Analysis of the in vitro cleavage products of the tomato black ring virus RNA-1-encoded 250K polyprotein.

Science.gov (United States)

Demangeat, G; Greif, C; Hemmer, O; Fritsch, C

1990-08-01

Tomato black ring virus RNA-1 was translated in a rabbit reticulocyte lysate. The primary translation product of Mr 250K, which corresponds to its whole coding capacity, was synthesized within 45 min and, during further incubation in the translation medium, was proteolytically processed. Essentially, four cleavage products (P190, P120, P60 and P50) were detected and located within P250 by pulse-chase and immunoprecipitation experiments. P190 is an intermediate cleavage product which is further cleaved to form P60 and P120. P120, which contains the region that has been assigned to the virus protease and the virus polymerase, was not further cleaved in vitro.

Characterization of a Non-Canonical Signal Peptidase Cleavage Site in a Replication Protein from Tomato Ringspot Virus.

Directory of Open Access Journals (Sweden)

Ting Wei

Full Text Available The NTB-VPg polyprotein from tomato ringspot virus is an integral membrane replication protein associated with endoplasmic reticulum membranes. A signal peptidase (SPase cleavage was previously detected in the C-terminal region of NTB-VPg downstream of a 14 amino acid (aa-long hydrophobic region (termed TM2. However, the exact location of the cleavage site was not determined. Using in vitro translation assays, we show that the SPase cleavage site is conserved in the NTB-VPg protein from various ToRSV isolates, although the rate of cleavage varies from one isolate to another. Systematic site-directed mutagenesis of the NTB-VPg SPase cleavage sites of two ToRSV isolates allowed the identification of sequences that affect cleavage efficiency. We also present evidence that SPase cleavage in the ToRSV-Rasp2 isolate occurs within a GAAGG sequence likely after the AAG (GAAG/G. Mutation of a downstream MAAV sequence to AAAV resulted in SPase cleavage at both the natural GAAG/G and the mutated AAA/V sequences. Given that there is a distance of seven aa between the two cleavage sites, this indicates that there is flexibility in the positioning of the cleavage sites relative to the inner surface of the membrane and the SPase active site. SPase cleavage sites are typically located 3-7 aa downstream of the hydrophobic region. However, the NTB-VPg GAAG/G cleavage site is located 17 aa downstream of the TM2 hydrophobic region, highlighting unusual features of the NTB-VPg SPase cleavage site. A putative 11 aa-long amphipathic helix was identified immediately downstream of the TM2 region and five aa upstream of the GAAG/G cleavage site. Based on these results, we present an updated topology model in which the hydrophobic and amphipathic domains form a long tilted helix or a bent helix in the membrane lipid bilayer, with the downstream cleavage site(s oriented parallel to the membrane inner surface.

Kinetics of phycocyanobilin cleavage from C-phycocyanin by methanolysis

DEFF Research Database (Denmark)

Malwade, Chandrakant Ramkrishna; Roda Serrat, Maria Cinta; Christensen, Knud Villy

2016-01-01

Phycocyanobilin (PCB) is an important linear tetrapyrrolic molecule for food as well as pharmaceutical industry. It is obtained from blue-green algae, where it is attached covalently to phycobiliproteins (C-PC and APC) present in the light harvesting complexes. In this work, cleavage of PCB from...

Separation of foot-and-mouth disease virus leader protein activities; identification of mutants that retain efficient self-processing activity but poorly induce eIF4G cleavage.

Science.gov (United States)

Guan, Su Hua; Belsham, Graham J

2017-04-01

Foot-and-mouth disease virus is a picornavirus and its RNA genome encodes a large polyprotein. The N-terminal part of this polyprotein is the leader protein, a cysteine protease, termed Lpro. The virus causes the rapid inhibition of host cell cap-dependent protein synthesis within infected cells. This results from the Lpro-dependent cleavage of the cellular translation initiation factor eIF4G. Lpro also releases itself from the virus capsid precursor by cleaving the L/P1 junction. Using site-directed mutagenesis of the Lpro coding sequence, we have investigated the role of 51 separate amino acid residues in the functions of this protein. These selected residues either are highly conserved or are charged and exposed on the protein surface. Using transient expression assays, within BHK-21 cells, it was found that residues around the active site (W52, L53 and A149) of Lpro and others located elsewhere (K38, K39, R44, H138 and W159) are involved in the induction of eIF4G cleavage but not in the processing of the L/P1 junction. Modified viruses, encoding such amino acid substitutions within Lpro, can replicate in BHK-21 cells but did not grow well in primary bovine thyroid cells. This study characterizes mutant viruses that are deficient in blocking host cell responses to infection (e.g. interferon induction) and can assist in the rational design of antiviral agents targeting this process and in the production of attenuated viruses.

Functional Insights Revealed by the Kinetic Mechanism of CRISPR/Cas9.

Science.gov (United States)

Raper, Austin T; Stephenson, Anthony A; Suo, Zucai

2018-02-28

The discovery of prokaryotic adaptive immunity prompted widespread use of the RNA-guided clustered regularly interspaced short palindromic repeat (CRISPR)-associated (Cas) endonuclease Cas9 for genetic engineering. However, its kinetic mechanism remains undefined, and details of DNA cleavage are poorly characterized. Here, we establish a kinetic mechanism of Streptococcus pyogenes Cas9 from guide-RNA binding through DNA cleavage and product release. Association of DNA to the binary complex of Cas9 and guide-RNA is rate-limiting during the first catalytic turnover, while DNA cleavage from a pre-formed ternary complex of Cas9, guide-RNA, and DNA is rapid. Moreover, an extremely slow release of DNA products essentially restricts Cas9 to be a single-turnover enzyme. By simultaneously measuring the contributions of the HNH and RuvC nuclease activities of Cas9 to DNA cleavage, we also uncovered the kinetic basis by which HNH conformationally regulates the RuvC cleavage activity. Together, our results provide crucial kinetic and functional details regarding Cas9 which will inform gene-editing experiments, guide future research to understand off-target DNA cleavage by Cas9, and aid in the continued development of Cas9 as a biotechnological tool.

Primary structure, gene organization and polypeptide expression of poliovirus RNA

Energy Technology Data Exchange (ETDEWEB)

Kitamura, N. (State Univ. of New York, Stony Brook); Semler, B.L.; Rothberg, P.G.

1981-06-18

The primary structure of the poliovirus genome has been determined. The RNA molecule is 7433 nucleotides long, polyadenylated at the 3' terminus, and covalently linked to a small protein (VPg) at the 5' terminus. An open reading frame of 2207 consecutive triplets spans over 89% of the nucleotide sequence and codes for the viral polyprotein NCVPOO. Twelve viral polypeptides have been mapped by amino acid sequence analysis and were found to be proteolytic cleavage products of the polyprotein, cleavages occurring predominantly at Gln-Gly pairs.

Metabolic cleavage of cell-penetrating peptides in contact with epithelial models

DEFF Research Database (Denmark)

Tréhin, Rachel; Nielsen, Hanne Mørck; Jahnke, Heinz-Georg

2004-01-01

We assessed the metabolic degradation kinetics and cleavage patterns of some selected CPP (cell-penetrating peptides) after incubation with confluent epithelial models. Synthesis of N-terminal CF [5(6)-carboxyfluorescein]-labelled CPP, namely hCT (human calcitonin)-derived sequences, Tat(47-57) a...

Microsolvated Model for the Kinetics and Thermodynamics of Glycosidic Bond Dissociative Cleavage of Nucleoside D4G.

Science.gov (United States)

Jiang, Yang; Xue, Ying; Zeng, Yi

2018-02-15

Using the microsolvated model that involves explicit water molecules and implicit solvent in the optimization, two proposed dissociative hydrolysis mechanisms of 2',3'-didehydro-2',3'-dideoxyguanosine (d4G) have been first investigated by means of M06-2X(CPCM, water)/6-31++G(d,p) method. The glycosidic bond dissociation for the generation of the oxacarbenium ion intermediate is the rate-determining step (RDS). The subsequent nucleophilic water attack from different side of the oxacarbenium ion intermediate gives either the α-product [(2S,5S)-5-(hydroxymethyl)-2,5-dihydrofuran-2-ol] or β-product [(2R,5S)-5-(hydroxymethyl)-2,5-dihydrofuran-2-ol] and is thus referred to as α-path (inversion) and β-path (retention). Two to five explicit water molecules (n = 2-5) are considered in the microsolvated model, and n = 3 or 4 is the smallest model capable of minimizing the activation energy for α-path and β-path, respectively. Our theoretical results suggest that α-path (n = 3) is more kinetically favorable with lower free energy barrier (RDS) of 27.7 kcal mol -1 , in contrast to that of 30.7 kcal mol -1 for the β-path (n = 4). The kinetic preference of the α-path is rationalized by NBO analysis. Whereas thte β-path is more thermodynamically favorable over the α-path, where the formation of β-product and α-product are exergonic and endergonic, respectively, providing theoretical support for the experimental observation that the β-cleavage product was the major one after sufficient reaction time. Comparisons of d4G with analogous cyclo-d4G and dG from kinetic free energy barriers and thermodynamic heterolytic dissociation energies were also carried out. Our kinetic and thermodynamic results manifest that the order of glycosidic bond stability should be d4G < cyclo-d4G < dG, which agrees well with the reported experimental stability order of d4G compounds and analogues and gives further understanding on the influence of 6-cyclopropylamino and unsaturated ribose to

Oxidative cleavage of erucic acid for the synthesis of brassylic acid

Energy Technology Data Exchange (ETDEWEB)

Mohammed J. Nasrullah; Pooja Thapliyal; Erica N. Pfarr; Nicholas S. Dusek; Kristofer L. Schiele; James A. Bahr

2010-10-29

The main focus of this work is to synthesize Brassylic Acid (BA) using oxidative cleavage of Erucic Acid (EA). Crambe (Crambe abyssinica) is an industrial oilseed grown in North Dakota. Crambe has potential as an industrial fatty acid feedstock as a source of Erucic acid (EA). It has approximately 50-60 % of EA, a C{sub 22} monounsaturated fatty acid. Oxidative cleavage of unsaturated fatty acids derived from oilseeds produces long chain (9, 11, and 13 carbon atoms) dibasic and monobasic acids. These acids are known commercial feedstocks for the preparation of nylons, polyesters, waxes, surfactants, and perfumes. Other sources of EA are Rapeseed seed oil which 50-60 % of EA. Rapeseed is grown outside USA. The oxidative cleavage of EA was done using a high throughput parallel pressure reactor system. Kinetics of the reaction shows that BA yields reach a saturation at 12 hours. H{sub 2}WO{sub 4} was found to be the best catalyst for the oxidative cleavage of EA. High yields of BA were obtained at 80 C with bubbling of O{sub 2} or 10 bar of O{sub 2} for 12 hours.

Electrochemical bond cleavage in pesticide ioxynil. Kinetic analysis by voltammetry and impedance spectroscopy

Czech Academy of Sciences Publication Activity Database

Sokolová, R.; Giannarelli, S.; Fanelli, N.; Pospíšil, Lubomír

2017-01-01

Roč. 49, SI C (2017), s. 134-138 ISSN 0324-1130 Institutional support: RVO:61388963 Keywords : electrochemical impedance spectroscopy * rate constant * self-protonation * faradaic phase angle * halogen cleavage * EC processes fitting Subject RIV: CG - Electrochemistry OBOR OECD: Electrochemistry (dry cells, batteries, fuel cells, corrosion metals, electrolysis) Impact factor: 0.238, year: 2016

Epitope mapping porcine reproductive and respiratory syndrome virus by phage display: the nsp2 fragment of the replicase polyprotein contains a cluster of B-cell epitopes

DEFF Research Database (Denmark)

Oleksiewicz, M.B.; Bøtner, Anette; Toft, P.

2001-01-01

to 53 amino acids in length. In the replicase polyprotein, a total of eight ES were identified, six of which localized to the Nsp2 replicase polyprotein processing end product, In the structural proteins, a total of two ES were identified, in the ORF3 and ORF4 minor envelope glycoproteins, The ORF4 ES...... screening with porcine sera and accounted for our failure to identify more than two ES in the structural genes of PRRSV, Genetic analysis showed that variable ES were also the most immunogenic in vivo. Serological analysis indicated differences in the immunoglobulin A responses between short-term and longer...

Electron spectroscopy of the interface carbon layer formation on the cleavage surfaces of the layered semiconductor In4Se3 crystals

International Nuclear Information System (INIS)

Galiy, P.V.; Musyanovych, A.V.; Nenchuk, T.M.

2005-01-01

The results of the quantitative X-ray photoelectron spectroscopy (XPS) and Auger electron spectroscopy (AES) of the interface carbon layer formation on the cleavage surfaces of the layered semiconductor In 4 Se 3 crystals are presented. The carbon coating formation occurs as the result of interaction of the air and residual gases atmosphere in ultra high vacuum (UHV) Auger spectrometer chamber with atomic clean interlayer cleavage surfaces of the crystals. The kinetics and peculiarities of interfacial carbon layer formation on the cleavage surfaces of the crystals, elemental and phase composition of the interface have been studied by quantitative XPS, AES and mass-spectroscopy

A Polyprotein-Expressing Salmonid Alphavirus Replicon Induces Modest Protection in Atlantic Salmon (Salmo Salar Against Infectious Pancreatic Necrosis

Directory of Open Access Journals (Sweden)

Azila Abdullah

2015-01-01

Full Text Available Vaccination is an important strategy for the control and prevention of infectious pancreatic necrosis (IPN in farmed Atlantic salmon (Salmo salar in the post-smolt stage in sea-water. In this study, a heterologous gene expression system, based on a replicon construct of salmonid alphavirus (SAV, was used for in vitro and in vivo expression of IPN virus proteins. The large open reading frame of segment A, encoding the polyprotein NH2-pVP2-VP4-VP3-COOH, as well as pVP2, were cloned and expressed by the SAV replicon in Chinook salmon embryo cells (CHSE-214 and epithelioma papulosum cyprini (EPC cells. The replicon constructs pSAV/polyprotein (pSAV/PP and pSAV/pVP2 were used to immunize Atlantic salmon (Salmo salar by a single intramuscular injection and tested in a subsequent IPN virus (IPNV challenge trial. A low to moderate protection against IPN was observed in fish immunized with the replicon vaccine that encoded the pSAV/PP, while the pSAV/pVP2 construct was not found to induce protection.

Pseudo-polyprotein translated from the full-length ORF1 of capillovirus is important for pathogenicity, but a truncated ORF1 protein without variable and CP regions is sufficient for replication.

Science.gov (United States)

Hirata, Hisae; Yamaji, Yasuyuki; Komatsu, Ken; Kagiwada, Satoshi; Oshima, Kenro; Okano, Yukari; Takahashi, Shuichiro; Ugaki, Masashi; Namba, Shigetou

2010-09-01

The first open-reading frame (ORF) of the genus Capillovirus encodes an apparently chimeric polyprotein containing conserved regions for replicase (Rep) and coat protein (CP), while other viruses in the family Flexiviridae have separate ORFs encoding these proteins. To investigate the role of the full-length ORF1 polyprotein of capillovirus, we generated truncation mutants of ORF1 of apple stem grooving virus by inserting a termination codon into the variable region located between the putative Rep- and CP-coding regions. These mutants were capable of systemic infection, although their pathogenicity was attenuated. In vitro translation of ORF1 produced both the full-length polyprotein and the smaller Rep protein. The results of in vivo reporter assays suggested that the mechanism of this early termination is a ribosomal -1 frame-shift occurring downstream from the conserved Rep domains. The mechanism of capillovirus gene expression and the very close evolutionary relationship between the genera Capillovirus and Trichovirus are discussed. Copyright (c) 2010. Published by Elsevier B.V.

A potyvirus vector efficiently targets recombinant proteins to chloroplasts, mitochondria and nuclei in plant cells when expressed at the amino terminus of the polyprotein.

Science.gov (United States)

Majer, Eszter; Navarro, José-Antonio; Daròs, José-Antonio

2015-09-01

Plant virus-based expression systems allow quick and efficient production of recombinant proteins in plant biofactories. Among them, a system derived from tobacco etch virus (TEV; genus potyvirus) permits coexpression of equimolar amounts of several recombinant proteins. This work analyzed how to target recombinant proteins to different subcellular localizations in the plant cell using this system. We constructed TEV clones in which green fluorescent protein (GFP), with a chloroplast transit peptide (cTP), a nuclear localization signal (NLS) or a mitochondrial targeting peptide (mTP) was expressed either as the most amino-terminal product or embedded in the viral polyprotein. Results showed that cTP and mTP mediated efficient translocation of GFP to the corresponding organelle only when present at the amino terminus of the viral polyprotein. In contrast, the NLS worked efficiently at both positions. Viruses expressing GFP in the amino terminus of the viral polyprotein produced milder symptoms. Untagged GFPs and cTP and NLS tagged amino-terminal GFPs accumulated to higher amounts in infected tissues. Finally, viral progeny from clones with internal GFPs maintained the extra gene better. These observations will help in the design of potyvirus-based vectors able to coexpress several proteins while targeting different subcellular localizations, as required in plant metabolic engineering. Copyright © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Resistance to pyridine-based inhibitor KF116 reveals an unexpected role of integrase in HIV-1 Gag-Pol polyprotein proteolytic processing.

Science.gov (United States)

Hoyte, Ashley C; Jamin, Augusta V; Koneru, Pratibha C; Kobe, Matthew J; Larue, Ross C; Fuchs, James R; Engelman, Alan N; Kvaratskhelia, Mamuka

2017-12-01

The pyridine-based multimerization selective HIV-1 integrase (IN) inhibitors (MINIs) are a distinct subclass of allosteric IN inhibitors. MINIs potently inhibit HIV-1 replication during virion maturation by inducing hyper- or aberrant IN multimerization but are largely ineffective during the early steps of viral replication. Here, we investigated the mechanism for the evolution of a triple IN substitution (T124N/V165I/T174I) that emerges in cell culture with a representative MINI, KF116. We show that HIV-1 NL4-3(IN T124N/V165I/T174I) confers marked (>2000-fold) resistance to KF116. Two IN substitutions (T124N/T174I) directly weaken inhibitor binding at the dimer interface of the catalytic core domain but at the same time markedly impair HIV-1 replication capacity. Unexpectedly, T124N/T174I IN substitutions inhibited proteolytic processing of HIV-1 polyproteins Gag and Gag-Pol, resulting in immature virions. Strikingly, the addition of the third IN substitution (V165I) restored polyprotein processing, virus particle maturation, and significant levels of replication capacity. These results reveal an unanticipated role of IN for polyprotein proteolytic processing during virion morphogenesis. The complex evolutionary pathway for the emergence of resistant viruses, which includes the need for the compensatory V165I IN substitution, highlights a relatively high genetic barrier exerted by MINI KF116. Additionally, we have solved the X-ray structure of the drug-resistant catalytic core domain protein, which provides means for rational development of second-generation MINIs. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

Optimizing electrode-attached redox-peptide systems for kinetic characterization of protease action on immobilized substrates. Observation of dissimilar behavior of trypsin and thrombin enzymes.

Science.gov (United States)

Anne, Agnès; Chovin, Arnaud; Demaille, Christophe

2012-06-12

In this work, we experimentally address the issue of optimizing gold electrode attached ferrocene (Fc)-peptide systems for kinetic measurements of protease action. Considering human α-thrombin and bovine trypsin as proteases of interest, we show that the recurring problem of incomplete cleavage of the peptide layer by these enzymes can be solved by using ultraflat template-stripped gold, instead of polished polycrystalline gold, as the Fc-peptide bearing electrode material. We describe how these fragile surfaces can be mounted in a rotating disk configuration so that enzyme mass transfer no longer limits the overall measured cleavage kinetics. Finally, we demonstrate that, once the system has been optimized, in situ real-time cyclic voltammetry monitoring of the protease action can yield high-quality kinetic data, showing no sign of interfering effects. The cleavage progress curves then closely match the Langmuirian variation expected for a kinetically controlled surface process. Global fit of the progress curves yield accurate values of the peptide cleavage rate for both trypsin and thrombin. It is shown that, whereas trypsin action on the surface-attached peptide closely follows Michaelis-Menten kinetics, thrombin displays a specific and unexpected behavior characterized by a nearly enzyme-concentration-independent cleavage rate in the subnanomolar enzyme concentration range. The reason for this behavior has still to be clarified, but its occurrence may limit the sensitivity of thrombin sensors based on Fc-peptide layers.

The large terminase DNA packaging motor grips DNA with its ATPase domain for cleavage by the flexible nuclease domain

Science.gov (United States)

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

Vertebrate Embryonic Cleavage Pattern Determination.

Science.gov (United States)

Hasley, Andrew; Chavez, Shawn; Danilchik, Michael; Wühr, Martin; Pelegri, Francisco

2017-01-01

The pattern of the earliest cell divisions in a vertebrate embryo lays the groundwork for later developmental events such as gastrulation, organogenesis, and overall body plan establishment. Understanding these early cleavage patterns and the mechanisms that create them is thus crucial for the study of vertebrate development. This chapter describes the early cleavage stages for species representing ray-finned fish, amphibians, birds, reptiles, mammals, and proto-vertebrate ascidians and summarizes current understanding of the mechanisms that govern these patterns. The nearly universal influence of cell shape on orientation and positioning of spindles and cleavage furrows and the mechanisms that mediate this influence are discussed. We discuss in particular models of aster and spindle centering and orientation in large embryonic blastomeres that rely on asymmetric internal pulling forces generated by the cleavage furrow for the previous cell cycle. Also explored are mechanisms that integrate cell division given the limited supply of cellular building blocks in the egg and several-fold changes of cell size during early development, as well as cytoskeletal specializations specific to early blastomeres including processes leading to blastomere cohesion. Finally, we discuss evolutionary conclusions beginning to emerge from the contemporary analysis of the phylogenetic distributions of cleavage patterns. In sum, this chapter seeks to summarize our current understanding of vertebrate early embryonic cleavage patterns and their control and evolution.

Separation of foot-and-mouth disease virus leader protein activities; identification of mutants that retain efficient self-processing activity but poorly induce eIF4G cleavage

DEFF Research Database (Denmark)

Belsham, Graham; Hua Guan, Su

2017-01-01

Foot-and-mouth disease virus (FMDV) is a picornavirus and its RNA genome encodes a large polyprotein. The N-terminal part of this polyprotein is the Leader protein, a cysteine protease, termed Lpro. The virus causes the rapid inhibition of host cell capdependent protein synthesis within infected...

Cleavage preference distinguishes the two-component NS2B-NS3 serine proteinases of Dengue and West Nile viruses.

Science.gov (United States)

Shiryaev, Sergey A; Kozlov, Igor A; Ratnikov, Boris I; Smith, Jeffrey W; Lebl, Michal; Strongin, Alex Y

2007-02-01

Regulated proteolysis of the polyprotein precursor by the NS2B-NS3 protease is required for the propagation of infectious virions. Unless the structural and functional parameters of NS2B-NS3 are precisely determined, an understanding of its functional role and the design of flaviviral inhibitors will be exceedingly difficult. Our objectives were to define the substrate recognition pattern of the NS2B-NS3 protease of West Nile and Dengue virises (WNV and DV respectively). To accomplish our goals, we used an efficient, 96-well plate format, method for the synthesis of 9-mer peptide substrates with the general P4-P3-P2-P1-P1'-P2'-P3'-P4'-Gly structure. The N-terminus and the constant C-terminal Gly of the peptides were tagged with a fluorescent tag and with a biotin tag respectively. The synthesis was followed by the proteolytic cleavage of the synthesized, tagged peptides. Because of the strict requirement for the presence of basic amino acid residues at the P1 and the P2 substrate positions, the analysis of approx. 300 peptide sequences was sufficient for an adequate representation of the cleavage preferences of the WNV and DV proteinases. Our results disclosed the strict substrate specificity of the WNV protease for which the (K/R)(K/R)R/GG amino acid motifs was optimal. The DV protease was less selective and it tolerated well the presence of a number of amino acid residue types at either the P1' or the P2' site, as long as the other position was occupied by a glycine residue. We believe that our data represent a valuable biochemical resource and a solid foundation to support the design of selective substrates and synthetic inhibitors of flaviviral proteinases.

Glycoside bond cleavage in the radiolysis of aqueous solutions of methylglycosides and disaccharides

International Nuclear Information System (INIS)

Shadyro, O.I.; Kisel', R.M.

2007-01-01

The kinetics of formation of methylglycoside and disaccharide radiolysis products resulting from the O-glycoside bond cleavage under the action of 137 Cs γ-radiation (0-2.5 kGy radiation doses, 0.28 Gy/s dose rate) was studied, and the yields of these products were determined. It was found that oxygen inhibits these processes. The findings suggest that the fragmentation reaction of C' 2 radicals plays an important role in the formation of carbohydrate degradation products in the radiolysis of aqueous carbohydrate solutions [ru

Cleavage and creep fracture of rock salt

International Nuclear Information System (INIS)

Chan, K.S.; Munson, D.E.; Bodner, S.R.

1996-01-01

The dominant failure mechanism in rock salt at ambient temperature is either cleavage or creep fracture. Since the transition of creep fracture to cleavage in a compressive stress field is not well understood, failure of rock salt by cleavage and creep fracture is analyzed in this paper to elucidate the effect of stress state on the competition between these two fracture mechanisms. For cleavage fracture, a shear crack is assumed to cause the formation and growth of a symmetric pair of wing cracks in a predominantly compressive stress field. The conditions for wing-crack instability are derived and presented as the cleavage fracture boundary in the fracture mechanism map. Using an existing creep fracture model, stress conditions for the onset of creep fracture and isochronous failure curves of specified times-to-rupture are calculated and incorporated into the fracture mechanism map. The regimes of dominance by cleavage and creep fracture are established and compared with experimental data. The result indicates that unstable propagation of cleavage cracks occurs only in the presence of tensile stress. The onset of creep fracture is promoted by a tensile stress, but can be totally suppressed by a high confining pressure. Transition of creep fracture to cleavage occurs when critical conditions of stress difference and tensile stress for crack instability are exceeded

Missed cleavage opportunities by FEN1 lead to Okazaki fragment maturation via the long-flap pathway

KAUST Repository

Zaher, Manal S.; Rashid, Fahad; Song, Bo; Joudeh, Luay I; Sobhy, Mohamed Abdelmaboud; Tehseen, Muhammad; Hingorani, Manju M; Hamdan, Samir

2018-01-01

RNA-DNA hybrid primers synthesized by low fidelity DNA polymerase α to initiate eukaryotic lagging strand synthesis must be removed efficiently during Okazaki fragment (OF) maturation to complete DNA replication. In this process, each OF primer is displaced and the resulting 5'-single-stranded flap is cleaved by structure-specific 5'-nucleases, mainly Flap Endonuclease 1 (FEN1), to generate a ligatable nick. At least two models have been proposed to describe primer removal, namely short- and long-flap pathways that involve FEN1 or FEN1 along with Replication Protein A (RPA) and Dna2 helicase/nuclease, respectively. We addressed the question of pathway choice by studying the kinetic mechanism of FEN1 action on short- and long-flap DNA substrates. Using single molecule FRET and rapid quench-flow bulk cleavage assays, we showed that unlike short-flap substrates, which are bound, bent and cleaved within the first encounter between FEN1 and DNA, long-flap substrates can escape cleavage even after DNA binding and bending. Notably, FEN1 can access both substrates in the presence of RPA, but bending and cleavage of long-flap DNA is specifically inhibited. We propose that FEN1 attempts to process both short and long flaps, but occasional missed cleavage of the latter allows RPA binding and triggers the long-flap OF maturation pathway.

Missed cleavage opportunities by FEN1 lead to Okazaki fragment maturation via the long-flap pathway

KAUST Repository

Zaher, Manal S.

2018-01-27

RNA-DNA hybrid primers synthesized by low fidelity DNA polymerase α to initiate eukaryotic lagging strand synthesis must be removed efficiently during Okazaki fragment (OF) maturation to complete DNA replication. In this process, each OF primer is displaced and the resulting 5\\'-single-stranded flap is cleaved by structure-specific 5\\'-nucleases, mainly Flap Endonuclease 1 (FEN1), to generate a ligatable nick. At least two models have been proposed to describe primer removal, namely short- and long-flap pathways that involve FEN1 or FEN1 along with Replication Protein A (RPA) and Dna2 helicase/nuclease, respectively. We addressed the question of pathway choice by studying the kinetic mechanism of FEN1 action on short- and long-flap DNA substrates. Using single molecule FRET and rapid quench-flow bulk cleavage assays, we showed that unlike short-flap substrates, which are bound, bent and cleaved within the first encounter between FEN1 and DNA, long-flap substrates can escape cleavage even after DNA binding and bending. Notably, FEN1 can access both substrates in the presence of RPA, but bending and cleavage of long-flap DNA is specifically inhibited. We propose that FEN1 attempts to process both short and long flaps, but occasional missed cleavage of the latter allows RPA binding and triggers the long-flap OF maturation pathway.

Fluorescence-based high-throughput screening of dicer cleavage activity.

Science.gov (United States)

Podolska, Katerina; Sedlak, David; Bartunek, Petr; Svoboda, Petr

2014-03-01

Production of small RNAs by ribonuclease III Dicer is a key step in microRNA and RNA interference pathways, which employ Dicer-produced small RNAs as sequence-specific silencing guides. Further studies and manipulations of microRNA and RNA interference pathways would benefit from identification of small-molecule modulators. Here, we report a study of a fluorescence-based in vitro Dicer cleavage assay, which was adapted for high-throughput screening. The kinetic assay can be performed under single-turnover conditions (35 nM substrate and 70 nM Dicer) in a small volume (5 µL), which makes it suitable for high-throughput screening in a 1536-well format. As a proof of principle, a small library of bioactive compounds was analyzed, demonstrating potential of the assay.

Reductive cleavage of chlorine from 6-chloronicotinic acid on mercury electrodes

International Nuclear Information System (INIS)

Ruiz Montoya, M.; Pintado, S.; Rodriguez Mellado, J.M.

2011-01-01

Highlights: → Dissociation constants (as pK) of 6 chloronicotinic acid (6CNA) obtained by UV-vis spectroscopy: -0.80 ± 0.05 (-COOH group) and 3.2 ± 0.1 (pyridinic nitrogen). → Electrolysis of 6CNA evidenced the reductive cleavage of chlorine from the molecule. → Kinetic parameters (Tafel slopes and reaction orders) determined at the foot of the waves. → Reduction pathways have been proposed. - Abstract: This paper presents polarographic (direct current, dc, and differential pulse, DP) and voltammetric (linear-sweep cyclic voltammetry) studies on the electroreduction of 6-chloronicotinic acid (6CNA) on mercury electrodes. In order to obtain the dissociation constants of 6CNA, UV-vis spectra were recorded as a function of pH. pK values of -0.80 ± 0.05 (-COOH group) and 3.2 ± 0.1 (pyridinic nitrogen) were obtained. The electrochemical studies were performed in the acidity range 6 M H 2 SO 4 to pH 8. Above the last pH value no signals were obtained. Electrolysis made at potentials corresponding to the limiting current of the first wave indicates that there is a reductive cleavage of chlorine from the molecule. This was confirmed by dc and DP polarografic results and also by voltammetric results. Kinetic parameters such as Tafel slopes and electrochemical reaction orders have been determined at potentials corresponding to the foot of the waves. From these results, together with those obtained by cyclic voltammetry, a reaction pathway is proposed, in which the rate-determining step of the process is the release of a chloride ion from the radical formed after the uptake of a H + ion and an electron.

Reductive cleavage of chlorine from 6-chloronicotinic acid on mercury electrodes

Energy Technology Data Exchange (ETDEWEB)

Ruiz Montoya, M., E-mail: mmontoya@uhu.e [Departamento de Ingenieria Quimica, Quimica Fisica y Quimica Organica, Universidad de Huelva, Campus El Carmen, Facultad de Ciencias Experimentales, E-21071 Huelva (Spain); Pintado, S., E-mail: q02pibes@uco.e [Departamento de Quimica Fisica y Termodinamica Aplicada, Universidad de Cordoba, Campus Universitario de Rabanales, edificio ' Marie Curie' ., E-14014 Cordoba (Spain); Rodriguez Mellado, J.M., E-mail: jmrodriguez@uco.e [Departamento de Quimica Fisica y Termodinamica Aplicada, Universidad de Cordoba, Campus Universitario de Rabanales, edificio ' Marie Curie' ., E-14014 Cordoba (Spain)

2011-04-30

Highlights: Dissociation constants (as pK) of 6 chloronicotinic acid (6CNA) obtained by UV-vis spectroscopy: -0.80 {+-} 0.05 (-COOH group) and 3.2 {+-} 0.1 (pyridinic nitrogen). Electrolysis of 6CNA evidenced the reductive cleavage of chlorine from the molecule. Kinetic parameters (Tafel slopes and reaction orders) determined at the foot of the waves. Reduction pathways have been proposed. - Abstract: This paper presents polarographic (direct current, dc, and differential pulse, DP) and voltammetric (linear-sweep cyclic voltammetry) studies on the electroreduction of 6-chloronicotinic acid (6CNA) on mercury electrodes. In order to obtain the dissociation constants of 6CNA, UV-vis spectra were recorded as a function of pH. pK values of -0.80 {+-} 0.05 (-COOH group) and 3.2 {+-} 0.1 (pyridinic nitrogen) were obtained. The electrochemical studies were performed in the acidity range 6 M H{sub 2}SO{sub 4} to pH 8. Above the last pH value no signals were obtained. Electrolysis made at potentials corresponding to the limiting current of the first wave indicates that there is a reductive cleavage of chlorine from the molecule. This was confirmed by dc and DP polarografic results and also by voltammetric results. Kinetic parameters such as Tafel slopes and electrochemical reaction orders have been determined at potentials corresponding to the foot of the waves. From these results, together with those obtained by cyclic voltammetry, a reaction pathway is proposed, in which the rate-determining step of the process is the release of a chloride ion from the radical formed after the uptake of a H{sup +} ion and an electron.

Kinetic analysis of the effects of target structure on siRNA efficiency

Science.gov (United States)

Chen, Jiawen; Zhang, Wenbing

2012-12-01

RNAi efficiency for target cleavage and protein expression is related to the target structure. Considering the RNA-induced silencing complex (RISC) as a multiple turnover enzyme, we investigated the effect of target mRNA structure on siRNA efficiency with kinetic analysis. The 4-step model was used to study the target cleavage kinetic process: hybridization nucleation at an accessible target site, RISC-mRNA hybrid elongation along with mRNA target structure melting, target cleavage, and enzyme reactivation. At this model, the terms accounting for the target accessibility, stability, and the seed and the nucleation site effects are all included. The results are in good agreement with that of experiments which show different arguments about the structure effects on siRNA efficiency. It shows that the siRNA efficiency is influenced by the integrated factors of target's accessibility, stability, and the seed effects. To study the off-target effects, a simple model of one siRNA binding to two mRNA targets was designed. By using this model, the possibility for diminishing the off-target effects by the concentration of siRNA was discussed.

Complete genome sequence of a proposed new tymovirus, tomato blistering mosaic virus.

Science.gov (United States)

Nicolini, Cícero; Inoue-Nagata, Alice Kazuko; Nagata, Tatsuya

2015-02-01

In a previous work, a distinct tymovirus infecting tomato plants in Brazil was reported and tentatively named tomato blistering mosaic virus (ToBMV). In this study, the complete genome sequence of ToBMV was determined and shown to have a size of 6277 nucleotides and three ORFs: ORF 1 encodes the replication-complex polyprotein, ORF 2 the movement protein, and ORF 3 the coat protein. The cleavage sites of the replication-complex polyprotein (GS/LP and VAG/QSP) of ToBMV were predicted by alignment analysis of amino acid sequences of other tymoviruses. In the phylogenetic tree, ToBMV clustered with the tymoviruses that infect solanaceous hosts.

The amino-terminus of the hepatitis C virus (HCV p7 viroporin and its cleavage from glycoprotein E2-p7 precursor determine specific infectivity and secretion levels of HCV particle types.

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Solène Denolly

2017-12-01

Full Text Available Viroporins are small transmembrane proteins with ion channel activities modulating properties of intracellular membranes that have diverse proviral functions. Hepatitis C virus (HCV encodes a viroporin, p7, acting during assembly, envelopment and secretion of viral particles (VP. HCV p7 is released from the viral polyprotein through cleavage at E2-p7 and p7-NS2 junctions by signal peptidase, but also exists as an E2p7 precursor, of poorly defined properties. Here, we found that ectopic p7 expression in HCVcc-infected cells reduced secretion of particle-associated E2 glycoproteins. Using biochemical assays, we show that p7 dose-dependently slows down the ER-to-Golgi traffic, leading to intracellular retention of E2, which suggested that timely E2p7 cleavage and p7 liberation are critical events to control E2 levels. By studying HCV mutants with accelerated E2p7 processing, we demonstrate that E2p7 cleavage controls E2 intracellular expression and secretion levels of nucleocapsid-free subviral particles and infectious virions. In addition, our imaging data reveal that, following p7 liberation, the amino-terminus of p7 is exposed towards the cytosol and coordinates the encounter between NS5A and NS2-based assembly sites loaded with E1E2 glycoproteins, which subsequently leads to nucleocapsid envelopment. We identify punctual mutants at p7 membrane interface that, by abrogating NS2/NS5A interaction, are defective for transmission of infectivity owing to decreased secretion of core and RNA and to increased secretion of non/partially-enveloped particles. Altogether, our results indicate that the retarded E2p7 precursor cleavage is essential to regulate the intracellular and secreted levels of E2 through p7-mediated modulation of the cell secretory pathway and to unmask critical novel assembly functions located at p7 amino-terminus.

Microstructure and cleavage in lath martensitic steels

International Nuclear Information System (INIS)

Morris, John W Jr; Kinney, Chris; Pytlewski, Ken; Adachi, Y

2013-01-01

In this paper we discuss the microstructure of lath martensitic steels and the mechanisms by which it controls cleavage fracture. The specific experimental example is a 9Ni (9 wt% Ni) steel annealed to have a large prior austenite grain size, then examined and tested in the as-quenched condition to produce a relatively coarse lath martensite. The microstructure is shown to approximate the recently identified ‘classic’ lath martensite structure: prior austenite grains are divided into packets, packets are subdivided into blocks, and blocks contain interleaved laths whose variants are the two Kurjumov–Sachs relations that share the same Bain axis of the transformation. When the steel is fractured in brittle cleavage, the laths in the block share {100} cleavage planes and cleave as a unit. However, cleavage cracks deflect or blunt at the boundaries between blocks with different Bain axes. It follows that, as predicted, the block size governs the effective grain size for cleavage. (paper)

Modeling and inferring cleavage patterns in proliferating epithelia.

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Ankit B Patel

2009-06-01

Full Text Available The regulation of cleavage plane orientation is one of the key mechanisms driving epithelial morphogenesis. Still, many aspects of the relationship between local cleavage patterns and tissue-level properties remain poorly understood. Here we develop a topological model that simulates the dynamics of a 2D proliferating epithelium from generation to generation, enabling the exploration of a wide variety of biologically plausible cleavage patterns. We investigate a spectrum of models that incorporate the spatial impact of neighboring cells and the temporal influence of parent cells on the choice of cleavage plane. Our findings show that cleavage patterns generate "signature" equilibrium distributions of polygonal cell shapes. These signatures enable the inference of local cleavage parameters such as neighbor impact, maternal influence, and division symmetry from global observations of the distribution of cell shape. Applying these insights to the proliferating epithelia of five diverse organisms, we find that strong division symmetry and moderate neighbor/maternal influence are required to reproduce the predominance of hexagonal cells and low variability in cell shape seen empirically. Furthermore, we present two distinct cleavage pattern models, one stochastic and one deterministic, that can reproduce the empirical distribution of cell shapes. Although the proliferating epithelia of the five diverse organisms show a highly conserved cell shape distribution, there are multiple plausible cleavage patterns that can generate this distribution, and experimental evidence suggests that indeed plants and fruitflies use distinct division mechanisms.

Imperfect DNA mirror repeats in the gag gene of HIV-1 (HXB2 identify key functional domains and coincide with protein structural elements in each of the mature proteins

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Lang Dorothy M

2007-10-01

Full Text Available Abstract Background A DNA mirror repeat is a sequence segment delimited on the basis of its containing a center of symmetry on a single strand, e.g. 5'-GCATGGTACG-3'. It is most frequently described in association with a functionally significant site in a genomic sequence, and its occurrence is regarded as noteworthy, if not unusual. However, imperfect mirror repeats (IMRs having ≥ 50% symmetry are common in the protein coding DNA of monomeric proteins and their distribution has been found to coincide with protein structural elements – helices, β sheets and turns. In this study, the distribution of IMRs is evaluated in a polyprotein – to determine whether IMRs may be related to the position or order of protein cleavage or other hierarchal aspects of protein function. The gag gene of HIV-1 [GenBank:K03455] was selected for the study because its protein motifs and structural components are well documented. Results There is a highly specific relationship between IMRs and structural and functional aspects of the Gag polyprotein. The five longest IMRs in the polyprotein translate a key functional segment in each of the five cleavage products. Throughout the protein, IMRs coincide with functionally significant segments of the protein. A detailed annotation of the protein, which combines structural, functional and IMR data illustrates these associations. There is a significant statistical correlation between the ends of IMRs and the ends of PSEs in each of the mature proteins. Weakly symmetric IMRs (≥ 33% are related to cleavage positions and processes. Conclusion The frequency and distribution of IMRs in HIV-1 Gag indicates that DNA symmetry is a fundamental property of protein coding DNA and that different levels of symmetry are associated with different functional aspects of the gene and its protein. The interaction between IMRs and protein structure and function is precise and interwoven over the entire length of the polyprotein. The

Intermolecular cleavage by UmuD-like mutagenesis proteins

Science.gov (United States)

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

Quantitative proteomics reveals the kinetics of trypsin-catalyzed protein digestion.

Science.gov (United States)

Pan, Yanbo; Cheng, Kai; Mao, Jiawei; Liu, Fangjie; Liu, Jing; Ye, Mingliang; Zou, Hanfa

2014-10-01

Trypsin is the popular protease to digest proteins into peptides in shotgun proteomics, but few studies have attempted to systematically investigate the kinetics of trypsin-catalyzed protein digestion in proteome samples. In this study, we applied quantitative proteomics via triplex stable isotope dimethyl labeling to investigate the kinetics of trypsin-catalyzed cleavage. It was found that trypsin cleaves the C-terminal to lysine (K) and arginine (R) residues with higher rates for R. And the cleavage sites surrounded by neutral residues could be quickly cut, while those with neighboring charged residues (D/E/K/R) or proline residue (P) could be slowly cut. In a proteome sample, a huge number of proteins with different physical chemical properties coexists. If any type of protein could be preferably digested, then limited digestion could be applied to reduce the sample complexity. However, we found that protein abundance and other physicochemical properties, such as molecular weight (Mw), grand average of hydropathicity (GRAVY), aliphatic index, and isoelectric point (pI) have no notable correlation with digestion priority of proteins.

Regioselectivity in the Reductive Bond Cleavage of Diarylalkylsulfonium Salts

DEFF Research Database (Denmark)

Kampmeier, Jack; Mansurul Hoque, AKM; D. Saeva, Franklin

2009-01-01

products vary from regiospecific alkyl cleavage to predominant aryl cleavage as a function of the potential of the reducing agent. We conclude that differences between the reductive cleavages of mono- and diarylsulfonium salts are direct consequences of the structures of the sulfuranyl radical......- tolylethylsulfonium and di-4-tolyl-2-phenylethylsulfonium salts by a variety of one-electron reducing agents ranging in potential from -0.77 to +2.5 eV (vs SCE) and including thermal reductants, indirect electrolyses mediated by a series of cyanoaromatics, and excited singlet states. We report that the cleavage...... intermediates and the bond dissociation energies of the alkyl and aryl bonds. Competitions between the rates of cleavage and oxidation of the intermediate sulfuranyl radicals and between concerted and stepwise mechanisms are discussed to explain the variations in bond cleavage products as a function...

The influence of junction conformation on RNA cleavage by the hairpin ribozyme in its natural junction form.

Science.gov (United States)

Thomson, J B; Lilley, D M

1999-01-01

In the natural form of the hairpin ribozyme the two loop-carrying duplexes that comprise the majority of essential bases for activity form two adjacent helical arms of a four-way RNA junction. In the present work we have manipulated the sequence around the junction in a way known to perturb the global folding properties. We find that replacement of the junction by a different sequence that has the same conformational properties as the natural sequence gives closely similar reaction rate and Arrhenius activation energy for the substrate cleavage reaction. By comparison, rotation of the natural sequence in order to alter the three-dimensional folding of the ribozyme leads to a tenfold reduction in the kinetics of cleavage. Replacement with the U1 four-way junction that is resistant to rotation into the antiparallel structure required to allow interaction between the loops also gives a tenfold reduction in cleavage rate. The results indicate that the conformation of the junction has a major influence on the catalytic activity of the ribozyme. The results are all consistent with a role for the junction in the provision of a framework by which the loops are presented for interaction in order to create the active form of the ribozyme. PMID:10024170

Identification of two auto-cleavage products of nonstructural protein 1 (nsp1) in porcine reproductive and respiratory syndrome virus infected cells: nsp1 function as interferon antagonist

International Nuclear Information System (INIS)

Chen, Z.; Lawson, S.; Sun, Z.; Zhou, X.; Guan, X.; Christopher-Hennings, J.; Nelson, E.A.; Fang, Y.

2010-01-01

The porcine reproductive and respiratory syndrome virus nsp1 is predicted to be auto-cleaved from the replicase polyprotein into nsp1α and nsp1β subunits. In infected cells, we detected the actual existence of nsp1α and nsp1β. Cleavage sites between nsp1α/nsp1β and nsp1β/nsp2 were identified by protein microsequencing analysis. Time course study showed that nsp1α and nsp1β mainly localize into the cell nucleus after 10 h post infection. Further analysis revealed that both proteins dramatically inhibited IFN-β expression. The nsp1β was observed to significantly inhibit expression from an interferon-stimulated response element promoter after Sendai virus infection or interferon treatment. It was further determined to inhibit nuclear translocation of STAT1 in the JAK-STAT signaling pathway. These results demonstrated that nsp1β has ability to inhibit both interferon synthesis and signaling, while nsp1α alone strongly inhibits interferon synthesis. These findings provide important insights into mechanisms of nsp1 in PRRSV pathogenesis and its impact in vaccine development.

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

Science.gov (United States)

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

2003-11-01

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

Analysis of embryo morphokinetics, multinucleation and cleavage anomalies using continuous time-lapse monitoring in blastocyst transfer cycles.

Science.gov (United States)

Desai, Nina; Ploskonka, Stephanie; Goodman, Linnea R; Austin, Cynthia; Goldberg, Jeffrey; Falcone, Tommaso

2014-06-20

Time-lapse imaging combined with embryo morphokinetics may offer a non-invasive means for improving embryo selection. Data from clinics worldwide are necessary to compare and ultimately develop embryo classifications models using kinetic data. The primary objective of this study was to determine if there were kinetic differences between embryos with limited potential and those more often associated with in vitro blastocyst formation and/or implantation. We also wanted to compare putative kinetic markers for embryo selection as proposed by other laboratories to what we were observing in our own laboratory setting. Kinetic data and cycle outcomes were retrospectively analyzed in patients age 39 and younger with 7 or more zygotes cultured in the Embryoscope. Timing of specific events from the point of insemination were determined using time-lapse (TL) imaging. The following kinetic markers were assessed: time to syngamy (tPNf), t2, time to two cells (c), 3c (t3), 4c ( t4), 5c (t5), 8c (t8), morula (tMor), start of blastulation (tSB); tBL, blastocyst (tBL); expanded blastocyst (tEBL). Durations of the second (cc2) and third (cc3) cell cycles, the t5-t2 interval as well as time to complete synchronous divisions s1, s2 and s3 were calculated. Incidence and impact on development of nuclear and cleavage anomalies were also assessed. A total of 648 embryos transferred on day 5 were analyzed. The clinical pregnancy and implantation rate were 72% and 50%, respectively. Morphokinetic data showed that tPNf, t2,t4, t8, s1, s2,s3 and cc2 were significantly different in embryos forming blastocysts (ET or frozen) versus those with limited potential either failing to blastulate or else forming poor quality blastocysts ,ultimately discarded. Comparison of embryo kinetics in cycles with all embryos implanting (KID+) versus no implantation (KID-) suggested that markers of embryo competence to implant may be different from ability to form a blastocyst. The incidence of multinucleation

Quantification of DNA cleavage specificity in Hi-C experiments.

Science.gov (United States)

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.

Dataset of cocoa aspartic protease cleavage sites

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Katharina Janek

2016-09-01

Full Text Available The data provide information in support of the research article, “The cleavage specificity of the aspartic protease of cocoa beans involved in the generation of the cocoa-specific aroma precursors” (Janek et al., 2016 [1]. Three different protein substrates were partially digested with the aspartic protease isolated from cocoa beans and commercial pepsin, respectively. The obtained peptide fragments were analyzed by matrix-assisted laser-desorption/ionization time-of-flight mass spectrometry (MALDI-TOF/TOF-MS/MS and identified using the MASCOT server. The N- and C-terminal ends of the peptide fragments were used to identify the corresponding in-vitro cleavage sites by comparison with the amino acid sequences of the substrate proteins. The same procedure was applied to identify the cleavage sites used by the cocoa aspartic protease during cocoa fermentation starting from the published amino acid sequences of oligopeptides isolated from fermented cocoa beans. Keywords: Aspartic protease, Cleavage sites, Cocoa, In-vitro proteolysis, Mass spectrometry, Peptides

Cleavage events and sperm dynamics in chick intrauterine embryos.

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Hyung Chul Lee

Full Text Available This study was undertaken to elucidate detailed event of early embryogenesis in chicken embryos using a noninvasive egg retrieval technique before oviposition. White Leghorn intrauterine eggs were retrieved from 95 cyclic hens aged up to 54-56 weeks and morphogenetic observation was made under both bright field and fluorescent image in a time course manner. Differing from mammals, asymmetric cleavage to yield preblastodermal cells was observed throughout early embryogenesis. The first two divisions occurred synchronously and four polarized preblastodermal cells resulted after cruciform cleavage. Then, asynchronous cleavage continued in a radial manner and overall cell size in the initial cleavage region was smaller than that in the distal area. Numerous sperms were visible, regardless of zygotic nuclei formation. Condensed sperm heads were present mainly in the perivitelline space and cytoplasm, and rarely in the yolk region, while decondensed sperm heads were only visible in the yolk. In conclusion, apparent differences in sperm dynamics and early cleavage events compared with mammalian embryos were detected in chick embryo development, which demonstrated polarized cleavage with penetrating supernumerary sperm into multiple regions.

New Insight into the Cleavage Reaction of Nostoc sp. Strain PCC 7120 Carotenoid Cleavage Dioxygenase in Natural and Nonnatural Carotenoids

Science.gov (United States)

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

In vivo analysis of the Notch receptor S1 cleavage.

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Robert J Lake

2009-08-01

Full Text Available A ligand-independent cleavage (S1 in the extracellular domain of the mammalian Notch receptor results in what is considered to be the canonical heterodimeric form of Notch on the cell surface. The in vivo consequences and significance of this cleavage on Drosophila Notch signaling remain unclear and contradictory. We determined the cleavage site in Drosophila and examined its in vivo function by a transgenic analysis of receptors that cannot be cleaved. Our results demonstrate a correlation between loss of cleavage and loss of in vivo function of the Notch receptor, supporting the notion that S1 cleavage is an in vivo mechanism of Notch signal control.

In vitro measurement of beta-carotene cleavage activity : methodological considerations and the effect of other carotenoids on beta-carotene cleavage

NARCIS (Netherlands)

Vliet, T. van; Schaik, F. van; Schreurs, W.H.P.; Berg, H. van den

1996-01-01

In view of controversies about assessment of the β-carotene cleavage activity, methodological aspects and problems of the dioxygenase assay are described. Using rat and hamster intestinal preparations the method was optimized on retinal formation, the only cleavage product we could demonstrate. It

Kinetics of Oxidation of Cobalt(III Complexes of a Acids by Hydrogen Peroxide in the Presence of Surfactants

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Mansur Ahmed

2008-01-01

Full Text Available Hydrogen peroxide oxidation of pentaamminecobalt(III complexes of α-hydroxy acids at 35°C in micellar medium has been attempted. In this reaction the rate of oxidation shows first order kinetics each in [cobalt(III] and [H2O2]. Hydrogen peroxide induced electron transfer in [(NH35 CoIII-L]2+ complexes of α-hydroxy acids readily yields 100% of cobalt(II with nearly 100% of C-C bond cleavage products suggesting that it behaves mainly as one equivalent oxidant in micellar medium. With unbound ligand also it behaves only as C-C cleavage agent rather than C-H cleavage agent. With increasing micellar concentration an increase in the rate is observed.

Pripper: prediction of caspase cleavage sites from whole proteomes

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Salmi Jussi

2010-06-01

Full Text Available Abstract Background Caspases are a family of proteases that have central functions in programmed cell death (apoptosis and inflammation. Caspases mediate their effects through aspartate-specific cleavage of their target proteins, and at present almost 400 caspase substrates are known. There are several methods developed to predict caspase cleavage sites from individual proteins, but currently none of them can be used to predict caspase cleavage sites from multiple proteins or entire proteomes, or to use several classifiers in combination. The possibility to create a database from predicted caspase cleavage products for the whole genome could significantly aid in identifying novel caspase targets from tandem mass spectrometry based proteomic experiments. Results Three different pattern recognition classifiers were developed for predicting caspase cleavage sites from protein sequences. Evaluation of the classifiers with quality measures indicated that all of the three classifiers performed well in predicting caspase cleavage sites, and when combining different classifiers the accuracy increased further. A new tool, Pripper, was developed to utilize the classifiers and predict the caspase cut sites from an arbitrary number of input sequences. A database was constructed with the developed tool, and it was used to identify caspase target proteins from tandem mass spectrometry data from two different proteomic experiments. Both known caspase cleavage products as well as novel cleavage products were identified using the database demonstrating the usefulness of the tool. Pripper is not restricted to predicting only caspase cut sites, but it gives the possibility to scan protein sequences for any given motif(s and predict cut sites once a suitable cut site prediction model for any other protease has been developed. Pripper is freely available and can be downloaded from http://users.utu.fi/mijopi/Pripper. Conclusions We have developed Pripper, a tool for

Quantitative characterization of cleavage and hydrogen-assisted quasi-cleavage fracture surfaces with the use of confocal laser scanning microscopy

International Nuclear Information System (INIS)

Merson, E.; Kudrya, A.V.; Trachenko, V.A.; Merson, D.; Danilov, V.; Vinogradov, A.

2016-01-01

“True” cleavage (TC) and quasi-cleavage (QC) fracture surfaces of low-carbon steel specimens tested in liquid nitrogen and after hydrogen charging respectively were investigated by quantitative confocal laser scanning microscopy (CLSM) and conventional scanning electron microscopy (SEM) with electron-backscattered diffraction (EBSD). Topological and crystallographic features of the TC fracture surface are found in good agreement with the generally accepted cleavage mechanism: TC facets diameters correspond to those of grains; the crack path strictly follows the crystallographic orientation of grains and the most of the cleavage cracks are parallel to {100} planes. On the 2D SEM images, the QC facets appeared resembling the TC ones in terms of river line patterns, shapes and sizes. However, the substantial differences between the topography of these two kinds of fracture surfaces were revealed by 3D CLSM: the average misorientation angle between QC facets and the roughness of the QC fracture surface were much lower than those measured for TC. It is demonstrated that all these features are attributed to the specific fracture mechanism operating during hydrogen-assisted cracking.

Quantitative characterization of cleavage and hydrogen-assisted quasi-cleavage fracture surfaces with the use of confocal laser scanning microscopy

Energy Technology Data Exchange (ETDEWEB)

Merson, E. [Institute of Advanced Technologies, Togliatti State University, 445667 (Russian Federation); Kudrya, A.V.; Trachenko, V.A. [Department of Physical Metallurgy and the Physics of Strength, NUST MISiS, Moscow 119490 (Russian Federation); Merson, D. [Institute of Advanced Technologies, Togliatti State University, 445667 (Russian Federation); Laboratory for Advanced Materials, Kazan Federal University, Naberezhnye Chelny 423812, Republic of Tatarstan (Russian Federation); Danilov, V. [Institute of Advanced Technologies, Togliatti State University, 445667 (Russian Federation); Vinogradov, A. [Institute of Advanced Technologies, Togliatti State University, 445667 (Russian Federation); Department of Engineering Design and Materials, Norwegian University of Science and Technology – NTNU, N-7491 Trondheim (Norway)

2016-05-17

“True” cleavage (TC) and quasi-cleavage (QC) fracture surfaces of low-carbon steel specimens tested in liquid nitrogen and after hydrogen charging respectively were investigated by quantitative confocal laser scanning microscopy (CLSM) and conventional scanning electron microscopy (SEM) with electron-backscattered diffraction (EBSD). Topological and crystallographic features of the TC fracture surface are found in good agreement with the generally accepted cleavage mechanism: TC facets diameters correspond to those of grains; the crack path strictly follows the crystallographic orientation of grains and the most of the cleavage cracks are parallel to {100} planes. On the 2D SEM images, the QC facets appeared resembling the TC ones in terms of river line patterns, shapes and sizes. However, the substantial differences between the topography of these two kinds of fracture surfaces were revealed by 3D CLSM: the average misorientation angle between QC facets and the roughness of the QC fracture surface were much lower than those measured for TC. It is demonstrated that all these features are attributed to the specific fracture mechanism operating during hydrogen-assisted cracking.

Hepatic oxidative stress in ovariectomized transgenic mice expressing the hepatitis C virus polyprotein is augmented through suppression of adenosine monophosphate-activated protein kinase/proliferator-activated receptor gamma co-activator 1 alpha signaling.

Science.gov (United States)

Tomiyama, Yasuyuki; Nishina, Sohji; Hara, Yuichi; Kawase, Tomoya; Hino, Keisuke

2014-10-01

Oxidative stress plays an important role in hepatocarcinogenesis of hepatitis C virus (HCV)-related chronic liver diseases. Despite the evidence of an increased proportion of females among elderly patients with HCV-related hepatocellular carcinoma (HCC), it remains unknown whether HCV augments hepatic oxidative stress in postmenopausal women. The aim of this study was to determine whether oxidative stress was augmented in ovariectomized (OVX) transgenic mice expressing the HCV polyprotein and to investigate its underlying mechanisms. OVX and sham-operated female transgenic mice expressing the HCV polyprotein and non-transgenic littermates were assessed for the production of reactive oxygen species (ROS), expression of inflammatory cytokines and antioxidant potential in the liver. Compared with OVX non-transgenic mice, OVX transgenic mice showed marked hepatic steatosis and ROS production without increased induction of inflammatory cytokines, but there was no increase in ROS-detoxifying enzymes such as superoxide dismutase 2 and glutathione peroxidase 1. In accordance with these results, OVX transgenic mice showed less activation of peroxisome proliferator-activated receptor-γ co-activator-1α (PGC-1α), which is required for the induction of ROS-detoxifying enzymes, and no activation of adenosine monophosphate-activated protein kinase-α (AMPKα), which regulates the activity of PGC-1α. Our study demonstrated that hepatic oxidative stress was augmented in OVX transgenic mice expressing the HCV polyprotein by attenuation of antioxidant potential through inhibition of AMPK/PGC-1α signaling. These results may account in part for the mechanisms by which HCV-infected women are at high risk for HCC development when some period has passed after menopause. © 2013 The Japan Society of Hepatology.

Functional analysis of coordinated cleavage in V(D)J recombination.

Science.gov (United States)

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.

Kinetic mechanism and nucleotide specificity of NADH peroxidase

International Nuclear Information System (INIS)

Stoll, V.S.; Blanchard, J.S.

1988-01-01

NADH peroxidase is a flavoprotein isolated from Streptococcus faecalis which catalyzes the pyridine nucleotide-dependent reduction of hydrogen peroxide to water. Initial velocity, product, and dead-end inhibition studies have been performed at pH 7.5 and support a ping-pong kinetic mechanism. In the absence of hydrogen peroxide, both transhydrogenation between NADH and thioNAD, and isotope exchange between [ 14 C]NADH and NAD, have been demonstrated, although in both these experiments, the maximal velocity of nucleotide exchange was less than 1.5% the maximal velocity of the peroxidatic reaction. We propose that NADH binds tightly to both oxidized and two-electron reduced enzyme. NADH oxidation proceeds stereospecifically with the transfer of the 4S hydrogen to enzyme, and then, via exchange, to water. No primary tritium kinetic isotope effect was observed, and no statistically significant primary deuterium kinetic isotope effects on V/K were determined, although primary deuterium kinetic isotope effects on V were observed in the presence and absence of sodium acetate. NADH peroxidase thus shares with other flavoprotein reductases striking kinetic, spectroscopic, and stereochemical similarities. On this basis, we propose a chemical mechanism for the peroxide cleaving reaction catalyzed by NADH peroxidase which involves the obligate formation of a flavinperoxide, and peroxo bond cleavage by nucleophilic attack by enzymatic dithiols

A new cultural cleavage in post-modern society

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Jan-Erik Lane

2007-09-01

Full Text Available The attitudes towards gender and homosexuality tend to be linked at the micro level (individuals, which explains the political saliency of this newly emerging cleavage. At the macro level (country, the main finding is that the value orientations towards gender and homosexuality are strongly embedded in the basic cultural or civilisation differences among countries. As developing countries modernise and enter post-modernity, they will also experience the gender cleavage, especially when they adhere to an individualistic culture. Cultural cleavages in the post-modern society, whether in rich or developing countries, can only be properly researched by the survey method. It opens up a large area for both micro and macro analyses in the social sciences.

In-capillary self-assembly and proteolytic cleavage of polyhistidine peptide capped quantum dots

Energy Technology Data Exchange (ETDEWEB)

Wang, Jianhao; Li, Jingyan; Li, Jinchen; Liu, Feifei [School of Pharmaceutical Engineering and Life Science, Changzhou University, Changzhou, Jiangsu, 213164 (China); Zhou, Xiang; Yao, Yi [Changzhou Qianhong Bio-pharma Co. Ltd, Changzhou 213164, Jiangsu (China); Wang, Cheli [School of Pharmaceutical Engineering and Life Science, Changzhou University, Changzhou, Jiangsu, 213164 (China); Qiu, Lin, E-mail: linqiupjj@gmail.com [School of Pharmaceutical Engineering and Life Science, Changzhou University, Changzhou, Jiangsu, 213164 (China); Jiang, Pengju, E-mail: pengju.jiang@gmail.com [School of Pharmaceutical Engineering and Life Science, Changzhou University, Changzhou, Jiangsu, 213164 (China); State Key Laboratory of Pharmaceutical Biotechnology, Nanjing, Jiangsu (China)

2015-10-01

A new method using fluorescence coupled capillary electrophoresis (CE-FL) for monitoring self-assembly and proteolytic cleavage of hexahistidine peptide capped quantum dots (QDs) inside a capillary has been developed in this report. QDs and the ATTO 590-labeled hexahistidine peptide (H6-ATTO) were injected into a capillary, sequentially. Their self-assembly inside the capillary was driven by a metal-affinity force which yielded a new fluorescence signal due to Förster resonance energy transfer (FRET). The highly efficient separation of fluorescent complexes and the FRET process were analyzed using CE-FL. The self-assembly of QDs and biomolecules was found to effectively take place inside the capillary. The kinetics of the assembly was monitored by CE-FL, and the approach was extended to the study of proteolytic cleavage of surface conjugated peptides. Being the first in-depth analysis of in-capillary nanoparticle–biomolecule assembly, the novel approach reported here provides inspiration to the development of QD-based FRET probes for biomedical applications. - Highlights: • We examined the self-assembly QDs with H6-ATTO inside a capillary. • We prove CE-FL to be a powerful method to resolve QDs-H6-ATTO complex. • We achieve chromatographic separation of QDs-H6-ATTO complex. • We discovered a novel strategy for the online detection of thrombin. • This technique integrated “injection, mixing, reaction, separation and detection”.

Unexpected tolerance of alpha-cleavage of the prion protein to sequence variations.

Directory of Open Access Journals (Sweden)

José B Oliveira-Martins

Full Text Available The cellular form of the prion protein, PrP(C, undergoes extensive proteolysis at the alpha site (109K [see text]H110. Expression of non-cleavable PrP(C mutants in transgenic mice correlates with neurotoxicity, suggesting that alpha-cleavage is important for PrP(C physiology. To gain insights into the mechanisms of alpha-cleavage, we generated a library of PrP(C mutants with mutations in the region neighbouring the alpha-cleavage site. The prevalence of C1, the carboxy adduct of alpha-cleavage, was determined for each mutant. In cell lines of disparate origin, C1 prevalence was unaffected by variations in charge and hydrophobicity of the region neighbouring the alpha-cleavage site, and by substitutions of the residues in the palindrome that flanks this site. Instead, alpha-cleavage was size-dependently impaired by deletions within the domain 106-119. Almost no cleavage was observed upon full deletion of this domain. These results suggest that alpha-cleavage is executed by an alpha-PrPase whose activity, despite surprisingly limited sequence specificity, is dependent on the size of the central region of PrP(C.

Comparative and phylogenetic perspectives of the cleavage process in tailed amphibians.

Science.gov (United States)

Desnitskiy, Alexey G; Litvinchuk, Spartak N

2015-10-01

The order Caudata includes about 660 species and displays a variety of important developmental traits such as cleavage pattern and egg size. However, the cleavage process of tailed amphibians has never been analyzed within a phylogenetic framework. We use published data on the embryos of 36 species concerning the character of the third cleavage furrow (latitudinal, longitudinal or variable) and the magnitude of synchronous cleavage period (up to 3-4 synchronous cell divisions in the animal hemisphere or a considerably longer series of synchronous divisions followed by midblastula transition). Several species from basal caudate families Cryptobranchidae (Andrias davidianus and Cryptobranchus alleganiensis) and Hynobiidae (Onychodactylus japonicus) as well as several representatives from derived families Plethodontidae (Desmognathus fuscus and Ensatina eschscholtzii) and Proteidae (Necturus maculosus) are characterized by longitudinal furrows of the third cleavage and the loss of synchrony as early as the 8-cell stage. By contrast, many representatives of derived families Ambystomatidae and Salamandridae have latitudinal furrows of the third cleavage and extensive period of synchronous divisions. Our analysis of these ontogenetic characters mapped onto a phylogenetic tree shows that the cleavage pattern of large, yolky eggs with short series of synchronous divisions is an ancestral trait for the tailed amphibians, while the data on the orientation of third cleavage furrows seem to be ambiguous with respect to phylogeny. Nevertheless, the midblastula transition, which is characteristic of the model species Ambystoma mexicanum (Caudata) and Xenopus laevis (Anura), might have evolved convergently in these two amphibian orders.

Efficient production of a bioactive Bevacizumab monoclonal antibody using the 2A self-cleavage peptide in transgenic rice callus

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Lei Chen

2016-08-01

Full Text Available Bevacizumab, a humanized monoclonal antibody (mAb targeting to the vascular endothelial growth factor (VEGF, has been widely used in clinical practice for the treatment of multiple cancers. Bevacizumab was mostly produced by the mammalian cell expression system. We here reported the first plant-derived Bevacizumab by using transgenic rice callus as an alternative gene expression system. Codon-optimized Bevacizumab light chain (BLC and heavy chain (BHC genes were designed, synthesized as a polyprotein with a 2A self-cleavage linker peptide from the Foot-and-mouth disease virus (FMDV, cloned into a plant binary vector under a constitutive maize ubiquitin promoter, and transformed into rice nuclear genome through Agrobacterium-mediated transformation. Southern blot and western blot analyses confirmed the integration and expression of BLC and BHC genes in transgenic rice callus. Enzyme linked immunosorbent assay (ELISA analysis indicated that the rice-derived Bevacizumab mAb was biologically active and the recombinant mAb was expressed at high levels (160.7-242.8 mg kg-1FW in transgenic rice callus. The mAb was purified by using protein A affinity chromatography and the purified antibody was tested for its binding affinity with its target hVEGF antigen by ELISA. Rice callus produced Bevacizumab and a commercial Bevacizumab (Avastin were shown to have similar binding affinity to hVEGF. These results indicated that rice callus produced Bevacizumab could have similar biological activity and might potentially be used as a cost-effective biosimilar molecule in future cancer treatment.

