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Sample records for polyprotein cleavage kinetics

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

    Directory of Open Access Journals (Sweden)

    Artur Kaul

    2009-08-01

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

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

    Science.gov (United States)

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

    2009-08-01

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

  3. Identification of Cleavage Sites Recognized by the 3C-Like Cysteine Protease within the Two Polyproteins of Strawberry Mottle Virus

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    Hélène Sanfaçon

    2017-04-01

    Full Text Available Strawberry mottle virus (SMoV, family Secoviridae, order Picornavirales is one of several viruses found in association with strawberry decline disease in Eastern Canada. The SMoV genome consists of two positive-sense single-stranded RNAs, each encoding one large polyprotein. The RNA1 polyprotein (P1 includes the domains for a putative helicase, a VPg, a 3C-like cysteine protease and an RNA-dependent RNA polymerase at its C-terminus, and one or two protein domains at its N-terminus. The RNA2 polyprotein (P2 is predicted to contain the domains for a movement protein (MP and one or several coat proteins at its N-terminus, and one or more additional domains for proteins of unknown function at its C-terminus. The RNA1-encoded 3C-like protease is presumed to cleave the two polyproteins in cis (P1 and in trans (P2. Using in vitro processing assays, we systematically scanned the two polyproteins for cleavage sites recognized by this protease. We identified five cis-cleavage sites in P1, with cleavage between the putative helicase and VPg domains being the most efficient. The presence of six protein domains in the SMoV P1, including two upstream of the putative helicase domain, is a feature shared with nepoviruses but not with comoviruses. Results from trans-cleavage assays indicate that the RNA1-encoded 3C-like protease recognized a single cleavage site, which was between the predicted MP and coat protein domains in the P2 polyprotein. The cleavage site consensus sequence for the SMoV 3C-like protease is AxE (E or Q/(G or S.

  4. Identification of the protease cleavage sites in a reconstituted Gag polyprotein of an HERV-K(HML-2 element

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    Kurth Reinhard

    2011-05-01

    Full Text Available Abstract Background The human genome harbors several largely preserved HERV-K(HML-2 elements. Although this retroviral family comes closest of all known HERVs to producing replication competent virions, mutations acquired during their chromosomal residence have rendered them incapable of expressing infectious particles. This also holds true for the HERV-K113 element that has conserved open reading frames (ORFs for all its proteins in addition to a functional LTR promoter. Uncertainty concerning the localization and impact of post-insertional mutations has greatly hampered the functional characterization of these ancient retroviruses and their proteins. However, analogous to other betaretroviruses, it is known that HERV-K(HML-2 virions undergo a maturation process during or shortly after release from the host cell. During this process, the subdomains of the Gag polyproteins are released by proteolytic cleavage, although the nature of the mature HERV-K(HML-2 Gag proteins and the exact position of the cleavage sites have until now remained unknown. Results By aligning the amino acid sequences encoded by the gag-pro-pol ORFs of HERV-K113 with the corresponding segments from 10 other well-preserved human specific elements we identified non-synonymous post-insertional mutations that have occurred in this region of the provirus. Reversion of these mutations and a partial codon optimization facilitated the large-scale production of maturation-competent HERV-K113 virus-like particles (VLPs. The Gag subdomains of purified mature VLPs were separated by reversed-phase high-pressure liquid chromatography and initially characterized using specific antibodies. Cleavage sites were identified by mass spectrometry and N-terminal sequencing and confirmed by mutagenesis. Our results indicate that the gag gene product Pr74Gag of HERV-K(HML-2 is processed to yield p15-MA (matrix, SP1 (spacer peptide of 14 amino acids, p15, p27-CA (capsid, p10-NC (nucleocapsid and two

  5. Identification of the protease cleavage sites in a reconstituted Gag polyprotein of an HERV-K(HML-2) element.

    Science.gov (United States)

    George, Maja; Schwecke, Torsten; Beimforde, Nadine; Hohn, Oliver; Chudak, Claudia; Zimmermann, Anja; Kurth, Reinhard; Naumann, Dieter; Bannert, Norbert

    2011-05-09

    The human genome harbors several largely preserved HERV-K(HML-2) elements. Although this retroviral family comes closest of all known HERVs to producing replication competent virions, mutations acquired during their chromosomal residence have rendered them incapable of expressing infectious particles. This also holds true for the HERV-K113 element that has conserved open reading frames (ORFs) for all its proteins in addition to a functional LTR promoter. Uncertainty concerning the localization and impact of post-insertional mutations has greatly hampered the functional characterization of these ancient retroviruses and their proteins. However, analogous to other betaretroviruses, it is known that HERV-K(HML-2) virions undergo a maturation process during or shortly after release from the host cell. During this process, the subdomains of the Gag polyproteins are released by proteolytic cleavage, although the nature of the mature HERV-K(HML-2) Gag proteins and the exact position of the cleavage sites have until now remained unknown. By aligning the amino acid sequences encoded by the gag-pro-pol ORFs of HERV-K113 with the corresponding segments from 10 other well-preserved human specific elements we identified non-synonymous post-insertional mutations that have occurred in this region of the provirus. Reversion of these mutations and a partial codon optimization facilitated the large-scale production of maturation-competent HERV-K113 virus-like particles (VLPs). The Gag subdomains of purified mature VLPs were separated by reversed-phase high-pressure liquid chromatography and initially characterized using specific antibodies. Cleavage sites were identified by mass spectrometry and N-terminal sequencing and confirmed by mutagenesis. Our results indicate that the gag gene product Pr74Gag of HERV-K(HML-2) is processed to yield p15-MA (matrix), SP1 (spacer peptide of 14 amino acids), p15, p27-CA (capsid), p10-NC (nucleocapsid) and two C-terminally encoded glutamine- and

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

    OpenAIRE

    Demangeat, Gerard; Greif, Charles; Hemmer, O; Fritsch, C

    1990-01-01

    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 furthe...

