-R transition upon proteolytic cleavage of the RCL hinting that the S-to-R transition observed in human CBG is not a prerequisite for CBG function in rat. This observation cautions against drawing general conclusions about molecular mechanisms by comparing and merging structural data from different species. PMID:23300763

Effects of dimethyl sulfoxide on asymmetric division and cytokinesis in mouse oocytes

PubMed Central

2014-01-01

Background Dimethyl sulfoxide (DMSO) is used extensively as a permeable cryoprotectant and is a common solvent utilized for several water-insoluble substances. DMSO has various biological and pharmacological activities; however, the effect of DMSO on mouse oocyte meiotic maturation remains unknown. Results In DMSO-treated oocytes, we observed abnormal MII oocytes that contained large polar bodies, including 2-cell–like MII oocytes, during in vitro maturation. Oocyte polarization did not occur, due to the absence of actin cap formation and spindle migration. These features are among the primary causes of abnormal symmetric division; however, analysis of the mRNA expression levels of genes related to asymmetric division revealed no significant difference in the expression of these factors between the 3% DMSO-treated group and the control group. After each “blastomere” of the 2-cell–like MII stage oocytes was injected by one sperm head respectively, the oocytes still possessed the ability to extrude the second polar body from each “blastomere” and to begin cleavage. However, MII oocytes with large polar bodies developed to the blastocyst stage after intracytoplasmic sperm injection (ICSI). Furthermore, other permeable cryoprotectants, such as ethylene glycol and glycerol, also caused asymmetric division failure. Conclusion Permeable cryoprotectants, such as DMSO, ethylene glycol, and glycerol, affect asymmetric division. DMSO disrupts cytokinesis completion by inhibiting cortical reorganization and polarization. Oocytes that undergo symmetric division maintain the ability to begin cleavage after ICSI. PMID:24953160

Elucidation of the trigonelline degradation pathway reveals previously undescribed enzymes and metabolites.

PubMed

Perchat, Nadia; Saaidi, Pierre-Loïc; Darii, Ekaterina; Pellé, Christine; Petit, Jean-Louis; Besnard-Gonnet, Marielle; de Berardinis, Véronique; Dupont, Maeva; Gimbernat, Alexandra; Salanoubat, Marcel; Fischer, Cécile; Perret, Alain

2018-05-08

Trigonelline (TG; N- methylnicotinate) is a ubiquitous osmolyte. Although it is known that it can be degraded, the enzymes and metabolites have not been described so far. In this work, we challenged the laboratory model soil-borne, gram-negative bacterium Acinetobacter baylyi ADP1 (ADP1) for its ability to grow on TG and we identified a cluster of catabolic, transporter, and regulatory genes. We dissected the pathway to the level of enzymes and metabolites, and proceeded to in vitro reconstruction of the complete pathway by six purified proteins. The four enzymatic steps that lead from TG to methylamine and succinate are described, and the structures of previously undescribed metabolites are provided. Unlike many aromatic compounds that undergo hydroxylation prior to ring cleavage, the first step of TG catabolism proceeds through direct cleavage of the C5-C6 bound, catalyzed by a flavin-dependent, two-component oxygenase, which yields ( Z )-2-(( N- methylformamido)methylene)-5-hydroxy-butyrolactone (MFMB). MFMB is then oxidized into ( E )-2-(( N- methylformamido) methylene) succinate (MFMS), which is split up by a hydrolase into carbon dioxide, methylamine, formic acid, and succinate semialdehyde (SSA). SSA eventually fuels up the TCA by means of an SSA dehydrogenase, assisted by a Conserved Hypothetical Protein. The cluster is conserved across marine, soil, and plant-associated bacteria. This emphasizes the role of TG as a ubiquitous nutrient for which an efficient microbial catabolic toolbox is available.

Microstructural study of a nitroxide-mediated poly(ethylene oxide)/polystyrene block copolymer (PEO-b-PS) by electrospray tandem mass spectrometry.

PubMed

Girod, Marion; Phan, Trang N T; Charles, Laurence

2008-08-01

Electrospray ionization tandem mass spectrometry has been used to characterize the microstructure of a nitroxide-mediated poly(ethylene oxide)/polystyrene block copolymer, called SG1-capped PEO-b-PS. The main dissociation route of co-oligomers adducted with lithium or silver cation was observed to proceed via the homolytic cleavage of a C-ON bond, aimed at undergoing reversible homolysis during nitroxide mediated polymerization. This cleavage results in the elimination of the terminal SG1 end-group as a radical, inducing a complete depolymerization process of the PS block from the so-formed radical cation. These successive eliminations of styrene molecules allowed a straightforward determination of the PS block size. An alternative fragmentation pathway of the radical cation was shown to provide structural information on the junction group between the two blocks. Proposed dissociation mechanisms were supported by accurate mass measurements. Structural information on the SG1 end-group could be reached from weak abundance fragment ions detected in the low m/z range of the MS/MS spectrum. Amongst fragments typically expected from PS dissociation, only beta ions were produced. Moreover, specific dissociation of the PEO block was not observed to occur in MS/MS, suggesting that these rearrangement reactions do not compete effectively with dissociations of the odd-electron fragment ions. Information about the PEO block length and the initiated end-group were obtained in MS(3) experiments.

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.

Identification of the protease cleavage sites in a reconstituted Gag polyprotein of an HERV-K(HML-2) element

PubMed Central

2011-01-01

Background The human genome harbors several largely preserved HERV-K(HML-2) elements. Although this retroviral family comes closest of all known HERVs to producing replication competent virions, mutations acquired during their chromosomal residence have rendered them incapable of expressing infectious particles. This also holds true for the HERV-K113 element that has conserved open reading frames (ORFs) for all its proteins in addition to a functional LTR promoter. Uncertainty concerning the localization and impact of post-insertional mutations has greatly hampered the functional characterization of these ancient retroviruses and their proteins. However, analogous to other betaretroviruses, it is known that HERV-K(HML-2) virions undergo a maturation process during or shortly after release from the host cell. During this process, the subdomains of the Gag polyproteins are released by proteolytic cleavage, although the nature of the mature HERV-K(HML-2) Gag proteins and the exact position of the cleavage sites have until now remained unknown. Results By aligning the amino acid sequences encoded by the gag-pro-pol ORFs of HERV-K113 with the corresponding segments from 10 other well-preserved human specific elements we identified non-synonymous post-insertional mutations that have occurred in this region of the provirus. Reversion of these mutations and a partial codon optimization facilitated the large-scale production of maturation-competent HERV-K113 virus-like particles (VLPs). The Gag subdomains of purified mature VLPs were separated by reversed-phase high-pressure liquid chromatography and initially characterized using specific antibodies. Cleavage sites were identified by mass spectrometry and N-terminal sequencing and confirmed by mutagenesis. Our results indicate that the gag gene product Pr74Gag of HERV-K(HML-2) is processed to yield p15-MA (matrix), SP1 (spacer peptide of 14 amino acids), p15, p27-CA (capsid), p10-NC (nucleocapsid) and two C-terminally encoded glutamine- and proline-rich peptides, QP1 and QP2, spanning 23 and 19 amino acids, respectively. Conclusions Expression of reconstituted sequences of original HERV elements is an important tool for studying fundamental aspects of the biology of these ancient viruses. The analysis of HERV-K(HML-2) Gag processing and the nature of the mature Gag proteins presented here will facilitate further studies of the discrete functions of these proteins and of their potential impact on the human host. PMID:21554716

Group 4 Metalloporphyrin diolato Complexes and Catalytic Application of Metalloporphyrins and Related Transition Metal Complexes

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

Du, Guodong

In this work, the first examples of group 4 metalloporphyrin 1,2-diolato complexes were synthesized through a number of strategies. In general, treatment of imido metalloporphyrin complexes, (TTP)M=NR, (M = Ti, Zr, Hf), with vicinal diols led to the formation of a series of diolato complexes. Alternatively, the chelating pinacolate complexes could be prepared by metathesis of (TTP)MCl 2 (M = Ti, Hf) with disodium pinacolate. These complexes were found to undergo C-C cleavage reactions to produce organic carbonyl compounds. For titanium porphyrins, treatment of a titanium(II) alkyne adduct, (TTP)Ti(η 2-PhC≡CPh), with aromatic aldehydes or aryl ketones resulted in reductive couplingmore » of the carbonyl groups to produce the corresponding diolato complexes. Aliphatic aldehydes or ketones were not reactive towards (TTP)Ti(η 2-PhC≡CPh). However, these carbonyl compounds could be incorporated into a diolato complex on reaction with a reactive precursor, (TTP)Ti[O(Ph) 2C(Ph) 2O] to provide unsymmetrical diolato complexes via cross coupling reactions. In addition, an enediolato complex (TTP)Ti(OCPhCPhO) was obtained from the reaction of (TTP)Ti(η 2-PhC≡CPh) with benzoin. Titanium porphyrin diolato complexes were found to be intermediates in the (TTP)Ti=O-catalyzed cleavage reactions of vicinal diols, in which atmospheric oxygen was the oxidant. Furthermore, (TTP)Ti=O was capable of catalyzing the oxidation of benzyl alcohol and α-hydroxy ketones to benzaldehyde and α-diketones, respectively. Other high valent metalloporphyrin complexes also can catalyze the oxidative diol cleavage and the benzyl alcohol oxidation reactions with dioxygen. A comparison of Ti(IV) and Sn(IV) porphyrin chemistry was undertaken. While chelated diolato complexes were invariably obtained for titanium porphyrins on treatment with 1,2-diols, the reaction of vicinal diols with tin porphyrins gave a number of products, including mono-, bis-alkoxo, and chelating diolato complexes, depending on the identity of diols and the stoichiometry employed. It was also found that tin porphyrin complexes promoted the oxidative cleavage of vicinal diols and the oxidation of α-ketols to α-diketones with dioxygen. In extending the chemistry of metalloporphyrins and analogous complexes, a series of chiral tetraaza macrocyclic ligands and metal complexes were designed and synthesized. Examination of iron(II) complexes showed that they were efficient catalysts for the cyclopropanation of styrene by diazo reagents. Good yields and high diastereoselectivity were obtained with modest enantioselectivity. A rationalization of the stereoselectivity was presented on the basis of structural factors in a carbene intermediate.« less

Identification of the protease cleavage sites in a reconstituted Gag polyprotein of an HERV-K(HML-2) element.

PubMed

George, Maja; Schwecke, Torsten; Beimforde, Nadine; Hohn, Oliver; Chudak, Claudia; Zimmermann, Anja; Kurth, Reinhard; Naumann, Dieter; Bannert, Norbert

2011-05-09

The human genome harbors several largely preserved HERV-K(HML-2) elements. Although this retroviral family comes closest of all known HERVs to producing replication competent virions, mutations acquired during their chromosomal residence have rendered them incapable of expressing infectious particles. This also holds true for the HERV-K113 element that has conserved open reading frames (ORFs) for all its proteins in addition to a functional LTR promoter. Uncertainty concerning the localization and impact of post-insertional mutations has greatly hampered the functional characterization of these ancient retroviruses and their proteins. However, analogous to other betaretroviruses, it is known that HERV-K(HML-2) virions undergo a maturation process during or shortly after release from the host cell. During this process, the subdomains of the Gag polyproteins are released by proteolytic cleavage, although the nature of the mature HERV-K(HML-2) Gag proteins and the exact position of the cleavage sites have until now remained unknown. By aligning the amino acid sequences encoded by the gag-pro-pol ORFs of HERV-K113 with the corresponding segments from 10 other well-preserved human specific elements we identified non-synonymous post-insertional mutations that have occurred in this region of the provirus. Reversion of these mutations and a partial codon optimization facilitated the large-scale production of maturation-competent HERV-K113 virus-like particles (VLPs). The Gag subdomains of purified mature VLPs were separated by reversed-phase high-pressure liquid chromatography and initially characterized using specific antibodies. Cleavage sites were identified by mass spectrometry and N-terminal sequencing and confirmed by mutagenesis. Our results indicate that the gag gene product Pr74Gag of HERV-K(HML-2) is processed to yield p15-MA (matrix), SP1 (spacer peptide of 14 amino acids), p15, p27-CA (capsid), p10-NC (nucleocapsid) and two C-terminally encoded glutamine- and proline-rich peptides, QP1 and QP2, spanning 23 and 19 amino acids, respectively. Expression of reconstituted sequences of original HERV elements is an important tool for studying fundamental aspects of the biology of these ancient viruses. The analysis of HERV-K(HML-2) Gag processing and the nature of the mature Gag proteins presented here will facilitate further studies of the discrete functions of these proteins and of their potential impact on the human host.

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.

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.

Bond cleavage of lignin model compounds into aromatic monomers using supported metal catalysts in supercritical water

PubMed Central

Yamaguchi, Aritomo; Mimura, Naoki; Shirai, Masayuki; Sato, Osamu

2017-01-01

More efficient use of lignin carbon is necessary for carbon-efficient utilization of lignocellulosic biomass. Conversion of lignin into valuable aromatic compounds requires the cleavage of C–O ether bonds and C–C bonds between lignin monomer units. The catalytic cleavage of C–O bonds is still challenging, and cleavage of C–C bonds is even more difficult. Here, we report cleavage of the aromatic C–O bonds in lignin model compounds using supported metal catalysts in supercritical water without adding hydrogen gas and without causing hydrogenation of the aromatic rings. The cleavage of the C–C bond in bibenzyl was also achieved with Rh/C as a catalyst. Use of this technique may greatly facilitate the conversion of lignin into valuable aromatic compounds. PMID:28387304

Embryonic expression of festina lente (fel), a novel maternal gene, in the oligochaete annelid Tubifex tubifex.

PubMed

Nakamura, Takuma; Shiomi, Inori; Shimizu, Takashi

2017-11-01

We have cloned and characterized the expression of a novel maternal gene festina lente (designated Ttu-fel) from the clitellate annelid Tubifex tubifex. Northern blot analyses have shown that Ttu-fel mRNA is approximately 8 kbp in length and that its expression is restricted to oocytes undergoing maturation division and early embryos up to 22-cell stage. Maternal transcripts of Ttu-fel are first detected in oocytes in the ovary of young adults (ca. 40 days after hatching); its expression continues in growing oocytes in the ovisac. Ttu-fel mRNA is distributed broadly throughout the egg undergoing maturation divisions. During the process of ooplasmic segregation that results in the pole plasm formation, Ttu-fel mRNA becomes concentrated to the animal and vegetal poles. The RNA in the animal hemisphere is distributed in a gradient with highest concentration in the cortical region. During the first two cleavages, Ttu-fel mRNA is segregated to CD cell then to D cell; it is subsequently inherited by the three D quadtrant micromeres, 1d, 2d and 3d. Around the time of transition to 22-cell stage, Ttu-fel mRNA becomes undetectable throughout the embryo. Copyright © 2017 Elsevier B.V. All rights reserved.

Direct Functionalization of an Acid-Terminated Nanodiamond with Azide: Enabling Access to 4-Substituted-1,2,3-Triazole-Functionalized Particles

DOE PAGES

Kennedy, Zachary C.; Barrett, Christopher A.; Warner, Marvin G.

2017-03-01

Azides on the periphery of nanodiamond materials (ND) are of great utility because they have been shown to undergo Cu-catalyzed and Cu-free cycloaddition reactions with structurally diverse alkynes, affording particles tailored for applications in biology and materials science. However, current methods employed to access ND featuring azide groups typically require either harsh pretreatment procedures or multiple synthesis steps and use surface linking groups that may be susceptible to undesirable cleavage. Here in this paper we demonstrate an alternative single-step approach to producing linker-free, azide-functionalized ND. Our method was applied to low-cost, detonation-derived ND powders where surface carbonyl groups undergo silver-mediatedmore » decarboxylation and radical substitution with azide. ND with directly grafted azide groups were then treated with a variety of aliphatic, aromatic, and fluorescent alkynes to afford 1-(ND)-4-substituted-1,2,3-triazole materials under standard copper-catalyzed cycloaddition conditions. Surface modification steps were verified by characteristic infrared absorptions and elemental analyses. High loadings of triazole surface groups (up to 0.85 mmol g –1) were obtained as determined from thermogravimetric analysis. The azidation procedure disclosed is envisioned to become a valuable initial transformation in numerous future applications of ND.« less

Kinetics and mechanism for the sonochemical degradation of a nonionic surfactant.