Fine-tuning alkyne cycloadditions: Insights into photochemistry responsible for the double-strand DNA cleavage via structural perturbations in diaryl alkyne conjugates

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Igor V. Alabugin

2011-06-01

Full Text Available Hybrid molecules combining photoactivated aryl acetylenes and a dicationic lysine moiety cause the most efficient double-strand (ds DNA cleavage known to date for a small molecule. In order to test the connection between the alkylating ability and the DNA-damaging properties of these compounds, we investigated the photoreactivity of three isomeric aryl–tetrafluoropyridinyl (TFP alkynes with amide substituents in different positions (o-, m-, and p- toward a model π-system. Reactions with 1,4-cyclohexadiene (1,4-CHD were used to probe the alkylating properties of the triplet excited states in these three isomers whilst Stern–Volmer quenching experiments were used to investigate the kinetics of photoinduced electron transfer (PET. The three analogous isomeric lysine conjugates cleaved DNA with different efficiencies (34, 15, and 0% of ds DNA cleavage for p-, m-, and o-substituted lysine conjugates, respectively consistent with the alkylating ability of the respective acetamides. The significant protecting effect of the hydroxyl radical and singlet oxygen scavengers to DNA cleavage was shown only with m-lysine conjugate. All three isomeric lysine conjugates inhibited human melanoma cell growth under photoactivation: The p-conjugate had the lowest CC50 (50% cell cytotoxicity value of 1.49 × 10−7 M.

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

Science.gov (United States)

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

2016-01-01

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

Kininogen Cleavage Assay: Diagnostic Assistance for Kinin-Mediated Angioedema Conditions.

Directory of Open Access Journals (Sweden)

Rémi Baroso

Full Text Available Angioedema without wheals (AE is a symptom characterised by localised episodes of oedema presumably caused by kinin release from kininogen cleavage. It can result from a hereditary deficiency in C1 Inhibitor (C1Inh, but it can present with normal level of C1Inh. These forms are typically difficult to diagnose although enhanced kinin production is suspected or demonstrated in some cases.We wanted to investigate bradykinin overproduction in all AE condition with normal C1Inh, excluding cases with enhanced kinin catabolism, and to propose this parameter as a disease biomarker.We retrospectively investigated high molecular weight kininogen (HK cleavage pattern, using gel electrophoresis and immunorevelation. Plasma samples were drawn using the same standardised procedure from blood donors or AE patients with normal C1Inh conditions, normal kinin catabolism, and without prophylaxis.Circulating native HK plasma concentrations were similar in the healthy men (interquartile range: 98-175μg/mL, n = 51 and in healthy women (90-176μg/mL, n = 74, while HK cleavage was lower (p14.4% HK cleavage for men; 33.0% HK cleavage for women, with >98% specificity achieved for all parameters. In plasma from patients undergoing recovery two months after oestrogen/progestin combination withdrawal (n = 13 or two weeks after AE attack (n = 2, HK cleavage was not fully restored, suggesting its use as a post-attack assay.As a diagnostic tool, HK cleavage can offer physicians supportive arguments for kinin production in suspected AE cases and improve patient follow-up in clinical trials or prophylactic management.

Detection of nucleic acid sequences by invader-directed cleavage

Science.gov (United States)

Brow, Mary Ann D.; Hall, Jeff Steven Grotelueschen; Lyamichev, Victor; Olive, David Michael; Prudent, James Robert

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 5' nuclease activity of a variety of enzymes is used to cleave the target-dependent cleavage structure, thereby indicating the presence of specific nucleic acid sequences or specific variations thereof. The present invention further relates to methods and devices for the separation of nucleic acid molecules based by charge.

A potyvirus-based gene vector allows producing active human S-COMT and animal GFP, but not human sorcin, in vector-infected plants.

Science.gov (United States)

Kelloniemi, Jani; Mäkinen, Kristiina; Valkonen, Jari P T

2006-05-01

Potato virus A (PVA), a potyvirus with a (+)ssRNA genome translated to a large polyprotein, was engineered and used as a gene vector for expression of heterologous proteins in plants. Foreign genes including jellyfish GFP (Aequorea victoria) encoding the green fluorescent protein (GFP, 27 kDa) and the genes of human origin (Homo sapiens) encoding a soluble resistance-related calcium-binding protein (sorcin, 22 kDa) and the catechol-O-methyltransferase (S-COMT; 25 kDa) were cloned between the cistrons for the viral replicase and coat protein (CP). The inserts caused no adverse effects on viral infectivity and virulence, and the inserted sequences remained intact in progeny viruses in the systemically infected leaves. The heterologous proteins were released from the viral polyprotein following cleavage by the main viral proteinase, NIa, at engineered proteolytic processing sites flanking the insert. Active GFP, as indicated by green fluorescence, and S-COMT with high levels of enzymatic activity were produced. In contrast, no sorcin was detected despite the expected equimolar amounts of the foreign and viral proteins being expressed as a polyprotein. These data reveal inherent differences between heterologous proteins in their suitability for production in plants.

Controllable laser thermal cleavage of sapphire wafers

Science.gov (United States)

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

2018-03-01

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

PENGKLONAN DAN PERUNUTAN NUKLEOTIDA GEN SELUBUNG PROTEIN DAN 3’UTR (untranslated region PEANUT STRIPE VIRUS

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Hasriadi Mat Akin

2011-10-01

Full Text Available Cloning and sequencing of coat protein gene and 3’UTR (untranslated region of peanut stripe virus. The cDNA of 3' terminal of peanut stripe virus genomic RNA was cloned and sequenced. The cDNA was ligated with plasmid vector pGEM-T Easy and transformed to competent cells of Escherichia coli. The 3' terminal of PstV genomic RNA contained 1195 nucleotides (nts.  The region included the nucleotide sequences of NIb (nuclear inclusion body (129 nts, CP gene (coat protein gene (861 nts, and 3'UTR (untranslated region (205 nts. The nucleotide sequence of a CP gene contained one long uninterrupted open reading frame (ORF without a start codon, which ended a UAG stop codon. The 287 amino acid residues of PStV coat protein were predicted from the CP gene.  The amino acid was analyzed for the presence of consensus polyprotein cleavage site for maturation of potyvirus polyprotein.  A putative cleavage site was found at position 43 (Q/S following the Valine (V residue at -4 position.  This isolate of PstV can be expected to be aphid transmissible because the coat protein contained a DAG triplet at position 53-55.

RNA2 of grapevine fanleaf virus: sequence analysis and coat protein cistron location.

Science.gov (United States)

Serghini, M A; Fuchs, M; Pinck, M; Reinbolt, J; Walter, B; Pinck, L

1990-07-01

The nucleotide sequence of the genomic RNA2 (3774 nucleotides) of grapevine fanleaf virus strain F13 was determined from overlapping cDNA clones and its genetic organization was deduced. Two rapid and efficient methods were used for cDNA cloning of the 5' region of RNA2. The complete sequence contained only one long open reading frame of 3555 nucleotides (1184 codons, 131K product). The analysis of the N-terminal sequence of purified coat protein (CP) and identification of its C-terminal residue have allowed the CP cistron to be precisely positioned within the polyprotein. The CP produced by proteolytic cleavage at the Arg/Gly site between residues 680 and 681 contains 504 amino acids (Mr 56019) and has hydrophobic properties. The Arg/Gly cleavage site deduced by N-terminal amino acid sequence analysis is the first for a nepovirus coat protein and for plant viruses expressing their genomic RNAs by polyprotein synthesis. Comparison of GFLV RNA2 with M RNA of cowpea mosaic comovirus and with RNA2 of two closely related nepoviruses, tomato black ring virus and Hungarian grapevine chrome mosaic virus, showed strong similarities among the 3' non-coding regions but less similarity among the 5' end non-coding sequences than reported among other nepovirus RNAs.

Kinetic Evaluation of Imidacloprid Degradation in Mice Organs Treated with Olive Oil Polyphenols Extract

OpenAIRE

Broznić, Dalibor; Marinić, Jelena; Tota, Marin; Čanadi Jurešić, Gordana; Milin, Čedomila

2008-01-01

Imidacloprid is a highly effective insecticide, acting as agonists at the insect nicotinic acetylcholine receptor. Nevertheless, imidacloprid itself or its metabolites could exhibit toxicity in mammals. Imidacloprid biotransformation involves oxidative cleavage, releasing the 6-chloronicotinic acid. Therefore, the concentration of imidacloprid and 6-chloronicotinic acid was used to characterize degradation kinetics and distribution of imidacloprid in mice liver, kidneys and lungs. Additionall...

Full Length Research Paper Curcumin induces cleavage of -catenin ...

African Journals Online (AJOL)

β-Catenin/Tcf-4 signaling pathway plays important roles in colorectal tumorigenesis. RT-PCR, western blotting and immunoprecipitation were used to study the effects of curcumin on β-catenin/Tcf-4 signaling pathway in HT-29 cells. Treatment of curcumin could induce cleavage of β-catenin and the cleavage could be ...

Altered cleavage patterns in human tripronuclear embryos and their association to fertilization method

DEFF Research Database (Denmark)

Joergensen, Mette Warming; Agerholm, Inge; Hindkjaer, Johnny

2014-01-01

PURPOSE: To analyze the cleavage patterns in dipronuclear (2PN) and tripronuclear (3PN) embryos in relation to fertilization method. METHOD: Time-lapse analysis. RESULTS: Compared to 2PN, more 3PN IVF embryos displayed early cleavage into 3 cells (p ... stage (p embryos, the 2nd and 3rd cleavage cycles were completed within the expected time frame. However, timing of the cell divisions within the cleavage cycles differed between the two groups. In contrast......, the completion of the 1st, 2nd, and 3rd cleavage cycle was delayed, but with a similar division pattern for 3PN ICSI compared with the 2PN ICSI embryos. 3PN, more often than 2PN ICSI embryos, displayed early cleavage into 3 cells (p = 0.03) and arrested development from the compaction stage and onwards (p = 0...

Incorporation of 12-methoxydodecanoate into the human immunodeficiency virus 1 gag polyprotein precursor inhibits its proteolytic processing and virus production in a chronically infected human lymphoid cell line.

OpenAIRE

Bryant, M L; Ratner, L; Duronio, R J; Kishore, N S; Devadas, B; Adams, S P; Gordon, J I

1991-01-01

Covalent linkage of myristate (tetradecanoate; 14:0) to the NH2-terminal glycine residue of the human immunodeficiency virus 1 (HIV-1) 55-kDa gag polyprotein precursor (Pr55gag) is necessary for its proteolytic processing and viral assembly. We have shown recently that several analogs of myristate in which a methylene group is replaced by a single oxygen or sulfur atom are substrates for Saccharomyces cerevisiae and mammalian myristoyl-CoA:protein N-myristoyltransferase (EC 2.3.1.97; NMT) des...

RecA-mediated cleavage reaction of Lambda repressor and DNA ...

African Journals Online (AJOL)

PRECIOUS

2010-01-11

Jan 11, 2010 ... hydrolyze ATP at all, but fulfills RecA functions such as cleavage of Lambda repressor and strand .... DNA binding properties of RecA and may result in an in- .... AMP-PNP there is no cleavage of Lambda repressor (Figure.

Construction of a molecular clone of ovine enzootic nasal tumor virus.

Science.gov (United States)

Walsh, Scott R; Gerpe, María Carla Rosales; Wootton, Sarah K

2016-12-30

Enzootic nasal tumor virus (ENTV-1) is an ovine betaretrovirus that has been linked to enzootic nasal adenocarcinoma (ENA), a contagious tumor of the ethmoid turbinates of sheep. Transmission experiments performed using virus isolated from cell free nasal tumor homogenates suggest that ENTV-1 is the causative agent of ENA; however, this etiological relationship has not been conclusively proven due to the fact that the virus cannot be propagated in vitro nor is there an infectious molecular clone of the virus. Here we report construction of a molecular clone of ENTV-1 and demonstrate that transfection of this molecular clone into HEK 293T cells produces mature virus particles. Analysis of recombinant virus particles derived from the initial molecular clone revealed a defect in the proteolytic processing of Gag; however, this defect could be corrected by co-expression of the Gag-Pro-Pol polyprotein from the highly related Jaagsiekte sheep retrovirus (JSRV) suggesting that the polyprotein cleavage sites in the ENTV-1 molecular clone were functional. Mutagenesis of the molecular clone to correct amino acid variants identified within the pro gene did not restore proteolytic processing; whereas deletion of one proline residue from a polyproline tract located in variable region 1 (VR1) of the matrix resulted in production of CA protein of the mature (cleaved) size strongly suggesting that normal virion morphogenesis and polyprotein cleavage took place. Finally, electron microscopy revealed the presence of spherical virus particles with an eccentric capsid and an average diameter of about 100 nm. In summary, we have constructed the first molecular clone of ENTV-1 from which mature virus particles can be produced. Future experiments using virus produced from this molecular clone can now be conducted to fulfill Koch's postulates and demonstrate that ENTV-1 is necessary and sufficient to induce ENA in sheep.

Acquisition of a novel eleven amino acid insertion directly N-terminal to a tetrabasic cleavage site confers intracellular cleavage of an H7N7 influenza virus hemagglutinin

International Nuclear Information System (INIS)

Hamilton, Brian S.; Sun, Xiangjie; Chung, Changik; Whittaker, Gary R.

2012-01-01

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 appear 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.

Acquisition of a novel eleven amino acid insertion directly N-terminal to a tetrabasic cleavage site confers intracellular cleavage of an H7N7 influenza virus hemagglutinin

Energy Technology Data Exchange (ETDEWEB)

Hamilton, Brian S.; Sun, Xiangjie; Chung, Changik [Department of Microbiology and Immunology, College of Veterinary Medicine, Cornell University, Ithaca NY 14853 (United States); New York Center of Excellence for Influenza Research and Surveillance, University of Rochester Medical Center, Rochester NY 14627 (United States); Whittaker, Gary R., E-mail: grw7@cornell.edu [Department of Microbiology and Immunology, College of Veterinary Medicine, Cornell University, Ithaca NY 14853 (United States); New York Center of Excellence for Influenza Research and Surveillance, University of Rochester Medical Center, Rochester NY 14627 (United States)

2012-12-05

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 appear 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.

Short RNA guides cleavage by eukaryotic RNase III.

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Bruno Lamontagne

Full Text Available In eukaryotes, short RNAs guide a variety of enzymatic activities that range from RNA editing to translation repression. It is hypothesized that pre-existing proteins evolved to bind and use guide RNA during evolution. However, the capacity of modern proteins to adopt new RNA guides has never been demonstrated. Here we show that Rnt1p, the yeast orthologue of the bacterial dsRNA-specific RNase III, can bind short RNA transcripts and use them as guides for sequence-specific cleavage. Target cleavage occurred at a constant distance from the Rnt1p binding site, leaving the guide RNA intact for subsequent cleavage. Our results indicate that RNase III may trigger sequence-specific RNA degradation independent of the RNAi machinery, and they open the road for a new generation of precise RNA silencing tools that do not trigger a dsRNA-mediated immune response.

Involvement of a cytosine side chain in proton transfer in the rate-determining step of ribozyme self-cleavage

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Shih, I-hung; Been, Michael D.

2001-01-01

Ribozymes of hepatitis delta virus have been proposed to use an active-site cytosine as an acid-base catalyst in the self-cleavage reaction. In this study, we have examined the role of cytosine in more detail with the antigenomic ribozyme. Evidence that proton transfer in the rate-determining step involved cytosine 76 (C76) was obtained from examining cleavage activity of the wild-type and imidazole buffer-rescued C76-deleted (C76Δ) ribozymes in D2O and H2O. In both reactions, a similar kinetic isotope effect and shift in the apparent pKa indicate that the buffer is functionally substituting for the side chain in proton transfer. Proton inventory of the wild-type reaction supported a mechanism of a single proton transfer at the transition state. This proton transfer step was further characterized by exogenous base rescue of a C76Δ mutant with cytosine and imidazole analogues. For the imidazole analogues that rescued activity, the apparent pKa of the rescue reaction, measured under kcat/KM conditions, correlated with the pKa of the base. From these data a Brønsted coefficient (β) of 0.51 was determined for the base-rescued reaction of C76Δ. This value is consistent with that expected for proton transfer in the transition state. Together, these data provide strong support for a mechanism where an RNA side chain participates directly in general acid or general base catalysis of the wild-type ribozyme to facilitate RNA cleavage. PMID:11171978

Construction of dengue virus protease expression plasmid and in vitro protease assay for screening antiviral inhibitors.

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Lai, Huiguo; Teramoto, Tadahisa; Padmanabhan, Radhakrishnan

2014-01-01

Dengue virus serotypes 1-4 (DENV1-4) are mosquito-borne human pathogens of global significance causing ~390 million cases annually worldwide. The virus infections cause in general a self-limiting disease, known as dengue fever, but occasionally also more severe forms, especially during secondary infections, dengue hemorrhagic fever and dengue shock syndrome causing ~25,000 deaths annually. The DENV genome contains a single-strand positive sense RNA, approximately 11 kb in length. The 5'-end has a type I cap structure. The 3'-end has no poly(A) tail. The viral RNA has a single long open reading frame that is translated by the host translational machinery to yield a polyprotein precursor. Processing of the polyprotein precursor occurs co-translationally by cellular proteases and posttranslationally by the viral serine protease in the endoplasmic reticulum (ER) to yield three structural proteins (capsid (C), precursor membrane (prM), and envelope (E) and seven nonstructural (NS) proteins (NS1, NS2A, NS2B, NS3, NS4A, NS4B, and NS5). The active viral protease consists of both NS2B, an integral membrane protein in the ER, and the N-terminal part of NS3 (180 amino acid residues) that contains the trypsin-like serine protease domain having a catalytic triad of H51, D75, and S135. The C-terminal part of NS3, ~170-618 amino acid residues, encodes an NTPase/RNA helicase and 5'-RNA triphosphatase activities; the latter enzyme is required for the first step in 5'-capping. The cleavage sites of the polyprotein by the viral protease consist of two basic amino acid residues such as KR, RR, or QR, followed by short chain amino acid residues, G, S, or T. Since the cleavage of the polyprotein by the viral protease is absolutely required for assembly of the viral replicase, blockage of NS2B/NS3pro activity provides an effective means for designing dengue virus (DENV) small-molecule therapeutics. Here we describe the screening of small-molecule inhibitors against DENV2 protease.

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

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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.

Effect of microstructure on the cleavage fracture strength of low carbon Mn-Ni-Mo bainitic steels

International Nuclear Information System (INIS)

Im, Young-Roc; Lee, Byeong-Joo; Oh, Yong Jun; Hong, Jun Hwa; Lee, Hu-Chul

2004-01-01

The effects of the microstructure on the cleavage fracture strength of low carbon Mn-Ni-Mo bainitic steels were examined. A four-point bend test and double-notched bend specimens were used to measure the cleavage fracture strength of the alloys and identify the cleavage initiating micro-cracks, respectively. The cleavage fracture strength and DBTT of Mn-Ni-Mo bainitic steels were strongly affected by the alloy carbon content. The decrease in the alloy carbon content resulted in a decrease in the inter-lath cementite-crowded layers and higher cleavage fracture strength. Micro-cracks that formed across the inter-lath cementite-crowded layers were observed to initiate cleavage fracture. The width of these inter-lath cementite-crowded layers was accepted as a cleavage initiating micro-crack size in the micro-mechanical modeling of the cleavage fracture, and the measured cleavage strength values of the bainitic Mn-Ni-Mo steels were well represented by the modified Griffith relationship

Genetic characterization of a novel Iflavirus associated with vomiting disease in the Chinese oak silkmoth Antheraea pernyi.

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Peng Geng

Full Text Available Larvae of the Chinese oak silkmoth (Antheraea pernyi are often affected by AVD (A. pernyi vomiting disease, whose causative agent has long been suspected to be a virus. In an unrelated project we discovered a novel positive sense single-stranded RNA virus that could reproduce AVD symptoms upon injection into healthy A. pernyi larvae. The genome of this virus is 10,163 nucleotides long, has a natural poly-A tail, and contains a single, large open reading frame flanked at the 5' and 3' ends by untranslated regions containing putative structural elements for replication and translation of the virus genome. The open reading frame is predicted to encode a 3036 amino acid polyprotein with four viral structural proteins (VP1-VP4 located in the N-terminal end and the non-structural proteins, including a helicase, RNA-dependent RNA polymerase and 3C-protease, located in the C-terminal end of the polyprotein. Putative 3C-protease and autolytic cleavage sites were identified for processing the polyprotein into functional units. The genome organization, amino acid sequence and phylogenetic analyses suggest that the virus is a novel species of the genus Iflavirus, with the proposed name of Antheraea pernyi Iflavirus (ApIV.

Characterization of SNARE Cleavage Products Generated by Formulated Botulinum Neurotoxin Type-A Drug Products

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Jack Xie

2010-08-01

Full Text Available The study evaluated substrate cleavage product(s generated by three botulinum neurotoxin serotype A (BoNT/A medicinal drug products utilizing a novel and highly specific, light-chain activity, high-performance liquid chromatography (LCA-HPLC method. Samples were reacted with a commercially available BoNT/A fluorescent substrate derived from the SNAP-25 sequence. Reaction products were separated by reversed-phase HPLC. The method detected an atypical cleavage pattern by one of the formulated drug products. IncobotulinumtoxinA produced two cleavage fragments rather than the single fragment typically generated by BoNT/A. Identification confirmed the secondary cleavage at a position corresponding to SNAP-25 Arg198–Ala199 (normal BoNT/A cleavage is Gln197–Arg198. Arg198–Ala199 is also the cleavage site for trypsin and serotype C toxin. Normal cleavage was observed for all other BoNT/A drug product samples, as well as 900-kD and 150-kD bulk toxin BoNT/A. The reason for this unexpected secondary cleavage pattern by one formulated BoNT/A drug product is unknown. Possible explanations include a contaminating protease and/or damage to the 150-kD type-A toxin causing nonspecific substrate recognition and subsequent cleavage uncharacteristic of type-A toxin. The BoNT/A drug products were also analyzed via the LCA-HPLC assay using a commercial BoNT/C fluorescent substrate derived from the syntaxin sequence. Cleavage of the serotype C substrate by incobotulinumtoxinA was also confirmed whilst neither of the other drug products cleaved the syntaxin substrate.

Post-transcription cleavage generates the 3' end of F17R transcripts in vaccinia virus

International Nuclear Information System (INIS)

D'Costa, Susan M.; Antczak, James B.; Pickup, David J.; Condit, Richard C.

2004-01-01

Most vaccinia virus intermediate and late mRNAs possess 3' ends that are extremely heterogeneous in sequence. However, late mRNAs encoding the cowpox A-type inclusion protein (ATI), the second largest subunit of the RNA polymerase, and the late telomeric transcripts possess homogeneous 3' ends. In the case of the ATI mRNA, it has been shown that the homogeneous 3' end is generated by a post-transcriptional endoribonucleolytic cleavage event. We have determined that the F17R gene also produces homogeneous transcripts generated by a post-transcriptional cleavage event. Mapping of in vivo mRNA shows that the major 3' end of the F17R transcript maps 1262 nt downstream of the F17R translational start site. In vitro transcripts spanning the in vivo 3' end are cleaved in an in vitro reaction using extracts from virus infected cells, and the site of cleavage is the same both in vivo and in vitro. Cleavage is not observed using extract from cells infected in the presence of hydroxyurea; therefore, the cleavage factor is either virus-coded or virus-induced during the post-replicative phase of virus replication. The cis-acting sequence responsible for cleavage is orientation specific and the factor responsible for cleavage activity has biochemical properties similar to the factor required for cleavage of ATI transcripts. Partially purified cleavage factor generates cleavage products of expected size when either the ATI or F17R substrates are used in vitro, strongly suggesting that cleavage of both transcripts is mediated by the same factor

Complete sequence of RNA1 of grapevine Anatolian ringspot virus.

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Digiaro, Michele; Nahdi, Sabrine; Elbeaino, Toufic

2012-10-01

The nucleotide sequence of RNA1 of grapevine Anatolian ringspot virus (GARSV), a nepovirus of subgroup B, was determined from cDNA clones. It is 7,288 nucleotides in length excluding the 3' terminal poly(A) tail and contains a large open reading frame (ORF), extending from nucleotides 272 to 7001, encoding a polypeptide of 2,243 amino acids with a predicted molecular mass of 250 kDa. The primary structure of the polyprotein, compared with that of other viral polyproteins, revealed the presence of all the characteristic domains of members of the order Picornavirales, i.e., the NTP-binding protein (1B(Hel)), the viral genome-linked protein (1C(VPg)), the proteinase (1D(Prot)), the RNA-dependent RNA polymerase (1E(Pol)), and of the protease cofactor (1A(Pro-cof)) shared by members of the subfamily Comovirinae within the family Secoviridae. The cleavage sites predicted within the polyprotein were found to be in agreement with those previously reported for nepoviruses of subgroup B, processing from 1A to 1E proteins of 67, 64, 3, 23 and 92 kDa, respectively. The RNA1-encoded polyprotein (p1) shared the highest amino acid sequence identity (66 %) with tomato black ring virus (TBRV) and beet ringspot virus (BRSV). The 5'- and 3'-noncoding regions (NCRs) of GARSV-RNA1 shared 89 % and 95 % nucleotide sequence identity respectively with the corresponding regions in RNA2. Phylogenetic analysis confirmed the close relationship of GARSV to members of subgroup B of the genus Nepovirus.

Impact of human milk pasteurization on the kinetics of peptide release during in vitro dynamic term newborn digestion.

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Deglaire, Amélie; De Oliveira, Samira C; Jardin, Julien; Briard-Bion, Valérie; Emily, Mathieu; Ménard, Olivia; Bourlieu, Claire; Dupont, Didier

2016-07-01

Holder pasteurization (62.5°C, 30 min) ensures sanitary quality of donor's human milk but also denatures beneficial proteins. Understanding whether this further impacts the kinetics of peptide release during gastrointestinal digestion of human milk was the aim of the present paper. Mature raw (RHM) or pasteurized (PHM) human milk were digested (RHM, n = 2; PHM, n = 3) by an in vitro dynamic system (term stage). Label-free quantitative peptidomics was performed on milk and digesta (ten time points). Ascending hierarchical clustering was conducted on "Pasteurization × Digestion time" interaction coefficients. Preproteolysis occurred in human milk (159 unique peptides; RHM: 91, PHM: 151), mostly on β-casein (88% of the endogenous peptides). The predicted cleavage number increased with pasteurization, potentially through plasmin activation (plasmin cleavages: RHM, 53; PHM, 76). During digestion, eight clusters resumed 1054 peptides from RHM and PHM, originating for 49% of them from β-casein. For seven clusters (57% of peptides), the kinetics of peptide release differed between RHM and PHM. The parent protein was significantly linked to the clustering (p-value = 1.4 E-09), with β-casein and lactoferrin associated to clusters in an opposite manner. Pasteurization impacted selectively gastric and intestinal kinetics of peptide release in term newborns, which may have further nutritional consequences. © 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Prediction of proteasome cleavage motifs by neural networks

DEFF Research Database (Denmark)

Kesimir, C.; Nussbaum, A.K.; Schild, H.

2002-01-01

physiological conditions. Our algorithm has been trained not only on in vitro data, but also on MHC Class I ligand data, which reflect a combination of immunoproteasome and constitutive proteasome specificity. This feature, together with the use of neural networks, a non-linear classification technique, make...... the prediction of MHC Class I ligand boundaries more accurate: 65% of the cleavage sites and 85% of the non-cleavage sites are correctly determined. Moreover, we show that the neural networks trained on the constitutive proteasome data learns a specificity that differs from that of the networks trained on MHC...

Remarkable weakness against cleavage stress for YBCO-coated conductors and its effect on the YBCO coil performance

International Nuclear Information System (INIS)

Yanagisawa, Y.; Nakagome, H.; Takematsu, T.; Takao, T.; Sato, N.; Takahashi, M.; Maeda, H.

2011-01-01

Cleavage strength for YBCO-coated conductor is extremely low, typically 0.5 MPa. The remarkable weakness is due to cracks on the slit edge of the conductor. The cleavage stress appears on YBCO double pancake coils impregnated with epoxy. The cleavage stress should be avoided in the coil winding. Cleavage strength for an YBCO-coated conductor at 77 K was investigated with a model experiment. The nominal cleavage strength for an YBCO-coated conductor is extremely low, typically 0.5 MPa. This low nominal cleavage strength is due to stress concentration on a small part of the YBCO-coated conductor in cleavage fracture. Debonding by the cleavage stress occurs at the interface between the buffer layer and the Hastelloy substrate. The nominal cleavage strength for a slit edge of the conductor is 2.5-times lower than that for the original edge of the conductor; cracks and micro-peel existing over the slit edge reduce the cleavage strength for the slit edge. Cleavage stress and peel stress should be avoided in coil winding, as they easily delaminate the YBCO-coated conductor, resulting in substantial degradation of coil performance. These problems are especially important for epoxy impregnated YBCO-coated conductor coils. It appears that effect of cleavage stress and peel stress are mostly negligible for paraffin impregnated YBCO-coated conductor coils or dry wound YBCO-coated conductor coils.

Bioremediation of 2-chlorophenol containing wastewater by aerobic granules-kinetics and toxicity

Energy Technology Data Exchange (ETDEWEB)

Khan, Mohammad Zain [Environmental Research Laboratory, Department of chemistry, Aligarh Muslim University, Faculty of Science, Aligarh 202002, UP (India); Mondal, Pijush Kanti, E-mail: pijushamu@gmail.com [Environmental Research laboratory, Department of Applied Chemistry, Faculty of Engineering and Technology, Aligarh Muslim University, Aligarh 202002, UP (India); Sabir, Suhail, E-mail: sabirsuhail09@gmail.com [Environmental Research Laboratory, Department of chemistry, Aligarh Muslim University, Faculty of Science, Aligarh 202002, UP (India)

2011-06-15

Highlights: {yields} 2-Chlorophenol degrading aerobic granules were cultivated in sequencing batch reactor in presence of glucose and the organic loading rate (OLR) was increased from 6.9 to 9.7 kg COD m{sup -3} d{sup -1} during the experiment. {yields} Spectral studies confirmed that the biodegradation occurs via chlorocatechol pathway and modified ortho-cleavage. {yields} Biodegradation kinetics of 2-CP followed the Haldane model with kinetic parameters (R{sup 2} > 0.9) V{sub max} = 840 mg2-CP gMLVSS{sup -1} d{sup -1}, K{sub s} = 24.61 mg L{sup -1}, K{sub i} = 315.02 mg L{sup -1}. {yields} Genotoxic examination by plasmid nicking assay confirmed that the effluent was non-toxic. - Abstract: 2-Chlorophenol (2-CP) degrading aerobic granules were cultivated in a sequencing batch reactor (SBR) in presence of glucose. The organic loading rate (OLR) was increased from 6.9 to 9.7 kg COD m{sup -3} d{sup -1} (1150-1617 mg L{sup -1}COD per cycle) during the experiment. The alkalinity (1000 mg L{sup -1} as CaCO{sub 3}) was maintained throughout the experiment. The specific cell growth rate was found to be 0.013 d{sup -1}. A COD removal efficiency of 94% was achieved after steady state at 8 h HRT (hydraulic retention time). FTIR, UV, GC, GC/MS studies confirmed that the biodegradation of 2-CP occurs via chlorocatechol (modified ortho-cleavage) pathway. Biodegradation kinetics followed the Haldane model with kinetic parameters: V{sub max} = 840 mg2-CP gMLVSS{sup -1} d{sup -1}, K{sub s} = 24.61 mg L{sup -1}, K{sub i} = 315.02 mg L{sup -1}. Abiotic losses of 2-CP due to volatilization and photo degradation by sunlight were less than 3% and the results of genotoxicity showed that the degradation products are eco-friendly.

Far-UV photochemical bond cleavage of n-amyl nitrite: bypassing a repulsive surface.

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Minitti, Michael P; Zhang, Yao; Rosenberg, Martin; Brogaard, Rasmus Y; Deb, Sanghamitra; Sølling, Theis I; Weber, Peter M

2012-01-19

We have investigated the deep-UV photoinduced, homolytic bond cleavage of amyl nitrite to form NO and pentoxy radicals. One-color multiphoton ionization with ultrashort laser pulses through the S(2) state resonance gives rise to photoelectron spectra that reflect ionization from the S(1) state. Time-resolved pump-probe photoionization measurements show that upon excitation at 207 nm, the generation of NO in the v = 2 state is delayed, with a rise time of 283 (16) fs. The time-resolved mass spectrum shows the NO to be expelled with a kinetic energy of 1.0 eV, which is consistent with dissociation on the S(1) state potential energy surface. Combined, these observations show that the first step of the dissociation reaction involves an internal conversion from the S(2) to the S(1) state, which is followed by the ejection of the NO radical on the predissociative S(1) state potential energy surface.

Foot-and-mouth disease virus capsid proteins; analysis of protein processing, assembly and utility as vaccines

DEFF Research Database (Denmark)

Belsham, Graham

Foot-and-mouth disease (FMD) remains one of the most economically important infectious diseases of production animals globally. The infection is caused by foot-and-mouth disease virus (FMDV), a member of the picornavirus family. The positive sense RNA genome of the virus includes a single, large......, open reading frame that encodes a polyprotein. The intact polyprotein is never observed as it is processed, both during and after translation, to 15 different mature proteins plus a variety of precursors. The FMDV capsid protein precursor, P1-2A, is cleaved by the virus encoded 3C protease (3Cpro......) to generate VP0, VP3, VP1 and the peptide 2A. Sixty copies of each of the capsid proteins “self-assemble” into empty capsid particles or with the RNA genome into infectious viruses. These particles normally lack 2A but it is possible to construct and isolate mutant FMDVs in which the cleavage of the VP1/2A...

Flanking signal and mature peptide residues influence signal peptide cleavage

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Ranganathan Shoba

2008-12-01

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

Staggering in the cleavage pattern of E. coli ABC-excinuclease

International Nuclear Information System (INIS)

Myles, G.M.; Van Houten, B.; Sancar, A.

1986-01-01

E. coli ABC excinuclease is a complex of three proteins encoded by the uvrA, uvrB, and uvrC genes. The enzyme repairs DNA mono and diadducts by the single strand cleavage of DNA eight phosphodiester bond 5' and four or five phosphodiester bonds 3' to a DNA lesion and facilitates the removal of the resulting twelve or thirteen nucleotide fragment. In this study, the authors have investigated the excision pattern for ultraviolet (UV) induced diadducts, i.e. cyclobutane pyrimidine dimers and pyrimidine-pyrimidone (6-4) photoproducts. Terminally (5' or 3') labeled DNA was irradiated with 254nm UV and treated with ABC excinuclease before and after photoreactivation of cyclobutane dimers by E. coli DNA photolyase. In this way, the authors were able to differentiate between the cleavage pattern of pyrimidine dimers and of (6-4) photoproducts. Their results show that certain TT cyclobutane dimers and rare TT (6-4) photoproducts are excised by cleavage seven and, less frequently, six phosphodiester bonds to the 5' side of the DNA lesion in addition to the primary cutting site at the eight 5' phosphodiester bond. The 3' cleavage sites are maintained at the fourth and fifth phosphodiester bonds for the these UV induced lesions. These data indicate that the cleavage pattern of the ABC excinuclease may be dependent upon both the type of DNA lesion as well as it surrounding nucleotide sequence. In addition, the authors analysis shows that (6-4) photoproducts are much better substrates for ABC excinuclease than are pyrimidine dimers

Downstream element determines RNase Y cleavage of the saePQRS operon in Staphylococcus aureus.

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Marincola, Gabriella; Wolz, Christiane

2017-06-02

In gram-positive bacteria, RNase J1, RNase J2 and RNase Y are thought to be major contributors to mRNA degradation and maturation. In Staphylococcus aureus, RNase Y activity is restricted to regulating the mRNA decay of only certain transcripts. Here the saePQRS operon was used as a model to analyze RNase Y specificity in living cells. A RNase Y cleavage site is located in an intergenic region between saeP and saeQ. This cleavage resulted in rapid degradation of the upstream fragment and stabilization of the downstream fragment. Thereby, the expression ratio of the different components of the operon was shifted towards saeRS, emphasizing the regulatory role of RNase Y activity. To assess cleavage specificity different regions surrounding the sae CS were cloned upstream of truncated gfp, and processing was analyzed in vivo using probes up- and downstream of CS. RNase Y cleavage was not determined by the cleavage site sequence. Instead a 24-bp double-stranded recognition structure was identified that was required to initiate cleavage 6 nt upstream. The results indicate that RNase Y activity is determined by secondary structure recognition determinants, which guide cleavage from a distance. © The Author(s) 2017. Published by Oxford University Press on behalf of Nucleic Acids Research.

Protein cleavage strategies for an improved analysis of the membrane proteome

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Poetsch Ansgar

2006-03-01

Full Text Available Abstract Background Membrane proteins still remain elusive in proteomic studies. This is in part due to the distribution of the amino acids lysine and arginine, which are less frequent in integral membrane proteins and almost absent in transmembrane helices. As these amino acids are cleavage targets for the commonly used protease trypsin, alternative cleavage conditions, which should improve membrane protein analysis, were tested by in silico digestion for the three organisms Saccharomyces cerevisiae, Halobacterium sp. NRC-1, and Corynebacterium glutamicum as hallmarks for eukaryotes, archea and eubacteria. Results For the membrane proteomes from all three analyzed organisms, we identified cleavage conditions that achieve better sequence and proteome coverage than trypsin. Greater improvement was obtained for bacteria than for yeast, which was attributed to differences in protein size and GRAVY. It was demonstrated for bacteriorhodopsin that the in silico predictions agree well with the experimental observations. Conclusion For all three examined organisms, it was found that a combination of chymotrypsin and staphylococcal peptidase I gave significantly better results than trypsin. As some of the improved cleavage conditions are not more elaborate than trypsin digestion and have been proven useful in practice, we suppose that the cleavage at both hydrophilic and hydrophobic amino acids should facilitate in general the analysis of membrane proteins for all organisms.

Cleavage and Cell Adhesion Properties of Human Epithelial Cell Adhesion Molecule (HEPCAM)*

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Tsaktanis, Thanos; Kremling, Heidi; Pavšič, Miha; von Stackelberg, Ricarda; Mack, Brigitte; Fukumori, Akio; Steiner, Harald; Vielmuth, Franziska; Spindler, Volker; Huang, Zhe; Jakubowski, Jasmine; Stoecklein, Nikolas H.; Luxenburger, Elke; Lauber, Kirsten; Lenarčič, Brigita; Gires, Olivier

2015-01-01

Human epithelial cell adhesion molecule (HEPCAM) is a tumor-associated antigen frequently expressed in carcinomas, which promotes proliferation after regulated intramembrane proteolysis. Here, we describe extracellular shedding of HEPCAM at two α-sites through a disintegrin and metalloprotease (ADAM) and at one β-site through BACE1. Transmembrane cleavage by γ-secretase occurs at three γ-sites to generate extracellular Aβ-like fragments and at two ϵ-sites to release human EPCAM intracellular domain HEPICD, which is efficiently degraded by the proteasome. Mapping of cleavage sites onto three-dimensional structures of HEPEX cis-dimer predicted conditional availability of α- and β-sites. Endocytosis of HEPCAM warrants acidification in cytoplasmic vesicles to dissociate protein cis-dimers required for cleavage by BACE1 at low pH values. Intramembrane cleavage sites are accessible and not part of the structurally important transmembrane helix dimer crossing region. Surprisingly, neither chemical inhibition of cleavage nor cellular knock-out of HEPCAM using CRISPR-Cas9 technology impacted the adhesion of carcinoma cell lines. Hence, a direct function of HEPCAM as an adhesion molecule in carcinoma cells is not supported and appears to be questionable. PMID:26292218

Calicivirus 3C-like proteinase inhibits cellular translation by cleavage of poly(A)-binding protein.

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Kuyumcu-Martinez, Muge; Belliot, Gaël; Sosnovtsev, Stanislav V; Chang, Kyeong-Ok; Green, Kim Y; Lloyd, Richard E

2004-08-01

Caliciviruses are single-stranded RNA viruses that cause a wide range of diseases in both humans and animals, but little is known about the regulation of cellular translation during infection. We used two distinct calicivirus strains, MD145-12 (genus Norovirus) and feline calicivirus (FCV) (genus Vesivirus), to investigate potential strategies used by the caliciviruses to inhibit cellular translation. Recombinant 3C-like proteinases (r3CL(pro)) from norovirus and FCV were found to cleave poly(A)-binding protein (PABP) in the absence of other viral proteins. The norovirus r3CL(pro) PABP cleavage products were indistinguishable from those generated by poliovirus (PV) 3C(pro) cleavage, while the FCV r3CL(pro) products differed due to cleavage at an alternate cleavage site 24 amino acids downstream of one of the PV 3C(pro) cleavage sites. All cleavages by calicivirus or PV proteases separated the C-terminal domain of PABP that binds translation factors eIF4B and eRF3 from the N-terminal RNA-binding domain of PABP. The effect of PABP cleavage by the norovirus r3CL(pro) was analyzed in HeLa cell translation extracts, and the presence of r3CL(pro) inhibited translation of both endogenous and exogenous mRNAs. Translation inhibition was poly(A) dependent, and replenishment of the extracts with PABP restored translation. Analysis of FCV-infected feline kidney cells showed that the levels of de novo cellular protein synthesis decreased over time as virus-specific proteins accumulated, and cleavage of PABP occurred in virus-infected cells. Our data indicate that the calicivirus 3CL(pro), like PV 3C(pro), mediates the cleavage of PABP as part of its strategy to inhibit cellular translation. PABP cleavage may be a common mechanism among certain virus families to manipulate cellular translation.

The potato carotenoid cleavage dioxygenase 4 catalyzes a single cleavage of β-ionone ring-containing carotenes and non-epoxidated xanthophylls

KAUST Repository

Bruno, Mark

2015-04-01

Down-regulation of the potato carotenoid cleavage dioxygenase 4 (StCCD4) transcript level led to tubers with altered morphology and sprouting activity, which also accumulated higher levels of violaxanthin and lutein leading to elevated carotenoid amounts. This phenotype indicates a role of this enzyme in tuber development, which may be exerted by a cleavage product. In this work, we investigated the enzymatic activity of StCCD4, by expressing the corresponding cDNA in carotenoid accumulating Escherichia coli strains and by performing in vitro assays with heterologously expressed enzyme. StCCD4 catalyzed the cleavage of all-. trans-β-carotene at the C9\\'-C10\\' double bond, leading to β-ionone and all-. trans-β-apo-10\\'-carotenal, both in vivo and in vitro. The enzyme also cleaved β,β-cryptoxanthin, zeaxanthin and lutein either at the C9\\'-C10\\' or the C9-C10 double bond in vitro. In contrast, we did not observe any conversion of violaxanthin and only traces of activity with 9-. cis-β-carotene, which led to 9-. cis-β-apo-10\\'-carotenal. Our data indicate that all-. trans-β-carotene is the likely substrate of StCCD4 in planta, and that this carotene may be precursor of an unknown compound involved in tuber development.

Real-Time Label-Free Direct Electronic Monitoring of Topoisomerase Enzyme Binding Kinetics on Graphene.

Science.gov (United States)

Zuccaro, Laura; Tesauro, Cinzia; Kurkina, Tetiana; Fiorani, Paola; Yu, Hak Ki; Knudsen, Birgitta R; Kern, Klaus; Desideri, Alessandro; Balasubramanian, Kannan

2015-11-24

Monolayer graphene field-effect sensors operating in liquid have been widely deployed for detecting a range of analyte species often under equilibrium conditions. Here we report on the real-time detection of the binding kinetics of the essential human enzyme, topoisomerase I interacting with substrate molecules (DNA probes) that are immobilized electrochemically on to monolayer graphene strips. By monitoring the field-effect characteristics of the graphene biosensor in real-time during the enzyme-substrate interactions, we are able to decipher the surface binding constant for the cleavage reaction step of topoisomerase I activity in a label-free manner. Moreover, an appropriate design of the capture probes allows us to distinctly follow the cleavage step of topoisomerase I functioning in real-time down to picomolar concentrations. The presented results are promising for future rapid screening of drugs that are being evaluated for regulating enzyme activity.

[Recent knowledge about intestinal absorption and cleavage of carotenoids].

Science.gov (United States)

Borel, P; Drai, J; Faure, H; Fayol, V; Galabert, C; Laromiguière, M; Le Moël, G

2005-01-01

Our knowledge about intestinal absorption and cleavage of carotenoids has rapidly grown during the last years. New facts about carotenoid absorption have emerged while some controversies about cleavage are close to end. The knowledge of the absorption and conversion processes is indispensable to understand and interpret the perturbations that can occur in the metabolism of carotenoids and vitamin A. Recently, it has been shown that the absorption of certain carotenoids is not passive - as believed for a long time - but is a facilitated process that requires, at least for lutein, the class B-type 1 scavenger receptor (SR-B1). Various epidemiological and clinical studies have shown wide variations in carotenoid absorption from one subject to another, such differences are now explained by the structure of the concerned carotenoid, by the nature of the food that is absorbed with the carotenoid, by diverse exogenous factors like the intake of medicines or interfering components, by diet factors, by genetic factors, and by the nutritional status of the subject. Recently, the precise mechanism of beta-carotene cleavage by betabeta-carotene 15,15' monooxygenase (EC 1.14.99.36) - formerly called beta-carotene 15,15' dioxygenase (ex EC 1.13.11.21) - has been discovered, and a second enzyme which cleaves asymmetrically the beta-carotene molecule has been found. beta-carotene 15,15' monooxygenase only acts on the 15,15' bond, thus forming two molecules of retinal from one molecule of beta-carotene by central cleavage. Even though the betabeta-carotene 15,15' monooxygenase is much more active on the beta-carotene molecule, a study has shown that it can act on all carotenoids. Searchers now agree that other enzymes that can catalyse an eccentric cleavage of carotenoids probably exist, but under physiological conditions the betabeta-carotene 15,15' monooxygenase is by far the most active, and it is mainly effective in the small bowel mucosa and in the liver. However the

Endocytic down-regulation of ErbB2 is stimulated by cleavage of its C-terminus

DEFF Research Database (Denmark)

Lerdrup, Mads; Bruun, Silas; Grandal, Michael Vibo

2007-01-01

inhibition of HSP90 with geldanamycin this cleavage is accompanied by proteasome-dependent endocytosis of ErbB2. However, it is unknown whether C-terminal cleavage is linked to endocytosis. To study ErbB2 cleavage and endocytic trafficking, we fused yellow fluorescent protein (YFP) and cyan fluorescent...

Effects of Cysteamine on Sheep Embryo Cleavage Rates

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Sinem Ö. ENGİNLER

2015-01-01

Full Text Available Oxidative stress during in vitro culture leads to defects in development of gametes and embryos. Several antioxidants such as cysteamine, L-ascorbic acid, beta mercaptoethanol, cysteine, glutathione, proteins, vitamins have been used to supplement culture media to counter the oxidative stress. This study was conducted to detect the effect of adding cysteamine to the maturation medium to subsequent cleavage rates of sheep embryos. Totally 604 ovaries were obtained by ten replica and 2060 oocytes were collected. The cumulus oocyte complexes were recovered by the slicing method. A total of 1818 selected oocytes were divided into two groups and used for maturation (88.25%. The first group was created as supplemented with cysteamine (Group A and second group (Group B, control without cysteamine in TCM-199. The two groups were incubated for 24 h at 38.8 °C in an atmosphere of 5% CO2 in humidified air for in vitro maturation (IVM. After IVM, oocytes were fertilized with 50 x 107 / mL fresh ram semen in BSOF medium for 18 h. After fertilization, maturation groups were divided into two subgroups with different culture media: Group AI-SOF (Synthetic Oviduct Fluid medium, Group AII-CR1aa (Charles Rosencrans medium, Group BI-SOF and Group BII-CR1aa were achieved. Cleavage rates were evaluated at day 2. post insemination. The rates of cleavage were detected as 59.54% (184/309, 55.44% (173/312, 65.34% (215/329, 59.34% (200/337 respectively, with showing no statistically significant difference between the groups at the level of P>0.05. In conclusion, supplementing cysteamine to maturation media in TCM-199 did not affect the cleavage rates of sheep embryos in SOF and CR1aa culture media.

Synthetic Applications and Mechanistic Studies of the Hydroxide-Mediated Cleavage of Carbon-Carbon Bonds in Ketones

DEFF Research Database (Denmark)

Mazziotta, Andrea; Makarov, Ilya S.; Fristrup, Peter

2017-01-01

The hydroxide-mediated cleavage of ketones into alkanes and carboxylic acids has been reinvestigated and the substrate scope extended to benzyl carbonyl compounds. The transformation is performed with a 0.05 M ketone solution in refluxing xylene in the presence of 10 equiv of potassium hydroxide....... The reaction constitutes a straightforward protocol for the synthesis of certain phenyl-substituted carboxylic acids from 2-phenylcycloalkanones. The mechanism was investigated by kinetic experiments which indicated a first order reaction in hydroxide and a full negative charge in the rate-determining step....... The studies were complemented by a theoretical investigation where two possible pathways were characterized by DFT/M06-2X. The calculations showed that the scission takes place by nucleophilic attack of hydroxide on the ketone followed by fragmentation of the resulting oxyanion into the carboxylic acid...

Synthetic Applications and Mechanistic Studies of the Hydroxide-Mediated Cleavage of Carbon-Carbon Bonds in Ketones.

Science.gov (United States)

Mazziotta, Andrea; Makarov, Ilya S; Fristrup, Peter; Madsen, Robert

2017-06-02

The hydroxide-mediated cleavage of ketones into alkanes and carboxylic acids has been reinvestigated and the substrate scope extended to benzyl carbonyl compounds. The transformation is performed with a 0.05 M ketone solution in refluxing xylene in the presence of 10 equiv of potassium hydroxide. The reaction constitutes a straightforward protocol for the synthesis of certain phenyl-substituted carboxylic acids from 2-phenylcycloalkanones. The mechanism was investigated by kinetic experiments which indicated a first order reaction in hydroxide and a full negative charge in the rate-determining step. The studies were complemented by a theoretical investigation where two possible pathways were characterized by DFT/M06-2X. The calculations showed that the scission takes place by nucleophilic attack of hydroxide on the ketone followed by fragmentation of the resulting oxyanion into the carboxylic acid and a benzyl anion.

Suboptimal inhibition of protease activity in human immunodeficiency virus type 1: Effects on virion morphogenesis and RNA maturation

International Nuclear Information System (INIS)

Moore, Michael D.; Fu, William; Soheilian, Ferri; Nagashima, Kunio; Ptak, Roger G.; Pathak, Vinay K.; Hu, Wei-Shau

2008-01-01

Protease activity within nascently released human immunodeficiency virus type 1 (HIV-1) particles is responsible for the cleavage of the viral polyproteins Gag and Gag-Pol into their constituent parts, which results in the subsequent condensation of the mature conical core surrounding the viral genomic RNA. Concomitant with viral maturation is a conformational change in the packaged viral RNA from a loosely associated dimer into a more thermodynamically stable form. In this study we used suboptimal concentrations of two protease inhibitors, lopinavir and atazanavir, to study their effects on Gag polyprotein processing and on the properties of the RNA in treated virions. Analysis of the treated virions demonstrated that even with high levels of inhibition of viral infectivity (IC 90 ), most of the Gag and Gag-Pol polyproteins were processed, although slight but significant increases in processing intermediates of Gag were detected. Drug treatments also caused a significant increase in the proportion of viruses displaying either immature or aberrant mature morphologies. The aberrant mature particles were characterized by an electron-dense region at the viral periphery and an electron-lucent core structure in the viral center, possibly indicating exclusion of the genomic RNA from these viral cores. Intriguingly, drug treatments caused only a slight decrease in overall thermodynamic stability of the viral RNA dimer, suggesting that the dimeric viral RNA was able to mature in the absence of correct core condensation

Phylogenetic analyses of the polyprotein coding sequences of serotype O foot-and-mouth disease viruses in East Africa: evidence for interserotypic recombination

Directory of Open Access Journals (Sweden)

Balinda Sheila N

2010-08-01

Full Text Available Abstract Background Foot-and-mouth disease (FMD is endemic in East Africa with the majority of the reported outbreaks attributed to serotype O virus. In this study, phylogenetic analyses of the polyprotein coding region of serotype O FMD viruses from Kenya and Uganda has been undertaken to infer evolutionary relationships and processes responsible for the generation and maintenance of diversity within this serotype. FMD virus RNA was obtained from six samples following virus isolation in cell culture and in one case by direct extraction from an oropharyngeal sample. Following RT-PCR, the single long open reading frame, encoding the polyprotein, was sequenced. Results Phylogenetic comparisons of the VP1 coding region showed that the recent East African viruses belong to one lineage within the EA-2 topotype while an older Kenyan strain, K/52/1992 is a representative of the topotype EA-1. Evolutionary relationships between the coding regions for the leader protease (L, the capsid region and almost the entire coding region are monophyletic except for the K/52/1992 which is distinct. Furthermore, phylogenetic relationships for the P2 and P3 regions suggest that the K/52/1992 is a probable recombinant between serotypes A and O. A bootscan analysis of K/52/1992 with East African FMD serotype A viruses (A21/KEN/1964 and A23/KEN/1965 and serotype O viral isolate (K/117/1999 revealed that the P2 region is probably derived from a serotype A strain while the P3 region appears to be a mosaic derived from both serotypes A and O. Conclusions Sequences of the VP1 coding region from recent serotype O FMDVs from Kenya and Uganda are all representatives of a specific East African lineage (topotype EA-2, a probable indication that hardly any FMD introductions of this serotype have occurred from outside the region in the recent past. Furthermore, evidence for interserotypic recombination, within the non-structural protein coding regions, between FMDVs of serotypes A

Variable context Markov chains for HIV protease cleavage site prediction.

Science.gov (United States)

Oğul, Hasan

2009-06-01

Deciphering the knowledge of HIV protease specificity and developing computational tools for detecting its cleavage sites in protein polypeptide chain are very desirable for designing efficient and specific chemical inhibitors to prevent acquired immunodeficiency syndrome. In this study, we developed a generative model based on a generalization of variable order Markov chains (VOMC) for peptide sequences and adapted the model for prediction of their cleavability by certain proteases. The new method, called variable context Markov chains (VCMC), attempts to identify the context equivalence based on the evolutionary similarities between individual amino acids. It was applied for HIV-1 protease cleavage site prediction problem and shown to outperform existing methods in terms of prediction accuracy on a common dataset. In general, the method is a promising tool for prediction of cleavage sites of all proteases and encouraged to be used for any kind of peptide classification problem as well.

Changes in Contact Area in Meniscus Horizontal Cleavage Tears Subjected to Repair and Resection.

Science.gov (United States)

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 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 contact pressure and reduced average contact area at all degrees of flexion compared with the intact state (P 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

Caspase-Dependent Apoptosis Induced by Telomere Cleavage and TRF2 Loss

Directory of Open Access Journals (Sweden)

Asha S. Multani

2000-07-01

Full Text Available Chromosomal abnormalities involving telomeric associations (TAs often precede replicative senescence and abnormal chromosome configurations. We report here that telomere cleavage following exposure to proapoptotic agents is an early event in apoptosis. Exposure of human and murine cancer cells to a variety of pro-apoptotic stimuli (staurosporine, thapsigargin, anti-Fas antibody, cancer chemotherapeutic agents resulted in telomere cleavage and aggregation, finally their extrusion from the nuclei. Telomere loss was associated with arrest of cells in G2/M phase and preceded DNA fragmentation. Telomere erosion and subsequent large-scale chromatin cleavage were inhibited by overexpression of the anti -apoptotic protein, bcl-2, two peptide caspase inhibitors (BACMK and zVADfmk, indicating that both events are regulated by caspase activation. The results demonstrate that telomere cleavage is an early chromatin alteration detected in various cancer cell lines leading to drug-induced apoptosis, suggest that this event contributes to mitotic catastrophe and induction of cell death. Results also suggest that the decrease of telomeric-repeat binding factor 2 (TRF2 may be the earliest event in the ara-C-induced telomere shortening, induction of endoreduplication and chromosomal fragmentation leading to cell death.

The potato carotenoid cleavage dioxygenase 4 catalyzes a single cleavage of β-ionone ring-containing carotenes and non-epoxidated xanthophylls.

Science.gov (United States)

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

2015-04-15

Down-regulation of the potato carotenoid cleavage dioxygenase 4 (StCCD4) transcript level led to tubers with altered morphology and sprouting activity, which also accumulated higher levels of violaxanthin and lutein leading to elevated carotenoid amounts. This phenotype indicates a role of this enzyme in tuber development, which may be exerted by a cleavage product. In this work, we investigated the enzymatic activity of StCCD4, by expressing the corresponding cDNA in carotenoid accumulating Escherichia coli strains and by performing in vitro assays with heterologously expressed enzyme. StCCD4 catalyzed the cleavage of all-trans-β-carotene at the C9'-C10' double bond, leading to β-ionone and all-trans-β-apo-10'-carotenal, both in vivo and in vitro. The enzyme also cleaved β,β-cryptoxanthin, zeaxanthin and lutein either at the C9'-C10' or the C9-C10 double bond in vitro. In contrast, we did not observe any conversion of violaxanthin and only traces of activity with 9-cis-β-carotene, which led to 9-cis-β-apo-10'-carotenal. Our data indicate that all-trans-β-carotene is the likely substrate of StCCD4 in planta, and that this carotene may be precursor of an unknown compound involved in tuber development. Copyright © 2015 Elsevier Inc. All rights reserved.

Mechanistic aspects of dinitrogen cleavage and hydrogenation to produce ammonia in catalysis and organometallic chemistry: relevance of metal hydride bonds and dihydrogen.

Science.gov (United States)

Jia, Hong-Peng; Quadrelli, Elsje Alessandra

2014-01-21

Dinitrogen cleavage and hydrogenation by transition-metal centers to produce ammonia is central in industry and in Nature. After an introductory section on the thermodynamic and kinetic challenges linked to N2 splitting, this tutorial review discusses three major classes of transition-metal systems (homogeneous, heterogeneous and biological) capable of achieving dissociation and hydrogenation of dinitrogen. Molecular complexes, solid-state Haber-Bosch catalytic systems, silica-supported tantalum hydrides and nitrogenase will be discussed. Emphasis is focused on the reaction mechanisms operating in the process of dissociation and hydrogenation of dinitrogen, and in particular on the key role played by metal hydride bonds and by dihydrogen in such reactions.

Positioning the 5'-flap junction in the active site controls the rate of flap endonuclease-1-catalyzed DNA cleavage

KAUST Repository

Song, Bo

2018-02-09

Flap endonucleases catalyze cleavage of single-stranded DNA flaps formed during replication, repair and recombination, and are therefore essential for genome processing and stability. Recent crystal structures of DNA-bound human flap endonuclease (hFEN1) offer new insights into how conformational changes in the DNA and hFEN1 may facilitate the reaction mechanism. For example, previous biochemical studies of DNA conformation performed under non-catalytic conditions with Ca2+ have suggested that base unpairing at the 5\\'-flap:template junction is an important step in the reaction, but the new structural data suggest otherwise. To clarify the role of DNA changes in the kinetic mechanism, we measured a series of transient steps - from substrate binding to product release - during the hFEN1-catalyzed reaction in the presence of Mg2+. We found that while hFEN1 binds and bends DNA at a fast, diffusion-limited rate, much slower Mg2+-dependent conformational changes in DNA around the active site are subsequently necessary and rate-limiting for 5\\'-flap cleavage. These changes are reported overall by fluorescence of 2-aminopurine at the 5\\'-flap:template junction, indicating that local DNA distortion (e.g., disruption of base stacking observed in structures), associated with positioning the 5\\'-flap scissile phosphodiester bond in the hFEN1 active site, controls catalysis. hFEN1 residues with distinct roles in the catalytic mechanism, including those binding metal ions (Asp-34, Asp-181), steering the 5\\'-flap through the active site and binding the scissile phosphate (Lys-93, Arg-100), and stacking against the base 5\\' to the scissile phosphate (Tyr-40), all contribute to these rate-limiting conformational changes, ensuring efficient and specific cleavage of 5\\'-flaps.

Positioning the 5'-flap junction in the active site controls the rate of flap endonuclease-1-catalyzed DNA cleavage

KAUST Repository

Song, Bo; Hamdan, Samir; Hingorani, Manju M

2018-01-01

Flap endonucleases catalyze cleavage of single-stranded DNA flaps formed during replication, repair and recombination, and are therefore essential for genome processing and stability. Recent crystal structures of DNA-bound human flap endonuclease (hFEN1) offer new insights into how conformational changes in the DNA and hFEN1 may facilitate the reaction mechanism. For example, previous biochemical studies of DNA conformation performed under non-catalytic conditions with Ca2+ have suggested that base unpairing at the 5'-flap:template junction is an important step in the reaction, but the new structural data suggest otherwise. To clarify the role of DNA changes in the kinetic mechanism, we measured a series of transient steps - from substrate binding to product release - during the hFEN1-catalyzed reaction in the presence of Mg2+. We found that while hFEN1 binds and bends DNA at a fast, diffusion-limited rate, much slower Mg2+-dependent conformational changes in DNA around the active site are subsequently necessary and rate-limiting for 5'-flap cleavage. These changes are reported overall by fluorescence of 2-aminopurine at the 5'-flap:template junction, indicating that local DNA distortion (e.g., disruption of base stacking observed in structures), associated with positioning the 5'-flap scissile phosphodiester bond in the hFEN1 active site, controls catalysis. hFEN1 residues with distinct roles in the catalytic mechanism, including those binding metal ions (Asp-34, Asp-181), steering the 5'-flap through the active site and binding the scissile phosphate (Lys-93, Arg-100), and stacking against the base 5' to the scissile phosphate (Tyr-40), all contribute to these rate-limiting conformational changes, ensuring efficient and specific cleavage of 5'-flaps.

Pressure modulates the self-cleavage step of the hairpin ribozyme

Science.gov (United States)

Schuabb, Caroline; Kumar, Narendra; Pataraia, Salome; Marx, Dominik; Winter, Roland

2017-03-01

The ability of certain RNAs, denoted as ribozymes, to not only store genetic information but also catalyse chemical reactions gave support to the RNA world hypothesis as a putative step in the development of early life on Earth. This, however, might have evolved under extreme environmental conditions, including the deep sea with pressures in the kbar regime. Here we study pressure-induced effects on the self-cleavage of hairpin ribozyme by following structural changes in real-time. Our results suggest that compression of the ribozyme leads to an accelerated transesterification reaction, being the self-cleavage step, although the overall process is retarded in the high-pressure regime. The results reveal that favourable interactions between the reaction site and neighbouring nucleobases are strengthened under pressure, resulting therefore in an accelerated self-cleavage step upon compression. These results suggest that properly engineered ribozymes may also act as piezophilic biocatalysts in addition to their hitherto known properties.

Computational analysis and modeling of cleavage by the immunoproteasome and the constitutive proteasome

Directory of Open Access Journals (Sweden)

Lafuente Esther M

2010-09-01

Full Text Available Abstract Background Proteasomes play a central role in the major histocompatibility class I (MHCI antigen processing pathway. They conduct the proteolytic degradation of proteins in the cytosol, generating the C-terminus of CD8 T cell epitopes and MHCI-peptide ligands (P1 residue of cleavage site. There are two types of proteasomes, the constitutive form, expressed in most cell types, and the immunoproteasome, which is constitutively expressed in mature dendritic cells. Protective CD8 T cell epitopes are likely generated by the immunoproteasome and the constitutive proteasome, and here we have modeled and analyzed the cleavage by these two proteases. Results We have modeled the immunoproteasome and proteasome cleavage sites upon two non-overlapping sets of peptides consisting of 553 CD8 T cell epitopes, naturally processed and restricted by human MHCI molecules, and 382 peptides eluted from human MHCI molecules, respectively, using N-grams. Cleavage models were generated considering different epitope and MHCI-eluted fragment lengths and the same number of C-terminal flanking residues. Models were evaluated in 5-fold cross-validation. Judging by the Mathew's Correlation Coefficient (MCC, optimal cleavage models for the proteasome (MCC = 0.43 ± 0.07 and the immunoproteasome (MCC = 0.36 ± 0.06 were obtained from 12-residue peptide fragments. Using an independent dataset consisting of 137 HIV1-specific CD8 T cell epitopes, the immunoproteasome and proteasome cleavage models achieved MCC values of 0.30 and 0.18, respectively, comparatively better than those achieved by related methods. Using ROC analyses, we have also shown that, combined with MHCI-peptide binding predictions, cleavage predictions by the immunoproteasome and proteasome models significantly increase the discovery rate of CD8 T cell epitopes restricted by different MHCI molecules, including A*0201, A*0301, A*2402, B*0702, B*2705. Conclusions We have developed models that are specific

A method for phenomenological and chemical kinetics study of autocatalytic reactive dissolution by optical microscopy. The case of uranium dioxide dissolution in nitric acid media

Science.gov (United States)

Marc, Philippe; Magnaldo, Alastair; Godard, Jérémy; Schaer, Éric

2018-03-01

Dissolution is a milestone of the head-end of hydrometallurgical processes, as the stabilization rates of the chemical elements determine the process performance and hold-up. This study aims at better understanding the chemical and physico-chemical phenomena of uranium dioxide dissolution reactions in nitric acid media in the Purex process, which separates the reusable materials and the final wastes of the spent nuclear fuels. It has been documented that the attack of sintering-manufactured uranium dioxide solids occurs through preferential attack sites, which leads to the development of cracks in the solids. Optical microscopy observations show that in some cases, the development of these cracks leads to the solid cleavage. It is shown here that the dissolution of the detached fragments is much slower than the process of the complete cleavage of the solid, and occurs with no disturbing phenomena, like gas bubbling. This fact has motivated the measurement of dissolution kinetics using optical microscopy and image processing. By further discriminating between external resistance and chemical reaction, the "true" chemical kinetics of the reaction have been measured, and the highly autocatalytic nature of the reaction confirmed. Based on these results, the constants of the chemical reactions kinetic laws have also been evaluated.

A method for phenomenological and chemical kinetics study of autocatalytic reactive dissolution by optical microscopy. The case of uranium dioxide dissolution in nitric acid media

Directory of Open Access Journals (Sweden)

Marc Philippe

2018-01-01

Full Text Available Dissolution is a milestone of the head-end of hydrometallurgical processes, as the stabilization rates of the chemical elements determine the process performance and hold-up. This study aims at better understanding the chemical and physico-chemical phenomena of uranium dioxide dissolution reactions in nitric acid media in the Purex process, which separates the reusable materials and the final wastes of the spent nuclear fuels. It has been documented that the attack of sintering-manufactured uranium dioxide solids occurs through preferential attack sites, which leads to the development of cracks in the solids. Optical microscopy observations show that in some cases, the development of these cracks leads to the solid cleavage. It is shown here that the dissolution of the detached fragments is much slower than the process of the complete cleavage of the solid, and occurs with no disturbing phenomena, like gas bubbling. This fact has motivated the measurement of dissolution kinetics using optical microscopy and image processing. By further discriminating between external resistance and chemical reaction, the “true” chemical kinetics of the reaction have been measured, and the highly autocatalytic nature of the reaction confirmed. Based on these results, the constants of the chemical reactions kinetic laws have also been evaluated.

Steady-state kinetics of substrate binding and iron release in tomato ACC oxidase.

Science.gov (United States)

Thrower, J S; Blalock, R; Klinman, J P

2001-08-14

1-Aminocyclopropane-1-carboxylate oxidase (ACC oxidase) catalyzes the last step in the biosynthetic pathway of the plant hormone, ethylene. This unusual reaction results in the oxidative ring cleavage of 1-aminocyclopropane carboxylate (ACC) into ethylene, cyanide, and CO2 and requires ferrous ion, ascorbate, and molecular oxygen for catalysis. A new purification procedure and assay method have been developed for tomato ACC oxidase that result in greatly increased enzymatic activity. This method allowed us to determine the rate of iron release from the enzyme and the effect of the activator, CO2, on this rate. Initial velocity studies support an ordered kinetic mechanism where ACC binds first followed by O2; ascorbate can bind after O2 or possibly before ACC. This kinetic mechanism differs from one recently proposed for the ACC oxidase from avocado.