  7. 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.

  8. 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...

  9. Tailored Polyproteins Using Sequential Staple and Cut.

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    Garg, Surbhi; Singaraju, Gayathri Sindhuri; Yengkhom, Sunanda; Rakshit, Sabyasachi

    2018-04-30

    Polyproteins, individual protein units joined covalently in tandem, have evolved as a promising tool for measuring the dynamic folding of biomacromolecules in single-molecule force spectroscopy. However, the synthetic routes to prepare polyproteins have been a bottleneck, and urge development of in vitro methods to knit individual protein units covalently into polyprotein. Employing two enzymes of orthogonal functionalities periodically in sequence, we synthesized monodispersed polyproteins on a solid surface. We used Sortase A (SrtA), the enzyme known for sequence specific transpeptidation, to staple protein units covalently through peptide bonds. Exploiting the sequence-specific peptide cleaving ability of TEV protease, we controlled the progress of the reaction to one attachment at a time. Finally, with unique design of the unit proteins we control the orientation of proteins in polyprotein. This simple conjugation has the potential to staple proteins with different functionalities and from different expression systems, in any number in the polyprotein and, above all, via irreversible peptide bonds. Multiple chimeric constructs can also be synthesized with interchangeable protein units.

  10. In vitro processing of the RNA-2-encoded polyprotein of two nepoviruses: tomato black ring virus and grapevine chrome mosaic virus.

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    Demangeat, G; Hemmer, O; Fritsch, C; Le Gall, O; Candresse, T

    1991-02-01

    In vitro translation of RNA-2 of each of two closely related nepoviruses, tomato black ring virus (TBRV) and grapevine chrome mosaic virus (GCMV), in a rabbit reticulocyte lysate resulted in the synthesis of single polypeptides of 150K and 146K respectively. Processing of these polyproteins occurred after the addition of translation products of homologous RNA-1. The positions of the cleavage products within the polyproteins were determined. From the N to the C terminus, Mr values for the proteins were 50K, 46K and 59K for TBRV and 44K, 46K and 56K for GCMV. TBRV RNA-1 translation products also cleaved the polyproteins encoded by GCMV RNA-2 which suggests that the cleavage sites in the two polyproteins are similar.

  11. 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

  12. Self-processing 2A-polyproteins--a system for co-ordinate expression of multiple proteins in transgenic plants.

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    Halpin, C; Cooke, S E; Barakate, A; El Amrani, A; Ryan, M D

    1999-02-01

    Achieving co-ordinate, high-level and stable expression of multiple transgenes in plants is currently difficult. Expression levels are notoriously variable and influenced by factors that act independently on transgenes at different genetic loci. Instability of expression due to loss, re-arrangement or silencing of transgenes may occur, and is exacerbated by increasing numbers of transgenic loci and repeated use of homologous sequences. Even linking two or more genes within a T-DNA does not necessarily result in co-ordinate expression. Linking proteins in a single open reading frame--a polyprotein--is a strategy for co-ordinate expression used by many viruses. After translation, polyproteins are processed into constituent polypeptides, usually by proteinases encoded within the polyprotein itself. However, in foot-and-mouth disease virus (FMDV), a sequence (2A) of just 16-20 amino acids appears to have the unique capability to mediate cleavage at its own C-terminus by an apparently enzyme-independent, novel type of reaction. This sequence can also mediate cleavage in a heterologous protein context in a range of eukaryotic expression systems. We have constructed a plasmid in which the 2A sequence is inserted between the reporter genes chloramphenicol acetyltransferase (CAT) and beta-glucuronidase (GUS), maintaining a single open reading frame. Here we report that expression of this construct in wheatgerm lysate and transgenic plants results in efficient cleavage of the polyprotein and co-ordinate expression of active CAT and GUS. Self-processing polyproteins using the FMDV 2A sequence could therefore provide a system for ensuring co-ordinated, stable expression of multiple introduced proteins in plant cells.

  13. 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

  14. Single-Molecule Force Spectroscopy Trajectories of a Single Protein and Its Polyproteins Are Equivalent: A Direct Experimental Validation Based on A Small Protein NuG2.

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    Lei, Hai; He, Chengzhi; Hu, Chunguang; Li, Jinliang; Hu, Xiaodong; Hu, Xiaotang; Li, Hongbin

    2017-05-22

    Single-molecule force spectroscopy (SMFS) has become a powerful tool in investigating the mechanical unfolding/folding of proteins at the single-molecule level. Polyproteins made of tandem identical repeats have been widely used in atomic force microscopy (AFM)-based SMFS studies, where polyproteins not only serve as fingerprints to identify single-molecule stretching events, but may also improve statistics of data collection. However, the inherent assumption of such experiments is that all the domains in the polyprotein are equivalent and one SMFS trajectory of stretching a polyprotein made of n domains is equivalent to n trajectories of stretching a single domain. Such an assumption has not been validated experimentally. Using a small protein NuG2 and its polyprotein (NuG2) 4 as model systems, here we use optical trapping (OT) to directly validate this assumption. Our results show that OT experiments on NuG2 and (NuG2) 4 lead to identical parameters describing the unfolding and folding kinetics of NuG2, demonstrating that indeed stretching a polyprotein of NuG2 is equivalent to stretching single NuG2 in force spectroscopy experiments and thus validating the use of polyproteins in SMFS experiments. © 2017 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.