PubMed

Singla, Ritu; Grieser, Franz; Ashokkumar, Muthupandian

2009-03-26

The sonolytic degradation of the nonionic surfactant, octaethylene glycol monododecyl ether (C(12)E(8)), has been studied at various initial concentrations below and above its critical micelle concentration (CMC). It has been observed that the degradation rate increases with an increase in the initial concentration of the surfactant until the CMC is reached. Above the CMC an almost constant degradation rate is observed, suggesting that the surfactant in its monomer form is involved in the degradation process. The degradation process of C(12)E(8) involves two distinct primary processes occurring at the bubble/solution interface: (a) hydroxylation/oxidation of the surfactant and (b) pyrolytic fragmentation of the surfactant. The oxidative cleavage of ethylene oxide units provides evidence for OH radical attack. Hydroxylation of the ethoxy chain gives rise to various short-chain carboxyalkyl-polyethylene glycol intermediates. The polyethylene glycol chain formed, due to the scission of the C(12)E(8) molecule, undergoes rapid hydroxylation/oxidation to yield simple compounds that have the potential to undergo further degradation. The detection of multiple intermediates indicates that several processes affect the complete degradation pathways of the surfactant molecule. TOC analysis, however, indicates that the sonolytic mineralization of the surfactant is difficult to achieve at reasonable rates due to the relatively low surface activity of the degradation products formed during sonolysis.

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.

Does Cleavage Work at Work? Men, but Not Women, Falsely Believe Cleavage Sells a Weak Product

ERIC Educational Resources Information Center

Glick, Peter; Chrislock, Karyna; Petersik, Korinne; Vijay, Madhuri; Turek, Aleksandra

2008-01-01

We examined whether men, but not women, would be distracted by a female sales representative's exposed cleavage, leading to greater perceived efficacy for a weak, but not for a strong product. A community sample of 88 men and 97 women viewed a video of a female pharmaceutical sales representative who (a) had exposed cleavage or dressed modestly…

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

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.

PrP(C) homodimerization stimulates the production of PrPC cleaved fragments PrPN1 and PrPC1.

PubMed

Béland, Maxime; Motard, Julie; Barbarin, Alice; Roucou, Xavier

2012-09-19

An endoproteolytic cleavage termed α-cleavage between residues 111/112 is a characteristic feature of the cellular prion protein (PrP(C)). This cleavage generates a soluble N-terminal fragment (PrPN1) and a glycosylphosphatidylinositol-anchored C-terminal fragment (PrPC1). Independent studies demonstrate that modulating PrP(C) α-cleavage represents a potential therapeutic strategy in prion diseases. The regulation of PrP(C) α-cleavage is unclear. The only known domain that is essential for the α-cleavage to occur is a hydrophobic domain (HD). Importantly, the HD is also essential for the formation of PrP(C) homodimers. To explore the role of PrP(C) homodimerization on the α-cleavage, we used a well described inducible dimerization strategy whereby a chimeric PrP(C) composed of a modified FK506-binding protein (Fv) fused with PrP(C) and termed Fv-PrP is incubated in the presence of a dimerizer AP20187 ligand. We show that homodimerization leads to a considerable increase of PrP(C) α-cleavage in cultured cells and release of PrPN1 and PrPC1. Interestingly, enforced homodimerization increased PrP(C) levels at the plasma membrane, and preventing PrP(C) trafficking to the cell surface inhibited dimerization-induced α-cleavage. These observations were confirmed in primary hippocampal neurons from transgenic mice expressing Fv-PrP. The proteases responsible for the α-cleavage are still elusive, and in contrast to initial studies we confirm more recent investigations that neither ADAM10 nor ADAM17 are involved. Importantly, PrPN1 produced after PrP(C) homodimerization protects against toxic amyloid-β (Aβ) oligomers. Thus, our results show that PrP(C) homodimerization is an important regulator of PrP(C) α-cleavage and may represent a potential therapeutic avenue against Aβ toxicity in Alzheimer's disease.

Functional analysis of coordinated cleavage in V(D)J recombination.

PubMed

Kim, D R; Oettinger, M A

1998-08-01

V(D)J recombination in vivo requires a pair of signals with distinct spacer elements of 12 and 23 bp that separate conserved heptamer and nonamer motifs. Cleavage in vitro by the RAG1 and RAG2 proteins can occur at individual signals when the reaction buffer contains Mn2+, but cleavage is restricted to substrates containing two signals when Mg2+ is the divalent cation. By using a novel V(D)J cleavage substrate, we show that while the RAG proteins alone establish a moderate preference for a 12/23 pair versus a 12/12 pair, a much stricter dependence of cleavage on the 12/23 signal pair is produced by the inclusion of HMG1 and competitor double-stranded DNA. The competitor DNA serves to inhibit the cleavage of substrates carrying a 12/12 or 23/23 pair, as well as the cutting at individual signals in 12/23 substrates. We show that a 23/33 pair is more efficiently recombined than a 12/33 pair, suggesting that the 12/23 rule can be generalized to a requirement for spacers that differ from each other by a single helical turn. Furthermore, we suggest that a fixed spatial orientation of signals is required for cleavage. In general, the same signal variants that can be cleaved singly can function under conditions in which a signal pair is required. However, a chemically modified substrate with one noncleavable signal enables us to show that formation of a functional cleavage complex is mechanistically separable from the cleavage reaction itself and that although cleavage requires a pair of signals, cutting does not have to occur simultaneously at both. The implications of these results are discussed with respect to the mechanism of V(D)J recombination and the generation of chromosomal translocations.

Hairpin ribozyme cleavage catalyzed by aminoglycoside antibiotics and the polyamine spermine in the absence of metal ions.

PubMed Central

Earnshaw, D J; Gait, M J

1998-01-01

The hairpin ribozyme is a small catalytic RNA that achieves an active configuration by docking of its two helical domains in an antiparallel fashion. Both docking and subsequent cleavage are dependent on the presence of divalent metal ions, such as magnesium, but there is no evidence to date for direct participation of such ions in the chemical cleavage step. We show that aminoglycoside antibiotics inhibit cleavage of the hairpin ribozyme in the presence of metal ions with the most effective being 5-epi-sisomicin and neomycin B. In contrast, in the absence of metal ions, a number of aminoglycoside antibiotics at 10 mM concentration promote hairpin cleavage with rates only 13-20-fold lower than the magnesium-dependent reaction. We show that neomycin B competes with metal ions by ion replacement with the postively charged amino groups of the antibiotic. In addition, we show that the polyamine spermine at 10 mM promotes efficient hairpin cleavage with rates similar to the magnesium-dependent reaction. Low concentrations of either spermine or the shorter polyamine spermidine synergize with 5 mM magnesium ions to boost cleavage rates considerably. In contrast, at 500 microM magnesium ions, 4 mM spermine, but not spermidine, boosts the cleavage rate. The results have significance both in understanding the role of ions in hairpin ribozyme cleavage and in potential therapeutic applications in mammalian cells. PMID:9837982

Constitutive α- and β-secretase cleavages of the amyloid precursor protein are partially coupled in neurons, but not in frequently used cell lines.

PubMed

Colombo, Alessio; Wang, Huanhuan; Kuhn, Peer-Hendrik; Page, Richard; Kremmer, Elisabeth; Dempsey, Peter J; Crawford, Howard C; Lichtenthaler, Stefan F

2013-01-01

Proteolytic cleavage of the amyloid precursor protein (APP) by the two proteases α- and β-secretases controls the generation of the amyloid β peptide (Aβ), a key player in Alzheimer's disease pathogenesis. The α-secretase ADAM10 and the β-secretase BACE1 have opposite effects on Aβ generation and are assumed to compete for APP as a substrate, such that their cleavages are inversely coupled. This concept was mainly demonstrated in studies using activation or overexpression of α- and β-secretases. Here, we report that this inverse coupling is not seen to the same extent upon inhibition of the endogenous proteases. Genetic and pharmacological inhibition of ADAM10 and BACE1 revealed that the endogenous, constitutive α-secretase cleavage of APP is largely uncoupled from β-secretase cleavage and Aβ generation in neuroglioma H4 cells and in neuronally differentiated SH-SY5Y cells. In contrast, inverse coupling was observed in primary cortical neurons. However, this coupling was not bidirectional. Inhibition of BACE1 increased ADAM10 cleavage of APP, but a reduction of ADAM10 activity did not increase the BACE1 cleavage of APP in the neurons. Our analysis shows that the inverse coupling of the endogenous α- and β-secretase cleavages depends on the cellular model and suggests that a reduction of ADAM10 activity is unlikely to increase the AD risk through increased β-secretase cleavage. Copyright © 2012 Elsevier Inc. All rights reserved.

Specific Cleavage of the Nucleoprotein of Fish Rhabdovirus.

PubMed

Zhou, G-Z; Yi, Y-J; Chen, Z-Y; Zhang, Q-Y

2015-11-01

Siniperca chuatsi rhabdovirus (SCRV) is one of myriad rhabdoviruses recorded in fish. Preliminary data show that inhibition of the SCRV nucleoprotein (N) could significantly reduce the progeny virus titers in infected Epithelioma papulosum cyprinid (EPC) cells. Here, the authors propose that cleavage of the viral 47-kDa N protein is caspase-mediated based on caspase inhibition experiments, transient expression in EPC transfection, and analysis of cleavage sites. Cleavage of the SCRV N protein in culture was prevented by a pan-caspase inhibitor, z-VAD-FMK (z-Val-Ala-DL-Asp-fluoromethyl ketone). Subsequently, N was transiently expressed in EPC cells, the results of which indicated that the specific cleavage of N also occurred in the cells transfected with N-GFP plasmid. Several truncated fragments of the N gene were constructed and transiently transfected into EPC cells. Immunoblotting results indicated that D324 and D374 are the cleavage sites of N by caspases. The authors also found that z-VAD-FMK could inhibit the cytopathic effect in SCRV-infected EPC cells but not affect the production of infectious progeny, suggesting that the caspase-mediated cleavage of N protein is not required for in vitro SCRV replication. To the authors' knowledge, this is the first report on the cleavage of rhabdovirus proteins. © The Author(s) 2015.

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

Quantification of DNA cleavage specificity in Hi-C experiments.

PubMed

Meluzzi, Dario; Arya, Gaurav

2016-01-08

Hi-C experiments produce large numbers of DNA sequence read pairs that are typically analyzed to deduce genomewide interactions between arbitrary loci. A key step in these experiments is the cleavage of cross-linked chromatin with a restriction endonuclease. Although this cleavage should happen specifically at the enzyme's recognition sequence, an unknown proportion of cleavage events may involve other sequences, owing to the enzyme's star activity or to random DNA breakage. A quantitative estimation of these non-specific cleavages may enable simulating realistic Hi-C read pairs for validation of downstream analyses, monitoring the reproducibility of experimental conditions and investigating biophysical properties that correlate with DNA cleavage patterns. Here we describe a computational method for analyzing Hi-C read pairs to estimate the fractions of cleavages at different possible targets. The method relies on expressing an observed local target distribution downstream of aligned reads as a linear combination of known conditional local target distributions. We validated this method using Hi-C read pairs obtained by computer simulation. Application of the method to experimental Hi-C datasets from murine cells revealed interesting similarities and differences in patterns of cleavage across the various experiments considered. © The Author(s) 2015. Published by Oxford University Press on behalf of Nucleic Acids Research.

Sequence specificity of the hammerhead ribozyme revisited; the NHH rule.

PubMed Central

Kore, A R; Vaish, N K; Kutzke, U; Eckstein, F

1998-01-01

The sequence specificity of hammerhead ribozyme cleavage has been re-evaluated with respect to the NUH rule. Contrary to previous reports it was found that substrates with GAC triplets were also cleaved. This was established in three different sequence contexts. The rate of cleavage under single turnover conditions was between 3 and 7% that of cleavage 3' of GUC. Specificity of cleavage of substrates containing a central A in the cleavable triplet can be described as NAH, where N can be any nucleotide and H any nucleotide but G. As cleavage 3' of NCH triplets has recently been described, the NUH rule can be reformulated to NHH. PMID:9722629

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.

Resolution of model Holliday junctions by yeast endonuclease: effect of DNA structure and sequence.

PubMed Central

Parsons, C A; Murchie, A I; Lilley, D M; West, S C

1989-01-01

The resolution of Holliday junctions in DNA involves specific cleavage at or close to the site of the junction. A nuclease from Saccharomyces cerevisiae cleaves model Holliday junctions in vitro by the introduction of nicks in regions of duplex DNA adjacent to the crossover point. In previous studies [Parsons and West (1988) Cell, 52, 621-629] it was shown that cleavage occurred within homologous arm sequences with precise symmetry across the junction. In contrast, junctions with heterologous arm sequences were cleaved asymmetrically. In this work, we have studied the effect of sequence changes and base modification upon the site of cleavage. It is shown that the specificity of cleavage is unchanged providing that perfect homology is maintained between opposing arm sequences. However, in the absence of homology, cleavage depends upon sequence context and is affected by minor changes such as base modification. These data support the proposed mechanism for cleavage of a Holliday junction, which requires homologous alignment of arm sequences in an enzyme--DNA complex as a prerequisite for symmetrical cleavage by the yeast endonuclease. Images PMID:2653810

Why Nature Uses Radical SAM Enzymes so Widely: Electron Nuclear Double Resonance Studies of Lysine 2,3-Aminomutase Show the 5'-dAdo• "Free Radical" Is Never Free.

PubMed

Horitani, Masaki; Byer, Amanda S; Shisler, Krista A; Chandra, Tilak; Broderick, Joan B; Hoffman, Brian M

2015-06-10

Lysine 2,3-aminomutase (LAM) is a radical S-adenosyl-L-methionine (SAM) enzyme and, like other members of this superfamily, LAM utilizes radical-generating machinery comprising SAM anchored to the unique Fe of a [4Fe-4S] cluster via a classical five-membered N,O chelate ring. Catalysis is initiated by reductive cleavage of the SAM S-C5' bond, which creates the highly reactive 5'-deoxyadenosyl radical (5'-dAdo•), the same radical generated by homolytic Co-C bond cleavage in B12 radical enzymes. The SAM surrogate S-3',4'-anhydroadenosyl-L-methionine (anSAM) can replace SAM as a cofactor in the isomerization of L-α-lysine to L-β-lysine by LAM, via the stable allylic anhydroadenosyl radical (anAdo•). Here electron nuclear double resonance (ENDOR) spectroscopy of the anAdo• radical in the presence of (13)C, (2)H, and (15)N-labeled lysine completes the picture of how the active site of LAM from Clostridium subterminale SB4 "tames" the 5'-dAdo• radical, preventing it from carrying out harmful side reactions: this "free radical" in LAM is never free. The low steric demands of the radical-generating [4Fe-4S]/SAM construct allow the substrate target to bind adjacent to the S-C5' bond, thereby enabling the 5'-dAdo• radical created by cleavage of this bond to react with its partners by undergoing small motions, ∼0.6 Å toward the target and ∼1.5 Å overall, that are controlled by tight van der Waals contact with its partners. We suggest that the accessibility to substrate and ready control of the reactive C5' radical, with "van der Waals control" of small motions throughout the catalytic cycle, is common within the radical SAM enzyme superfamily and is a major reason why these enzymes are the preferred means of initiating radical reactions in nature.

Why Nature Uses Radical SAM Enzymes so Widely: Electron Nuclear Double Resonance Studies of Lysine 2,3-Aminomutase Show the 5′-dAdo• “Free Radical” Is Never Free

PubMed Central

Horitani, Masaki; Byer, Amanda S.; Shisler, Krista A.; Chandra, Tilak; Broderick, Joan B.; Hoffman, Brian M.

2015-01-01

Lysine 2,3-aminomutase (LAM) is a radical S-adenosyl-L-methionine (SAM) enzyme and, like other members of this superfamily, LAM utilizes radical-generating machinery comprising SAM anchored to the unique Fe of a [4Fe-4S] cluster via a classical five-membered N,O chelate ring. Catalysis is initiated by reductive cleavage of the SAM S–C5′ bond, which creates the highly reactive 5′-deoxyadenosyl radical (5′-dAdo•), the same radical generated by homolytic Co–C bond cleavage in B12 radical enzymes. The SAM surrogate S-3′,4′-anhydroadenosyl-L-methionine (anSAM) can replace SAM as a cofactor in the isomerization of L-α-lysine to L-β-lysine by LAM, via the stable allylic anhydroadenosyl radical (anAdo•). Here electron nuclear double resonance (ENDOR) spectroscopy of the anAdo• radical in the presence of 13C, 2H, and 15N-labeled lysine completes the picture of how the active site of LAM from Clostridium subterminale SB4 “tames” the 5′-dAdo• radical, preventing it from carrying out harmful side reactions: this “free radical” in LAM is never free. The low steric demands of the radical-generating [4Fe-4S]/SAM construct allow the substrate target to bind adjacent to the S–C5′ bond, thereby enabling the 5′-dAdo• radical created by cleavage of this bond to react with its partners by undergoing small motions, ~0.6 Å toward the target and ~1.5 Å overall, that are controlled by tight van der Waals contact with its partners. We suggest that the accessibility to substrate and ready control of the reactive C5′ radical, with “van der Waals control” of small motions throughout the catalytic cycle, is common within the radical SAM enzyme superfamily and is a major reason why these enzymes are the preferred means of initiating radical reactions in nature. PMID:25923449

[Correlation between IVF outcomes and Ureaplasma urealyticum infection in male reproductive tract].