On the temperature independence of statistical model parameters for cleavage fracture in ferritic steels

Science.gov (United States)

Qian, Guian; Lei, Wei-Sheng; Niffenegger, M.; González-Albuixech, V. F.

2018-04-01

The work relates to the effect of temperature on the model parameters in local approaches (LAs) to cleavage fracture. According to a recently developed LA model, the physical consensus of plastic deformation being a prerequisite to cleavage fracture enforces any LA model of cleavage fracture to observe initial yielding of a volume element as its threshold stress state to incur cleavage fracture in addition to the conventional practice of confining the fracture process zone within the plastic deformation zone. The physical consistency of the new LA model to the basic LA methodology and the differences between the new LA model and other existing models are interpreted. Then this new LA model is adopted to investigate the temperature dependence of LA model parameters using circumferentially notched round tensile specimens. With the published strength data as input, finite element (FE) calculation is conducted for elastic-perfectly plastic deformation and the realistic elastic-plastic hardening, respectively, to provide stress distributions for model calibration. The calibration results in temperature independent model parameters. This leads to the establishment of a 'master curve' characteristic to synchronise the correlation between the nominal strength and the corresponding cleavage fracture probability at different temperatures. This 'master curve' behaviour is verified by strength data from three different steels, providing a new path to calculate cleavage fracture probability with significantly reduced FE efforts.

Fractographic observations of cleavage initiation in the ductile-brittle transition region of a reactor-pressure-vessel steel

International Nuclear Information System (INIS)

Rosenfield, A.R.; Shetty, D.K.; Skidmore, A.J.

1983-01-01

This note reports the results of a fractographic study conducted on a group of 1T compact fracture toughness specimens of a heavy-section A508 steel denoted TSE6 tested in the ductile-brittle transition region (22 and 82 0 C). The fatigue-precracked specimens were loaded at a rapid rate (760 or 550 mm per second) to promote cleavage-crack growth and lower-bound toughness behavior. All specimens experienced unstable cleavage fracture prior to reaching a maximum in the load displacement curve. Some ductile crack growth occurred in half of the specimens. The objective of fractographic examinations was to understand the observed statistical variations in cleavage initiation by (a) locating the origins of unstable cleavage fracture in the vicinity of the fatigue-precrack or ductilerupture crack fronts, (b) identifying microstructural features associated with the triggering of cleavage, and (c) documenting characteristic fracture surface dimensions such as the extent of stable-crack growth prior to unstable cleavage (Δα) and the distance of the cleavage origin from the ductilerupture front, /chi/ (or fatigue-crack front when Δα = 0)

Sensitive and fast mutation detection by solid phase chemical cleavage

DEFF Research Database (Denmark)

Hansen, Lise Lotte; Justesen, Just; Kruse, Torben A

1996-01-01

We have developed a solid phase chemical cleavage method (SpCCM) for screening large DNA fragments for mutations. All reactions can be carried out in microtiterwells from the first amplification of the patient (or test) DNA through the search for mutations. The reaction time is significantly...... reduced compared to the conventional chemical cleavage method (CCM), and even by using a uniformly labelled probe, the exact position and nature of the mutation can be revealed. The SpCCM is suitable for automatization using a workstation to carry out the reactions and a fluorescent detection-based DNA...

On the formation and nature of quasi-cleavage fracture surfaces in hydrogen embrittled steels

Energy Technology Data Exchange (ETDEWEB)

Martin, May L.; Fenske, Jamey A.; Liu, Grace S.; Sofronis, Petros [University of Illinois, Dept. of Materials Science and Engineering, 1304 W. Green St., Urbana, IL 61801 (United States); Robertson, Ian M., E-mail: ianr@illinois.edu [University of Illinois, Dept. of Materials Science and Engineering, 1304 W. Green St., Urbana, IL 61801 (United States)

2011-02-15

Quasi-cleavage, a common feature of hydrogen-induced fracture surfaces, is generally taken as being cleavage-like but not along a known cleavage plane. Despite the frequency with which this surface is observed, the relationship to the underlying microstructure remains unknown. Through a combination of topographical reconstruction of secondary electron microscope fractographs and a transmission electron microscopy study of the microstructure from site-specific locations, it will be shown that the features on quasi-cleavage surfaces are ridges that can be correlated with sub-surface intense and highly localized deformation bands. It will be demonstrated that the fracture surface arises from the growth and coalescence of voids that initiate at and extend along slip band intersections. This mechanism and process is fully consistent with hydrogen enhancing and localizing plastic processes.

Plum Pox Virus 6K1 Protein Is Required for Viral Replication and Targets the Viral Replication Complex at the Early Stage of Infection.

Science.gov (United States)

Cui, Hongguang; Wang, Aiming

2016-05-15

The potyviral RNA genome encodes two polyproteins that are proteolytically processed by three viral protease domains into 11 mature proteins. Extensive molecular studies have identified functions for the majority of the viral proteins. For example, 6K2, one of the two smallest potyviral proteins, is an integral membrane protein and induces the endoplasmic reticulum (ER)-originated replication vesicles that target the chloroplast for robust viral replication. However, the functional role of 6K1, the other smallest protein, remains uncharacterized. In this study, we developed a series of recombinant full-length viral cDNA clones derived from a Canadian Plum pox virus (PPV) isolate. We found that deletion of any of the short motifs of 6K1 (each of which ranged from 5 to 13 amino acids), most of the 6K1 sequence (but with the conserved sequence of the cleavage sites being retained), or all of the 6K1 sequence in the PPV infectious clone abolished viral replication. The trans expression of 6K1 or the cis expression of a dislocated 6K1 failed to rescue the loss-of-replication phenotype, suggesting the temporal and spatial requirement of 6K1 for viral replication. Disruption of the N- or C-terminal cleavage site of 6K1, which prevented the release of 6K1 from the polyprotein, either partially or completely inhibited viral replication, suggesting the functional importance of the mature 6K1. We further found that green fluorescent protein-tagged 6K1 formed punctate inclusions at the viral early infection stage and colocalized with chloroplast-bound viral replicase elements 6K2 and NIb. Taken together, our results suggest that 6K1 is required for viral replication and is an important viral element of the viral replication complex at the early infection stage. Potyviruses account for more than 30% of known plant viruses and consist of many agriculturally important viruses. The genomes of potyviruses encode two polyproteins that are proteolytically processed into 11 mature

The potato carotenoid cleavage dioxygenase 4 catalyzes a single cleavage of β-ionone ring-containing carotenes and non-epoxidated xanthophylls

KAUST Repository

Bruno, Mark; Beyer, Peter D.; Al-Babili, Salim

2015-01-01

amounts. This phenotype indicates a role of this enzyme in tuber development, which may be exerted by a cleavage product. In this work, we investigated the enzymatic activity of StCCD4, by expressing the corresponding cDNA in carotenoid accumulating

Numerical modeling of ductile tearing effects on cleavage fracture toughness

International Nuclear Information System (INIS)

Dodds, R.H. Jr.; Tang, M.; Anderson, T.L.

1994-05-01

Experimental studies demonstrate a significant effect of specimen size, a/W ratio and prior ductile tearing on cleavage fracture toughness values (J c ) measured in the ductile-to-brittle transition region of ferritic materials. In the lower-transition region, cleavage fracture often occurs under conditions of large-scale yielding but without prior ductile crack extension. The increased toughness develops when plastic zones formed at the crack tip interact with nearby specimen surfaces which relaxes crack-tip constraint (stress triaxiality). In the mid-to-upper transition region, small amounts of ductile crack extension (often c -values. Previous work by the authors described a micromechanics fracture model to correct measured J c -values for the mechanistic effects of large-scale yielding. This new work extends the model to also include the influence of ductile crack extension prior to cleavage. The paper explores development of the new model, provides necessary graphs and procedures for its application and demonstrates the effects of the model on fracture data sets for two pressure vessel steels (A533B and A515)

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

Science.gov (United States)

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.

Critical cleavage fracture stress characterization of A508 nuclear pressure vessel steels

International Nuclear Information System (INIS)

Wu, Sujun; Jin, Huijin; Sun, Yanbin; Cao, Luowei

2014-01-01

The critical cleavage fracture stress of SA508 Gr.4N and SA508 Gr.3 low alloy reactor pressure vessel (RPV) steels was studied through the combination of experiments and finite element method (FEM) analysis. The results showed that the value of the local cleavage fracture stress, σ F , of SA508 Gr.4N steel was significantly higher than that of SA508 Gr.3 steel. Detailed microstructural analysis was carried out using FEGSEM which revealed much smaller grains, finer and more homogenous carbide particles formed in SA508 Gr.4N steel. Compared with the SA508 Gr.3 steel currently used in the nuclear industry, the SA508 Gr.4N steel possesses higher strength and notch toughness as well as improved cleavage fracture behavior, and is considered a better candidate RPV steel for the next generation nuclear reactors. - Highlights: • Critical cleavage fracture stress was calculated through experiments and FEM. • Effects of both grain and carbide particle sizes on σ F were discussed. • The SA508 Gr.4N steel is a better candidate for the next generation nuclear reactors

A set of simple cell processes is sufficient to model spiral cleavage.

Science.gov (United States)

Brun-Usan, Miguel; Marín-Riera, Miquel; Grande, Cristina; Truchado-Garcia, Marta; Salazar-Ciudad, Isaac

2017-01-01

During cleavage, different cellular processes cause the zygote to become partitioned into a set of cells with a specific spatial arrangement. These processes include the orientation of cell division according to: an animal-vegetal gradient; the main axis (Hertwig's rule) of the cell; and the contact areas between cells or the perpendicularity between consecutive cell divisions (Sachs' rule). Cell adhesion and cortical rotation have also been proposed to be involved in spiral cleavage. We use a computational model of cell and tissue biomechanics to account for the different existing hypotheses about how the specific spatial arrangement of cells in spiral cleavage arises during development. Cell polarization by an animal-vegetal gradient, a bias to perpendicularity between consecutive cell divisions (Sachs' rule), cortical rotation and cell adhesion, when combined, reproduce the spiral cleavage, whereas other combinations of processes cannot. Specifically, cortical rotation is necessary at the 8-cell stage to direct all micromeres in the same direction. By varying the relative strength of these processes, we reproduce the spatial arrangement of cells in the blastulae of seven different invertebrate species. © 2017. Published by The Company of Biologists Ltd.

Next generation sequencing and molecular analysis of artichoke Italian latent virus.

Science.gov (United States)

Elbeaino, Toufic; Belghacem, Imen; Mascia, Tiziana; Gallitelli, Donato; Digiaro, Michele

2017-06-01

Next-generation sequencing (NGS) allowed the assembly of the complete RNA-1 and RNA-2 sequences of a grapevine isolate of artichoke Italian latent virus (AILV). RNA-1 and RNA-2 are 7,338 and 4,630 nucleotides in length excluding the 3' terminal poly(A) tail, and encode two putative polyproteins of 255.8 kDa (p1) and 149.6 kDa (p2), respectively. All conserved motifs and predicted cleavage sites, typical for nepovirus polyproteins, were found in p1 and p2. AILV p1 and p2 share high amino acid identity with their homologues in beet ringspot virus (p1, 81% and p2, 71%), tomato black ring virus (p1, 79% and p2, 63%), grapevine Anatolian ringspot virus (p1, 65% and p2, 63%), and grapevine chrome mosaic virus (p1, 60% and p2, 54%), and to a lesser extent with other grapevine nepoviruses of subgroup A and C. Phylogenetic and sequence analyses, all confirmed the strict relationship of AILV with members classified in subgroup B of genus Nepovirus.

Boron-doped diamond electrodes for the electrochemical oxidation and cleavage of peptides.

Science.gov (United States)

Roeser, Julien; Alting, Niels F A; Permentier, Hjalmar P; Bruins, Andries P; Bischoff, Rainer

2013-07-16

Electrochemical oxidation of peptides and proteins is traditionally performed on carbon-based electrodes. Adsorption caused by the affinity of hydrophobic and aromatic amino acids toward these surfaces leads to electrode fouling. We compared the performance of boron-doped diamond (BDD) and glassy carbon (GC) electrodes for the electrochemical oxidation and cleavage of peptides. An optimal working potential of 2000 mV was chosen to ensure oxidation of peptides on BDD by electron transfer processes only. Oxidation by electrogenerated OH radicals took place above 2500 mV on BDD, which is undesirable if cleavage of a peptide is to be achieved. BDD showed improved cleavage yield and reduced adsorption for a set of small peptides, some of which had been previously shown to undergo electrochemical cleavage C-terminal to tyrosine (Tyr) and tryptophan (Trp) on porous carbon electrodes. Repeated oxidation with BDD electrodes resulted in progressively lower conversion yields due to a change in surface termination. Cathodic pretreatment of BDD at a negative potential in an acidic environment successfully regenerated the electrode surface and allowed for repeatable reactions over extended periods of time. BDD electrodes are a promising alternative to GC electrodes in terms of reduced adsorption and fouling and the possibility to regenerate them for consistent high-yield electrochemical cleavage of peptides. The fact that OH-radicals can be produced by anodic oxidation of water at elevated positive potentials is an additional advantage as they allow another set of oxidative reactions in analogy to the Fenton reaction, thus widening the scope of electrochemistry in protein and peptide chemistry and analytics.

Cleavage mechanoluminescence in elemental and III-V semiconductors

International Nuclear Information System (INIS)

Chandra, B.P.; Patel, R.P.; Gour, Anubha S.; Chandra, V.K.; Gupta, R.K.

2003-01-01

The present paper reports the theory of mechanoluminescence (ML) produced during cleavage of elemental and III-V semiconductors. It seems that the formation of crack-induced localized states is responsible for the ML excitation produced during the cleavage of elemental and III-V semiconductors. According to this mechanism, as the atoms are drawn away from each other in an advancing crack tip, the decreasing wave function overlap across the crack may result in localized states which is associated with increasing electron energy. If the energy of these localized states approach that of the conduction band, transition to the conduction band via tunnelling would be possible, creating minority carriers, and consequently the electron-hole recombination may give rise to mechanoluminescence. When an elemental or III-V semiconductor is cleaved, initially the ML intensity increases with time, attains a peak value I m at the time t m corresponding to completion of the cleavage of the semiconductor, and then it decreases following power law decay. Expressions are derived for the ML intensity I m corresponding to the peak of the ML intensity versus time curve and for the total ML intensity I T . It is shown that both I m and I T should increase directly with the area of the newly created surfaces of the crystals. From the measurements of the ML intensity, the velocity of crack propagation in material can be determined by using the relation v=H/t m

Secondary. cap alpha. -deuterium kinetic isotope effects in solvolyses of ferrocenylmethyl acetate and benzoate in ethanol

Energy Technology Data Exchange (ETDEWEB)

Sutic, D. (Univ. of Zagreb, Yugoslavia); Asperger, S.; Borcic, S.

1982-12-17

Secondary ..cap alpha..-deuterium kinetic isotope effects (KIE) in solvolyses of ferrocenyldideuteriomethyl acetate and benzoate were determined in 96% (v/v) ethanol, at 25/sup 0/C, as k/sub H//k/sub D/ = 1.24 and 1.26, respectively. The KIEs were also determined in the presence of 0.1 mol dm/sup -3/ lithium perchlorate: the k/sub H//k/ sub D/ values were 1.23 and 1.22 for acetate and benzoate complexes, respectively. The maximum KIE for the C-O bond cleavage of a primary substrate is as large as, or larger than, that of secondary derivatives, which is estimated to be 1.23 per deuterium. The measured KIE of about 12% per D therefore represents a strongly reduced effect relative to its maximum. The solvolyses exhibit ''a special salt effect''. This effect indicates the presence of solvent-separated ion pairs and the return to tight pairs. As the maximum KIE is expected in solvolyses involving transformation of one type of ion pair into another, the strongly reduced ..cap alpha..-D KIE supports the structure involving direct participation of electrons that in the ground state are localized at the iron atom. The alkyl-oxygen cleavage is accompanied by 10-15% acyl-oxygen cleavage.

Implementation of a combinatorial cleavage and deprotection scheme

DEFF Research Database (Denmark)

Nielsen, John; Rasmussen, Palle H.

1996-01-01

Phthalhydrazide libraries are synthesized in solution from substituted hydrazines and phthalimides in several different library formats including single compounds, indexed sub-libraries and a full library. When carried out during solid-phase synthesis, this combinatorial cleavage and deprotection...

Genetic stability of attenuated mengovirus vectors with duplicate primary cleavage sequences

International Nuclear Information System (INIS)

Binder, J.J.; Hoffman, M.A.; Palmenberg, A.C.

2003-01-01

Short poly(C)-tract Mengoviruses have proven vaccine efficacy in many species of animals. A novel vector for the delivery of foreign proteins was created by insertion of a second autoproteolytic primary cleavage cassette linked to a multiple cloning site (MCS) into an attenuated variant of Mengo. Nineteen cDNAs from foreign sequences that ranged from 39 to 1653 bases were cloned into the MCS. The viral reading frame was maintained and translation resulted in dual, autocatalytic excision of the foreign peptides without disruption of any Mengo proteins. All cDNAs except those with the largest insertions produced viable virus. Active proteins such as GFP, CAT, and SIV p27 were expressed within infected cells. Relative to parental Mengo, the growth kinetics and genetic stability of each vector was inversely proportional to the size of the inserted sequence. While segments up to 1000 bases could be carried, inserts greater than 500-600 bases were usually reduced in size during serial passage. The limit on carrying capacity was probably due to difficulties in virion assembly or particle stability. Yet for inserts less than 500-600 bases, the Mengo vectors provided an effective system for the delivery of foreign epitopes into cells and mice

Structural and kinetic isotope effect studies of nicotinamidase (Pnc1) from Saccharomyces cerevisiae.

Science.gov (United States)

Smith, Brian C; Anderson, Mark A; Hoadley, Kelly A; Keck, James L; Cleland, W Wallace; Denu, John M

2012-01-10

Nicotinamidases catalyze the hydrolysis of nicotinamide to nicotinic acid and ammonia. Nicotinamidases are absent in mammals but function in NAD(+) salvage in many bacteria, yeast, plants, protozoa, and metazoans. We have performed structural and kinetic investigations of the nicotinamidase from Saccharomyces cerevisiae (Pnc1). Steady-state product inhibitor analysis revealed an irreversible reaction in which ammonia is the first product released, followed by nicotinic acid. A series of nicotinamide analogues acting as inhibitors or substrates were examined, revealing that the nicotinamide carbonyl oxygen and ring nitrogen are critical for binding and reactivity. X-ray structural analysis revealed a covalent adduct between nicotinaldehyde and Cys167 of Pnc1 and coordination of the nicotinamide ring nitrogen to the active-site zinc ion. Using this structure as a guide, the function of several residues was probed via mutagenesis and primary (15)N and (13)C kinetic isotope effects (KIEs) on V/K for amide bond hydrolysis. The KIE values of almost all variants were increased, indicating that C-N bond cleavage is at least partially rate limiting; however, a decreased KIE for D51N was indicative of a stronger commitment to catalysis. In addition, KIE values using slower alternate substrates indicated that C-N bond cleavage is at least partially rate limiting with nicotinamide to highly rate limiting with thionicotinamide. A detailed mechanism involving nucleophilic attack of Cys167, followed by elimination of ammonia and then hydrolysis to liberate nicotinic acid, is discussed. These results will aid in the design of mechanism-based inhibitors to target pathogens that rely on nicotinamidase activity.

Structural and Kinetic Isotope Effect Studies of Nicotinamidase (Pnc1) from S. cerevisiae†

Science.gov (United States)

Smith, Brian C.; Anderson, Mark A.; Hoadley, Kelly A.; Keck, James L.; Cleland, W. Wallace; Denu, John M.

2011-01-01

Nicotinamidases catalyze the hydrolysis of nicotinamide to nicotinic acid and ammonia. Nicotinamidases are absent in mammals but function in NAD+ salvage in many bacteria, yeast, plants, protozoa, and metazoans. We have performed structural and kinetic investigations of the nicotinamidase from S. cerevisiae (Pnc1). Steady-state product inhibitor analysis revealed an irreversible reaction where ammonia is the first product released, followed by nicotinic acid. A series of nicotinamide analogs acting as inhibitors or substrates were examined revealing that the nicotinamide carbonyl oxygen and ring nitrogen are critical for binding and reactivity. X-ray structural analysis revealed a covalent adduct between nicotinaldehyde and Cys167 of Pnc1 and coordination of the nicotinamide ring nitrogen to the active-site zinc ion. Using this structure as a guide, the function of several residues was probed via mutagenesis and primary 15N and 13C kinetic isotope effects (KIE) on V/K for amide bond hydrolysis. The KIE values of almost all variants were increased indicating that C-N bond cleavage is at least partially rate limiting; however, a decreased KIE for D51N was observed indicative of a higher commitment to catalysis. In addition, KIE values using slower alternate substrates indicated that C-N bond cleavage is at least partially rate limiting with nicotinamide to highly rate limiting with thionicotinamide. A detailed mechanism is discussed involving nucleophilic attack of Cys167, followed by elimination of ammonia and then hydrolysis to liberate nicotinic acid. These results will aid design of mechanism-based inhibitors to target pathogens that rely on nicotinamidase activity. PMID:22229411

Structural and Kinetic Isotope Effect Studies of Nicotinamidase (Pnc1) from Saccharomyces cerevisiae

International Nuclear Information System (INIS)

Smith, Brian C.; Anderson, Mark A.; Hoadley, Kelly A.; Keck, James L.; Cleland, W. Wallace; Denu, John M.

2012-01-01

Nicotinamidases catalyze the hydrolysis of nicotinamide to nicotinic acid and ammonia. Nicotinamidases are absent in mammals but function in NAD + salvage in many bacteria, yeast, plants, protozoa, and metazoans. We have performed structural and kinetic investigations of the nicotinamidase from Saccharomyces cerevisiae (Pnc1). Steady-state product inhibitor analysis revealed an irreversible reaction in which ammonia is the first product released, followed by nicotinic acid. A series of nicotinamide analogues acting as inhibitors or substrates were examined, revealing that the nicotinamide carbonyl oxygen and ring nitrogen are critical for binding and reactivity. X-ray structural analysis revealed a covalent adduct between nicotinaldehyde and Cys167 of Pnc1 and coordination of the nicotinamide ring nitrogen to the active-site zinc ion. Using this structure as a guide, the function of several residues was probed via mutagenesis and primary 15 N and 13 C kinetic isotope effects (KIEs) on V/K for amide bond hydrolysis. The KIE values of almost all variants were increased, indicating that C-N bond cleavage is at least partially rate limiting; however, a decreased KIE for D51N was indicative of a stronger commitment to catalysis. In addition, KIE values using slower alternate substrates indicated that C-N bond cleavage is at least partially rate limiting with nicotinamide to highly rate limiting with thionicotinamide. A detailed mechanism involving nucleophilic attack of Cys167, followed by elimination of ammonia and then hydrolysis to liberate nicotinic acid, is discussed. These results will aid in the design of mechanism-based inhibitors to target pathogens that rely on nicotinamidase activity.

Mutation in Spike Protein Cleavage Site and Pathogenesis of Feline Coronavirus

Science.gov (United States)

Licitra, Beth N.; Millet, Jean K.; Regan, Andrew D.; Hamilton, Brian S.; Rinaldi, Vera D.; Duhamel, Gerald E.

2013-01-01

Feline coronaviruses (FCoV) exist as 2 biotypes: feline enteric coronavirus (FECV) and feline infectious peritonitis virus (FIPV). FECV causes subclinical infections; FIPV causes feline infectious peritonitis (FIP), a systemic and fatal disease. It is thought that mutations in FECV enable infection of macrophages, causing FIP. However, the molecular basis for this biotype switch is unknown. We examined a furin cleavage site in the region between receptor-binding (S1) and fusion (S2) domains of the spike of serotype 1 FCoV. FECV sequences were compared with FIPV sequences. All FECVs had a conserved furin cleavage motif. For FIPV, there was a correlation with the disease and >1 substitution in the S1/S2 motif. Fluorogenic peptide assays confirmed that the substitutions modulate furin cleavage. We document a functionally relevant S1/S2 mutation that arises when FIP develops in a cat. These insights into FIP pathogenesis may be useful in development of diagnostic, prevention, and treatment measures against coronaviruses. PMID:23763835

Trichomonas vaginalis Metalloproteinase Induces mTOR Cleavage of SiHa Cells

Science.gov (United States)

Quan, Juan-Hua; Choi, In-Wook; Yang, Jung-Bo; Zhou, Wei; Cha, Guang-Ho; Zhou, Yu; Ryu, Jae-Sook

2014-01-01

Trichomonas vaginalis secretes a number of proteases which are suspected to be the cause of pathogenesis; however, little is understood how they manipulate host cells. The mammalian target of rapamycin (mTOR) regulates cell growth, cell proliferation, cell motility, cell survival, protein synthesis, and transcription. We detected various types of metalloproteinases including GP63 protein from T. vaginalis trophozoites, and T. vaginalis GP63 metalloproteinase was confirmed by sequencing and western blot. When SiHa cells were stimulated with live T. vaginalis, T. vaginalis excretory-secretory products (ESP) or T. vaginalis lysate, live T. vaginalis and T. vaginalis ESP induced the mTOR cleavage in both time- and parasite load-dependent manner, but T. vaginalis lysate did not. Pretreatment of T. vaginalis with a metalloproteinase inhibitor, 1,10-phenanthroline, completely disappeared the mTOR cleavage in SiHa cells. Collectively, T. vaginalis metallopeptidase induces host cell mTOR cleavage, which may be related to survival of the parasite. PMID:25548410

Condensed tannins: A novel rearrangement of procyanidins and prodelphinidins in thiolytic cleavage

Science.gov (United States)

G. Wayne McGraw; Jan P. Steynberg; Richard W. Hemingway

1993-01-01

Conditions commonly used for the thiolytic cleavage of interflavanoid bonds of condensed tannins also result in cleavage of the C4 to C10 bond of flavan units. Subsequenet lectrophilic attack of the C4 carbocation on the C2' or C6' of the B-ring, and loss of phloroglucinol (the A-ring), result in the formation of a mixture of 1,3-dithiobenzyl-2,4,s,6-...

Yeast ribonuclease III uses a network of multiple hydrogen bonds for RNA binding and cleavage.

Science.gov (United States)

Lavoie, Mathieu; Abou Elela, Sherif

2008-08-19

Members of the bacterial RNase III family recognize a variety of short structured RNAs with few common features. It is not clear how this group of enzymes supports high cleavage fidelity while maintaining a broad base of substrates. Here we show that the yeast orthologue of RNase III (Rnt1p) uses a network of 2'-OH-dependent interactions to recognize substrates with different structures. We designed a series of bipartite substrates permitting the distinction between binding and cleavage defects. Each substrate was engineered to carry a single or multiple 2'- O-methyl or 2'-fluoro ribonucleotide substitutions to prevent the formation of hydrogen bonds with a specific nucleotide or group of nucleotides. Interestingly, introduction of 2'- O-methyl ribonucleotides near the cleavage site increased the rate of catalysis, indicating that 2'-OH are not required for cleavage. Substitution of nucleotides in known Rnt1p binding site with 2'- O-methyl ribonucleotides inhibited cleavage while single 2'-fluoro ribonucleotide substitutions did not. This indicates that while no single 2'-OH is essential for Rnt1p cleavage, small changes in the substrate structure are not tolerated. Strikingly, several nucleotide substitutions greatly increased the substrate dissociation constant with little or no effect on the Michaelis-Menten constant or rate of catalysis. Together, the results indicate that Rnt1p uses a network of nucleotide interactions to identify its substrate and support two distinct modes of binding. One mode is primarily mediated by the dsRNA binding domain and leads to the formation of stable RNA/protein complex, while the other requires the presence of the nuclease and N-terminal domains and leads to RNA cleavage.

Nuclear reprogramming: kinetics of cell cycle and metabolic progression as determinants of success.

Directory of Open Access Journals (Sweden)

Sebastian Thomas Balbach

Full Text Available Establishment of totipotency after somatic cell nuclear transfer (NT requires not only reprogramming of gene expression, but also conversion of the cell cycle from quiescence to the precisely timed sequence of embryonic cleavage. Inadequate adaptation of the somatic nucleus to the embryonic cell cycle regime may lay the foundation for NT embryo failure and their reported lower cell counts. We combined bright field and fluorescence imaging of histone H(2b-GFP expressing mouse embryos, to record cell divisions up to the blastocyst stage. This allowed us to quantitatively analyze cleavage kinetics of cloned embryos and revealed an extended and inconstant duration of the second and third cell cycles compared to fertilized controls generated by intracytoplasmic sperm injection (ICSI. Compared to fertilized embryos, slow and fast cleaving NT embryos presented similar rates of errors in M phase, but were considerably less tolerant to mitotic errors and underwent cleavage arrest. Although NT embryos vary substantially in their speed of cell cycle progression, transcriptome analysis did not detect systematic differences between fast and slow NT embryos. Profiling of amino acid turnover during pre-implantation development revealed that NT embryos consume lower amounts of amino acids, in particular arginine, than fertilized embryos until morula stage. An increased arginine supplementation enhanced development to blastocyst and increased embryo cell numbers. We conclude that a cell cycle delay, which is independent of pluripotency marker reactivation, and metabolic restraints reduce cell counts of NT embryos and impede their development.

Polycystin-1 C-terminal Cleavage Is Modulated by Polycystin-2 Expression*

Science.gov (United States)

Bertuccio, Claudia A.; Chapin, Hannah C.; Cai, Yiqiang; Mistry, Kavita; Chauvet, Veronique; Somlo, Stefan; Caplan, Michael J.

2009-01-01

Autosomal dominant polycystic kidney disease is caused by mutations in the genes encoding polycystin-1 (PC-1) and polycystin-2 (PC-2). PC-1 cleavage releases its cytoplasmic C-terminal tail (CTT), which enters the nucleus. To determine whether PC-1 CTT cleavage is influenced by PC-2, a quantitative cleavage assay was utilized, in which the DNA binding and activation domains of Gal4 and VP16, respectively, were appended to PC-1 downstream of its CTT domain (PKDgalvp). Cells cotransfected with the resultant PKDgalvp fusion protein and PC-2 showed an increase in luciferase activity and in CTT expression, indicating that the C-terminal tail of PC-1 is cleaved and enters the nucleus. To assess whether CTT cleavage depends upon Ca2+ signaling, cells transfected with PKDgalvp alone or together with PC-2 were incubated with several agents that alter intracellular Ca2+ concentrations. PC-2 enhancement of luciferase activity was not altered by any of these treatments. Using a series of PC-2 C-terminal truncated mutations, we identified a portion of the PC-2 protein that is required to stimulate PC-1 CTT accumulation. These data demonstrate that release of the CTT from PC-1 is influenced and stabilized by PC-2. This effect is independent of Ca2+ but is regulated by sequences contained within the PC-2 C-terminal tail, suggesting a mechanism through which PC-1 and PC-2 may modulate a novel signaling pathway. PMID:19491093

Restriction enzyme cleavage of ultraviolet-damaged Simian virus 40 and pBR322 DNA

International Nuclear Information System (INIS)

Cleaver, J.E.

1983-01-01

Cleavage of specific DNA sequences by the restriction enzymes EcoRI, HindIII and TaqI was prevented when the DNA was irradiated with ultraviolet light. Most of the effects were attributed to cyclobutane pyrimidine dimers in the recognition sequences; the effectiveness of irradiation was directly proportional to the number of potential dimer sites in the DNA. Combining EcoRI with dimer-specific endonuclease digestion revealed that pyrimidine dimers blocked cleavage within one base-pair on the strand opposite to the dimer but did not block cleavage three to four base-pairs away on the same strand. These are the probable limits for the range of influence of pyrimidine dimers along the DNA, at least for this enzyme. The effect of irradiation on cleavage by TaqI seemed far greater than expected for the cyclobutane dimer yield, possibly because of effects from photoproducts flanking the tetranucleotide recognition sequence and the effect of non-cyclobutane (6-4)pyrimidine photoproducts involving adjacent T and C bases. (author)

Control of extracellular cleavage of ProBDNF by high frequency neuronal activity

OpenAIRE

Nagappan, Guhan; Zaitsev, Eugene; Senatorov, Vladimir V.; Yang, Jianmin; Hempstead, Barbara L.; Lu, Bai

2009-01-01

Pro- and mature neurotrophins often elicit opposing biological effects. For example, mature brain-derived neurotrophic factor (mBDNF) is critical for long-term potentiation induced by high-frequency stimulation, whereas proBDNF facilitate long-term depression induced by low-frequency stimulation. Because mBDNF is derived from proBDNF by endoproteolytic cleavage, mechanisms regulating the cleavage of proBDNF may control the direction of BDNF regulation. Using methods that selectively detect pr...

Stimulation of NADH-dependent microsomal DNA strand cleavage by rifamycin SV.

Science.gov (United States)

Kukiełka, E; Cederbaum, A I

1995-04-15

Rifamycin SV is an antibiotic anti-bacterial agent used in the treatment of tuberculosis. This drug can autoxidize, especially in the presence of metals, and generate reactive oxygen species. A previous study indicated that rifamycin SV can increase NADH-dependent microsomal production of reactive oxygen species. The current study evaluated the ability of rifamycin SV to interact with iron and increase microsomal production of hydroxyl radical, as detected by conversion of supercoiled plasmid DNA into the relaxed open circular state. The plasmid used was pBluescript II KS(-), and the forms of DNA were separated by agarose-gel electrophoresis. Incubation of rat liver microsomes with plasmid plus NADH plus ferric-ATP caused DNA strand cleavage. The addition of rifamycin SV produced a time- and concentration-dependent increase in DNA-strand cleavage. No stimulation by rifamycin SV occurred in the absence of microsomes, NADH or ferric-ATP. Stimulation occurred with other ferric complexes besides ferric-ATP, e.g. ferric-histidine, ferric-citrate, ferric-EDTA, and ferric-(NH4)2SO4. Rifamycin SV did not significantly increase the high rates of DNA strand cleavage found with NADPH as the microsomal reductant. The stimulation of NADH-dependent microsomal DNA strand cleavage was completely blocked by catalase, superoxide dismutase, GSH and a variety of hydroxyl-radical-scavenging agents, but not by anti-oxidants that prevent microsomal lipid peroxidation. Redox cycling agents, such as menadione and paraquat, in contrast with rifamycin SV, stimulated the NADPH-dependent reaction; menadione and rifamycin SV were superior to paraquat in stimulating the NADH-dependent reaction. These results indicate that rifamycin SV can, in the presence of an iron catalyst, increase microsomal production of reactive oxygen species which can cause DNA-strand cleavage. In contrast with other redox cycling agents, the stimulation by rifamycin SV is more pronounced with NADH than with NADPH as the

Cloning and sequencing of full-length cDNAs of RNA1 and RNA2 of a Tomato black ring virus isolate from Poland.

Science.gov (United States)

Jończyk, M; Le Gall, O; Pałucha, A; Borodynko, N; Pospieszny, H

2004-04-01

Full-length cDNA clones corresponding to the RNA1 and RNA2 of the Polish isolate MJ of Tomato black ring virus (TBRV, genus Nepovirus) were obtained using a direct recombination strategy in yeast, and their complete nucleotide sequences were established. RNA1 is 7358 nucleotides and RNA2 is 4633 nucleotides in length, excluding the poly(A) tails. Both RNAs contain a single open reading frame encoding polyproteins of 254 kDa and 149 kDa for RNA1 and RNA2 respectively. Putative cleavage sites were identified, and the relationships between TBRV and related nepoviruses were studied by sequence comparison.

Efficient expression and purification of a protease from the common cold virus, human rhinovirus type 14

Science.gov (United States)

Leong, L. E.-C.; Walker, P. A.; Porter, A. G.

1992-08-01

The protease (3C pro) from human rhinovirus serotype-14 (HRV-14) has been cloned and efficiently expressed in E. coli. A straightforward single-step purification of the recombinant 3C pro has been achieved by fusing the protein to the car☐y-terminus of the glutathione-S-transferase from Schistosoma japonicum. Modifications made to the 5' end of the PCR fragment coding for the 3C pro have allowed the specific cleavage of the fusion protein using thrombin to yield mature 3C pro with the correct amino-terminal amino acid. This protease has been shown to be active when assayed using synthetic peptides corresponding to the natural cleavage recognition sequences within the polyprotein. Other substrates are being developed for this protease for possible use in the screening of inhibitors of 3C pro. Sufficient protease 3C pro has been purified for initial attempts at crystallization.

Proximity-activated nanoparticles: in vitro performance of specific structural modification by enzymatic cleavage

Science.gov (United States)

Adam Smith, R; Sewell, Sarah L; Giorgio, Todd D

2008-01-01

The development and in vitro performance of a modular nanoscale system capable of specific structural modification by enzymatic activity is described in this work. Due to its small physical size and adaptable characteristics, this system has the potential for utilization in targeted delivery systems and biosensing. Nanoparticle probes were synthesized containing two distinct fluorescent species including a quantum dot base particle and fluorescently labeled cleavable peptide substrate. Activity of these probes was monitored by gel electrophoresis with quantitative cleavage measurements made by fluorometric analysis. The model proximity-activated nanoparticles studied here exhibit significant susceptibility to cleavage by matrix metalloprotease-7 (MMP-7) at physiologically relevant concentrations, with nearly complete cleavage of available substrate molecules after 24 hours. This response is specific to MMP-7 enzyme activity, as cleavage is completely inhibited with the addition of EDTA. Utilization of enzyme-specific modification is a sensitive approach with broad applications for targeted therapeutics and biosensing. The versatility of this nanoparticle system is highlighted in its modular design, as it has the capability to integrate characteristics for detection, biosensing, targeting, and payload delivery into a single, multifunctional nanoparticle structure. PMID:18488420

PROSPER: an integrated feature-based tool for predicting protease substrate cleavage sites.

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Jiangning Song

Full Text Available The ability to catalytically cleave protein substrates after synthesis is fundamental for all forms of life. Accordingly, site-specific proteolysis is one of the most important post-translational modifications. The key to understanding the physiological role of a protease is to identify its natural substrate(s. Knowledge of the substrate specificity of a protease can dramatically improve our ability to predict its target protein substrates, but this information must be utilized in an effective manner in order to efficiently identify protein substrates by in silico approaches. To address this problem, we present PROSPER, an integrated feature-based server for in silico identification of protease substrates and their cleavage sites for twenty-four different proteases. PROSPER utilizes established specificity information for these proteases (derived from the MEROPS database with a machine learning approach to predict protease cleavage sites by using different, but complementary sequence and structure characteristics. Features used by PROSPER include local amino acid sequence profile, predicted secondary structure, solvent accessibility and predicted native disorder. Thus, for proteases with known amino acid specificity, PROSPER provides a convenient, pre-prepared tool for use in identifying protein substrates for the enzymes. Systematic prediction analysis for the twenty-four proteases thus far included in the database revealed that the features we have included in the tool strongly improve performance in terms of cleavage site prediction, as evidenced by their contribution to performance improvement in terms of identifying known cleavage sites in substrates for these enzymes. In comparison with two state-of-the-art prediction tools, PoPS and SitePrediction, PROSPER achieves greater accuracy and coverage. To our knowledge, PROSPER is the first comprehensive server capable of predicting cleavage sites of multiple proteases within a single substrate

Fe-Catalyzed Oxidative Cleavage of Unsaturated Fatty Acids

NARCIS (Netherlands)

Spannring, P.

2013-01-01

The oxidative cleavage of unsaturated fatty acids into aldehydes or carboxylic acids gives access to valuable products. The products can be used as chemical building blocks, as emulsifiers or in the paint or polymer industry. Ozonolysis is applied industrially to cleave the fatty acid oleic acid

Signal peptide discrimination and cleavage site identification using SVM and NN.

Science.gov (United States)

Kazemian, H B; Yusuf, S A; White, K

2014-02-01

About 15% of all proteins in a genome contain a signal peptide (SP) sequence, at the N-terminus, that targets the protein to intracellular secretory pathways. Once the protein is targeted correctly in the cell, the SP is cleaved, releasing the mature protein. Accurate prediction of the presence of these short amino-acid SP chains is crucial for modelling the topology of membrane proteins, since SP sequences can be confused with transmembrane domains due to similar composition of hydrophobic amino acids. This paper presents a cascaded Support Vector Machine (SVM)-Neural Network (NN) classification methodology for SP discrimination and cleavage site identification. The proposed method utilises a dual phase classification approach using SVM as a primary classifier to discriminate SP sequences from Non-SP. The methodology further employs NNs to predict the most suitable cleavage site candidates. In phase one, a SVM classification utilises hydrophobic propensities as a primary feature vector extraction using symmetric sliding window amino-acid sequence analysis for discrimination of SP and Non-SP. In phase two, a NN classification uses asymmetric sliding window sequence analysis for prediction of cleavage site identification. The proposed SVM-NN method was tested using Uni-Prot non-redundant datasets of eukaryotic and prokaryotic proteins with SP and Non-SP N-termini. Computer simulation results demonstrate an overall accuracy of 0.90 for SP and Non-SP discrimination based on Matthews Correlation Coefficient (MCC) tests using SVM. For SP cleavage site prediction, the overall accuracy is 91.5% based on cross-validation tests using the novel SVM-NN model. © 2013 Published by Elsevier Ltd.

Sequence specificity of DNA cleavage by Micrococcus luteus γ endonuclease

International Nuclear Information System (INIS)

Hentosh, P.; Henner, W.D.; Reynolds, R.J.

1985-01-01

DNA fragments of defined sequence have been used to determine the sites of cleavage by γ-endonuclease activity in extracts prepared from Micrococcus luteus. End-labeled DNA restriction fragments of pBR322 DNA that had been irradiated under nitrogen in the presence of potassium iodide or t-butanol were treated with M. luteus γ endonuclease and analyzed on irradiated DNA preferentially at the positions of cytosines and thymines. DNA cleavage occurred immediately to the 3' side of pyrimidines in irradiated DNA and resulted in fragments that terminate in a 5'-phosphoryl group. These studies indicate that both altered cytosines and thymines may be important DNA lesions requiring repair after exposure to γ radiation

Oxidation of trimethoprim by ferrate(VI): kinetics, products, and antibacterial activity.

Science.gov (United States)

Anquandah, George A K; Sharma, Virender K; Knight, D Andrew; Batchu, Sudha Rani; Gardinali, Piero R

2011-12-15

Kinetics, stoichiometry, and products of the oxidation of trimethoprim (TMP), one of the most commonly detected antibacterial agents in surface waters and municipal wastewaters, by ferrate(VI) (Fe(VI)) were determined. The pH dependent second-order rate constants of the reactions of Fe(VI) with TMP were examined using acid-base properties of Fe(VI) and TMP. The kinetics of reactions of diaminopyrimidine (DAP) and trimethoxytoluene (TMT) with Fe(VI) were also determined to understand the reactivity of Fe(VI) with TMP. Oxidation products of the reactions of Fe(VI) with TMP and DAP were identified by liquid chromatography-tandem mass spectrometry (LC-MS/MS). Reaction pathways of oxidation of TMP by Fe(VI) are proposed to demonstrate the cleavage of the TMP molecule to ultimately result in 3,4,5,-trimethoxybenzaldehyde and 2,4-dinitropyrimidine as among the final identified products. The oxidized products mixture exhibited no antibacterial activity against E. coli after complete consumption of TMP. Removal of TMP in the secondary effluent by Fe(VI) was achieved.

Model for how type I restriction enzymes select cleavage sites in DNA

International Nuclear Information System (INIS)

Studier, F.W.; Bandyopadhyay, P.K.

1988-01-01

Under appropriate conditions, digestion of phage T7 DNA by the type I restriction enzyme EcoK produces an orderly progression of discrete DNA fragments. All details of the fragmentation pattern can be explained on the basis of the known properties of type I enzymes, together with two further assumptions: (i) in the ATP-stimulated translocation reaction, the enzyme bound at the recognition sequence translocates DNA toward itself from both directions simultaneously; and (ii) when translocation causes neighboring enzymes to meet, they cut the DNA between them. The kinetics of digestion at 37 degree C indicates that the rate of translocation of DNA from each side of a bound enzyme is about 200 base pairs per second, and the cuts are completed within 15-25 sec of the time neighboring enzymes meet. The resulting DNA fragments each contain a single recognition site with an enzyme (or subunit) remaining bound to it. At high enzyme concentrations, such fragments can bu further degraded, apparently by cooperation between the specifically bound and excess enzymes. This model is consistent with a substantial body of previous work on the nuclease activity of EcoB and EcoK, and it explains in a simple way how cleavage sites are selected

DNA Cleavage Activity of Diazonium Salts: Chemical Nucleases

OpenAIRE

KIZIL, Murat

2014-01-01

4-Fenoldiazonium tetrafluoroborate and 4-benzoicaciddiazonium tetrafluoroborate was prepared and was shown to be an effective DNA cleavage agent in the presence of the 1-electron donor copper(II) chloride. Its mechanism involves the generation of the aryl radical cleaving DNA by hydrogen atom abstraction from deoxyribose sugar.

Autoactivation of mouse trypsinogens is regulated by chymotrypsin C via cleavage of the autolysis loop.

Science.gov (United States)

Németh, Balázs Csaba; Wartmann, Thomas; Halangk, Walter; Sahin-Tóth, Miklós

2013-08-16

Chymotrypsin C (CTRC) is a proteolytic regulator of trypsinogen autoactivation in humans. CTRC cleavage of the trypsinogen activation peptide stimulates autoactivation, whereas cleavage of the calcium binding loop promotes trypsinogen degradation. Trypsinogen mutations that alter these regulatory cleavages lead to increased intrapancreatic trypsinogen activation and cause hereditary pancreatitis. The aim of this study was to characterize the regulation of autoactivation of mouse trypsinogens by mouse Ctrc. We found that the mouse pancreas expresses four trypsinogen isoforms to high levels, T7, T8, T9, and T20. Only the T7 activation peptide was cleaved by mouse Ctrc, causing negligible stimulation of autoactivation. Surprisingly, mouse Ctrc poorly cleaved the calcium binding loop in all mouse trypsinogens. In contrast, mouse Ctrc readily cleaved the Phe-150-Gly-151 peptide bond in the autolysis loop of T8 and T9 and inhibited autoactivation. Mouse chymotrypsin B also cleaved the same peptide bond but was 7-fold slower. T7 was less sensitive to chymotryptic regulation, which involved slow cleavage of the Leu-149-Ser-150 peptide bond in the autolysis loop. Modeling indicated steric proximity of the autolysis loop and the activation peptide in trypsinogen, suggesting the cleaved autolysis loop may directly interfere with activation. We conclude that autoactivation of mouse trypsinogens is under the control of mouse Ctrc with some notable differences from the human situation. Thus, cleavage of the trypsinogen activation peptide or the calcium binding loop by Ctrc is unimportant. Instead, inhibition of autoactivation via cleavage of the autolysis loop is the dominant mechanism that can mitigate intrapancreatic trypsinogen activation.

Analysis of Network Topologies Underlying Ethylene Growth Response Kinetics

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Aaron M. Prescott

2016-08-01

Full Text Available Most models for ethylene signaling involve a linear pathway. However, measurements of seedling growth kinetics when ethylene is applied and removed have resulted in more complex network models that include coherent feedforward, negative feedback, and positive feedback motifs. However, the dynamical responses of the proposed networks have not been explored in a quantitative manner. Here, we explore (i whether any of the proposed models are capable of producing growth-response behaviors consistent with experimental observations and (ii what mechanistic roles various parts of the network topologies play in ethylene signaling. To address this, we used computational methods to explore two general network topologies: The first contains a coherent feedforward loop that inhibits growth and a negative feedback from growth onto itself (CFF/NFB. In the second, ethylene promotes the cleavage of EIN2, with the product of the cleavage inhibiting growth and promoting the production of EIN2 through a positive feedback loop (PFB. Since few network parameters for ethylene signaling are known in detail, we used an evolutionary algorithm to explore sets of parameters that produce behaviors similar to experimental growth response kinetics of both wildtype and mutant seedlings. We generated a library of parameter sets by independently running the evolutionary algorithm many times. Both network topologies produce behavior consistent with experimental observations and analysis of the parameter sets allows us to identify important network interactions and parameter constraints. We additionally screened these parameter sets for growth recovery in the presence of sub-saturating ethylene doses, which is an experimentally-observed property that emerges in some of the evolved parameter sets. Finally, we probed simplified networks maintaining key features of the CFF/NFB and PFB topologies. From this, we verified observations drawn from the larger networks about mechanisms

Analysis of Network Topologies Underlying Ethylene Growth Response Kinetics.

Science.gov (United States)

Prescott, Aaron M; McCollough, Forest W; Eldreth, Bryan L; Binder, Brad M; Abel, Steven M

2016-01-01

Most models for ethylene signaling involve a linear pathway. However, measurements of seedling growth kinetics when ethylene is applied and removed have resulted in more complex network models that include coherent feedforward, negative feedback, and positive feedback motifs. The dynamical responses of the proposed networks have not been explored in a quantitative manner. Here, we explore (i) whether any of the proposed models are capable of producing growth-response behaviors consistent with experimental observations and (ii) what mechanistic roles various parts of the network topologies play in ethylene signaling. To address this, we used computational methods to explore two general network topologies: The first contains a coherent feedforward loop that inhibits growth and a negative feedback from growth onto itself (CFF/NFB). In the second, ethylene promotes the cleavage of EIN2, with the product of the cleavage inhibiting growth and promoting the production of EIN2 through a positive feedback loop (PFB). Since few network parameters for ethylene signaling are known in detail, we used an evolutionary algorithm to explore sets of parameters that produce behaviors similar to experimental growth response kinetics of both wildtype and mutant seedlings. We generated a library of parameter sets by independently running the evolutionary algorithm many times. Both network topologies produce behavior consistent with experimental observations, and analysis of the parameter sets allows us to identify important network interactions and parameter constraints. We additionally screened these parameter sets for growth recovery in the presence of sub-saturating ethylene doses, which is an experimentally-observed property that emerges in some of the evolved parameter sets. Finally, we probed simplified networks maintaining key features of the CFF/NFB and PFB topologies. From this, we verified observations drawn from the larger networks about mechanisms underlying ethylene

Analysis by the reductive-cleavage method of linkage positions in a polysaccharide containing 4-linked D-glucopyranosyluronic residues.

Science.gov (United States)

Vodonik, S A; Gray, G R

1988-04-01

The fate of 4-linked D-glucopyranosyluronic residues under reductive-cleavage conditions was investigated by using the Klebsiella aerogenes type 54 strain A3 capsular polysaccharide. Treatment of the fully methylated polysaccharide with triethylsilane and trimethylsilyl trifluoromethanesulfonate in dichloromethane, followed by in situ acetylation, yielded 1,5-anhydro-2,3,4,6-tetra-O-methyl-D-glucitol, 3,4-di-O-acetyl-1,5-anhydro-2,6-di-O-methyl-D-glucitol, and 3-O-acetyl-1,5-anhydro-2,4-di-O-methyl-L-fucitol, as expected, but the expected product of reductive cleavage of the 4-linked D-glucopyranosyluronic residue, namely, methyl 3-O-acetyl-2,6-anhydro-4,5-di-O-methyl-L-gulonate, was not observed. Instead, methyl 2-O-acetyl-3,6-anhydro-4,5-di-O-methyl-L-gulonate (6) was identified as the sole product of reductive cleavage of the 4-linked D-glucopyranosyluronic residue. That compound 6 arose as a result of rearrangement during reductive cleavage rather than by reductive cleavage of a 5-linked D-glucofuranosyluronic residue, was established by reductive cleavage of the fully methylated polysaccharide following reduction of its ester groups with either lithium aluminum hydride or lithium aluminum deuteride. The products of the latter reductive cleavage were the same as before, except for the absence of 6 and the presence of 4,6-di-O-acetyl-1,5-anhydro-2,3-di-O-methyl-D-glucitol, or its 6,6-dideuterio isomer. Although the reductive-cleavage technique is suitable for the direct analysis of polysaccharides containing 4-linked D-glucopyranosyluronic residues, it does not establish whether the uronic residue is a 4-linked pyranoside or a 5-linked furanoside. The expected product is, however, derived from the 4-linked D-glucopyranosyluronic residue after sequential methylation, reduction of its ester group and reductive cleavage.

Programmable RNA recognition and cleavage by CRISPR/Cas9.

Science.gov (United States)

O'Connell, Mitchell R; Oakes, Benjamin L; Sternberg, Samuel H; East-Seletsky, Alexandra; Kaplan, Matias; Doudna, Jennifer A

2014-12-11

The CRISPR-associated protein Cas9 is an RNA-guided DNA endonuclease that uses RNA-DNA complementarity to identify target sites for sequence-specific double-stranded DNA (dsDNA) cleavage. In its native context, Cas9 acts on DNA substrates exclusively because both binding and catalysis require recognition of a short DNA sequence, known as the protospacer adjacent motif (PAM), next to and on the strand opposite the twenty-nucleotide target site in dsDNA. Cas9 has proven to be a versatile tool for genome engineering and gene regulation in a large range of prokaryotic and eukaryotic cell types, and in whole organisms, but it has been thought to be incapable of targeting RNA. Here we show that Cas9 binds with high affinity to single-stranded RNA (ssRNA) targets matching the Cas9-associated guide RNA sequence when the PAM is presented in trans as a separate DNA oligonucleotide. Furthermore, PAM-presenting oligonucleotides (PAMmers) stimulate site-specific endonucleolytic cleavage of ssRNA targets, similar to PAM-mediated stimulation of Cas9-catalysed DNA cleavage. Using specially designed PAMmers, Cas9 can be specifically directed to bind or cut RNA targets while avoiding corresponding DNA sequences, and we demonstrate that this strategy enables the isolation of a specific endogenous messenger RNA from cells. These results reveal a fundamental connection between PAM binding and substrate selection by Cas9, and highlight the utility of Cas9 for programmable transcript recognition without the need for tags.

Structural and Kinetic Isotope Effect Studies of Nicotinamidase (Pnc1) from Saccharomyces cerevisiae

Energy Technology Data Exchange (ETDEWEB)

Smith, Brian C.; Anderson, Mark A.; Hoadley, Kelly A.; Keck, James L.; Cleland, W. Wallace; Denu, John M. (UW)

2012-05-08

Nicotinamidases catalyze the hydrolysis of nicotinamide to nicotinic acid and ammonia. Nicotinamidases are absent in mammals but function in NAD{sup +} salvage in many bacteria, yeast, plants, protozoa, and metazoans. We have performed structural and kinetic investigations of the nicotinamidase from Saccharomyces cerevisiae (Pnc1). Steady-state product inhibitor analysis revealed an irreversible reaction in which ammonia is the first product released, followed by nicotinic acid. A series of nicotinamide analogues acting as inhibitors or substrates were examined, revealing that the nicotinamide carbonyl oxygen and ring nitrogen are critical for binding and reactivity. X-ray structural analysis revealed a covalent adduct between nicotinaldehyde and Cys167 of Pnc1 and coordination of the nicotinamide ring nitrogen to the active-site zinc ion. Using this structure as a guide, the function of several residues was probed via mutagenesis and primary {sup 15}N and {sup 13}C kinetic isotope effects (KIEs) on V/K for amide bond hydrolysis. The KIE values of almost all variants were increased, indicating that C-N bond cleavage is at least partially rate limiting; however, a decreased KIE for D51N was indicative of a stronger commitment to catalysis. In addition, KIE values using slower alternate substrates indicated that C-N bond cleavage is at least partially rate limiting with nicotinamide to highly rate limiting with thionicotinamide. A detailed mechanism involving nucleophilic attack of Cys167, followed by elimination of ammonia and then hydrolysis to liberate nicotinic acid, is discussed. These results will aid in the design of mechanism-based inhibitors to target pathogens that rely on nicotinamidase activity.

Conservation of a proteinase cleavage site between an insect retrovirus (gypsy) Env protein and a baculovirus envelope fusion protein

International Nuclear Information System (INIS)

Pearson, Margot N.; Rohrmann, George F.

2004-01-01

The predicted Env protein of insect retroviruses (errantiviruses) is related to the envelope fusion protein of a major division of the Baculoviridae. The highest degree of homology is found in a region that contains a furin cleavage site in the baculovirus proteins and an adjacent sequence that has the properties of a fusion peptide. In this investigation, the homologous region in the Env protein of the gypsy retrovirus of Drosophila melanogaster (DmegypV) was investigated. Alteration of the predicted DmegypV Env proteinase cleavage site from RIAR to AIAR significantly reduced cleavage of Env in both Spodoptera frugiperda (Sf-9) and D. melanogaster (S2) cell lines. When the predicted DmegypV Env cleavage site RIAR was substituted for the cleavage sequence RRKR in the Lymantria dispar nucleopolyhedrovirus fusion protein (LD130) sequence, cleavage of the hybrid LD130 molecules still occurred, although at a reduced level. The conserved 21-amino acid sequence just downstream of the cleavage site, which is thought to be the fusion peptide in LD130, was also characterized. When this sequence from DmegypV Env was substituted for the homologous sequence in LD130, cleavage still occurred, but no fusion was observed in either cell type. In addition, although a DmegypV-Env-green fluorescent protein construct localized to cell membranes, no cell fusion was observed

Drosha regulates gene expression independently of RNA cleavage function

DEFF Research Database (Denmark)

Gromak, Natalia; Dienstbier, Martin; Macias, Sara

2013-01-01

Drosha is the main RNase III-like enzyme involved in the process of microRNA (miRNA) biogenesis in the nucleus. Using whole-genome ChIP-on-chip analysis, we demonstrate that, in addition to miRNA sequences, Drosha specifically binds promoter-proximal regions of many human genes in a transcription......-dependent manner. This binding is not associated with miRNA production or RNA cleavage. Drosha knockdown in HeLa cells downregulated nascent gene transcription, resulting in a reduction of polyadenylated mRNA produced from these gene regions. Furthermore, we show that this function of Drosha is dependent on its N......-terminal protein-interaction domain, which associates with the RNA-binding protein CBP80 and RNA Polymerase II. Consequently, we uncover a previously unsuspected RNA cleavage-independent function of Drosha in the regulation of human gene expression....

Polycystin-1 Cleavage and the Regulation of Transcriptional Pathways

Science.gov (United States)

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

Cell-surface acceleration of urokinase-catalyzed receptor cleavage

DEFF Research Database (Denmark)

Høyer-Hansen, G; Ploug, M; Behrendt, N

1997-01-01

by a prior incubation of the cells with uPA inactivated by diisopropyl fluorophosphate, demonstrating a requirement for specific receptor binding of the active uPA to obtain the high-efficiency cleavage of cell-bound uPAR. Furthermore, amino-terminal sequence analysis revealed that uPAR(2+3), purified from U...

Fetal hemoglobin is much less prone to DNA cleavage compared to the adult protein

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Sandeep Chakane

2017-08-01

Full Text Available Hemoglobin (Hb is well protected inside the red blood cells (RBCs. Upon hemolysis and when free in circulation, Hb can be involved in a range of radical generating reactions and may thereby attack several different biomolecules. In this study, we have examined the potential damaging effects of cell-free Hb on plasmid DNA (pDNA. Hb induced cleavage of supercoiled pDNA (sc pDNA which was proportional to the concentration of Hb applied. Almost 70% of sc pDNA was converted to open circular or linear DNA using 10 µM of Hb in 12 h. Hb can be present in several different forms. The oxy (HbO2 and met forms are most reactive, while the carboxy-protein shows only low hydrolytic activity. Hemoglobin A (HbA could easily induce complete pDNA cleavage while fetal hemoglobin (HbF was three-fold less reactive. By inserting, a redox active cysteine residue on the surface of the alpha chain of HbF by site-directed mutagenesis, the DNA cleavage reaction was enhanced by 82%. Reactive oxygen species were not directly involved in the reaction since addition of superoxide dismutase and catalase did not prevent pDNA cleavage. The reactivity of Hb with pDNA can rather be associated with the formation of protein based radicals. Keywords: Adult hemoglobin, Fetal hemoglobin, Supercoiled plasmid DNA, DNA cleavage, Cysteine, Protein radicals

Abyssal fiction: common shares, colonial cleavages

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Alexandre Montaury

2016-12-01

Full Text Available The paper aims to develop a reflection on the interaction between the legacies of colonialism and traditional symbolic and cultural practices in African Portuguese-speaking spaces. From a preliminary analysis of fictional texts of wide circulation in Brazil, aims to examine the cleavages, or “abyssal lines” that constitute experiences printed in the daily life of the former Portuguese colony of Cape Verde, Mozambique and Angola.---DOI: http://dx.doi.org/10.21881/abriluff.2016n17a378

Transcriptional down-regulation and rRNA cleavage in Dictyostelium discoideum mitochondria during Legionella pneumophila infection.

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Chenyu Zhang

2009-05-01

Full Text Available Bacterial pathogens employ a variety of survival strategies when they invade eukaryotic cells. The amoeba Dictyostelium discoideum is used as a model host to study the pathogenic mechanisms that Legionella pneumophila, the causative agent of Legionnaire's disease, uses to kill eukaryotic cells. Here we show that the infection of D. discoideum by L. pneumophila results in a decrease in mitochondrial messenger RNAs, beginning more than 8 hours prior to detectable host cell death. These changes can be mimicked by hydrogen peroxide treatment, but not by other cytotoxic agents. The mitochondrial large subunit ribosomal RNA (LSU rRNA is also cleaved at three specific sites during the course of infection. Two LSU rRNA fragments appear first, followed by smaller fragments produced by additional cleavage events. The initial LSU rRNA cleavage site is predicted to be on the surface of the large subunit of the mitochondrial ribosome, while two secondary sites map to the predicted interface with the small subunit. No LSU rRNA cleavage was observed after exposure of D. discoideum to hydrogen peroxide, or other cytotoxic chemicals that kill cells in a variety of ways. Functional L. pneumophila type II and type IV secretion systems are required for the cleavage, establishing a correlation between the pathogenesis of L. pneumophila and D. discoideum LSU rRNA destruction. LSU rRNA cleavage was not observed in L. pneumophila infections of Acanthamoeba castellanii or human U937 cells, suggesting that L. pneumophila uses distinct mechanisms to interrupt metabolism in different hosts. Thus, L. pneumophila infection of D. discoideum results in dramatic decrease of mitochondrial RNAs, and in the specific cleavage of mitochondrial rRNA. The predicted location of the cleavage sites on the mitochondrial ribosome suggests that rRNA destruction is initiated by a specific sequence of events. These findings suggest that L. pneumophila specifically disrupts mitochondrial

Cleavage strain in the Variscan fold belt, County Cork, Ireland, estimated from stretched arsenopyrite rosettes

Science.gov (United States)

Ford, M.; Ferguson, C.C.

1985-01-01

In south-west Ireland, hydrothermally formed arsenopyrite crystals in a Devonian mudstone have responded to Variscan deformation by brittle extension fracture and fragment separation. The interfragment gaps and terminal extension zones of each crystal are infilled with fibrous quartz. Stretches within the cleavage plane have been calculated by the various methods available, most of which can be modified to incorporate terminal extension zones. The Strain Reversal Method is the most accurate currently available but still gives a minimum estimate of the overall strain. The more direct Hossain method, which gives only slightly lower estimates with this data, is more practical for field use. A strain ellipse can be estimated from each crystal rosette composed of three laths (assuming the original interlimb angles were all 60??) and, because actual rather than relative stretches are estimated, this provides a lower bound to the area increase in the plane of cleavage. Based on the average of our calculated strain ellipses this area increase is at least 114% and implies an average shortening across the cleavage of at least 53%. However, several lines of evidence suggest that the cleavage deformation was more intense and more oblate than that calculated, and we argue that a 300% area increase in the cleavage plane and 75% shortening across the cleavage are more realistic estimates of the true strain. Furthermore, the along-strike elongation indicated is at least 80%, which may be regionally significant. Estimates of orogenic contraction derived from balanced section construction should therefore take into account the possibility of a substantial strike elongation, and tectonic models that can accommodate such elongations need to be developed. ?? 1985.

Cleavage specificity analysis of six type II transmembrane serine proteases (TTSPs using PICS with proteome-derived peptide libraries.

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Olivier Barré

Full Text Available Type II transmembrane serine proteases (TTSPs are a family of cell membrane tethered serine proteases with unclear roles as their cleavage site specificities and substrate degradomes have not been fully elucidated. Indeed just 52 cleavage sites are annotated in MEROPS, the database of proteases, their substrates and inhibitors.To profile the active site specificities of the TTSPs, we applied Proteomic Identification of protease Cleavage Sites (PICS. Human proteome-derived database searchable peptide libraries were assayed with six human TTSPs (matriptase, matriptase-2, matriptase-3, HAT, DESC and hepsin to simultaneously determine sequence preferences on the N-terminal non-prime (P and C-terminal prime (P' sides of the scissile bond. Prime-side cleavage products were isolated following biotinylation and identified by tandem mass spectrometry. The corresponding non-prime side sequences were derived from human proteome databases using bioinformatics. Sequencing of 2,405 individual cleaved peptides allowed for the development of the family consensus protease cleavage site specificity revealing a strong specificity for arginine in the P1 position and surprisingly a lysine in P1' position. TTSP cleavage between R↓K was confirmed using synthetic peptides. By parsing through known substrates and known structures of TTSP catalytic domains, and by modeling the remainder, structural explanations for this strong specificity were derived.Degradomics analysis of 2,405 cleavage sites revealed a similar and characteristic TTSP family specificity at the P1 and P1' positions for arginine and lysine in unfolded peptides. The prime side is important for cleavage specificity, thus making these proteases unusual within the tryptic-enzyme class that generally has overriding non-prime side specificity.

Change in radiosensitivity of sea-urchin eggs during early cleavage stages

International Nuclear Information System (INIS)

Nakamura, I.

1977-01-01

When sea-urchin eggs were irradiated with 137 Cs γ-rays, their radiosensitivity, expressed by the percentage which formed pluteus larvae, fluctuated during the early cleavage cycle. Split-dose irradiations were made both in the sensitive and resistant phases. For eggs in the sensitive phase, the effect of the first exposure of 500 rad was not diminished during the interval before the second exposure. Eggs irradiated in the resistant phase were only slightly damaged. Results implied that fluctuations in radiosensitivity of sea-urchin eggs were caused mainly by different degrees of non-repairable damage in each phase of cleavage rather than by different recovery abilities. (author)

Caspase Cleavages of the Lymphocyte-oriented Kinase Prevent Ezrin, Radixin, and Moesin Phosphorylation during Apoptosis*

Science.gov (United States)

Leroy, Catherine; Belkina, Natalya V.; Long, Thavy; Deruy, Emeric; Dissous, Colette; Shaw, Stephen; Tulasne, David

2016-01-01

The lymphocyte-oriented kinase (LOK), also called serine threonine kinase 10 (STK10), is synthesized mainly in lymphocytes. It is involved in lymphocyte migration and polarization and can phosphorylate ezrin, radixin, and moesin (the ERM proteins). In a T lymphocyte cell line and in purified human lymphocytes, we found LOK to be cleaved by caspases during apoptosis. The first cleavage occurs at aspartic residue 332, located between the kinase domain and the coiled-coil regulation domain. This cleavage generates an N-terminal fragment, p50 N-LOK, containing the kinase domain and a C-terminal fragment, which is further cleaved during apoptosis. Although these cleavages preserve the entire kinase domain, p50 N-LOK displays no kinase activity. In apoptotic lymphocytes, caspase cleavages of LOK are concomitant with a decrease in ERM phosphorylation. When non-apoptotic lymphocytes from mice with homozygous and heterozygous LOK knockout were compared, the latter showed a higher level of ERM phosphorylation, but when apoptosis was induced, LOK−/− and LOK+/− lymphocytes showed the same low level, confirming in vivo that LOK-induced ERM phosphorylation is prevented during lymphocyte apoptosis. Our results demonstrate that cleavage of LOK during apoptosis abolishes its kinase activity, causing a decrease in ERM phosphorylation, crucial to the role of the ERM proteins in linking the plasma membrane to actin filaments. PMID:26945071

Caspase Cleavages of the Lymphocyte-oriented Kinase Prevent Ezrin, Radixin, and Moesin Phosphorylation during Apoptosis.