  15. Functional characterization of the proteolytic activity of the tomato black ring nepovirus RNA-1-encoded polyprotein.

    Science.gov (United States)

    Hemmer, O; Greif, C; Dufourcq, P; Reinbolt, J; Fritsch, C

    1995-01-10

    Translation of tomato black ring virus (TBRV) RNA-1 in a rabbit reticulocyte lysate leads to the synthesis of a 250K polyprotein which cleaves itself into smaller proteins of 50, 60, 120, and 190K. Polypeptides synthesized from synthetic transcripts corresponding to different regions of TBRV RNA-1 are processed only when they encode the 23K protein delimited earlier by sequence homology with the cowpea mosaic virus 24K protease. The proteolytic activity of this protein is completely lost by mutating residues C170 (to I) or L188 (to H), residues which align with conserved residues of the viral serine-like proteases. The 120K protein is generated by cleavage of the dipeptide K/A localized in front of the VPg but is not further cleaved in vitro at the K/S site (at the C terminus of the VPg) or between the protease and polymerase domains. However, both the protein VPgProPol (120K) and the protein ProPol (117K) produced in vitro from synthetic transcripts can cleave in trans the RNA-2-encoded 150K polyprotein, but they cannot cleave in trans polypeptides containing a cleavage site expressed from RNA-1 transcripts in which the protease cistron is absent or modified.

  16. Modifications to the Foot-and-Mouth Disease Virus 2A Peptide: Influence on Polyprotein Processing and Virus Replication.

    Science.gov (United States)

    Kjær, Jonas; Belsham, Graham J

    2018-04-15

    Foot-and-mouth disease virus (FMDV) has a positive-sense single-stranded RNA (ssRNA) genome that includes a single, large open reading frame encoding a polyprotein. The cotranslational "cleavage" of this polyprotein at the 2A/2B junction is mediated by the 2A peptide (18 residues in length) using a nonproteolytic mechanism termed "ribosome skipping" or "StopGo." Multiple variants of the 2A polypeptide with this property among the picornaviruses share a conserved C-terminal motif [D(V/I)E(S/T)NPG↓P]. The impact of 2A modifications within this motif on FMDV protein synthesis, polyprotein processing, and virus viability were investigated. Amino acid substitutions are tolerated at residues E 14 , S 15 , and N 16 within the 2A sequences of infectious FMDVs despite their reported "cleavage" efficiencies at the 2A/2B junction of only ca. 30 to 50% compared to that of the wild type (wt). In contrast, no viruses containing substitutions at residue P 17 , G 18 , or P 19 , which displayed little or no "cleavage" activity in vitro , were rescued, but wt revertants were obtained. The 2A substitutions impaired the replication of an FMDV replicon. Using transient-expression assays, it was shown that certain amino acid substitutions at residues E 14 , S 15 , N 16 , and P 19 resulted in partial "cleavage" of a protease-free polyprotein, indicating that these specific residues are not essential for cotranslational "cleavage." Immunofluorescence studies, using full-length FMDV RNA transcripts encoding mutant 2A peptides, indicated that the 2A peptide remained attached to adjacent proteins, presumably 2B. These results show that efficient "cleavage" at the 2A/2B junction is required for optimal virus replication. However, maximal StopGo activity does not appear to be essential for the viability of FMDV. IMPORTANCE Foot-and-mouth disease virus (FMDV) causes one of the most economically important diseases of farm animals. Cotranslational "cleavage" of the FMDV polyprotein precursor at

  17. Novel ATPase activity of the polyprotein intermediate, Viral Protein genome-linked-Nuclear Inclusion-a protease, of Pepper vein banding potyvirus

    International Nuclear Information System (INIS)

    Mathur, Chhavi; Savithri, Handanahal S.

    2012-01-01

    Highlights: ► Pepper vein banding potyvirus VPg harbors Walker motifs. ► VPg exhibits ATPase activity in the presence of NIa-Pro. ► Plausible structural and functional interplay between VPg and NIa-Pro. ► Functional relevance of prolonged presence of VPg-Pro during infection. -- Abstract: Potyviruses temporally regulate their protein function by polyprotein processing. Previous studies have shown that VPg (Viral Protein genome-linked) of Pepper vein banding virus interacts with the NIa-Pro (Nuclear Inclusion-a protease) domain, and modulates the kinetics of the protease. In the present study, we report for the first time that VPg harbors the Walker motifs A and B, and the presence of NIa-Pro, especially in cis (cleavage site (E191A) VPg-Pro mutant), is essential for manifestation of the ATPase activity. Mutation of Lys47 (Walker motif A) and Asp88:Glu89 (Walker motif B) to alanine in E191A VPg-Pro lead to reduced ATPase activity, confirming that this activity was inherent to VPg. We propose that potyviral VPg, established as an intrinsically disordered domain, undergoes plausible structural alterations upon interaction with globular NIa-Pro which induces the ATPase activity.