PubMed

Fan, Yu-Ping; Pan, Jia-Ping; Hu, Ye; Huang, Wen-Qiang; Wang, Yu; Ruan, Jing-Ling; Li, Yun; Teng, Xiao-Ming

2014-01-01

To investigate the influence of Ureaplasma urealyticum (Uu) infection in the male reproductive tract on the outcomes of IVF and the clinical significance of preoperative Uu test by analyzing the correlation between the results of Uu culture before IVF-ET and the outcomes of IVF-ET. Among 1,059 couples undergoing IVF-ET, we selected 973 after excluding genetic factors and divided them into a Uu negative and a Uu positive group according to the results of culture of Uu in the semen of the males. We compared the rates of IVF fertilization, oocyte cleavage, clinical pregnancy and abortion between the two groups, and analyzed the influence of Uu infection on IVF outcomes. Among the 973 selected subjects, 836 were Uu negative (group A) and 137 Uu positive (group B), and of the latter, 130 were restored to Uu negative after treatment (group B1) and the other 7 remained unchanged (group B2). No significant differences were found between groups A and B in the rates of IVF fertilization (81.6% vs 79.8%, P = 0.13), abnormal fertilization (11.8% vs 12.4%, P = 0.58) and oocyte cleavage (92.0% vs 92.1%, P = 0.94), nor between groups A and B2 (81.6% vs 89.8%, P = 0.10; 11.8% vs 13.2%, P = 0.75; 92.0% vs 92.5%, P = 0.10). Totally, 747 of the patients underwent embryo transfer, including 643 in group A and 104 in group B. There were no significant differences between groups A and B in the rates of clinical pregnancy (38.6% vs 34.7%, P = 0.44) and abortion (16.5% vs 22.2%, P = 0.39), nor between groups A and B2 (38.6% vs 33.3%, P = 0.79; 16.5% vs 0, P = 0.53). Uu infection in the male reproductive tract does not significantly affect the rates of IVF fertilization, oocyte cleavage, clinical pregnancy and abortion. However, more investigations with larger sample sizes of the cases restored from Uu positive to Uu negative are needed to lend further support to our findings.

Water-Soluble Fe(II)−H2O Complex with a Weak O−H Bond Transfers a Hydrogen Atom via an Observable Monomeric Fe(III)−OH

PubMed Central

Brines, Lisa M.; Coggins, Michael K.; Poon, Penny Chaau Yan; Toledo, Santiago; Kaminsky, Werner; Kirk, Martin L.

2015-01-01

Understanding the metal ion properties that favor O−H bond formation versus cleavage should facilitate the development of catalysts tailored to promote a specific reaction, e.g., C−H activation or H2O oxidation. The first step in H2O oxidation involves the endothermic cleavage of a strong O−H bond (BDFE = 122.7 kcal/mol), promoted by binding the H2O to a metal ion, and by coupling electron transfer to proton transfer (PCET). This study focuses on details regarding how a metal ion’s electronic structure and ligand environment can tune the energetics of M(HO−H) bond cleavage. The synthesis and characterization of an Fe(II)−H2O complex, 1, that undergoes PCET in H2O to afford a rare example of a monomeric Fe(III)−OH, 7, is described. High-spin 7 is also reproducibly generated via the addition of H2O to {[FeIII(OMe2N4(tren))]2-(µ-O)}2+ (8). The O−H bond BDFE of Fe(II)−H2O (1) (68.6 kcal/mol) is calculated using linear fits to its Pourbaix diagram and shown to be 54.1 kcal/mol less than that of H2O and 10.9 kcal/mol less than that of [Fe(II)(H2O)6]2+. The O−H bond of 1 is noticeably weaker than the majority of reported Mn+(HxO−H) (M = Mn, Fe; n+ = 2+, 3+; x = 0, 1) complexes. Consistent with their relative BDFEs, Fe(II)−H2O (1) is found to donate a H atom to TEMPO•, whereas the majority of previously reported Mn+−O(H) complexes, including [MnIII(SMe2N4(tren))(OH)]+ (2), have been shown to abstract H atoms from TEMPOH. Factors responsible for the weaker O−H bond of 1, such as differences in the electron-donating properties of the ligand, metal ion Lewis acidity, and electronic structure, are discussed. PMID:25611075

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.

Identification of Proteolytic Cleavage Sites of EphA2 by Membrane Type 1 Matrix Metalloproteinase on the Surface of Cancer Cells.

PubMed

Kikuchi, Keiji; Kozuka-Hata, Hiroko; Oyama, Masaaki; Seiki, Motoharu; Koshikawa, Naohiko

2018-01-01

Proteolytic cleavage of membrane proteins can alter their functions depending on the cleavage sites. We recently demonstrated that membrane type 1 matrix metalloproteinase (MT1-MMP ) converts the tumor suppressor EphA2 into an oncogenic signal transducer through EphA2 cleavage. The cleaved EphA2 fragment that remains at the cell surface may be a better target for cancer therapy than intact EphA2. To analyze the cleavage site(s) of EphA2, we purified the fragments from tumor cells expressing MT1-MMP and Myc- and 6× His-tagged EphA2 by two-step affinity purification . The purified fragment was digested with trypsin to generate proteolytic peptides , and the amino acid sequences of these peptides were determined by nano-LC-mass spectrometry to identify the MT1-MMP-mediated cleavage site(s) of EphA2.

The action of the bacterial toxin microcin B17. Insight into the cleavage-religation reaction of DNA gyrase.

PubMed

Pierrat, Olivier A; Maxwell, Anthony

2003-09-12

We have examined the effects of the bacterial toxin microcin B17 (MccB17) on the reactions of Escherichia coli DNA gyrase. MccB17 slows down but does not completely inhibit the DNA supercoiling and relaxation reactions of gyrase. A kinetic analysis of the cleavage-religation equilibrium of gyrase was performed to determine the effect of the toxin on the forward (cleavage) and reverse (religation) reactions. A simple mechanism of two consecutive reversible reactions with a nicked DNA intermediate was used to simulate the kinetics of cleavage and religation. The action of MccB17 on the kinetics of cleavage and religation was compared with that of the quinolones ciprofloxacin and oxolinic acid. With relaxed DNA as substrate, only a small amount of gyrase cleavage complex is observed with MccB17 in the absence of ATP, whereas the presence of the nucleotide significantly enhances the effect of the toxin on both the cleavage and religation reactions. In contrast, ciprofloxacin, oxolinic acid, and Ca2+ show lesser dependence on ATP to stabilize the cleavage complex. MccB17 enhances the overall rate of DNA cleavage by increasing the forward rate constant (k2) of the second equilibrium. In contrast, ciprofloxacin increases the amount of cleaved DNA by a combined effect on the forward and reverse rate constants of both equilibria. Based on these results and on the observations that MccB17 only slowly inhibits the supercoiling and relaxation reactions, we suggest a model of the interaction of MccB17 with gyrase.

Reduction-Triggered Transformation of Crosslinking Modules of Disulfide-Containing Micelles with Chemically Tunable Rates.

PubMed

Deng, Zhengyu; Yuan, Shuai; Xu, Ronald X; Liang, Haojun; Liu, Shiyong

2018-05-16

A dilemma exists between the circulation stability and cargo release/mass diffusion at desired sites for designing delivery nanocarriers and in vivo nanoreactors. We herein report disulfide-crosslinked (DCL) micelles exhibiting reduction-triggered switching of crosslinking modules and synchronized hydrophobic-to-hydrophilic transition. Tumor cell-targeted DCL micelles undergo cytoplasmic milieu-triggered disulfide cleavage and cascade self-immolative decaging reactions at chemically adjustable rates, generating primary amine moieties. Extensive amidation reactions with neighboring ester moieties then occur due to high local concentrations and suppression of apparent amine pKa within hydrophobic cores, leading to the transformation of crosslinking modules and formation of tracelessly crosslinked (TCL) micelles with hydrophilic cores inside live cells. We further integrate this design principle with theranostic nanocarriers for selective intracellular drug transport guided by enhanced magnetic resonance (MR) imaging performance. © 2018 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

The investigation of substituent effects on the fragmentation pathways of pentacoordinated phenoxyspirophosphoranes by ESI-MSn.

PubMed

Cui, Xiaoyan; Sun, Can; Zhao, Pei; Wang, Yanyan; Guo, Yanchun; Zhao, Yufen; Cao, Shuxia

2018-04-01

The fragmentation pathways of pentacoordinated phenoxyspirophosphoranes were investigated in the positive mode by electrospray ionization multistage mass spectrometry. The results demonstrate that the sodium adducts of the title compounds undergo two competitive fragmentation pathways, and the fragmentation patterns are heavily dependent on the various substituent patterns at the phenolic group. An electron-withdrawing substituent at the ortho-position always results in the removal of a corresponding phenol analogue, while cleavage by spiroring opening becomes the predominant fragmentation pathway if an electron-donating substituent is at the phenolic group. The substituent effects on the competitive fragmentation pathways were further elucidated by theoretical calculations, single crystal structure analysis, and high-resolution mass spectrometry. The results contribute to the understanding of the gas-phase fragmentation reactions and the structure identification of spirophosphorane analogues by electrospray ionization multistage mass spectrometry. Copyright © 2018 John Wiley & Sons, Ltd.

Nuclear Export of Messenger RNA

PubMed Central

Katahira, Jun

2015-01-01

Transport of messenger RNA (mRNA) from the nucleus to the cytoplasm is an essential step of eukaryotic gene expression. In the cell nucleus, a precursor mRNA undergoes a series of processing steps, including capping at the 5' ends, splicing and cleavage/polyadenylation at the 3' ends. During this process, the mRNA associates with a wide variety of proteins, forming a messenger ribonucleoprotein (mRNP) particle. Association with factors involved in nuclear export also occurs during transcription and processing, and thus nuclear export is fully integrated into mRNA maturation. The coupling between mRNA maturation and nuclear export is an important mechanism for providing only fully functional and competent mRNA to the cytoplasmic translational machinery, thereby ensuring accuracy and swiftness of gene expression. This review describes the molecular mechanism of nuclear mRNA export mediated by the principal transport factors, including Tap-p15 and the TREX complex. PMID:25836925

Significance of Brain Tissue Oxygenation and the Arachidonic Acid Cascade in Stroke

PubMed Central

Rink, Cameron

2011-01-01

Abstract The significance of the hypoxia component of stroke injury is highlighted by hypermetabolic brain tissue enriched with arachidonic acid (AA), a 22:6n-3 polyunsaturated fatty acid. In an ischemic stroke environment in which cerebral blood flow is arrested, oxygen-starved brain tissue initiates the rapid cleavage of AA from the membrane phospholipid bilayer. Once free, AA undergoes both enzyme-independent and enzyme-mediated oxidative metabolism, resulting in the formation of number of biologically active metabolites which themselves contribute to pathological stroke outcomes. This review is intended to examine two divergent roles of molecular dioxygen in brain tissue as (1) a substrate for life-sustaining homeostatic metabolism of glucose and (2) a substrate for pathogenic metabolism of AA under conditions of stroke. Recent developments in research concerning supplemental oxygen therapy as an intervention to correct the hypoxic component of stroke injury are discussed. Antioxid. Redox Signal. 14, 1889–1903. PMID:20673202

CNS Injury: Posttranslational Modification of the Tau Protein as a Biomarker.

PubMed

Caprelli, Mitchell T; Mothe, Andrea J; Tator, Charles H

2017-11-01

The ideal biomarker for central nervous system (CNS) trauma in patients would be a molecular marker specific for injured nervous tissue that would provide a consistent and reliable assessment of the presence and severity of injury and the prognosis for recovery. One candidate biomarker is the protein tau, a microtubule-associated protein abundant in the axonal compartment of CNS neurons. Following axonal injury, tau becomes modified primarily by hyperphosphorylation of its various amino acid residues and cleavage into smaller fragments. These posttrauma products can leak into the cerebrospinal fluid or bloodstream and become candidate biomarkers of CNS injury. This review examines the primary molecular changes that tau undergoes following traumatic brain injury and spinal cord injury, and reviews the current literature in traumatic CNS biomarker research with a focus on the potential for hyperphosphorylated and cleaved tau as sensitive biomarkers of injury.

A Light-Induced Reaction with Oxygen Leads to Chromophore Decomposition and Irreversible Photobleaching in GFP-Type Proteins.

PubMed

Grigorenko, Bella L; Nemukhin, Alexander V; Polyakov, Igor V; Khrenova, Maria G; Krylov, Anna I

2015-04-30

Photobleaching and photostability of proteins of the green fluorescent protein (GFP) family are crucially important for practical applications of these widely used biomarkers. On the basis of simulations, we propose a mechanism for irreversible bleaching in GFP-type proteins under intense light illumination. The key feature of the mechanism is a photoinduced reaction of the chromophore with molecular oxygen (O2) inside the protein barrel leading to the chromophore's decomposition. Using quantum mechanics/molecular mechanics (QM/MM) modeling we show that a model system comprising the protein-bound Chro(-) and O2 can be excited to an electronic state of the intermolecular charge-transfer (CT) character (Chro(•)···O2(-•)). Once in the CT state, the system undergoes a series of chemical reactions with low activation barriers resulting in the cleavage of the bridging bond between the phenolic and imidazolinone rings and disintegration of the chromophore.

Catabolism of exogenous deoxyinosine in cultured epithelial amniotic cells.

PubMed

Carta, M C; Mattana, A; Camici, M; Allegrini, S; Tozzi, M G; Sgarrella, F

2001-10-03

Uptake and catabolism of purine nucleosides have been commonly considered as means to salvage the purine ring for nucleic acid synthesis, usually neglecting the destiny of the pentose moiety. With the aim to ascertain if deoxyribose derived from exogenous DNA can be utilised as a carbon and energy source, we studied the catabolism of exogenous deoxyinosine in a cell line derived from human amnion epithelium (WISH). Intact WISH cells catabolise deoxyinosine by conversion into hypoxanthine. The nucleoside enters the cell through a nitrobenzylthioinosine-insensitive equilibrative transport. Deoxyinosine undergoes a phosphorolytic cleavage inside the cell. The purine base diffuses back to the external medium, while the phosphorylated pentose moiety can be further catabolised to glycolysis and citric acid cycle intermediates. Our results indicate that the catabolism of the deoxynucleoside can be considered mainly as a means to meet the carbon and energy requirements of growing cells.

The genome-wide DNA sequence specificity of the anti-tumour drug bleomycin in human cells.

PubMed

Murray, Vincent; Chen, Jon K; Tanaka, Mark M

2016-07-01

The cancer chemotherapeutic agent, bleomycin, cleaves DNA at specific sites. For the first time, the genome-wide DNA sequence specificity of bleomycin breakage was determined in human cells. Utilising Illumina next-generation DNA sequencing techniques, over 200 million bleomycin cleavage sites were examined to elucidate the bleomycin genome-wide DNA selectivity. The genome-wide bleomycin cleavage data were analysed by four different methods to determine the cellular DNA sequence specificity of bleomycin strand breakage. For the most highly cleaved DNA sequences, the preferred site of bleomycin breakage was at 5'-GT* dinucleotide sequences (where the asterisk indicates the bleomycin cleavage site), with lesser cleavage at 5'-GC* dinucleotides. This investigation also determined longer bleomycin cleavage sequences, with preferred cleavage at 5'-GT*A and 5'- TGT* trinucleotide sequences, and 5'-TGT*A tetranucleotides. For cellular DNA, the hexanucleotide DNA sequence 5'-RTGT*AY (where R is a purine and Y is a pyrimidine) was the most highly cleaved DNA sequence. It was striking that alternating purine-pyrimidine sequences were highly cleaved by bleomycin. The highest intensity cleavage sites in cellular and purified DNA were very similar although there were some minor differences. Statistical nucleotide frequency analysis indicated a G nucleotide was present at the -3 position (relative to the cleavage site) in cellular DNA but was absent in purified DNA.

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.

Cleavage reaction of HDV ribozymes in the presence of Mg2+ is accompanied by a conformational change.

PubMed

Tanaka, Yoichiro; Tagaya, Mitsuhiro; Hori, Tamaki; Sakamoto, Taiichi; Kurihara, Yasuyuki; Katahira, Masato; Uesugi, Seiichi

2002-06-01

Hepatitis delta virus (HDV) ribozymes cleave RNA in the presence of divalent metal ions. We have previously elucidated the solution conformation of a minimized trans-acting HDV ribozyme and obtained evidence by NMR study that an Mg2+ ion binds to a site close to the cleavage site. We examined two ribozyme systems: a pre-cleavage complex with a non-cleavable substrate analogue (mS8) and a post-cleavage complex with a 3' cleavage product (P7). Upon titration with MgCl2, the complex with P7 showed a profound spectral change, while that with mS8 showed broadening of the signals. Analysis of the NOESY spectra of the P7 complex at high Mg2+ concentration revealed that a G:U pair is formed within the L3 loop, and the P1 and P4 stems are stabilized with respect to those of the pre-cleavage complex. The present analysis indicates that the cleavage reaction of the HDV ribozyme produces a big conformational change. Furthermore, presence of the 5'-terminal cytidine residue prevents this conformational change and its absence stabilizes the product-ribozyme complex in the presence of Mg2+. The structure of the Mg2+-bound P7 complex is similar to the crystal structure found for a product-ribozyme complex but is different from the pre-cleavage structure.