Science.gov (United States)

Leroy, Catherine; Belkina, Natalya V; Long, Thavy; Deruy, Emeric; Dissous, Colette; Shaw, Stephen; Tulasne, David

2016-05-06

The lymphocyte-oriented kinase (LOK), also called serine threonine kinase 10 (STK10), is synthesized mainly in lymphocytes. It is involved in lymphocyte migration and polarization and can phosphorylate ezrin, radixin, and moesin (the ERM proteins). In a T lymphocyte cell line and in purified human lymphocytes, we found LOK to be cleaved by caspases during apoptosis. The first cleavage occurs at aspartic residue 332, located between the kinase domain and the coiled-coil regulation domain. This cleavage generates an N-terminal fragment, p50 N-LOK, containing the kinase domain and a C-terminal fragment, which is further cleaved during apoptosis. Although these cleavages preserve the entire kinase domain, p50 N-LOK displays no kinase activity. In apoptotic lymphocytes, caspase cleavages of LOK are concomitant with a decrease in ERM phosphorylation. When non-apoptotic lymphocytes from mice with homozygous and heterozygous LOK knockout were compared, the latter showed a higher level of ERM phosphorylation, but when apoptosis was induced, LOK(-/-) and LOK(+/-) lymphocytes showed the same low level, confirming in vivo that LOK-induced ERM phosphorylation is prevented during lymphocyte apoptosis. Our results demonstrate that cleavage of LOK during apoptosis abolishes its kinase activity, causing a decrease in ERM phosphorylation, crucial to the role of the ERM proteins in linking the plasma membrane to actin filaments. © 2016 by The American Society for Biochemistry and Molecular Biology, Inc.

Caspase-2 cleavage of tau reversibly impairs memory.

Science.gov (United States)

Zhao, Xiaohui; Kotilinek, Linda A; Smith, Benjamin; Hlynialuk, Chris; Zahs, Kathleen; Ramsden, Martin; Cleary, James; Ashe, Karen H

2016-11-01

In Alzheimer's disease (AD) and other tauopathies, the tau protein forms fibrils, which are believed to be neurotoxic. However, fibrillar tau has been dissociated from neuron death and network dysfunction, suggesting the involvement of nonfibrillar species. Here we describe a novel pathological process in which caspase-2 cleavage of tau at Asp314 impairs cognitive and synaptic function in animal and cellular models of tauopathies by promoting the missorting of tau to dendritic spines. The truncation product, Δtau314, resists fibrillation and is present at higher levels in brains from cognitively impaired mice and humans with AD. The expression of tau mutants that resisted caspase-2 cleavage prevented tau from infiltrating spines, dislocating glutamate receptors and impairing synaptic function in cultured neurons, and it prevented memory deficits and neurodegeneration in mice. Decreasing the levels of caspase-2 restored long-term memory in mice that had existing deficits. Our results suggest an overall treatment strategy for re-establishing synaptic function and restoring memory in patients with AD by preventing tau from accumulating in dendritic spines.

AID-induced decrease in topoisomerase 1 induces DNA structural alteration and DNA cleavage for class switch recombination.

Science.gov (United States)

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.

Carotenoid-cleavage activities of crude enzymes from Pandanous amryllifolius.

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Ningrum, Andriati; Schreiner, Matthias

2014-11-01

Carotenoid degradation products, known as norisoprenoids, are aroma-impact compounds in several plants. Pandan wangi is a common name of the shrub Pandanus amaryllifolius. The genus name 'Pandanus' is derived from the Indonesian name of the tree, pandan. In Indonesia, the leaves from the plant are used for several purposes, e.g., as natural colorants and flavor, and as traditional treatments. The aim of this study was to determine the cleavage of β-carotene and β-apo-8'-carotenal by carotenoid-cleavage enzymes isolated from pandan leaves, to investigate dependencies of the enzymatic activities on temperature and pH, to determine the enzymatic reaction products by using Headspace Solid Phase Microextraction Gas Chromatography/Mass Spectrophotometry (HS-SPME GC/MS), and to investigate the influence of heat treatment and addition of crude enzyme on formation of norisoprenoids. Crude enzymes from pandan leaves showed higher activity against β-carotene than β-apo-8'-carotenal. The optimum temperature of crude enzymes was 70°, while the optimum pH value was 6. We identified β-ionone as the major volatile reaction product from the incubations of two different carotenoid substrates, β-carotene and β-apo-8'-carotenal. Several treatments, e.g., heat treatment and addition of crude enzymes in pandan leaves contributed to the norisoprenoid content. Our findings revealed that the crude enzymes from pandan leaves with carotenoid-cleavage activity might provide a potential application, especially for biocatalysis, in natural-flavor industry. Copyright © 2014 Verlag Helvetica Chimica Acta AG, Zürich.

Caspase activation increases beta-amyloid generation independently of caspase cleavage of the beta-amyloid precursor protein (APP).

Science.gov (United States)

Tesco, Giuseppina; Koh, Young Ho; Tanzi, Rudolph E

2003-11-14

The amyloid precursor protein (APP) undergoes "alternative" proteolysis mediated by caspases. Three major caspase recognition sites have been identified in the APP, i.e. one at the C terminus (Asp720) and two at the N terminus (Asp197 and Asp219). Caspase cleavage at Asp720 has been suggested as leading to increased production of Abeta. Thus, we set out to determine which putative caspase sites in APP, if any, are cleaved in Chinese hamster ovary cell lines concurrently with the increased Abeta production that occurs during apoptosis. We found that cleavage at Asp720 occurred concurrently with caspase 3 activation and the increased production of total secreted Abeta and Abeta1-42 in association with staurosporine- and etoposide-induced apoptosis. To investigate the contribution of caspase cleavage of APP to Abeta generation, we expressed an APP mutant truncated at Asp720 that mimics APP caspase cleavage at the C-terminal site. This did not increase Abeta generation but, in contrast, dramatically decreased Abeta production in Chinese hamster ovary cells. Furthermore, the ablation of caspase-dependent cleavage at Asp720, Asp197, and Asp219 (by site-directed mutagenesis) did not prevent enhanced Abeta production following etoposide-induced apoptosis. These findings indicate that the enhanced Abeta generation associated with apoptosis does not require cleavage of APP at its C-terminal (Asp720) and/or N-terminal caspase sites.

The complete genome sequence of a south Indian isolate of Rice tungro spherical virus reveals evidence of genetic recombination between distinct isolates.

Science.gov (United States)

Sailaja, B; Anjum, Najreen; Patil, Yogesh K; Agarwal, Surekha; Malathi, P; Krishnaveni, D; Balachandran, S M; Viraktamath, B C; Mangrauthia, Satendra K

2013-12-01

In this study, complete genome of a south Indian isolate of Rice tungro spherical virus (RTSV) from Andhra Pradesh (AP) was sequenced, and the predicted amino acid sequence was analysed. The RTSV RNA genome consists of 12,171 nt without the poly(A) tail, encoding a putative typical polyprotein of 3,470 amino acids. Furthermore, cleavage sites and sequence motifs of the polyprotein were predicted. Multiple alignment with other RTSV isolates showed a nucleotide sequence identity of 95% to east Indian isolates and 90% to Philippines isolates. A phylogenetic tree based on complete genome sequence showed that Indian isolates clustered together, while Vt6 and PhilA isolates of Philippines formed two separate clusters. Twelve recombination events were detected in RNA genome of RTSV using the Recombination Detection Program version 3. Recombination analysis suggested significant role of 5' end and central region of genome in virus evolution. Further, AP and Odisha isolates appeared as important RTSV isolates involved in diversification of this virus in India through recombination phenomenon. The new addition of complete genome of first south Indian isolate provided an opportunity to establish the molecular evolution of RTSV through recombination analysis and phylogenetic relationship.

SOCIAL CLEAVAGES IN THE AMERICAN SOCIETY AS A FACTOR OF 2016 PRESIDENTIAL CAMPAIGN

Directory of Open Access Journals (Sweden)

P. S. Kanevskiy

2017-01-01

Full Text Available Current article is dedicated to analysis of social cleavages in the American elections and the ways they influenced on presidential election in 2016. Originally developed by S. Rokkan and S.M. Lipset, social cleavages became a classic theme for contemporary political sociology. However, despite the fact that the theory has been developing primarily by Americans, it has been rarely used to analyze electoral system in the USA. Traditionally it’s been aimed at European and developing countries where electoral fragmentation is seen more clearly. But recent changes in the American society and the political system demonstrate the emergence of social cleavages that had not been inherent before. The article shows how American electoral space transformed since the 1980s and how it became more fragmented under the influence of social, economic and ideological factors. Elections in 2016 became a watershed for social cleavages that accumulated through time and aggravated even more considering internal crises in the Democratic and more so in the Republican parties. Donald Trump’s victory is an impersonation of the American party system crisis and of the mainstream politicians’ inability to find proper explanation of the changing electorate. Author shows that American society today is polarized even more than many European countries while group identification determines vectors of political change.

Electrostatic instability of some jellium model lattices of high symmetry to their plane cleavage

International Nuclear Information System (INIS)

Kholopov, Eugene V; Kalashnikova, Vita V

2007-01-01

Jellium model structures composed of regular lattices of equal point charges immersed in a neutralizing uniform background are considered. The symmetric description eliminating the effect of potentials without transverse structural modulation is extended to the events specified by alternating distances between point-charge planes. Based on modulated potentials typical of plane-wise lattice summation, the energy of interaction between two semi-infinite hemi-crystals divided by a plane is obtained for cubic and hexagonal crystals, where all the characteristic orientations of the cleavage plane are taken into account. We found that simple cubic and hexagonal lattices, as well as cubic and hexagonal diamond structures, turn out to be unstable for certain cleavage planes. The most favourable cleavage planes for the bcc, fcc and hcp structures are also emphasized

Stimulation of topoisomerase II mediated DNA cleavage at specific sequence elements by the 2-nitroimidazole Ro 15-0216

International Nuclear Information System (INIS)

Sorensen, B.S.; Jensen, P.S.; Andersen, A.H.; Christiansen, K.; Alsner, J.; Thomsen, B.; Westergaard, O.

1990-01-01

The effect of the 2-nitroimidazole Ro 15-0216 upon the interaction between purified topoisomerase II and its DNA substrate was investigated. The cleavage reaction in the presence of this DNA-nonintercalative drug took place with the hallmarks of a regular topoisomerase II mediated cleavage reaction, including covalent linkage of the enzyme to the cleaved DNA. In the presence of Ro 15-0216, topoisomerase II mediated cleavage was extensively stimulated at major cleavage sites of which only one existed in the 4363 base pair pBR322 molecule. The sites stimulated by Ro 15-0216 shared a pronounced sequence homology, indicating that a specific nucleotide sequence is crucial for the action of this drug. The effect of Ro 15-0216 thus differs from that of the clinically important topoisomerase II targeted agents such as mAMSA, VM26, and VP16, which enhance enzyme-mediated cleavage at a multiple number of sites. In contrast to the previous described drugs, Ro 15-0216 did not exert any inhibitory effect on the enzyme's catalytic activity. This observation might be ascribed to the low stability of the cleavage complexes formed in the presence of Ro 15-0216 as compared to the stability of the ones formed in the presence of traditional topoisomerase II targeted drugs

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

Science.gov (United States)

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.

The Conserved ATM Kinase RAG2-S365 Phosphorylation Site Limits Cleavage Events in Individual Cells Independent of Any Repair Defect

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Susannah L. Hewitt

2017-10-01

Full Text Available Many DNA lesions associated with lymphoid malignancies are linked to off-target cleavage by the RAG1/2 recombinase. However, off-target cleavage has mostly been analyzed in the context of DNA repair defects, confounding any mechanistic understanding of cleavage deregulation. We identified a conserved SQ phosphorylation site on RAG2 365 to 366 that is involved in feedback control of RAG cleavage. Mutation of serine 365 to a non-phosphorylatable alanine permits bi-allelic and bi-locus RAG-mediated breaks in the same cell, leading to reciprocal translocations. This phenomenon is analogous to the phenotype we described for ATM kinase inactivation. Here, we establish deregulated cleavage itself as a driver of chromosomal instability without the associated repair defect. Intriguingly, a RAG2-S365E phosphomimetic rescues the deregulated cleavage of ATM inactivation, reducing the incidence of reciprocal translocations. These data support a model in which feedback control of cleavage and maintenance of genome stability involves ATM-mediated phosphorylation of RAG2.

Coupling fibroblast growth factor 23 production and cleavage: iron deficiency, rickets, and kidney disease.

Science.gov (United States)

Wolf, Myles; White, Kenneth E

2014-07-01

High levels of fibroblast growth factor 23 (FGF23) cause the rare disorders of hypophosphatemic rickets and are a risk factor for cardiovascular disease and death in patients with chronic kidney disease (CKD). Despite major advances in understanding FGF23 biology, fundamental aspects of FGF23 regulation in health and in CKD remain mostly unknown. Autosomal dominant hypophosphatemic rickets (ADHR) is caused by gain-of-function mutations in FGF23 that prevent its proteolytic cleavage, but affected individuals experience a waxing and waning course of phosphate wasting. This led to the discovery that iron deficiency is an environmental trigger that stimulates FGF23 expression and hypophosphatemia in ADHR. Unlike osteocytes in ADHR, normal osteocytes couple increased FGF23 production with commensurately increased FGF23 cleavage to ensure that normal phosphate homeostasis is maintained in the event of iron deficiency. Simultaneous measurement of FGF23 by intact and C-terminal assays supported these breakthroughs by providing minimally invasive insight into FGF23 production and cleavage in bone. These findings also suggest a novel mechanism of FGF23 elevation in patients with CKD, who are often iron deficient and demonstrate increased FGF23 production and decreased FGF23 cleavage, consistent with an acquired state that mimics the molecular pathophysiology of ADHR. Iron deficiency stimulates FGF23 production, but normal osteocytes couple increased FGF23 production with increased cleavage to maintain normal circulating levels of biologically active hormone. These findings uncover a second level of FGF23 regulation within osteocytes, failure of which culminates in elevated levels of biologically active FGF23 in ADHR and perhaps CKD.

Developing a capillary electrophoresis based method for dynamically monitoring enzyme cleavage activity using quantum dots-peptide assembly.

Science.gov (United States)

Wang, Jianhao; Fan, Jie; Liu, Li; Ding, Shumin; Liu, Xiaoqian; Wang, Jianpeng; Gao, Liqian; Chattopadhaya, Souvik; Miao, Peng; Xia, Jiang; Qiu, Lin; Jiang, Pengju

2017-10-01

Herein, a novel assay has been developed for monitoring PreScission protease (His-PSP) mediated enzyme cleavage of ATTO 590 labeled peptide substrate (ATTO-LEV). This novel method is based on combining the use of capillary electrophoresis and fluorescence detection (CE-FL) to dynamically monitor the enzyme cleavage activity. A multivalent peptide substrate was first constructed by immobilizing His-tagged ATTO 590 labeled peptide substrate (ATTO-LEVH6) onto the surface of CdSe/ZnS quantum dots (QDs). Once successfully immobilized, the novel multivalent peptide substrate resulted in the Förster resonance energy transfer (FRET) from QDs to ATTO 590. The ATTO-LEVH6-QD assembly was then incubated with His-PSP to study the proteolytic cleavage of surface bound ATTO-LEVH6 by CE-FL. Our data suggests that PreScission-mediated proteolytic cleavage is enzyme concentration- and incubation time-dependent. By combining capillary electrophoresis, QDs and FRET, our study herein not only provides a new method for the detection and dynamically monitoring of PSP enzyme cleavage activity, but also can be extended to the detection of many other enzymes and proteases. © 2017 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Metabolic Engineering to Develop a Pathway for the Selective Cleavage of Carbon-Nitrogen Bonds

Energy Technology Data Exchange (ETDEWEB)

John J. Kilbane II

2005-10-01

The objective of the project is to develop a biochemical pathway for the selective cleavage of C-N bonds in molecules found in petroleum. Specifically a novel biochemical pathway will be developed for the selective cleavage of C-N bonds in carbazole. The cleavage of the first C-N bond in carbazole is accomplished by the enzyme carbazole dioxygenase, that catalyzes the conversion of carbazole to 2-aminobiphenyl-2,3-diol. The genes encoding carbazole dioxygenase were cloned from Sphingomonas sp. GTIN11 and from Pseudomonas resinovorans CA10. The selective cleavage of the second C-N bond has been challenging, and efforts to overcome that challenge have been the focus of recent research in this project. Enrichment culture experiments succeeded in isolating bacterial cultures that can metabolize 2-aminobiphenyl, but no enzyme capable of selectively cleaving the C-N bond in 2-aminobiphenyl has been identified. Aniline is very similar to the structure of 2-aminobiphenyl and aniline dioxygenase catalyzes the conversion of aniline to catechol and ammonia. For the remainder of the project the emphasis of research will be to simultaneously express the genes for carbazole dioxygenase and for aniline dioxygenase in the same bacterial host and then to select for derivative cultures capable of using carbazole as the sole source of nitrogen.

Influence of the austenitizing temperature in the cleavage facet size of Niocor 2

International Nuclear Information System (INIS)

Darwish, F.A.I.; Teixeira, J.C.G.; Fernandes, R.A.; Juer, S.

1983-01-01

Convetional Charpy specimens of Niocor 2 steel cooled in air from various austenitizing temperatures were fractured at -196 0 C so as to insure failure by cleavage. The cleavage facet size distribution was determined and then correlated with the grain size and other aspects of the microstructure. The results that the average facet size can be increased through a coarsening of the microstructure. For the case where the γ→α transformation products are predominantely acicular, the facet size is shown to depend on substructural aspects primarily the lath packet size. (Author) [pt

[Cleavage of DNA fragments induced by UV nanosecond laser excitation at 193 nm].

Science.gov (United States)

Vtiurina, N N; Grokhovskiĭ, S L; Filimonov, I V; Medvedkov, O I; Nechipurenko, D Iu; Vasil'ev, S A; Nechipurenko, Iu D

2011-01-01

The cleavage of dsDNA fragments in aqueous solution after irradiation with UV laser pulses at 193 nm has been studied. Samples were investigated using polyacrylamide gel electrophoresis. The intensity of damage of particular phosphodiester bond after hot alkali treatment was shown to depend on the base pair sequence. It was established that the probability of cleavage is twice higher for sites of DNA containing two or more successively running guanine residues. A possible mechanism of damage to the DNA molecule connected with the migration of holes along the helix is discussed.

Affect of Presenilin Mutations on APP Cleavage; Insights into the Pathogenesis of FAD

Directory of Open Access Journals (Sweden)

Nuomin eLi

2016-03-01

Full Text Available Alzheimer disease (AD is characterized by progressive memory loss, reduction in cognitive functions, and damage to the brain. The β-amyloid precursor protein can be sequentially cleaved by β- secretase and γ-secretase. Mutations in the presenilin1（PS1） are the most common cause of Familial Alzheimer’s disease ( FAD. PS1 mutations can alter the activity of γ-secretase on the cleavage of the β-amyloid precursor protein, causing increased Aβ production. Previous studies show that the βAPP-C-terminal fragment is first cleaved by β-scretase, primarily generating long fragments of Aβ48 and Aβ49, followed by the stepwise cleavage of every three amino acid residues at the C terminus, resulting in Aβ48-, 45-, 42 line and Aβ49-, 46-, 43-, 40 line. Here, we used LC-MS/MS to analyze unique peptides IAT, VVIA, ITL，TVI，IVI through sequential cleavage, combined with ELISA to test the level of Aβ42 and Aβ40 for validation. The results show that most FAD mutant PS1 can alter the level of Aβ42 and Aβ40 monitored by the Aβ42/Aβ40 ratio. Among them, 6 mutants (I143T, H163P, S170F, Q223R, M233V and G384A affect the Aβ42/40 ratio through both Aβ49-40 and Aβ48-38 lines; L166P through decreasing the Aβ49-40 line, 6 mutants (I143V, M146V, G217A, E280A, L381V and L392V through increasing the Aβ48-42 line. More importantly, we found some mutations can affect the γ-secretase cleavage preference of α-CTF and β-CTF. In conclusion, we found that the FAD PS1 mutations mainly increase the generation of Aβ42 by decreasing the cleavage of Aβ42-Aβ38 and Aβ43-Aβ40.

Elucidation of reaction mechanism for m -cresol hydrodeoxygenation over Fe based catalysts: A kinetic study

Energy Technology Data Exchange (ETDEWEB)

Hong, Yongchun; Wang, Yong

2017-09-01

Fe based catalysts are promising for hydrodeoxygenation (HDO) of lignin derived phenolics due to their high selectivity for aromatics. In this work, the reaction mechanism of m-cresol HDO on Fe catalysts and the kinetic consequence with Pd addition were elucidated by examining the effect of H2, H2O and m-cresol pressures on toluene formation rate on Fe and PdFe catalysts. A direct CO bond cleavage mechanism is proposed for HDO catalysis on both Fe and PdFe catalysts, while Pd provides a facilitated reaction pathway at the PdFe interface and therefore promotes the catalysis on Fe without changing the high selectivity towards aromatics.

High-resolution characterization of sequence signatures due to non-random cleavage of cell-free DNA.

Science.gov (United States)

Chandrananda, Dineika; Thorne, Natalie P; Bahlo, Melanie

2015-06-17

High-throughput sequencing of cell-free DNA fragments found in human plasma has been used to non-invasively detect fetal aneuploidy, monitor organ transplants and investigate tumor DNA. However, many biological properties of this extracellular genetic material remain unknown. Research that further characterizes circulating DNA could substantially increase its diagnostic value by allowing the application of more sophisticated bioinformatics tools that lead to an improved signal to noise ratio in the sequencing data. In this study, we investigate various features of cell-free DNA in plasma using deep-sequencing data from two pregnant women (>70X, >50X) and compare them with matched cellular DNA. We utilize a descriptive approach to examine how the biological cleavage of cell-free DNA affects different sequence signatures such as fragment lengths, sequence motifs at fragment ends and the distribution of cleavage sites along the genome. We show that the size distributions of these cell-free DNA molecules are dependent on their autosomal and mitochondrial origin as well as the genomic location within chromosomes. DNA mapping to particular microsatellites and alpha repeat elements display unique size signatures. We show how cell-free fragments occur in clusters along the genome, localizing to nucleosomal arrays and are preferentially cleaved at linker regions by correlating the mapping locations of these fragments with ENCODE annotation of chromatin organization. Our work further demonstrates that cell-free autosomal DNA cleavage is sequence dependent. The region spanning up to 10 positions on either side of the DNA cleavage site show a consistent pattern of preference for specific nucleotides. This sequence motif is present in cleavage sites localized to nucleosomal cores and linker regions but is absent in nucleosome-free mitochondrial DNA. These background signals in cell-free DNA sequencing data stem from the non-random biological cleavage of these fragments. This

Embryo apoptosis identification: Oocyte grade or cleavage stage?

Science.gov (United States)

Bakri, Noraina Mohd; Ibrahim, Siti Fatimah; Osman, Nurul Atikah; Hasan, Nurhaslina; Jaffar, Farah Hanan Fathihah; Rahman, Zulaiha Abdul; Osman, Khairul

2015-01-01

Apoptosis is a programed cell death that is vital for tissue homeostasis. However, embryo apoptosis had been known to be related to embryo fragmentation which should be avoided in in vitro fertilization (IVF). The purpose of this study was to evaluate the relationship of embryo apoptosis with the grade of immature oocytes and cleavage stage of in vitro produced (IVP) cattle embryos. This study consisted of 345 oocytes collected through ovary slicing. Immature oocytes were graded as A, B and C. This grading was based on cumulus cell thickness and compactness. All oocytes then underwent an in vitro maturation (IVM) procedure. An IVF was done 24 h after IVM culture. Prior to staining, stage of cleaved embryos was determined and classified as either 2, 4, 8 or >8-cell embryo stage. Apoptosis status of cleaved IVP embryos was determined by using annexin V-FITC staining technique at 48 and 72 h post insemination (hpi). Apoptosis status for each embryo was classified as either early or late. The result showed that there was no significant difference (p > 0.05) of apoptosis status among grade A, B and C embryos. All grades of oocytes showed embryo apoptosis where 1.5% late apoptosis for grade A, 4.5% and 10.4% of early and late apoptosis for grade B and grade C. Early apoptosis was not seen in grade A embryo. We also noted no significant difference (p > 0.05) of apoptosis status between 2, 4, 8 and >8-cell embryo stage. Early apoptosis was also not seen in >8-cell stage. Even though there were no differences in apoptosis expression between the three classes, the cleavage rate of grade A oocytes was significantly higher (p < 0.01) than grade B and grade C. In conclusion, the apoptosis expression in the embryo can occur regardless of the oocyte quality and the cleavage stage of the embryo produced. PMID:26858565

AcEST: BP919742 [AcEST

Lifescience Database Archive (English)

Full Text Available VVT Structural polyprotein OS=Venezuelan equine... 30 7.6 sp|P36332|POLS_EEVVP St...ructural polyprotein OS=Venezuelan equine... 30 7.6 sp|P36331|POLS_EEVVM Structural polyprotein OS=Venezuelan equine...... 30 7.6 sp|P36330|POLS_EEVVE Structural polyprotein OS=Venezuelan equine... 30 7.6 sp|P05674|POLS..._EEVV8 Structural polyprotein OS=Venezuelan equine... 30 7.6 sp|P36329|POLS_EEVV3 Structural polyprotein OS=Venezuelan equine...09592|POLS_EEVVT Structural polyprotein OS=Venezuelan equine encephalitis virus (

Branched-chain amino acids reduce hepatic iron accumulation and oxidative stress in hepatitis C virus polyprotein-expressing mice

Science.gov (United States)

Korenaga, Masaaki; Nishina, Sohji; Korenaga, Keiko; Tomiyama, Yasuyuki; Yoshioka, Naoko; Hara, Yuichi; Sasaki, Yusuke; Shimonaka, Yasushi; Hino, Keisuke

2015-01-01

Background & Aims Branched-chain amino acids (BCAA) reduce the incidence of hepatocellular carcinoma (HCC) in patients with cirrhosis. However, the mechanisms that underlie these effects remain unknown. Previously, we reported that oxidative stress in male transgenic mice that expressed hepatitis C virus polyprotein (HCVTgM) caused hepatic iron accumulation by reducing hepcidin transcription, thereby leading to HCC development. This study investigated whether long-term treatment with BCAA reduced hepatic iron accumulation and oxidative stress in iron-overloaded HCVTgM and in patients with HCV-related advanced fibrosis. Methods Male HCVTgM were fed an excess-iron diet that comprised either casein or 3.0% BCAA, or a control diet, for 6 months. Results For HCVTgM, BCAA supplementation increased the serum hepcidin-25 levels and antioxidant status [ratio of biological antioxidant potential (BAP) relative to derivatives of reactive oxygen metabolites (dROM)], decreased the hepatic iron contents, attenuated reactive oxygen species generation, and restored mitochondrial superoxide dismutase expression and mitochondrial complex I activity in the liver compared with mice fed the control diet. After 48 weeks of BCAA supplementation in patients with HCV-related advanced fibrosis, BAP/dROM and serum hepcidin-25 increased and serum ferritin decreased compared with the pretreatment levels. Conclusions BCAA supplementation reduced oxidative stress by restoring mitochondrial function and improved iron metabolism by increasing hepcidin-25 in both iron-overloaded HCVTgM and patients with HCV-related advanced fibrosis. These activities of BCAA may partially account for their inhibitory effects on HCC development in cirrhosis patients. PMID:25156780

Dual CRISPR-Cas9 Cleavage Mediated Gene Excision and Targeted Integration in Yarrowia lipolytica.

Science.gov (United States)

Gao, Difeng; Smith, Spencer; Spagnuolo, Michael; Rodriguez, Gabriel; Blenner, Mark

2018-05-29

CRISPR-Cas9 technology has been successfully applied in Yarrowia lipolytica for targeted genomic editing including gene disruption and integration; however, disruptions by existing methods typically result from small frameshift mutations caused by indels within the coding region, which usually resulted in unnatural protein. In this study, a dual cleavage strategy directed by paired sgRNAs is developed for gene knockout. This method allows fast and robust gene excision, demonstrated on six genes of interest. The targeted regions for excision vary in length from 0.3 kb up to 3.5 kb and contain both non-coding and coding regions. The majority of the gene excisions are repaired by perfect nonhomologous end-joining without indel. Based on this dual cleavage system, two targeted markerless integration methods are developed by providing repair templates. While both strategies are effective, homology mediated end joining (HMEJ) based method are twice as efficient as homology recombination (HR) based method. In both cases, dual cleavage leads to similar or improved gene integration efficiencies compared to gene excision without integration. This dual cleavage strategy will be useful for not only generating more predictable and robust gene knockout, but also for efficient targeted markerless integration, and simultaneous knockout and integration in Y. lipolytica. © 2018 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Porcine deltacoronavirus nsp5 inhibits interferon-β production through the cleavage of NEMO.

Science.gov (United States)

Zhu, Xinyu; Fang, Liurong; Wang, Dang; Yang, Yuting; Chen, Jiyao; Ye, Xu; Foda, Mohamed Frahat; Xiao, Shaobo

2017-02-01

Porcine deltacoronavirus (PDCoV) causes acute enteric disease and mortality in seronegative neonatal piglets. Previously we have demonstrated that PDCoV infection suppresses the production of interferon-beta (IFN-β), while the detailed mechanisms are poorly understood. Here, we demonstrate that nonstructural protein 5 (nsp5) of PDCoV, the 3C-like protease, significantly inhibits Sendai virus (SEV)-induced IFN-β production by targeting the NF-κB essential modulator (NEMO), confirmed by the diminished function of NEMO cleaved by PDCoV. The PDCoV nsp5 cleavage site in the NEMO protein was identified as glutamine 231, and was identical to the porcine epidemic diarrhea virus nsp5 cleavage site, revealing the likelihood of a common target in NEMO for coronaviruses. Furthermore, this cleavage impaired the ability of NEMO to activate the IFN response and downstream signaling. Taken together, our findings reveal PDCoV nsp5 to be a newly identified IFN antagonist and enhance the understanding of immune evasion by deltacoronaviruses. Copyright © 2016 Elsevier Inc. All rights reserved.

Cas9-catalyzed DNA Cleavage Generates Staggered Ends: Evidence from Molecular Dynamics Simulations

Science.gov (United States)

Zuo, Zhicheng; Liu, Jin

2016-11-01

The CRISPR-associated endonuclease Cas9 from Streptococcus pyogenes (spCas9) along with a single guide RNA (sgRNA) has emerged as a versatile toolbox for genome editing. Despite recent advances in the mechanism studies on spCas9-sgRNA-mediated double-stranded DNA (dsDNA) recognition and cleavage, it is still unclear how the catalytic Mg2+ ions induce the conformation changes toward the catalytic active state. It also remains controversial whether Cas9 generates blunt-ended or staggered-ended breaks with overhangs in the DNA. To investigate these issues, here we performed the first all-atom molecular dynamics simulations of the spCas9-sgRNA-dsDNA system with and without Mg2+ bound. The simulation results showed that binding of two Mg2+ ions at the RuvC domain active site could lead to structurally and energetically favorable coordination ready for the non-target DNA strand cleavage. Importantly, we demonstrated with our simulations that Cas9-catalyzed DNA cleavage produces 1-bp staggered ends rather than generally assumed blunt ends.

Hydrodenitrogenation mechanism of aromatic amines. Kinetic study and simulation

International Nuclear Information System (INIS)

D'Araujo, P.A.P.

1994-06-01

The decomposition of model molecules reacting alone or in competition was studied in a fixed bed reactor at 623 K and 7 MPa over a sulfided NiMo/Al 2 O 3 catalyst. The inhibiting effect of H 2 S and some nitrogen molecules, namely quinoline type compounds plays a major role in the transformation of anilines intermediates. On the other hand H 2 S acts as a cocatalyst and promote carbon-nitrogen bond cleavage, specially at low H 2 S partial pressure. When the H 2 S partial pressure is greater than the nitrogen compound partial pressure an inhibiting effect of H 2 S occurs and its promoting effect on carbon-nitrogen bond cleavage is cancelled. Hydrogen has a positive but moderate effect in hydrogenation steps. The mechanism of carbon-nitrogen bond scission depends on the structure of the nitrogen molecule namely on the hybridization of the carbon atom bearing the nitrogen atom. If the carbon a with respect to the nitrogen is monosubstituted the mechanism is essentially a nucleophilic substitution. When the degree of substitution increases the elimination mechanism becomes more important and the two mechanisms are in competition. With a sulfided catalyst, H 2 S from the gas phase doesn't change the importance of each mechanism, it just increases the rate of the reaction. In the presence of an oxide catalyst the contribution of the two mechanisms change. This result shows the importance of the sulphur species from the surface. Using isotopic exchange we could demonstrate that the sites able to dissociate H 2 S and H 2 are the same, and that the dissociation is of heterolytic nature. The kinetic modeling of hydrotreatment reactions using the CHEMKIN/SURFACE CHEMKIN package seems to be a convenient method in order to understand the kinetic and mechanistic phenomena in hydrodenitrogenation. The preliminary simulations in the case of 2.6 diethylaniline showed that only one type of site is not sufficient in order to account for the experimental results. Further simulations

Predictors of hepatitis B cure using gene therapy to deliver DNA cleavage enzymes: a mathematical modeling approach.

Directory of Open Access Journals (Sweden)

Joshua T Schiffer

Full Text Available Most chronic viral infections are managed with small molecule therapies that inhibit replication but are not curative because non-replicating viral forms can persist despite decades of suppressive treatment. There are therefore numerous strategies in development to eradicate all non-replicating viruses from the body. We are currently engineering DNA cleavage enzymes that specifically target hepatitis B virus covalently closed circular DNA (HBV cccDNA, the episomal form of the virus that persists despite potent antiviral therapies. DNA cleavage enzymes, including homing endonucleases or meganucleases, zinc-finger nucleases (ZFNs, TAL effector nucleases (TALENs, and CRISPR-associated system 9 (Cas9 proteins, can disrupt specific regions of viral DNA. Because DNA repair is error prone, the virus can be neutralized after repeated cleavage events when a target sequence becomes mutated. DNA cleavage enzymes will be delivered as genes within viral vectors that enter hepatocytes. Here we develop mathematical models that describe the delivery and intracellular activity of DNA cleavage enzymes. Model simulations predict that high vector to target cell ratio, limited removal of delivery vectors by humoral immunity, and avid binding between enzyme and its DNA target will promote the highest level of cccDNA disruption. Development of de novo resistance to cleavage enzymes may occur if DNA cleavage and error prone repair does not render the viral episome replication incompetent: our model predicts that concurrent delivery of multiple enzymes which target different vital cccDNA regions, or sequential delivery of different enzymes, are both potentially useful strategies for avoiding multi-enzyme resistance. The underlying dynamics of cccDNA persistence are unlikely to impact the probability of cure provided that antiviral therapy is given concurrently during eradication trials. We conclude by describing experiments that can be used to validate the model, which

Active site mutations change the cleavage specificity of neprilysin.

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Travis Sexton

Full Text Available Neprilysin (NEP, a member of the M13 subgroup of the zinc-dependent endopeptidase family is a membrane bound peptidase capable of cleaving a variety of physiological peptides. We have generated a series of neprilysin variants containing mutations at either one of two active site residues, Phe(563 and Ser(546. Among the mutants studied in detail we observed changes in their activity towards leucine(5-enkephalin, insulin B chain, and amyloid β(1-40. For example, NEP(F563I displayed an increase in preference towards cleaving leucine(5-enkephalin relative to insulin B chain, while mutant NEP(S546E was less discriminating than neprilysin. Mutants NEP(F563L and NEP(S546E exhibit different cleavage site preferences than neprilysin with insulin B chain and amyloid ß(1-40 as substrates. These data indicate that it is possible to alter the cleavage site specificity of neprilysin opening the way for the development of substrate specific or substrate exclusive forms of the enzyme with enhanced therapeutic potential.

Cleavage of thymine N3-H bonds by low-energy electrons attached to base π* orbitals

International Nuclear Information System (INIS)

Theodore, Magali; Sobczyk, Monika; Simons, Jack

2006-01-01

In this work, we extend our earlier studies on single strand break (SSB) formation in DNA to consider the possibility of cleaving a thymine N 3 -H bond to generate a nitrogen-centered anion and a hydrogen radical which might proceed to induce further bond cleavages. In earlier studies, we considered SSBs induced by low-energy electrons that attach to DNA bases' π* orbitals or to phosphate P=O π* orbitals to cleave sugar-phosphate C-O bonds or base-sugar N 1 -C bonds. We also studied the effects of base π-stacking on the rates of such bond cleavages. To date, our results suggest that sugar-phosphate C-O bonds have the lowest barriers to cleavage, that attachment of electrons with energies below 2 eV most likely occurs at the base π* orbitals, that electrons with energy above 2 eV can also attach to phosphate P=O π* orbitals, and that base π stacking has a modest but slowing effect on the rates of SSB formation. However, we had not yet examined the possibility that base N 3 -H bonds could rupture subsequent to base π* orbital capture. In the present work, the latter possibility is considered and it is found that the barrier to cleavage of the N 3 -H bond in thymine is considerably higher than for cleaving sugar-phosphate C-O bonds, so our prediction that SSB formation is dominated by C-O bond cleavage remains intact

Effects of Olive Metabolites on DNA Cleavage Mediated by Human Type II Topoisomerases

Science.gov (United States)

2016-01-01

Several naturally occurring dietary polyphenols with chemopreventive or anticancer properties are topoisomerase II poisons. To identify additional phytochemicals that enhance topoisomerase II-mediated DNA cleavage, a library of 341 Mediterranean plant extracts was screened for activity against human topoisomerase IIα. An extract from Phillyrea latifolia L., a member of the olive tree family, displayed high activity against the human enzyme. On the basis of previous metabolomics studies, we identified several polyphenols (hydroxytyrosol, oleuropein, verbascoside, tyrosol, and caffeic acid) as potential candidates for topoisomerase II poisons. Of these, hydroxytyrosol, oleuropein, and verbascoside enhanced topoisomerase II-mediated DNA cleavage. The potency of these olive metabolites increased 10–100-fold in the presence of an oxidant. Hydroxytyrosol, oleuropein, and verbascoside displayed hallmark characteristics of covalent topoisomerase II poisons. (1) The activity of the metabolites was abrogated by a reducing agent. (2) Compounds inhibited topoisomerase II activity when they were incubated with the enzyme prior to the addition of DNA. (3) Compounds were unable to poison a topoisomerase IIα construct that lacked the N-terminal domain. Because hydroxytyrosol, oleuropein, and verbascoside are broadly distributed across the olive family, extracts from the leaves, bark, and fruit of 11 olive tree species were tested for activity against human topoisomerase IIα. Several of the extracts enhanced enzyme-mediated DNA cleavage. Finally, a commercial olive leaf supplement and extra virgin olive oils pressed from a variety of Olea europea subspecies enhanced DNA cleavage mediated by topoisomerase IIα. Thus, olive metabolites appear to act as topoisomerase II poisons in complex formulations intended for human dietary consumption. PMID:26132160

Maximizing Selective Cleavages at Aspartic Acid and Proline Residues for the Identification of Intact Proteins

Science.gov (United States)

Foreman, David J.; Dziekonski, Eric T.; McLuckey, Scott A.

2018-04-01

A new approach for the identification of intact proteins has been developed that relies on the generation of relatively few abundant products from specific cleavage sites. This strategy is intended to complement standard approaches that seek to generate many fragments relatively non-selectively. Specifically, this strategy seeks to maximize selective cleavage at aspartic acid and proline residues via collisional activation of precursor ions formed via electrospray ionization (ESI) under denaturing conditions. A statistical analysis of the SWISS-PROT database was used to predict the number of arginine residues for a given intact protein mass and predict a m/z range where the protein carries a similar charge to the number of arginine residues thereby enhancing cleavage at aspartic acid residues by limiting proton mobility. Cleavage at aspartic acid residues is predicted to be most favorable in the m/z range of 1500-2500, a range higher than that normally generated by ESI at low pH. Gas-phase proton transfer ion/ion reactions are therefore used for precursor ion concentration from relatively high charge states followed by ion isolation and subsequent generation of precursor ions within the optimal m/z range via a second proton transfer reaction step. It is shown that the majority of product ion abundance is concentrated into cleavages C-terminal to aspartic acid residues and N-terminal to proline residues for ions generated by this process. Implementation of a scoring system that weights both ion fragment type and ion fragment area demonstrated identification of standard proteins, ranging in mass from 8.5 to 29.0 kDa. [Figure not available: see fulltext.

The Generation of Dehydroalanine Residues in Protonated Polypeptides: Ion/Ion Reactions for Introducing Selective Cleavages

Science.gov (United States)

Peng, Zhou; Bu, Jiexun; McLuckey, Scott A.

2017-09-01

We examine a gas-phase approach for converting a subset of amino acid residues in polypeptide cations to dehydroalanine (Dha). Subsequent activation of the modified polypeptide ions gives rise to specific cleavage N-terminal to the Dha residue. This process allows for the incorporation of selective cleavages in the structural characterization of polypeptide ions. An ion/ion reaction within the mass spectrometer between a multiply protonated polypeptide and the sulfate radical anion introduces a radical site into the multiply protonated polypeptide reactant. Subsequent collisional activation of the polypeptide radical cation gives rise to radical side chain loss from one of several particular amino acid side chains (e.g., leucine, asparagine, lysine, glutamine, and glutamic acid) to yield a Dha residue. The Dha residues facilitate preferential backbone cleavages to produce signature c- and z-ions, demonstrated with cations derived from melittin, mechano growth factor (MGF), and ubiquitin. The efficiencies for radical side chain loss and for subsequent generation of specific c- and z-ions have been examined as functions of precursor ion charge state and activation conditions using cations of ubiquitin as a model for a small protein. It is noted that these efficiencies are not strongly dependent on ion trap collisional activation conditions but are sensitive to precursor ion charge state. Moderate to low charge states show the greatest overall yields for the specific Dha cleavages, whereas small molecule losses (e.g., water/ammonia) dominate at the lowest charge states and proton catalyzed amide bond cleavages that give rise to b- and y-ions tend to dominate at high charge states. [Figure not available: see fulltext.

ATP-Dependent C–F Bond Cleavage Allows the Complete Degradation of 4-Fluoroaromatics without Oxygen

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Oliver Tiedt

2016-08-01

Full Text Available Complete biodegradation of the abundant and persistent fluoroaromatics requires enzymatic cleavage of an arylic C–F bond, probably the most stable single bond of a biodegradable organic molecule. While in aerobic microorganisms defluorination of fluoroaromatics is initiated by oxygenases, arylic C–F bond cleavage has never been observed in the absence of oxygen. Here, an oxygen-independent enzymatic aryl fluoride bond cleavage is described during the complete degradation of 4-fluorobenzoate or 4-fluorotoluene to CO2 and HF in the denitrifying Thauera aromatica: the ATP-dependent defluorination of 4-fluorobenzoyl-coenzyme A (4-F-BzCoA to benzoyl-coenzyme A (BzCoA and HF, catalyzed by class I BzCoA reductase (BCR. Adaptation to growth with the fluoroaromatics was accomplished by the downregulation of a promiscuous benzoate-CoA ligase and the concomitant upregulation of 4-F-BzCoA-defluorinating/dearomatizing BCR on the transcriptional level. We propose an unprecedented mechanism for reductive arylic C–F bond cleavage via a Birch reduction-like mechanism resulting in a formal nucleophilic aromatic substitution. In the proposed anionic 4-fluorodienoyl-CoA transition state, fluoride elimination to BzCoA is favored over protonation to a fluorinated cyclic dienoyl-CoA.

On the distinction of the mechanisms of DNA cleavage by restriction enzymes—The I-, II-, and III-type molecular motors

Science.gov (United States)

Pikin, S. A.

2008-09-01

A comparative physical description is given for the functioning of various restriction enzymes and for their processes of DNA cleavage. The previously proposed model system of kinetic equations is applied to the I-and III-type enzymes, which use ATP molecules as an energy source, while the II-type enzymes work thanks to catalytic reactions with participation of an electric field. All the enzymes achieved bending and twisting DNA, providing for either the linear motion of the II-type enzyme along the DNA chain or the DNA translocation by the I-and III-type enzymes due to moving chiral kinks. A comparative estimation of the considered linear and angular velocities is performed. The role of stalling forces for enzyme-DNA complexes, which induce the observed cutting of the DNA either inside the enzyme (II) or in some “weak” places outside enzymes I and III, which results in the supercoiling of the DNA, is shown. The role of ionic screening for the described processes is discussed.

Mechanisms for lymphocytic choriomeningitis virus glycoprotein cleavage, transport, and incorporation into virions

International Nuclear Information System (INIS)

Kunz, Stefan; Edelmann, Kurt H.; Torre, Juan-Carlos de la; Gorney, Robert; Oldstone, Michael B.A.

2003-01-01

The glycoprotein (GP) of lymphocytic choriomeningitis virus (LCMV) serves as virus attachment protein to its receptor on host cells and is a key determinant for cell tropism, pathogenesis, and epidemiology of the virus. The GP of LCMV is posttranslationally cleaved by the subtilase SKI-1/S1P into two subunits, the peripheral GP1, which is implicated in receptor binding, and the transmembrane GP2 that is structurally similar to the fusion active membrane proximal portions of the glycoproteins of other enveloped viruses. The present study shows that cleavage by SKI-1/S1P is not required for cell surface expression of LCMVGP on infected cells but is essential for its incorporation into virions and for the production of infectious virus particles. In absence of SKI-1/S1P cleavage, cell-to-cell propagation of the virus was markedly reduced. Further, proteolytic processing of LCMVGP depends on the presence of a cluster of basic amino acids at the C-terminus of the cytoplasmic domain of GP2, a structural motif that is conserved in Old World arenaviruses. The effect of the truncation of the cytoplasmic tail on cleavage suggests a structural interdependence between the cytoplasmic domain and the ectodomains of LCMVGP

Sequence specific inhibition of DNA restriction enzyme cleavage by PNA

DEFF Research Database (Denmark)

Nielsen, P.E.; Egholm, M.; Berg, R.H.

1993-01-01

Plasmids containing double-stranded 10-mer PNA (peptide nucleic acid chimera) targets proximally flanked by two restriction enzyme sites were challenged with the complementary PNA or PNAs having one or two mismatches, and the effect on the restriction enzyme cleavage of the flanking sites was ass...

Cleavage of desmin by cysteine proteases: Calpains and cathepsin B

DEFF Research Database (Denmark)

Baron, Caroline; Jacobsen, S.; Purslow, P.P.

2004-01-01

The intermediate filament protein, desmin, was purified from pork longissimus dorsi and incubated with either P-calpain, m-calpain or cathepsin B. Proteolysis of desmin was followed using SDS-PAGE and Western blotting. After incubation of desmin with the proteases, cleavage sites on the desmin mo...

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

Science.gov (United States)

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

2005-11-01

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

Reaction kinetics of resveratrol with thiyl and alkoxyl radicals

International Nuclear Information System (INIS)

Dzeba, I.; Mihaljevic, B.

2011-01-01

Complete text of publication follows. Plant derived resveratrol (trans-3,5,4'-trihydroxystilbene) possesses a broad spectrum of biological activities, one of them are very well known its antioxidative properties. Our work aims to provide kinetic data with regard to the reactivity of resveratrol with uninvestigated short-lived bioradicals, identified as mediators in oxidative lipid degradation processes. Radicals of our interest are alkoxyl radicals, well known propagators of the chain free radical reactions in lipids, and thiyl radicals which protect lipids from their degradation pathway, but at the same time cause the isomerization of the double bonds. In order to investigate these reactions of resveratrol laser flash photolysis was used. On the basis of competitive kinetics the rate constants were determined under pseudo-first order conditions in acetonitrile solutions at room temperature. Thiyl radicals were generated indirectly in solution containing 1-octadecanthiol and photosensitive benzophenone in acetonitrile using the light pulses at 347 nm from ruby laser. Tert-butoxyl radicals were generated directly by peroxide bond cleavage from di-tert-butyl peroxide in acetonitrile by light pulses of Nd:YAG at 355 nm, and ruby at 347 nm. Obtained rate constants for the reactions of resveratrol and radicals generated by laser flash photolysis will be summarized and compared with rare literature data for the rate constants of investigated reactions of resveratrol and other radicals generated by pulse radiolysis.

Combined TGA-MS kinetic analysis of multistep processes. Thermal decomposition and ceramification of polysilazane and polysiloxane preceramic polymers.

Science.gov (United States)

García-Garrido, C; Sánchez-Jiménez, P E; Pérez-Maqueda, L A; Perejón, A; Criado, José M

2016-10-26

The polymer-to-ceramic transformation kinetics of two widely employed ceramic precursors, 1,3,5,7-tetramethyl-1,3,5,7-tetravinylcyclotetrasiloxane (TTCS) and polyureamethylvinylsilazane (CERASET), have been investigated using coupled thermogravimetry and mass spectrometry (TG-MS), Raman, XRD and FTIR. The thermally induced decomposition of the pre-ceramic polymer is the critical step in the synthesis of polymer derived ceramics (PDCs) and accurate kinetic modeling is key to attaining a complete understanding of the underlying process and to attempt any behavior predictions. However, obtaining a precise kinetic description of processes of such complexity, consisting of several largely overlapping physico-chemical processes comprising the cleavage of the starting polymeric network and the release of organic moieties, is extremely difficult. Here, by using the evolved gases detected by MS as a guide it has been possible to determine the number of steps that compose the overall process, which was subsequently resolved using a semiempirical deconvolution method based on the Frasier-Suzuki function. Such a function is more appropriate that the more usual Gaussian or Lorentzian functions since it takes into account the intrinsic asymmetry of kinetic curves. Then, the kinetic parameters of each constituent step were independently determined using both model-free and model-fitting procedures, and it was found that the processes obey mostly diffusion models which can be attributed to the diffusion of the released gases through the solid matrix. The validity of the obtained kinetic parameters was tested not only by the successful reconstruction of the original experimental curves, but also by predicting the kinetic curves of the overall processes yielded by different thermal schedules and by a mixed TTCS-CERASET precursor.

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

Science.gov (United States)

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.

Can laccases catalyze bond cleavage in lignin?

DEFF Research Database (Denmark)

Munk, Line; Sitarz, Anna Katarzyna; Kalyani, Dayanand

2015-01-01

illustrations of the putative laccase catalyzed reactions, including the possible reactions of the reactive radical intermediates taking place after the initial oxidation of the phenol-hydroxyl groups, we show that i) Laccase activity is able to catalyze bond cleavage in low molecular weight phenolic lignin......-substituted phenols, benzenethiols, polyphenols, and polyamines, which may be oxidized. In addition, the currently available analytical methods that can be used to detect enzyme catalyzed changes in lignin are summarized, and an improved nomenclature for unequivocal interpretation of the action of laccases on lignin...

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

International Nuclear Information System (INIS)

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

1986-01-01

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

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

Energy Technology Data Exchange (ETDEWEB)

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

1986-08-01

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

Effects of mutations in the VP2/VP4 cleavage site of Swine vesicular disease virus on RNA encapsidation and viral infectivity

NARCIS (Netherlands)

Rebel, J.M.J.; Leendertse, C.H.; Dekker, A.; Moormann, R.J.M.

2003-01-01

We studied VP0 cleavage of Swine vesicular disease virus (SVDV), a member of the Picornaviridae using a full-length cDNA copy of the Dutch SVDV isolate. The influences of mutations, introduced at the cleavage site of SVDV, on VP0 cleavage, RNA encapsidation and viral infection were studied. Double

Proteolytic cleavage orchestrates cofactor insertion and protein assembly in [NiFe]-hydrogenase biosynthesis.

Science.gov (United States)

Senger, Moritz; Stripp, Sven T; Soboh, Basem

2017-07-14

Metalloenzymes catalyze complex and essential processes, such as photosynthesis, respiration, and nitrogen fixation. For example, bacteria and archaea use [NiFe]-hydrogenases to catalyze the uptake and release of molecular hydrogen (H 2 ). [NiFe]-hydrogenases are redox enzymes composed of a large subunit that harbors a NiFe(CN) 2 CO metallo-center and a small subunit with three iron-sulfur clusters. The large subunit is synthesized with a C-terminal extension, cleaved off by a specific endopeptidase during maturation. The exact role of the C-terminal extension has remained elusive; however, cleavage takes place exclusively after assembly of the [NiFe]-cofactor and before large and small subunits form the catalytically active heterodimer. To unravel the functional role of the C-terminal extension, we used an enzymatic in vitro maturation assay that allows synthesizing functional [NiFe]-hydrogenase-2 of Escherichia coli from purified components. The maturation process included formation and insertion of the NiFe(CN) 2 CO cofactor into the large subunit, endoproteolytic cleavage of the C-terminal extension, and dimerization with the small subunit. Biochemical and spectroscopic analysis indicated that the C-terminal extension of the large subunit is essential for recognition by the maturation machinery. Only upon completion of cofactor insertion was removal of the C-terminal extension observed. Our results indicate that endoproteolytic cleavage is a central checkpoint in the maturation process. Here, cleavage temporally orchestrates cofactor insertion and protein assembly and ensures that only cofactor-containing protein can continue along the assembly line toward functional [NiFe]-hydrogenase. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

On the mechanism of action of ribonucleases: dinucleotide cleavage catalyzed by imidazole and Zn2+.

OpenAIRE

Breslow, R; Huang, D L; Anslyn, E

1989-01-01

Cyclization/cleavage of the 2-(p-nitrophenyl) phosphate ester of propylene glycol is catalyzed by imidazole and, much more effectively, by Zn2+ with imidazole. In the latter case, the mechanism involves simultaneous Lewis acid/base catalysis. Similar Zn2+ and imidazole catalysis of cyclization/cleavage is seen with the dinucleotide 3',5'-UpU (uridylyluridine). Again, the zinc system is much more effective than is catalysis by imidazole alone, and in this case simultaneous Lewis acid/base cata...

Force-Induced Calpain Cleavage of Talin Is Critical for Growth, Adhesion Development, and Rigidity Sensing.

Science.gov (United States)

Saxena, Mayur; Changede, Rishita; Hone, James; Wolfenson, Haguy; Sheetz, Michael P

2017-12-13

Cell growth depends upon formation of cell-matrix adhesions, but mechanisms detailing the transmission of signals from adhesions to control proliferation are still lacking. Here, we find that the scaffold protein talin undergoes force-induced cleavage in early adhesions to produce the talin rod fragment that is needed for cell cycle progression. Expression of noncleavable talin blocks cell growth, adhesion maturation, proper mechanosensing, and the related property of EGF activation of motility. Further, the expression of talin rod in the presence of noncleavable full-length talin rescues cell growth and other functions. The cleavage of talin is found in early adhesions where there is also rapid turnover of talin that depends upon calpain and TRPM4 activity as well as the generation of force on talin. Thus, we suggest that an important function of talin is its control over cell cycle progression through its cleavage in early adhesions.

A Subset of Membrane-Altering Agents and γ-Secretase Modulators Provoke Nonsubstrate Cleavage by Rhomboid Proteases

Directory of Open Access Journals (Sweden)

Siniša Urban

2014-09-01

Full Text Available Rhomboid proteases are integral membrane enzymes that regulate cell signaling, adhesion, and organelle homeostasis pathways, making substrate specificity a key feature of their function. Interestingly, we found that perturbing the membrane pharmacologically in living cells had little effect on substrate processing but induced inappropriate cleavage of nonsubstrates by rhomboid proteases. A subclass of drugs known to modulate γ-secretase activity acted on the membrane directly and induced nonsubstrate cleavage by rhomboid proteases but left true substrate cleavage sites unaltered. These observations highlight an active role for the membrane in guiding rhomboid selectivity and caution that membrane-targeted drugs should be evaluated for cross-activity against membrane-resident enzymes that are otherwise unrelated to the intended drug target. Furthermore, some γ-secretase-modulating activity or toxicity could partly result from global membrane effects.

DNA cleavage enzymes for treatment of persistent viral infections: Recent advances and the pathway forward

Energy Technology Data Exchange (ETDEWEB)

Weber, Nicholas D., E-mail: nweber@fhcrc.org [Vaccine and Infectious Disease Division, Fred Hutchinson Cancer Research Center, 1100 Fairview Ave N, E5-110, Seattle, WA 98109 (United States); Department of Laboratory Medicine, University of Washington, Seattle, WA 98195 (United States); Aubert, Martine, E-mail: maubert@fhcrc.org [Vaccine and Infectious Disease Division, Fred Hutchinson Cancer Research Center, 1100 Fairview Ave N, E5-110, Seattle, WA 98109 (United States); Dang, Chung H., E-mail: cdang@fhcrc.org [Vaccine and Infectious Disease Division, Fred Hutchinson Cancer Research Center, 1100 Fairview Ave N, E5-110, Seattle, WA 98109 (United States); Stone, Daniel, E-mail: dstone2@fhcrc.org [Vaccine and Infectious Disease Division, Fred Hutchinson Cancer Research Center, 1100 Fairview Ave N, E5-110, Seattle, WA 98109 (United States); Jerome, Keith R., E-mail: kjerome@fhcrc.org [Vaccine and Infectious Disease Division, Fred Hutchinson Cancer Research Center, 1100 Fairview Ave N, E5-110, Seattle, WA 98109 (United States); Department of Laboratory Medicine, University of Washington, Seattle, WA 98195 (United States); Department of Microbiology, University of Washington, Seattle, WA 98195 (United States)

2014-04-15

Treatment for most persistent viral infections consists of palliative drug options rather than curative approaches. This is often because long-lasting viral DNA in infected cells is not affected by current antivirals, providing a source for viral persistence and reactivation. Targeting latent viral DNA itself could therefore provide a basis for novel curative strategies. DNA cleavage enzymes can be used to induce targeted mutagenesis of specific genes, including those of exogenous viruses. Although initial in vitro and even in vivo studies have been carried out using DNA cleavage enzymes targeting various viruses, many questions still remain concerning the feasibility of these strategies as they transition into preclinical research. Here, we review the most recent findings on DNA cleavage enzymes for human viral infections, consider the most relevant animal models for several human viral infections, and address issues regarding safety and enzyme delivery. Results from well-designed in vivo studies will ideally provide answers to the most urgent remaining questions, and allow continued progress toward clinical application. - Highlights: • Recent in vitro and in vivo results for DNA cleavage enzymes targeting persistent viral infections. • Analysis of the best animal models for testing enzymes for HBV, HSV, HIV and HPV. • Challenges facing in vivo delivery of therapeutic enzymes for persistent viral infections. • Safety issues to be addressed with proper animal studies.

Hemoglobin cleavage site-specificity of the Plasmodium falciparum cysteine proteases falcipain-2 and falcipain-3.

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Shoba Subramanian

Full Text Available The Plasmodium falciparum cysteine proteases falcipain-2 and falcipain-3 degrade host hemoglobin to provide free amino acids for parasite protein synthesis. Hemoglobin hydrolysis has been described as an ordered process initiated by aspartic proteases, but cysteine protease inhibitors completely block the process, suggesting that cysteine proteases can also initiate hemoglobin hydrolysis. To characterize the specific roles of falcipains, we used three approaches. First, using random P(1 - P(4 amino acid substrate libraries, falcipain-2 and falcipain-3 demonstrated strong preference for cleavage sites with Leu at the P(2 position. Second, with overlapping peptides spanning alpha and beta globin and proteolysis-dependent (18O labeling, hydrolysis was seen at many cleavage sites. Third, with intact hemoglobin, numerous cleavage products were identified. Our results suggest that hemoglobin hydrolysis by malaria parasites is not a highly ordered process, but rather proceeds with rapid cleavage by falcipains at multiple sites. However, falcipain-2 and falcipain-3 show strong specificity for P(2 Leu in small peptide substrates, in agreement with the specificity in optimized small molecule inhibitors that was identified previously. These results are consistent with a principal role of falcipain-2 and falcipain-3 in the hydrolysis of hemoglobin by P. falciparum and with the possibility of developing small molecule inhibitors with optimized specificity as antimalarial agents.

In vitro protease cleavage and computer simulations reveal the HIV-1 capsid maturation pathway

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Ning, Jiying; Erdemci-Tandogan, Gonca; Yufenyuy, Ernest L.; Wagner, Jef; Himes, Benjamin A.; Zhao, Gongpu; Aiken, Christopher; Zandi, Roya; Zhang, Peijun

2016-12-01

HIV-1 virions assemble as immature particles containing Gag polyproteins that are processed by the viral protease into individual components, resulting in the formation of mature infectious particles. There are two competing models for the process of forming the mature HIV-1 core: the disassembly and de novo reassembly model and the non-diffusional displacive model. To study the maturation pathway, we simulate HIV-1 maturation in vitro by digesting immature particles and assembled virus-like particles with recombinant HIV-1 protease and monitor the process with biochemical assays and cryoEM structural analysis in parallel. Processing of Gag in vitro is accurate and efficient and results in both soluble capsid protein and conical or tubular capsid assemblies, seemingly converted from immature Gag particles. Computer simulations further reveal probable assembly pathways of HIV-1 capsid formation. Combining the experimental data and computer simulations, our results suggest a sequential combination of both displacive and disassembly/reassembly processes for HIV-1 maturation.

Glutamic Acid Selective Chemical Cleavage of Peptide Bonds.

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Nalbone, Joseph M; Lahankar, Neelam; Buissereth, Lyssa; Raj, Monika

2016-03-04

Site-specific hydrolysis of peptide bonds at glutamic acid under neutral aqueous conditions is reported. The method relies on the activation of the backbone amide chain at glutamic acid by the formation of a pyroglutamyl (pGlu) imide moiety. This activation increases the susceptibility of a peptide bond toward hydrolysis. The method is highly specific and demonstrates broad substrate scope including cleavage of various bioactive peptides with unnatural amino acid residues, which are unsuitable substrates for enzymatic hydrolysis.

The Role of G-Protein-Coupled Receptor Proteolysis Site Cleavage of Polycystin-1 in Renal Physiology and Polycystic Kidney Disease

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Marie Trudel

2016-01-01

Full Text Available Polycystin-1 (PC1 plays an essential role in renal tubular morphogenesis, and PC1 dysfunction causes human autosomal dominant polycystic kidney disease. A fundamental characteristic of PC1 is post-translational modification via cleavage at the juxtamembrane GPCR proteolysis site (GPS motif that is part of the larger GAIN domain. Given the considerable biochemical complexity of PC1 molecules generated in vivo by this process, GPS cleavage has several profound implications on the intracellular trafficking and localization in association with their particular function. The critical nature of GPS cleavage is further emphasized by the increasing numbers of PKD1 mutations that significantly affect this cleavage process. The GAIN domain with the GPS motif therefore represents the key structural element with fundamental importance for PC1 and might be polycystic kidney disease’s (PKD Achilles’ heel in a large spectrum of PKD1 missense mutations. We highlight the central roles of PC1 cleavage for the regulation of its biogenesis, intracellular trafficking and function, as well as its significance in polycystic kidney disease.

N-Terminomics TAILS Identifies Host Cell Substrates of Poliovirus and Coxsackievirus B3 3C Proteinases That Modulate Virus Infection

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Jagdeo, Julienne M.; Dufour, Antoine; Klein, Theo; Solis, Nestor; Kleifeld, Oded; Kizhakkedathu, Jayachandran; Luo, Honglin; Overall, Christopher M.

2018-01-01

ABSTRACT Enteroviruses encode proteinases that are essential for processing of the translated viral polyprotein. In addition, viral proteinases also target host proteins to manipulate cellular processes and evade innate antiviral responses to promote replication and infection. Although some host protein substrates of enterovirus proteinases have been identified, the full repertoire of targets remains unknown. We used a novel quantitative in vitro proteomics-based approach, termed terminal amine isotopic labeling of substrates (TAILS), to identify with high confidence 72 and 34 new host protein targets of poliovirus and coxsackievirus B3 (CVB3) 3C proteinases (3Cpros) in HeLa cell and cardiomyocyte HL-1 cell lysates, respectively. We validated a subset of candidate substrates that are targets of poliovirus 3Cpro in vitro including three common protein targets, phosphoribosylformylglycinamidine synthetase (PFAS), hnRNP K, and hnRNP M, of both proteinases. 3Cpro-targeted substrates were also cleaved in virus-infected cells but not noncleavable mutant proteins designed from the TAILS-identified cleavage sites. Knockdown of TAILS-identified target proteins modulated infection both negatively and positively, suggesting that cleavage by 3Cpro promotes infection. Indeed, expression of a cleavage-resistant mutant form of the endoplasmic reticulum (ER)-Golgi vesicle-tethering protein p115 decreased viral replication and yield. As the first comprehensive study to identify and validate functional enterovirus 3Cpro substrates in vivo, we conclude that N-terminomics by TAILS is an effective strategy to identify host targets of viral proteinases in a nonbiased manner. IMPORTANCE Enteroviruses are positive-strand RNA viruses that encode proteases that cleave the viral polyprotein into the individual mature viral proteins. In addition, viral proteases target host proteins in order to modulate cellular pathways and block antiviral responses in order to facilitate virus infection

Cell elongation is an adaptive response for clearing long chromatid arms from the cleavage plane

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Kotadia, Shaila; Montembault, Emilie; Sullivan, William

2012-01-01

Chromosome segregation must be coordinated with cell cleavage to ensure correct transmission of the genome to daughter cells. Here we identify a novel mechanism by which Drosophila melanogaster neuronal stem cells coordinate sister chromatid segregation with cleavage furrow ingression. Cells adapted to a dramatic increase in chromatid arm length by transiently elongating during anaphase/telophase. The degree of cell elongation correlated with the length of the trailing chromatid arms and was concomitant with a slight increase in spindle length and an enlargement of the zone of cortical myosin distribution. Rho guanine-nucleotide exchange factor (Pebble)–depleted cells failed to elongate during segregation of long chromatids. As a result, Pebble-depleted adult flies exhibited morphological defects likely caused by cell death during development. These studies reveal a novel pathway linking trailing chromatid arms and cortical myosin that ensures the clearance of chromatids from the cleavage plane at the appropriate time during cytokinesis, thus preserving genome integrity. PMID:23185030

Carotenoid Cleavage Oxygenases from Microbes and Photosynthetic Organisms: Features and Functions

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Oussama Ahrazem

2016-10-01

Full Text Available Apocarotenoids are carotenoid-derived compounds widespread in all major taxonomic groups, where they play important roles in different physiological processes. In addition, apocarotenoids include compounds with high economic value in food and cosmetics industries. Apocarotenoid biosynthesis starts with the action of carotenoid cleavage dioxygenases (CCDs, a family of non-heme iron enzymes that catalyze the oxidative cleavage of carbon–carbon double bonds in carotenoid backbones through a similar molecular mechanism, generating aldehyde or ketone groups in the cleaving ends. From the identification of the first CCD enzyme in plants, an increasing number of CCDs have been identified in many other species, including microorganisms, proving to be a ubiquitously distributed and evolutionarily conserved enzymatic family. This review focuses on CCDs from plants, algae, fungi, and bacteria, describing recent progress in their functions and regulatory mechanisms in relation to the different roles played by the apocarotenoids in these organisms.

Base substitutions at scissile bond sites are sufficient to alter RNA-binding and cleavage activity of RNase III.

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Kim, Kyungsub; Sim, Se-Hoon; Jeon, Che Ok; Lee, Younghoon; Lee, Kangseok

2011-02-01

RNase III, a double-stranded RNA-specific endoribonuclease, degrades bdm mRNA via cleavage at specific sites. To better understand the mechanism of cleavage site selection by RNase III, we performed a genetic screen for sequences containing mutations at the bdm RNA cleavage sites that resulted in altered mRNA stability using a transcriptional bdm'-'cat fusion construct. While most of the isolated mutants showed the increased bdm'-'cat mRNA stability that resulted from the inability of RNase III to cleave the mutated sequences, one mutant sequence (wt-L) displayed in vivo RNA stability similar to that of the wild-type sequence. In vivo and in vitro analyses of the wt-L RNA substrate showed that it was cut only once on the RNA strand to the 5'-terminus by RNase III, while the binding constant of RNase III to this mutant substrate was moderately increased. A base substitution at the uncleaved RNase III cleavage site in wt-L mutant RNA found in another mutant lowered the RNA-binding affinity by 11-fold and abolished the hydrolysis of scissile bonds by RNase III. Our results show that base substitutions at sites forming the scissile bonds are sufficient to alter RNA cleavage as well as the binding activity of RNase III. © 2010 Federation of European Microbiological Societies. Published by Blackwell Publishing Ltd. All rights reserved.

Snake venom serine proteinases specificity mapping by proteomic identification of cleavage sites.