  18. Cleavage sites within the poliovirus capsid protein precursors

    International Nuclear Information System (INIS)

    Larsen, G.R.; Anderson, C.W.; Dorner, A.J.; Semler, B.L.; Wimmer, E.

    1982-01-01

    Partial amino-terminal sequence analysis was performed on radiolabeled poliovirus capsid proteins VP1, VP2, and VP3. A computer-assisted comparison of the amino acid sequences obtained with that predicted by the nucleotide sequence of the poliovirus genome allows assignment of the amino terminus of each capsid protein to a unique position within the virus polyprotein. Sequence analysis of trypsin-digested VP4, which has a blocked amino terminus, demonstrates that VP4 is encoded at or very near to the amino terminus of the polyprotein. The gene order of the capsid proteins is VP4-VP2-VP3-VP1. Cleavage of VP0 to VP4 and VP2 is shown to occur between asparagine and serine, whereas the cleavages that separate VP2/VP3 and VP3/VP1 occur between glutamine and glycine residues. This finding supports the hypothesis that the cleavage of VP0, which occurs during virion morphogenesis, is distinct from the cleavages that separate functional regions of the polyprotein

  19. Cleavage sites in the polypeptide precursors of poliovirus protein P2-X

    International Nuclear Information System (INIS)

    Selmer, B.L.; Hanecak, R.; Anderson, C.W.; Wimmer, E.

    1981-01-01

    Partial amino-terminal sequence analysis has been performed on the three major polypeptide products (P2-3b, P2-5b, and P2-X) from the central region (P2) of the poliovirus polyprotein, and this analysis precisely locates the amino termini of these products with respect to the nucleotide sequence of the poliovirus RNA genome. Like most of the products of the replicase region (P3), the amino termini of P2-5b and P2-X are generated by cleavage between glutamine and glycine residues. Thus, P2-5b and P2-X are probably both produced by the action of a singly (virus-encoded.) proteinase. The amino terminus of P2-3b, on the other hand, is produced by a cleavage between the carboxy-terminal tyrosine of VP1 and the glycine encoded by nucleotides 3381-3383. This result may suggest that more than one proteolytic activity is required for the complete processing of the poliovirus polyprotein

  20. Coronavirus 3CLpro proteinase cleavage sites: Possible relevance to SARS virus pathology

    Directory of Open Access Journals (Sweden)

    Blom Nikolaj

    2004-06-01

    Full Text Available Abstract Background Despite the passing of more than a year since the first outbreak of Severe Acute Respiratory Syndrome (SARS, efficient counter-measures are still few and many believe that reappearance of SARS, or a similar disease caused by a coronavirus, is not unlikely. For other virus families like 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. Prompted by this, we set out to analyse and predict cleavage by the coronavirus main proteinase using computational methods. Results We retrieved sequence data on seven fully sequenced coronaviruses and identified the main 3CL proteinase cleavage sites in polyproteins using alignments. A neural network was trained to recognise the cleavage sites in the genomes obtaining a sensitivity of 87.0% and a specificity of 99.0%. Several proteins known to be cleaved by other viruses were submitted to prediction as well as proteins suspected relevant in coronavirus pathology. Cleavage sites were predicted in proteins such as the cystic fibrosis transmembrane conductance regulator (CFTR, transcription factors CREB-RP and OCT-1, and components of the ubiquitin pathway. Conclusions Our prediction method NetCorona predicts coronavirus cleavage sites with high specificity and several potential cleavage candidates were identified which might be important to elucidate coronavirus pathology. Furthermore, the method might assist in design of proteinase inhibitors for treatment of SARS and possible future diseases caused by coronaviruses. It is made available for public use at our website: http://www.cbs.dtu.dk/services/NetCorona/.

  1. Uncoupling of Protease trans-Cleavage and Helicase Activities in Pestivirus NS3.

    Science.gov (United States)

    Zheng, Fengwei; Lu, Guoliang; Li, Ling; Gong, Peng; Pan, Zishu

    2017-11-01

    The nonstructural protein NS3 from the Flaviviridae family is a multifunctional protein that contains an N-terminal protease and a C-terminal helicase, playing essential roles in viral polyprotein processing and genome replication. Here we report a full-length crystal structure of the classical swine fever virus (CSFV) NS3 in complex with its NS4A protease cofactor segment (PCS) at a 2.35-Å resolution. The structure reveals a previously unidentified ∼2,200-Å 2 intramolecular protease-helicase interface comprising three clusters of interactions, representing a "closed" global conformation related to the NS3-NS4A cis -cleavage event. Although this conformation is incompatible with protease trans -cleavage, it appears to be functionally important and beneficial to the helicase activity, as the mutations designed to perturb this conformation impaired both the helicase activities in vitro and virus production in vivo Our work reveals important features of protease-helicase coordination in pestivirus NS3 and provides a key basis for how different conformational states may explicitly contribute to certain functions of this natural protease-helicase fusion protein. IMPORTANCE Many RNA viruses encode helicases to aid their RNA genome replication and transcription by unwinding structured RNA. Being naturally fused to a protease participating in viral polyprotein processing, the NS3 helicases encoded by the Flaviviridae family viruses are unique. Therefore, how these two enzyme modules coordinate in a single polypeptide is of particular interest. Here we report a previously unidentified conformation of pestivirus NS3 in complex with its NS4A protease cofactor segment (PCS). This conformational state is related to the protease cis -cleavage event and is optimal for the function of helicase. This work provides an important basis to understand how different enzymatic activities of NS3 may be achieved by the coordination between the protease and helicase through different

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

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    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.