The large terminase DNA packaging motor grips DNA with its ATPase domain for cleavage by the flexible nuclease domain

PubMed Central

Hilbert, Brendan J.; Hayes, Janelle A.; Stone, Nicholas P.; Xu, Rui-Gang

2017-01-01

Abstract Many viruses use a powerful terminase motor to pump their genome inside an empty procapsid shell during virus maturation. The large terminase (TerL) protein contains both enzymatic activities necessary for packaging in such viruses: the adenosine triphosphatase (ATPase) that powers DNA translocation and an endonuclease that cleaves the concatemeric genome at both initiation and completion of genome packaging. However, how TerL binds DNA during translocation and cleavage remains mysterious. Here we investigate DNA binding and cleavage using TerL from the thermophilic phage P74-26. We report the structure of the P74-26 TerL nuclease domain, which allows us to model DNA binding in the nuclease active site. We screened a large panel of TerL variants for defects in binding and DNA cleavage, revealing that the ATPase domain is the primary site for DNA binding, and is required for nuclease activity. The nuclease domain is dispensable for DNA binding but residues lining the active site guide DNA for cleavage. Kinetic analysis of DNA cleavage suggests flexible tethering of the nuclease domains during DNA cleavage. We propose that interactions with the procapsid during DNA translocation conformationally restrict the nuclease domain, inhibiting cleavage; TerL release from the capsid upon completion of packaging unlocks the nuclease domains to cleave DNA. PMID:28082398

Differential Sensitivities of Pulmonary and Coronary Arteries to Hemoglobin-Based Oxygen Carriers and Nitrovasodilators: Study in a Bovine Ex Vivo Model of Vascular Strips

DTIC Science & Technology

2010-01-01

dependent manner, with a relatively high average IC50 of8.5 J.lM (Table 1 ). For bovine pulmonary artery, the JC50 for sodium nitrite was more than 1... dependent on nitrovasodilator concentration, suggesting SNP and sodium nitrite -induced autocatalytic conversion of oxyhemoglobin to methemoglobin at...Gladwin, M.T., Kim-Shapiro, D.R., 2008. The functional nitrite reductase activity of the heme -globins. Blood 112, 2636-2647. Hart, j.L, Ledvina, M.A

Guidance Document: Surface Soils Sampling for Munitions Residues in Military Live Fire Training Ranges: Canadian Protocol

DTIC Science & Technology

2012-12-01

NA Boron B 0.5 NA Cadmium Cd 0.3 22 Calcium Ca 4.0 NA Chromium Cr 0.5 87 Cobalt Co 0.5 300 Copper Cu 0.4 91 Iron Fe 0.6 NA Lead Pb 2.5 600...on their susceptibility to initiation. Primary explosives, which include lead azide, lead styphnate, and mercury fulminate, are highly susceptible...ballistic properties. The degradation of NC leads to substances which speed up the degradation process, or else an autocatalytic reaction. To counteract

The evolution of the protein synthesis system. I - A model of a primitive protein synthesis system

NASA Technical Reports Server (NTRS)

Mizutani, H.; Ponnamperuma, C.

1977-01-01

A model is developed to describe the evolution of the protein synthesis system. The model is comprised of two independent autocatalytic systems, one including one gene (A-gene) and two activated amino acid polymerases (O and A-polymerases), and the other including the addition of another gene (N-gene) and a nucleotide polymerase. Simulation results have suggested that even a small enzymic activity and polymerase specificity could lead the system to the most accurate protein synthesis, as far as permitted by transitions to systems with higher accuracy.

Site-specific Phosphorylation Protects Glycogen Synthase Kinase-3β from Calpain-mediated Truncation of Its N and C Termini*

PubMed Central

Ma, Shanshan; Liu, Shaojun; Huang, Qiaoying; Xie, Bo; Lai, Bingquan; Wang, Chong; Song, Bin; Li, Mingtao

2012-01-01

Glycogen synthase kinase-3β (GSK-3β), a key regulator of neuronal apoptosis, is inhibited by the phosphorylation of Ser-9/Ser-389 and was recently shown to be cleaved by calpain at the N terminus, leading to its subsequent activation. In this study calpain was found to cleave GSK-3β not only at the N terminus but also at the C terminus, and cleavage sites were identified at residues Thr-38–Thr-39 and Ile-384–Gln-385. Furthermore, the cleavage of GSK-3β occurred in tandem with Ser-9 dephosphorylation during cerebellar granule neuron apoptosis. Increasing Ser-9 phosphorylation of GSK-3β by inhibiting phosphatase 1/2A or pretreating with purified active Akt inhibited calpain-mediated cleavage of GSK-3β at both N and C termini, whereas non-phosphorylatable mutant GSK-3β S9A facilitated its cleavage. In contrast, Ser-389 phosphorylation selectively inhibited the cleavage of GSK-3β at the C terminus but not the N terminus. Calpain-mediated cleavage resulted in three truncated products, all of which contained an intact kinase domain: ΔN-GSK-3β (amino acids 39–420), ΔC-GSK-3β (amino acids 1–384), and ΔN/ΔC-GSK-3β (amino acids 39–384). All three truncated products showed increased kinase and pro-apoptotic activity, with ΔN/ΔC-GSK-3β being the most active form. This observation suggests that the GSK-3β C terminus acts as an autoinhibitory domain similar to the N terminus. Taken together, these findings demonstrate that calpain-mediated cleavage activates GSK-3β by removing its N- and C-terminal autoinhibitory domains and that Ser-9 phosphorylation inhibits the cleavage of GSK-3β at both termini. In contrast, Ser-389 phosphorylation inhibits only C-terminal cleavage but not N-terminal cleavage. These findings also identify a mechanism by which site-specific phosphorylation and calpain-mediated cleavage operate in concert to regulate GSK-3β activity. PMID:22496446

Site-specific phosphorylation protects glycogen synthase kinase-3β from calpain-mediated truncation of its N and C termini.

PubMed

Ma, Shanshan; Liu, Shaojun; Huang, Qiaoying; Xie, Bo; Lai, Bingquan; Wang, Chong; Song, Bin; Li, Mingtao

2012-06-29

Glycogen synthase kinase-3β (GSK-3β), a key regulator of neuronal apoptosis, is inhibited by the phosphorylation of Ser-9/Ser-389 and was recently shown to be cleaved by calpain at the N terminus, leading to its subsequent activation. In this study calpain was found to cleave GSK-3β not only at the N terminus but also at the C terminus, and cleavage sites were identified at residues Thr-38-Thr-39 and Ile-384-Gln-385. Furthermore, the cleavage of GSK-3β occurred in tandem with Ser-9 dephosphorylation during cerebellar granule neuron apoptosis. Increasing Ser-9 phosphorylation of GSK-3β by inhibiting phosphatase 1/2A or pretreating with purified active Akt inhibited calpain-mediated cleavage of GSK-3β at both N and C termini, whereas non-phosphorylatable mutant GSK-3β S9A facilitated its cleavage. In contrast, Ser-389 phosphorylation selectively inhibited the cleavage of GSK-3β at the C terminus but not the N terminus. Calpain-mediated cleavage resulted in three truncated products, all of which contained an intact kinase domain: ΔN-GSK-3β (amino acids 39-420), ΔC-GSK-3β (amino acids 1-384), and ΔN/ΔC-GSK-3β (amino acids 39-384). All three truncated products showed increased kinase and pro-apoptotic activity, with ΔN/ΔC-GSK-3β being the most active form. This observation suggests that the GSK-3β C terminus acts as an autoinhibitory domain similar to the N terminus. Taken together, these findings demonstrate that calpain-mediated cleavage activates GSK-3β by removing its N- and C-terminal autoinhibitory domains and that Ser-9 phosphorylation inhibits the cleavage of GSK-3β at both termini. In contrast, Ser-389 phosphorylation inhibits only C-terminal cleavage but not N-terminal cleavage. These findings also identify a mechanism by which site-specific phosphorylation and calpain-mediated cleavage operate in concert to regulate GSK-3β activity.

The structure and stratigraphy of the Pen Argyl Member of the Martinsburg Formation in Lehigh and Berks counties, Pennsylvania

USGS Publications Warehouse

Lash, Gary George

1978-01-01

The Pen Argyl Member, the upper claystone slate member of the Martinsburg Formation, was studied in three quadrangles in Lehigh and Berks Counties, Pennsylvania. Graptolites collected from the Pen Argyl Member at Lehigh Gap indicate a lower Upper Ordovician (Edenian-Maysvillian) age for the Pen Argyl Member. The Pen Argyl Member in this area is located on the normal limb and in the brow of the large, recumbent Musconetcong nappe. It is a deep water flysch deposit emplaced by turbidity currents from a southeasterly source. Sedimentologic and structural evidence show that the Pen Argyl member overlies the sandy middle Ramseyburg Member, thus supporting the tripartite subdivision of the Martinsburg Formation. Field and thin section study indicates that the penetrative slaty cleavage formed in an indurated rock probably by pressure solution and neocrystallization under lower greenschist facies metamorphism. Strain-slip cleavage formed as a result of a stress couple operating parallel to the slaty cleavage that transposed the slaty cleavage into a more spaced cleavage. Both cleavages are believed to have formed within the same stress continuum and in close succession. Analysis of the folds in the Pen Argyl Member indicate six phases of major and minor folding. The earliest folding, F1, resulted in the development of the recumbent nappe. F2 folds can only be determined statistically; these axes plunge either northeast or southwest Asymmetric folds, F3, and associated F4 crenulations formed within the same stress continuum. F5 folds are large open folds and are exemplified by the Mosservi!le anticline. Kink folds, F6 and associated crenulations are fault related and were the last folds to form. Faults in the Pen Argyl Member range from small displacements along slaty cleavage to large reverse faults. The largest of these, the Eckville fault, is recognized throughout the three quadrangle area. It is a high angle reverse fault that separates the Shochary sequence from the Pen Argyl member to the north. Detailed fabric analysis of the Pen Argyl Member indicates that (1) the strike of the slaty cleavage is consistent throughout the study area, (2) bedding strikes are undulose indicating that the rocks were folded prior to slaty cleavage development, (3) slaty cleavage-bedding intersections indicate an early northeast-southwest fold set and a later east-west trend of fold axes, and (4) slaty cleavage-strain-slip cleavage intersections indicate two periods of strain-slip cleavage development, the later period being fault related. Synthesis of field work and fabric data suggest that the Pen Argyl Member was deposited in the waning stages of flysch deposition during the Taconic orogeny. The nappe, F1, was formed at this time as a result of stress generated by plate convergence to the southeast. Further Taconian deformation of the normal limb of the nappe resulted in the northeast-southwest plunging F2 folds. Initial Alleghenian deformation resulted in the F3 asymmetric folds and slaty cleavage, S1. Later in the same stress continuum the F4 crenulations and strain-slip cleavage, S2, formed. Subsequently, F5 open folding occurred. Kink folds and crenulations, F6, and strain-slip cleavage, S3, formed in conjunction with late Alleghenian reverse faults such as the Eckville fault.

New Insight into the Cleavage Reaction of Nostoc sp. Strain PCC 7120 Carotenoid Cleavage Dioxygenase in Natural and Nonnatural Carotenoids

PubMed Central

Heo, Jinsol; Kim, Se Hyeuk

2013-01-01

Carotenoid cleavage dioxygenases (CCDs) are enzymes that catalyze the oxidative cleavage of carotenoids at a specific double bond to generate apocarotenoids. In this study, we investigated the activity and substrate preferences of NSC3, a CCD of Nostoc sp. strain PCC 7120, in vivo and in vitro using natural and nonnatural carotenoid structures. NSC3 cleaved β-apo-8′-carotenal at 3 positions, C-13C-14, C-15C-15′, and C-13′C-14′, revealing a unique cleavage pattern. NSC3 cleaves the natural structure of carotenoids 4,4′-diaponeurosporene, 4,4′-diaponeurosporen-4′-al, 4,4′-diaponeurosporen-4′-oic acid, 4,4′-diapotorulene, and 4,4′-diapotorulen-4′-al to generate novel cleavage products (apo-14′-diaponeurosporenal, apo-13′-diaponeurosporenal, apo-10′-diaponeurosporenal, apo-14′-diapotorulenal, and apo-10′-diapotorulenal, respectively). The study of carotenoids with natural or nonnatural structures produced by using synthetic modules could provide information valuable for understanding the cleavage reactions or substrate preferences of other CCDs in vivo and in vitro. PMID:23524669

Cleavage of nucleic acids

DOEpatents

Prudent, James R.; Hall, Jeff G.; Lyamichev, Victor L.; Brow, Mary Ann D.; Dahlberg, James E.

2007-12-11

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.

Invasive cleavage of nucleic acids

DOEpatents

Prudent, James R.; Hall, Jeff G.; Lyamichev, Victor I.; Brow, Mary Ann D.; Dahlberg, James E.

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.

Invasive cleavage of nucleic acids

DOEpatents

Prudent, James R.; Hall, Jeff G.; Lyamichev, Victor I.; Brow, Mary Ann D.; Dahlberg, James E.

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.

Cleavage of nucleic acids

DOEpatents

Prudent, James R.; Hall, Jeff G.; Lyamichev, Victor I.; Brow; Mary Ann D.; Dahlberg, James E.

2010-11-09

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.

Cleavage of nucleic acids

DOEpatents

Prudent, James R.; Hall, Jeff G.; Lyamichev, Victor I.; Brow, Mary Ann D.; Dahlberg, James E.

2000-01-01

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 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.

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.

Effects of Single Amino Acid Substitution on the Collision-Induced Dissociation of Intact Protein Ions: Turkey Ovomucoid Third Domain

PubMed Central

Newton, Kelly A.; Pitteri, Sharon J.; Laskowski, Michael; McLuckey, Scott A.

2005-01-01

Expanded understanding of the factors that direct polypeptide ion fragmentation can lead to improved specificity in the use of tandem mass spectrometry for the identification and characterization of proteins. Like the fragmentation of peptide cations, the dissociation of whole protein cations shows several preferred cleavages, the likelihood for which is parent ion charge dependent. While such cleavages are often observed, they are far from universally observed, despite the presence of the residues known to promote them. Furthermore, cleavages at residues not noted to be common in a variety of proteins can be dominant for a particular protein or protein ion charge state. Motivated by the ability to study a small protein, turkey ovomucoid third domain, for which a variety of single amino acid variants are available, the effects of changing the identity of one amino acid in the protein sequence on its dissociation behavior were examined. In particular, changes in amino acids associated with C-terminal aspartic acid cleavage and N-terminal proline cleavage were emphasized. Consistent with previous studies, the product ion spectra were found to be dependent upon the parent ion charge state. Furthermore, the fraction of possible C-terminal aspartic acid cleavages observed to occur for this protein was significantly larger than the fraction of possible N-terminal proline cleavages. In fact, very little N-terminal proline cleavage was noted for the wild-type protein despite the presence of three proline residues in the protein. The addition/removal of proline and aspartic acids was studied along with changes in selected residues adjacent to proline residues. Evidence for inhibition of proline cleavage by the presence of nearby basic residues was noted, particularly if the basic residue was likely to be protonated. PMID:15473693

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

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

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

Determination of Double Bond Positions in Polyunsaturated Fatty Acids Using the Photochemical Paternò-Büchi Reaction with Acetone and Tandem Mass Spectrometry.

PubMed

Murphy, Robert C; Okuno, Toshiaki; Johnson, Christopher A; Barkley, Robert M

2017-08-15

The positions of double bonds along the carbon chain of methylene interrupted polyunsaturated fatty acids are unique identifiers of specific fatty acids derived from biochemical reactions that occur in cells. It is possible to obtain direct structural information as to these double bond positions using tandem mass spectrometry after collisional activation of the carboxylate anions of an acetone adduct at each of the double bond positions formed by the photochemical Paternò-Büchi reaction with acetone. This reaction can be carried out by exposing a small portion of an inline fused silica capillary to UV photons from a mercury vapor lamp as the sample is infused into the electrospray ion source of a mass spectrometer. Collisional activation of [M - H] - yields a series of reverse Paternò-Büchi reaction product ions that essentially are derived from cleavage of the original carbon-carbon double bonds that yield an isopropenyl carboxylate anion corresponding to each double bond location. Aldehydic reverse Paternò-Büchi product ions are much less abundant as the carbon chain length and number of double bonds increase. The use of a mixture of D 0 /D 6 -acetone facilitates identification of these double bonds indicating product ions as shown for arachidonic acid. If oxygen is present in the solvent stream undergoing UV photoactivation, ozone cleavage ions are also observed without prior collisional activation. This reaction was used to determine the double bond positions in a 20:3 fatty acid that accumulated in phospholipids of RAW 264.7 cells cultured for 3 days.