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Zelanis, André; Huesgen, Pitter F; Oliveira, Ana Karina; Tashima, Alexandre K; Serrano, Solange M T; Overall, Christopher M

2015-01-15

Many snake venom toxins are serine proteases but their specific in vivo targets are mostly unknown. Various act on components of the coagulation cascade, and fibrinolytic and kallikrein-kinin systems to trigger various pathological effects observed in the envenomation. Despite showing high similarity in terms of primary structure snake venom serine proteinases (SVSPs) show exquisite specificity towards macromolecular substrates. Therefore, the characterization of their peptide bond specificity is important for understanding the active site preference associated with effective proteolysis as well as for the design of peptide substrates and inhibitors. Bothrops jararaca contains various SVSPs among which Bothrops protease A is a specific fibrinogenolytic agent and PA-BJ is a platelet-activating enzyme. In this study we used proteome derived peptide libraries in the Proteomic Identification of protease Cleavage Sites (PICS) approach to explore the peptide bond specificity of Bothrops protease A and PA-BJ in order to determine their individual peptide cleavage sequences. A total of 371 cleavage sites (208 for Bothrops protease A and 163 for PA-BJ) were detected and both proteinases displayed a clear preference for arginine at the P1 position. Moreover, the analysis of the specificity profiles of Bothrops protease A and PA-BJ revealed subtle differences in the preferences along P6-P6', despite a common yet unusual preference for Pro at P2. Taken together, these results map the subsite specificity of both SVSPs and shed light in the functional differences between these proteinases. Proteolysis is key to various pathological effects observed upon envenomation by viperid snakes. The use of the Proteomic Identification of protease Cleavage Sites (PICS) approach for the easy mapping of proteinase subsite preferences at both the prime- and non-prime sides concurrently gives rise to a fresh understanding of the interaction of the snake venom serine proteinases with peptide and

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

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

2015-01-01

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

Monoclonal antibodies to the reactive centre loop (RCL) of human corticosteroid-binding globulin (CBG) can protect against proteolytic cleavage.

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Lewis, John G; Elder, Peter A

2017-07-01

Corticosteroid-binding globulin (CBG) binds most of the cortisol in circulation and is a non-functional member of the family of serine protease inhibitors (serpins) with an exposed elastase sensitive reactive centre loop (RCL). The RCL can be cleaved by human neutrophil elastase, released from activated neutrophils, and can also be cleaved at nearby site(s) by elastase released by Pseudomonas aeruginosa, and at two further sites, also within the RCL, by bovine chymotrypsin. Cleavage of the RCL results in a conformational change accompanied by a marked decrease in affinity for cortisol and hence its release at the site of proteolysis. These cleavages are irreversible and the similar half-lives of cleaved and intact CBG could mean that there may be some advantage in slowing the rate of CBG cleavage in acute inflammation thereby increasing the proportion of intact CBG in circulation. Here we show, for the first time, that pre-incubation of tethered human CBG with two monoclonal antibodies to the RCL of CBG protects against cleavage by all three enzymes. Furthermore, in plasma, pre-incubation with both RCL monoclonal antibodies delays neutrophil elastase cleavage of the RCL and one of these RCL monoclonal antibodies also delays bovine chymotrypsin cleavage of the RCL. These findings may provide a basis and rationale for the concept of the use of RCL antibodies as therapeutic agents to effectively increase the proportion of intact CBG in circulation which may be of benefit in acute inflammation. Copyright © 2017 Elsevier Ltd. All rights reserved.

Photoenhanced Oxidative DNA Cleavage with Non-Heme Iron(II) Complexes

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Li, Qian; Browne, Wesley R.; Roelfes, Gerard

2010-01-01

The DNA cleavage activity of iron(II) complexes of a series of monotopic pentadentate N,N-bis(2-pyridylmethyl)-N-bis(2-pyridyl)methylamine (N4Py)-derived ligands (1-5) was investigated under laser irradiation at 473, 400.8, and 355 nm in the absence of a reducing agent and compared to that under

Determination of polyphenolic content, HPLC analyses and DNA cleavage activity of Malaysian Averrhoa carambola L. fruit extracts

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Zakia Khanam

2015-10-01

Full Text Available In developing countries, the increasing gap between population growth and food supply has created renewed interest in finding reliable and cheap natural resources of nutraceutical value and health promoting properties. Therefore, the present study deals with the phytochemical analyses and DNA cleavage activity of Averrhoa carambola L. fruit (starfruit extracts. The phytochemical studies involve colour tests and quantification of phenolics and flavonoids of the prepared ethanolic and aqueous extracts. Identification of phenolic acids and flavonoids present in the extracts were conducted by high performance liquid chromatography (HPLC equipped with diode array detector (DAD. DNA cleavage activity of the extracts was evaluated through gel electrophoresis against plasmid Escherichia coli DNA at different concentrations (0.125–0.60 μg/μl. The results of the study exhibited that the starfruit is a rich source of polyphenols and all the extracts exhibited a dose dependent DNA cleavage activity, whereas ethanolic extract induced more cleavage as compared to the aqueous extract. In conclusion, the present study provides preliminary evidence with regard to nutraceutical value of the fruit. So, further extensive study is a prerequisite to exploit DNA cleaving properties of the fruit extracts for therapeutic application.

Supplementary data for the mechanism for cleavage of three typical glucosidic bonds induced by hydroxyl free radical

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Yujie Dai

2017-12-01

Full Text Available The data presented in this article are related to the research article entitled “The mechanism for cleavage of three typical glucosidic bonds induced by hydroxyl free radical” (Dai et al., 2017 [1]. This article includes the structures of three kinds of disaccharides such as maltose, fructose and cellobiose, the diagrammatic sketch of the hydrogen abstraction reaction of the disaccharides by hydroxyl radical, the structure of the transition states for pyran ring opening of moiety A and cleavage of α(1→2 glycosidic bond starting from the hydrogen abstraction of C6–H in moiety A of sucrose, the transition state structure for cleavage of α(1→2 glycosidic bond starting from the hydrogen abstraction of C1′-H in moiety B of sucrose, the transition state structure, sketch for the reaction process and relative energy change of the reaction pathway for direct cleavage of α(1→4 glycosidic bond starting from hydrogen abstraction of C6′–H of moiety B of maltose.

Single-stranded DNA cleavage by divergent CRISPR-Cas9 enzymes

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Ma, Enbo; Harrington, Lucas B.; O’Connell, Mitchell R.; Zhou, Kaihong; Doudna, Jennifer A.

2015-01-01

Summary Double-stranded DNA (dsDNA) cleavage by Cas9 is a hallmark of type II CRISPR-Cas immune systems. Cas9–guide RNA complexes recognize 20-base-pair sequences in DNA and generate a site-specific double-strand break, a robust activity harnessed for genome editing. DNA recognition by all studied Cas9 enzymes requires a protospacer adjacent motif (PAM) next to the target site. We show that Cas9 enzymes from evolutionarily divergent bacteria can recognize and cleave single-stranded DNA (ssDNA) by an RNA-guided, PAM-independent recognition mechanism. Comparative analysis shows that in contrast to the type II-A S. pyogenes Cas9 that is widely used for genome engineering, the smaller type II-C Cas9 proteins have limited dsDNA binding and unwinding activity and promiscuous guide-RNA specificity. These results indicate that inefficiency of type II-C Cas9 enzymes for genome editing results from a limited ability to cleave dsDNA, and suggest that ssDNA cleavage was an ancestral function of the Cas9 enzyme family. PMID:26545076

Two-dimensional NMR evidence for cleavage of lignin and xylan substituents in wheat straw through hydrothermal pretreatment and enzymatic hydrolysis

DEFF Research Database (Denmark)

Yelle, Daniel J.; Kaparaju, Laxmi-Narasimha Prasad; Hunt, Christopher G.

2013-01-01

correlation spectroscopy, via an heteronuclear single quantum coherence experiment, revealed substantial lignin β-aryl ether cleavage, deacetylation via cleavage of the natural acetates at the 2-O- and 3-O-positions of xylan, and uronic acid depletion via cleavage of the (1 → 2)-linked 4-O....... g., further deacylation revealed by the depletion in ferulate and p-coumarate structures). Supplementary chemical analyses showed that the hydrothermal pretreatment increased the cellulose and lignin concentration with partial removal of extractives and hemicelluloses. The subsequent enzymatic...

Evolution of inhibitor-resistant natural mutant forms of HIV-1 protease probed by pre-steady state kinetic analysis.

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Zakharova, Maria Yu; Kuznetsova, Alexandra A; Kaliberda, Elena N; Dronina, Maria A; Kolesnikov, Alexander V; Kozyr, Arina V; Smirnov, Ivan V; Rumsh, Lev D; Fedorova, Olga S; Knorre, Dmitry G; Gabibov, Alexander G; Kuznetsov, Nikita A

2017-11-01

Pre-steady state kinetic analysis of mechanistic features of substrate binding and processing is crucial for insight into the evolution of inhibitor-resistant forms of HIV-1 protease. These data may provide a correct vector for rational drug design assuming possible intrinsic dynamic effects. These data should also give some clues to the molecular mechanism of protease action and resistance to inhibitors. Here we report pre-steady state kinetics of the interaction of wild type or mutant forms of HIV-1 protease with a FRET-labeled peptide. The three-stage "minimal" kinetic scheme with first and second reversible steps of substrate binding and with following irreversible peptide cleavage step adequately described experimental data. For the first time, a set of "elementary" kinetic parameters of wild type HIV-1 protease and its natural mutant inhibitor-resistant forms MDR-HM, ANAM-11 and prDRV4 were compared. Inhibitors of the first and second generation were used to estimate the inhibitory effects on HIV-1 protease activity. The resulting set of kinetic data supported that the mutant forms are kinetically unaffected by inhibitors of the first generation, proving their functional resistance to these compounds. The second generation inhibitor darunavir inhibited mutant forms MDR-HM and ANAM-11, but was ineffective against prDRV4. Our kinetic data revealed that these inhibitors induced different conformational changes in the enzyme and, thereby they have different mode of binding in the enzyme active site. These data confirmed hypothesis that the driving force of the inhibitor-resistance evolution is disruption of enzyme-inhibitor complex by changing of the contact network in the inhibitor binding site. Copyright © 2017 Elsevier B.V. and Société Française de Biochimie et Biologie Moléculaire (SFBBM). All rights reserved.

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

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

2017-09-13

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

The type of GnRH analogue used during controlled ovarian stimulation influences early embryo developmental kinetics

DEFF Research Database (Denmark)

Muñoz, Manuel; Cruz, María; Humaidan, Peter

2013-01-01

in the Instituto Valenciano de Infertilidad (IVI) Alicante and the Instituto Universitario-IVI Valencia, Spain, 2817 embryos deriving from 400 couples undergoing oocyte donation were analysed. After controlled ovarian stimulation and IVF/intracytoplamic sperm injection, the timing of embryonic cleavages......OBJECTIVE: To explore if the GnRH analogue used for controlled ovarian stimulation (COS) and the ovulation triggering factor (GnRH agonist+hCG triggering versus GnRH antagonist+GnRH agonist triggering) affect embryo development and kinetics. STUDY DESIGN: In a retrospective cohort study...... was assessed by a video time-lapse system. The results were analysed using Student's t test for comparison of timings (hours) and Chi-squared test for comparison of proportions. A p-value...

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

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Lindquist Jeffrey N

2005-11-01

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

ChloroP, a neural network-based method for predicting chloroplast transitpeptides and their cleavage sites

DEFF Research Database (Denmark)

Emanuelsson, O.; Nielsen, Henrik; von Heijne, Gunnar

1999-01-01

the cleavage sites given in SWISS-PROT. An analysis of 715 Arabidopsis thaliana sequences from SWISS-PROT suggests that the ChloroP method should be useful for the identification of putative transit peptides in genome-wide sequence data. The ChloroP predictor is available as a web-server at http......We present a neural network based method (ChloroP) for identifying chloroplast transit peptides and their cleavage sites. Using cross-validation, 88% of the sequences in our homology reduced training set were correctly classified as transit peptides or nontransit peptides. This performance level...

Functional analyses of the three simian hemorrhagic fever virus nonstructural protein 1 papain-like proteases.

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Vatter, Heather A; Di, Han; Donaldson, Eric F; Radu, Gertrud U; Maines, Taronna R; Brinton, Margo A

2014-08-01

The N-terminal region of simian hemorrhagic fever virus (SHFV) nonstructural polyprotein 1a is predicted to encode three papain-like proteases (PLP1α, PLP1β, and PLP1γ). Catalytic residues and cleavage sites for each of the SHFV PLP1s were predicted by alignment of the SHFV PLP1 region sequences with each other as well as with those of other arteriviruses, and the predicted catalytic residues were shown to be proximal by homology modeling of the SHFV nsp1s on porcine respiratory and reproductive syndrome virus (PRRSV) nsp1 crystal structures. The functionality of the predicted catalytic Cys residues and cleavage sites was tested by analysis of the autoproteolytic products generated in in vitro transcription/translation reactions done with wild-type or mutant SHFV nsp1 constructs. Cleavage sites were also analyzed by mass spectroscopy analysis of selected immunoprecipitated cleavage products. The data showed that each of the three SHFV PLP1s is an active protease. Cys63 was identified as the catalytic Cys of SHFV PLP1α and is adjacent to an Ala instead of the canonical Tyr observed in other arterivirus PLP1s. SHFV PLP1γ is able to cleave at both downstream and upstream nsp1 junction sites. Although intermediate precursor polyproteins as well as alternative products generated by each of the SHFV PLP1s cleaving at sites within the N-terminal region of nsp1β were produced in the in vitro reactions, Western blotting of SHFV-infected, MA104 cell lysates with SHFV nsp1 protein-specific antibodies detected only the three mature nsp1 proteins. SHFV is unique among arteriviruses in having three N-terminal papain-like protease 1 (PLP1) domains. Other arteriviruses encode one or two active PLP1s. This is the first functional study of the SHFV PLP1s. Analysis of the products of in vitro autoprocessing of an N-terminal SHFV nonstructural 1a polypeptide fragment showed that each of the three SHFV PLP1s is active, and the predicted catalytic Cys residues and cleavage sites

AcEST: DK944418 [AcEST

Lifescience Database Archive (English)

Full Text Available 7|POLS_EEEVF Structural polyprotein OS=Eastern equine encephalitis virus (strain Florida 91-469) Align lengt...icant alignments: (bits) Value sp|Q4QXJ7|POLS_EEEVF Structural polyprotein OS=Eastern equine en... 30 4.9 sp...|P08768|POLS_EEEV Structural polyprotein OS=Eastern equine enc... 30 4.9 sp|P27284|POLS_EEEV3 Structural polyprotein OS=Eastern equin...e en... 29 6.5 >sp|Q4QXJ7|POLS_EEEVF Structural polyprotein OS=Eastern equine...68|POLS_EEEV Structural polyprotein OS=Eastern equine encephalitis virus PE=2 SV=1 Length = 1239 Score = 29.

Fast cleavage of phycocyanobilin from phycocyanin for use in food colouring.

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Roda-Serrat, Maria Cinta; Christensen, Knud Villy; El-Houri, Rime Bahij; Fretté, Xavier; Christensen, Lars Porskjær

2018-02-01

Phycocyanins from cyanobacteria are possible sources for new natural blue colourants. Their chromophore, phycocyanobilin (PCB), was cleaved from the apoprotein by solvolysis in alcohols and alcoholic aqueous solutions. In all cases two PCB isomers were obtained, while different solvent adducts were formed upon the use of different reagents. The reaction is believed to take place via two competing pathways, a concerted E2 elimination and a S N 2 nucleophilic substitution. Three cleavage methods were compared in terms of yield and purity: conventional reflux, sealed vessel heated in an oil bath, and microwave assisted reaction. The sealed vessel method is a new approach for fast cleavage of PCB from phycocyanin and gave at 120°C the same yield within 30min compared to 16h by the conventional reflux method (P<0.05). In addition the sealed vessel method resulted in improved purity compared to the other methods. Microwave irradiation increased product degradation. Copyright © 2017 Elsevier Ltd. All rights reserved.

TRAIL-induced cleavage and inactivation of SPAK sensitizes cells to apoptosis

International Nuclear Information System (INIS)

Polek, Tara C.; Talpaz, Moshe; Spivak-Kroizman, Taly R.

2006-01-01

Ste20-related proline-alanine-rich kinase (SPAK) has been linked to various cellular processes, including proliferation, differentiation, and ion transport regulation. Recently, we showed that SPAK mediates signaling by the TNF receptor, RELT. The presence of a caspase cleavage site in SPAK prompted us to study its involvement in apoptotic signaling induced by another TNF member, TRAIL. We show that TRAIL stimulated caspase 3-like proteases that cleaved SPAK at two distinct sites. Cleavage had little effect on the activity of SPAK but removed its substrate-binding domain. In addition, TRAIL reduced the activity of SPAK in HeLa cells in a caspase-independent manner. Thus, TRAIL inhibited SPAK by two mechanisms: activation of caspases, which removed its substrate-binding domain, and caspase-independent down-regulation of SPAK activity. Furthermore, reducing the amount of SPAK by siRNA increased the sensitivity of HeLa cells to TRAIL-induced apoptosis. Thus, TRAIL down-regulation of SPAK is an important event that enhances its apoptotic effects

Inhibition of influenza virus infection and hemagglutinin cleavage by the protease inhibitor HAI-2

Energy Technology Data Exchange (ETDEWEB)

Hamilton, Brian S.; Chung, Changik; Cyphers, Soreen Y.; Rinaldi, Vera D.; Marcano, Valerie C.; Whittaker, Gary R., E-mail: grw7@cornell.edu

2014-07-25

Highlights: • Biochemical and cell biological analysis of HAI-2 as an inhibitor of influenza HA cleavage activation. • Biochemical and cell biological analysis of HAI-2 as an inhibitor of influenza virus infection. • Comparative analysis of HAI-2 for vesicular stomatitis virus and human parainfluenza virus type-1. • Analysis of the activity of HAI-2 in a mouse model of influenza. - Abstract: Influenza virus remains a significant concern to public health, with the continued potential for a high fatality pandemic. Vaccination and antiviral therapeutics are effective measures to circumvent influenza virus infection, however, multiple strains have emerged that are resistant to the antiviral therapeutics currently on the market. With this considered, investigation of alternative antiviral therapeutics is being conducted. One such approach is to inhibit cleavage activation of the influenza virus hemagglutinin (HA), which is an essential step in the viral replication cycle that permits viral-endosome fusion. Therefore, targeting trypsin-like, host proteases responsible for HA cleavage in vivo may prove to be an effective therapeutic. Hepatocyte growth factor activator inhibitor 2 (HAI-2) is naturally expressed in the respiratory tract and is a potent inhibitor of trypsin-like serine proteases, some of which have been determined to cleave HA. In this study, we demonstrate that HAI-2 is an effective inhibitor of cleavage of HA from the human-adapted H1 and H3 subtypes. HAI-2 inhibited influenza virus H1N1 infection in cell culture, and HAI-2 administration showed protection in a mouse model of influenza. HAI-2 has the potential to be an effective, alternative antiviral therapeutic for influenza.

Inhibition of influenza virus infection and hemagglutinin cleavage by the protease inhibitor HAI-2

International Nuclear Information System (INIS)

Hamilton, Brian S.; Chung, Changik; Cyphers, Soreen Y.; Rinaldi, Vera D.; Marcano, Valerie C.; Whittaker, Gary R.

2014-01-01

Highlights: • Biochemical and cell biological analysis of HAI-2 as an inhibitor of influenza HA cleavage activation. • Biochemical and cell biological analysis of HAI-2 as an inhibitor of influenza virus infection. • Comparative analysis of HAI-2 for vesicular stomatitis virus and human parainfluenza virus type-1. • Analysis of the activity of HAI-2 in a mouse model of influenza. - Abstract: Influenza virus remains a significant concern to public health, with the continued potential for a high fatality pandemic. Vaccination and antiviral therapeutics are effective measures to circumvent influenza virus infection, however, multiple strains have emerged that are resistant to the antiviral therapeutics currently on the market. With this considered, investigation of alternative antiviral therapeutics is being conducted. One such approach is to inhibit cleavage activation of the influenza virus hemagglutinin (HA), which is an essential step in the viral replication cycle that permits viral-endosome fusion. Therefore, targeting trypsin-like, host proteases responsible for HA cleavage in vivo may prove to be an effective therapeutic. Hepatocyte growth factor activator inhibitor 2 (HAI-2) is naturally expressed in the respiratory tract and is a potent inhibitor of trypsin-like serine proteases, some of which have been determined to cleave HA. In this study, we demonstrate that HAI-2 is an effective inhibitor of cleavage of HA from the human-adapted H1 and H3 subtypes. HAI-2 inhibited influenza virus H1N1 infection in cell culture, and HAI-2 administration showed protection in a mouse model of influenza. HAI-2 has the potential to be an effective, alternative antiviral therapeutic for influenza

Sequential reduction–oxidation for photocatalytic degradation of tetrabromobisphenol A: Kinetics and intermediates

International Nuclear Information System (INIS)

Guo, Yaoguang; Lou, Xiaoyi; Xiao, Dongxue; Xu, Lei; Wang, Zhaohui; Liu, Jianshe

2012-01-01

Highlights: ► Sequential photocatalytic reduction–oxidation degradation of TBBPA was firstly examined. ► Different atmospheres were found to have significant effect on debromination reaction. ► A possible sequential photocatalytic reduction–oxidation pathway was proposed. - Abstract: C-Br bond cleavage is considered as a key step to reduce their toxicities and increase degradation rates for most brominated organic pollutants. Here a sequential reduction/oxidation strategy (i.e. debromination followed by photocatalytic oxidation) for photocatalytic degradation of tetrabromobisphenol A (TBBPA), one of the most frequently used brominated flame retardants, was proposed on the basis of kinetic analysis and intermediates identification. The results demonstrated that the rates of debromination and even photodegradation of TBBPA strongly depended on the atmospheres, initial TBBPA concentrations, pH of the reaction solution, hydrogen donors, and electron acceptors. These kinetic data and byproducts identification obtained by GC–MS measurement indicated that reductive debromination reaction by photo-induced electrons dominated under N 2 -saturated condition, while oxidation reaction by photoexcited holes or hydroxyl radicals played a leading role when air was saturated. It also suggested that the reaction might be further optimized for pretreatment of TBBPA-contaminated wastewater by a two-stage reductive debromination/subsequent oxidative decomposition process in the UV-TiO 2 system by changing the reaction atmospheres.

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

Science.gov (United States)

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

2015-12-15

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

Relationship between synthesis and cleavage of poliovirus-specific proteins.

OpenAIRE

Thomas, A A; Voorma, H O; Boeye, A

1983-01-01

Poliovirus proteinase was studied in vitro in lysates from poliovirus-infected HeLa cells. Preincubation of these lysates caused (i) a reduction in poliovirus proteinase activity and (ii) a partial dependence on exogenous mRNA for optimal translation. Proteins translated from endogenous poliovirus RNA in preincubated extracts from virus-infected HeLa cells are poorly cleaved. This cleavage deficiency is alleviated by adding fresh poliovirus RNA to the translation system, thus, allowing re-ini...

Cleavage of influenza RNA by using a human PUF-based artificial RNA-binding protein–staphylococcal nuclease hybrid

International Nuclear Information System (INIS)

Mori, Tomoaki; Nakamura, Kento; Masaoka, Keisuke; Fujita, Yusuke; Morisada, Ryosuke; Mori, Koichi; Tobimatsu, Takamasa; Sera, Takashi

2016-01-01

Various viruses infect animals and humans and cause a variety of diseases, including cancer. However, effective methodologies to prevent virus infection have not yet been established. Therefore, development of technologies to inactivate viruses is highly desired. We have already demonstrated that cleavage of a DNA virus genome was effective to prevent its replication. Here, we expanded this methodology to RNA viruses. In the present study, we used staphylococcal nuclease (SNase) instead of the PIN domain (PilT N-terminus) of human SMG6 as an RNA-cleavage domain and fused the SNase to a human Pumilio/fem-3 binding factor (PUF)-based artificial RNA-binding protein to construct an artificial RNA restriction enzyme with enhanced RNA-cleavage rates for influenzavirus. The resulting SNase-fusion nuclease cleaved influenza RNA at rates 120-fold greater than the corresponding PIN-fusion nuclease. The cleaving ability of the PIN-fusion nuclease was not improved even though the linker moiety between the PUF and RNA-cleavage domain was changed. Gel shift assays revealed that the RNA-binding properties of the PUF derivative used was not as good as wild type PUF. Improvement of the binding properties or the design method will allow the SNase-fusion nuclease to cleave an RNA target in mammalian animal cells and/or organisms. - Highlights: • A novel RNA restriction enzyme using SNase was developed tor cleave viral RNA. • Our enzyme cleaved influenza RNA with rates >120-fold higher rates a PIN-fusion one. • Our artificial enzyme with the L5 linker showed the highest RNA cleavage rate. • Our artificial enzyme site-selectively cleaved influenza RNA in vitro.

Cleavage of spike protein of SARS coronavirus by protease factor Xa is associated with viral infectivity

International Nuclear Information System (INIS)

Du, Lanying; Kao, Richard Y.; Zhou, Yusen; He, Yuxian; Zhao, Guangyu; Wong, Charlotte; Jiang, Shibo; Yuen, Kwok-Yung; Jin, Dong-Yan; Zheng, Bo-Jian

2007-01-01

The spike (S) protein of SARS coronavirus (SARS-CoV) has been known to recognize and bind to host receptors, whose conformational changes then facilitate fusion between the viral envelope and host cell membrane, leading to viral entry into target cells. However, other functions of SARS-CoV S protein such as proteolytic cleavage and its implications to viral infection are incompletely understood. In this study, we demonstrated that the infection of SARS-CoV and a pseudovirus bearing the S protein of SARS-CoV was inhibited by a protease inhibitor Ben-HCl. Also, the protease Factor Xa, a target of Ben-HCl abundantly expressed in infected cells, was able to cleave the recombinant and pseudoviral S protein into S1 and S2 subunits, and the cleavage was inhibited by Ben-HCl. Furthermore, this cleavage correlated with the infectivity of the pseudovirus. Taken together, our study suggests a plausible mechanism by which SARS-CoV cleaves its S protein to facilitate viral infection

AcEST: DK952708 [AcEST

Lifescience Database Archive (English)

Full Text Available form 1 OS=Drosophila melano... 30 9.4 sp|P09592|POLS_EEVVT Structural polyprotein OS=Venezuelan equine... 30... 9.4 sp|P36332|POLS_EEVVP Structural polyprotein OS=Venezuelan equine... 30 9.4 sp|P36330|POLS_EEVVE Structu...ral polyprotein OS=Venezuelan equine... 30 9.4 sp|P05674|POLS_EEVV8 Structural po...lyprotein OS=Venezuelan equine... 30 9.4 sp|P36329|POLS_EEVV3 Structural polyprotein OS=Venezuelan equine...

Cleavage/alteration of interleukin-8 by matrix metalloproteinase-9 in the female lower genital tract.

Science.gov (United States)

Zariffard, M Reza; Anastos, Kathryn; French, Audrey L; Munyazesa, Elisaphane; Cohen, Mardge; Landay, Alan L; Spear, Gregory T

2015-01-01

Interleukin-8 (IL-8, CXCL8) plays important roles in immune responses at mucosal sites including in the lower genital tract. Since several types of bacteria produce proteases that cleave IL-8 and many types of bacteria can be present in lower genital tract microbiota, we assessed genital fluids for IL-8 cleavage/alteration. Genital fluids collected by lavage from 200 women (23 HIV-seronegative and 177 HIV-seropositive) were tested for IL-8 cleavage/alteration by ELISA. IL-8 cleaving/altering activity was observed in fluids from both HIV-positive (28%) and HIV-negative women (35%). There was no clear relationship between the activity and the types of bacteria present in the lower genital tract as determined by high-throughput sequencing of the 16S rRNA gene. Protease inhibitors specific for matrix metalloproteinases (MMPs) reduced the activity and a multiplex assay that detects both inactive and active MMPs showed the presence of multiple MMPs, including MMP-1, -3, -7, -8, -9, -10 and -12 in genital secretions from many of the women. The IL-8-cleaving/altering activity significantly correlated with active MMP-9 as well as with cleavage of a substrate that is acted on by several active MMPs. These studies show that multiple MMPs are present in the genital tract of women and strongly suggest that MMP-9 in genital secretions can cleave IL-8 at this mucosal site. These studies suggest that MMP-mediated cleavage of IL-8 can modulate inflammatory responses in the lower genital tract.

AcEST: DK944496 [AcEST

Lifescience Database Archive (English)

Full Text Available it_id Q4QXJ7 Definition sp|Q4QXJ7|POLS_EEEVF Structural polyprotein OS=Eastern equine encephalitis virus (st...ore E Sequences producing significant alignments: (bits) Value sp|Q4QXJ7|POLS_EEEVF Structural polyprotein OS=Eastern equine... en... 32 1.3 sp|P08768|POLS_EEEV Structural polyprotein OS=Eastern equine enc... 32 1.3 sp|...P27284|POLS_EEEV3 Structural polyprotein OS=Eastern equine en... 31 1.7 sp|Q86925...|POLS_AURAV Structural polyprotein OS=Aura virus PE=3 SV=2 31 2.2 sp|Q306W7|POLS_EEEV8 Structural polyprotein OS=Eastern equine

Two tandem RNase III cleavage sites determine betT mRNA stability in response to osmotic stress in Escherichia coli.

Directory of Open Access Journals (Sweden)

Minji Sim

Full Text Available While identifying genes regulated by ribonuclease III (RNase III in Escherichia coli, we observed that steady-state levels of betT mRNA, which encodes a transporter mediating the influx of choline, are dependent on cellular concentrations of RNase III. In the present study, we also observed that steady-state levels of betT mRNA are dependent on RNase III activity upon exposure to osmotic stress, indicating the presence of cis-acting elements controlled by RNase III in betT mRNA. Primer extension analyses of betT mRNA revealed two tandem RNase III cleavage sites in its stem-loop region, which were biochemically confirmed via in vitro cleavage assays. Analyses of cleavage sites suggested the stochastic selection of cleavage sites by RNase III, and mutational analyses indicated that RNase III cleavage at either site individually is insufficient for efficient betT mRNA degradation. In addition, both the half-life and abundance of betT mRNA were significantly increased in association with decreased RNase III activity under hyper-osmotic stress conditions. Our findings demonstrate that betT mRNA stability is controlled by RNase III at the post-transcriptional level under conditions of osmotic stress.

A camel-derived MERS-CoV with a variant spike protein cleavage site and distinct fusion activation properties

Science.gov (United States)

Millet, Jean Kaoru; Goldstein, Monty E; Labitt, Rachael N; Hsu, Hung-Lun; Daniel, Susan; Whittaker, Gary R

2016-01-01

Middle East respiratory syndrome coronavirus (MERS-CoV) continues to circulate in both humans and camels, and the origin and evolution of the virus remain unclear. Here we characterize the spike protein of a camel-derived MERS-CoV (NRCE-HKU205) identified in 2013, early in the MERS outbreak. NRCE-HKU205 spike protein has a variant cleavage motif with regard to the S2′ fusion activation site—notably, a novel substitution of isoleucine for the otherwise invariant serine at the critical P1′ cleavage site position. The substitutions resulted in a loss of furin-mediated cleavage, as shown by fluorogenic peptide cleavage and western blot assays. Cell–cell fusion and pseudotyped virus infectivity assays demonstrated that the S2′ substitutions decreased spike-mediated fusion and viral entry. However, cathepsin and trypsin-like protease activation were retained, albeit with much reduced efficiency compared with the prototypical EMC/2012 human strain. We show that NRCE-HKU205 has more limited fusion activation properties possibly resulting in more restricted viral tropism and may represent an intermediate in the complex pattern of MERS-CoV ecology and evolution. PMID:27999426

Discrete population balance models of random agglomeration and cleavage in polymer pyrolysis

Directory of Open Access Journals (Sweden)

John E. J. Staggs

2017-05-01

Full Text Available The processes of random agglomeration and cleavage (both of which are important for the development of new models of polymer combustion, but are also applicable in a wide range of fields including atmospheric physics, radiation modelling and astrophysics are analysed using population balance methods. The evolution of a discrete distribution of particles is considered within this framework, resulting in a set of ordinary differential equations for the individual particle concentrations. Exact solutions for these equations are derived, together with moment generating functions. Application of the discrete Laplace transform (analogous to the Z-transform is found to be effective in these problems, providing both exact solutions for particle concentrations and moment generating functions. The combined agglomeration-cleavage problem is also considered. Unfortunately, it has been impossible to find an exact solution for the full problem, but a stable steady state has been identified and computed.

Evidence of Alternative Cystatin C Signal Sequence Cleavage Which Is Influenced by the A25T Polymorphism.

Directory of Open Access Journals (Sweden)

Annie Nguyen

Full Text Available Cystatin C (Cys C is a small, potent, cysteine protease inhibitor. An Ala25Thr (A25T polymorphism in Cys C has been associated with both macular degeneration and late-onset Alzheimer's disease. Previously, studies have suggested that this polymorphism may compromise the secretion of Cys C. Interestingly, we found that untagged A25T, A25T tagged C-terminally with FLAG, or A25T FLAG followed by green fluorescent protein (GFP, were all secreted as efficiently from immortalized human cells as their wild-type (WT counterparts (e.g., 112%, 100%, and 88% of WT levels from HEK-293T cells, respectively. Supporting these observations, WT and A25T Cys C variants also showed similar intracellular steady state levels. Furthermore, A25T Cys C did not activate the unfolded protein response and followed the same canonical endoplasmic reticulum (ER-Golgi trafficking pathway as WT Cys C. WT Cys C has been shown to undergo signal sequence cleavage between residues Gly26 and Ser27. While the A25T polymorphism did not affect Cys C secretion, we hypothesized that it may alter where the Cys C signal sequence is preferentially cleaved. Under normal conditions, WT and A25T Cys C have the same signal sequence cleavage site after Gly26 (referred to as 'site 2' cleavage. However, in particular circumstances when the residues around site 2 are modified (such as by the presence of an N-terminal FLAG tag immediately after Gly26, or by a Gly26Lys (G26K mutation, A25T has a significantly higher likelihood than WT Cys C of alternative signal sequence cleavage after Ala20 ('site 1' or even earlier in the Cys C sequence. Overall, our results indicate that the A25T polymorphism does not cause a significant reduction in Cys C secretion, but instead predisposes the protein to be cleaved at an alternative signal sequence cleavage site if site 2 is hindered. Additional N-terminal amino acids resulting from alternative signal sequence cleavage may, in turn, affect the protease

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

Science.gov (United States)

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

2015-01-01

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

Micromechanisms and toughness for cleavage fracture of steel

International Nuclear Information System (INIS)

Rosenfield, A.R.; Majumdar, B.S.

1987-01-01

A complete understanding of the fracture mechanisms of steel in the ductile/brittle transition region requires analysis not only of crack initiation, but also of crack propagation. This paper reviews micrographic and fractographic experiments that give insight into both phenomena, and suggests a frame-work through which both may be related. Unstable cleavage crack initiation can occur after some blunting of the original fatigue precrack or after some stable crack growth. In either event, instability appears to be triggered by the fracture of a brittle micro-constituent ahead of the precrack. The large scatter in reported K IC values within the transition region reflects the size distribution and relative scarcity of these 'trigger' particles. While a large number of models have attempted to correlate toughness in the ductile/brittle transition regime to events occurring ahead of the crack tip, surprisingly little attention has been paid to events occurring behind the crack front. Fractographic evidence as well as metallographic sectioning of arrested cracks show that the mechanism of rapid crack propagation by cleavage is affected strongly by partial crack-plane deflection which leaves unbroken ligaments in its wake. The tearing of these ligaments by dimple-rupture is the dominant energy-absorbing mechanism. Etch-pit experiments using an Fe-Si alloy show that the crack-tip stress intensity based on plastic zone size is extremely low. It is suggested that the mechanism of crack arrest should be modeled using a sharp crack which is restrained by a distribution of discrete pinching forces along its faces. The same model is applied to crack initiation. (orig.)

Cleavage and protection of locked nucleic acid-modified DNA by restriction endonucleases

DEFF Research Database (Denmark)

Crouzier, Lucile; Dubois, Camille; Wengel, Jesper

2012-01-01

Locked nucleic acid (LNA) is one of the most prominent nucleic acid analogues reported so far. We herein for the first time report cleavage by restriction endonuclease of LNA-modified DNA oligonucleotides. The experiments revealed that RsaI is an efficient enzyme capable of recognizing and cleaving...

Non-kinetic capabilities: complementing the kinetic prevalence to targeting

OpenAIRE

Ducheine, P.

2014-01-01

Targeting is used in military doctrine to describe a military operational way, using (military) means to influence a target (or addressee) in order to achieve designated political and/or military goals. The four factors italicized are used to analyse non-kinetic targeting, complementing our knowledge and understanding of the kinetic prevalence. Paradoxically, non-kinetic targeting is not recognized as a separate concept: kinetic and non-kinetic are intertwined facets of targeting. Kinetic tar...

RecA-mediated cleavage activates UmuD for mutagenesis: Mechanistic relationship between transcriptional derepression and posttranslational activation

International Nuclear Information System (INIS)

Nohmi, Takehiko; Battista, J.R.; Dodson, L.A.; Walker, G.C.

1988-01-01

The products of the SOS-regulated umuDC operon are required for most UV and chemical mutagenesis in Escherichia coli. It has been shown that the UmuD protein shares homology with LexA, the repressor of the SOS genes. In this paper the authors describe a series of genetic experiments that indicate that the purpose of RecA-mediated cleavage of UmuD at its bond between Cys-24 and Gly-25 is to activate UmuD for its role in mutagenesis and that the COOH-terminal fragment of UmuD is necessary and sufficient for the role of UmuD in UV mutagenesis. Other genetic experiments are presented that (i) support the hypothesis that the primary role of Ser-60 in UmuD function is to act as a nucleophile in the RecA-mediated cleavage reaction and (ii) raise the possibility that RecA has a third role in UV mutagenesis besides mediating the cleavage of LexA and UmuD

Targeted cleavage of hepatitis E virus 3' end RNA mediated by hammerhead ribozymes inhibits viral RNA replication

International Nuclear Information System (INIS)

Sriram, Bandi; Thakral, Deepshi; Panda, Subrat Kumar

2003-01-01

The 3' end of hepatitis E virus (HEV) contains cis-acting regulatory element, which plays an important role in viral replication. To develop specific replication inhibitor at the molecular level, mono- and di-hammerhead ribozymes (Rz) were designed and synthesized against the conserved 3' end sequences of HEV, which cleave at nucleotide positions 7125 and 7112/7125, respectively. Di-hammerhead ribozyme with two catalytic motifs in tandem was designed to cleave simultaneously at two sites spaced 13 nucleotides apart, which increases the overall cleavage efficiency and prevents the development of escape mutants. Specific cleavage products were obtained with both the ribozymes in vitro at physiological conditions. The inactive control ribozymes showed no cleavage. The ribozymes showed specific inhibition of HEV 3' end fused-luciferase reporter gene expression by ∼37 and ∼60%, respectively in HepG2 cells. These results demonstrate a feasible approach to inhibit the HEV replication to a limited extent by targeting the cis-acting 3' end of HEV with hammerhead ribozymes

Effect of Albendazole at different concentrations on fertilization and early cleavage in Tetrapygus niger “sea urchin”

Directory of Open Access Journals (Sweden)

Gina Zavaleta Espejo

2013-09-01

Full Text Available In this study we evaluated the effect of Albendazole at different concentrations and exposure times on the process of fertilization and early cleavage in Tetrapygus niger "sea urchin". Each experimental group consisted of 200 mL, of previously filtered seawater at pH 7.3 and temperature of 20 ± 2 °C, plus five drops of eggs and two drops of spermatozoa exposed to different concentrations of Albendazole 400 ppm, 800 ppm and 1200 ppm. Determining the effect of Albendazole was conducted by counting the number of cones of fertilization, as well as the number of cleaving embryos with normal and abnormal. The ANOVA and multiple comparison test Tukey averages showed significant differences between the treatments, namely that increasing the concentration of Albendazole fertilization rate decreases and increases the percentage of embryos with abnormal cleavage, so therefore concluded that the Albendazole at different concentrations and exposure times affects the process of fertilization and early cleavage in T, niger "sea urchin"

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

Science.gov (United States)

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

2017-03-01

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

Reversible Heterolytic Cleavage of the H-H Bond by Molybdenum Complexes: Controlling the Dynamics of Exchange Between Proton and Hydride

Energy Technology Data Exchange (ETDEWEB)

Zhang, Shaoguang; Appel, Aaron M.; Bullock, R. Morris

2017-05-18

Controlling the heterolytic cleavage of the H-H bond of dihydrogen is critically important in catalytic hydrogenations and in the catalytic oxidation of H2. We show how the rate of reversible heterolytic cleavage of H2 can be controlled over nearly four orders of magnitude at 25 °C, from 2.1 × 103 s-1 to ≥107 s-1. Bifunctional Mo complexes, [CpMo(CO)(κ3-P2N2)]+ (P2N2 = 1,5-diaza-3,7-diphosphacyclooctane with alkyl/aryl groups on N and P), have been developed for heterolytic cleavage of H2 into a proton and a hydride, akin to Frustrated Lewis Pairs. The H-H bond cleavage is enabled by the basic amine in the second coordination sphere. The products of heterolytic cleavage of H2, Mo hydride complexes bearing protonated amines, [CpMo(H)(CO)(P2N2H)]+, were characterized by spectroscopic studies and by X-ray crystallography. Variable temperature 1H, 15N and 2-D 1H-1H ROESY NMR spectra indicated rapid exchange of the proton and hydride. The exchange rates are in the order [CpMo(H)(CO)(PPh2NPh2H)]+ > [CpMo(H)(CO)(PtBu2NPh2H)]+ > [CpMo(H)(CO)(PPh2NBn2H)]+ > [CpMo(H)(CO)(PtBu2NBn2H)]+ > [CpMo(H)(CO)(PtBu2NtBu2H)]+. The pKa values determined in acetonitrile range from 9.3 to 17.7, and show a linear correlation with the logarithm of the exchange rates. Thus the exchange dynamics are controlled through the relative acidity of the [CpMo(H)(CO)(P2N2H)]+ and [CpMo(H2)(CO)(P2N2)]+ isomers, providing a design principle for controlling heterolytic cleavage of H2.

Roles of the Picornaviral 3C Proteinase in the Viral Life Cycle and Host Cells

Directory of Open Access Journals (Sweden)

Di Sun

2016-03-01

Full Text Available The Picornaviridae family comprises a large group of non-enveloped viruses that have a major impact on human and veterinary health. The viral genome contains one open reading frame encoding a single polyprotein that can be processed by viral proteinases. The crucial 3C proteinases (3Cpros of picornaviruses share similar spatial structures and it is becoming apparent that 3Cpro plays a significant role in the viral life cycle and virus host interaction. Importantly, the proteinase and RNA-binding activity of 3Cpro are involved in viral polyprotein processing and the initiation of viral RNA synthesis. In addition, 3Cpro can induce the cleavage of certain cellular factors required for transcription, translation and nucleocytoplasmic trafficking to modulate cell physiology for viral replication. Due to interactions between 3Cpro and these essential factors, 3Cpro is also involved in viral pathogenesis to support efficient infection. Furthermore, based on the structural conservation, the development of irreversible inhibitors and discovery of non-covalent inhibitors for 3Cpro are ongoing and a better understanding of the roles played by 3Cpro may provide insights into the development of potential antiviral treatments. In this review, the current knowledge regarding the structural features, multiple functions in the viral life cycle, pathogen host interaction, and development of antiviral compounds for 3Cpro is summarized.

AcEST: DK961630 [AcEST

Lifescience Database Archive (English)

Full Text Available racterized protein OS=Rhodo... 36 0.85 tr|Q0ZQM0|Q0ZQM0_9PICO Polyprotein OS=Duck hepatitis virus 1 PE=... 3...5 2.5 tr|Q0ZQL9|Q0ZQL9_9PICO Polyprotein OS=Duck hepatitis virus 1 PE=... 35 2.5 tr|Q0GH72|Q0GH72_9PICO Polyprotein OS=Duck hepatitis... virus 1 PE=... 35 2.5 tr|Q0GA17|Q0GA17_9PICO Polyprotein OS=Duck hepatitis virus 1... PE=... 35 2.5 tr|Q078N6|Q078N6_9PICO Polyprotein OS=Duck hepatitis virus 1 PE=...... 35 2.5 tr|Q076B4|Q076B4_9PICO Polyprotein OS=Duck hepatitis virus 1 PE=... 35 2.5 tr|B8Y8E4|B8Y8E4_9PICO Polyprotein OS=Duck hepati

Kinetic modeling and exploratory numerical simulation of chloroplastic starch degradation

Directory of Open Access Journals (Sweden)

Nag Ambarish

2011-06-01

lower coupling between metabolic subsystems in different compartments. Conclusions We find that in the reference steady state, starch cleavage is the most significant determinant of carbon flux, with turnover of oligosaccharides playing a secondary role. Independence of stationary point with respect to initial dynamic variable values confirms a unique stationary point in the phase space of dynamically varying concentrations of the model network. Stromal maltooligosaccharide metabolism was highly coupled to the available starch concentration. From the most highly converged trajectories, distances between unique fixed points of phase spaces show that cytosolic maltose levels depend on the total concentrations of arabinogalactan and glucose present in the cytosol. In addition, cellular compartmentalization serves to dampen much, but not all, of the effects of one subnetwork on another, such that kinetic modeling of single compartments would likely capture most dynamics that are fast on the timescale of the transport reactions.

Alkali metal control over N-N cleavage in iron complexes.

Science.gov (United States)

Grubel, Katarzyna; Brennessel, William W; Mercado, Brandon Q; Holland, Patrick L

2014-12-03

Though N2 cleavage on K-promoted Fe surfaces is important in the large-scale Haber-Bosch process, there is still ambiguity about the number of Fe atoms involved during the N-N cleaving step and the interactions responsible for the promoting ability of K. This work explores a molecular Fe system for N2 reduction, particularly focusing on the differences in the results obtained using different alkali metals as reductants (Na, K, Rb, Cs). The products of these reactions feature new types of Fe-N2 and Fe-nitride cores. Surprisingly, adding more equivalents of reductant to the system gives a product in which the N-N bond is not cleaved, indicating that the reducing power is not the most important factor that determines the extent of N2 activation. On the other hand, the results suggest that the size of the alkali metal cation can control the number of Fe atoms that can approach N2, which in turn controls the ability to achieve N2 cleavage. The accumulated results indicate that cleaving the triple N-N bond to nitrides is facilitated by simultaneous approach of least three low-valent Fe atoms to a single molecule of N2.

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

Science.gov (United States)

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

2014-12-01

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

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

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Tong Guo

2012-10-01

Full Text Available Abstract 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 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.

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

Science.gov (United States)

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.

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

Science.gov (United States)

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

Coronavirus 3CL(pro) proteinase cleavage sites: Possible relevance to SARS virus pathology

DEFF Research Database (Denmark)

Kiemer, Lars; Lund, Ole; Brunak, Søren

2004-01-01

the picornaviruses it is known that pathology is related to proteolytic cleavage of host proteins by viral proteinases. Furthermore, several studies indicate that virus proliferation can be arrested using specific proteinase inhibitors supporting the belief that proteinases are indeed important during infection...

Recent Advances in Ring-Opening Functionalization of Cycloalkanols by C-C σ-Bond Cleavage.

Science.gov (United States)

Wu, Xinxin; Zhu, Chen

2018-06-01

Cycloalkanols prove to be privileged precursors for the synthesis of distally substituted alkyl ketones and polycyclic aromatic hydrocarbons (PAHs) by virtue of cleavage of their cyclic C-C bonds. Direct functionalization of cyclobutanols to build up other chemical bonds (e. g., C-F, C-Cl, C-Br, C-N, C-S, C-Se, C-C, etc.) has been achieved by using the ring-opening strategy. Mechanistically, the C-C cleavage of cyclobutanols can be involved in two pathways: (a) transition-metal catalyzed β-carbon elimination; (b) radical-mediated 'radical clock'-type ring opening. The recent advances of our group for the ring-opening functionalization of tertiary cycloalkanols are described in this account. © 2018 The Chemical Society of Japan & Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.

Urokinase links plasminogen activation and cell adhesion by cleavage of the RGD motif in vitronectin.

Science.gov (United States)

De Lorenzi, Valentina; Sarra Ferraris, Gian Maria; Madsen, Jeppe B; Lupia, Michela; Andreasen, Peter A; Sidenius, Nicolai

2016-07-01

Components of the plasminogen activation system including urokinase (uPA), its inhibitor (PAI-1) and its cell surface receptor (uPAR) have been implicated in a wide variety of biological processes related to tissue homoeostasis. Firstly, the binding of uPA to uPAR favours extracellular proteolysis by enhancing cell surface plasminogen activation. Secondly, it promotes cell adhesion and signalling through binding of the provisional matrix protein vitronectin. We now report that uPA and plasmin induces a potent negative feedback on cell adhesion through specific cleavage of the RGD motif in vitronectin. Cleavage of vitronectin by uPA displays a remarkable receptor dependence and requires concomitant binding of both uPA and vitronectin to uPAR Moreover, we show that PAI-1 counteracts the negative feedback and behaves as a proteolysis-triggered stabilizer of uPAR-mediated cell adhesion to vitronectin. These findings identify a novel and highly specific function for the plasminogen activation system in the regulation of cell adhesion to vitronectin. The cleavage of vitronectin by uPA and plasmin results in the release of N-terminal vitronectin fragments that can be detected in vivo, underscoring the potential physiological relevance of the process. © 2016 The Authors.

ADAM13 cleavage of cadherin-11 promotes CNC migration independently of the homophilic binding site.

Science.gov (United States)

Abbruzzese, Genevieve; Becker, Sarah F; Kashef, Jubin; Alfandari, Dominique

2016-07-15

The cranial neural crest (CNC) is a highly motile population of cells that is responsible for forming the face and jaw in all vertebrates and perturbing their migration can lead to craniofacial birth defects. Cell motility requires a dynamic modification of cell-cell and cell-matrix adhesion. In the CNC, cleavage of the cell adhesion molecule cadherin-11 by ADAM13 is essential for cell migration. This cleavage generates a shed extracellular fragment of cadherin-11 (EC1-3) that possesses pro-migratory activity via an unknown mechanism. Cadherin-11 plays an important role in modulating contact inhibition of locomotion (CIL) in the CNC to regulate directional cell migration. Here, we show that while the integral cadherin-11 requires the homophilic binding site to promote CNC migration in vivo, the EC1-3 fragment does not. In addition, we show that increased ADAM13 activity or expression of the EC1-3 fragment increases CNC invasiveness in vitro and blocks the repulsive CIL response in colliding cells. This activity requires the presence of an intact homophilic binding site on the EC1-3 suggesting that the cleavage fragment may function as a competitive inhibitor of cadherin-11 adhesion in CIL but not to promote cell migration in vivo. Copyright © 2015. Published by Elsevier Inc.

Analysis of the Proteolytic Processing of ABCA3: Identification of Cleavage Site and Involved Proteases.

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Nicole Hofmann

Full Text Available ABCA3 is a lipid transporter in the limiting membrane of lamellar bodies in alveolar type II cells. Mutations in the ABCA3 gene cause respiratory distress syndrome in new-borns and childhood interstitial lung disease. ABCA3 is N-terminally cleaved by an as yet unknown protease, a process believed to regulate ABCA3 activity.The exact site where ABCA3 is cleaved was localized using mass spectrometry (MS. Proteases involved in ABCA3 processing were identified using small molecule inhibitors and siRNA mediated gene knockdown. Results were verified by in vitro digestion of a synthetic peptide substrate mimicking ABCA3's cleavage region, followed by MS analysis.We found that cleavage of ABCA3 occurs after Lys174 which is located in the proteins' first luminal loop. Inhibition of cathepsin L and, to a lesser extent, cathepsin B resulted in attenuation of ABCA3 cleavage. Both enzymes showed activity against the ABCA3 peptide in vitro with cathepsin L being more active.We show here that, like some other proteins of the lysosomal membrane, ABCA3 is a substrate of cathepsin L. Therefore, cathepsin L may represent a potential target to therapeutically influence ABCA3 activity in ABCA3-associated lung disease.

DNA-binding, DNA cleavage and cytotoxicity studies of two anthraquinone derivatives.

Science.gov (United States)

Gholivand, M B; Kashanian, S; Peyman, H

2012-02-15

The interaction of native calf thymus DNA (CT-DNA) with two anthraquinones including quinizarin (1,4-dihydroxy anthraquinone) and danthron (1,8-dihydroxy anthraquinone) in a mixture of 0.04M Brittone-Robinson buffer and 50% of ethanol were studied at physiological pH by spectrofluorometric and cyclic voltammetry techniques. The former technique was used to calculate the binding constants of anthraquinones-DNA complexes at different temperatures. Thermodynamic study indicated that the reactions of both anthraquinone-DNA systems are predominantly entropically driven. Furthermore, the binding mechanisms on the reaction of the two anthraquinones with DNA and the effect of ionic strength on the fluorescence property of the system have also been investigated. The results of the experiments indicated that the binding modes of quinizarin and danthron with DNA were evaluated to be groove binding. Moreover, the cytotoxic activity of both compounds against human chronic myelogenous leukemia K562 cell line and DNA cleavage were investigated. The results indicated that these compounds slightly cleavage pUC18 plasmid DNA and showed minor antitumor activity against K562 (human chronic myeloid leukemia) cell line. Copyright © 2011 Elsevier B.V. All rights reserved.

Copper-Catalyzed Oxidative Reaction of β-Keto Sulfones with Alcohols via C-S Bond Cleavage: Reaction Development and Mechanism Study.

Science.gov (United States)

Du, Bingnan; Wang, Wenmin; Wang, Yang; Qi, Zhenghang; Tian, Jiaqi; Zhou, Jie; Wang, Xiaochen; Han, Jianlin; Ma, Jing; Pan, Yi

2018-02-16

A Cu-catalyzed cascade oxidative radical process of β-keto sulfones with alcohols has been achieved by using oxygen as an oxidant. In this reaction, β-keto sulfones were converted into sulfinate esters under the oxidative conditions via cleavage of C-S bond. Experimental and computational studies demonstrate that a new pathway is involved in this reaction, which proceeds through the formation of the key four-coordinated Cu II intermediate, O-O bond homolysis induced C-S bond cleavage and Cu-catalyzed esterification to form the final products. This reaction provides a new strategy to sulfonate esters and enriches the research content of C-S bond cleavage and transformations. © 2018 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.

Effect of oxygen and nitroaromatic cell radiosensitizers on radiation-induced cleavage of internucleotide bonds: ApA, dApA, and poly(A)

International Nuclear Information System (INIS)

Raleigh, J.A.; Kremers, W.; Whitehouse, R.

1975-01-01

Irradiation of the dinucleoside monophosphates ApA and dApA in deoxygenated solution leads to a preferential cleavage of the 3' end of the internucleotide bond. Cleavage at the 3' bond is favored to the extent of 2 to 1 over 5' cleavage. Oxygen and nitroaromatic compounds inhibit 3' bond breaking in ApA and dApA in agreement with earlier findings from studies of 3'- and 5'-mononucleotides. In contrast to the mononucleotide results, no enhancement of 5' cleavage is observed for ApA and dApA irradiated in the presence of oxygen or the nitroaromatic additives. The over-all effect of the additives is to decrease the combined (3' and 5') yield of internucleotide bond breaking in ApA and dApA. This phenomenon is also observed for polyadenylic acid in the presence of the nitroaromatics. Oxygen marginally enhances internucleotide bond breaking in polyadenylic acid (factor 1.1) over that seen in deoxygenated solution. Postirradiation alkaline hydrolysis of dApA leads to further ester cleavage revealing the presence of radiation-induced alkali-labile bonds. The number of these bonds decreases in the order oxygen greater than nitrofurans greater than nitrobenzenes approximately irradiation in the absence of additives

Regulation of proteolytic cleavage of brain-derived neurotrophic factor precursor by antidepressants in human neuroblastoma cells

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Lin PY

2015-10-01

Full Text Available Pao-Yen Lin1,2 1Department of Psychiatry, Kaohsiung Chang Gung Memorial Hospital, Chang Gung University College of Medicine, 2Center for Translational Research in Biomedical Sciences, Kaohsiung Chang Gung Memorial Hospital, Kaohsiung, Taiwan Abstract: Evidence has supported the role of brain-derived neurotrophic factor (BDNF in antidepressant effect. The precursor of BDNF (proBDNF often exerts opposing biological effects on mature BDNF (mBDNF. Hence, the balance between proBDNF and mBDNF might be critical in total neurotrophic effects, leading to susceptibility to or recovery from depression. In the current study, we measured the protein expression levels of proBDNF, and its proteolytic products, truncated BDNF, and mBDNF, in human SH-SY5Y cells treated with different antidepressants. We found that the treatment significantly increased the production of mBDNF, but decreased the production of truncated BDNF and proBDNF. These results support that antidepressants can promote proBDNF cleavage. Further studies are needed to clarify whether proBDNF cleavage plays a role in antidepressant mechanisms. Keywords: antidepressant, mature BDNF, neurotrophic effect, proBDNF cleavage 

Infectious Maize rayado fino virus from Cloned cDNA.

Science.gov (United States)

Edwards, Michael C; Weiland, John J; Todd, Jane; Stewart, Lucy R

2015-06-01

A full-length cDNA clone was produced from a U.S. isolate of Maize rayado fino virus (MRFV), the type member of the genus Marafivirus within the family Tymoviridae. Infectivity of transcripts derived from cDNA clones was demonstrated by infection of maize plants and protoplasts, as well as by transmission via the known leafhopper vectors Dalbulus maidis and Graminella nigrifrons that transmit the virus in a persistent-propagative manner. Infection of maize plants through vascular puncture inoculation of seed with transcript RNA resulted in the induction of fine stipple stripe symptoms typical of those produced by wild-type MRFV and a frequency of infection comparable with that of the wild type. Northern and Western blotting confirmed the production of MRFV-specific RNAs and proteins in infected plants and protoplasts. An unanticipated increase in subgenomic RNA synthesis over levels in infected plants was observed in protoplasts infected with either wild-type or cloned virus. A conserved cleavage site motif previously demonstrated to function in both Oat blue dwarf virus capsid protein and tymoviral nonstructural protein processing was identified near the amino terminus of the MRFV replicase polyprotein, suggesting that cleavage at this site also may occur.

Identification of prokaryotic and eukaryotic signal peptides and prediction of their cleavage sites

DEFF Research Database (Denmark)

Nielsen, Henrik; Engelbrecht, Jacob; Brunak, Søren

1997-01-01

We have developed a new method for the identification of signal peptides and their cleavage based on neural networks trained on separate sets of prokaryotic and eukaryotic sequence. The method performs significantly better than previous prediction schemes and can easily be applied on genome...

Change in radiosensitivity on the development of sea urchin eggs during the early cleavage stage, 2

International Nuclear Information System (INIS)

Nakamura, Izumi

1975-01-01

The effect of cysteamine on the fluctuation of X-ray sensitivity during early cleavage stage of sea urchin eggs expressed by pluteus formation rate was examined. Sea urchin eggs were very resistant to radiation immediately after insemination or after S phase of the cleavage. When irradiation was given just prior to S phase or in the phase of cytokinesis, the eggs were very sensitive. In these sensitive stages, existence of cysteamine during X-irradiation apparently protected eggs against radiation effect. Dose modifying factor increased linearly from 1.8 to 3.4 with increasing dose of cysteamine (25mM-75mM) added. (auth.)

Multi scale study of the plasticity at low temperature in α-iron: application for the cleavage

International Nuclear Information System (INIS)

Chaussidon, J.

2007-10-01

An accident inside a nuclear power plant may lead to the cleavage of the nuclear vessel made of bainitic steel. In order to understand the origin of this fracture, we studied BCC-iron plasticity at low temperature using numerical simulations at different scales. Molecular Dynamics simulations show the high dependency of screw dislocation motion with temperature and stress. Results from these simulations were added to experiment data to develop a new Dislocation Dynamics code dedicated to BCC iron at low temperature. The code was used to model plasticity into a ferritic lath for various temperatures. This work confirms that cleavage is favoured by low temperatures. (author)

Enhancing Protein Disulfide Bond Cleavage by UV Excitation and Electron Capture Dissociation for Top-Down Mass Spectrometry.

Science.gov (United States)

Wongkongkathep, Piriya; Li, Huilin; Zhang, Xing; Loo, Rachel R Ogorzalek; Julian, Ryan R; Loo, Joseph A

2015-11-15

The application of ion pre-activation with 266 nm ultraviolet (UV) laser irradiation combined with electron capture dissociation (ECD) is demonstrated to enhance top-down mass spectrometry sequence coverage of disulfide bond containing proteins. UV-based activation can homolytically cleave a disulfide bond to yield two separated thiol radicals. Activated ECD experiments of insulin and ribonuclease A containing three and four disulfide bonds, respectively, were performed. UV-activation in combination with ECD allowed the three disulfide bonds of insulin to be cleaved and the overall sequence coverage to be increased. For the larger sized ribonuclease A with four disulfide bonds, irradiation from an infrared laser (10.6 µm) to disrupt non-covalent interactions was combined with UV-activation to facilitate the cleavage of up to three disulfide bonds. Preferences for disulfide bond cleavage are dependent on protein structure and sequence. Disulfide bonds can reform if the generated radicals remain in close proximity. By varying the time delay between the UV-activation and the ECD events, it was determined that disulfide bonds reform within 10-100 msec after their UV-homolytic cleavage.

Mechanisms of Bond Cleavage during Manganese Oxide and UV Degradation of Glyphosate: Results from Phosphate Oxygen Isotopes and Molecular Simulations.

Science.gov (United States)

Jaisi, Deb P; Li, Hui; Wallace, Adam F; Paudel, Prajwal; Sun, Mingjing; Balakrishna, Avula; Lerch, Robert N

2016-11-16

Degradation of glyphosate in the presence of manganese oxide and UV light was analyzed using phosphate oxygen isotope ratios and density function theory (DFT). The preference of C-P or C-N bond cleavage was found to vary with changing glyphosate/manganese oxide ratios, indicating the potential role of sorption-induced conformational changes on the composition of intermediate degradation products. Isotope data confirmed that one oxygen atom derived solely from water was incorporated into the released phosphate during glyphosate degradation, and this might suggest similar nucleophilic substitution at P centers and C-P bond cleavage both in manganese oxide- and UV light-mediated degradation. The DFT results reveal that the C-P bond could be cleaved by water, OH - or • OH, with the energy barrier opposing bond dissociation being lowest in the presence of the radical species, and that C-N bond cleavage is favored by the formation of both nitrogen- and carbon-centered radicals. Overall, these results highlight the factors controlling the dominance of C-P or C-N bond cleavage that determines the composition of intermediate/final products and ultimately the degradation pathway.

Di-µ-hydroxo Bridge Cleavage Reactions between [Co(nta)(µ-OH)] 2 ...

African Journals Online (AJOL)

NJD

2004-04-22

Apr 22, 2004 ... subsequent rate determining steps to form presumably a ligand-substituted, mono-bridged complex, [(nta)(OH)Co-µ-. OH-Co(nta)(L)]2– (L = py/dmap). The latter decomposes rapidly to form the products. The preferred pathway for these bridge cleavage seemed to be the reaction of the mono-µ-hydroxo-.

Aqueous photodegradation of antibiotic florfenicol: kinetics and degradation pathway studies.

Science.gov (United States)

Zhang, Ya; Li, Jianhua; Zhou, Lei; Wang, Guoqing; Feng, Yanhong; Wang, Zunyao; Yang, Xi

2016-04-01

The occurrence of antibacterial agents in natural environment was of scientific concern in recent years. As endocrine disrupting chemicals, they had potential risk on ecology system and human beings. In the present study, the photodegradation kinetics and pathways of florfenicol were investigated under solar and xenon lamp irradiation in aquatic systems. Direct photolysis half-lives of florfenicol were determined as 187.29 h under solar irradiation and 22.43 h under xenon lamp irradiation, respectively. Reactive oxygen species (ROS), such as hydroxyl radical (·OH) and singlet oxygen ((1)O2) were found to play an important role in indirect photolysis process. The presence of nitrate and dissolved organic matters (DOMs) could affect photolysis of florfenicol in solutions through light screening effect, quenching effect, and photoinduced oxidization process. Photoproducts of florfenicol in DOMs solutions were identified by solid phase extraction-liquid chromatography-mass spectrometry (SPE-LC-MS) analysis techniques, and degradation pathways were proposed, including photoinduced hydrolysis, oxidation by (1)O2 and ·OH, dechlorination, and cleavage of the side chain.

A study of complex defects failing by fatigue, ductile tearing and cleavage

International Nuclear Information System (INIS)

Bezensek, B.; Ren, Z.; Hancock, J.W.

2001-01-01

Defect assessment procedures ensure the structural integrity of plant, which may contain complex defects. The present work addresses complex defects with re-entrant sectors, which develop from the interaction of two co-planar surface breaking defects in fatigue. Experimental studies show rapid fatigue growth and amplified crack driving forces in the re-entrant sector. This leads to the rapid evolution of the complex crack into a bounding semielliptical defect. Experiments involving ductile tearing of cracks with a re-entrant sector show that tearing initiates in the re-entrant sector and that the defect evolves into a bounding semielliptical defect. Cleavage failures of defects with re-entrant sectors indicate the re-characterisation procedure is only conservative after invoking constraint arguments. The study confirms the conservatism inherent in the re-characterisation rules of assessment procedures, such as BS 7910 [1] and ASME Section XI [2] for complex defects extending by fatigue or ductile tearing. A potentially non-conservative situation exists for defects with re-entrant sectors failing by cleavage at small fractions of the limit load.(author)

AcEST: BP911943 [AcEST

Lifescience Database Archive (English)

Full Text Available ructural polyprotein OS=Eastern equine encephalitis virus (strain PE-3.0815) Align length 24 Score (bit) 30....alue sp|Q306W7|POLS_EEEV8 Structural polyprotein OS=Eastern equine en... 30 3.7 sp|Q306W5|POLS_EEEV1 Structu...ral polyprotein OS=Eastern equine en... 30 3.7 sp|Q74HV0|LUXS_LACJO S-ribosylhomocysteine lyase OS=Lactobaci...llu... 30 4.8 sp|Q4QXJ7|POLS_EEEVF Structural polyprotein OS=Eastern equine en...... 29 8.3 sp|P27284|POLS_EEEV3 Structural polyprotein OS=Eastern equine en... 29 8.3 sp|P08768|POLS_EEEV Struc

Degradation of tropoelastin by matrix metalloproteinases--cleavage site specificities and release of matrikines

DEFF Research Database (Denmark)

Heinz, Andrea; Jung, Michael C; Duca, Laurent

2010-01-01

To provide a basis for the development of approaches to treat elastin-degrading diseases, the aim of this study was to investigate the degradation of the natural substrate tropoelastin by the elastinolytic matrix metalloproteinases MMP-7, MMP-9, and MMP-12 and to compare the cleavage site...

AcEST: DK958683 [AcEST

Lifescience Database Archive (English)

Full Text Available nome polyprotein OS=Hepatitis C virus gen... 33 0.92 sp|Q8AZM0|POLS_BSNV Structural polyprotein OS=Blotched snake...KSPPDEPDDSEAGSVSSMPPLEG 2401 >sp|Q8AZM0|POLS_BSNV Structural polyprotein OS=Blotched snake

High-fat diet feeding causes rapid, non-apoptotic cleavage of caspase-3 in astrocytes.