  3. Specific in vitro cleavage of Mason-Pfizer monkey virus capsid protein: evidence for a potential role of retroviral protease in early stages of infection

    International Nuclear Information System (INIS)

    Rumlova, Michaela; Ruml, Tomas; Pohl, Jan; Pichova, Iva

    2003-01-01

    Processing of Gag polyproteins by viral protease (PR) leads to reorganization of immature retroviral particles and formation of a ribonucleoprotein core. In some retroviruses, such as HIV and RSV, cleavage of a spacer peptide separating capsid and nucleocapsid proteins is essential for the core formation. We show here that no similar spacer peptide is present in the capsid-nucleocapsid (CA-NC) region of Mason-Pfizer monkey virus (M-PMV) and that the CA protein is cleaved in vitro by the PR within the major homology region (MHR) and the NC protein in several sites at the N-terminus. The CA cleavage product was also identified shortly after penetration of M-PMV into COS cells, suggesting that the protease-catalyzed cleavage is involved in core disintegration

  4. Secondary isotope effects on alpha-cleavage reactions

    International Nuclear Information System (INIS)

    Ingemann, S.; Hammerum, S.

    1980-01-01

    Kinetic deuterium isotope effects on mass spectral reactions have in several instances been utilized to provide structural information and to answer mechanistic questions. Typically, the influence of the deuterium label on the rate of one of a number of competing reactions has been studied. Secondary isotope effects have usually been assumed to be relatively insignificant in comparison with the observed kinetic effects, even though various workers have shown that secondary isotope effects may indeed exert a considerable influence on the rates of competing simple cleavages. Recent studies have provided quantitative data to show that the mere presence of deuterium atoms up to six bonds away may influence the rate of a simple cleavage reaction. In relation to an investigation of rearrangements accompanying simple cleavage reactions, a semi-quantitative measure was needed of the variation of the secondary isotope effect with the number of bonds between the deuterium label and the point of rupture. The influence has therefore been examined of the presence of remote deuterium atoms on a typical simple cleavage reaction, the α-cleavage of aliphatic amines. As a model compound, N-methyldipentylamine was chosen, systematically labelled with deuterium. (author)

  5. 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.

  6. 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.

  7. 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...

  8. 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...

  9. 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

  10. 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)

  11. 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.

  12. Disruption of TLR3 signaling due to cleavage of TRIF by the hepatitis A virus protease-polymerase processing intermediate, 3CD.

    Directory of Open Access Journals (Sweden)

    Lin Qu

    2011-09-01

    Full Text Available Toll-like receptor 3 (TLR3 and cytosolic RIG-I-like helicases (RIG-I and MDA5 sense viral RNAs and activate innate immune signaling pathways that induce expression of interferon (IFN through specific adaptor proteins, TIR domain-containing adaptor inducing interferon-β (TRIF, and mitochondrial antiviral signaling protein (MAVS, respectively. Previously, we demonstrated that hepatitis A virus (HAV, a unique hepatotropic human picornavirus, disrupts RIG-I/MDA5 signaling by targeting MAVS for cleavage by 3ABC, a precursor of the sole HAV protease, 3C(pro, that is derived by auto-processing of the P3 (3ABCD segment of the viral polyprotein. Here, we show that HAV also disrupts TLR3 signaling, inhibiting poly(I:C-stimulated dimerization of IFN regulatory factor 3 (IRF-3, IRF-3 translocation to the nucleus, and IFN-β promoter activation, by targeting TRIF for degradation by a distinct 3ABCD processing intermediate, the 3CD protease-polymerase precursor. TRIF is proteolytically cleaved by 3CD, but not by the mature 3C(pro protease or the 3ABC precursor that degrades MAVS. 3CD-mediated degradation of TRIF depends on both the cysteine protease activity of 3C(pro and downstream 3D(pol sequence, but not 3D(pol polymerase activity. Cleavage occurs at two non-canonical 3C(pro recognition sequences in TRIF, and involves a hierarchical process in which primary cleavage at Gln-554 is a prerequisite for scission at Gln-190. The results of mutational studies indicate that 3D(pol sequence modulates the substrate specificity of the upstream 3C(pro protease when fused to it in cis in 3CD, allowing 3CD to target cleavage sites not normally recognized by 3C(pro. HAV thus disrupts both RIG-I/MDA5 and TLR3 signaling pathways through cleavage of essential adaptor proteins by two distinct protease precursors derived from the common 3ABCD polyprotein processing intermediate.

  13. 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

  14. 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.

  15. 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...

  16. 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

  17. Dataset of cocoa aspartic protease cleavage sites

    Directory of Open Access Journals (Sweden)

    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

  18. 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

  19. 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.

  20. 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.

  1. In vivo analysis of the Notch receptor S1 cleavage.

    Directory of Open Access Journals (Sweden)

    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.

  2. 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.

  3. 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...

  4. Abyssal fiction: common shares, colonial cleavages

    Directory of Open Access Journals (Sweden)

    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

  5. 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...

  6. 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

  7. 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

  8. 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.

  9. 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.

    Directory of Open Access Journals (Sweden)

    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.

  10. 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 ...

  11. Modeling and inferring cleavage patterns in proliferating epithelia.

    Directory of Open Access Journals (Sweden)

    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.