Confinement of caspase-12 proteolytic activity to autoprocessing

PubMed Central

Roy, Sophie; Sharom, Jeffrey R.; Houde, Caroline; Loisel, Thomas P.; Vaillancourt, John P.; Shao, Wei; Saleh, Maya; Nicholson, Donald W.

2008-01-01

Caspase-12 is a dominant-negative regulator of caspase-1 (IL-1β-converting enzyme) and an attenuator of cytokine responsiveness to septic infections. This molecular role for caspase-12 appears to be akin to the role of cFLIP in regulating caspase-8 in the extrinsic cell death pathway; however, unlike cFLIP/Usurpin, we demonstrate here that caspase-12 is catalytically competent. To examine these catalytic properties, rat caspase-12 was cloned, and the recombinant enzyme was used to examine the cleavage of macromolecular and synthetic fluorogenic substrates. Although caspase-12 could mediate autoproteolytic maturation of its own proenzyme, in both cis and trans, it was not able to cleave any other polypeptide substrate, including other caspase proenzymes, apoptotic substrates, cytokine precursors, or proteins in the endoplasmic reticulum that normally undergo caspase-mediated proteolysis. The dearth of potential substrates for caspase-12 also was confirmed by whole-cell diagonal-gel analysis. Autolytic cleavage within the caspase-12 proenzyme was mapped to a single site at the large–small subunit junction, ATAD319, and this motif was recognized by caspase-12 when incorporated into synthetic fluorogenic substrates. The specific activity of caspase-12 with these substates was several orders of magnitude lower than caspases-1 and -3, highlighting its relative catalytic paucity. In intact cells, caspase-12 autoproteolysis occurred in the inhibitory complex containing caspase-1. We propose that the proteolytic activity of caspase-12 is confined to its own proenzyme and that autocleavage within the caspase-1 complex may be a means for temporal limitation of the inhibitory effects of caspase-12 on proinflammatory cytokine maturation. PMID:18332441

Culture media influenced laboratory outcomes but not neonatal birth weight in assisted reproductive technology.

PubMed

Yin, Tai-lang; Zhang, Yi; Li, Sai-jiao; Zhao, Meng; Ding, Jin-li; Xu, Wang-ming; Yang, Jing

2015-12-01

Whether the type of culture media utilized in assisted reproductive technology has impacts on laboratory outcomes and birth weight of newborns in in-vitro fertilization (IVF)/intracytoplasmic sperm injection (ICSI) was investigated. A total of 673 patients undergoing IVF/ICSI and giving birth to live singletons after fresh embryo transfer on day 3 from Jan. 1, 2010 to Dec. 31, 2012 were included. Three types of culture media were used during this period: Quinn's Advantage (QA), Single Step Medium (SSM), and Continuous Single Culture medium (CSC). Fertilization rate (FR), normal fertilization rate (NFR), cleavage rate (CR), normal cleavage rate (NCR), good-quality embryo rate (GQER) and neonatal birth weight were compared using one-way ANOVA and χ (2) tests. Multiple linear regression analysis was performed to determine the impact of culture media on laboratory outcomes and birth weight. In IVF cycles, GQER was significantly decreased in SSM medium group as compared with QA or CSC media groups (63.6% vs. 69.0% in QA; vs. 71.3% in CSC, P=0.011). In ICSI cycles, FR, NFR and CR were significantly lower in CSC medium group than in other two media groups. No significant difference was observed in neonatal birthweight among the three groups (P=0.759). Multiple linear regression analyses confirmed that the type of culture medium was correlated with FR, NFR, CR and GQER, but not with neonatal birth weight. The type of culture media had potential influences on laboratory outcomes but did not exhibit an impact on the birth weight of singletons in ART.

Constitutive Endocytosis of VEGFR2 Protects the Receptor against Shedding.

PubMed

Basagiannis, Dimitris; Christoforidis, Savvas

2016-08-05

VEGFR2 plays a fundamental role in blood vessel formation and in life threatening diseases, such as cancer angiogenesis and cardiovascular disorders. Although inactive growth factor receptors are mainly localized at the plasma membrane, VEGFR2 undergoes constitutive endocytosis (in the absence of ligand) and recycling. Intriguingly, the significance of these futile transport cycles of VEGFR2 remains unclear. Here we found that, unexpectedly, the function of constitutive endocytosis of VEGFR2 is to protect the receptor against plasma membrane cleavage (shedding), thereby preserving the functional state of the receptor until the time of activation by VEGF. Inhibition of constitutive endocytosis of VEGFR2, by interference with the function of clathrin, dynamin, or Rab5, increases dramatically the cleavage/shedding of VEGFR2. Shedding of VEGFR2 produces an N-terminal soluble fragment (100 kDa, s100), which is released in the extracellular space, and a residual C-terminal part (130 kDa, p130) that remains integrated at the plasma membrane. The released soluble fragment (s100) co-immunoprecipitates with VEGF, in line with the topology of the VEGF-binding domain at the N terminus of VEGFR2. Increased shedding of VEGFR2 (via inhibition of constitutive endocytosis) results in reduced response to VEGF, consistently with the loss of the VEGF-binding domain from the membrane remnant of VEGFR2. These data suggest that constitutive internalization of VEGFR2 protects the receptor against shedding and provides evidence for an unprecedented mechanism via which endocytosis can regulate the fate and activity of growth factor receptors. © 2016 by The American Society for Biochemistry and Molecular Biology, Inc.

An active-site phenylalanine directs substrate binding and C-H cleavage in the alpha-ketoglutarate-dependent dioxygenase TauD.

PubMed

McCusker, Kevin P; Klinman, Judith P

2010-04-14

Enzymes that cleave C-H bonds are often found to depend on well-packed hydrophobic cores that influence the distance between the hydrogen donor and acceptor. Residue F159 in taurine alpha-ketoglutarate dioxygenase (TauD) is demonstrated to play an important role in the binding and orientation of its substrate, which undergoes a hydrogen atom transfer to the active site Fe(IV)=O. Mutation of F159 to smaller hydrophobic side chains (L, V, A) leads to substantially reduced rates for substrate binding and for C-H bond cleavage, as well as increased contribution of the chemical step to k(cat) under steady-state turnover conditions. The greater sensitivity of these substrate-dependent processes to mutation at position 159 than observed for the oxygen activation process supports a previous conclusion of modularity of function within the active site of TauD (McCusker, K. P.; Klinman, J. P. Proc. Natl. Acad. Sci. U.S.A. 2009, 106, 19791-19795). Extraction of intrinsic deuterium kinetic isotope effects (KIEs) using single turnover transients shows 2- to 4-fold increase in the size of the KIE for F159V in relation to wild-type and F159L. It appears that there is a break in behavior following removal of a single methylene from the side chain of F159L to generate F159V, whereby the protein active site loses its ability to restore the internuclear distance between substrate and Fe(IV)=O that supports optimal hydrogenic wave function overlap.

A Single Mutation Unlocks Cascading Exaptations in the Origin of a Potent Pitviper Neurotoxin.

PubMed

Whittington, A Carl; Mason, Andrew J; Rokyta, Darin R

2017-04-01

Evolutionary innovations and complex phenotypes seemingly require an improbable amount of genetic change to evolve. Rattlesnakes display two dramatically different venom phenotypes. Type I venoms are hemorrhagic with low systemic toxicity and high expression of tissue-destroying snake venom metalloproteinases. Type II venoms are highly neurotoxic and lack snake venom metalloproteinase expression and associated hemorrhagic activity. This dichotomy hinges on Mojave toxin (MTx), a phospholipase A2 (PLA2) based β-neurotoxin expressed in Type II venoms. MTx is comprised of a nontoxic acidic subunit that undergoes extensive proteolytic processing and allosterically regulates activity of a neurotoxic basic subunit. Evolution of the acidic subunit presents an evolutionary challenge because the need for high expression of a nontoxic venom component and the proteolytic machinery required for processing suggests genetic changes of seemingly little immediate benefit to fitness. We showed that MTx evolved through a cascading series of exaptations unlocked by a single nucleotide change. The evolution of one new cleavage site in the acidic subunit unmasked buried cleavage sites already present in ancestral PLA2s, enabling proteolytic processing. Snake venom serine proteases, already present in the venom to disrupt prey hemostasis, possess the requisite specificities for MTx acidic subunit proteolysis. The dimerization interface between MTx subunits evolved by exploiting a latent, but masked, hydrophobic interaction between ancestral PLA2s. The evolution of MTx through exaptation of existing functional and structural features suggests complex phenotypes that depend on evolutionary innovations can arise from minimal genetic change enabled by prior evolution.

Constitutive Endocytosis of VEGFR2 Protects the Receptor against Shedding*

PubMed Central

Basagiannis, Dimitris; Christoforidis, Savvas

2016-01-01

VEGFR2 plays a fundamental role in blood vessel formation and in life threatening diseases, such as cancer angiogenesis and cardiovascular disorders. Although inactive growth factor receptors are mainly localized at the plasma membrane, VEGFR2 undergoes constitutive endocytosis (in the absence of ligand) and recycling. Intriguingly, the significance of these futile transport cycles of VEGFR2 remains unclear. Here we found that, unexpectedly, the function of constitutive endocytosis of VEGFR2 is to protect the receptor against plasma membrane cleavage (shedding), thereby preserving the functional state of the receptor until the time of activation by VEGF. Inhibition of constitutive endocytosis of VEGFR2, by interference with the function of clathrin, dynamin, or Rab5, increases dramatically the cleavage/shedding of VEGFR2. Shedding of VEGFR2 produces an N-terminal soluble fragment (100 kDa, s100), which is released in the extracellular space, and a residual C-terminal part (130 kDa, p130) that remains integrated at the plasma membrane. The released soluble fragment (s100) co-immunoprecipitates with VEGF, in line with the topology of the VEGF-binding domain at the N terminus of VEGFR2. Increased shedding of VEGFR2 (via inhibition of constitutive endocytosis) results in reduced response to VEGF, consistently with the loss of the VEGF-binding domain from the membrane remnant of VEGFR2. These data suggest that constitutive internalization of VEGFR2 protects the receptor against shedding and provides evidence for an unprecedented mechanism via which endocytosis can regulate the fate and activity of growth factor receptors. PMID:27298320

Cancer Cell-derived Exosomes Induce Mitogen-activated Protein Kinase-dependent Monocyte Survival by Transport of Functional Receptor Tyrosine Kinases*

PubMed Central

Song, Xiao; Ding, Yanping; Liu, Gang; Yang, Xiao; Zhao, Ruifang; Zhang, Yinlong; Zhao, Xiao; Anderson, Gregory J.; Nie, Guangjun

2016-01-01

Tumor-associated macrophages (TAM) play pivotal roles in cancer initiation and progression. Monocytes, the precursors of TAMs, normally undergo spontaneous apoptosis within 2 days, but can subsist in the inflammatory tumor microenvironment for continuous survival and generation of sufficient TAMs. The mechanisms underlying tumor-driving monocyte survival remain obscure. Here we report that cancer cell-derived exosomes were crucial mediators for monocyte survival in the inflammatory niche. Analysis of the survival-promoting molecules in monocytes revealed that cancer cell-derived exosomes activated Ras and extracellular signal-regulated kinases in the mitogen-activated protein kinase (MAPK) pathway, resulting in the prevention of caspase cleavage. Phosphorylated receptor tyrosine kinases (RTKs), such as phosphorylated epidermal growth factor receptor (EGFR) and human epidermal growth factor receptor 2 (HER-2), were abundantly expressed in cancer cell-derived exosomes. Knock-out of EGFR or/and HER-2, or alternatively, inhibitors against their phosphorylation significantly disturbed the exosome-mediated activation of the MAPK pathway, inhibition of caspase cleavage, and increase in survival rate in monocytes. Moreover, the deprived survival-stimulating activity of exosomes due to null expression of EGFR and HER-2 could be restored by activation of another RTK, insulin receptor. Overall, our study uncovered a mechanism of tumor-associated monocyte survival and demonstrated that cancer cell-derived exosomes can stimulate the MAPK pathway in monocytes through transport of functional RTKs, leading to inactivation of apoptosis-related caspases. This work provides insights into the long sought question on monocyte survival prior to formation of plentiful TAMs in the tumor microenvironment. PMID:26895960

Mutations in the Parainfluenza Virus 5 Fusion Protein Reveal Domains Important for Fusion Triggering and Metastability

PubMed Central

Bose, Sayantan; Heath, Carissa M.; Shah, Priya A.; Alayyoubi, Maher; Jardetzky, Theodore S.

2013-01-01

Paramyxovirus membrane glycoproteins F (fusion protein) and HN, H, or G (attachment protein) are critical for virus entry, which occurs through fusion of viral and cellular envelopes. The F protein folds into a homotrimeric, metastable prefusion form that can be triggered by the attachment protein to undergo a series of structural rearrangements, ultimately folding into a stable postfusion form. In paramyxovirus-infected cells, the F protein is activated in the Golgi apparatus by cleavage adjacent to a hydrophobic fusion peptide that inserts into the target membrane, eventually bringing the membranes together by F refolding. However, it is not clear how the attachment protein, known as HN in parainfluenza virus 5 (PIV5), interacts with F and triggers F to initiate fusion. To understand the roles of various F protein domains in fusion triggering and metastability, single point mutations were introduced into the PIV5 F protein. By extensive study of F protein cleavage activation, surface expression, and energetics of fusion triggering, we found a role for an immunoglobulin-like (Ig-like) domain, where multiple hydrophobic residues on the PIV5 F protein may mediate F-HN interactions. Additionally, destabilizing mutations of PIV5 F that resulted in HN trigger-independent mutant F proteins were identified in a region along the border of F trimer subunits. The positions of the potential HN-interacting region and the region important for F stability in the lower part of the PIV5 F prefusion structure provide clues to the receptor-binding initiated, HN-mediated F trigger. PMID:24089572

High-performance and versatile electrochemical aptasensor based on self-supported nanoporous gold microelectrode and enzyme-induced signal amplification.

PubMed

Shi, Lei; Rong, Xiaojiao; Wang, Yan; Ding, Shiming; Tang, Wanying

2018-04-15

Herein, novel and versatile electrochemical aptasensors were constructed on a self-supported nanoporous gold (np-Au) microelectrode, integrating with an exonuclease III (Exo III) induced signal amplification strategy. Self-supported np-Au microelectrode with 3D bicontinuous nanoporous structures possesses tremendously large specific area, clean surface, high stability and biocompatibility, bringing about significant advantages in both molecular recognition and signal response. As paradigms, two analytes of bisphenol A (BPA) and ochratoxin A (OTA) were selected to demonstrate the superiority and versatility of designed aptasensors. Trace amounts of mDNA (associated with BPA or OTA concentration) hybridized with cDNA strands assembled on np-Au microelectrode, activating the cleavage reaction with Exo III. Thus, cDNA was digested and mDNA was released to undergo a new hybridization and cleavage cycle. Finally, residual cDNA strands were recognized by methylene blue labelled rDNA/AuNPs with the assistance of hDNA to generate the electrochemical signals, which were used to quantitatively monitor targets. Under the optimized conditions, prepared aptasensors exhibited wide linear ranges (25pg/mL to 2ng/mL for BPA, 10pg/mL to 5ng/mL for OTA) with ultralow detection limits (10pg/mL for BPA, 5pg/mL for OTA), excellent selectivity and stability, and reliable detection in real samples. This work opens a new horizon for constructing promising electrochemical aptasensors for environmental monitoring, medical diagnostics and food safety. Copyright © 2017 Elsevier B.V. All rights reserved.

Binding and cleavage of nucleic acids by the "hairpin" ribozyme.

PubMed

Chowrira, B M; Burke, J M

1991-09-03

The "hairpin" ribozyme derived from the minus strand of tobacco ringspot virus satellite RNA [(-)sTRSV] efficiently catalyzes sequence-specific RNA hydrolysis in trans (Feldstein et al., 1989; Hampel & Triz, 1989; Haseloff & Gerlach, 1989). The ribozyme does not cleave DNA. An RNA substrate analogue containing a single deoxyribonucleotide residue 5' to the cleavage site (A-1) binds to the ribozyme efficiently but cannot be cleaved. A DNA substrate analogue with a ribonucleotide at A-1 is cleaved; thus A-1 provides the only 2'-OH required for cleavage. These results support cleavage via a transphosphorylation mechanism initiated by attack of the 2'-OH of A-1 on the scissile phosphodiester. The ribozyme discriminates between DNA and RNA in both binding and cleavage. Results indicate that the 2'-OH of A-1 functions in complex stabilization as well as cleavage. The ribozyme efficiently cleaves a phosphorothioate diester linkage, suggesting that the pro-Rp oxygen at the scissile phosphodiester does not coordinate Mg2+.

Autotransporter structure reveals intra-barrel cleavage followed by conformational changes.