Science.gov (United States)

Guyenet, Stephan J; Nguyen, Hong T; Hwang, Bang H; Schwartz, Michael W; Baskin, Denis G; Thaler, Joshua P

2013-05-28

Astrocytes respond to multiple forms of central nervous system (CNS) injury by entering a reactive state characterized by morphological changes and a specific pattern of altered protein expression. Termed astrogliosis, this response has been shown to strongly influence the injury response and functional recovery of CNS tissues. This pattern of CNS inflammation and injury associated with astrogliosis has recently been found to occur in the energy homeostasis centers of the hypothalamus during diet-induced obesity (DIO) in rodent models, but the characterization of the astrocyte response remains incomplete. Here, we report that astrocytes in the mediobasal hypothalamus respond robustly and rapidly to purified high-fat diet (HFD) feeding by cleaving caspase-3, a protease whose cleavage is often associated with apoptosis. Although obesity develops in HFD-fed rats by day 14, caspase-3 cleavage occurs by day 3, prior to the development of obesity, suggesting the possibility that it could play a causal role in the hypothalamic neuropathology and fat gain observed in DIO. Caspase-3 cleavage is not associated with an increase in the rate of apoptosis, as determined by TUNEL staining, suggesting it plays a non-apoptotic role analogous to the response to excitotoxic neuron injury. Our results indicate that astrocytes in the mediobasal hypothalamus respond rapidly and robustly to HFD feeding, activating caspase-3 in the absence of apoptosis, a process that has the potential to influence the course of DIO. Copyright © 2013 Elsevier B.V. All rights reserved.

KLONING GEN PUTATIVE CLEAVAGE PROTEIN 1 (PCP-1 PADA UDANG VANAME (Litopenaeus vannamei YANG TERSERANG INFECTIOUS MYONECROSIS VIRUS

Directory of Open Access Journals (Sweden)

Hessy Novita

2016-12-01

Full Text Available Penanggulangan penyakit ikan dapat dilakukan dengan cara meningkatkan kekebalan tubuh ikan melalui program vaksinasi. Namun vaksinasi tidak tepat untuk udang, karena udang tidak mempunyai immunological memory seperti ikan. Oleh karena itu, perlindungan udang terhadap serangan penyakit viral dengan menggunakan RNA interference (RNAi. Teknologi RNAi digunakan untuk menghalangi (interfere proses replikasi infectious myonecrosis virus (IMNV pada udang vaname dengan cara menon-aktifkan gen putative cleavage protein 1 (PCP-1, yang berfungsi dalam pembentukan capsid dan proses transkripsi RNA IMNV. Penelitian ini bertujuan untuk melakukan kloning gen putative cleavage protein 1 dalam rangka perakitan teknologi RNAi untuk pengendalian penyakit IMNV pada udang vaname. Tahapan penelitian meliputi koleksi sampel, isolasi RNA, sintesis cDNA, amplifikasi PCR, purifikasi DNA, transformasi, isolasi plasmid, serta sekuensing dan analisis data. Hasil isolasi plasmid cDNA PCP-1 memperlihatkan semua koloni bakteri terseleksi ternyata membawa plasmid hasil insersi DNA gen PCP–1, hasil sekuen dengan nilai homologinya mencapai 100% dan 99% yang dibandingkan dengan sekuen di Genebank. Hasil penelitian menunjukkan bahwa kloning gen putative cleavage protein 1 (PCP-1 dari udang vaname yang terserang Infectious Myonecrosis Virus berhasil dikloning yang nantinya digunakan untuk perakitan RNAi. The prevention of fish diseases can be done by increasing of the fish immune through vaccination programs. However, the vaccination can not be done for the shrimp,due to the absence of  immunological memory. Therefore, the protection of shrimp against viral diseases was done by using of RNA interference (RNAi. RNAi technology is used to interfere infectious myonecrosis virus (IMNV replication process on white shrimp by disabling of putative cleavage protein 1 (PCP-1gene, which functions in capsid formation and RNA transcription process. The study was conducted to perform putative

Evolutionary relationships within the human rhinovirus genus: comparison of serotypes 89, 2, and 14

International Nuclear Information System (INIS)

Duechler, M.; Skern, T.; Sommergruber, W.; Neubauer, C.; Gruendler, P.; Fogy, I.; Blaas, D.; Kuechler, E.

1987-01-01

The complete nucleotide sequence of the genome of human rhinovirus type 89 was determined from the cDNA that had been cloned into Escherichia coli. The genome is 7152 nucleotides long and contains a single large open reading frame of 2164 codons. Translation commences at position 619 and ends 42 nucleotides before the poly(a) tract. The positions of three proteolytic cleavage sites in the polyprotein were determined by N-terminal amino acid sequencing of the capsid proteins; the remainder were predicted from comparisons with other picornaviruses. Extensive similarity between the derived amino acid sequences of human rhinovirus types 89 and 2 was found, whereas the similarity between human rhinovirus types 89 and 14 was considerably less. It is apparent that human rhinoviruses may be more closely related than has been previously thought

Partial sequence determination of metabolically labeled radioactive proteins and peptides

International Nuclear Information System (INIS)

Anderson, C.W.

1982-01-01

The author has used the sequence analysis of radioactive proteins and peptides to approach several problems during the past few years. They, in collaboration with others, have mapped precisely several adenovirus proteins with respect to the nucleotide sequence of the adenovirus genome; identified hitherto missed proteins encoded by bacteriophage MS2 and by simian virus 40; analyzed the aminoterminal maturation of several virus proteins; determined the cleavage sites for processing of the poliovirus polyprotein; and analyzed the mechanism of frameshifting by excess normal tRNAs during cell-free protein synthesis. This chapter is designed to aid those without prior experience at protein sequence determinations. It is based primarily on the experience gained in the studies cited above, which made use of the Beckman 890 series automated protein sequencers

The mitochondrion-like organelle of Trimastix pyriformis contains the complete glycine cleavage system

Czech Academy of Sciences Publication Activity Database

Zubáčová, Z.; Novák, L.; Bublíková, J.; Vacek, V.; Fousek, Jan; Rídl, Jakub; Tachezy, J.; Doležal, P.; Vlček, Čestmír; Hampl, V.

2013-01-01

Roč. 8, č. 3 (2013), e55417 E-ISSN 1932-6203 R&D Projects: GA ČR GAP506/12/1010 Institutional support: RVO:68378050 Keywords : transcriptome sequencing * Trimastix * mitochondrion -like organelle * glycine cleavage complex Subject RIV: EB - Genetics ; Molecular Biology Impact factor: 3.534, year: 2013

The timing of pronuclear formation, DNA synthesis and cleavage in the human 1-cell embryo.

Science.gov (United States)

Capmany, G; Taylor, A; Braude, P R; Bolton, V N

1996-05-01

The timing of pronuclear formation and breakdown, DNA synthesis and cleavage during the first cell cycle of human embryogenesis are described. Pronuclei formed between 3 and 10 h post-insemination (hpi; median 8 hpi). S-phase commenced between 8 and 14 hpi, and was completed between 10 and 18 hpi. M-phase was observed between 22 and 31 hpi (median duration 3 h), and cleavage to the 2-cell stage took place between 25 and 33 hpi. The timing of the same events was determined in 1-cell embryos derived from re-inseminated human oocytes that had failed to fertilize during therapeutic in-vitro fertilization (IVF). In these embryos, pronuclei formed between 3 and 8 h post-re-insemination (hpr-i), coinciding with the beginning of S-phase. While S-phase was completed as early as 10 hpr-i in some embryos, it extended until at least 16 hpr-i in others. Pronuclear breakdown and cleavage occurred from 23 and 26 hpr-i respectively; however, they did not occur in some embryos until after 46 hpr-i. The results demonstrate a markedly greater degree of variation in the timing of these events in embryos derived from re-inseminated oocytes compared with embryos derived from conventional IVF, and thus throw into question the validity of using the former as models for studies of the first cell cycle of human embryogenesis.

Novel carotenoid cleavage dioxygenase catalyzes the first dedicated step in saffron crocin biosynthesis

KAUST Repository

Frusciante, Sarah; Diretto, Gianfranco; Bruno, Mark; Ferrante, Paola; Pietrella, Marco; Prado-Cabrero, Alfonso; Rubio-Moraga, Á ngela L.; Beyer, Peter D.; Gó mez-Gó mez, Lourdes; Al-Babili, Salim; Giuliano, Giovanni

2014-01-01

Crocus sativus stigmas are the source of the saffron spice and accumulate the apocarotenoids crocetin, crocins, picrocrocin, and safranal, responsible for its color, taste, and aroma. Through deep transcriptome sequencing, we identified a novel dioxygenase, carotenoid cleavage dioxygenase 2 (CCD2), expressed early during stigma development and closely related to, but distinct from, the CCD1 dioxygenase family. CCD2 is the only identified member of a novel CCD clade, presents the structural features of a bona fide CCD, and is able to cleave zeaxanthin, the presumed precursor of saffron apocarotenoids, both in Escherichia coli and in maize endosperm. The cleavage products, identified through high-resolution mass spectrometry and comigration with authentic standards, are crocetin dialdehyde and crocetin, respectively. In vitro assays show that CCD2 cleaves sequentially the 7,8 and 7′,8′ double bonds adjacent to a 3-OH-β-ionone ring and that the conversion of zeaxanthin to crocetin dialdehyde proceeds via the C30 intermediate 3-OH-β-apo-8′-carotenal. In contrast, zeaxanthin cleavage dioxygenase (ZCD), an enzyme previously claimed to mediate crocetin formation, did not cleave zeaxanthin or 3-OH-β-apo-8′-carotenal in the test systems used. Sequence comparison and structure prediction suggest that ZCD is an N-truncated CCD4 form, lacking one blade of the β-propeller structure conserved in all CCDs. These results constitute strong evidence that CCD2 catalyzes the first dedicated step in crocin biosynthesis. Similar to CCD1, CCD2 has a cytoplasmic localization, suggesting that it may cleave carotenoids localized in the chromoplast outer envelope.

Novel carotenoid cleavage dioxygenase catalyzes the first dedicated step in saffron crocin biosynthesis

KAUST Repository

Frusciante, Sarah

2014-08-05

Crocus sativus stigmas are the source of the saffron spice and accumulate the apocarotenoids crocetin, crocins, picrocrocin, and safranal, responsible for its color, taste, and aroma. Through deep transcriptome sequencing, we identified a novel dioxygenase, carotenoid cleavage dioxygenase 2 (CCD2), expressed early during stigma development and closely related to, but distinct from, the CCD1 dioxygenase family. CCD2 is the only identified member of a novel CCD clade, presents the structural features of a bona fide CCD, and is able to cleave zeaxanthin, the presumed precursor of saffron apocarotenoids, both in Escherichia coli and in maize endosperm. The cleavage products, identified through high-resolution mass spectrometry and comigration with authentic standards, are crocetin dialdehyde and crocetin, respectively. In vitro assays show that CCD2 cleaves sequentially the 7,8 and 7′,8′ double bonds adjacent to a 3-OH-β-ionone ring and that the conversion of zeaxanthin to crocetin dialdehyde proceeds via the C30 intermediate 3-OH-β-apo-8′-carotenal. In contrast, zeaxanthin cleavage dioxygenase (ZCD), an enzyme previously claimed to mediate crocetin formation, did not cleave zeaxanthin or 3-OH-β-apo-8′-carotenal in the test systems used. Sequence comparison and structure prediction suggest that ZCD is an N-truncated CCD4 form, lacking one blade of the β-propeller structure conserved in all CCDs. These results constitute strong evidence that CCD2 catalyzes the first dedicated step in crocin biosynthesis. Similar to CCD1, CCD2 has a cytoplasmic localization, suggesting that it may cleave carotenoids localized in the chromoplast outer envelope.

PE2 cleavage mutants of Sindbis virus : Correlation between viral infectivity and pH-dependent membrane fusion activation of the spike heterodimer

NARCIS (Netherlands)

Smit, JM; Klimstra, WB; Ryman, KD; Bittman, R; Johnston, RE; Wilschut, J

2001-01-01

The spike glycoprotein E2 of Sindbis virus (SIN) is synthesized in the infected cell as a PE2 precursor protein, which matures through cleavage by a cellular furin-like protease. Previous work has shown that SIN mutants impaired in PE2 cleavage are noninfectious on BHK-21 cells, the block in

Morphogenesis of bacteriophage phi29 of Bacillus subtilis: cleavage and assembly of the neck appendage protein

International Nuclear Information System (INIS)

Tosi, M.E.; Reilly, B.E.; Anderson, D.L.

1975-01-01

Each of the 12 neck appendages of the Bacillus subtilis bacteriophage phi 29 consists of a single protein molecular weight of about 75,000, and on the mature virion the appendages are assembled to the lower of two collars. The appendage protein is cleaved from a percursor protein, P(J), with a molecular weight of about 88,000. This cleavage is independent of neck assembly, occurring during infection by mutants that cannot synthesize the proteins of the upper and lower collars of the neck. The cleaved form of the appendage protein is efficiently complemented in vitro to particles lacking appendages. Thus, cleavage of the appendage precursor protein apparently does not occur in situ on the maturing virus

Photocatalytic degradation kinetics and mechanism of environmental pharmaceuticals in aqueous suspension of TiO{sub 2}: A case of {beta}-blockers

Energy Technology Data Exchange (ETDEWEB)

Yang Hai [State Key Laboratory of Organic Geochemistry and Guangdong Key Laboratory of Environmental Resources Utilization and Protection, Guangzhou Institute of Geochemistry, Chinese Academy of Sciences, Kehua Street, Tianhe District, Guangzhou 510640 (China); Graduate School of Chinese Academy of Sciences, Beijing 100049 (China); An Taicheng, E-mail: antc99@gig.ac.cn [State Key Laboratory of Organic Geochemistry and Guangdong Key Laboratory of Environmental Resources Utilization and Protection, Guangzhou Institute of Geochemistry, Chinese Academy of Sciences, Kehua Street, Tianhe District, Guangzhou 510640 (China); Li Guiying [State Key Laboratory of Organic Geochemistry and Guangdong Key Laboratory of Environmental Resources Utilization and Protection, Guangzhou Institute of Geochemistry, Chinese Academy of Sciences, Kehua Street, Tianhe District, Guangzhou 510640 (China); Song Weihua; Cooper, William J. [Urban Water Research Center, Department of Civil and Environmental Engineering, University of California, Irvine, CA 92697-2175 (United States); Luo Haiying [State Key Laboratory of Organic Geochemistry and Guangdong Key Laboratory of Environmental Resources Utilization and Protection, Guangzhou Institute of Geochemistry, Chinese Academy of Sciences, Kehua Street, Tianhe District, Guangzhou 510640 (China); Graduate School of Chinese Academy of Sciences, Beijing 100049 (China); Guangzhou Product Quality Supervision and Testing Institute, National Centre for Quality Supervision and Testing of Processed Food (Guangzhou), Guangzhou 510110 (China); Guo Xindong [Guangzhou Product Quality Supervision and Testing Institute, National Centre for Quality Supervision and Testing of Processed Food (Guangzhou), Guangzhou 510110 (China)

2010-07-15

This study investigated the photocatalytic degradation of three {beta}-blockers in TiO{sub 2} suspensions. The disappearance of the compounds followed pseudo-first-order kinetics according to the Langmuir-Hinshelwood model and the rate constants were 0.075, 0.072 and 0.182 min{sup -1} for atenolol, metoprolol and propranolol, respectively. After 240 min irradiation, the reaction intermediates were completely mineralized to CO{sub 2} and the nitrogen was predominantly as NH{sub 4}{sup +}. The influence of initial pH and {beta}-blocker concentration on the kinetics was also studied. From adsorption studies it appears that the photocatalytic degradation occurred mainly on the surface of TiO{sub 2}. Further studies indicated that surface reaction with {center_dot}OH radical was principally responsible for the degradation of these three {beta}-blockers. The major degradation intermediates were identified by HPLC/MS analysis. Cleavage of the side chain and the addition of the hydroxyl group to the parent compounds were found to be the two main degradation pathways for all three {beta}-blockers.

Photo-induced DNA cleavage and cytotoxicity of a ruthenium(II) arene anticancer complex

Czech Academy of Sciences Publication Activity Database

Brabec, Viktor; Prachařová, J.; Štěpánková, Jana; Sadler, P. J.; Kašpárková, Jana

2016-01-01

Roč. 160, JUL2016 (2016), s. 149-155 ISSN 0162-0134 R&D Projects: GA ČR(CZ) GA14-21053S; GA MŠk(CZ) LD14019 Institutional support: RVO:68081707 Keywords : Ruthenium anticancer complex * DNA cleavage * Phototoxicity Subject RIV: BO - Biophysics Impact factor: 3.348, year: 2016

Cleavage of cohesin rings coordinates the separation of centrioles and chromatids.

Science.gov (United States)

Schöckel, Laura; Möckel, Martin; Mayer, Bernd; Boos, Dominik; Stemmann, Olaf

2011-07-10

Cohesin pairs sister chromatids by forming a tripartite Scc1-Smc1-Smc3 ring around them. In mitosis, cohesin is removed from chromosome arms by the phosphorylation-dependent prophase pathway. Centromeric cohesin is protected by shugoshin 1 and protein phosphatase 2A (Sgo1-PP2A) and opened only in anaphase by separase-dependent cleavage of Scc1 (refs 4-6). Following chromosome segregation, centrioles loosen their tight orthogonal arrangement, which licenses later centrosome duplication in S phase. Although a role of separase in centriole disengagement has been reported, the molecular details of this process remain enigmatic. Here, we identify cohesin as a centriole-engagement factor. Both premature sister-chromatid separation and centriole disengagement are induced by ectopic activation of separase or depletion of Sgo1. These unscheduled events are suppressed by expression of non-cleavable Scc1 or inhibition of the prophase pathway. When endogenous Scc1 is replaced by artificially cleavable Scc1, the corresponding site-specific protease triggers centriole disengagement. Separation of centrioles can alternatively be induced by ectopic cleavage of an engineered Smc3. Thus, the chromosome and centrosome cycles exhibit extensive parallels and are coordinated with each other by dual use of the cohesin ring complex.

A transcript cleavage factor of Mycobacterium tuberculosis important for its survival.

Directory of Open Access Journals (Sweden)

Arnab China

Full Text Available After initiation of transcription, a number of proteins participate during elongation and termination modifying the properties of the RNA polymerase (RNAP. Gre factors are one such group conserved across bacteria. They regulate transcription by projecting their N-terminal coiled-coil domain into the active center of RNAP through the secondary channel and stimulating hydrolysis of the newly synthesized RNA in backtracked elongation complexes. Rv1080c is a putative gre factor (MtbGre in the genome of Mycobacterium tuberculosis. The protein enhanced the efficiency of promoter clearance by lowering abortive transcription and also rescued arrested and paused elongation complexes on the GC rich mycobacterial template. Although MtbGre is similar in domain organization and shares key residues for catalysis and RNAP interaction with the Gre factors of Escherichia coli, it could not complement an E. coli gre deficient strain. Moreover, MtbGre failed to rescue E. coli RNAP stalled elongation complexes, indicating the importance of specific protein-protein interactions for transcript cleavage. Decrease in the level of MtbGre reduced the bacterial survival by several fold indicating its essential role in mycobacteria. Another Gre homolog, Rv3788 was not functional in transcript cleavage activity indicating that a single Gre is sufficient for efficient transcription of the M. tuberculosis genome.

Microbial mineralization of ring-substituted anilines through an ortho-cleavage pathway.

Science.gov (United States)

Zeyer, J; Wasserfallen, A; Timmis, K N

1985-08-01

Moraxella sp. strain G is able to utilize as sole source of carbon and nitrogen aniline, 4-fluoroaniline, 2-chloroaniline, 3-chloroaniline, 4-chloroaniline (PCA), and 4-bromoaniline but not 4-iodoaniline, 4-methylaniline, 4-methoxyaniline, or 3,4-dichloroaniline. The generation time on PCA was 6 h. The pathway for the degradation of PCA was investigated by analysis of catabolic intermediates and enzyme activities. Mutants of strain G were isolated to enhance the accumulation of specific pathway intermediates. PCA was converted by an aniline oxygenase to 4-chlorocatechol, which in turn was degraded via a modified ortho-cleavage pathway. Synthesis of the aniline oxygenase was inducible by various anilines. This enzyme exhibited a broad substrate specificity. Its specific activity towards substituted anilines seemed to be correlated more with the size than with the electron-withdrawing effect of the substituent and was very low towards anilines having substituents larger than iodine or a methyl group. The initial enzyme of the modified ortho-cleavage pathway, catechol 1,2-dioxygenase, had similar characteristics to those of corresponding enzymes of pathways for the degradation of chlorobenzoic acid and chlorophenol, that is, a broad substrate specificity and high activity towards chlorinated and methylated catechols.

AcEST: DK960462 [AcEST

Lifescience Database Archive (English)

Full Text Available nome polyprotein OS=Hepatitis C virus gen... 33 0.91 sp|Q8AZM0|POLS_BSNV Structural polyprotein OS=Blotched snake...|POLS_BSNV Structural polyprotein OS=Blotched snakehead virus PE=1 SV=1 Length = 1069 Score = 31.2 bits (69)

Probing Electron-Induced Bond Cleavage at the Single-Molecule Level Using DNA Origami Templates

DEFF Research Database (Denmark)

Keller, Adrian Clemens; Bald, Ilko; Rotaru, Alexandru

2012-01-01

Low-energy electrons (LEEs) play an important role in nanolithography, atmospheric chemistry, and DNA radiation damage. Previously, the cleavage of specific chemical bonds triggered by LEEs has been demonstrated in a variety of small organic molecules such as halogenated benzenes and DNA nucleoba...

A structural and kinetic study on myofibrils prevented from shortening by chemical cross-linking.

Science.gov (United States)

Herrmann, C; Sleep, J; Chaussepied, P; Travers, F; Barman, T

1993-07-20

In previous work, we studied the early steps of the Mg(2+)-ATPase activity of Ca(2+)-activated myofibrils [Houadjeto, M., Travers, F., & Barman, T. (1992) Biochemistry 31, 1564-1569]. The myofibrils were free to contract, and the results obtained refer to the ATPase cycle of myofibrils contracting with no external load. Here we studied the ATPase of myofibrils contracting isometrically. To prevent shortening, we cross-linked them with 1-ethyl-3-[3-(dimethylamino)propyl]carbodiimide (EDC). SDS-PAGE and Western blot analyses showed that the myosin rods were extensively cross-linked and that 8% of the myosin heads were cross-linked to the thin filament. The transient kinetics of the cross-linked myofibrils were studied in 0.1 M potassium acetate, pH 7.4 and 4 degrees C, by the rapid-flow quench method. The ATP binding steps were studied by the cold ATP chase and the cleavage and release of products steps by the Pi burst method. In Pi burst experiments, the sizes of the bursts were equal within experimental error to the ATPase site concentrations (as determined by the cold ATP chase methods) for both cross-linked (isometric) and un-cross-linked (isotonic) myofibrils. This shows that in both cases the rate-limiting step is after the cleavage of ATP. When cross-linked, the kcat of Ca(2+)-activated myofibrils was reduced from 1.7 to 0.8 s-1. This is consistent with the observation that fibers shortening at moderate velocity have a higher ATPase activity than isometric fibers.(ABSTRACT TRUNCATED AT 250 WORDS)

The impact of envelope glycoprotein cleavage on the antigenicity, infectivity, and neutralization sensitivity of Env-pseudotyped human immunodeficiency virus type 1 particles

International Nuclear Information System (INIS)

Herrera, Carolina; Klasse, Per Johan; Michael, Elizabeth; Kake, Shivani; Barnes, Kelly; Kibler, Christopher W.; Campbell-Gardener, Lila; Si, Zhihai; Sodroski, Joseph; Moore, John P.; Beddows, Simon

2005-01-01

Endoproteolytic processing of the human immunodeficiency virus type 1 (HIV-1) envelope (Env) glycoproteins is an obligate part of the biosynthetic pathway that generates functional, fusion-competent Env complexes, which are then incorporated into infectious virions. We have examined the influence of cleavage on Env-specific antibody reactivity, Env incorporation into pseudovirions, and the infectivity and neutralization sensitivity of Env-pseudotyped viruses. To do so, we have used both incompletely processed wild-type (Wt) Env and engineered, cleavage-defective Env mutants. We find that there is no simple association between antibody reactivity to cell surface-expressed Env, and the ability of the same antibody to neutralize virus pseudotyped with the same Env proteins. One explanation for the absence of such an association is the diverse array of Env species present on the surface of transiently transfected cells. We also confirm that cleavage-defective mutants are antigenically different from Wt Env. These findings have implications for the use of Env binding assays as predictors of neutralizing activity, and for the development of cleavage-defective Env trimers for use as subunit immunogens

Neutrophil Protease Cleavage of Von Willebrand Factor in Glomeruli – An Anti-thrombotic Mechanism in the Kidney

Directory of Open Access Journals (Sweden)

Ramesh Tati

2017-02-01

Full Text Available Adequate cleavage of von Willebrand factor (VWF prevents formation of thrombi. ADAMTS13 is the main VWF-cleaving protease and its deficiency results in development of thrombotic microangiopathy. Besides ADAMTS13 other proteases may also possess VWF-cleaving activity, but their physiological importance in preventing thrombus formation is unknown. This study investigated if, and which, proteases could cleave VWF in the glomerulus. The content of the glomerular basement membrane (GBM was studied as a reflection of processes occurring in the subendothelial glomerular space. VWF was incubated with human GBMs and VWF cleavage was assessed by multimer structure analysis, immunoblotting and mass spectrometry. VWF was cleaved into the smallest multimers by the GBM, which contained ADAMTS13 as well as neutrophil proteases, elastase, proteinase 3 (PR3, cathepsin-G and matrix-metalloproteinase 9. The most potent components of the GBM capable of VWF cleavage were in the serine protease or metalloprotease category, but not ADAMTS13. Neutralization of neutrophil serine proteases inhibited GBM-mediated VWF-cleaving activity, demonstrating a marked contribution of elastase and/or PR3. VWF-platelet strings formed on the surface of primary glomerular endothelial cells, in a perfusion system, were cleaved by both elastase and the GBM, a process blocked by elastase inhibitor. Ultramorphological studies of the human kidney demonstrated neutrophils releasing elastase into the GBM. Neutrophil proteases may contribute to VWF cleavage within the subendothelium, adjacent to the GBM, and thus regulate thrombus size. This anti-thrombotic mechanism would protect the normal kidney during inflammation and could also explain why most patients with ADAMTS13 deficiency do not develop severe kidney failure.

Kallistatin Ameliorates Influenza Virus Pathogenesis by Inhibition of Kallikrein-Related Peptidase 1-Mediated Cleavage of Viral Hemagglutinin

Science.gov (United States)

Leu, Chia-Hsing; Yang, Mei-Lin; Chung, Nai-Hui; Huang, Yen-Jang; Su, Yu-Chu; Chen, Yi-Cheng; Lin, Chia-Cheng; Shieh, Gia-Shing; Chang, Meng-Ya; Wang, Shainn-Wei; Chang, Yao; Chao, Julie; Chao, Lee

2015-01-01

Proteolytic cleavage of the hemagglutinin (HA) of influenza virus by host trypsin-like proteases is required for viral infectivity. Some serine proteases are capable of cleaving influenza virus HA, whereas some serine protease inhibitors (serpins) inhibit the HA cleavage in various cell types. Kallikrein-related peptidase 1 (KLK1, also known as tissue kallikrein) is a widely distributed serine protease. Kallistatin, a serpin synthesized mainly in the liver and rapidly secreted into the circulation, forms complexes with KLK1 and inhibits its activity. Here, we investigated the roles of KLK1 and kallistatin in influenza virus infection. We show that the levels of KLK1 increased, whereas those of kallistatin decreased, in the lungs of mice during influenza virus infection. KLK1 cleaved H1, H2, and H3 HA molecules and consequently enhanced viral production. In contrast, kallistatin inhibited KLK1-mediated HA cleavage and reduced viral production. Cells transduced with the kallistatin gene secreted kallistatin extracellularly, which rendered them more resistant to influenza virus infection. Furthermore, lentivirus-mediated kallistatin gene delivery protected mice against lethal influenza virus challenge by reducing the viral load, inflammation, and injury in the lung. Taking the data together, we determined that KLK1 and kallistatin contribute to the pathogenesis of influenza virus by affecting the cleavage of the HA peptide and inflammatory responses. This study provides a proof of principle for the potential therapeutic application of kallistatin or other KLK1 inhibitors for influenza. Since proteolytic activation also enhances the infectivity of some other viruses, kallistatin and other kallikrein inhibitors may be explored as antiviral agents against these viruses. PMID:26149981

Thermodynamics and kinetics of RNA tertiary structure formation in the junctionless hairpin ribozyme.

Science.gov (United States)

White, Neil A; Hoogstraten, Charles G

2017-09-01

The hairpin ribozyme consists of two RNA internal loops that interact to form the catalytically active structure. This docking transition is a rare example of intermolecular formation of RNA tertiary structure without coupling to helix annealing. We have used temperature-dependent surface plasmon resonance (SPR) to characterize the thermodynamics and kinetics of RNA tertiary structure formation for the junctionless form of the ribozyme, in which loops A and B reside on separate molecules. We find docking to be strongly enthalpy-driven and to be accompanied by substantial activation barriers for association and dissociation, consistent with the structural reorganization of both internal loops upon complex formation. Comparisons with the parallel analysis of a ribozyme variant carrying a 2'-O-methyl modification at the self-cleavage site and with published data in other systems reveal a surprising diversity of thermodynamic signatures, emphasizing the delicate balance of contributions to the free energy of formation of RNA tertiary structure. Copyright © 2017 Elsevier B.V. All rights reserved.

Molecular pathogenesis of H5 highly pathogenic avian influenza: the role of the haemagglutinin cleavage site motif

Science.gov (United States)

Luczo, Jasmina M.; Stambas, John; Durr, Peter A.; Michalski, Wojtek P.

2015-01-01

Summary The emergence of H5N1 highly pathogenic avian influenza has caused a heavy socio‐economic burden through culling of poultry to minimise human and livestock infection. Although human infections with H5N1 have to date been limited, concerns for the pandemic potential of this zoonotic virus have been greatly intensified following experimental evidence of aerosol transmission of H5N1 viruses in a mammalian infection model. In this review, we discuss the dominance of the haemagglutinin cleavage site motif as a pathogenicity determinant, the host‐pathogen molecular interactions driving cleavage activation, reverse genetics manipulations and identification of residues key to haemagglutinin cleavage site functionality and the mechanisms of cell and tissue damage during H5N1 infection. We specifically focus on the disease in chickens, as it is in this species that high pathogenicity frequently evolves and from which transmission to the human population occurs. With >75% of emerging infectious diseases being of zoonotic origin, it is necessary to understand pathogenesis in the primary host to explain spillover events into the human population. © 2015 The Authors. Reviews in Medical Virology published by John Wiley & Sons Ltd. PMID:26467906

Kinetic Typography

DEFF Research Database (Denmark)

van Leeuwen, Theo; Djonov, Emilia

2014-01-01

After discussing broad cultural drivers behind the development of kinetic typography, the chapter outlines an approach to analysing kinetic typography which is based on Halliday's theory of transitivity, as applied by Kress and Van Leeuwen to visual images.......After discussing broad cultural drivers behind the development of kinetic typography, the chapter outlines an approach to analysing kinetic typography which is based on Halliday's theory of transitivity, as applied by Kress and Van Leeuwen to visual images....

Sequence motif upstream of the Hendra virus fusion protein cleavage site is not sufficient to promote efficient proteolytic processing

International Nuclear Information System (INIS)

Craft, Willie Warren; Dutch, Rebecca Ellis

2005-01-01

The Hendra virus fusion (HeV F) protein is synthesized as a precursor, F 0 , and proteolytically cleaved into the mature F 1 and F 2 heterodimer, following an HDLVDGVK 109 motif. This cleavage event is required for fusogenic activity. To determine the amino acid requirements for processing of the HeV F protein, we constructed multiple mutants. Individual and simultaneous alanine substitutions of the eight residues immediately upstream of the cleavage site did not eliminate processing. A chimeric SV5 F protein in which the furin site was substituted for the VDGVK 109 motif of the HeV F protein was not processed but was expressed on the cell surface. Another chimeric SV5 F protein containing the HDLVDGVK 109 motif of the HeV F protein underwent partial cleavage. These data indicate that the upstream region can play a role in protease recognition, but is neither absolutely required nor sufficient for efficient processing of the HeV F protein

Fracture mechanics and physics approach to cleavage analysis in bcc monocrystals

International Nuclear Information System (INIS)

Ivanova, V.S.; Plastinin, V.M.

1980-01-01

On monocrystals of molybdenum obtained by electron--beam zone melting studied are the bonds between micro-and macroparameters of fracture controlling the limit state. Monocrystals of three orientations have been studied, namely >001 110 111<. Confirmed is an important role of plastic deformation in the (110) family planes at cleavage forming in the (100) family planes. A correlation connection is established between threshold value of the stress intensity coefficient and activation energy of plastic deformation

Carbon–carbon bond cleavage for Cu-mediated aromatic trifluoromethylations and pentafluoroethylations

Directory of Open Access Journals (Sweden)

Tsuyuka Sugiishi

2015-12-01

Full Text Available This short review highlights the copper-mediated fluoroalkylation using perfluoroalkylated carboxylic acid derivatives. Carbon–carbon bond cleavage of perfluoroalkylated carboxylic acid derivatives takes place in fluoroalkylation reactions at high temperature (150–200 °C or under basic conditions to generate fluoroalkyl anion sources for the formation of fluoroalkylcopper species. The fluoroalkylation reactions, which proceed through decarboxylation or tetrahedral intermediates, are useful protocols for the synthesis of fluoroalkylated aromatics.

Insights into the Reaction Mechanism of Aromatic Ring Cleavage by Homogentisate Dioxygenase: A Quantum Mechanical/Molecular Mechanical Study.

Science.gov (United States)

Qi, Yue; Lu, Jiarui; Lai, Wenzhen

2016-05-26

To elucidate the reaction mechanism of the ring cleavage of homogentisate by homogentisate dioxygenase, quantum mechanical/molecular mechanical (QM/MM) calculations were carried out by using two systems in different protonation states of the substrate C2 hydroxyl group. When the substrate C2 hydroxyl group is ionized (the ionized pathway), the superoxo attack on the substrate is the rate-limiting step in the catalytic cycle, with a barrier of 15.9 kcal/mol. Glu396 was found to play an important role in stabilizing the bridge species and its O-O cleavage product by donating a proton via a hydrogen-bonded water molecule. When the substrate C2 hydroxyl group is not ionized (the nonionized pathway), the O-O bond cleavage of the bridge species is the rate-limiting step, with a barrier of 15.3 kcal/mol. The QM/MM-optimized geometries for the dioxygen and alkylperoxo complexes using the nonionized model (for the C2 hydroxyl group) are in agreement with the experimental crystal structures, suggesting that the C2 hydroxyl group is more likely to be nonionized.

Racial Cleavage in Local Voting: The Case of School and Tax Issue Referendums.

Science.gov (United States)

Button, James

1993-01-01

Explores voting behavior of African Americans and whites in local school and tax referenda to determine whether racial conflict is still a primal factor in noncandidate elections. Results for voters in 5 counties in Florida (over 1,699,000 voters) reveal African-American underregistration and the continuing importance of racial cleavage. (SLD)

Dienone-phenol Rearrangement of C-9 Oxygenated Decalinic Dienone and Analogs through B-Ring Cleavage

Institute of Scientific and Technical Information of China (English)

无

2003-01-01

Dehydrogenation of 9-hydroxy decalinic enones and analogs with DDQ resulted in a formal dienone-phenol type rearrangement via B-ring cleavage, while the corresponding dienone acetates underwent base-catalyzed formal dienone-phenol type rearrangement analogously.

The Path of Carbon in Photosynthesis XVI. Kinetic Relationships of the Intermediates in Steady State Photosynthesis

Science.gov (United States)

Benson, A. A.; Kawaguchi, S.; Hayes, P.; Calvin, M.

1952-06-05

A kinetic study of the accumulation of C{sup 14} in the intermediates of steady state photosynthesis in C{sup 14}O{sub 2} provides information regarding the sequence of reactions involved. The work described applied the radio-chromatographic technique for analysis of the labeled early products. The simultaneous carboxylation reaction resulting in malic acid as well as phosphoglycerate is demonstrated in experiments at high light intensity. A comparison of radioactivities in a number of phosphorylated sugars as a function of time reveals concurrent synthesis of fructose and sedoheptulose phosphates followed by that of ribulose phosphates and later by that of glucose phosphates. The possibility that the cleavage of C{sub 4} compounds to C{sub 2} carbon dioxide acceptors may involve C{sub 7} and C{sub 5} sugars and evidence for this mechanism is presented.

Andrographolide downregulates the v-Src and Bcr-Abl oncoproteins and induces Hsp90 cleavage in the ROS-dependent suppression of cancer malignancy.

Science.gov (United States)

Liu, Sheng-Hung; Lin, Chao-Hsiung; Liang, Fong-Ping; Chen, Pei-Fen; Kuo, Cheng-Deng; Alam, Mohd Mujahid; Maiti, Barnali; Hung, Shih-Kai; Chi, Chin-Wen; Sun, Chung-Ming; Fu, Shu-Ling

2014-01-15

Andrographolide is a diterpenoid compound isolated from Andrographis paniculata that exhibits anticancer activity. We previously reported that andrographolide suppressed v-Src-mediated cellular transformation by promoting the degradation of Src. In the present study, we demonstrated the involvement of Hsp90 in the andrographolide-mediated inhibition of Src oncogenic activity. Using a proteomics approach, a cleavage fragment of Hsp90α was identified in andrographolide-treated cells. The concentration- and time-dependent induction of Hsp90 cleavage that accompanied the reduction in Src was validated in RK3E cells transformed with either v-Src or a human truncated c-Src variant and treated with andrographolide. In cancer cells, the induction of Hsp90 cleavage by andrographolide and its structural derivatives correlated well with decreased Src levels, the suppression of transformation, and the induction of apoptosis. Moreover, the andrographolide-induced Hsp90 cleavage, Src degradation, inhibition of transformation, and induction of apoptosis were abolished by a ROS inhibitor, N-acetyl-cysteine. Notably, Hsp90 cleavage, decreased levels of Bcr-Abl (another known Hsp90 client protein), and the induction of apoptosis were also observed in human K562 leukemia cells treated with andrographolide or its active derivatives. Together, we demonstrated a novel mechanism by which andrographolide suppressed cancer malignancy that involved inhibiting Hsp90 function and reducing the levels of Hsp90 client proteins. Our results broaden the molecular basis of andrographolide-mediated anticancer activity. Copyright © 2013 Elsevier Inc. All rights reserved.

Extensional collapse in the Neoproterozoic Araçuaí orogen, eastern Brazil: a setting for reactivation of asymmetric crenulation cleavage

Science.gov (United States)

Marshak, Stephen; Alkmim, Fernando F.; Whittington, Alan; Pedrosa-Soares, Antônio Carlos

2006-01-01

The Araçuaí orogen of eastern Brazil is one of many Brasiliano/Pan African orogens formed during the Neoproterozoic assembly of Gondwana. Its western edge, bordering the São Francisco craton, is the Serra do Espinhaço fold-thrust belt, in which top-up-to-the-west (reverse-sense) faults, west-verging folds (F 1), and east-dipping spaced to phyllitic cleavage (S 1) developed. We have found that the kinematics of deformation changes markedly at the hinterland margin of this fold-thrust belt. Here, beneath a plateau known as the Chapada Acauã, metadiamictite and fine-grained pelitic schist comprise an east-dipping belt that contains an assemblage of structures indicative of top-down-to-the-east (normal-sense) shear. This assemblage includes a cascade of F 2 folds that refold F 1 folds and verge down the dip of the belt's enveloping surfaces, vertical tension gashes, and top-down-to-the-east rotated clasts. Based on the presence of these structures, we propose that the plateau exposes a regional-scale normal-sense shear zone, here called the Chapada Acauã shear zone (CASZ). Because F 2 folds refold F 1 folds, normal-sense shear in the CASZ occurred subsequent to initial west-verging thrusting. Considering this timing of motion in the CASZ, we suggest that the zone accommodated displacement of the internal zone of the Araçuaí orogen down, relative to its foreland fold-thrust belt, and thus played a role in extensional collapse of the orogen. The CASZ trends parallel to preserved thrusts to the west, and thus may represent an inverted thrust fault. Notably, throughout the CASZ, S 1 schistosity has been overprinted by a pervasive, west-dipping asymmetric crenulation cleavage (S 2). The sigmoid shape of S 1 surfaces in S 2 microlithons require that slip on each S 2 surface was top-down-to-the-west. S 2 cleavage is axial-planar to the down-dip verging F 2 folds. Based on its geometry, we suggest that S 2 cleavage initiated either as an antithetic extensional

Electrophilic Cleavage and Functionalization of Polyisobutylenes Bearing Unsaturations in the Backbone and Synthesis of Polymers for this Process

Science.gov (United States)

Campbell, Christopher Garrett

weight multifunctional PIBs. In the third project, we attempted to synthesize a random copolymer, previously reported by Kennedy et al., of isobutylene and 2,4-dimethyl-1,3-pentadiene (DMPD). The interest in this copolymer was based on its structural similarity to the coupled PIB mentioned in the first project. However, we found that these two monomers are not well suited to the creation of random copolymers due to a large difference in reactivity ratios. The project presented in this chapter was then redirected toward the structural characterization of the products of attempted copolymerization and of the homo-polymerization of DMPD. In the fourth project, we investigated the copolymer of isobutylene and beta-pinene as a substrate for the aforementioned cleavage/functionalization reaction. We were able to synthesize high molecular weight copolymers of these two monomers via slurry polymerization catalyzed by either TiCl4 or ethylaluminum dichloride (EADC), and though the degradation and functionalization kinetics were much slower than for butyl rubber, we did observe a drastic decrease in molecular weight accompanied by functionalization of the polymer, thus proving this chemistry is applicable to copolymers of isobutylene other than butyl rubber.

Synthesis of a designed transmembrane protein by thioether ligation of solubilised segments : Nα-haloacetylated peptides survived resin cleavage using TFA with EDT as scavenger

NARCIS (Netherlands)

Englebretsen, D.R; Choma, C.T.; Robillard, G.T.

1998-01-01

Nα-haloacetylated peptides made by Fmoc solid phase synthesis survived cleavage when EDT was used as a cleavage component. Two segments of a desgned transmembrane protein, one bromoacetylated, the other containing a cysteine, and each bearing a "solubilising tail" peptide, were synthesised by Fmoc

Kinetic modeling of the polymer-derived ceramics route: investigation of the thermal decomposition kinetics of poly[B-(methylamino)borazine] precursors into boron nitride.

Science.gov (United States)

Bernard, Samuel; Fiaty, Koffi; Cornu, David; Miele, Philippe; Laurent, Pierre

2006-05-11

A complete kinetic modeling of the polymer-derived ceramics (PDCs) route is achieved for the first time through the investigation of the solid-state decomposition of a typical melt-spinnable poly[B-(methylamino)borazine] into boron nitride fibers at various heating rates. Through the use of the Lorentz fitting approach, it is shown that the two-step weight loss associated with the polymer-to-ceramic conversion is governed by a complex interplay of five diffusion-type transport mechanisms that are independent of the applied heating schedule. The application of the Friedman method to dynamic thermogravimetry data yields Ea and ln A values that are seen to increase with the extent of the ceramic conversion from region one (Ea = 38.73 kJ mol(-1)) to region five (Ea = 146.64 kJ mol(-1)). This fact indicates that both the mechanisms within those regions are parallel routes to the formation of the final solid-state material and their complexity increases with the conversion progress. The cross-linking process (first weight loss) of the polymer is governed by three dependent poorly energetic mechanisms. The first weight loss is activated by ammonolysis reactions that provide a modified polymer capable of undergoing condensation reactions in regions two and three to yield a highly cross-linked polymer. A large evolution of methylamine is identified during this process. Mineralization (region four) and ceramization (region five) steps are represented by two highly energetic multistep mechanisms. The mineralization step is associated with a large evolution of methylamine and occurs during the transition between the cross-linking and ceramization processes through the cleavage of the inter-ring B-N bonds. Ceramization represents the end of the polymer-to-ceramic conversion in which the planar consolidation of BN hexagons occurs through complex structural rearrangements of the borazine units (cleavage of the intraring B-N bonds) accompanied with an ammonia evolution. Finally

AcEST: DK960274 [AcEST

Lifescience Database Archive (English)

Full Text Available _HCVBB Genome polyprotein OS=Hepatitis C virus gen... 33 0.93 sp|Q8AZM0|POLS_BSNV Structural polyprotein OS=Blotched snake...sp|Q8AZM0|POLS_BSNV Structural polyprotein OS=Blotched snakehead virus PE=1 SV=1 Length = 1069 Score = 31.2

AcEST: DK949578 [AcEST

Lifescience Database Archive (English)

Full Text Available |POLG_HCVBB Genome polyprotein OS=Hepatitis C virus gen... 33 1.0 sp|Q8AZM0|POLS_BSNV Structural polyprotein OS=Blotched snake...1 >sp|Q8AZM0|POLS_BSNV Structural polyprotein OS=Blotched snakehead virus PE=1 SV=1 Length = 1069 Score = 31

AcEST: DK961950 [AcEST

Lifescience Database Archive (English)

Full Text Available VBB Genome polyprotein OS=Hepatitis C virus gen... 33 0.92 sp|Q8AZM0|POLS_BSNV Structural polyprotein OS=Blotched snake...Q8AZM0|POLS_BSNV Structural polyprotein OS=Blotched snakehead virus PE=1 SV=1 Length = 1069 Score = 31.2 bit

AcEST: DK948648 [AcEST

Lifescience Database Archive (English)

Full Text Available OLG_HCVBB Genome polyprotein OS=Hepatitis C virus gen... 33 1.0 sp|Q8AZM0|POLS_BSNV Structural polyprotein OS=Blotched snake...sp|Q8AZM0|POLS_BSNV Structural polyprotein OS=Blotched snakehead virus PE=1 SV=1 Length = 1069 Score = 31.2

Potent Inhibition of Feline Coronaviruses with Peptidyl Compounds Targeting Coronavirus 3C-like Protease

Science.gov (United States)

Kim, Yunjeong; Mandadapu, Sivakoteswara Rao; Groutas, William C.; Chang, Kyeong-Ok

2012-01-01

Feline coronavirus infection is common among domestic and exotic felid species and usually associated with mild or asymptomatic enteritis; however, feline infectious peritonitis (FIP) is a fatal disease of cats that is caused by systemic infection with a feline infectious peritonitis virus (FIPV), a variant of feline enteric coronavirus (FECV). Currently, there is no specific treatment approved for FIP despite the importance of FIP as the leading infectious cause of death in young cats. During the replication process, coronavirus produces viral polyproteins that are processed into mature proteins by viral proteases, the main protease (3C-like [3CL] protease) and the papain-like protease. Since the cleavages of viral polyproteins are an essential step for virus replication, blockage of viral protease is an attractive target for therapeutic intervention. Previously, we reported the generation of broad-spectrum peptidyl inhibitors against viruses that possess a 3C or 3CL protease. In this study, we further evaluated the antiviral effects of the peptidyl inhibitors against feline coronaviruses, and investigated the interaction between our protease inhibitor and a cathepsin B inhibitor, an entry blocker, against feline coronaviruses in cell culture. Herein we report that our compounds behave as reversible, competitive inhibitors of 3CL protease, potently inhibited the replication of feline coronaviruses (EC50 in a nanomolar range) and, furthermore, the combination of cathepsin B and 3CL protease inhibitors led to a strong synergistic interaction against feline coronaviruses in cell culture systems. PMID:23219425

Amino acid sequence requirements in the human IgA1 hinge for cleavage by streptococcal IgA1 proteases

DEFF Research Database (Denmark)

Senior, BW; Batten, MR; Kilian, Mogens

2002-01-01

All the IgA1 proteases of the different pathogenic species of Streptococcus cleave the hinge of the alpha chain of human IgA1 only at one proline-threonine peptide bond. In order to study the importance of these amino acids for cleavage, several hinge mutant recombinant IgA1 antibodies were const...... constructed. The mutations were found to be without major effect upon the structure or functional abilities of the antibodies. However, they had a major effect upon their sensitivity to cleavage by some of the IgA1 proteases....

Cleavage and synthesis function of high and low redox potential laccases towards 4-morpholinoaniline and aminated as well as chlorinated phenols.

Science.gov (United States)

Hahn, Veronika; Mikolasch, Annett; Schauer, Frieder

2014-02-01

Laccases are able to mediate both cleavage and synthesis processes. The basis for this dual reaction capability lies in the property of the enzyme laccase to oxidize phenolic, and to some extent non-phenolic substances, to reactive radicals which can undergo on the one hand separations of small substitutents or large molecule parts from the parent compound and on the other hand coupling reactions with other radicals or molecules which are not themselves oxidizable by laccase. The cleavage of the non-phenolic compound 4-morpholinoaniline as well as the deamination of 4-aminophenol and the dechlorination of 4-chlorophenol resulted in the formation of 1,4-hydroquinone which is immediately oxidized by laccase to 1,4-benzoquinone. The formation of the 1,4-hydroquinone/1,4-benzoquinone is the rate limiting step for the synthesis of the heteromolecular dimers and trimers composed of 1,4-benzoquinone and one or two molecules of morpholine. In addition to the synthesis of new compounds from the cleavage products, 4-morpholinoaniline polymerized probably via azo groups and C-N bonds to a homomolecular dimer and trimer. Similarities and differences in cleavage and synthesis reactions catalyzed by the low redox potential laccase of Myceliophthora thermophila (0.46 V) and the high redox potential laccase of Pycnoporus cinnabarinus (0.79 V) were determined. In addition, the dependency of the cleavage and synthesis efficiencies on the (a) structure and redox potential of the laccase, (b) structure and redox potential of the substrate, (c) pH value of the buffer used, (d) incubation temperature, (e) solvent concentration, and (f) laccase activity is discussed in general.

The use of a xylosylated plant glycoprotein as an internal standard accounting for N-linked glycan cleavage and sample preparation variability.

Science.gov (United States)

Walker, S Hunter; Taylor, Amber D; Muddiman, David C

2013-06-30

Traditionally, free oligosaccharide internal standards are used to account for variability in glycan relative quantification experiments by mass spectrometry. However, a more suitable internal standard would be a glycoprotein, which could also control for enzymatic cleavage efficiency, allowing for more accurate quantitative experiments. Hydrophobic, hydrazide N-linked glycan reagents (both native and stable-isotope labeled) are used to derivatize and differentially label N-linked glycan samples for relative quantification, and the samples are analyzed by a reversed-phase liquid chromatography chip system coupled online to a Q-Exactive mass spectrometer. The inclusion of two internal standards, maltoheptaose (previously used) and horseradish peroxidase (HRP) (novel), is studied to demonstrate the effectiveness of using a glycoprotein as an internal standard in glycan relative quantification experiments. HRP is a glycoprotein containing a xylosylated N-linked glycan, which is unique from mammalian N-linked glycans. Thus, the internal standard xylosylated glycan could be detected without interference to the sample. Additionally, it was shown that differences in cleavage efficiency can be detected by monitoring the HRP glycan. In a sample where cleavage efficiency variation is minimal, the HRP glycan performs as well as maltoheptaose. Because the HRP glycan performs as well as maltoheptaose but is also capable of correcting and accounting for cleavage variability, it is a more versatile internal standard and will be used in all subsequent biological studies. Because of the possible lot-to-lot variation of an enzyme, differences in biological matrix, and variable enzyme activity over time, it is a necessity to account for glycan cleavage variability in glycan relative quantification experiments. Copyright © 2013 John Wiley & Sons, Ltd.

Identification of Rbd2 as a candidate protease for sterol regulatory element binding protein (SREBP) cleavage in fission yeast

Energy Technology Data Exchange (ETDEWEB)

Kim, Jinsil; Ha, Hye-Jeong [Aging Research Center, Korea Research Institute of Bioscience and Biotechnology (KRIBB), 125 Gwahak-ro, Yuseong-gu, Daejeon 34141 (Korea, Republic of); Kim, Sujin [Aging Research Center, Korea Research Institute of Bioscience and Biotechnology (KRIBB), 125 Gwahak-ro, Yuseong-gu, Daejeon 34141 (Korea, Republic of); Department of Functional Genomics, University of Science and Technology (UST), 217 Gajeong-ro, Yuseong-gu, Daejeon 34113 (Korea, Republic of); Choi, Ah-Reum [Aging Research Center, Korea Research Institute of Bioscience and Biotechnology (KRIBB), 125 Gwahak-ro, Yuseong-gu, Daejeon 34141 (Korea, Republic of); Lee, Sook-Jeong [Department of New Drug Discovery and Development, Chungnam National University, 99 Daehak-ro, Yuseong-gu, Daejeon 34134 (Korea, Republic of); Hoe, Kwang-Lae, E-mail: kwanghoe@cnu.ac.kr [Department of New Drug Discovery and Development, Chungnam National University, 99 Daehak-ro, Yuseong-gu, Daejeon 34134 (Korea, Republic of); Kim, Dong-Uk, E-mail: kimdongu@kribb.re.kr [Aging Research Center, Korea Research Institute of Bioscience and Biotechnology (KRIBB), 125 Gwahak-ro, Yuseong-gu, Daejeon 34141 (Korea, Republic of)

2015-12-25

Lipid homeostasis in mammalian cells is regulated by sterol regulatory element-binding protein (SREBP) transcription factors that are activated through sequential cleavage by Golgi Site-1 and Site-2 proteases. Fission yeast SREBP, Sre1, engages a different mechanism involving the Golgi Dsc E3 ligase complex, but it is not clearly understood exactly how Sre1 is proteolytically cleaved and activated. In this study, we screened the Schizosaccharomyces pombe non-essential haploid deletion collection to identify missing components of the Sre1 cleavage machinery. Our screen identified an additional component of the SREBP pathway required for Sre1 proteolysis named rhomboid protein 2 (Rbd2). We show that an rbd2 deletion mutant fails to grow under hypoxic and hypoxia-mimetic conditions due to lack of Sre1 activity and that this growth phenotype is rescued by Sre1N, a cleaved active form of Sre1. We found that the growth inhibition phenotype under low oxygen conditions is specific to the strain with deletion of rbd2, not any other fission yeast rhomboid-encoding genes. Our study also identified conserved residues of Rbd2 that are required for Sre1 proteolytic cleavage. All together, our results suggest that Rbd2 is a functional SREBP protease with conserved residues required for Sre1 cleavage and provide an important piece of the puzzle to understand the mechanisms for Sre1 activation and the regulation of various biological and pathological processes involving SREBPs. - Highlights: • An rbd2-deleted yeast strain shows defects in growth in response to low oxygen levels. • rbd2-deficient cells fail to generate cleaved Sre1 (Sre1N) under hypoxic conditions. • Expression of Sre1N rescues the rbd2 deletion mutant growth phenotype. • Rbd2 contains conserved residues potentially critical for catalytic activity. • Mutation of the conserved Rbd2 catalytic residues leads to defects in Sre1 cleavage.

Impaired Cleavage of Preproinsulin Signal Peptide Linked to Autosomal-Dominant Diabetes

Science.gov (United States)

Liu, Ming; Lara-Lemus, Roberto; Shan, Shu-ou; Wright, Jordan; Haataja, Leena; Barbetti, Fabrizio; Guo, Huan; Larkin, Dennis; Arvan, Peter

2012-01-01

Recently, missense mutations upstream of preproinsulin’s signal peptide (SP) cleavage site were reported to cause mutant INS gene-induced diabetes of youth (MIDY). Our objective was to understand the molecular pathogenesis using metabolic labeling and assays of proinsulin export and insulin and C-peptide production to examine the earliest events of insulin biosynthesis, highlighting molecular mechanisms underlying β-cell failure plus a novel strategy that might ameliorate the MIDY syndrome. We find that whereas preproinsulin-A(SP23)S is efficiently cleaved, producing authentic proinsulin and insulin, preproinsulin-A(SP24)D is inefficiently cleaved at an improper site, producing two subpopulations of molecules. Both show impaired oxidative folding and are retained in the endoplasmic reticulum (ER). Preproinsulin-A(SP24)D also blocks ER exit of coexpressed wild-type proinsulin, accounting for its dominant-negative behavior. Upon increased expression of ER–oxidoreductin-1, preproinsulin-A(SP24)D remains blocked but oxidative folding of wild-type proinsulin improves, accelerating its ER export and increasing wild-type insulin production. We conclude that the efficiency of SP cleavage is linked to the oxidation of (pre)proinsulin. In turn, impaired (pre)proinsulin oxidation affects ER export of the mutant as well as that of coexpressed wild-type proinsulin. Improving oxidative folding of wild-type proinsulin may provide a feasible way to rescue insulin production in patients with MIDY. PMID:22357960

Structural and functional characterization of cleavage and inactivation of human serine protease inhibitors by the bacterial SPATE protease EspPα from enterohemorrhagic E. coli.

Directory of Open Access Journals (Sweden)

André Weiss

Full Text Available EspPα and EspI are serine protease autotransporters found in enterohemorrhagic Escherichia coli. They both belong to the SPATE autotransporter family and are believed to contribute to pathogenicity via proteolytic cleavage and inactivation of different key host proteins during infection. Here, we describe the specific cleavage and functional inactivation of serine protease inhibitors (serpins by EspPα and compare this activity with the related SPATE EspI. Serpins are structurally related proteins that regulate vital protease cascades, such as blood coagulation and inflammatory host response. For the rapid determination of serpin cleavage sites, we applied direct MALDI-TOF-MS or ESI-FTMS analysis of coincubations of serpins and SPATE proteases and confirmed observed cleavage positions using in-gel-digest of SDS-PAGE-separated degradation products. Activities of both serpin and SPATE protease were assessed in a newly developed photometrical assay using chromogenic peptide substrates. EspPα cleaved the serpins α1-protease inhibitor (α1-PI, α1-antichymotrypsin, angiotensinogen, and α2-antiplasmin. Serpin cleavage led to loss of inhibitory function as demonstrated for α1-PI while EspPα activity was not affected. Notably, EspPα showed pronounced specificity and cleaved procoagulatory serpins such as α2-antiplasmin while the anticoagulatory antithrombin III was not affected. Together with recently published research, this underlines the interference of EspPα with hemostasis or inflammatory responses during infection, while the observed interaction of EspI with serpins is likely to be not physiologically relevant. EspPα-mediated serpin cleavage occurred always in flexible loops, indicating that this structural motif might be required for substrate recognition.

AcEST: DK955248 [AcEST

Lifescience Database Archive (English)

Full Text Available _BSNV Structural polyprotein OS=Blotched snakehead virus Align length 54 Score (b...|POLS_BSNV Structural polyprotein OS=Blotched snakehead... 31 1.7 sp|Q7XWS7|FH12_...sphatase non-receptor ... 29 8.4 >sp|Q8AZM0|POLS_BSNV Structural polyprotein OS=Blotched snakehead virus PE=

NCBI nr-aa BLAST: CBRC-TGUT-37-0394 [SEVENS

Lifescience Database Archive (English)

Full Text Available CBRC-TGUT-37-0394 ref|NP_056885.1| gPr95 polyprotein precursor [Rous sarcoma virus]... gb|AAB59934.1| env-Pr95 polyprotein precursor [Rous sarcoma virus - Prague C] gb|AAC82562.1| gPr95 polyprotein precursor [Rous sarcoma virus] NP_056885.1 8e-49 43% ...

Intracellular Cleavage of the Cx43 C-Terminal Domain by Matrix-Metalloproteases: A Novel Contributor to Inflammation?

Directory of Open Access Journals (Sweden)

Marijke De Bock

2015-01-01

Full Text Available The coordination of tissue function is mediated by gap junctions (GJs that enable direct cell-cell transfer of metabolic and electric signals. GJs are formed by connexin (Cx proteins of which Cx43 is most widespread in the human body. Beyond its role in direct intercellular communication, Cx43 also forms nonjunctional hemichannels (HCs in the plasma membrane that mediate the release of paracrine signaling molecules in the extracellular environment. Both HC and GJ channel function are regulated by protein-protein interactions and posttranslational modifications that predominantly take place in the C-terminal domain of Cx43. Matrix metalloproteases (MMPs are a major group of zinc-dependent proteases, known to regulate not only extracellular matrix remodeling, but also processing of intracellular proteins. Together with Cx43 channels, both GJs and HCs, MMPs contribute to acute inflammation and a small number of studies reports on an MMP-Cx43 link. Here, we build further on these reports and present a novel hypothesis that describes proteolytic cleavage of the Cx43 C-terminal domain by MMPs and explores possibilities of how such cleavage events may affect Cx43 channel function. Finally, we set out how aberrant channel function resulting from cleavage can contribute to the acute inflammatory response during tissue injury.

Birth of normal infants after transfer of embryos that were twice vitrified/warmed at cleavage stages: report of two cases.

Science.gov (United States)

Valle, Marcello; Guimarães, Fernando; Cavagnoli, Melissa; Sampaio, Marcos; Geber, Selmo

2012-12-01

The role of cryopreservation in assisted reproductive technology programs has increased within the last years allowing the transfer of a limited number of embryos and the storage of the remaining for future use. The reduction in the number of transferred embryos decreases the frequency of multiple pregnancy rates and of ovarian hyperstimulation syndrome while the cumulative pregnancy rate can be maximized. Moreover, as not all embryos will survive the warming process more cleavage stage embryos are warmed to improve selection for transfer. Therefore, surplus good quality cleavage stage embryos and/or blastocysts must be re-vitrified for further transfer to achieve pregnancy. To our knowledge, there have been no reports demonstrating that human embryos can be successfully vitrified/warmed twice at the cleavage stage. Thus we report two successful pregnancies and deliveries of healthy babies after transfer of embryos that were twice vitrified/warmed at 2-4 cells stage. Copyright © 2012 Elsevier Inc. All rights reserved.

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

Science.gov (United States)

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

2017-10-04

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

Cesium removal and kinetics equilibrium: Precipitation kinetics

International Nuclear Information System (INIS)

Barnes, M.J.

1999-01-01

This task consisted of both non-radioactive and radioactive (tracer) tests examining the influence of potentially significant variables on cesium tetraphenylborate precipitation kinetics. The work investigated the time required to reach cesium decontamination and the conditions that affect the cesium precipitation kinetics

Fast cleavage of phycocyanobilin from phycocyanin for use in food colouring

DEFF Research Database (Denmark)

Roda-Serrat, Maria Cinta; Christensen, Knud Villy; El-Houri, Rime

2018-01-01

Phycocyanins from cyanobacteria are possible sources for new natural blue colourants. Their chromophore, phycocyanobilin (PCB), was cleaved from the apoprotein by solvolysis in alcohols and alcoholic aqueous solutions. In all cases two PCB isomers were obtained, while different solvent adducts were......, and microwave assisted reaction. The sealed vessel method is a new approach for fast cleavage of PCB from phycocyanin and gave at 120°C the same yield within 30 min compared to 16 hours by the conventional reflux method (P

Abnormal early cleavage events predict early embryo demise: sperm oxidative stress and early abnormal cleavage.

Science.gov (United States)

Burruel, Victoria; Klooster, Katie; Barker, Christopher M; Pera, Renee Reijo; Meyers, Stuart

2014-10-13

Human embryos resulting from abnormal early cleavage can result in aneuploidy and failure to develop normally to the blastocyst stage. The nature of paternal influence on early embryo development has not been directly demonstrated although many studies have suggested effects from spermatozoal chromatin packaging, DNA damage, centriolar and mitotic spindle integrity, and plasma membrane integrity. The goal of this study was to determine whether early developmental events were affected by oxidative damage to the fertilizing sperm. Survival analysis was used to compare patterns of blastocyst formation based on P2 duration. Kaplan-Meier survival curves demonstrate that relatively few embryos with short (P2 times reached blastocysts, and the two curves diverged beginning on day 4, with nearly all of the embryos with longer P2 times reaching blastocysts by day 6 (p < .01). We determined that duration of the 2nd to 3rd mitoses were sensitive periods in the presence of spermatozoal oxidative stress. Embryos that displayed either too long or too short cytokineses demonstrated an increased failure to reach blastocyst stage and therefore survive for further development. Although paternal-derived gene expression occurs later in development, this study suggests a specific role in early mitosis that is highly influenced by paternal factors.

Beta-scission of alkoxyl radicals on peptides and proteins can give rise to backbone cleavage and loss of side-chains

International Nuclear Information System (INIS)

Headlam, H.A.; Davies, M.J.; Mortimer, A.; Easton, C.J.

2000-01-01

Full text: Exposure of proteins to radicals in the presence of O 2 brings about multiple changes including side-chain oxidation, backbone fragmentation, cross-linking, unfolding, changes in hydrophobicity and conformation, altered susceptibility to proteolytic enzymes and formation of new reactive groups (e.g. hydroperoxides and 3,4-dihydroxyphenylalanine). All of these processes can result in loss of structural or enzymatic activity. The mechanisms that give rise to backbone cleavage are only partly understood. Whilst it is known that direct hydrogen atom abstraction at a-carbon sites gives backbone cleavages it has also been proposed that initial attack at side-chain sites might also give rise to backbone cleavage. In this study we have examined whether initial attack at the β- (C-3) position can give rise to α-carbon radicals (and hence backbone cleavage) via the formation, and subsequent β- scission, of C-3 alkoxyl radicals. This process has been observed previously with protected amino acids in organic solvents (J. Chem. Soc. Perkin Trans. 2, 1997, 503-507) but the occurrence of such reactions with proteins in aqueous solution has not been explored. Alkoxyl radicals were generated at the C-3 position of a variety of protected amino acids and small peptides by two methods: metal-ion catalysed decomposition of hydroperoxides formed as a result of γ-radiolysis in the presence of O 2 , and UV photolysis of nitrate esters. In most cases radicals have been detected by EPR spectroscopy using nitroso and nitrone spin traps, which can be assigned by comparison with literature data to α-carbon radicals; in some case assignments were confirmed by the generation of the putative species by other routes. With Ala peptide hydroperoxides and nitrate esters, and MNP as the spin trap, the major radical detected in each case has been assigned to the adduct of an α-carbon radical with partial structure - NH- . CH-C(O) - consistent with the rapid occurrence of the above

Ultrafast spectroscopy on DNA-cleavage by endonuclease in molecular crowding.

Science.gov (United States)

Singh, Priya; Choudhury, Susobhan; Dutta, Shreyasi; Adhikari, Aniruddha; Bhattacharya, Siddhartha; Pal, Debasish; Pal, Samir Kumar

2017-10-01

The jam-packed intracellular environments differ the activity of a biological macromolecule from that in laboratory environments (in vitro) through a number of mechanisms called molecular crowding related to structure, function and dynamics of the macromolecule. Here, we have explored the structure, function and dynamics of a model enzyme protein DNase I in molecular crowing of polyethylene glycol (PEG; MW 3350). We have used steady state and picosecond resolved dynamics of a well-known intercalator ethidium bromide (EB) in a 20-mer double-stranded DNA (dsDNA) to monitor the DNA-cleavage by the enzyme in absence and presence PEG. We have also labelled the enzyme by a well-known fluorescent probe 8-anilino-1-naphthalenesulfonic acid ammonium salt (ANS) to study the molecular mechanism of the protein-DNA association through exited state relaxation of the probe in absence (dictated by polarity) and presence of EB in the DNA (dictated by Förster resonance energy transfer (FRET)). The overall and local structures of the protein in presence of PEG have been followed by circular dichroism and time resolved polarization gated spectroscopy respectively. The enhanced dynamical flexibility of protein in presence of PEG as revealed from excited state lifetime and polarization gated anisotropy of ANS has been correlated with the stronger DNA-binding for the higher nuclease activity. We have also used conventional experimental strategy of agarose gel electrophoresis to monitor DNA-cleavage and found consistent results of enhanced nuclease activities both on synthetic 20-mer oligonucleotide and long genomic DNA from calf thymus. Copyright © 2017 Elsevier B.V. All rights reserved.

Effect of women's age on embryo morphology, cleavage rate and competence-A multicenter cohort study

DEFF Research Database (Denmark)

Grøndahl, Marie Louise; Christiansen, Sofie Lindgren; Kesmodel, Ulrik Schiøler

2017-01-01

This multicenter cohort study on embryo assessment and outcome data from 11,744 IVF/ICSI cycles with 104,830 oocytes and 42,074 embryos, presents the effect of women's age on oocyte, zygote, embryo morphology and cleavage parameters, as well as cycle outcome measures corrected for confounding.......0001) with increasing age. Maternal age had no effect on cleavage parameters or on the morphology of the embryo day 2 post insemination. Interestingly, initial hCG value after single embryo transfer followed by ongoing pregnancy was increased with age in both IVF (p = 0.007) and ICSI (p = 0.001) cycles. For the first...... time, we show that a woman's age does impose a significant footprint on early embryo morphological development (3PN). In addition, the developmentally competent embryos were associated with increased initial hCG values as the age of the women increased. Further studies are needed to elucidate...