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

    Directory of Open Access Journals (Sweden)

    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.

  13. 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....

  14. 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.

  15. 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.

  16. 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.

  17. 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.

  18. 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.

  19. 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.

  20. 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...

  1. 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

  2. Cleavage events and sperm dynamics in chick intrauterine embryos.

    Directory of Open Access Journals (Sweden)

    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.

  3. 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.

  4. Nucleophilic addition to olefins. 5. Reaction of 1,1-dinitro-2,2-diphenylethylene with water and hydroxide ion in 50% Me2SO-50% water. Complete kinetic analysis of hydrolytic cleavage of the C=C double bond in acidic and basic solution

    International Nuclear Information System (INIS)

    Bernasconi, C.F.; Carre, D.J.; Kanavarioti, A.

    1981-01-01

    Hydrolysis of 1,1-dinitro-2,2-diphenylethylene (2) to form benzophenone and dinitromethane (or its anion) was studied in 50% Me 2 SO-50% H 2 O and also in 50% Me 2 SO-50% D 2 O at pHs of 1 to 16. Solvent isotope effects general acid and general base catalysis, and structure-reactivity relationships were used to study the kinetics. The conclusions are the following: (1) the equilibrium constants for OH - and water addition to 2 to form T/sub OH/ - are comparable to those for the corresponding reactions of benzylidene Meldrum's acid (1), but the rate constants are much lower for 2 than for 1; (2) carbon protonation of T/sub OH/ - follows an Eigen curve similar to that for 1,1-dinitroethane anion but which is displaced upward by nearly 1 log unit. This indicates a higher intrinsic protonation rate because of a smaller charge delocalization in T/sub OH/ - owing to an enhanced steric hindrance to coplanarity of the nitro groups in T/sub OH/ - ; (3) intramolecular proton transfer from the OH group to the carbanionic site in T/sub OH/ - is insignificant, which is in contrast to the behavior of the addition complex between 2 and morpholine; (4) the base-catalyzed breakdown of T/sub OH/ 0 into benzophenone and dinitromethane anion occurs by rate-limiting oxygen deprotonation, which implies that k 4 for CH(NO 2 ) 2 - departure from T/sub OH/ 0- is much greater than 2 x 10 9 s -1 , a remarkably high rate for a carbanionic leaving group. The water-catalyzed breakdown of T/sub OH/ 0 proceeds by a different mechanism, which is most likely concerted, with a transition state; (5) the acid-catalyzed breakdown of T/sub OH/ 2- occurs by rate-limiting carbon protonation (k 6 /sup BH/), but the water-catalyzed breakdown follows a different mechanism. Various possibilities are discussed, and a slight preference is given to a preassociation mechanism

  5. 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.

  6. 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

  7. 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.)

  8. 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

  9. 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”.

  10. Pripper: prediction of caspase cleavage sites from whole proteomes

    Directory of Open Access Journals (Sweden)

    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

  11. 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.

  12. 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.

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

    Science.gov (United States)

    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.

  14. 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.

  15. 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.

  16. 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....

  17. A new cultural cleavage in post-modern society

    Directory of Open Access Journals (Sweden)

    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.

  18. 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.

  19. Short RNA guides cleavage by eukaryotic RNase III.

    Directory of Open Access Journals (Sweden)

    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.

  20. 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

  1. 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...

  2. 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...

  3. 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.

  4. 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

  5. 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.

  6. 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...

  7. 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.

  8. 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

  9. 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...

  10. 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...

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

    Czech Academy of Sciences Publication Activity Database

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

    2017-01-01

    Roč. 49, C (2017), s. 134-138 ISSN 0324-1130 R&D Projects: GA ČR(CZ) GA14-05180S Institutional support: RVO:61388955 Keywords : electrochemical impedance spectroscopy * rate constant * self-protonation Subject RIV: CG - Electrochemistry OBOR OECD: Physical chemistry Impact factor: 0.238, year: 2016

  12. Granulocyte kinetics

    International Nuclear Information System (INIS)

    Peters, A.M.; Lavender, J.P.; Saverymuttu, S.H.

    1985-01-01

    By using density gradient materials enriched with autologous plasma, the authors have been able to isolate granulocutes from other cellular elements and label them with In-111 without separation from a plasma environment. The kinetic behavior of these cells suggests that phenomena attributed to granulocyte activation are greatly reduced by this labeling. Here, they review their study of granulocyte kinetics in health and disease in hope of quantifying sites of margination and identifying principal sites of destruction. The three principle headings of the paper are distribution, life-span, and destruction

  13. Flanking signal and mature peptide residues influence signal peptide cleavage

    Directory of Open Access Journals (Sweden)

    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.

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

    Science.gov (United States)

    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.

  15. 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

  16. 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....

  17. 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

  18. Physisorption kinetics

    CERN Document Server

    Kreuzer, Hans Jürgen

    1986-01-01

    This monograph deals with the kinetics of adsorption and desorption of molecules physisorbed on solid surfaces. Although frequent and detailed reference is made to experiment, it is mainly concerned with the theory of the subject. In this, we have attempted to present a unified picture based on the master equation approach. Physisorption kinetics is by no means a closed and mature subject; rather, in writing this monograph we intended to survey a field very much in flux, to assess its achievements so far, and to give a reasonable basis from which further developments can take off. For this reason we have included many papers in the bibliography that are not referred to in the text but are of relevance to physisorption. To keep this monograph to a reasonable size, and also to allow for some unity in the presentation of the material, we had to omit a number of topics related to physisorption kinetics. We have not covered to any extent the equilibrium properties of physisorbed layers such as structures, phase tr...