PubMed

Barnard, Travis J; Dautin, Nathalie; Lukacik, Petra; Bernstein, Harris D; Buchanan, Susan K

2007-12-01

Autotransporters are virulence factors produced by Gram-negative bacteria. They consist of two domains, an N-terminal 'passenger' domain and a C-terminal beta-domain. beta-domains form beta-barrel structures in the outer membrane while passenger domains are translocated into the extracellular space. In some autotransporters, the two domains are separated by proteolytic cleavage. Using X-ray crystallography, we solved the 2.7-A structure of the post-cleavage state of the beta-domain of EspP, an autotransporter produced by Escherichia coli strain O157:H7. The structure consists of a 12-stranded beta-barrel with the passenger domain-beta-domain cleavage junction located inside the barrel pore, approximately midway between the extracellular and periplasmic surfaces of the outer membrane. The structure reveals an unprecedented intra-barrel cleavage mechanism and suggests that two conformational changes occur in the beta-domain after cleavage, one conferring increased stability on the beta-domain and another restricting access to the barrel pore.

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.

A role for exosomes in the constitutive and stimulus-induced ectodomain cleavage of L1 and CD44.

PubMed

Stoeck, Alexander; Keller, Sascha; Riedle, Svenja; Sanderson, Michael P; Runz, Steffen; Le Naour, Francois; Gutwein, Paul; Ludwig, Andreas; Rubinstein, Eric; Altevogt, Peter

2006-02-01

Ectodomain shedding is a proteolytic mechanism by which transmembrane molecules are converted into a soluble form. Cleavage is mediated by metalloproteases and proceeds in a constitutive or inducible fashion. Although believed to be a cell-surface event, there is increasing evidence that cleavage can take place in intracellular compartments. However, it is unknown how cleaved soluble molecules get access to the extracellular space. By analysing L1 (CD171) and CD44 in ovarian carcinoma cells, we show in the present paper that the cleavage induced by ionomycin, APMA (4-aminophenylmercuric acetate) or MCD (methyl-beta-cyclodextrin) is initiated in an endosomal compartment that is subsequently released in the form of exosomes. Calcium influx augmented the release of exosomes containing functionally active forms of ADAM10 (a disintegrin and metalloprotease 10) and ADAM17 [TACE (tumour necrosis factor a-converting enzyme)] as well as CD44 and L1 cytoplasmic cleavage fragments. Cleavage could also proceed in released exosomes, but only depletion of ADAM10 by small interfering RNA blocked cleavage under constitutive and induced conditions. In contrast, cleavage of L1 in response to PMA occurred at the cell surface and was mediated by ADAM17. We conclude that different ADAMs are involved in distinct cellular compartments and that ADAM10 is responsible for shedding in vesicles. Our findings open up the possibility that exosomes serve as a platform for ectodomain shedding and as a vehicle for the cellular export of soluble molecules.

Biological Cleavage of the C–P Bond in Perfluoroalkyl Phosphinic Acids in Male Sprague-Dawley Rats and the Formation of Persistent and Reactive Metabolites

PubMed Central

Yeung, Leo W.Y.; Mabury, Scott A.

2017-01-01

Background: Perfluoroalkyl phosphinic acids (PFPiAs) have been detected in humans, wildlife, and various environmental matrices. These compounds have been used with perfluoroalkyl phosphonic acids (PFPAs) as surfactants in consumer products and as nonfoaming additives in pesticide formulations. Unlike the structurally related perfluoroalkyl sulfonic and carboxylic acids, little is known about the biological fate of PFPiAs. Objectives: We determined the biotransformation products of PFPiAs and some pharmacokinetic parameters in a rat model. Methods: Male Sprague-Dawley rats received an oral gavage dose of either C6/C8PFPiA, C8/C8PFPiA, or C8PFPA. Blood was sampled over time, and livers were harvested upon sacrifice. Analytes were quantified using ultra-high-performance liquid chromatography–tandem mass spectrometry or gas chromatography–mass spectrometry. Results: PFPiAs were metabolized to the corresponding PFPAs and 1H-perfluoroalkanes (1H-PFAs), with 70% and 75% biotransformation 2 wk after a single bolus dose for C6/C8PFPiA and C8/C8PFPiA, respectively. This is the first reported cleavage of a C-P bond in mammals, and the first attempt, with a single-dose exposure, to characterize the degradation of any perfluoroalkyl acid. Elimination half-lives were 1.9±0.5 and 2.8±0.8 days for C6/C8PFPiA and C8/C8PFPiA, respectively, and 0.95±0.17 days for C8PFPA. Although elimination half-lives were not determined for 1H-PFAs, concentrations were higher than the corresponding PFPAs 48 h after rats were dosed with PFPiAs, suggestive of slower elimination. Conclusions: PFPiAs were metabolized in Sprague-Dawley rats to form persistent PFPAs as well as 1H-PFAs, which contain a labile hydrogen that may undergo further metabolism. These results in rats produced preliminary findings of the pharmacokinetics and metabolism of PFPiAs, which should be further investigated in humans. If there is a parallel between the disposition of these chemicals in humans and rats, then humans with detectable amounts of PFPiAs in their blood may be undergoing continuous exposure. https://doi.org/10.1289/EHP1841 PMID:29135439

The nature of the structural phase transition from the hexagonal (4H) phase to the cubic (3C) phase of silver.

PubMed

Chakraborty, Indrani; Shirodkar, Sharmila N; Gohil, Smita; Waghmare, Umesh V; Ayyub, Pushan

2014-03-19

The phase transition from the hexagonal 4H polytype of silver to the commonly known 3C (fcc) phase was studied in detail using x-ray diffraction, electron microscopy, differential scanning calorimetry and Raman spectroscopy. The phase transition is irreversible and accompanied by extensive microstructural changes and grain growth. Detailed scanning and isothermal calorimetric analysis suggests that it is an autocatalytic transformation. Though the calorimetric data suggest an exothermic first-order phase transition with an onset at 155.6 °C (for a heating rate of 2 K min(-1)) and a latent heat of 312.9 J g(-1), the microstructure and the electrical resistance appear to change gradually from much lower temperatures. The 4H phase shows a Raman active mode at 64.3 cm(-1) (at 4 K) that undergoes mode softening as the 4H → 3C transformation temperature is approached. A first-principles density functional theory calculation shows that the stacking fault energy of 4H-Ag increases monotonically with temperature. That 4H-Ag has a higher density of stacking faults than 3C-Ag, implies the metastability of the former at higher temperatures. Energetically, the 4H phase is intermediate between the hexagonal 2H phase and the 3C ground state, as indicated by the spontaneous transformation of the 2H to the 4H phase at -4 °C. Our data appear to indicate that the 4H-Ag phase is stabilized at reduced dimensions and thermally induced grain growth is probably responsible for triggering the irreversible transformation to cubic Ag.

Dual color microscopic imagery of cells expressing the green fluorescent protein and a red-shifted variant.

PubMed

Yang, T T; Kain, S R; Kitts, P; Kondepudi, A; Yang, M M; Youvan, D C

1996-01-01

The green fluorescent protein (GFP) from the jellyfish, Aequorea victoria, has become a versatile reporter for monitoring gene expression and protein localization in a variety of cells and organisms. GFP emits bright green light (lambda max = 510 nm) when excited with ultraviolet (UV) or blue light (lambda max = 395 nm, minor peak at 470 nm). The chromophore in GFP is intrinsic to the primary structure of the protein, and fluorescence from GFP does not require additional gene products, substrates or other factors. GFP fluorescence is stable, species-independent and can be monitored noninvasively using the techniques of fluorescence microscopy and flow cytometry [Chalfie et al., Science 263 (1994) 802-805; Stearns, Curr. Biol. 5 (1995) 262-264]. The protein appears to undergo an autocatalytic reaction to create the fluorophore [Heim et al., Proc. Natl. Acad. Sci. USA 91 (1994) 12501-12504] in a process involving cyclization of a Tyr66 aa residue. Recently [Delagrave et al., Bio/Technology 13 (1995) 151-154], a combinatorial mutagenic strategy was targeted at aa 64 through 69, which spans the chromophore of A. victoria GFP, yielding a number of different mutants with red-shifted fluorescence excitation spectra. One of these, RSGFP4, retains the characteristic green emission spectra (lambda max = 505 nm), but has a single excitation peak (lambda max = 490 nm). The fluorescence properties of RSGFP4 are similar to those of another naturally occurring GFP from the sea pansy, Renilla reniformis [Ward and Cormier, Photobiochem. Photobiol. 27 (1978) 389-396]. In the present study, we demonstrate by fluorescence microscopy that selective excitation of A. victoria GFP and RSGFP4 allows for spectral separation of each fluorescent signal, and provides the means to image these signals independently in a mixed population of bacteria or mammalian cells.

The C terminus of the catalytic domain of type A botulinum neurotoxin may facilitate product release from the active site.

PubMed

Mizanur, Rahman M; Frasca, Verna; Swaminathan, Subramanyam; Bavari, Sina; Webb, Robert; Smith, Leonard A; Ahmed, S Ashraf

2013-08-16

Botulinum neurotoxins are the most toxic of all compounds. The toxicity is related to a poor zinc endopeptidase activity located in a 50-kDa domain known as light chain (Lc) of the toxin. The C-terminal tail of Lc is not visible in any of the currently available x-ray structures, and it has no known function but undergoes autocatalytic truncations during purification and storage. By synthesizing C-terminal peptides of various lengths, in this study, we have shown that these peptides competitively inhibit the normal catalytic activity of Lc of serotype A (LcA) and have defined the length of the mature LcA to consist of the first 444 residues. Two catalytically inactive mutants also inhibited LcA activity. Our results suggested that the C terminus of LcA might interact at or near its own active site. By using synthetic C-terminal peptides from LcB, LcC1, LcD, LcE, and LcF and their respective substrate peptides, we have shown that the inhibition of activity is specific only for LcA. Although a potent inhibitor with a Ki of 4.5 μm, the largest of our LcA C-terminal peptides stimulated LcA activity when added at near-stoichiometric concentration to three versions of LcA differing in their C-terminal lengths. The result suggested a product removal role of the LcA C terminus. This suggestion is supported by a weak but specific interaction determined by isothermal titration calorimetry between an LcA C-terminal peptide and N-terminal product from a peptide substrate of LcA. Our results also underscore the importance of using a mature LcA as an inhibitor screening target.

Approaches to the origin of life on Earth.

PubMed

Kauffman, Stuart A

2011-11-18

I discuss briefly the history of the origin of life field, focusing on the "Miller" era of prebiotic synthesis, through the "Orgel" era seeking enzyme free template replication of single stranded RNA or similar polynucleotides, to the RNA world era with one of its foci on a ribozyme with the capacity to act as a polymerase able to copy itself. I give the history of the independent invention in 1971 by T. Ganti, M. Eigen and myself of three alternative theories of the origin of molecular replication: the Chemotron, the Hypercycle, and Collectively Autocatalytic Sets, CAS, respectively. To date, only collectively autocatalytic DNA, RNA, and peptide sets have achieved molecular reproduction of polymers. Theoretical work and experimental work on CAS both support their plausibility as models of openly evolvable protocells, if housed in dividing compartments such as dividing liposomes. My own further hypothesis beyond that of CAS in themselves, of their formation as a phase transition in complex chemical reaction systems of substrates, reactions and products, where the molecules in the system are candidates to catalyze the very same reactions, now firmly established as theorems, awaits experimental proof using combinatorial chemistry to make libraries of stochastic DNA, RNA and/or polypeptides, or other classes of molecules to test the hypothesis that molecular polymer reproduction has emerged as a true phase transition in complex chemical reaction systems. I remark that my colleague Marc Ballivet of the University of Geneva and I, may have issued the first publications discussing what became combinatorial chemistry, in published issued patents in 1987, 1989 and later, in this field.

Crystallization kinetics of orthorhombic paracetamol from supercooled melts studied by non-isothermal DSC.

PubMed

Nikolakakis, Ioannis; Kachrimanis, Kyriakos

2017-02-01

A simple and highly reproducible procedure was established for the study of orthorhombic paracetamol crystallization kinetics, comprising melting, quench-cooling of the melt and scanning the formed glass by DSC at different heating rates. Results were analyzed on the basis of the mean as well as local values of the Avrami exponent, n, the energy of activation, as well as the Šesták-Berggren two-parameter autocatalytic kinetic model. The mean value of the Avrami kinetic exponent, n, ranged between 3 and 5, indicating deviation from the nucleation and growth mechanism underlying the Johnson-Mehl, Avrami-Kolmogorov (JMAK) model. To verify the extent of the deviation, local values of the Avrami exponent as a function of the volume fraction transformed were calculated. Inspection of the local exponent values indicates that the crystallization mechanism changes over time, possibly reflecting the uncertainty of crystallization onset, instability of nucleation due to an autocatalytic effect of the crystalline phase, and growth anisotropy due to impingement of spherulites in the last stages of crystallization. The apparent energy of activation, E a , has a rather low mean value, close to 81 kJ/mol, which is in agreement with the observed instability of glassy-state paracetamol. Isoconversional methods revealed that E a tends to decrease with the volume fraction transformed, possibly because of the different energy demands of nucleation and growth. The exponents of the Šesták-Berggren two-parameter model showed that the crystallized fraction influences the process, confirming the complexity of the crystallization mechanism.

A critical tryptophan and Ca2+ in activation and catalysis of TPPI, the enzyme deficient in classic late-infantile neuronal ceroid lipofuscinosis.

PubMed

Kuizon, Salomon; DiMaiuta, Kathleen; Walus, Marius; Jenkins, Edmund C; Kuizon, Marisol; Kida, Elizabeth; Golabek, Adam A; Espinoza, Daniel O; Pullarkat, Raju K; Junaid, Mohammed A

2010-08-03

Tripeptidyl aminopeptidase I (TPPI) is a crucial lysosomal enzyme that is deficient in the fatal neurodegenerative disorder called classic late-infantile neuronal ceroid lipofuscinosis (LINCL). It is involved in the catabolism of proteins in the lysosomes. Recent X-ray crystallographic studies have provided insights into the structural/functional aspects of TPPI catalysis, and indicated presence of an octahedrally coordinated Ca(2+). Purified precursor and mature TPPI were used to study inhibition by NBS and EDTA using biochemical and immunological approaches. Site-directed mutagenesis with confocal imaging technique identified a critical W residue in TPPI activity, and the processing of precursor into mature enzyme. NBS is a potent inhibitor of the purified TPPI. In mammalian TPPI, W542 is critical for tripeptidyl peptidase activity as well as autocatalysis. Transfection studies have indicated that mutants of the TPPI that harbor residues other than W at position 542 have delayed processing, and are retained in the ER rather than transported to lysosomes. EDTA inhibits the autocatalytic processing of the precursor TPPI. We propose that W542 and Ca(2+) are critical for maintaining the proper tertiary structure of the precursor proprotein as well as the mature TPPI. Additionally, Ca(2+) is necessary for the autocatalytic processing of the precursor protein into the mature TPPI. We have identified NBS as a potent TPPI inhibitor, which led in delineating a critical role for W542 residue. Studies with such compounds will prove valuable in identifying the critical residues in the TPPI catalysis and its structure-function analysis.

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

Argonaute-based programmable RNase as a tool for cleavage of highly-structured RNA.

PubMed

Dayeh, Daniel M; Cantara, William A; Kitzrow, Jonathan P; Musier-Forsyth, Karin; Nakanishi, Kotaro

2018-06-12

The recent identification and development of RNA-guided enzymes for programmable cleavage of target nucleic acids offers exciting possibilities for both therapeutic and biotechnological applications. However, critical challenges such as expensive guide RNAs and inability to predict the efficiency of target recognition, especially for highly-structured RNAs, remain to be addressed. Here, we introduce a programmable RNA restriction enzyme, based on a budding yeast Argonaute (AGO), programmed with cost-effective 23-nucleotide (nt) single-stranded DNAs as guides. DNA guides offer the advantage that diverse sequences can be easily designed and purchased, enabling high-throughput screening to identify optimal recognition sites in the target RNA. Using this DNA-induced slicing complex (DISC) programmed with 11 different guide DNAs designed to span the sequence, sites of cleavage were identified in the 352-nt human immunodeficiency virus type 1 5'-untranslated region. This assay, coupled with primer extension and capillary electrophoresis, allows detection and relative quantification of all DISC-cleavage sites simultaneously in a single reaction. Comparison between DISC cleavage and RNase H cleavage reveals that DISC not only cleaves solvent-exposed sites, but also sites that become more accessible upon DISC binding. This study demonstrates the advantages of the DISC system for programmable cleavage of highly-structured, functional RNAs.

Association of a peptoid ligand with the apical loop of pri-miR-21 inhibits cleavage by Drosha

PubMed Central

Diaz, Jason P.; Chirayil, Rachel; Chirayil, Sara; Tom, Martin; Head, Katie J.; Luebke, Kevin J.