Ultrarapid mutation detection by multiplex, solid-phase chemical cleavage

Energy Technology Data Exchange (ETDEWEB)

Rowley, G.; Saad, S.; Giannelli, F.; Green, P.M. [Guy`s & St. Thomas`s Hospitals, London (United Kingdom)

1995-12-10

The chemical cleavage of mismatches in heteroduplexes formed by probe and test DNA detects and locates any sequence change in long DNA segments ({approximately}1.8 kb), and its efficiency has been well tested in the analysis of both average (e.g., coagulation factor IX) and large, complex genes (e.g., coagulation factor VIII and dystrophin). In the latter application RT/PCR products allow the examination of all essential sequences of the gene in a minimum number of reactions. We use two specific chemical reactants (hydroxylamine and osmium tetroxide) and piperidine cleavage of the above procedure to develop a very fast mutation screening method. This is based on: (1) 5{prime} or internal fluorescent labeling to allow concurrent screening of three to four DNA fragments and (2) solid-phase chemistry to use a microliter format and reduce the time required for the procedure, from amplification of sequence to gel loading inclusive, to one person-working-day. We test the two variations of the method, one entailing 5{prime} labeling of probe DNA and the other uniform labeling of both probe and target DNA, by detecting 114 known hemophilia B (coagulation factor IX) mutations and by analyzing 129 new patients. Uniform labeling of both probe and target DNA prior to formation of the heteroduplexes leads to almost twofold redundancy in the ability to detect mutations. Alternatively, the latter procedure may offer very efficient though less than 100% screening for sequence changes with only hydroxylamine. The full method with two chemical reactions (hydroxylamine and osmium tetroxide) should allow one person to screen with virtually 100% accuracy more than 300 kb of sequence in three ABI 373 gels in 1 day. 26 refs., 7 figs., 1 tab.

Photooxidative cleavage of 4(1H)-quinolinones to 2-acylaminobenzoic acids and derivatives

Energy Technology Data Exchange (ETDEWEB)

Staskun, B. (University of the Witwatersrand, Johannesburg (South Africa). Dept. of Chemistry); Foote, C.S. (California Univ., Los Angeles (USA). Dept. of Chemistry)

1984-12-01

4(1H)-Quinolinones undergo oxidative cleavage to afford the corresponding 2-acylaminobenzoic acids when subjected to dye-sensitized photooxygenation in methanol-aqueous sodium hydroxide solution. The derived 2-aminobenzoic acid was the predominant product in certain instances. The reaction, with singlet oxygen suggested as the active species, provides an alternative methodology for access to nuclear- substituted anthranilic acids and derivatives.

Viral persistence, liver disease and host response in Hepatitis C-like virus rat model

DEFF Research Database (Denmark)

Trivedi, Sheetal; Murthy, Satyapramod; Sharma, Himanshu

2018-01-01

The lack of a relevant, tractable, and immunocompetent animal model for hepatitis C virus (HCV) has severely impeded investigations of viral persistence, immunity and pathogenesis. In the absence of immunocompetent models with robust HCV infection, homolog hepaciviruses in their natural host could...... potentially provide useful surrogate models. We isolated a rodent hepacivirus (RHV) from wild rats (Rattus norvegicus), RHV-rn1, acquired the complete viral genome sequence and developed an infectious reverse genetics system. RHV-rn1 resembles HCV in genomic features including the pattern of polyprotein...... cleavage sites and secondary structures in the viral 5' and 3' UTRs. We used site-directed and random mutagenesis to determine that only the first of the two miR-122 seed sites in viral 5'UTR is required for viral replication and persistence in rats. Next, we used the clone derived virus progeny to infect...

Atorvastatin prevents Aβ oligomer-induced neurotoxicity in cultured rat hippocampal neurons by inhibiting Tau cleavage

Science.gov (United States)

Sui, Hai-juan; Zhang, Ling-ling; Liu, Zhou; Jin, Ying

2015-01-01

Aim: The proteolytic cleavage of Tau is involved in Aβ-induced neuronal dysfunction and cell death. In this study, we investigated whether atorvastatin could prevent Tau cleavage and hence prevent Aβ1–42 oligomer (AβO)-induced neurotoxicity in cultured cortical neurons. Methods: Cultured rat hippocampal neurons were incubated in the presence of AβOs (1.25 μmol/L) with or without atorvastatin pretreatment. ATP content and LDH in the culture medium were measured to assess the neuronal viability. Caspase-3/7 and calpain protease activities were detected. The levels of phospho-Akt, phospho-Erk1/2, phospho-GSK3β, p35 and Tau proteins were measured using Western blotting. Results: Treatment of the neurons with AβO significantly decreased the neuronal viability, induced rapid activation of calpain and caspase-3/7 proteases, accompanied by Tau degradation and relatively stable fragments generated in the neurons. AβO also suppressed Akt and Erk1/2 kinase activity, while increased GSK3β and Cdk5 activity in the neurons. Pretreatment with atorvastatin (0.5, 1, 2.5 μmol/L) dose-dependently inhibited AβO-induced activation of calpain and caspase-3/7 proteases, and effectively diminished the generation of Tau fragments, attenuated synaptic damage and increased neuronal survival. Atorvastatin pretreatment also prevented AβO-induced decreases in Akt and Erk1/2 kinase activity and the increases in GSK3β and Cdk5 kinase activity. Conclusion: Atorvastatin prevents AβO-induced neurotoxicity in cultured rat hippocampal neurons by inhibiting calpain- and caspase-mediated Tau cleavage. PMID:25891085

Transformation of benzophenone-type UV filters by chlorine: Kinetics, products identification and toxicity assessments

International Nuclear Information System (INIS)

Li, Jian; Ma, Li-yun; Xu, Li

2016-01-01

Highlights: • Chlorination kinetics of three benzophenone-type UV filters (BPs) was studied. • Chlorination of BPs followed second-order reaction. • The transformation products (TPs) of six BPs were identified. • Several transformation pathways were proposed. • Mostly enhanced toxicity of TPs after chlorination was observed. - Abstract: The present study focused on the kinetics, transformation pathways and toxicity of several benzophenone-type ultraviolet filters (BPs) during the water chlorination disinfection process. The transformation kinetics of the studied three BPs was found to be second-order reaction, which was dependent on the concentration of BPs and chlorine. The second-order rate constants increased from 86.7 to 975 M"−"1 s"−"1 for oxybenzone, 49.6–261.7 M"−"1 s"−"1 for 4-hydroxybenzophenone and 51.7–540 M"−"1 s"−"1 for 2-hydroxy-4-methoxybenzophenone-5-sulfonic acid with the increasing pH value from 6 to 8 of the chlorination disinfection condition. Then the transformation products (TPs) of these BPs were identified by HPLC-QTof analysis. Several transformation pathways, including electrophilic substitution, methoxyl substitution, ketone groups oxidation, hydrolysis, decarboxylation and ring cleavage reaction, were speculated to participate in the chlorination transformation process. Finally, according to the toxicity experiment on luminescent bacteria, Photobacterium phosphoreum, enhanced toxicity was observed for almost all the TPs of the studied BPs except for 2,2′-dihydroxy-4,4′-dimethoxybenzophenone; it suggested the formation of TPs with more toxic than the parent compounds during the chlorination process. The present study provided a foundation to understand the transformation of BPs during chlorination disinfection process, and was of great significance to the drinking water safety.

Transformation of benzophenone-type UV filters by chlorine: Kinetics, products identification and toxicity assessments

Energy Technology Data Exchange (ETDEWEB)

Li, Jian; Ma, Li-yun; Xu, Li, E-mail: xulpharm@mails.tjmu.edu.cn

2016-07-05

Highlights: • Chlorination kinetics of three benzophenone-type UV filters (BPs) was studied. • Chlorination of BPs followed second-order reaction. • The transformation products (TPs) of six BPs were identified. • Several transformation pathways were proposed. • Mostly enhanced toxicity of TPs after chlorination was observed. - Abstract: The present study focused on the kinetics, transformation pathways and toxicity of several benzophenone-type ultraviolet filters (BPs) during the water chlorination disinfection process. The transformation kinetics of the studied three BPs was found to be second-order reaction, which was dependent on the concentration of BPs and chlorine. The second-order rate constants increased from 86.7 to 975 M{sup −1} s{sup −1} for oxybenzone, 49.6–261.7 M{sup −1} s{sup −1} for 4-hydroxybenzophenone and 51.7–540 M{sup −1} s{sup −1} for 2-hydroxy-4-methoxybenzophenone-5-sulfonic acid with the increasing pH value from 6 to 8 of the chlorination disinfection condition. Then the transformation products (TPs) of these BPs were identified by HPLC-QTof analysis. Several transformation pathways, including electrophilic substitution, methoxyl substitution, ketone groups oxidation, hydrolysis, decarboxylation and ring cleavage reaction, were speculated to participate in the chlorination transformation process. Finally, according to the toxicity experiment on luminescent bacteria, Photobacterium phosphoreum, enhanced toxicity was observed for almost all the TPs of the studied BPs except for 2,2′-dihydroxy-4,4′-dimethoxybenzophenone; it suggested the formation of TPs with more toxic than the parent compounds during the chlorination process. The present study provided a foundation to understand the transformation of BPs during chlorination disinfection process, and was of great significance to the drinking water safety.

AcEST: DK954773 [AcEST

Lifescience Database Archive (English)

Full Text Available y: 436 K 438 K Sbjct: 364 K 364 >sp|Q9WJC7|POLN_EEVVM Non-structural polyprotein OS=Venezuelan equine enceph...ILFSPK 1648 >sp|Q8V294|POLN_EEVVC Non-structural polyprotein OS=Venezuelan equine encephalitis virus (strain... polyprotein OS=Venezuelan equine encephalitis virus PE=4 SV=1 Length = 2506 Scor

Disclosure of key stereoelectronic factors for efficient H2 binding and cleavage in the active site of [NiFe]-hydrogenases.

Science.gov (United States)

Bruschi, Maurizio; Tiberti, Matteo; Guerra, Alessandro; De Gioia, Luca

2014-02-05

A comparative analysis of a series of DFT models of [NiFe]-hydrogenases, ranging from minimal NiFe clusters to very large systems including both the first and second coordination sphere of the bimetallic cofactor, was carried out with the aim of unraveling which stereoelectronic properties of the active site of [NiFe]-hydrogenases are crucial for efficient H2 binding and cleavage. H2 binding to the Ni-SIa redox state is energetically favored (by 4.0 kcal mol(-1)) only when H2 binds to Ni, the NiFe metal cluster is in a low spin state, and the Ni cysteine ligands have a peculiar seesaw coordination geometry, which in the enzyme is stabilized by the protein environment. The influence of the Ni coordination geometry on the H2 binding affinity was then quantitatively evaluated and rationalized analyzing frontier molecular orbitals and populations. Several plausible reaction pathways leading to H2 cleavage were also studied. It turned out that a two-step pathway, where H2 cleavage takes place on the Ni-SIa redox state of the enzyme, is characterized by very low reaction barriers and favorable reaction energies. More importantly, the seesaw coordination geometry of Ni was found to be a key feature for facile H2 cleavage. The discovery of the crucial influence of the Ni coordination geometry on H2 binding and activation in the active site of [NiFe]-hydrogenases could be exploited in the design of novel biomimetic synthetic catalysts.

Heparin kinetics

International Nuclear Information System (INIS)

Swart, C.A.M. de.

1983-01-01

The author has studied the kinetics of heparin and heparin fractions after intravenous administration in humans and in this thesis the results of this study are reported. Basic knowledge about the physico-chemical properties of heparin and its interactions with proteins resulting in anticoagulant and lipolytic effects are discussed in a review (chapter II), which also comprises some clinical aspects of heparin therapy. In chapter III the kinetics of the anticoagulant effect are described after intravenous administration of five commercial heparin preparations. A mathematical model is presented that fits best to these kinetics. The kinetics of the anticoagulant and lipolytic effects after intravenous injection of various 35 S-radiolabelled heparin fractions and their relationship with the disappearance of the radiolabel are described in chapter IV. Chapter V gives a description of the kinetics of two radiolabels after injection of in vitro formed complexes consisting of purified, 125 I-radiolabelled antithrombin III and various 35 S-radiolabelled heparin fractions. (Auth.)

The shallow flaw effect and the local approach to cleavage fracture

International Nuclear Information System (INIS)

Moinereau, D.

1996-10-01

The capability of Beremin model to explain the shallow flaw effect in cleavage fracture is evaluated. Numerous two-dimensional finite element calculations are performed on several cracked specimens (cladded and un-cladded specimens with different values of a/W ratio) submitted to mechanical or thermal loading. The behavior of different specimens is examined using the Weibull stress σ w versus stress intensity factor K J curves. The stress fields and plastic zones at the crack tip are also compared on respective cracked specimens. (K.A.)

L = spiro-N3P3[O2C12H8][N(CH3)NH2

Indian Academy of Sciences (India)

Unknown

over a million-fold, the rate of hydrolysis of the latter also is enhanced considerably. Detailed kinetic evaluation of these reactions has been carried out and all the kinetic parameters including the Michaelis– ... cleavage experiments.

Deletion of the thrombin cleavage domain of osteopontin mediates breast cancer cell adhesion, proteolytic activity, tumorgenicity, and metastasis

International Nuclear Information System (INIS)

Beausoleil, Michel S; Schulze, Erika B; Goodale, David; Postenka, Carl O; Allan, Alison L

2011-01-01

Osteopontin (OPN) is a secreted phosphoprotein often overexpressed at high levels in the blood and primary tumors of breast cancer patients. OPN contains two integrin-binding sites and a thrombin cleavage domain located in close proximity to each other. To study the role of the thrombin cleavage site of OPN, MDA-MB-468 human breast cancer cells were stably transfected with either wildtype OPN (468-OPN), mutant OPN lacking the thrombin cleavage domain (468-ΔTC) or an empty vector (468-CON) and assessed for in vitro and in vivo functional differences in malignant/metastatic behavior. All three cell lines were found to equivalently express thrombin, tissue factor, CD44, αvβ5 integrin and β1 integrin. Relative to 468-OPN and 468-CON cells, 468-ΔTC cells expressing OPN with a deleted thrombin cleavage domain demonstrated decreased cell adhesion (p < 0.001), decreased mRNA expression of MCAM, maspin and TRAIL (p < 0.01), and increased uPA expression and activity (p < 0.01) in vitro. Furthermore, injection of 468-ΔTC cells into the mammary fat pad of nude mice resulted in decreased primary tumor latency time (p < 0.01) and increased primary tumor growth and lymph node metastatic burden (p < 0.001) compared to 468-OPN and 468-CON cells. The results presented here suggest that expression of thrombin-uncleavable OPN imparts an early tumor formation advantage as well as a metastatic advantage for breast cancer cells, possibly due to increased proteolytic activity and decreased adhesion and apoptosis. Clarification of the mechanisms responsible for these observations and the translation of this knowledge into the clinic could ultimately provide new therapeutic opportunities for combating breast cancer

A catalytic metal ion interacts with the cleavage site G•U wobble in the HDV ribozyme†

Science.gov (United States)

Chen, Jui-Hui; Gong, Bo; Bevilacqua, Philip C.; Carey, Paul R.; Golden, Barbara L.

2009-01-01

The HDV ribozyme self-cleaves by a chemical mechanism involving general acid-base catalysis to generate a 2′,3′-cyclic phosphate and a 5′-hydroxyl termini. Biochemical studies from several laboratories have implicated C75 as the general acid and hydrated magnesium as the general base. We have previously shown that C75 has a pKa shifted > 2 pH units toward neutrality [Gong, B., Chen, J. H., Chase, E., Chadalavada, D. M., Yajima, R., Golden, B. L., Bevilacqua, P. C., and Carey, P. R. (2007) J. Am. Chem. Soc. 129, 13335–13342.], while in crystal structures, it is well-positioned for proton transfer. However no crystallographic evidence for a hydrated magnesium poised to serve as a general base in the reaction has been observed in high-resolution crystal structures of various reaction states and mutants. Herein, we use solution kinetic experiments and parallel Raman crystallographic studies to examine the effects of pH on rate and Mg2+-binding properties of wild-type and 7-deazaguanosine mutants of the HDV ribozyme. These data suggest that a previously-unobserved hydrated magnesium ion interacts with the N7 of the cleavage site G•U wobble base pair. Integrating this metal ion binding site with the available crystal structures provides a new three-dimensional model for the active site of the ribozyme that accommodates all available biochemical data and appears competent for catalysis. The position of this metal is consistent with a role of a magnesium-bound hydroxide as a general base as dictated by biochemical data. PMID:19178151

Nickel-Catalyzed Synthesis of Primary Aryl and Heteroaryl Amines via C–O Bond Cleavage

KAUST Repository

Yue, Huifeng

2017-03-13

A nickel-catalyzed protocol for the conversion of aryl and heteroaryl alcohol derivatives to primary and secondary aromatic amines via C(sp2)-O bond cleavage is described. The new amination protocol can be applied to a range of substrates bearing diverse functional groups and uses readily available benzophenone imines as an effective nitrogen source.

Nickel-Catalyzed Synthesis of Primary Aryl and Heteroaryl Amines via C–O Bond Cleavage

KAUST Repository

Yue, Huifeng; Guo, Lin; Liu, Xiangqian; Rueping, Magnus

2017-01-01

A nickel-catalyzed protocol for the conversion of aryl and heteroaryl alcohol derivatives to primary and secondary aromatic amines via C(sp2)-O bond cleavage is described. The new amination protocol can be applied to a range of substrates bearing diverse functional groups and uses readily available benzophenone imines as an effective nitrogen source.

Bond cleavage reactions of the bridge structure in coal in the presence of hydrogen donating compounds; Suiso kyoyosei kagobutsu sonzaika deno sekitanchu no kakyo kozo no kairetsu hanno

Energy Technology Data Exchange (ETDEWEB)

Bando, N.; Kidena, K.; Murata, S.; Nomura, M. [Osaka University, Osaka (Japan). Faculty of Engineering

1996-10-28

In this paper, bond cleavage reactions are discussed in relation to the softening and solubilization of coal. Were used 9,10-dihydroanthracene (DHA) and 9,10-dihydrophenanthrene (DHP) as models of hydrogen donating compounds in coal, and bibenzyl, 1,2-diethane, benzylphenylether, and 1,5-dibenzylnaphthalene were used as models of bridge structure compounds. They were compared mutually, as to reactivity of coal against DHA and DHP. For the homolytic cleavage of bridges, DHA with excellent radical supplement performance provided excellent hydrogen donating performance. While, for the ipso-position cleavage of bridges, it was found that DHP can act as an effective hydrogen donor. For the reaction between coal and hydrogenated aromatic compounds, cleavage of relatively weak bonds, such as ether linkage and dimethylene linkage, occurred at about 380{degree}C, and hydrogen from DHA or DHP was consumed. On the other hand, the results suggested that the cleavage reaction at ipso-position affected by hydrogen donating solvent is also important at temperature range around 420{degree}C. 2 refs., 3 figs., 1 tab.

Efficient Construction of Energetic Materials via Nonmetallic Catalytic Carbon-Carbon Cleavage/Oxime-Release-Coupling Reactions.

Science.gov (United States)

Zhao, Gang; He, Chunlin; Yin, Ping; Imler, Gregory H; Parrish, Damon A; Shreeve, Jean'ne M

2018-03-14

The exploitation of C-C activation to facilitate chemical reactions is well-known in organic chemistry. Traditional strategies in homogeneous media rely upon catalyst-activated or metal-mediated C-C bonds leading to the design of new processes for applications in organic chemistry. However, activation of a C-C bond, compared with C-H bond activation, is a more challenging process and an underdeveloped area because thermodynamics does not favor insertion into a C-C bond in solution. Carbon-carbon bond cleavage through loss of an oxime moiety has not been reported. In this paper, a new observation of self-coupling via C-C bond cleavage with concomitant loss of oxime in the absence of metals (either metal-complex mediation or catalysis) results in dihydroxylammonium 5,5-bistetrazole-1,10-diolate (TKX-50) as well as N, N'-([3,3'-bi(1,2,4-oxadiazole)]-5,5'-diyl)dinitramine, a potential candidate for a new generation of energetic materials.

EMMPRIN Modulates Epithelial Barrier Function through a MMP–Mediated Occludin Cleavage

Science.gov (United States)

Huet, Eric; Vallée, Benoit; Delbé, Jean; Mourah, Samia; Prulière-Escabasse, Virginie; Tremouilleres, Magali; Kadomatsu, Kenji; Doan, Serge; Baudouin, Christophe; Menashi, Suzanne; Gabison, Eric E.

2011-01-01

Dry eye is a common disease that develops as a result of alteration of tear fluid, leading to osmotic stress and a perturbed epithelial barrier. Matrix metalloproteinase-9 (MMP-9) may be important in dry eye disease, as its genetic knockout conferred resistance to the epithelial disruption. We show that extracellular matrix metalloproteinase inducer (EMMPRIN; also termed CD147), an inducer of MMP expression, participates in the pathogenesis of dry eye through MMP-mediated cleavage of occludin, an important component of tight junctions. EMMPRIN expression was increased on the ocular surface of dry eye patients and correlated with those of MMP-9. High osmolarity in cell culture, mimicking dry eye conditions, increased both EMMPRIN and MMP-9 and resulted in the disruption of epithelial junctions through the cleavage of occludin. Exogenously added recombinant EMMPRIN had similar effects that were abrogated in the presence of the MMP inhibitor marimastat. Membrane occludin immunostaining was markedly increased in the apical corneal epithelium of both EMMPRIN and MMP-9 knock-out mice. Furthermore, an inverse correlation between EMMPRIN and occludin membrane staining was consistently observed both in vitro and in vivo as a function of corneal epithelial cells differentiation. These data suggest a possible role of EMMPRIN in regulating the amount of occludin at the cell surface in homeostasis beyond pathological situations such as dry eye disease, and EMMPRIN may be essential for the formation and maintenance of organized epithelial structure. PMID:21777561

In vitro maturation of Drosophila melanogaster Spätzle protein with refolded Easter reveals a novel cleavage site within the prodomain.

Science.gov (United States)

Ursel, Christian; Fandrich, Uwe; Hoffmann, Anita; Sieg, Torsten; Ihling, Christian; Stubbs, Milton T

2013-08-01

Dorsoventral patterning during Drosophila melanogaster embryogenesis is mediated by a well-defined gradient of the mature NGF-like ligand Spätzle. Easter, the ultimate protease of a ventrally-restricted serine protease cascade, plays a key role in the regulation of the morphogenic gradient, catalyzing the activation cleavage of proSpätzle. As a result of alternative splicing, proSpätzle exists in multiple isoforms, almost all of which differ only in their prodomain. Although this domain is unstructured in isolation, it has a stabilizing influence on the mature cystine knot domain and is involved in the binding to the Toll receptor. Here, we report the expression and refolding of Easter, and show that the renatured enzyme performs the activation cleavage of two Spätzle isoforms. We determine the affinity of the prodomain for the cystine knot domain, and show that Easter performs a previously unknown secondary cleavage in each prodomain.

Characterization of cleavage events in the multifunctional cilium adhesin Mhp684 (P146) reveals a mechanism by which Mycoplasma hyopneumoniae regulates surface topography.

Science.gov (United States)

Bogema, Daniel R; Deutscher, Ania T; Woolley, Lauren K; Seymour, Lisa M; Raymond, Benjamin B A; Tacchi, Jessica L; Padula, Matthew P; Dixon, Nicholas E; Minion, F Chris; Jenkins, Cheryl; Walker, Mark J; Djordjevic, Steven P

2012-01-01

Mycoplasma hyopneumoniae causes enormous economic losses to swine production worldwide by colonizing the ciliated epithelium in the porcine respiratory tract, resulting in widespread damage to the mucociliary escalator, prolonged inflammation, reduced weight gain, and secondary infections. Protein Mhp684 (P146) comprises 1,317 amino acids, and while the N-terminal 400 residues display significant sequence identity to the archetype cilium adhesin P97, the remainder of the molecule is novel and displays unusual motifs. Proteome analysis shows that P146 preprotein is endogenously cleaved into three major fragments identified here as P50(P146), P40(P146), and P85(P146) that reside on the cell surface. Liquid chromatography with tandem mass spectrometry (LC-MS/MS) identified a semitryptic peptide that delineated a major cleavage site in Mhp684. Cleavage occurred at the phenylalanine residue within sequence (672)ATEF↓QQ(677), consistent with a cleavage motif resembling S/T-X-F↓X-D/E recently identified in Mhp683 and other P97/P102 family members. Biotinylated surface proteins recovered by avidin chromatography and separated by two-dimensional gel electrophoresis (2-D GE) showed that more-extensive endoproteolytic cleavage of P146 occurs. Recombinant fragments F1(P146)-F3(P146) that mimic P50(P146), P40(P146), and P85(P146) were constructed and shown to bind porcine epithelial cilia and biotinylated heparin with physiologically relevant affinity. Recombinant versions of F3(P146) generated from M. hyopneumoniae strain J and 232 sequences strongly bind porcine plasminogen, and the removal of their respective C-terminal lysine and arginine residues significantly reduces this interaction. These data reveal that P146 is an extensively processed, multifunctional adhesin of M. hyopneumoniae. Extensive cleavage coupled with variable cleavage efficiency provides a mechanism by which M. hyopneumoniae regulates protein topography. Vaccines used to control Mycoplasma

Molecular mechanism of the intramembrane cleavage of the β-carboxyl terminal fragment of amyloid precursor protein by γ-secretase

Directory of Open Access Journals (Sweden)

Maho eMorishima-Kawashima

2014-11-01

Full Text Available Amyloid β-protein (Aβ plays a central role in the pathogenesis of Alzheimer’s disease, the most common age-associated neurodegenerative disorder. Aβ is generated through intramembrane proteolysis of the β-carboxyl terminal fragment (βCTF of β-amyloid precursor protein (APP by γ-secretase. The initial cleavage by γ-secretase occurs in the membrane/cytoplasm boundary of the βCTF, liberating the APP intracellular domain (AICD. The remaining βCTFs, which are truncated at the C-terminus (longer Aβs, are then cropped sequentially in a stepwise manner, predominantly at three residue intervals, to generate Aβ. There are two major Aβ product lines which generate Aβ40 and Aβ42 with concomitant release of three and two tripeptides, respectively. Additionally, many alternative cleavages occur, releasing peptides with three to six residues. These modulate the Aβ product lines and define the species and quantity of Aβ generated. Here, we review our current understanding of the intramembrane cleavage of the βCTF by γ-secretase, which may contribute to the future goal of developing an efficient therapeutic strategy for Alzheimer’s disease.

Dynamics of bleomycin interaction with a strongly bound hairpin DNA substrate, and implications for cleavage of the bound DNA.

Science.gov (United States)

Bozeman, Trevor C; Nanjunda, Rupesh; Tang, Chenhong; Liu, Yang; Segerman, Zachary J; Zaleski, Paul A; Wilson, W David; Hecht, Sidney M

2012-10-31

Recent studies involving DNAs bound strongly by bleomycins have documented that such DNAs are degraded by the antitumor antibiotic with characteristics different from those observed when studying the cleavage of randomly chosen DNAs in the presence of excess Fe·BLM. In the present study, surface plasmon resonance has been used to characterize the dynamics of BLM B(2) binding to a strongly bound hairpin DNA, to define the effects of Fe(3+), salt, and temperature on BLM-DNA interaction. One strong primary DNA binding site, and at least one much weaker site, were documented. In contrast, more than one strong cleavage site was found, an observation also made for two other hairpin DNAs. Evidence is presented for BLM equilibration between the stronger and weaker binding sites in a way that renders BLM unavailable to other, less strongly bound DNAs. Thus, enhanced binding to a given site does not necessarily result in increased DNA degradation at that site; i.e., for strongly bound DNAs, the facility of DNA cleavage must involve other parameters in addition to the intrinsic rate of C-4' H atom abstraction from DNA sugars.

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

Science.gov (United States)

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

Ku-mediated coupling of DNA cleavage and repair during programmed genome rearrangements in the ciliate Paramecium tetraurelia.

Directory of Open Access Journals (Sweden)

Antoine Marmignon

2014-08-01

Full Text Available During somatic differentiation, physiological DNA double-strand breaks (DSB can drive programmed genome rearrangements (PGR, during which DSB repair pathways are mobilized to safeguard genome integrity. Because of their unique nuclear dimorphism, ciliates are powerful unicellular eukaryotic models to study the mechanisms involved in PGR. At each sexual cycle, the germline nucleus is transmitted to the progeny, but the somatic nucleus, essential for gene expression, is destroyed and a new somatic nucleus differentiates from a copy of the germline nucleus. In Paramecium tetraurelia, the development of the somatic nucleus involves massive PGR, including the precise elimination of at least 45,000 germline sequences (Internal Eliminated Sequences, IES. IES excision proceeds through a cut-and-close mechanism: a domesticated transposase, PiggyMac, is essential for DNA cleavage, and DSB repair at excision sites involves the Ligase IV, a specific component of the non-homologous end-joining (NHEJ pathway. At the genome-wide level, a huge number of programmed DSBs must be repaired during this process to allow the assembly of functional somatic chromosomes. To understand how DNA cleavage and DSB repair are coordinated during PGR, we have focused on Ku, the earliest actor of NHEJ-mediated repair. Two Ku70 and three Ku80 paralogs are encoded in the genome of P. tetraurelia: Ku70a and Ku80c are produced during sexual processes and localize specifically in the developing new somatic nucleus. Using RNA interference, we show that the development-specific Ku70/Ku80c heterodimer is essential for the recovery of a functional somatic nucleus. Strikingly, at the molecular level, PiggyMac-dependent DNA cleavage is abolished at IES boundaries in cells depleted for Ku80c, resulting in IES retention in the somatic genome. PiggyMac and Ku70a/Ku80c co-purify as a complex when overproduced in a heterologous system. We conclude that Ku has been integrated in the Paramecium

Airway protease/antiprotease imbalance in atopic asthmatics contributes to increased influenza A virus cleavage and replication

Science.gov (United States)

Asthmatics are more susceptible to influenza infections, yet mechanisms mediating this enhanced susceptibility are unknown. Influenza virus hemagglutinin (HA) protein binds to sialic add residues on the host cells. HA requires cleavage to allow fusion of the viral HA with host ce...

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

Science.gov (United States)

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.

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

DEFF Research Database (Denmark)

Gullberg, Maria; Polacek, Charlotta; Belsham, Graham

2014-01-01

The foot-and-mouth disease virus (FMDV) capsid protein precursor P1-2A is cleaved by the virus-encoded 3C protease to VP0, VP3, VP1 and 2A. It was shown previously that modification of a single amino acid residue (K210E) within the VP1 protein and close to the VP1/2A cleavage site, inhibited...... cleavage of this junction and produced 'self-tagged' virus particles. A second site substitution (E83K) within VP1 was also observed within the rescued virus [Gullberg et al. (2013). J Virol 87: , 11591-11603]. It was shown here that introduction of this E83K change alone into a serotype O virus resulted...... in the rapid accumulation of a second site substitution within the 2A sequence (L2P), which also blocked VP1/2A cleavage. This suggests a linkage between the E83K change in VP1 and cleavage of the VP1/2A junction. Cells infected with viruses containing the VP1 K210E or the 2A L2P substitutions contained...

AcEST: DK944561 [AcEST

Lifescience Database Archive (English)

Full Text Available hit_id Q306W7 Definition sp|Q306W7|POLS_EEEV8 Structural polyprotein OS=Eastern equine encephalitis virus (s...E Sequences producing significant alignments: (bits) Value sp|Q306W7|POLS_EEEV8 Structural polyprotein OS=Eastern equine...domain-conta... 29 6.3 >sp|Q306W7|POLS_EEEV8 Structural polyprotein OS=Eastern equine encephalitis virus (st

Changes in radiosensitivity of the in vitro fertilized mouse ova during zygotic stage from fertilization to first cleavage

International Nuclear Information System (INIS)

Yamada, Takeshi; Yukawa, Osami; Matsuda, Yoichi; Ohkawa, Akiko

1982-01-01

The radiosensitivity of mouse zygotes fertilized in vitro (BC3F 1 ovum x ICR sperm) to X-rays has been measured as a function of time from fertilization to first cleavage. The dose of X-rays (LD 50 ) required to prevent development of 50% of the zygotes to the blastocyst stage in vitro varied markedly depending on the time of irradiation from about 40 to 400 R. Sensitivity is relatively low shortly after sperm entry and becomes extremely high (LD 50 , 40 R) at the stage just before pronuclear formation (4 to 6 hr after insemination). In the later pronuclear stage (12 hr of fertilization) the sensitivity becomes low (LD 50 , about 400 R), and it increases again just before first cleavage. (author)

Detection of siRNA Mediated Target mRNA Cleavage Activities in Human Cells by a Novel Stem-Loop Array RT-PCR Analysis

Science.gov (United States)

2016-09-07

sequences of the target mRNA, and a double stranded stem at the 5′ end that forms a stem -loop to function as a forceps to stabilize the secondary...E-mjournal homepage: www.elsevier.com/locate/bbrepDetection of siRNA-mediated target mRNA cleavage activities in human cells by a novel stem -loop...challenges for the accurate and efficient detection and verification of cleavage sites on target mRNAs. Here we used a sensitive stem -loop array reverse

Decomposition mechanisms and kinetics of novel energetic molecules BNFF-1 and ANFF-1: quantum-chemical modeling.

Science.gov (United States)

Tsyshevsky, Roman V; Kuklja, Maija M

2013-07-18

Decomposition mechanisms, activation barriers, Arrhenius parameters, and reaction kinetics of the novel explosive compounds, 3,4-bis(4-nitro-1,2,5-oxadiazol-3-yl)-1,2,5-oxadiazole (BNFF-1), and 3-(4-amino-1,2,5-oxadiazol-3-yl)-4-(4-nitro-1,2,5-oxadiazol-3-yl)-1,2,5-oxadiazole (ANFF-1) were explored by means of density functional theory with a range of functionals combined with variational transition state theory. BNFF-1 and ANFF-1 were recently suggested to be good candidates for insensitive high energy density materials. Our modeling reveals that the decomposition initiation in both BNFF-1 and ANFF-1 molecules is triggered by ring cleavage reactions while the further process is defined by a competition between two major pathways, the fast C-NO₂ homolysis and slow nitro-nitrite isomerization releasing NO. We discuss insights on design of new energetic materials with targeted properties gained from our modeling.

Decomposition Mechanisms and Kinetics of Novel Energetic Molecules BNFF-1 and ANFF-1: Quantum-Chemical Modeling

Directory of Open Access Journals (Sweden)

Maija M. Kuklja

2013-07-01

Full Text Available Decomposition mechanisms, activation barriers, Arrhenius parameters, and reaction kinetics of the novel explosive compounds, 3,4-bis(4-nitro-1,2,5-oxadiazol-3-yl-1,2,5-oxadiazole (BNFF-1, and 3-(4-amino-1,2,5-oxadiazol-3-yl-4-(4-nitro-1,2,5-oxadiazol-3-yl-1,2,5-oxadiazole (ANFF-1 were explored by means of density functional theory with a range of functionals combined with variational transition state theory. BNFF-1 and ANFF-1 were recently suggested to be good candidates for insensitive high energy density materials. Our modeling reveals that the decomposition initiation in both BNFF-1 and ANFF-1 molecules is triggered by ring cleavage reactions while the further process is defined by a competition between two major pathways, the fast C-NO2 homolysis and slow nitro-nitrite isomerization releasing NO. We discuss insights on design of new energetic materials with targeted properties gained from our modeling.

A Kinetic Model Explains Why Shorter and Less Affine Enzyme-recruiting Oligonucleotides Can Be More Potent

Directory of Open Access Journals (Sweden)

Lykke Pedersen

2014-01-01

Full Text Available Antisense oligonucleotides complementary to RNA targets promise generality and ease of drug design. The first systemically administered antisense drug was recently approved for treatment and others are in clinical development. Chemical modifications that increase the hybridization affinity of oligonucleotides are reasoned to confer higher potency, i.e., modified oligonucleotides can be dosed at lower concentrations to achieve the same effect. Surprisingly, shorter and less affine oligonucleotides sometimes display increased potency. To explain this apparent contradiction, increased uptake or decreased propensity to form structures have been suggested as possible mechanisms. Here, we provide an alternative explanation that invokes only the kinetics behind oligonucleotide-mediated cleavage of RNA targets. A model based on the law of mass action predicts, and experiments support, the existence of an optimal binding affinity. Exaggerated affinity, and not length per se, is detrimental to potency. This finding clarifies how to optimally apply high-affinity modifications in the discovery of potent antisense oligonucleotide drugs.

Blocking Internalization of Phosphatidylethanolamine at Cleavage Furrow of Mitosis as a Novel Mechanism of Anti-Breast Cancer Strategy

National Research Council Canada - National Science Library

Cui, Zhen

2002-01-01

During the formation of cleavage furrow of mitosis, phosphatidylethanolamine (PE) flips from inner leaflet of the plasma membrane to the outer leaflet specifically in the furrow region near the contractile ring...

Blocking Internalization of Phosphatidylethanolamine at Cleavage Furrow of Mitosis as a Novel Mechanism of Anti-Breast-Cancer Strategy

National Research Council Canada - National Science Library

Cui, Zheng

2003-01-01

During the formation of cleavage furrow of mitosis, phosphatidylethanolamine (PE) flips from inner leaflet of the plasma membrane to the outer leaflet specifically in the furrow region near the contractile ring...

MODELS FOR MOUSE CHIMERA PRODUCTION: AGGREGATION OF ES CELLS WITH CLEAVAGE STAGE EMBRYOS

Directory of Open Access Journals (Sweden)

STANCA CLAUDIA

2007-01-01

Full Text Available In a mutant ES cells↔ wild-type embryo chimera, ES cells behave more like epiblastcells. They can contribute to the primitive ectoderm layers, which give rise to all theembryonic tissues and some extraembryonic tissues (Beddington and Robertson,1989, but not to trophectoderm or primitive endoderm. Using transgenic ES celllines, aggregated with cleavage stage host embryo, ES cells can integrate randomlyin the embryo proper. If they will be take part in the formation of ICM (inner cellmass, it will be possible to obtain germline chimera animals. To generate ES cells↔ cleavage stage host embryo chimeras, we used (CD-1 mice as donors of hostembryos as well as recipients of manipulated embryos. For chimera production, weused fluorescent-labeled ES cell line (CD1/EGFP, because in this case we canfollow the fate of ES cells during the embryonic development. We produced thechimers using “aggregation chimera technique”. 8 cells stage zona pellucida free,mouse embryos were aggregated in an aggregation plates, with a clump of ES cells(10 – 15 cells. The chimera embryos were cultivated for 24 hours in the incubator(at 37 °C, 5% CO2 in air. The chimera blastocysts resulted after cultivation, weretransferred to the uterus of the 2.5-dpc pseudo pregnant females.

MODELS FOR MOUSE CHIMERA PRODUCTION: AGGREGATION OF ES CELLS WITH CLEAVAGE STAGE EMBRYOS

Directory of Open Access Journals (Sweden)

CLAUDIA STANCA

2007-05-01

Full Text Available In a mutant ES cells↔ wild-type embryo chimera, ES cells behave more like epiblastcells. They can contribute to the primitive ectoderm layers, which give rise to all theembryonic tissues and some extraembryonic tissues (Beddington and Robertson,1989, but not to trophectoderm or primitive endoderm. Using transgenic ES celllines, aggregated with cleavage stage host embryo, ES cells can integrate randomlyin the embryo proper. If they will be take part in the formation of ICM (inner cellmass, it will be possible to obtain germline chimera animals. To generate ES cells↔ cleavage stage host embryo chimeras, we used (CD-1 mice as donors of hostembryos as well as recipients of manipulated embryos. For chimera production, weused fluorescent-labeled ES cell line (CD1/EGFP, because in this case we canfollow the fate of ES cells during the embryonic development. We produced thechimers using “aggregation chimera technique”. 8 cells stage zona pellucida free,mouse embryos were aggregated in an aggregation plates, with a clump of ES cells(10 – 15 cells. The chimera embryos were cultivated for 24 hours in the incubator(at 37 °C, 5% CO2 in air. The chimera blastocysts resulted after cultivation, weretransferred to the uterus of the 2.5-dpc pseudo pregnant females.

Inhibition of Cellular Adhesion by Immunological Targeting of Osteopontin Neoepitopes Generated through Matrix Metalloproteinase and Thrombin Cleavage.

Science.gov (United States)

Jürets, Alexander; Le Bras, Marie; Staffler, Günther; Stein, Gesine; Leitner, Lukas; Neuhofer, Angelika; Tardelli, Matteo; Turkof, Edvin; Zeyda, Maximilian; Stulnig, Thomas M

2016-01-01

Osteopontin (OPN), a secreted protein involved in inflammatory processes and cancer, induces cell adhesion, migration, and activation of inflammatory pathways in various cell types. Cells bind OPN via integrins at a canonical RGD region in the full length form as well as to a contiguous cryptic site that some have shown is unmasked upon thrombin or matrix metalloproteinase cleavage. Thus, the adhesive capacity of osteopontin is enhanced by proteolytic cleavage that may occur in inflammatory conditions such as obesity, atherosclerosis, rheumatoid arthritis, tumor growth and metastasis. Our aim was to inhibit cellular adhesion to recombinant truncated proteins that correspond to the N-terminal cleavage products of thrombin- or matrix metalloproteinase-cleaved OPN in vitro. We specifically targeted the cryptic integrin binding site with monoclonal antibodies and antisera induced by peptide immunization of mice. HEK 293 cells adhered markedly stronger to truncated OPN proteins than to full length OPN. Without affecting cell binding to the full length form, the raised monoclonal antibodies specifically impeded cellular adhesion to the OPN fragments. Moreover, we show that the peptides used for immunization were able to induce antisera, which impeded adhesion either to all OPN forms, including the full-length form, or selectively to the corresponding truncated recombinant proteins. In conclusion, we developed immunological tools to selectively target functional properties of protease-cleaved OPN forms, which could find applications in treatment and prevention of various inflammatory diseases and cancers.

Stochastic theory of interfacial enzyme kinetics: A kinetic Monte Carlo study

International Nuclear Information System (INIS)

Das, Biswajit; Gangopadhyay, Gautam

2012-01-01

Graphical abstract: Stochastic theory of interfacial enzyme kinetics is formulated. Numerical results of macroscopic phenomenon of lag-burst kinetics is obtained by using a kinetic Monte Carlo approach to single enzyme activity. Highlights: ► An enzyme is attached with the fluid state phospholipid molecules on the Langmuir monolayer. ► Through the diffusion, the enzyme molecule reaches the gel–fluid interface. ► After hydrolysing a phospholipid molecule it predominantly leaves the surface in the lag phase. ► The enzyme is strictly attached to the surface with scooting mode of motion and the burst phase appears. - Abstract: In the spirit of Gillespie’s stochastic approach we have formulated a theory to explore the advancement of the interfacial enzyme kinetics at the single enzyme level which is ultimately utilized to obtain the ensemble average macroscopic feature, lag-burst kinetics. We have provided a theory of the transition from the lag phase to the burst phase kinetics by considering the gradual development of electrostatic interaction among the positively charged enzyme and negatively charged product molecules deposited on the phospholipid surface. It is shown that the different diffusion time scales of the enzyme over the fluid and product regions are responsible for the memory effect in the correlation of successive turnover events of the hopping mode in the single trajectory analysis which again is reflected on the non-Gaussian distribution of turnover times on the macroscopic kinetics in the lag phase unlike the burst phase kinetics.

Modelling of ductile and cleavage fracture by local approach

International Nuclear Information System (INIS)

Samal, M.K.; Dutta, B.K.; Kushwaha, H.S.

2000-08-01

This report describes the modelling of ductile and cleavage fracture processes by local approach. It is now well known that the conventional fracture mechanics method based on single parameter criteria is not adequate to model the fracture processes. It is because of the existence of effect of size and geometry of flaw, loading type and rate on the fracture resistance behaviour of any structure. Hence, it is questionable to use same fracture resistance curves as determined from standard tests in the analysis of real life components because of existence of all the above effects. So, there is need to have a method in which the parameters used for the analysis will be true material properties, i.e. independent of geometry and size. One of the solutions to the above problem is the use of local approaches. These approaches have been extensively studied and applied to different materials (including SA33 Gr.6) in this report. Each method has been studied and reported in a separate section. This report has been divided into five sections. Section-I gives a brief review of the fundamentals of fracture process. Section-II deals with modelling of ductile fracture by locally uncoupled type of models. In this section, the critical cavity growth parameters of the different models have been determined for the primary heat transport (PHT) piping material of Indian pressurised heavy water reactor (PHWR). A comparative study has been done among different models. The dependency of the critical parameters on stress triaxiality factor has also been studied. It is observed that Rice and Tracey's model is the most suitable one. But, its parameters are not fully independent of triaxiality factor. For this purpose, a modification to Rice and Tracery's model is suggested in Section-III. Section-IV deals with modelling of ductile fracture process by locally coupled type of models. Section-V deals with the modelling of cleavage fracture process by Beremins model, which is based on Weibulls

AcEST: DK958823 [AcEST

Lifescience Database Archive (English)

Full Text Available MPPLEG 2401 >sp|Q8AZM0|POLS_BSNV Structural polyprotein OS=Blotched snakehead virus PE=1 SV=1 Length = 1069 ...p|Q68749|POLG_HCVBB Genome polyprotein OS=Hepatitis C virus gen... 33 0.98 sp|Q8AZM0|POLS_BSNV Structural polyprotein OS=Blotched sna...kehead... 31 4.8 sp|Q7XWS7|FH12_ORYSJ Formin-like protein 12 OS=Oryza sativa subs..

AcEST: BP918286 [AcEST

Lifescience Database Archive (English)

Full Text Available L LIM domain-containing protein pin-2 OS=Caen... 30 6.9 sp|Q4QXJ8|POLN_EEEVF Non-structural polyprotein OS=Easter...n equin... 26 8.0 sp|Q306W8|POLN_EEEV8 Non-structural polyprotein OS=Eastern equin... 26 8.0 sp|Q306W6|...POLN_EEEV1 Non-structural polyprotein OS=Eastern equin... 26 8.0 >sp|P43236|CATK_

Rendering one autolysis site in Bacillus subtilis neutral protease resistant to cleavage reveals a new fission

NARCIS (Netherlands)

Van den Burg, B; Eijsink, VGH; Vriend, G; Veltman, OR; Venema, G

Autolytic degradation of the thermolysin-like proteinase of Bacillus subtilis (TLP-sub) is responsible for the irreversible inactivation of the enzyme at elevated temperatures. Previously we have reported five cleavage sites in Tip-sub [Van den Burg et al, (1990) Biochem. J. 272, 93-97]. In an

High cleavage efficiency of a 2A peptide derived from porcine teschovirus-1 in human cell lines, zebrafish and mice.

Directory of Open Access Journals (Sweden)

Jin Hee Kim

Full Text Available When expression of more than one gene is required in cells, bicistronic or multicistronic expression vectors have been used. Among various strategies employed to construct bicistronic or multicistronic vectors, an internal ribosomal entry site (IRES has been widely used. Due to the large size and difference in expression levels between genes before and after IRES, however, a new strategy was required to replace IRES. A self-cleaving 2A peptide could be a good candidate to replace IRES because of its small size and high cleavage efficiency between genes upstream and downstream of the 2A peptide. Despite the advantages of the 2A peptides, its use is not widespread because (i there are no publicly available cloning vectors harboring a 2A peptide gene and (ii comprehensive comparison of cleavage efficiency among various 2A peptides reported to date has not been performed in different contexts. Here, we generated four expression plasmids each harboring different 2A peptides derived from the foot-and-mouth disease virus, equine rhinitis A virus, Thosea asigna virus and porcine teschovirus-1, respectively, and evaluated their cleavage efficiency in three commonly used human cell lines, zebrafish embryos and adult mice. Western blotting and confocal microscopic analyses revealed that among the four 2As, the one derived from porcine teschovirus-1 (P2A has the highest cleavage efficiency in all the contexts examined. We anticipate that the 2A-harboring cloning vectors we generated and the highest efficiency of the P2A peptide we demonstrated would help biomedical researchers easily adopt the 2A technology when bicistronic or multicistronic expression is required.

Perturbation with intrabodies reveals that calpain cleavage is required for degradation of huntingtin exon 1.

Directory of Open Access Journals (Sweden)

Amber L Southwell

2011-01-01

Full Text Available Proteolytic processing of mutant huntingtin (mHtt, the protein that causes Huntington's disease (HD, is critical for mHtt toxicity and disease progression. mHtt contains several caspase and calpain cleavage sites that generate N-terminal fragments that are more toxic than full-length mHtt. Further processing is then required for the degradation of these fragments, which in turn, reduces toxicity. This unknown, secondary degradative process represents a promising therapeutic target for HD.We have used intrabodies, intracellularly expressed antibody fragments, to gain insight into the mechanism of mutant huntingtin exon 1 (mHDx-1 clearance. Happ1, an intrabody recognizing the proline-rich region of mHDx-1, reduces the level of soluble mHDx-1 by increasing clearance. While proteasome and macroautophagy inhibitors reduce turnover of mHDx-1, Happ1 is still able to reduce mHDx-1 under these conditions, indicating Happ1-accelerated mHDx-1 clearance does not rely on these processes. In contrast, a calpain inhibitor or an inhibitor of lysosomal pH block Happ1-mediated acceleration of mHDx-1 clearance. These results suggest that mHDx-1 is cleaved by calpain, likely followed by lysosomal degradation and this process regulates the turnover rate of mHDx-1. Sequence analysis identifies amino acid (AA 15 as a potential calpain cleavage site. Calpain cleavage of recombinant mHDx-1 in vitro yields fragments of sizes corresponding to this prediction. Moreover, when the site is blocked by binding of another intrabody, V(L12.3, turnover of soluble mHDx-1 in living cells is blocked.These results indicate that calpain-mediated removal of the 15 N-terminal AAs is required for the degradation of mHDx-1, a finding that may have therapeutic implications.

A novel carotenoid cleavage activity involved in the biosynthesis of Citrus fruit-specific apocarotenoid pigments

KAUST Repository

Rodrigo, María J.

2013-09-04

Citrus is the first tree crop in terms of fruit production. The colour of Citrus fruit is one of the main quality attributes, caused by the accumulation of carotenoids and their derivative C30 apocarotenoids, mainly ?-citraurin (3-hydroxy-?-apo-8?-carotenal), which provide an attractive orange-reddish tint to the peel of oranges and mandarins. Though carotenoid biosynthesis and its regulation have been extensively studied in Citrus fruits, little is known about the formation of C30 apocarotenoids. The aim of this study was to the identify carotenoid cleavage enzyme(s) [CCD(s)] involved in the peel-specific C30 apocarotenoids. In silico data mining revealed a new family of five CCD4-type genes in Citrus. One gene of this family, CCD4b1, was expressed in reproductive and vegetative tissues of different Citrus species in a pattern correlating with the accumulation of C30 apocarotenoids. Moreover, developmental processes and treatments which alter Citrus fruit peel pigmentation led to changes of ?-citraurin content and CCD4b1 transcript levels. These results point to the involvement of CCD4b1 in ?-citraurin formation and indicate that the accumulation of this compound is determined by the availability of the presumed precursors zeaxanthin and ?-cryptoxanthin. Functional analysis of CCD4b1 by in vitro assays unequivocally demonstrated the asymmetric cleavage activity at the 7?,8? double bond in zeaxanthin and ?-cryptoxanthin, confrming its role in C30 apocarotenoid biosynthesis. Thus, a novel plant carotenoid cleavage activity targeting the 7?,8? double bond of cyclic C40 carotenoids has been identified. These results suggest that the presented enzyme is responsible for the biosynthesis of C30 apocarotenoids in Citrus which are key pigments in fruit coloration. The Author 2013.

A novel carotenoid cleavage activity involved in the biosynthesis of Citrus fruit-specific apocarotenoid pigments

KAUST Repository

Rodrigo, Marí a J.; Alqué zar, Berta; Aló s, Enriqueta; Medina, Ví ctor; Carmona, Lourdes; Bruno, Mark; Al-Babili, Salim; Zacarí as, Lorenzo

2013-01-01

Citrus is the first tree crop in terms of fruit production. The colour of Citrus fruit is one of the main quality attributes, caused by the accumulation of carotenoids and their derivative C30 apocarotenoids, mainly ?-citraurin (3-hydroxy-?-apo-8?-carotenal), which provide an attractive orange-reddish tint to the peel of oranges and mandarins. Though carotenoid biosynthesis and its regulation have been extensively studied in Citrus fruits, little is known about the formation of C30 apocarotenoids. The aim of this study was to the identify carotenoid cleavage enzyme(s) [CCD(s)] involved in the peel-specific C30 apocarotenoids. In silico data mining revealed a new family of five CCD4-type genes in Citrus. One gene of this family, CCD4b1, was expressed in reproductive and vegetative tissues of different Citrus species in a pattern correlating with the accumulation of C30 apocarotenoids. Moreover, developmental processes and treatments which alter Citrus fruit peel pigmentation led to changes of ?-citraurin content and CCD4b1 transcript levels. These results point to the involvement of CCD4b1 in ?-citraurin formation and indicate that the accumulation of this compound is determined by the availability of the presumed precursors zeaxanthin and ?-cryptoxanthin. Functional analysis of CCD4b1 by in vitro assays unequivocally demonstrated the asymmetric cleavage activity at the 7?,8? double bond in zeaxanthin and ?-cryptoxanthin, confrming its role in C30 apocarotenoid biosynthesis. Thus, a novel plant carotenoid cleavage activity targeting the 7?,8? double bond of cyclic C40 carotenoids has been identified. These results suggest that the presented enzyme is responsible for the biosynthesis of C30 apocarotenoids in Citrus which are key pigments in fruit coloration. The Author 2013.

Physical kinetics

International Nuclear Information System (INIS)

Lifschitz, E.M.; Pitajewski, L.P.

1983-01-01

The textbook covers the subject under the following headings: kinetic gas theory, diffusion approximation, collisionless plasma, collisions within the plasma, plasma in the magnetic field, theory of instabilities, dielectrics, quantum fluids, metals, diagram technique for nonequilibrium systems, superconductors, and kinetics of phase transformations

Cu(II)-catalyzed esterification reaction via aerobic oxidative cleavage of C(CO)-C(alkyl) bonds.

Science.gov (United States)

Ma, Ran; He, Liang-Nian; Liu, An-Hua; Song, Qing-Wen

2016-02-04

A novel Cu(II)-catalyzed aerobic oxidative esterification of simple ketones for the synthesis of esters has been developed with wide functional group tolerance. This process is assumed to go through a tandem sequence consisting of α-oxygenation/esterification/nucleophilic addition/C-C bond cleavage and carbon dioxide is released as the only byproduct.

Cleavage of the SUN-domain protein Mps3 at its N-terminus regulates centrosome disjunction in budding yeast meiosis.

Directory of Open Access Journals (Sweden)

Ping Li

2017-06-01

Full Text Available Centrosomes organize microtubules and are essential for spindle formation and chromosome segregation during cell division. Duplicated centrosomes are physically linked, but how this linkage is dissolved remains unclear. Yeast centrosomes are tethered by a nuclear-envelope-attached structure called the half-bridge, whose components have mammalian homologues. We report here that cleavage of the half-bridge protein Mps3 promotes accurate centrosome disjunction in budding yeast. Mps3 is a single-pass SUN-domain protein anchored at the inner nuclear membrane and concentrated at the nuclear side of the half-bridge. Using the unique feature in yeast meiosis that centrosomes are linked for hours before their separation, we have revealed that Mps3 is cleaved at its nucleus-localized N-terminal domain, the process of which is regulated by its phosphorylation at serine 70. Cleavage of Mps3 takes place at the yeast centrosome and requires proteasome activity. We show that noncleavable Mps3 (Mps3-nc inhibits centrosome separation during yeast meiosis. In addition, overexpression of mps3-nc in vegetative yeast cells also inhibits centrosome separation and is lethal. Our findings provide a genetic mechanism for the regulation of SUN-domain protein-mediated activities, including centrosome separation, by irreversible protein cleavage at the nuclear periphery.

Cleavage of the SUN-domain protein Mps3 at its N-terminus regulates centrosome disjunction in budding yeast meiosis

Science.gov (United States)

Koch, Bailey A.; Han, Xuemei

2017-01-01

Centrosomes organize microtubules and are essential for spindle formation and chromosome segregation during cell division. Duplicated centrosomes are physically linked, but how this linkage is dissolved remains unclear. Yeast centrosomes are tethered by a nuclear-envelope-attached structure called the half-bridge, whose components have mammalian homologues. We report here that cleavage of the half-bridge protein Mps3 promotes accurate centrosome disjunction in budding yeast. Mps3 is a single-pass SUN-domain protein anchored at the inner nuclear membrane and concentrated at the nuclear side of the half-bridge. Using the unique feature in yeast meiosis that centrosomes are linked for hours before their separation, we have revealed that Mps3 is cleaved at its nucleus-localized N-terminal domain, the process of which is regulated by its phosphorylation at serine 70. Cleavage of Mps3 takes place at the yeast centrosome and requires proteasome activity. We show that noncleavable Mps3 (Mps3-nc) inhibits centrosome separation during yeast meiosis. In addition, overexpression of mps3-nc in vegetative yeast cells also inhibits centrosome separation and is lethal. Our findings provide a genetic mechanism for the regulation of SUN-domain protein-mediated activities, including centrosome separation, by irreversible protein cleavage at the nuclear periphery. PMID:28609436

The Conformational Dynamics of Cas9 Governing DNA Cleavage Are Revealed by Single-Molecule FRET.

Science.gov (United States)

Yang, Mengyi; Peng, Sijia; Sun, Ruirui; Lin, Jingdi; Wang, Nan; Chen, Chunlai

2018-01-09

Off-target binding and cleavage by Cas9 pose major challenges in its application. How the conformational dynamics of Cas9 govern its nuclease activity under on- and off-target conditions remains largely unknown. Here, using intra-molecular single-molecule fluorescence resonance energy transfer measurements, we revealed that Cas9 in apo, sgRNA-bound, and dsDNA/sgRNA-bound forms spontaneously transits among three major conformational states, mainly reflecting significant conformational mobility of the catalytic HNH domain. We also uncovered surprising long-range allosteric communication between the HNH domain and the RNA/DNA heteroduplex at the PAM-distal end to ensure correct positioning of the catalytic site, which demonstrated that a unique proofreading mechanism served as the last checkpoint before DNA cleavage. Several Cas9 residues were likely to mediate the allosteric communication and proofreading step. Modulating interactions between Cas9 and heteroduplex at the PAM-distal end by introducing mutations on these sites provides an alternative route to improve and optimize the CRISPR/Cas9 toolbox. Copyright © 2017 The Author(s). Published by Elsevier Inc. All rights reserved.

AcEST: BP916713 [AcEST

Lifescience Database Archive (English)

Full Text Available OS=Homo ... 33 0.91 sp|P03315|POLS_SFV Structural polyprotein OS=Semliki forest viru... 32 1.5 sp|P25386|USO...ATK 206 Query: 8 PL 3 L Sbjct: 207 KL 208 >sp|P03315|POLS_SFV Structural polyprotein OS=Semliki forest...r|Q8JMP5|Q8JMP5_SFV Structural polyprotein OS=Semliki forest vi... 34 4.4 tr|A9BD99|A9BD99_PROM4 Trigger fac... Predicted protein OS=Monosiga brevicollis... 34 4.4 tr|Q87049|Q87049_SFV Virion protein OS=Semliki forest... virus GN=C... 34 5.8 tr|Q809B6|Q809B6_SFV Structural polyprotein OS=Semliki forest

Biochemical and kinetic analysis of the GH3 family beta-xylosidase from Aspergillus awamori X-100.

Science.gov (United States)

Eneyskaya, Elena V; Ivanen, Dina R; Bobrov, Kirill S; Isaeva-Ivanova, Lyudmila S; Shabalin, Konstantin A; Savel'ev, Andrew N; Golubev, Alexander M; Kulminskaya, Anna A

2007-01-15

The beta-xylosidase from Aspergillus awamori X-100 belonging to the family 3 glycoside hydrolase revealed a distinctive transglycosylating ability to produce xylooligosaccharides with degree of polymerization more than 7. In order to explain this fact, the enzyme has been subjected to the detailed biochemical study. The enzymatic hydrolysis of p-nitrophenyl beta-D-xylopyranoside was found to occur with overall retention of substrate anomeric configuration suggesting cleavage of xylosidic bonds through a double-displacement mechanism. Kinetic study with aryl beta-xylopyranosides substrates, in which leaving group pK(a)s were in the range of 3.96-10.32, revealed monotonic function of log(k(cat)) and no correlation of log(k(cat)/Km) versus pKa values indicating deglycosylation as a rate-limiting step for the enzymatic hydrolysis. The classical bell-shaped pH dependence of k(cat)/Km indicated two ionizable groups in the beta-xylosidase active site with apparent pKa values of 2.2 and 6.4. The kinetic parameters of hydrolysis, Km and k(cat), of p-nitrophenyl beta-D-1,4-xylooligosaccharides were very close to those for hydrolysis of p-nitrophenyl-beta-D-xylopyranoside. Increase of p-nitrophenyl-beta-D-xylopyranoside concentration up to 80 mM led to increasing of the reaction velocity resulting in k(cat)(app)=81 s(-1). Addition of alpha-methyl D-xylopyranoside to the reaction mixture at high concentration of p-nitrophenyl-beta-D-xylopyranoside (50 mM) caused an acceleration of the beta-xylosidase-catalyzed reactions and appearance of a new transglycosylation product, alpha-methyl D-xylopyranosyl-1,4-beta-D-xylopyranoside, that was identified by 1H NMR spectroscopy. The kinetic model suggested for the enzymatic reaction was consistent with the results obtained.

Selective C(sp2)-C(sp) bond cleavage: the nitrogenation of alkynes to amides.

Science.gov (United States)

Qin, Chong; Feng, Peng; Ou, Yang; Shen, Tao; Wang, Teng; Jiao, Ning

2013-07-22

Breakthrough: A novel catalyzed direct highly selective C(sp2)-C(sp) bond functionalization of alkynes to amides has been developed. Nitrogenation is achieved by the highly selective C(sp2)-C(sp) bond cleavage of aryl-substituted alkynes. The oxidant-free and mild conditions and wide substrate scope make this method very practical. Copyright © 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Molecular cloning and characterization of cDNAs encoding carotenoid cleavage dioxygenase in bitter melon (Momordica charantia).

Science.gov (United States)

Tuan, Pham Anh; Park, Sang Un

2013-01-01

Carotenoid cleavage dioxygenases (CCDs) are a family of enzymes that catalyze the oxidative cleavage of carotenoids at various chain positions to form a broad spectrum of apocarotenoids, including aromatic substances, pigments and phytohormones. Using the rapid amplification of cDNA ends (RACE) PCR method, we isolated three cDNA-encoding CCDs (McCCD1, McCCD4, and McNCED) from Momordica charantia. Amino acid sequence alignments showed that they share high sequence identity with other orthologous genes. Quantitative real-time RT PCR (reverse transcriptase PCR) analysis revealed that the expression of McCCD1 and McCCD4 was highest in flowers, and lowest in roots and old leaves (O-leaves). During fruit maturation, the two genes displayed differential expression, with McCCD1 peaking at mid-stage maturation while McCCD4 showed the lowest expression at that stage. The mRNA expression level of McNCED, a key enzyme involved in abscisic acid (ABA) biosynthesis, was high during fruit maturation and further increased at the beginning of seed germination. When first-leaf stage plants of M. charantia were exposed to dehydration stress, McNCED mRNA expression was induced primarily in the leaves and, to a lesser extend, in roots and stems. McNCED expression was also induced by high temperature and salinity, while treatment with exogenous ABA led to a decrease. These results should be helpful in determining the substrates and cleavage sites catalyzed by CCD genes in M. charantia, and also in defining the roles of CCDs in growth and development, and in the plant's response to environmental stress. Copyright © 2012 Elsevier GmbH. All rights reserved.

Rh(III)-Catalyzed Synthesis of N-Unprotected Indoles from Imidamides and Diazo Ketoesters via C-H Activation and C-C/C-N Bond Cleavage.

Science.gov (United States)

Qi, Zisong; Yu, Songjie; Li, Xingwei

2016-02-19

The synthesis of N-unprotected indoles has been realized via Rh(III)-catalyzed C-H activation/annulation of imidamides with α-diazo β-ketoesters. The reaction occurs with the release of an amide coproduct, which originates from both the imidamide and the diazo as a result of C═N cleavage of the imidamide and C-C(acyl) cleavage of the diazo. A rhodacyclic intermediate has been isolated and a plausible mechanism has been proposed.

C-Terminally modified peptides via cleavage of the HMBA linker by O-, N- or S-nucleophiles

DEFF Research Database (Denmark)

Hansen, Jonas; Diness, Frederik; Meldal, Morten Peter

2016-01-01

A large variety of C-terminally modified peptides was obtained by nucleophilic cleavage of the ester bond in solid phase linked peptide esters of 4-hydroxymethyl benzamide (HMBA). The developed methods provided peptides, C-terminally functionalized as esters, amides and thioesters, with high purity...... directly from the resin in a single reaction step. A comprehensive screening of the reaction conditions and scope for nucleophilic cleavage of peptides from the HMBA linker was performed....

Deciphering the Mechanism of Alternative Cleavage and Polyadenylation in Mantle Cell Lymphoma (MCL)

Science.gov (United States)

2014-10-01

Kubo , T., Wada, T., Yamaguchi, Y., Shimizu, A. & Handa, H. Knock-down of 25 kDa subunit of cleavage factor Im inHela cells alters alternative...usage was calculated as 62normalized DDDCT. Oligonucleotides used for qRT–PCR. Cyclin D1 common forward, 59-CTGC CAGGAGCAGATCGAAG; reverse, 59...CTdeviation of either amplicon at all of the dilutions was calculated as a correction factor. d, The experiment shown in c was repeated for DICER1 and

Kinetic isotope effect studies on milk xanthine oxidase and on chicken liver xanthine dehydrogenase

International Nuclear Information System (INIS)

D'Ardenne, S.C.; Edmondson, D.E.