  19. 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...

  20. Glutamic Acid Selective Chemical Cleavage of Peptide Bonds.

    Science.gov (United States)

    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.

  1. Effects of Cysteamine on Sheep Embryo Cleavage Rates

    Directory of Open Access Journals (Sweden)

    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.

  2. 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)

  3. 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

  4. [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

  5. 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

  6. Stochastic kinetics

    International Nuclear Information System (INIS)

    Colombino, A.; Mosiello, R.; Norelli, F.; Jorio, V.M.; Pacilio, N.

    1975-01-01

    A nuclear system kinetics is formulated according to a stochastic approach. The detailed probability balance equations are written for the probability of finding the mixed population of neutrons and detected neutrons, i.e. detectrons, at a given level for a given instant of time. Equations are integrated in search of a probability profile: a series of cases is analyzed through a progressive criterium. It tends to take into account an increasing number of physical processes within the chosen model. The most important contribution is that solutions interpret analytically experimental conditions of equilibrium (moise analysis) and non equilibrium (pulsed neutron measurements, source drop technique, start up procedures)

  7. Tolrestat kinetics

    International Nuclear Information System (INIS)

    Hicks, D.R.; Kraml, M.; Cayen, M.N.; Dubuc, J.; Ryder, S.; Dvornik, D.

    1984-01-01

    The kinetics of tolrestat, a potent inhibitor of aldose reductase, were examined. Serum concentrations of tolrestat and of total 14 C were measured after dosing normal subjects and subjects with diabetes with 14 C-labeled tolrestat. In normal subjects, tolrestat was rapidly absorbed and disappearance from serum was biphasic. Distribution and elimination t 1/2s were approximately 2 and 10 to 12 hr, respectively, after single and multiple doses. Unchanged tolrestat accounted for the major portion of 14 C in serum. Radioactivity was rapidly and completely excreted in urine and feces in an approximate ratio of 2:1. Findings were much the same in subjects with diabetes. In normal subjects, the kinetics of oral tolrestat were independent of dose in the 10 to 800 mg range. Repetitive dosing did not result in unexpected cumulation. Tolrestat was more than 99% bound to serum protein; it did not compete with warfarin for binding sites but was displaced to some extent by high concentrations of tolbutamide or salicylate

  8. 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...

  9. 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

  10. 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...

  11. 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.

  12. Carotenoid-cleavage activities of crude enzymes from Pandanous amryllifolius.

    Science.gov (United States)

    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.

  13. 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.

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

    Science.gov (United States)

    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

  15. 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.

  16. 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

  17. 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

  18. 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.

  19. Active site mutations change the cleavage specificity of neprilysin.

    Directory of Open Access Journals (Sweden)

    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.

  20. 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...

  1. 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.

  2. 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...

  3. 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.

  4. 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.

  5. 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

  6. 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.

  7. 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.

  8. 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

  9. 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.)

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

    Directory of Open Access Journals (Sweden)

    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.

  11. 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

  12. 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.

  13. 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.

  14. 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.

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

    Directory of Open Access Journals (Sweden)

    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.

  16. 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.

  17. 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

  18. Diaphanous gene mutation affects spiral cleavage and chirality in snails

    Science.gov (United States)

    Kuroda, Reiko; Fujikura, Kohei; Abe, Masanori; Hosoiri, Yuji; Asakawa, Shuichi; Shimizu, Miho; Umeda, Shin; Ichikawa, Futaba; Takahashi, Hiromi

    2016-01-01

    L-R (left and right) symmetry breaking during embryogenesis and the establishment of asymmetric body plan are key issues in developmental biology, but the onset including the handedness-determining gene locus still remains unknown. Using pure dextral (DD) and sinistral (dd) strains of the pond snail Lymnaea stagnalis as well as its F2 through to F10 backcrossed lines, the single handedness-determining-gene locus was mapped by genetic linkage analysis, BAC cloning and chromosome walking. We have identified the actin-related diaphanous gene Lsdia1 as the strongest candidate. Although the cDNA and derived amino acid sequences of the tandemly duplicated Lsdia1 and Lsdia2 genes are very similar, we could discriminate the two genes/proteins in our molecular biology experiments. The Lsdia1 gene of the sinistral strain carries a frameshift mutation that abrogates full-length LsDia1 protein expression. In the dextral strain, it is already translated prior to oviposition. Expression of Lsdia1 (only in the dextral strain) and Lsdia2 (in both chirality) decreases after the 1-cell stage, with no asymmetric localization throughout. The evolutionary relationships among body handedness, SD/SI (spiral deformation/spindle inclination) at the third cleavage, and expression of diaphanous proteins are discussed in comparison with three other pond snails (L. peregra, Physa acuta and Indoplanorbis exustus). PMID:27708420

  19. Sox11 Reduces Caspase-6 Cleavage and Activity.

    Directory of Open Access Journals (Sweden)