2014-01-01

We have found a small molecule that specifically inhibits cleavage of a precursor to the oncogenic miRNA, miR-21, by the microprocessor complex of Drosha and DGCR8. We identified novel ligands for the apical loop of this precursor from a screen of 14,024 N-substituted oligoglycines (peptoids) in a microarray format. Eight distinct compounds with specific affinity were obtained, three having affinities for the targeted loop in the low micromolar range and greater than 15-fold discrimination against a closely related hairpin. One of these compounds completely inhibits microprocessor cleavage of a miR-21 primary transcript at concentrations at which cleavage of another miRNA primary transcript, pri-miR-16, is little affected. The apical loop of pri-miR-21, placed in the context of pri-miR-16, is sufficient for inhibition of microprocessor cleavage by the peptoid. This compound also inhibits cleavage of pri-miR-21 containing the pri-miR-16 apical loop, suggesting an additional site of association within pri-miR-21. The reported peptoid is the first example of a small molecule that inhibits microprocessor cleavage by binding to the apical loop of a pri-miRNA. PMID:24497550

Nicked-site substrates for a serine recombinase reveal enzyme-DNA communications and an essential tethering role of covalent enzyme-DNA linkages.

PubMed

Olorunniji, Femi J; McPherson, Arlene L; Pavlou, Hania J; McIlwraith, Michael J; Brazier, John A; Cosstick, Richard; Stark, W Marshall

2015-07-13

To analyse the mechanism and kinetics of DNA strand cleavages catalysed by the serine recombinase Tn3 resolvase, we made modified recombination sites with a single-strand nick in one of the two DNA strands. Resolvase acting on these sites cleaves the intact strand very rapidly, giving an abnormal half-site product which accumulates. We propose that these reactions mimic second-strand cleavage of an unmodified site. Cleavage occurs in a synapse of two sites, held together by a resolvase tetramer; cleavage at one site stimulates cleavage at the partner site. After cleavage of a nicked-site substrate, the half-site that is not covalently linked to a resolvase subunit dissociates rapidly from the synapse, destabilizing the entire complex. The covalent resolvase-DNA linkages in the natural reaction intermediate thus perform an essential DNA-tethering function. Chemical modifications of a nicked-site substrate at the positions of the scissile phosphodiesters result in abolition or inhibition of resolvase-mediated cleavage and effects on resolvase binding and synapsis, providing insight into the serine recombinase catalytic mechanism and how resolvase interacts with the substrate DNA. © The Author(s) 2015. Published by Oxford University Press on behalf of Nucleic Acids Research.

Thermo- and light-regulated formation and disintegration of double hydrophilic block copolymer assemblies with tunable fluorescence emissions.

PubMed

Wu, Yonghao; Hu, Huamin; Hu, Jinming; Liu, Tao; Zhang, Guoying; Liu, Shiyong

2013-03-19

We report on thermo- and light-regulated formation and disintegration of double hydrophilic block copolymer (DHBC) micelles associated with tunable fluorescence emissions by employing two types of DHBCs covalently labeled with fluorescence resonance energy transfer (FRET) donor and acceptor moieties, respectively, within the light and temperature dually responsive block. Both DHBCs are molecularly soluble at room temperature in their aqueous mixture, whereas, upon heating to above the critical micellization temperature (CMT, ~31 °C), they coassemble into mixed micelles possessing hydrophilic coronas and mixed cores containing FRET donors and acceptors. Accordingly, the closer spatial proximity between the FRET pair (NBDAE and RhBEA moieties) within micellar cores leads to substantially enhanced FRET efficiency, compared to that in the non-aggregated unimer state. Moreover, upon UV irradiation, the light-reactive moieties undergo light-cleavage reaction and transform into negatively charged carboxylate residues, leading to elevated CMT (∼46 °C). Thus, thermo-induced mixed micelles in the intermediate temperature range (31 °C < T < 46 °C) undergo light-triggered disintegration into unimers, accompanied with the decrease of FRET efficiency. Overall, the coassembly and disassembly occurring in the mixed DHBC solution can be dually regulated by temperature and UV irradiation, and most importantly, these processes can be facilely monitored via changes in FRET efficiency and distinct emission colors.

Fundamental study of hydrogen-attachment-induced peptide fragmentation occurring in the gas phase and during the matrix-assisted laser desorption/ionization process.

PubMed

Asakawa, Daiki; Takahashi, Hidenori; Iwamoto, Shinichi; Tanaka, Koichi

2018-05-09

Mass spectrometry with hydrogen-radical-mediated fragmentation techniques has been used for the sequencing of proteins/peptides. The two methods, matrix-assisted laser desorption/ionization in-source decay (MALDI-ISD) and hydrogen attachment/abstraction dissociation (HAD) are known as hydrogen-radical-mediated fragmentation techniques. MALDI-ISD occurs during laser induced desorption processes, whereas HAD utilizes the association of hydrogen with peptide ions in the gas phase. In this study, the general mechanisms of MALDI-ISD and HAD of peptides were investigated. We demonstrated the fragmentation of four model peptides and investigated the fragment formation pathways using density functional theory (DFT) calculations. The current experimental and computational joint study indicated that MALDI-ISD and HAD produce aminoketyl radical intermediates, which immediately undergo radical-induced cleavage at the N-Cα bond located on the C-terminal side of the radical site, leading to the c'/z˙ fragment pair. In the case of MALDI-ISD, the z˙ fragments undergo a subsequent reaction with the matrix to give z' and matrix adducts of the z fragments. In contrast, the c' and z˙ fragments react with hydrogen atoms during the HAD processes, and various fragment species, such as c˙, c', z˙ and z', were observed in the HAD-MS/MS mass spectra.

Direct Photolysis Rates and Transformation Pathways of the Lampricides TFM and Niclosamide in Simulated Sunlight.

PubMed

McConville, Megan B; Hubert, Terrance D; Remucal, Christina K

2016-09-20

The lampricides 3-trifluoromethyl-4-nitrophenol (TFM) and 2',5-dichloro-4'-nitrosalicylanilide (niclosamide) are directly added to many tributaries of the Great Lakes that harbor the invasive parasitic sea lamprey. Despite their long history of use, the fate of lampricides is not well understood. This study evaluates the rate and pathway of direct photodegradation of both lampricides under simulated sunlight. The estimated half-lives of TFM range from 16.6 ± 0.2 h (pH 9) to 32.9 ± 1.0 h (pH 6), while the half-lives of niclosamide range from 8.88 ± 0.52 days (pH 6) to 382 ± 83 days (pH 9) assuming continuous irradiation over a water depth of 55 cm. Both compounds degrade to form a series of aromatic intermediates, simple organic acids, ring cleavage products, and inorganic ions. Experimental data were used to construct a kinetic model which demonstrates that the aromatic products of TFM undergo rapid photolysis and emphasizes that niclosamide degradation is the rate-limiting step to dehalogenation and mineralization of the lampricide. This study demonstrates that TFM photodegradation is likely to occur on the time scale of lampricide applications (2-5 days), while niclosamide, the less selective lampricide, will undergo minimal direct photodegradation during its passage to the Great Lakes.

Direct photolysis rates and transformation pathways of the lampricides TFM and niclosamide in simulated sunlight

USGS Publications Warehouse

McConville, Megan B.; Hubert, Terrance D.; Remucal, Christina K.

2016-01-01

The lampricides 3-trifluoromethyl-4-nitrophenol (TFM) and 2′,5-dichloro-4′-nitrosalicylanilide (niclosamide) are directly added to many tributaries of the Great Lakes that harbor the invasive parasitic sea lamprey. Despite their long history of use, the fate of lampricides is not well understood. This study evaluates the rate and pathway of direct photodegradation of both lampricides under simulated sunlight. The estimated half-lives of TFM range from 16.6 ± 0.2 h (pH 9) to 32.9 ± 1.0 h (pH 6), while the half-lives of niclosamide range from 8.88 ± 0.52 days (pH 6) to 382 ± 83 days (pH 9) assuming continuous irradiation over a water depth of 55 cm. Both compounds degrade to form a series of aromatic intermediates, simple organic acids, ring cleavage products, and inorganic ions. Experimental data were used to construct a kinetic model which demonstrates that the aromatic products of TFM undergo rapid photolysis and emphasizes that niclosamide degradation is the rate-limiting step to dehalogenation and mineralization of the lampricide. This study demonstrates that TFM photodegradation is likely to occur on the time scale of lampricide applications (2–5 days), while niclosamide, the less selective lampricide, will undergo minimal direct photodegradation during its passage to the Great Lakes.

The Zebrafish G12 Gene is required for Nuclear Positioning and Cell Migrations during Early Development

NASA Technical Reports Server (NTRS)

Reinsch, S. S.; Conway, G. C.

2003-01-01

After fertilization Zebrafish embryos undergo synchronous cleavage to form a blastula of cells sitting upon a single multinucleate yolk cell. At the beginning of gastrulation these cells undergo extensive cell migrations to form the major body axes. We have discovered a gene, G12, which is required for cell migrations and positioning of nuclei in the large syncytial yolk cell. Overexpression of a G12-GFP fusion protein is not toxic and shows that the protein localizes inside the yolk cell to the yolk nuclei, microtubules, and to the margin between the blastomeres and the large yolk cell. Morpholino (MO) injection into the 1-cell embryo or into just the yolk syncytium conipletely inhibits cell migrations, doming of the yolk cell, and positioning of nuclei around the margin. This effect can be partially rescued by injection of G12-GFP encoding RNA. Given the known role of microtubules in nuclear positioning of yolk nuclei in Zebrafish, we investigated the microtubules in morpholiiio injected and rescued embryos. We find that microtubules are sparse and disorganized in MO-injected embryos and are restored to normal organization upon G12-GFP rescue. G12 plays a pivotal role in organization of inicrotubules during early development. G12 is highly conserved in vertebrates and two homologues exist in the human genome. One of the human hoinologues is amplified in aggressive breast tumors.

Mechanism of Intramembrane Cleavage of Alcadeins by γ-Secretase

PubMed Central

Piao, Yi; Kimura, Ayano; Urano, Satomi; Saito, Yuhki; Taru, Hidenori; Yamamoto, Tohru; Hata, Saori; Suzuki, Toshiharu

2013-01-01

Background Alcadein proteins (Alcs; Alcα, Alcβand Alcγ) are predominantly expressed in neurons, as is Alzheimer's β-amyloid (Aβ) precursor protein (APP). Both Alcs and APP are cleaved by primary α- or β-secretase to generate membrane-associated C-terminal fragments (CTFs). Alc CTFs are further cleaved by γ-secretase to secrete p3-Alc peptide along with the release of intracellular domain fragment (Alc ICD) from the membrane. In the case of APP, APP CTFβ is initially cleaved at the ε-site to release the intracellular domain fragment (AICD) and consequently the γ-site is determined, by which Aβ generates. The initial ε-site is thought to define the final γ-site position, which determines whether Aβ40/43 or Aβ42 is generated. However, initial intracellular ε-cleavage sites of Alc CTF to generate Alc ICD and the molecular mechanism that final γ-site position is determined remains unclear in Alcs. Methodology Using HEK293 cells expressing Alcs plus presenilin 1 (PS1, a catalytic unit of γ-secretase) and the membrane fractions of these cells, the generation of p3-Alc possessing C-terminal γ-cleavage site and Alc ICD possessing N-terminal ε-cleavage site were analysed with MALDI-TOF/MS. We determined the initial ε-site position of all Alcα, Alcβ and Alcγ, and analyzed the relationship between the initially determined ε-site position and the final γ-cleavage position. Conclusions The initial ε-site position does not always determine the final γ-cleavage position in Alcs, which differed from APP. No additional γ-cleavage sites are generated from artificial/non-physiological positions of ε-cleavage for Alcs, while the artificial ε-cleavage positions can influence in selection of physiological γ-site positions. Because alteration of γ-secretase activity is thought to be a pathogenesis of sporadic Alzheimer's disease, Alcs are useful and sensitive substrate to detect the altered cleavage of substrates by γ-secretase, which may be induced by malfunction of γ-secretase itself or changes of membrane environment for enzymatic reaction. PMID:23658629

Changes in Contact Area in Meniscus Horizontal Cleavage Tears Subjected to Repair and Resection.

PubMed

Beamer, Brandon S; Walley, Kempland C; Okajima, Stephen; Manoukian, Ohan S; Perez-Viloria, Miguel; DeAngelis, Joseph P; Ramappa, Arun J; Nazarian, Ara

2017-03-01

To assess the changes in tibiofemoral contact pressure and contact area in human knees with a horizontal cleavage tear before and after treatment. Ten human cadaveric knees were tested. Pressure sensors were placed under the medial meniscus and the knees were loaded at twice the body weight for 20 cycles at 0°, 10°, and 20° of flexion. Contact area and pressure were recorded for the intact meniscus, the meniscus with a horizontal cleavage tear, after meniscal repair, after partial meniscectomy (single leaflet), and after subtotal meniscectomy (double leaflet). The presence of a horizontal cleavage tear significantly increased average peak contact pressure and reduced effective average tibiofemoral contact area at all flexion angles tested compared with the intact state (P < .03). There was approximately a 70% increase in contact pressure after creation of the horizontal cleavage tear. Repairing the horizontal cleavage tear restored peak contact pressures and areas to within 15% of baseline, statistically similar to the intact state at all angles tested (P < .05). Partial meniscectomy and subtotal meniscectomy significantly increased average peak contact pressure and reduced average contact area at all degrees of flexion compared with the intact state (P < .05). The presence of a horizontal cleavage tear in the medial meniscus causes a significant reduction in contact area and a significant elevation in contact pressure. These changes may accelerate joint degeneration. A suture-based repair of these horizontal cleavage tears returns the contact area and contact pressure to nearly normal, whereas both partial and subtotal meniscectomy lead to significant reductions in contact area and significant elevations in contact pressure within the knee. Repairing horizontal cleavage tears may lead to improved clinical outcomes by preserving meniscal tissue and the meniscal function. Understanding contact area and peak contact pressure resulting from differing strategies for treating horizontal cleavage tears will allow the surgeon to evaluate the best strategy for treating his or her patients who present with this meniscal pathology. Copyright © 2016 Arthroscopy Association of North America. Published by Elsevier Inc. All rights reserved.

Thrombin specificity. Requirement for apolar amino acids adjacent to the thrombin cleavage site of polypeptide substrate.

PubMed

Chang, J Y

1985-09-02

alpha-Thrombin cleavage of 30 polypeptide hormones and their derivatives were analysed by quantitative amino-terminal analysis. The polypeptides included secretin, vasoactive intestinal polypeptide, cholecystokinin fragment, dynorphin A, somatostatins, gastrin-releasing peptide, calcitonins and human parathyroid hormone fragment. Most of them were selected mainly on the ground that they contain sequence structures homologous to the well known tripeptide substrates of alpha-thrombin. All selected polypeptides have one single major cleavage site and both Arg-Xaa and Lys-Xaa bonds were found to be selectively cleaved by alpha-thrombin. Under fixed conditions (1 nmol polypeptide/0.5 NIH unit alpha-thrombin in 20 microliters of 50 mM ammonium bicarbonate at 25 degrees C), the time required for 50% cleavage ranges from less than 1 min to longer than 24 h. Heparin invariably enhanced thrombin cleavage on all polypeptide analysed. The optimum cleavage site for alpha-thrombin has the structures of (a) P4-P3-Pro-Arg-P1'-P2', where P3 and P4 are hydrophobic amino acid and P1', P2' are nonacidic amino acids and (b) P2-Arg-P1', where P2 or P1' are Gly. The requirement for hydrophobic P3 and P4 was further demonstrated by the drastic decrease of thrombin cleavage rates in both gastrin-releasing peptide and calcitonins after chemical removal of hydrophobic P3 and P4 residues. The requirement for nonacidic P1' and P2' residues was demonstrated by the drastic increase of thrombin cleavage rates in both calcitonin and parathyroid hormone fragments, after specific chemical modification of acidic P1' and P2' residues. These findings confirm the importance of hydrophobic P2-P4 residues for thrombin specificity and provide new evidence to indicate that apolar P1' and P2' residues are also crucial for thrombin specificity. It is concluded that specific cleavage of polypeptides by alpha-thrombin can be reasonably predicted and that chemical modification can be a useful tool in enhancing thrombin cleavage.

RNase P cleaves transient structures in some riboswitches.

PubMed

Altman, Sidney; Wesolowski, Donna; Guerrier-Takada, Cecilia; Li, Yong

2005-08-09

RNase P from Escherichia coli cleaves the coenzyme B12 riboswitch from E. coli and a similar one from Bacillus subtilis. The cleavage sites do not occur in any recognizable structure, as judged from theoretical schemes that have been drawn for these 5' UTRs. However, it is possible to draw a scheme that is a good representation of the E. coli cleavage site for RNase P and for the cleavage site in B. subtilis. These data indicate that transient structures are important in RNase P cleavage and in riboswitch function. Coenzyme B12 has a small inhibitory effect on E. coli RNase P cleavage of the E. coli riboswitch. Both E. coli RNase P and a partially purified RNase P from Aspergillus nidulans mycelia succeeded in cleaving a putative arginine riboswitch from A. nidulans. The cleavage site may be a representative of another model substrate for eukaryotic RNase P. This 5' UTR controls splicing of the arginase mRNA in A. nidulans. Four other riboswitches in E. coli were not cleaved by RNase P under the conditions tested.