1990-01-01

The effect of isotopic substitution of the 8-H of xanthine (with 2 H and 3 H) on the rate of oxidation by bovine xanthine oxidase and by chicken xanthine dehydrogenase has been measured. V/K isotope effects were determined from competition experiments. No difference in H/T (V/K) values was observed between xanthine oxidase and xanthine dehydrogenase. Xanthine dehydrogenase exhibited a larger T/D (V/K) value than that observed for xanthine oxidase. Observed H/T (V/K) values for either enzyme are less than those H/T (V/K) values calculated with D/T (V/K) data. These discrepancies are suggested to arise from the presence of a rate-limiting step(s) prior to the irreversible C-H bond cleavage step in the mechanistic pathways of both enzymes. These kinetic complexities preclude examination of whether tunneling contributes to the reaction coordinate for the H-transfer step in each enzyme. No observable exchange of tritium with solvent is observed during the anaerobic incubation of [8- 3 H]xanthine with either enzyme, which suggests the reverse commitment to catalysis (C r ) is essentially zero. With the assumption of adherence to reduced mass relationships, the intrinsic deuterium isotope effect ( D k) for xanthine oxidation is calculated. By the use of these values and steady-state kinetic data, the minimal rate for the hydrogen-transfer step is calculated to be ∼75-fold faster than k cat for xanthine oxidase and ∼10-fold faster than k cat for xanthine dehydrogenase. Values calculated for each enzyme were found to be identical within experimental uncertainty

Effect of Organic Solvents and Biologically Relevant Ions on the Light-Induced DNA Cleavage by Pyrene and Its Amino and Hydroxy Derivatives

Directory of Open Access Journals (Sweden)

Hongtao Yu

2002-09-01

Full Text Available Abstract: Polycyclic aromatic hydrocarbons (PAHs are a class of carcinogenic compounds that are both naturally and artificially produced. Many PAHs are pro-carcinogens that require metabolic activation. Recently, it has been shown that PAH can induce DNA single strand cleavage and formation of PAH-DNA covalent adduct upon irradiation with UVA light. The light-induced DNA cleavage parallels phototoxicity in one instance. The DNA photocleavage efficiency depends on the structure of the PAHs. This article reports the effect of both organic solvents and the presence of biologically relevant ions, Na+, Mg2+, Ca2+, K+, Fe3+, Cu2+, Zn+2, Mn2+, and I-, on the light-induced DNA cleavage by pyrene, 1-hydroxypyrene and 1-aminopyrene. Since both 1-hydroxypyrene (0.6 μM and 1-aminopyrene (6 μM dissolve well in the minimum organic solvents used (2% methanol, dimethylsulfoxide, and dimethylformamide, increasing the amount of the organic solvent resulted in the decrease of the amount of DNA single strand cleavage caused by the combination effect of 1-hydroxy or 1-aminopyrene and UVA light. The result with the less watersoluble pyrene shows that increase of the amount of the organic solvent can increase the amount of DNA single strand DNA photocleavage cause by the combination of pyrene and UVA light. Therefore, there are two effects by the organic solvents: (i to dissolve PAH and (ii to quench DNA photocleavage. The presence of Fe3+ and Zn2+ enhances, while the presence of Ca2+ and Mn2+ inhibits the DNA photocleavage caused by 1-aminopyrene and UVA light. Other metal ions have minimal effect. This means that the effect of ions on DNA photocleavage by PAHs is complex. The presence of KI enhances DNA photocleavage. This indicates that the triplet-excited state of 1-aminopyrene is involved in causing DNA cleavage

Kinetic Studies on the Selective Oxidation of Benzyl Alcohols in Organic Medium under Phase Transfer Catalysis

Directory of Open Access Journals (Sweden)

K. Bijudas

2014-07-01

Full Text Available Kinetic studies on the oxidation of benzyl alcohol and substituted benzyl alcohols in benzene as the reaction medium have been studied by using potassium dichromate under phase transfer catalysis (PTC. The phase transfer catalysts (PT catalysts used were tetrabutylammonium bromide (TBAB and tetrabutylphosphonium bromide (TBPB.  Benzyl alcohols were selectively oxidised to corresponding benzaldehydes in good yield (above 90%.  The order of reactivity among the studied benzyl alcohols is p - OCH3 > p - CH3 > - H > p - Cl.  Plots of log k2 versus Hammett's substituent constant (s has been found to be curve shaped and this suggests that there should be a continuous change in transition state with changes in substituent present in the substrate from electron donating to electron withdrawing. A suitable mechanism has been suggested in which the rate determining step involves both C - H bond cleavage and C - O bond formations in concerted manner. © 2014 BCREC UNDIP. All rights reserved.Received: 16th March 2014; Revised: 18th May 2014; Accepted: 18th May 2014[How to Cite: Bijudas, K., Bashpa, P., Nair, T.D.R. (2014. Kinetic Studies on the Selective Oxidation of Benzyl Alcohol and Substituted Benzyl Alcohols in Organic Medium under Phase Transfer Catalysis. Bulletin of Chemical Reaction Engineering & Catalysis, 9 (2: 142-147. (doi:10.9767/bcrec.9.2.6476.142-147][Permalink/DOI: http://dx.doi.org/10.9767/bcrec.9.2.6476.142-147] 

Kinetics of phase transformations

International Nuclear Information System (INIS)

Thompson, M.O.; Aziz, M.J.; Stephenson, G.B.

1992-01-01

This volume contains papers presented at the Materials Research Society symposium on Kinetics of Phase Transformations held in Boston, Massachusetts from November 26-29, 1990. The symposium provided a forum for research results in an exceptionally broad and interdisciplinary field. Presentations covered nearly every major class of transformations including solid-solid, liquid-solid, transport phenomena and kinetics modeling. Papers involving amorphous Si, a dominant topic at the symposium, are collected in the first section followed by sections on four major areas of transformation kinetics. The symposium opened with joint sessions on ion and electron beam induced transformations in conjunction with the Surface Chemistry and Beam-Solid Interactions: symposium. Subsequent sessions focused on the areas of ordering and nonlinear diffusion kinetics, solid state reactions and amorphization, kinetics and defects of amorphous silicon, and kinetics of melting and solidification. Seven internationally recognized invited speakers reviewed many of the important problems and recent results in these areas, including defects in amorphous Si, crystal to glass transformations, ordering kinetics, solid-state amorphization, computer modeling, and liquid/solid transformations

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

Science.gov (United States)

Gloor, Jason W; Balakrishnan, Lata; Campbell, Judith L; Bambara, Robert A

2012-08-01

In eukaryotic Okazaki fragment processing, the RNA primer is displaced into a single-stranded flap prior to removal. Evidence suggests that some flaps become long before they are cleaved, and that this cleavage involves the sequential action of two nucleases. Strand displacement characteristics of the polymerase show that a short gap precedes the flap during synthesis. Using biochemical techniques, binding and cleavage assays presented here indicate that when the flap is ∼ 30 nt long the nuclease Dna2 can bind with high affinity to the flap and downstream double strand and begin cleavage. When the polymerase idles or dissociates the Dna2 can reorient for additional contacts with the upstream primer region, allowing the nuclease to remain stably bound as the flap is further shortened. The DNA can then equilibrate to a double flap that can bind Dna2 and flap endonuclease (FEN1) simultaneously. When Dna2 shortens the flap even more, FEN1 can displace the Dna2 and cleave at the flap base to make a nick for ligation.

AcEST: BP913167 [AcEST

Lifescience Database Archive (English)

Full Text Available n 2 OS=Homo ... 33 0.91 sp|P03315|POLS_SFV Structural polyprotein OS=Semliki forest viru... 32 1.5 sp|P25386...ESSAATK 206 Query: 8 PL 3 L Sbjct: 207 KL 208 >sp|P03315|POLS_SFV Structural polyprotein OS=Semliki forest....6 tr|Q8JMP5|Q8JMP5_SFV Structural polyprotein OS=Semliki forest vi... 34 4.4 tr|A9BD99|A9BD99_PROM4 Trigger...ion protein OS=Semliki forest virus GN=C... 34 5.8 tr|Q809B6|Q809B6_SFV Structural polyprotein OS=Semliki forest...ONBE Predicted protein OS=Monosiga brevicollis... 34 4.4 tr|Q87049|Q87049_SFV Vir

Nucleotide sequence of Hungarian grapevine chrome mosaic nepovirus RNA1.

Science.gov (United States)

Le Gall, O; Candresse, T; Brault, V; Dunez, J

1989-10-11

The nucleotide sequence of the RNA1 of hungarian grapevine chrome mosaic virus, a nepovirus very closely related to tomato black ring virus, has been determined from cDNA clones. It is 7212 nucleotides in length excluding the 3' terminal poly(A) tail and contains a large open reading frame extending from nucleotides 216 to 6971. The presumably encoded polyprotein is 2252 amino acids in length with a molecular weight of 250 kDa. The primary structure of the polyprotein was compared with that of other viral polyproteins, revealing the same general genetic organization as that of other picorna-like viruses (comoviruses, potyviruses and picornaviruses), except that an additional protein is suspected to occupy the N-terminus of the polyprotein.

Effect of pyrimido[1,6-a]benzimidazoles, quinolones, and Ca2+ on the DNA gyrase-mediated cleavage reaction.

Science.gov (United States)

Gmünder, H; Kuratli, K; Keck, W

1995-01-01

The quinolones inhibit the A subunit of DNA gyrase in the presence of Mg2+ by interrupting the DNA breakage and resealing steps, and the latter step is also retarded without quinolones if Mg2+ is replaced by Ca2+. Pyrimido[1,6-a]benzimidazoles have been found to represent a new class of potent DNA gyrase inhibitors which also act at the A subunit. To determine alterations in the DNA sequence specificity of DNA gyrase for cleavage sites in the presence of inhibitors of both classes or in the presence of Ca2+, we used DNA restriction fragments of 164, 85, and 71 bp from the pBR322 plasmid as model substrates. Each contained, at a different position, the 20-bp pBR322 sequence around position 990, where DNA gyrase preferentially cleaves in the presence of quinolones. Our results show that pyrimido[1,6-a]benzimidazoles have a mode of action similar to that of quinolones; they inhibit the resealing step and influence the DNA sequence specificity of DNA gyrase in the same way. Differences between inhibitors of both classes could be observed only in the preferences of DNA gyrase for these cleavage sites. The 20-bp sequence appeared to have some properties that induced DNA gyrase to cleave all three DNA fragments in the presence of inhibitors within this sequence, whereas cleavage in the presence of Ca2+ was in addition dependent on the length of the DNA fragments. PMID:7695300

Rhenium-Promoted C-C Bond-Cleavage Reactions of Internal Propargyl Alcohols.

Science.gov (United States)

Lee, Kui Fun; Bai, Wei; Sung, Herman H Y; Williams, Ian D; Lin, Zhenyang; Jia, Guochen

2018-06-07

The first examples of C-C bond cleavage reactions of internal propargyl alcohols to give vinylidene complexes are described. Treatment of [Re(dppm) 3 ]I with RC≡CC(OH)R'R'' (R=aryl, alkyl; C(OH)R'R''=C(OH)Ph 2, C(OH)Me 2 , C(OH)HPh, C(OH)H 2 ) produced the vinylidene complexes ReI(=C=CHR)(dppm) 2 with the elimination of C(O)R'R''. Computational studies support that the reactions proceed through a β-alkynyl elimination of alkoxide intermediates Re{OC(R')(R'')C≡CR}(dppm) 2 . © 2018 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.

Effect of Presenilin Mutations on APP Cleavage; Insights into the Pathogenesis of FAD

OpenAIRE

Li, Nuomin; Liu, Kefu; Qiu, Yunjie; Ren, Zehui; Dai, Rongji; Deng, Yulin; Qing, Hong

2016-01-01

Alzheimer disease (AD) is characterized by progressive memory loss, reduction in cognitive functions, and damage to the brain. The β-amyloid precursor protein can be sequentially cleaved by β- secretase and γ-secretase. Mutations in the presenilin1(PS1) are the most common cause of Familial Alzheimer’s disease (FAD). PS1 mutations can alter the activity of γ-secretase on the cleavage of the β-amyloid precursor protein, causing increased Aβ production. Previous studies show that the βAPP-C-ter...

Deciphering the Mechanism of Alternative Cleavage and Polyadenylation in Mantle Cell Lymphoma (MCL)

Science.gov (United States)

2015-12-01

event. The discovery that transformed and rapidly proliferating cells use alternative cleavage and polyadenylation ( APA ) to shorten the 3´UTR of their... APA . However, the mechanism that APA is still unknown. The goal of this project is to identify the mechanism of cyclin D1 APA regulation in cancer...for APA in MCL. In addition, by using RNA Seq. CFIm25 has been identified as an important global regulator of shortening of cyclin D1 mRNA and other

Kinetics in radiation chemistry

International Nuclear Information System (INIS)

Hummel, A.

1987-01-01

In this chapter the authors first briefly review the kinetics of first- and second-order processes for continuous and pulsed irradiation, without taking the effects of nonhomogeneous formation of the species into consideration. They also discuss diffusion controlled reactions under conditions where interactions of more than two particles can be neglected, first the kinetics of the diffusion-controlled reaction of randomly generated species (homogeneous reaction) and then that of isolated pairs of reactants. The latter is often called geminate kinetics when dealing with pairs of oppositely charged species; they shall use this term for the kinetics of isolated pairs in general. In the last section they discuss briefly the kinetics of groups of more than two reactants

Cathepsin-Mediated Cleavage of Peptides from Peptide Amphiphiles Leads to Enhanced Intracellular Peptide Accumulation

Energy Technology Data Exchange (ETDEWEB)

Acar, Handan [Institute; Department; Samaeekia, Ravand [Institute; Department; Schnorenberg, Mathew R. [Institute; Department; Medical; Sasmal, Dibyendu K. [Institute; Huang, Jun [Institute; Tirrell, Matthew V. [Institute; Institute; LaBelle, James L. [Department

2017-08-24

Peptides synthesized in the likeness of their native interaction domain(s) are natural choices to target protein protein interactions (PPIs) due to their fidelity of orthostatic contact points between binding partners. Despite therapeutic promise, intracellular delivery of biofunctional peptides at concentrations necessary for efficacy remains a formidable challenge. Peptide amphiphiles (PAs) provide a facile method of intracellular delivery and stabilization of bioactive peptides. PAs consisting of biofunctional peptide headgroups linked to hydrophobic alkyl lipid-like tails prevent peptide hydrolysis and proteolysis in circulation, and PA monomers are internalized via endocytosis. However, endocytotic sequestration and steric hindrance from the lipid tail are two major mechanisms that limit PA efficacy to target intracellular PPIs. To address these problems, we have constructed a PA platform consisting of cathepsin-B cleavable PAs in which a selective p53-based inhibitory peptide is cleaved from its lipid tail within endosomes, allowing for intracellular peptide accumulation and extracellular recycling of the lipid moiety. We monitor for cleavage and follow individual PA components in real time using a resonance energy transfer (FRET)-based tracking system. Using this platform, components in real time using a Forster we provide a better understanding and quantification of cellular internalization, trafficking, and endosomal cleavage of PAs and of the ultimate fates of each component.

The effects of the local fracture stress and carbides on the cleavage fracture characteristics of Mn-Mo-Ni low alloy steels in the transition region

International Nuclear Information System (INIS)

Yang, Won Jon; Huh, Moo Young; Roh, Sung Joo; Lee, Bong Sang; Oh, Yong Jun; Hong, Jun Hwa

2000-01-01

In the ductile-brittle transition temperature region of SA508 C1.3 Mn-Mo-Ni low alloy steels, the relationship of the local fracture stress and carbides influencing the cleavage fracture behavior was investigated. Based on the ASTM E1921-97 standard method, the reference transition temperatures were determined by three point bending fracture toughness tests. A local fracture stress σ f * , was determined from a theoretical stress distribution in front of crack tip using the cleavage initiation distance measured in each fractured specimen surface. The local fracture stress values showed a strong relationship with toughness characteristics of the materials and those were larger in the materials of smaller carbide size. Quantitative analysis of carbides showed that carbides larger than a certain size are mainly responsible for the cleavage fracture in the ductile-brittle transition temperature region. (author)

3' RNA ligase mediated rapid amplification of cDNA ends for validating viroid induced cleavage at the 3' extremity of the host mRNA.

Science.gov (United States)

Adkar-Purushothama, Charith Raj; Bru, Pierrick; Perreault, Jean-Pierre

2017-12-01

5' RNA ligase-mediated rapid amplification of cDNA ends (5' RLM-RACE) is a widely-accepted method for the validation of direct cleavage of a target gene by a microRNA (miRNA) and viroid-derived small RNA (vd-sRNA). However, this method cannot be used if cleavage takes place in the 3' extremity of the target RNA, as this gives insufficient sequence length to design nested PCR primers for 5' RLM RACE. To overcome this hurdle, we have developed 3' RNA ligase-mediated rapid amplification of cDNA ends (3' RLM RACE). In this method, an oligonucleotide adapter having 5' adenylated and 3' blocked is ligated to the 3' end of the cleaved RNA followed by PCR amplification using gene specific primers. In other words, in 3' RLM RACE, 3' end is mapped using 5' fragment instead of small 3' fragment. The method developed here was verified by examining the bioinformatics predicted and parallel analysis of RNA ends (PARE) proved cleavage sites of chloride channel protein CLC-b-like mRNA in Potato spindle tuber viroid infected tomato plants. The 3' RLM RACE developed in this study has the potential to validate the miRNA and vd-sRNA mediated cleavage of mRNAs at its 3' untranslated region (3' UTR). Copyright © 2017 Elsevier B.V. All rights reserved.

Poly(ADP-ribose) polymerase-1 and its cleavage products differentially modulate cellular protection through NF-kB-dependent signaling

Science.gov (United States)

Castri, Paola; Lee, Yang-ja; Ponzio, Todd; Maric, Dragan; Spatz, Maria; Bembry, Joliet; Hallenbeck, John

2014-01-01

Poly(ADP-ribose) polymerase-1 (PARP-1) and its cleavage products regulate cell viability and NF-kB activity when expressed in neurons. PARP-1 cleavage generates a 24kDa (PARP-124) and an 89kDa fragment (PARP-189). Compared to WT (PARP-1WT), the expression of an uncleavable PARP-1 (PARP-1UNCL) or of PARP-124 conferred protection from oxygen/glucose deprivation (OGD) or OGD/restoration of oxygen and glucose (ROG) damage in vitro, whereas expression of PARP-189 was cytotoxic. Viability experiments were performed in SH-SY5Y, a human neuroblastoma cell line, as well as in rat primary cortical neurons. Following OGD, the higher viability in the presence of PARP-1UNCL or PARP-124 was not accompanied with decreased formation of poly(ADP-riboses) or higher NAD levels. PARP-1 is a known cofactor for NF-kB, hence we investigated whether PARP-1 cleavage influences the inflammatory response. All PARP-1 constructs mimicked PARP-1WT in regards to induction of NF-kB translocation into the nucleus and its increased activation during ischemic challenge. However, expression of PARP-189 construct induced significantly higher NF-kB activity than PARP-1WT; and the same was true for NF-kB-dependent iNOS promoter binding activity. At a protein level, PARP-1UNCL and PARP-124 decreased iNOS (and lower levels of iNOS transcript) and COX-2, and increased Bcl-xL. The increased levels of NF-kB and iNOS transcriptional activities, seen with cytotoxic PARP-189, were accompanied by higher protein expression of COX-2 and iNOS (and higher levels of iNOS transcript) and lower protein expression of Bcl-xL. Taken together, these findings suggest that PARP-1 cleavage products may regulate cellular viability and inflammatory responses in opposing ways during in vitro models of “ischemia”. PMID:24333653

Triboluminescence and crystal structure of the complex [Eu(NО3 )3 (HMPA)3 ]: role of cleavage planes.

Science.gov (United States)

Bukvetskii, B V; Mirochnik, A G; Zhikhareva, P A

2017-05-01

The atomic structure of crystals of the [Eu(NО 3 ) 3 (HMPA) 3 ] [hexamethylphosphotriamide (HMPA)] complex characterized by an intensive luminescence and triboluminescence was determined using X-ray structural analysis. Noncentrosymmetric crystals have a monoclinic syngony: a = 16.0686 (3), b = 11.0853 (2), c = 20.9655 Å (4), β = 93.232° (1), space group P2 1 , Z = 4, ρ calc  = 1.560 g/cm 3 . The crystal structure is represented by individual С 18 Н 54 EuN 12 O 12 P 3 complexes linked through van der Waals interactions with clearly expressed cleavage planes. The Eu(III) atom coordination polyhedron reflected the state of a distorted square antiprism. Structural aspects of the suggested model, including formation of triboluminescence properties, were considered and the role of the cleavage planes was discussed. Copyright © 2016 John Wiley & Sons, Ltd.

Oxidative cleavage of non-phenolic β-O-4 lignin model dimers by an extracellular aromatic peroxygenase

Science.gov (United States)

Matthias Kinne; Marzena Poraj-Kobielska; Rene Ullrich; Paula Nousiainen; Jussi Sipilä; Katrin Scheibner; Kenneth E. Hammel; Martin Hofrichter

2011-01-01

The extracellular aromatic peroxygenase of the agaric fungus Agrocybe aegerita catalyzed the H2O2-dependent cleavage of non-phenolic arylgiycerol-ß-aryl ethers (ß-O-4 ethers). For instance 1-(3,4-dimethoxyphenyl)-2-(2-methoxy-phenoxy)propane-1,3-diol, a recalcitrant dimeric lignin model compound that represents the major...

Design and kinetic analysis of hammerhead ribozyme and DNAzyme that specifically cleave TEL-AML1 chimeric mRNA

International Nuclear Information System (INIS)

Choi, Woo-Hyung; Choi, Bo-Ra; Kim, Jae Hyun; Yeo, Woon-Seok; Oh, Sangtaek; Kim, Dong-Eun

2008-01-01

In order to develop the oligonucleotides to abolish an expression of TEL-AML1 chimeric RNA, which is a genetic aberration that causes the acute lymphoblastic leukemia (ALL), hammerhead ribozymes and deoxyoligoribozymes that can specifically cleave TEL-AML1 fusion RNA were designed. Constructs of the deoxyribozyme with an asymmetric substrate binding arm (Dz26) and the hammerhead ribozyme with a 4 nt-bulged substrate binding arm in the stem III (buRz28) were able to cleave TEL-AML1 chimeric RNA specifically at sites close to the junction in vitro, without cleaving the normal TEL and AML1 RNA. Single-turnover kinetic analysis under enzyme-excess condition revealed that the buRz28 is superior to the Dz26 in terms of substrate binding and RNA-cleavage. In conjunction with current progress in a gene-delivery technology, the designed oligonucleotides that specifically cleave the TEL-AML1 chimeric mRNA are hoped to be applicable for the treatment of ALL in vivo

Antibacterial and DNA cleavage activity of carbonyl functionalized N-heterocyclic carbene-silver(I) and selenium compounds

Science.gov (United States)

Haque, Rosenani A.; Iqbal, Muhammad Adnan; Mohamad, Faisal; Razali, Mohd R.

2018-03-01

The article describes syntheses and characterizations of carbonyl functionalized benzimidazolium salts, I-IV. While salts I-III are unstable at room temperature, salt IV remained stable and was further utilised to form N-heterocyclic carbene (NHC) compounds of silver(I), V and VI, and selenium compound, VII respectively. Compounds IV-VII were tested for their antibacterial potential against Escherichia coli (E. coli) and Staphylococcus aureus (S. aureus). Salt IV shows a very low inhibition potential (minimum inhibitory concentration, MIC 500 μg/mL) compared to the respective silver(I)-NHC, V and VI (MIC 31.25 μg/mL against both, E. coli and S. aureus) and selenium compound, VII (MIC 125 μg/mL against E. coli and 62.50 μg/mL against S. aureus). In DNA cleavage abilities, all the test compounds cleave DNA in which the VII cleaves the DNA at the faster rate. Meanwhile, the silver(I)-NHC complexes V and VI act at the same mode and pattern of DNA cleavage while VII is similar to IV.

Isolation and Functional Characterization of Carotenoid Cleavage Dioxygenase-1 from Laurus nobilis L. (Bay Laurel) Fruits.

Science.gov (United States)

Yahyaa, Mosaab; Berim, Anna; Isaacson, Tal; Marzouk, Sally; Bar, Einat; Davidovich-Rikanati, Rachel; Lewinsohn, Efraim; Ibdah, Mwafaq

2015-09-23

Bay laurel (Laurus nobilis L.) is an agriculturally important tree used in food, drugs, and the cosmetics industry. Many of the health beneficial properties of bay laurel are due to volatile terpene metabolites that they contain, including various norisoprenoids. Despite their importance, little is known about the norisoprenoid biosynthesis in Laurus nobilis fruits. We found that the volatile norisoprenoids 6-methyl-5-hepten-2-one, pseudoionone, and β-ionone accumulated in Laurus nobilis fruits in a pattern reflecting their carotenoid content. A full-length cDNA encoding a potential carotenoid cleavage dioxygenase (LnCCD1) was isolated. The LnCCD1 gene was overexpressed in Escherichia coli, and recombinant protein was assayed for its cleavage activity with an array of carotenoid substrates. The LnCCD1 protein was able to cleave a variety of carotenoids at the 9,10 (9',10') and 5,6 (5',6') positions to produce 6-methyl-5-hepten-2-one, pseudoionone, β-ionone, and α-ionone. Our results suggest a role for LnCCD1 in Laurus nobilis fruit flavor biosynthesis.

Kinetic equation solution by inverse kinetic method

International Nuclear Information System (INIS)

Salas, G.

1983-01-01

We propose a computer program (CAMU) which permits to solve the inverse kinetic equation. The CAMU code is written in HPL language for a HP 982 A microcomputer with a peripheral interface HP 9876 A ''thermal graphic printer''. The CAMU code solves the inverse kinetic equation by taking as data entry the output of the ionization chambers and integrating the equation with the help of the Simpson method. With this program we calculate the evolution of the reactivity in time for a given disturbance

The Oxygenase CAO-1 of Neurospora crassa Is a Resveratrol Cleavage Enzyme

KAUST Repository

Diaz-Sanchez, V.; F. Estrada, A.; Limon, M. C.; Al-Babili, Salim; Avalos, J.

2013-01-01

The genome of the ascomycete Neurospora crassa encodes CAO-1 and CAO-2, two members of the carotenoid cleavage oxygenase family that target double bonds in different substrates. Previous studies demonstrated the role of CAO-2 in cleaving the C40 carotene torulene, a key step in the synthesis of the C35 apocarotenoid pigment neurosporaxanthin. In this work, we investigated the activity of CAO-1, assuming that it may provide retinal, the chromophore of the NOP-1 rhodopsin, by cleaving β-carotene. For this purpose, we tested CAO-1 activity with carotenoid substrates that were, however, not converted. In contrast and consistent with its sequence similarity to family members that act on stilbenes, CAO-1 cleaved the interphenyl Cα-Cβ double bond of resveratrol and its derivative piceatannol. CAO-1 did not convert five other similar stilbenes, indicating a requirement for a minimal number of unmodified hydroxyl groups in the stilbene background. Confirming its biological function in converting stilbenes, adding resveratrol led to a pronounced increase in cao-1 mRNA levels, while light, a key regulator of carotenoid metabolism, did not alter them. Targeted Δcao-1 mutants were not impaired by the presence of resveratrol, a phytoalexin active against different fungi, which did not significantly affect the growth and development of wild-type Neurospora. However, under partial sorbose toxicity, the Δcao-1 colonies exhibited faster radial growth than control strains in the presence of resveratrol, suggesting a moderate toxic effect of resveratrol cleavage products.

Looking for exceptions on knowledge rules induced from HIV cleavage data set

Directory of Open Access Journals (Sweden)

Ronaldo Cristiano Prati

2004-01-01

Full Text Available The aim of data mining is to find useful knowledge inout of databases. In order to extract such knowledge, several methods can be used, among them machine learning (ML algorithms. In this work we focus on ML algorithms that express the extracted knowledge in a symbolic form, such as rules. This representation may allow us to ''explain'' the data. Rule learning algorithms are mainly designed to induce classification rules that can predict new cases with high accuracy. However, these sorts of rules generally express common sense knowledge, resulting in many interesting and useful rules not being discovered. Furthermore, the domain independent biases, especially those related to the language used to express the induced knowledge, could induce rules that are difficult to understand. Exceptions might be used in order to overcome these drawbacks. Exceptions are defined as rules that contradict common believebeliefs. This kind of rules can play an important role in the process of understanding the underlying data as well as in making critical decisions. By contradicting the user's common beliefves, exceptions are bound to be interesting. This work proposes a method to find exceptions. In order to illustrate the potential of our approach, we apply the method in a real world data set to discover rules and exceptions in the HIV virus protein cleavage process. A good understanding of the process that generates this data plays an important role oin the research of cleavage inhibitors. We consider believe that the proposed approach may help the domain expert to further understand this process.

The Oxygenase CAO-1 of Neurospora crassa Is a Resveratrol Cleavage Enzyme

KAUST Repository

Diaz-Sanchez, V.

2013-07-26

The genome of the ascomycete Neurospora crassa encodes CAO-1 and CAO-2, two members of the carotenoid cleavage oxygenase family that target double bonds in different substrates. Previous studies demonstrated the role of CAO-2 in cleaving the C40 carotene torulene, a key step in the synthesis of the C35 apocarotenoid pigment neurosporaxanthin. In this work, we investigated the activity of CAO-1, assuming that it may provide retinal, the chromophore of the NOP-1 rhodopsin, by cleaving β-carotene. For this purpose, we tested CAO-1 activity with carotenoid substrates that were, however, not converted. In contrast and consistent with its sequence similarity to family members that act on stilbenes, CAO-1 cleaved the interphenyl Cα-Cβ double bond of resveratrol and its derivative piceatannol. CAO-1 did not convert five other similar stilbenes, indicating a requirement for a minimal number of unmodified hydroxyl groups in the stilbene background. Confirming its biological function in converting stilbenes, adding resveratrol led to a pronounced increase in cao-1 mRNA levels, while light, a key regulator of carotenoid metabolism, did not alter them. Targeted Δcao-1 mutants were not impaired by the presence of resveratrol, a phytoalexin active against different fungi, which did not significantly affect the growth and development of wild-type Neurospora. However, under partial sorbose toxicity, the Δcao-1 colonies exhibited faster radial growth than control strains in the presence of resveratrol, suggesting a moderate toxic effect of resveratrol cleavage products.

The nuclear inclusion a (NIa protease of turnip mosaic virus (TuMV cleaves amyloid-β.

Directory of Open Access Journals (Sweden)

Hye-Eun Han

Full Text Available BACKGROUND: The nuclear inclusion a (NIa protease of turnip mosaic virus (TuMV is responsible for the processing of the viral polyprotein into functional proteins. NIa was previously shown to possess a relatively strict substrate specificity with a preference for Val-Xaa-His-Gln↓, with the scissile bond located after Gln. The presence of the same consensus sequence, Val(12-His-His-Gln(15, near the presumptive α-secretase cleavage site of the amyloid-β (Aβ peptide led us to hypothesize that NIa could possess activity against Aβ. METHODOLOGY/PRINCIPAL FINDINGS: Western blotting results showed that oligomeric as well as monomeric forms of Aβ can be degraded by NIa in vitro. The specific cleavage of Aβ was further confirmed by mass spectrometry analysis. NIa was shown to exist predominantly in the cytoplasm as observed by immunofluorescence microscopy. The overexpression of NIa in B103 neuroblastoma cells resulted in a significant reduction in cell death caused by both intracellularly generated and exogenously added Aβ. Moreover, lentiviral-mediated expression of NIa in APP(sw/PS1 transgenic mice significantly reduced the levels of Aβ and plaques in the brain. CONCLUSIONS/SIGNIFICANCE: These results indicate that the degradation of Aβ in the cytoplasm could be a novel strategy to control the levels of Aβ, plaque formation, and the associated cell death.

Introduction to chemical kinetics

CERN Document Server

Soustelle, Michel

2013-01-01

This book is a progressive presentation of kinetics of the chemical reactions. It provides complete coverage of the domain of chemical kinetics, which is necessary for the various future users in the fields of Chemistry, Physical Chemistry, Materials Science, Chemical Engineering, Macromolecular Chemistry and Combustion. It will help them to understand the most sophisticated knowledge of their future job area. Over 15 chapters, this book present the fundamentals of chemical kinetics, its relations with reaction mechanisms and kinetic properties. Two chapters are then devoted to experimental re

Supercritical kinetic analysis in simplified system of fuel debris using integral kinetic model

International Nuclear Information System (INIS)

Tuya, Delgersaikhan; Obara, Toru

2016-01-01

Highlights: • Kinetic analysis in simplified weakly coupled fuel debris system was performed. • The integral kinetic model was used to simulate criticality accidents. • The fission power and released energy during simulated accident were obtained. • Coupling between debris regions and its effect on the fission power was obtained. - Abstract: Preliminary prompt supercritical kinetic analyses in a simplified coupled system of fuel debris designed to roughly resemble a melted core of a nuclear reactor were performed using an integral kinetic model. The integral kinetic model, which can describe region- and time-dependent fission rate in a coupled system of arbitrary geometry, was used because the fuel debris system is weakly coupled in terms of neutronics. The results revealed some important characteristics of coupled systems, such as the coupling between debris regions and the effect of the coupling on the fission rate and released energy in each debris region during the simulated criticality accident. In brief, this study showed that the integral kinetic model can be applied to supercritical kinetic analysis in fuel debris systems and also that it can be a useful tool for investigating the effect of the coupling on consequences of a supercritical accident.

Inhibition of Human Cytomegalovirus pUL89 Terminase Subunit Blocks Virus Replication and Genome Cleavage.

Science.gov (United States)

Wang, Yan; Mao, Lili; Kankanala, Jayakanth; Wang, Zhengqiang; Geraghty, Robert J

2017-02-01

The human cytomegalovirus terminase complex cleaves concatemeric genomic DNA into unit lengths during genome packaging and particle assembly. This process is an attractive drug target because cleavage of concatemeric DNA is not required in mammalian cell DNA replication, indicating that drugs targeting the terminase complex could be safe and selective. One component of the human cytomegalovirus terminase complex, pUL89, provides the endonucleolytic activity for genome cleavage, and the domain responsible is reported to have an RNase H-like fold. We hypothesize that the pUL89 endonuclease activity is inhibited by known RNase H inhibitors. Using a novel enzyme-linked immunosorbent assay (ELISA) format as a screening assay, we found that a hydroxypyridonecarboxylic acid compound, previously reported to be an inhibitor of human immunodeficiency virus RNase H, inhibited pUL89 endonuclease activity at low-micromolar concentrations. Further characterization revealed that this pUL89 endonuclease inhibitor blocked human cytomegalovirus replication at a relatively late time point, similarly to other reported terminase complex inhibitors. Importantly, this inhibitor also prevented the cleavage of viral genomic DNA in infected cells. Taken together, these results substantiate our pharmacophore hypothesis and validate our ligand-based approach toward identifying novel inhibitors of pUL89 endonuclease. Human cytomegalovirus infection in individuals lacking a fully functioning immune system, such as newborns and transplant patients, can have severe and debilitating consequences. The U.S. Food and Drug Administration-approved anti-human cytomegalovirus drugs mainly target the viral polymerase, and resistance to these drugs has appeared. Therefore, anti-human cytomegalovirus drugs from novel targets are needed for use instead of, or in combination with, current polymerase inhibitors. pUL89 is a viral ATPase and endonuclease and is an attractive target for anti-human cytomegalovirus

Principles of chemical kinetics

CERN Document Server

House, James E

2007-01-01

James House's revised Principles of Chemical Kinetics provides a clear and logical description of chemical kinetics in a manner unlike any other book of its kind. Clearly written with detailed derivations, the text allows students to move rapidly from theoretical concepts of rates of reaction to concrete applications. Unlike other texts, House presents a balanced treatment of kinetic reactions in gas, solution, and solid states. The entire text has been revised and includes many new sections and an additional chapter on applications of kinetics. The topics covered include quantitative rela

Catalytic diastereoselective tandem conjugate addition-elimination reaction of Morita-Baylis-Hillman C adducts by C-C bond cleavage

KAUST Repository

Yang, Wenguo; Tan, Davin; Lee, Richmond; Li, Lixin; Pan, Yuanhang; Huang, Kuo-Wei; Tan, Choonhong; Jiang, Zhiyong

2012-01-01

Through the cleavage of the C-C bond, the first catalytic tandem conjugate addition-elimination reaction of Morita-Baylis-Hillman C adducts has been presented. Various S N2′-like C-, S-, and P-allylic compounds could be obtained with exclusive E

Single-Molecule Analysis for RISC Assembly and Target Cleavage.

Science.gov (United States)

Sasaki, Hiroshi M; Tadakuma, Hisashi; Tomari, Yukihide

2018-01-01

RNA-induced silencing complex (RISC) is a small RNA-protein complex that mediates silencing of complementary target RNAs. Biochemistry has been successfully used to characterize the molecular mechanism of RISC assembly and function for nearly two decades. However, further dissection of intermediate states during the reactions has been warranted to fill in the gaps in our understanding of RNA silencing mechanisms. Single-molecule analysis with total internal reflection fluorescence (TIRF) microscopy is a powerful imaging-based approach to interrogate complex formation and dynamics at the individual molecule level with high sensitivity. Combining this technique with our recently established in vitro reconstitution system of fly Ago2-RISC, we have developed a single-molecule observation system for RISC assembly. In this chapter, we summarize the detailed protocol for single-molecule analysis of chaperone-assisted assembly of fly Ago2-RISC as well as its target cleavage reaction.

Crack blunting, cleavage fracture in transition area and stable crack growth - investigated using the nonlinear fracture mechanics method

International Nuclear Information System (INIS)

Heerens, J.

1990-01-01

A procedure is developed which allows to estimate crack tip blunting using the stress-strain curve of the material and the J-integral. The second part deals with cleavage fracture in a quenched and tempered pressure vessel steel. It was found that within the ductile to brittle transition regime the fracture toughness is controlled by cleavage initiated at 'weak spots of the material' and by the normal stresses at the weak spots. In the last part of the paper the influence of specimen size on J-, Jm- and δ 5 -R-curves for side grooved CT-specimens under fully plastic condition is investigated. In order to characterize constraint-effects the necking of the specimens was measured. For specimens having similar constraint the parameters Jm and δ 5 yielded size independent R-curves over substantial larger amounts of crack extension than the J-integral. (orig.) With 114 figs., 10 tabs [de

Reaction kinetics and oxidation products formation in the degradation of ciprofloxacin and ibuprofen by ferrate(VI).

Science.gov (United States)

Zhou, Zhengwei; Jiang, Jia-Qian

2015-01-01

The treatment of ciprofloxacin (CIP) and ibuprofen (IBU) in test solutions by ferrate(VI) was investigated in this study. A series of jar test was performed in bench-scale at pH 6-9 and ferrate(VI) dose of 1-5 mg L(-1). Results demonstrated that ferrate(VI) removed CIP from test solutions efficiently, with above 70% of reduction under study conditions. In contrary, the removal rates of IBU were very low, less than 25% in all conditions. Raising ferrate(VI) dose improved the treatment performance, while the influence of solution pH was not significant at pH 6-9 compared with that of ferrate(VI) dose. In addition, kinetic studies of ferrate(VI) with both compounds were carried out at pH 8 and pH 9 (20 °C). Ferrate(VI) had a much higher reactivity with CIP than IBU at pH 8 and pH 9, with CIP's apparent second-order rate constants of 113.7±6.3 M(-1) s(-1) and 64.1±1.0 M(-1) s(-1), respectively. The rate constants of ferrate(VI) with IBU were less than 0.2 M(-1) s(-1) at pH 8 and pH 9. Furthermore, seven oxidation products (OPs) were formed during CIP degradation by ferrate(VI). The attack on the piperazinyl ring of the CIP by ferrate(VI) appeared to lead to the cleavage or hydroxylation of the rings, and the attack on the quinolone moiety by ferrate(VI) might lead to the cleavage of the double bond at the six-member heterocyclic ring. No OPs of IBU were detected during ferrate(VI) oxidation due to very small part of IBU was degraded by ferrate(VI). Copyright © 2014 Elsevier Ltd. All rights reserved.

Structural and functional basis for RNA cleavage by Ire1

Directory of Open Access Journals (Sweden)

Stroud Robert M

2011-07-01

Full Text Available Abstract Background The unfolded protein response (UPR controls the protein folding capacity of the endoplasmic reticulum (ER. Central to this signaling pathway is the ER-resident bifunctional transmembrane kinase/endoribonuclease Ire1. The endoribonuclease (RNase domain of Ire1 initiates a non-conventional mRNA splicing reaction, leading to the production of a transcription factor that controls UPR target genes. The mRNA splicing reaction is an obligatory step of Ire1 signaling, yet its mechanism has remained poorly understood due to the absence of substrate-bound crystal structures of Ire1, the lack of structural similarity between Ire1 and other RNases, and a scarcity of quantitative enzymological data. Here, we experimentally define the active site of Ire1 RNase and quantitatively evaluate the contribution of the key active site residues to catalysis. Results This analysis and two new crystal structures suggest that Ire1 RNase uses histidine H1061 and tyrosine Y1043 as the general acid-general base pair contributing ≥ 7.6 kcal/mol and 1.4 kcal/mol to transition state stabilization, respectively, and asparagine N1057 and arginine R1056 for coordination of the scissile phosphate. Investigation of the stem-loop recognition revealed that additionally to the stem-loops derived from the classic Ire1 substrates HAC1 and Xbp1 mRNA, Ire1 can site-specifically and rapidly cleave anticodon stem-loop (ASL of unmodified tRNAPhe, extending known substrate specificity of Ire1 RNase. Conclusions Our data define the catalytic center of Ire1 RNase and suggest a mechanism of RNA cleavage: each RNase monomer apparently contains a separate catalytic apparatus for RNA cleavage, whereas two RNase subunits contribute to RNA stem-loop docking. Conservation of the key residues among Ire1 homologues suggests that the mechanism elucidated here for yeast Ire1 applies to Ire1 in metazoan cells, and to the only known Ire1 homologue RNase L.

Increased cleavage and blastocyst rate in ewes treated with bovine somatotropin 5 days before the end of progestin-based estrous synchronization.

Science.gov (United States)

Montero-Pardo, A; Hernández-Cerón, J; Rojas-Maya, S; Valencia, J; Rodríguez-Cortez, A; Gutiérrez, C G

2011-05-01

Treatment with bovine somatotropin (bST) during estrous synchronization increased fertility and prolificacy in sheep. In the present study, a single dose of bST 5 days before the end of progestin treatment improved cleavage and embryo development. Stage of estrous cycle was synchronized in ewes (n=32) with progestin and superovulation was induced by use of FSH. Five days before the end of progestin treatment, ewes were randomly assigned to two groups: bST group (n=16) received a depot injection of 125 mg of bST sc (Lactotropina, Elanco, México) and the control group (n=16) received saline solution. Estrous was detected with rams fitted with an apron every 2 h and estrous sheep were mated every 8 h whilst in estrous. Embryos were recovered on Day 7 post mating, assessed microscopically and fixed in 4% paraformaldehyde. Cell number in blastocysts was counted after Hoechst 33342 staining. Plasma concentrations of IGF-I, insulin and progesterone were determined in eight sheep per group from the day of bST treatment to the day of embryo recovery. Cleavage rate, percentage of transferable embryos (transferable embryos/cleaved) and percentage of embryos reaching the blastocyst stage (blastocyst/cleaved) were compared between groups by logistic regression. IGF-I, insulin and progesterone plasma concentrations were analyzed by ANOVA for repeated measurements and cell number by ANOVA. Cleavage rate was greater (Psheep. Plasma concentrations of IGF-I and insulin were greater (Pprogesterone concentrations (P=0.5). It is concluded that bST injection 5 days before progestin removal increases cleavage rate and the proportion of embryos that reach the blastocyst stage. These effects are associated with an increase in IGF-I and insulin concentrations. Copyright © 2011 Elsevier B.V. All rights reserved.

DNA binding and cleavage studies of new sulfasalazine-derived dipeptide Zn(II) complex: Validation for specific recognition with 5 Prime -TMP

Energy Technology Data Exchange (ETDEWEB)

Tabassum, Sartaj [Department of Chemistry, Aligarh Muslim University, Aligarh, UP 202002 (India); Al-Asbahy, Waddhaah M.; Afzal, Mohd.; Shamsi, Manal; Arjmand, Farukh [Department of Chemistry, Aligarh Muslim University, Aligarh, UP 202002 (India)

2012-11-15

A new water soluble complex [Zn(glygly)(ssz)(H{sub 2}O)]{center_dot}6H{sub 2}O, 1 derived from dipeptide (glycyl glycine) and sulfasalazine was synthesized and characterized by spectroscopic (IR, UV-vis, NMR, ESI-MS) and analytical methods. The in vitro DNA binding studies of complex 1 with calf-thymus DNA were carried out by employing various biophysical methods and molecular docking technique which reveals strong electrostatic binding via phosphate backbone of DNA helix, in addition to partial intercalation. To gain further insight into the molecular recognition at the target site, interaction studies of complex 1 with 5 Prime -TMP and 5 Prime -GMP were carried out by UV-vis titration which was validated by {sup 1}H and {sup 31}P NMR with 5 Prime -TMP, which implicate the preferential selectivity of 1 towards N3 of thymine. Complex 1 is accessible to minor groove of DNA and cleaved pBR322 DNA via hydrolytic pathway (validated by T4 ligase assay). - Graphical abstract: Synthesis, characterization, DNA binding and cleavage studies of [Zn(glygly)(ssz)(H{sub 2}O)]{center_dot}6H{sub 2}O (1) containing glycyl glycine and sulfasalazine ligand. Complex 1 recognize minor groove of DNA and show hydrolytic DNA cleavage. Highlights: Black-Right-Pointing-Pointer Novel Zn(II) complex 1 bearing bioactive glycyl glycine and sulfasalazine ligand scaffold. Black-Right-Pointing-Pointer Cleavage activity of 1 was enhanced in presence of activators: H{sub 2}O{sub 2}>MPA>GSH>Asc. Black-Right-Pointing-Pointer Complex 1 recognize minor groove as depicted in the cleavage pattern and molecular docking. Black-Right-Pointing-Pointer Complex 1 cleaves pBR322 DNA via hydrolytic mechanism and validated by T4 DNA ligase experiments.

Plasma kinetic theory

International Nuclear Information System (INIS)

Elliott, J.A.

1993-01-01

Plasma kinetic theory is discussed and a comparison made with the kinetic theory of gases. The plasma is described by a modified set of fluid equations and it is shown how these fluid equations can be derived. (UK)

Enzymatic carotenoid cleavage in star fruit (Averrhoa carambola).

Science.gov (United States)

Fleischmann, Peter; Watanabe, Naoharu; Winterhalter, Peter

2003-05-01

This paper presents the first description of an enzyme fraction exhibiting carotenoid cleavage activity isolated from fruit skin of Averrhoa carambola. Partial purification of the enzyme could be achieved by acetone precipitation, ultrafiltration (300 kDa, 50 kDa), isoelectric focusing (pH 3-10) and sodium dodecyl sulfate polyacrylamide gel electrophoresis (7.5%). In this way, an enzymatically active protein fraction was obtained, consisting of four proteins in the molecular weight range of between 12 and 90 kDa. Using beta-carotene as substrate, the enzyme activity was detected spectrophotometrically at 505 nm. The main reaction product, detected by GC analysis, was beta-ionone. This proves that the isolated enzymes are closely related to aroma metabolism and release of star fruit. The time constant of the reaction was 16.6 min, the Michaelis Constant K(m)=3.6 micromol 1(-1) and the maximum velocity V(max)=10.5 x 10(-3) micromol l(-1) s(-1) mg((Protein))(-1). The optimum temperature was 45 degrees C.

DNA binding and cleavage studies of new sulfasalazine-derived dipeptide Zn(II) complex: Validation for specific recognition with 5′–TMP

International Nuclear Information System (INIS)

Tabassum, Sartaj; Al–Asbahy, Waddhaah M.; Afzal, Mohd.; Shamsi, Manal; Arjmand, Farukh

2012-01-01

A new water soluble complex [Zn(glygly)(ssz)(H 2 O)]·6H 2 O, 1 derived from dipeptide (glycyl glycine) and sulfasalazine was synthesized and characterized by spectroscopic (IR, UV–vis, NMR, ESI–MS) and analytical methods. The in vitro DNA binding studies of complex 1 with calf–thymus DNA were carried out by employing various biophysical methods and molecular docking technique which reveals strong electrostatic binding via phosphate backbone of DNA helix, in addition to partial intercalation. To gain further insight into the molecular recognition at the target site, interaction studies of complex 1 with 5′-TMP and 5′-GMP were carried out by UV–vis titration which was validated by 1 H and 31 P NMR with 5′-TMP, which implicate the preferential selectivity of 1 towards N3 of thymine. Complex 1 is accessible to minor groove of DNA and cleaved pBR322 DNA via hydrolytic pathway (validated by T4 ligase assay). - Graphical abstract: Synthesis, characterization, DNA binding and cleavage studies of [Zn(glygly)(ssz)(H 2 O)]·6H 2 O (1) containing glycyl glycine and sulfasalazine ligand. Complex 1 recognize minor groove of DNA and show hydrolytic DNA cleavage. Highlights: ► Novel Zn(II) complex 1 bearing bioactive glycyl glycine and sulfasalazine ligand scaffold. ► Cleavage activity of 1 was enhanced in presence of activators: H 2 O 2 >MPA>GSH>Asc. ► Complex 1 recognize minor groove as depicted in the cleavage pattern and molecular docking. ► Complex 1 cleaves pBR322 DNA via hydrolytic mechanism and validated by T4 DNA ligase experiments.

Mechanisms of catalytic cleavage of benzyl phenyl ether in aqueous and apolar phases

Energy Technology Data Exchange (ETDEWEB)

He, Jiayue; Lu, Lu; Zhao, Chen; Mei, Donghai; Lercher, Johannes A.

2014-03-01

Catalytic pathways for the cleavage of ether bonds in benzyl phenyl ether (BPE) in liquid phase using Ni- and zeolite-based catalysts are explored. In the absence of catalysts, the C-O bond is selectively cleaved in water by hydrolysis, forming phenol and benzyl alcohol as intermediates, followed by alkylation. The hydronium ions catalyzing the reactions are provided by the dissociation of water at 523 K. Upon addition of HZSM-5, rates of hydrolysis and alkylation are markedly increased in relation to proton concentrations. In the presence of Ni/SiO₂, the selective hydrogenolysis dominates for cleaving the C_aliphatic-O bond. Catalyzed by the dual-functional Ni/HZSM-5, hydrogenolysis occurs as the major route rather than hydrolysis (minor route). In apolar undecane, the non-catalytic thermal pyrolysis route dominates. Hydrogenolysis of BPE appears to be the major reaction pathway in undecane in the presence of Ni/SiO₂ or Ni/HZSM-5, almost completely suppressing radical reactions. Density functional theory (DFT) calculations strongly support the proposed C-O bond cleavage mechanisms on BPE in aqueous and apolar phases. These calculations show that BPE is initially protonated and subsequently hydrolyzed in the aqueous phase. Finally, DFT calculations suggest that the radical reactions in non-polar solvents lead to primary benzyl and phenoxy radicals in undecane, which leads to heavier condensation products as long as metals are absent for providing dissociated hydrogen.

A neural network method for identification of prokaryotic and eukaryotic signal peptides and prediction of their cleavage sites

DEFF Research Database (Denmark)

Nielsen, Henrik; Engelbrecht, Jacob; Brunak, Søren

1997-01-01

We have developed a new method for the identication of signal peptides and their cleavage sites based on neural networks trained on separate sets of prokaryotic and eukaryotic sequences. The method performs signicantly better than previous prediction schemes, and can easily be applied to genome...

Essential Structural and Functional Roles of the Cmr4 Subunit in RNA Cleavage by the Cmr CRISPR-Cas Complex

Directory of Open Access Journals (Sweden)

Nancy F. Ramia

2014-12-01

Full Text Available Summary: The Cmr complex is the multisubunit effector complex of the type III-B clustered regularly interspaced short palindromic repeats (CRISPR-Cas immune system. The Cmr complex recognizes a target RNA through base pairing with the integral CRISPR RNA (crRNA and cleaves the target at multiple regularly spaced locations within the complementary region. To understand the molecular basis of the function of this complex, we have assembled information from electron microscopic and X-ray crystallographic structural studies and mutagenesis of a complete Pyrococcus furiosus Cmr complex. Our findings reveal that four helically packed Cmr4 subunits, which make up the backbone of the Cmr complex, act as a platform to support crRNA binding and target RNA cleavage. Interestingly, we found a hook-like structural feature associated with Cmr4 that is likely the site of target RNA binding and cleavage. Our results also elucidate analogies in the mechanisms of crRNA and target molecule binding by the distinct Cmr type III-A and Cascade type I-E complexes. : Ramia et al. show that the helical core of the type III-B Cmr CRISPR-Cas effector complex, made up of multiple Cmr4 subunits, forms the platform for a corresponding number of cleavages of the target RNA. Comparison with the type I-E Cascade structure reveals strikingly similar mechanisms of crRNA and target binding.

Characterization and inhibition of norovirus proteases of genogroups I and II using a fluorescence resonance energy transfer assay

International Nuclear Information System (INIS)

Chang, Kyeong-Ok; Takahashi, Daisuke; Prakash, Om; Kim, Yunjeong

2012-01-01

Noroviruses are the major cause of food- or water-borne gastroenteritis outbreaks in humans. The norovirus protease that cleaves a large viral polyprotein to nonstructural proteins is essential for virus replication and an attractive target for antiviral drug development. Noroviruses show high genetic diversity with at least five genogroups, GI–GV, of which GI and GII are responsible for the majority of norovirus infections in humans. We cloned and expressed proteases of Norwalk virus (GI) and MD145 virus (GII) and characterized the enzymatic activities with fluorescence resonance energy transfer substrates. We demonstrated that the GI and GII proteases cleaved the substrates derived from the naturally occurring cleavage site in the open reading frame (ORF) 1 of G1 norovirus with similar efficiency, and that enzymatic activity of both proteases was inhibited by commercial protease inhibitors including chymostatin. The interaction of chymostatin to Norwalk virus protease was validated by nuclear magnetic resonance (NMR) spectroscopy.

An evaluation of analysis methodologies for predicting cleavage arrest of a deep crack in an RPV subjected to PTS loading conditions

International Nuclear Information System (INIS)

Keeney-Walker, J.; Bass, B.R.

1992-01-01

Several calculational procedures are compared for predicting cleavage arrest of a deep crack in the wall of a prototypical reactor pressure vessel (RPV) subjected to pressurized-thermal-shock (PTS) types of loading conditions. Three procedures examined in this study utilized the following models: (1) a static finite-element model (full bending); (2) a radially constrained static model; and (3) a thermoelastic dynamic finite-element model. A PTS transient loading condition was selected that produced a deep arrest of an axially-oriented initially shallow crack according to calculational results obtained from the static (full-bending) model. Results from the two static models were compared with those generated from the detailed thermoelastic dynamic finite-element analysis. The dynamic analyses modeled cleavage-crack propagation using node-release technique and an application-mode methodology based on dynamic fracture toughness curves generated from measured data. Comparisons presented here indicate that the degree to which dynamic solutions can be approximated by static models is highly dependent on several factors, including the material dynamic fracture curves and the propensity for cleavage reinitiation of the arrested crack under PTS loading conditions. Additional work is required to develop and validate a satisfactory dynamic fracture toughness model applicable to postcleavage arrest conditions in an RPV

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

International Nuclear Information System (INIS)

Wanitchang, Asawin; Narkpuk, Jaraspim; Jongkaewwattana, Anan

2013-01-01

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

Possible cleavage sites of glutelin partial degradation confirmed by immunological analysis in globulin-less mutants of rice (Oryza sativa L.).

Science.gov (United States)

Khan, Nadar; Yamaguchi, Satoru; Katsube-Tanaka, Tomoyuki

2017-10-01

Proteolytic cleavage or partial degradation of proteins is one of the important post-translational modifications for various biological processes, but it is difficult to analyze. Previously, we demonstrated that some subunits of the major rice (Oryza sativa L.) seed storage protein glutelin are partially degraded to produce newly identified polypeptides X1-X5 in mutants in which another major seed storage protein globulin is absent. In this study, the new polypeptides X3 and X4/X5 were immunologically confirmed to be derived from GluA3 and GluA1/GluA2 subunits, respectively. Additionally, the new polypeptides X1 and X2 were at least in part the α polypeptides of the GluB4 subunit partially degraded at the C-terminus. Simulated 2D-PAGE migration patterns of intact and partially degraded α polypeptides based on the calculation of their MWs and pIs enabled us to narrow or predict the possible locations of cleavage sites. The predicted cleavage sites were also verified by the comparison of 2D-PAGE patterns between seed-extracted and E. coli-expressed proteins of the intact and truncated α polypeptides. The results and methodologies demonstrated here would be useful for analyses of partial degradation of proteins and the structure-function relationships of rice seed protein bodies. © 2017 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

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

Energy Technology Data Exchange (ETDEWEB)

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

2013-08-15

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

Cleavage of a recombinant human immunoglobulin A2 (IgA2)-IgA1 hybrid antibody by certain bacterial IgA1 proteases

DEFF Research Database (Denmark)

Senior, B; Dunlop, JI; Batten, MR

2000-01-01

, Streptococcus pneumoniae, S. sanguis, Neisseria meningitidis types 1 and 2, N. gonorrhoeae types 1 and 2, and Haemophilus influenzae type 2. Thus, for these enzymes the recognition site for IgA1 cleavage is contained within half of the IgA1 hinge region; additional distal elements, if required, are provided...... by either an IgA1 or an IgA2 framework. In contrast, the IgA2/A1 hybrid appeared to be resistant to cleavage with S. oralis and some H. influenzae type 1 IgA1 proteases, suggesting these enzymes require additional determinants for efficient substrate recognition....

Kinetic Interface

DEFF Research Database (Denmark)

2009-01-01

A kinetic interface for orientation detection in a video training system is disclosed. The interface includes a balance platform instrumented with inertial motion sensors. The interface engages a participant's sense of balance in training exercises.......A kinetic interface for orientation detection in a video training system is disclosed. The interface includes a balance platform instrumented with inertial motion sensors. The interface engages a participant's sense of balance in training exercises....

AcEST: DK946532 [AcEST

Lifescience Database Archive (English)

Full Text Available FV Structural polyprotein OS=Barmah forest virus... 29 6.3 >sp|Q12594|DMAW_CLAFS ...+ + + C++ PPS S L HF Sbjct: 218 HCAVSTTHQTPDWFPLLCVQSPPSTSPFLGHF 249 >sp|P89946|POLS_BFV Structural polyprotein OS=Barmah forest

AcEST: DK948241 [AcEST

Lifescience Database Archive (English)

Full Text Available se [glutamine-hydrolyzing] OS=Xyl... 30 4.4 sp|P13896|POLN_WEEV Non-structural polyprotein OS=Western equine...+ Sbjct: 466 VIALRAVETVDFMTAHWAPLPYDFLSTVSNRIINELRGV 504 >sp|P13896|POLN_WEEV Non-structural polyprotein OS=Western equine

NCBI nr-aa BLAST: CBRC-TBEL-01-0399 [SEVENS

Lifescience Database Archive (English)

Full Text Available CBRC-TBEL-01-0399 ref|YP_224218.1| replicase-associated polyprotein [Citrus sudden ...death-associated virus] gb|AAW88343.1| replicase-associated polyprotein [Citrus sudden death-associated virus] YP_224218.1 2.3 37% ...

Two combinatorial optimization problems for SNP discovery using base-specific cleavage and mass spectrometry.

Science.gov (United States)

Chen, Xin; Wu, Qiong; Sun, Ruimin; Zhang, Louxin

2012-01-01

The discovery of single-nucleotide polymorphisms (SNPs) has important implications in a variety of genetic studies on human diseases and biological functions. One valuable approach proposed for SNP discovery is based on base-specific cleavage and mass spectrometry. However, it is still very challenging to achieve the full potential of this SNP discovery approach. In this study, we formulate two new combinatorial optimization problems. While both problems are aimed at reconstructing the sample sequence that would attain the minimum number of SNPs, they search over different candidate sequence spaces. The first problem, denoted as SNP - MSP, limits its search to sequences whose in silico predicted mass spectra have all their signals contained in the measured mass spectra. In contrast, the second problem, denoted as SNP - MSQ, limits its search to sequences whose in silico predicted mass spectra instead contain all the signals of the measured mass spectra. We present an exact dynamic programming algorithm for solving the SNP - MSP problem and also show that the SNP - MSQ problem is NP-hard by a reduction from a restricted variation of the 3-partition problem. We believe that an efficient solution to either problem above could offer a seamless integration of information in four complementary base-specific cleavage reactions, thereby improving the capability of the underlying biotechnology for sensitive and accurate SNP discovery.

Criterion of cleavage crack propagation and arrest in a nuclear PWR vessel steel

International Nuclear Information System (INIS)

Bousquet, Amaury

2013-01-01

The purpose of this PhD thesis is to understand physical mechanisms of cleavage crack propagation and arrest in the 16MND5 PWR vessel steel and to propose a robust predicting model based on a brittle fracture experimental campaign of finely instrumented laboratory specimens associated with numerical computations. First, experiments were carried out on thin CT25 specimens at five temperatures (-150 C, -125 C, -100 C, -7 C, -50 C). Two kinds of crack path, straight or branching path, have been observed. To characterize crack propagation and to measure crack speed, a high-speed framing camera system was used, combined with the development of an experimental protocol which allowed to observe CT surface without icing inside the thermal chamber and on the specimen. The framing camera (520 000 fps) has allowed to have a very accurate estimation of crack speed on the complete ligament of CT (∼ 25 mm). Besides, to analyse experiments and to study the impact of viscosity on the mechanical response around the crack tip, the elastic-viscoplastic behavior of the ferritic steel has been studied up to a strain rate of 104 s -1 for the tested temperatures.The extended Finite Element Method (X-FEM) was used in CAST3M FE software to model crack propagation. Numerical computations combine a local non linear dynamic approach with a RKR type fracture stress criterion to a characteristic distance. The work carried out has confirmed the form of the criterion proposed by Prabel at -125 C, and has identified the dependencies of the criterion on temperature and strain rate. From numerical analyzes in 2D and 3D, a multi-temperature fracture stress criterion, increasing function of the strain rate, was proposed. Predictive modeling were used to confirm the identified criterion on two specimen geometries (CT and compressive ring) in mode I at different temperatures. SEM observations and 3D analyzes made with optical microscope showed that the fracture mechanism was the cleavage associated

AcEST: DK957075 [AcEST

Lifescience Database Archive (English)

Full Text Available patitis C virus gen... 33 0.77 sp|Q8AZM0|POLS_BSNV Structural polyprotein OS=Blotched snake...jct: 2362 ---------SGDPGHSTGGGTTGETSKSPPDEPDDSEAGSVSSMPPLEG 2401 >sp|Q8AZM0|POLS_BSNV Structural polyprotein OS=Blotched snake

AcEST: DK956161 [AcEST

Lifescience Database Archive (English)

Full Text Available otein OS=Hepatitis C virus gen... 33 0.89 sp|Q8AZM0|POLS_BSNV Structural polyprotein OS=Blotched snakehead.....OLS_BSNV Structural polyprotein OS=Blotched snakehead virus PE=1 SV=1 Length = 10

AcEST: DK957443 [AcEST

Lifescience Database Archive (English)

Full Text Available Hepatitis C virus gen... 33 0.85 sp|Q8AZM0|POLS_BSNV Structural polyprotein OS=Blotched snakehead... 31 4.3 ...Structural polyprotein OS=Blotched snakehead virus PE=1 SV=1 Length = 1069 Score

AcEST: BP917834 [AcEST

Lifescience Database Archive (English)

Full Text Available ntrin-specific protease 7 OS=Mus musculu... 31 2.2 sp|P32537|POLG_HE701 Genome polyprotein OS=Human enterovirus...7 GQEPDASAASGRASSPNKSLESSASSEVSENSSV 400 >sp|P32537|POLG_HE701 Genome polyprotein OS=Human enterovirus

RISC-interacting clearing 3'- 5' exoribonucleases (RICEs) degrade uridylated cleavage fragments to maintain functional RISC in Arabidopsis thaliana.

Science.gov (United States)

Zhang, Zhonghui; Hu, Fuqu; Sung, Min Woo; Shu, Chang; Castillo-González, Claudia; Koiwa, Hisashi; Tang, Guiliang; Dickman, Martin; Li, Pingwei; Zhang, Xiuren

2017-05-02

RNA-induced silencing complex (RISC) is composed of miRNAs and AGO proteins. AGOs use miRNAs as guides to slice target mRNAs to produce truncated 5' and 3' RNA fragments. The 5' cleaved RNA fragments are marked with uridylation for degradation. Here, we identified novel cofactors of Arabidopsis AGOs, named RICE1 and RICE2. RICE proteins specifically degraded single-strand (ss) RNAs in vitro; but neither miRNAs nor miRNA*s in vivo. RICE1 exhibited a DnaQ-like exonuclease fold and formed a homohexamer with the active sites located at the interfaces between RICE1 subunits. Notably, ectopic expression of catalytically-inactive RICE1 not only significantly reduced miRNA levels; but also increased 5' cleavage RISC fragments with extended uridine tails. We conclude that RICEs act to degrade uridylated 5' products of AGO cleavage to maintain functional RISC. Our study also suggests a possible link between decay of cleaved target mRNAs and miRNA stability in RISC.

Cleavage of olefinic double bonds by mediated anodic oxidation

International Nuclear Information System (INIS)

Baeumer, U.-St.; Schaefer, H.J.

2003-01-01

Seven alkenes, e.g. 1-decene, methyl oleate, cyclododecene, norbornene, are cleaved by indirect anodic oxidation with IO 4 - /RuCl 3 as mediator to carboxylic acids. The best performance was achieved with two alternative ex cell-methods. Periodate is regenerated from iodate in a divided cell at a PbO 2 /Ti-anode. In the chemical reactor alkene and the produced carboxylic acid are immobilized in a chromatography column on Chromosorb W and oxidized with IO 4 - /RuO 4 in CH 3 CN/water. In the alternative version the alkene is oxidized in an emulsion generated by sonication and the organic phase is retained in the reactor by a separator. Acids and diacids are obtained in 61-91% chemical yield and good current yields. The amount of consumed periodate can be reduced to less than 5% of the amount needed for the chemical oxidation. The mediated anodic cleavage of alkenes is altogether an interesting alternative to ozonolysis

Kinetics and

Directory of Open Access Journals (Sweden)

Mojtaba Ahmadi

2016-11-01

Full Text Available The aqueous degradation of Reactive Yellow 84 (RY84 by potassium peroxydisulfate (K2S2O8 has been studied in laboratory scale experiments. The effect of the initial concentrations of potassium peroxydisulfate and RY84, pH and temperature on RY84 degradation were also examined. Experimental data were analyzed using first and second-order kinetics. The degradation kinetics of RY84 of the potassium peroxydisulfate process followed the second-order reaction kinetics. These rate constants have an extreme values similar to of 9.493 mM−1min−1 at a peroxydisulfate dose of 4 mmol/L. Thermodynamic parameters such as activation (Ea and Gibbs free energy (ΔG° were also evaluated. The negative value of ΔGo and Ea shows the spontaneous reaction natural conditions and exothermic nature.

Kinetic α secondary deuterium isotope effects for O-ethyl S-phenyl benzaldehyde acetal hydrolysis

International Nuclear Information System (INIS)

Ferraz, J.P.; Cordes, E.H.

1979-01-01

The rate of hydrolysis of O-ethyl S-phenyl benzaldehyde acetal at 25 0 C in 20% dioxane--80% water is independent of pH over the range pH6-12; k/sub obsd/ = 1.9 x 10 -7 s -1 . Under more acidic conditions, the rate increases linearly with the activity of the hydrated proton; k 2 = 2.95 x 10 -2 M -1 s -1 . The kinetic α secondary deuterium isotope effect for acid-catalyzed hydrolysis of O-ethyl S-phenyl benzaldehyde acetal, measured at 25 0 C in 20% aqueous dioxane containing 0.05 M HCl, is k/sub H//k/sub D/ = 1.038 +- 0.008, a value consistent with a transition state in which the C--S bond is stretched rather little. In contrast, the corresponding isotope effect for the pH-independent hydrolysis of this substrate, measured at 42.5 0 C in 20% dioxane, is 1.13 +- 0.02, a value consistent with complete C--S bond cleavage in the transition state and rate-determining diffusion apart of the ion-pair formed as the initial intermediate, in accord with the suggestion of Jensen and Jencks. 1 figure, 4 tables

SVM-based prediction of propeptide cleavage sites in spider toxins identifies toxin innovation in an Australian tarantula.

Directory of Open Access Journals (Sweden)

Emily S W Wong

Full Text Available Spider neurotoxins are commonly used as pharmacological tools and are a popular source of novel compounds with therapeutic and agrochemical potential. Since venom peptides are inherently toxic, the host spider must employ strategies to avoid adverse effects prior to venom use. It is partly for this reason that most spider toxins encode a protective proregion that upon enzymatic cleavage is excised from the mature peptide. In order to identify the mature toxin sequence directly from toxin transcripts, without resorting to protein sequencing, the propeptide cleavage site in the toxin precursor must be predicted bioinformatically. We evaluated different machine learning strategies (support vector machines, hidden Markov model and decision tree and developed an algorithm (SpiderP for prediction of propeptide cleavage sites in spider toxins. Our strategy uses a support vector machine (SVM framework that combines both local and global sequence information. Our method is superior or comparable to current tools for prediction of propeptide sequences in spider toxins. Evaluation of the SVM method on an independent test set of known toxin sequences yielded 96% sensitivity and 100% specificity. Furthermore, we sequenced five novel peptides (not used to train the final predictor from the venom of the Australian tarantula Selenotypus plumipes to test the accuracy of the predictor and found 80% sensitivity and 99.6% 8-mer specificity. Finally, we used the predictor together with homology information to predict and characterize seven groups of novel toxins from the deeply sequenced venom gland transcriptome of S. plumipes, which revealed structural complexity and innovations in the evolution of the toxins. The precursor prediction tool (SpiderP is freely available on ArachnoServer (http://www.arachnoserver.org/spiderP.html, a web portal to a comprehensive relational database of spider toxins. All training data, test data, and scripts used are available from