RNase P cleaves transient structures in some riboswitches

PubMed Central

Altman, Sidney; Wesolowski, Donna; Guerrier-Takada, Cecilia; Li, Yong

2005-01-01

RNase P from Escherichia coli cleaves the coenzyme B12 riboswitch from E. coli and a similar one from Bacillus subtilis. The cleavage sites do not occur in any recognizable structure, as judged from theoretical schemes that have been drawn for these 5′ UTRs. However, it is possible to draw a scheme that is a good representation of the E. coli cleavage site for RNase P and for the cleavage site in B. subtilis. These data indicate that transient structures are important in RNase P cleavage and in riboswitch function. Coenzyme B12 has a small inhibitory effect on E. coli RNase P cleavage of the E. coli riboswitch. Both E. coli RNase P and a partially purified RNase P from Aspergillus nidulans mycelia succeeded in cleaving a putative arginine riboswitch from A. nidulans. The cleavage site may be a representative of another model substrate for eukaryotic RNase P. This 5′ UTR controls splicing of the arginase mRNA in A. nidulans. Four other riboswitches in E. coli were not cleaved by RNase P under the conditions tested. PMID:16061811

Classical Swine Fever Virus Glycoprotein Erns Is an Endoribonuclease with an Unusual Base Specificity

PubMed Central

Hausmann, Yvonne; Roman-Sosa, Gleyder; Thiel, Heinz-Jürgen; Rümenapf, Till

2004-01-01

The glycoprotein Erns of pestiviruses is a virion-associated and -secreted RNase that is involved in virulence. The requirements at the cleavage site in heteropolymeric RNA substrates were studied for Erns. Limited digestion of heteropolymeric RNA substrates indicated a cleavage 5′ of uridine residues irrespective of the preceding nucleotide (Np/U). To further study specificity radiolabeled RNA, molecules of 45 to 56 nucleotides in length were synthesized that contained no or a single Np/U cleavage site. Cleavage was only observed in substrates containing an ApU, CpU, GpU, or UpU dinucleotide and occurred in two steps, an initial NpU-specific and a consecutive unspecific degradation. The NpU-specific cleavage was resistant to 7 M urea while the second-order cleavage was sensitive to denaturation. Kinetic analyses revealed that Erns is a highly active endoribonuclease (kcat/Km = 2 × 106 to 10 × 106 M−1 s−1) with a strong affinity to NpU containing single-stranded RNA substrates (Km = 85 to 260 nM). PMID:15113930

AID-induced decrease in topoisomerase 1 induces DNA structural alteration and DNA cleavage for class switch recombination.

PubMed

Kobayashi, Maki; Aida, Masatoshi; Nagaoka, Hitoshi; Begum, Nasim A; Kitawaki, Yoko; Nakata, Mikiyo; Stanlie, Andre; Doi, Tomomitsu; Kato, Lucia; Okazaki, Il-mi; Shinkura, Reiko; Muramatsu, Masamichi; Kinoshita, Kazuo; Honjo, Tasuku

2009-12-29

To initiate class switch recombination (CSR) activation-induced cytidine deaminase (AID) induces staggered nick cleavage in the S region, which lies 5' to each Ig constant region gene and is rich in palindromic sequences. Topoisomerase 1 (Top1) controls the supercoiling of DNA by nicking, rotating, and religating one strand of DNA. Curiously, Top1 reduction or AID overexpression causes the genomic instability. Here, we report that the inactivation of Top1 by its specific inhibitor camptothecin drastically blocked both the S region cleavage and CSR, indicating that Top1 is responsible for the S region cleavage in CSR. Surprisingly, AID expression suppressed Top1 mRNA translation and reduced its protein level. In addition, the decrease in the Top1 protein by RNA-mediated knockdown augmented the AID-dependent S region cleavage, as well as CSR. Furthermore, Top1 reduction altered DNA structure of the Smu region. Taken together, AID-induced Top1 reduction alters S region DNA structure probably to non-B form, on which Top1 can introduce nicks but cannot religate, resulting in S region cleavage.

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

Cleavage Entropy as Quantitative Measure of Protease Specificity

PubMed Central

Fuchs, Julian E.; von Grafenstein, Susanne; Huber, Roland G.; Margreiter, Michael A.; Spitzer, Gudrun M.; Wallnoefer, Hannes G.; Liedl, Klaus R.

2013-01-01

A purely information theory-guided approach to quantitatively characterize protease specificity is established. We calculate an entropy value for each protease subpocket based on sequences of cleaved substrates extracted from the MEROPS database. We compare our results with known subpocket specificity profiles for individual proteases and protease groups (e.g. serine proteases, metallo proteases) and reflect them quantitatively. Summation of subpocket-wise cleavage entropy contributions yields a measure for overall protease substrate specificity. This total cleavage entropy allows ranking of different proteases with respect to their specificity, separating unspecific digestive enzymes showing high total cleavage entropy from specific proteases involved in signaling cascades. The development of a quantitative cleavage entropy score allows an unbiased comparison of subpocket-wise and overall protease specificity. Thus, it enables assessment of relative importance of physicochemical and structural descriptors in protease recognition. We present an exemplary application of cleavage entropy in tracing substrate specificity in protease evolution. This highlights the wide range of substrate promiscuity within homologue proteases and hence the heavy impact of a limited number of mutations on individual substrate specificity. PMID:23637583

LaSota fusion (F) cleavage motif-mediated fusion activity is affected by other regions of the F protein from different genotype Newcastle disease virus in a chimeric virus: implication for virulence attenuation.

PubMed

Kim, Shin-Hee; Xiao, Sa; Collins, Peter L; Samal, Siba K

2016-06-01

The cleavage site sequence of the fusion (F) protein contributes to a wide range of virulence of Newcastle disease virus (NDV). In this study, we identified other important amino acid sequences of the F protein that affect cleavage and modulation of fusion. We generated chimeric Beaudette C (BC) viruses containing the cleavage site sequence of avirulent strain LaSota (Las-Fc) together with various regions of the F protein of another virulent strain AKO. We found that the F1 subunit is important for cleavage inhibition. Further dissection of the F1 subunit showed that replacement of four amino acids in the BC/Las-Fc protein with their AKO counterparts (T341S, M384I, T385A and I386L) resulted in an increase in fusion and replication in vitro. In contrast, the mutation N403D greatly reduced cleavage and viral replication, and affected protein conformation. These findings will be useful in developing improved live NDV vaccines and vaccine vectors.

Real-time single cell analysis of Bid cleavage and translocation in cisplatin-induced apoptosis

NASA Astrophysics Data System (ADS)

Liu, Lei; Xing, Da; Pei, Yihui; Chen, Wei R.

2007-02-01

Cancer cell apoptosis can be induced by cisplatin, an efficient anticancer agent. However, its mechanism is not fully understood. Bcl-2 homology domain (BH) 3-only proteins couple stress signals to mitochondrial apoptotic pathways. Calpain-mediated cleavage of the BH3-only protein Bid into a 14 kD truncated protein (tBid) has been implicated in cisplatin-induced apoptotic pathway. We utilized a recombinant fluorescence resonance energy transfer (FRET) Bid probe to determine the kinetics of Bid cleavage during cisplatin-induced apoptosis in ASTC-a-1 cells. The cells were also co-transfected with Bid-CFP and DsRed-Mit to dynamically detect tBid translocation. Cells showed a cleavage of the Bid-FRET probe occurring at about 4-5 h after treated with 20 µM cisplatin. Cleavage of the Bid-FRET probe coincided with a translocation of tBid from the cytosolic to the mitochondria, and the translocation lasted about 1.5 h. Using real-time single-cell analysis, we first observed the kinetics of Bid cleavage and translocation to mitochondria in living cells during cisplatin-induced apoptosis.

Role of cleavage at the core-E1 junction of hepatitis C virus polyprotein in viral morphogenesis.

PubMed

Pène, Véronique; Lemasson, Matthieu; Harper, Francis; Pierron, Gérard; Rosenberg, Arielle R

2017-01-01

In hepatitis C virus (HCV) polyprotein sequence, core protein terminates with E1 envelope signal peptide. Cleavage by signal peptidase (SP) separates E1 from the complete form of core protein, anchored in the endoplasmic reticulum (ER) membrane by the signal peptide. Subsequent cleavage of the signal peptide by signal-peptide peptidase (SPP) releases the mature form of core protein, which preferentially relocates to lipid droplets. Both of these cleavages are required for the HCV infectious cycle, supporting the idea that HCV assembly begins at the surface of lipid droplets, yet SPP-catalyzed cleavage is dispensable for initiation of budding in the ER. Here we have addressed at what step(s) of the HCV infectious cycle SP-catalyzed cleavage at the core-E1 junction is required. Taking advantage of the sole system that has allowed visualization of HCV budding events in the ER lumen of mammalian cells, we showed that, unexpectedly, mutations abolishing this cleavage did not prevent but instead tended to promote the initiation of viral budding. Moreover, even though no viral particles were released from Huh-7 cells transfected with a full-length HCV genome bearing these mutations, intracellular viral particles containing core protein protected by a membrane envelope were formed. These were visualized by electron microscopy as capsid-containing particles with a diameter of about 70 nm and 40 nm before and after delipidation, respectively, comparable to intracellular wild-type particle precursors except that they were non-infectious. Thus, our results show that SP-catalyzed cleavage is dispensable for HCV budding per se, but is required for the viral particles to acquire their infectivity and secretion. These data support the idea that HCV assembly occurs in concert with budding at the ER membrane. Furthermore, capsid-containing particles did not accumulate in the absence of SP-catalyzed cleavage, suggesting the quality of newly formed viral particles is controlled before secretion.

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.

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.

Degradation of neurotensin by rat brain synaptic membranes: involvement of a thermolysin-like metalloendopeptidase (enkephalinase), angiotensin-converting enzyme, and other unidentified peptidases.

PubMed

Checler, F; Vincent, J P; Kitabgi, P

1983-08-01

Neurotensin was inactivated by membrane-bound and soluble degrading activities present in purified preparations of rat brain synaptic membranes. Degradation products were identified by HPLC and amino acid analysis. The major points of cleavage of neurotensin were the Arg8-Arg9, Pro10-Tyr11, and Tyr11-Ile12 peptide bonds with the membrane-bound activity and the Arg8-Arg9 and Pro10-Tyr11 bonds with the soluble activity. Several lines of evidence indicated that the cleavage of the Arg8-Arg9 bond by the membrane-bound activity resulted mainly from the conversion of neurotensin1-10 to neurotensin1-8 by a dipeptidyl carboxypeptidase. In particular, captopril inhibited this cleavage with an IC50 (5.7 nM) close to its K1 (7 nM) for angiotensin-converting enzyme. Thiorphan inhibited the cleavage at the Tyr11-Ile12 bond by the membrane-bound activity with an IC50 (17 nM) similar to its K1 (4.7 nM) for enkephalinase. Both cleavages were inhibited by 1,10-phenanthroline. These and other data suggested that angiotensin-converting enzyme and a thermolysin-like metalloendopeptidase (enkephalinase) were the membrane-bound peptidases responsible for cleavages at the Arg8-Arg9 and Tyr11-Ile12 bonds, respectively. In contrast, captopril had no effect on the cleavage at the Arg8-Arg9 bond by the soluble activity, indicating that the enzyme responsible for this cleavage was different from angiotensin-converting enzyme. The cleavage at the Pro10-Tyr11 bond by both the membrane-bound and the soluble activities appeared to be catalyzed by an endopeptidase different from known brain proline endopeptidases. The possibility is discussed that the enzymes described here participate in physiological mechanisms of neurotensin inactivation at the synaptic level.

Role of cleavage at the core-E1 junction of hepatitis C virus polyprotein in viral morphogenesis

PubMed Central

Pène, Véronique; Lemasson, Matthieu; Harper, Francis; Pierron, Gérard; Rosenberg, Arielle R.

2017-01-01

In hepatitis C virus (HCV) polyprotein sequence, core protein terminates with E1 envelope signal peptide. Cleavage by signal peptidase (SP) separates E1 from the complete form of core protein, anchored in the endoplasmic reticulum (ER) membrane by the signal peptide. Subsequent cleavage of the signal peptide by signal-peptide peptidase (SPP) releases the mature form of core protein, which preferentially relocates to lipid droplets. Both of these cleavages are required for the HCV infectious cycle, supporting the idea that HCV assembly begins at the surface of lipid droplets, yet SPP-catalyzed cleavage is dispensable for initiation of budding in the ER. Here we have addressed at what step(s) of the HCV infectious cycle SP-catalyzed cleavage at the core-E1 junction is required. Taking advantage of the sole system that has allowed visualization of HCV budding events in the ER lumen of mammalian cells, we showed that, unexpectedly, mutations abolishing this cleavage did not prevent but instead tended to promote the initiation of viral budding. Moreover, even though no viral particles were released from Huh-7 cells transfected with a full-length HCV genome bearing these mutations, intracellular viral particles containing core protein protected by a membrane envelope were formed. These were visualized by electron microscopy as capsid-containing particles with a diameter of about 70 nm and 40 nm before and after delipidation, respectively, comparable to intracellular wild-type particle precursors except that they were non-infectious. Thus, our results show that SP-catalyzed cleavage is dispensable for HCV budding per se, but is required for the viral particles to acquire their infectivity and secretion. These data support the idea that HCV assembly occurs in concert with budding at the ER membrane. Furthermore, capsid-containing particles did not accumulate in the absence of SP-catalyzed cleavage, suggesting the quality of newly formed viral particles is controlled before secretion. PMID:28437468

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.

Experiments on schistosity and slaty cleavage

USGS Publications Warehouse

Becker, George Ferdinand

1904-01-01

Schistosity as a structure is important, and it is a part of the business of geologists to explain its origin. Slaty cleavage has further and greater importance as a possible tectonic feature. Scarcely a great mountain range exists, or has existed, along the course of which belts of slaty rock are not found, the dip of the cleavage usually approaching verticality. Are these slate belts equivalent to minutely distributed step faults of great total throw, or do they indicate compression perpendicular to the cleavage without attendant relative dislocation? Evidently the answer to this question is of first importance in the interpretation of orogenic phenomena.

Antibodies to H2a and H2b histones from the sera of HIV-infected patients catalyze site-specific degradation of these histones.

PubMed

Baranova, Svetlana V; Dmitrienok, Pavel S; Ivanisenko, Nikita V; Buneva, Valentina N; Nevinsky, Georgy A

2017-06-01

Histones and their post-translational modifications have key roles in chromatin remodeling and gene transcription. Besides intranuclear functions, histones act as damage-associated molecules when they are released into the extracellular space. Administration of histones to animals leads to systemic inflammatory and toxic responses. Autoantibodies with enzymatic activities (abzymes) are distinctive features of some autoimmune and viral diseases. Electrophoretically homogeneous IgGs containing no canonical enzymes were isolated from the sera of HIV-infected patients by chromatography on several affinity sorbents including anti-histone Sepharose. In contrast to canonical proteases (trypsin, chymotrypsin, proteinase K), IgGs from HIV-infected patients specifically hydrolyzed only histones but not many other tested globular proteins. Using MALDI mass spectrometry the sites of H2a and H2b histone cleavage by anti-histone IgGs were determined for the first time. One cluster of H2a hydrolysis contains two major (↕) and four moderate (↓) cleavage sites: 31-H↓R↓L↓L↓R↕K G↕N-38. One major and two moderate sites of cleavage were revealed in the second cluster: 14-A↕KSRS↓SRA↓G-22. The third cluster corresponding to the H2a C-terminal part contains only five minor (†) sites of cleavage: 82-H†LQLAIRNDEELN†KLLG†RV†T†I-102. It was shown that two major and four moderate sites of cleavage were present in the main cluster of H2b hydrolysis: 46-K↕QvhpD↓TgiS↓SkA↓M↕GiM↓N-63. Two moderate sites of cleavage correspond to a relatively short 6-mer cluster: 12-K↓GskK↓A-17. The third relatively long 9-mer cluster contains one major and two minor sites of H2b cleavage: 80-L↕AHYN†KRS†T-88. In the nucleosome core particle, most of the major and moderate cleavage sites are located at the H2a/H2b interaction interface. Minor cleavage sites of H2a are involved in binding with H3 in the nucleosome core. Two moderate cleavage sites of H2b and one major cleavage site of H2a are located in the disordered N-terminal region interacting with DNA. According to the crystal structure of the nucleosome core particle, all identified cleavage sites are expected to affect H2a and H2b folding, nucleosome assembly, and binding of H2a and H2b with DNA. The existence of H2a and H2b hydrolyzing abzymes may be very important for the further understanding of unknown possibilities of immune systems and biological functions of antibodies.

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.

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