    Elaine Waldron-Roby

    Full Text Available The apoptotic cascade is an orchestrated event, whose final stages are mediated by effector caspases. Regulatory binding proteins have been identified for caspases such as caspase-3, -7, -8, and -9. Many of these proteins belong to the inhibitor of apoptosis (IAP family. By contrast, caspase-6 is not believed to be influenced by IAPs, and little is known about its regulation. We therefore performed a yeast-two-hybrid screen using a constitutively inactive form of caspase-6 for bait in order to identify novel regulators of caspase-6 activity. Sox11 was identified as a potential caspase-6 interacting protein. Sox11 was capable of dramatically reducing caspase-6 activity, as well as preventing caspase-6 self- cleavage. Several regions, including amino acids 117-214 and 362-395 within sox11 as well as a nuclear localization signal (NLS all contributed to the reduction in caspase-6 activity. Furthermore, sox11 was also capable of decreasing other effector caspase activity but not initiator caspases -8 and -9. The ability of sox11 to reduce effector caspase activity was also reflected in its capacity to reduce cell death following toxic insult. Interestingly, other sox proteins also had the ability to reduce caspase-6 activity but to a lesser extent than sox11.

  20. 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.

  1. 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

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

    Science.gov (United States)

    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.

  3. 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.

  4. 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.

  5. 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...

  6. 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)

  7. Dynamic propagation and cleavage crack arrest in bainitic steel

    International Nuclear Information System (INIS)

    Hajjaj, M.

    2006-06-01

    In complement of the studies of harmfulness of defects, generally realized in term of initiation, the concept of crack arrest could be used as complementary analyses to the studies of safety. The stop occurs when the stress intensity factor becomes lower than crack arrest toughness (KIa) calculated in elasto-statics (KI ≤ KIa). The aim of this thesis is to understand and predict the stop of a crack propagating at high speed in a 18MND5 steel used in the pressure water reactor (PWR). The test chosen to study crack arrest is the disc thermal shock test. The observations under the scanning electron microscope of the fracture surface showed that the crack arrest always occurs in cleavage mode and that the critical microstructural entity with respect to the propagation and crack arrest corresponds to at least the size of the prior austenitic grain. The numerical analyses in elasto-statics confirm the conservatism of the codified curve of the RCC-M with respect to the values of KIa. The dynamic numerical analyses show that the deceleration of the crack measured at the end of the propagation is related to the global dynamic of the structure (vibrations). The transferability to components of crack arrest toughness obtained from tests analysed in static is thus not assured. The disc thermal shock tests were also modelled by considering a criterion of propagation and arrest of the type 'RKR' characterized by a critical stress sc which depends on the temperature. The results obtained account well for the crack jump measured in experiments as well as the shape of the crack arrest front. (author)

  8. 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.

  9. 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

  10. 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...

  11. 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-...

  12. 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.

  13. 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.

  14. Social economy partnerships and the public/private cleavages

    Directory of Open Access Journals (Sweden)

    Joxerramon Bengoetxea

    2012-06-01

    Full Text Available Public/Private Partnerships can be seen as one particular topos where the divide between the public domain, all levels of the Public Administration and the private initiative and private property is turned into a joint venture rather than a confrontation or a cleavage. Some of the possible combinations of public and private and where public/private partnerships might fit are displayed analytically. The importance of political theory or ideology in conceiving the relationships between ‘public’ and ‘private’, and the conceptions of a market economy as opposed to a social market economy cannot be exaggerated enough, but equally important are the legal or regulatory framework and the underlying dominant legal culture and legal principles, and of course the economic and financial situation. Public/private partnerships thrive in some conditions, but seem to wane in others, and the current predicament is not favourable, taking into account that only the regulatory framework is supportive of these ventures. Los partenariados público-privados se pueden entender como un espacio particular, en el que el sector público, todos los niveles de la administración pública, y la iniciativa privada y la propiedad privada, abordan una empresa conjunta, en lugar mantener posturas contrapuestas. Se muestran algunas de las posibles combinaciones del sector público y privado, en las que tendrían cabida los partenariados público/privados. Es patente la importancia de la teoría o la ideología política para entender las relaciones entre lo público y lo privado, y las concepciones de una economía de mercado frente a una economía social, pero tampoco se puede negar la importancia del marco legal o reglamentario y la cultura jurídica dominante subyacente, y los principios jurídicos, sin olvidar la situación económica y financiera. Los partenariados público-privados prosperan en algunas condiciones, pero no lo hacen siempre, y la situación econ

  15. 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

  16. 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.

  17. 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.

  18. 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

  19. The mechanism and kinetics of the electrochemical cleavage of azo bond of 2-hydroxy-5-sulfophenyl-azo-benzoic acids

    International Nuclear Information System (INIS)

    Mandic, Zoran; Nigovic, Biljana; Simunic, Branimir

    2004-01-01

    The electrochemical reduction of 2-hydroxy-5-[(4-sulfophenyl)azo]benzoic acid, 2-hydroxy-5-[(3-sulfophenyl)azo]benzoic acid, 2-hydroxy-5-[(2-sulfophenyl)azo]benzoic acid and 2-hydroxy-5-azo-benzoic acid has been carried out in aqueous solutions at glassy carbon electrode using cyclic voltammetry and chronoamperometry. The position of sulfo substituent relative to azo bridge as well as pH of the solution have significant impact on the electrochemical behavior of these compounds. It has been proposed that these compounds are reduced predominantly as hydrazone tautomers resulting in corresponding hydrazo compounds. The overall electrochemical reduction follows DISP2 mechanism, ultimately leading to the 5-amino salicylic acid and sulfanilic acid. The rate determining step is the homogenous redox reaction between intermediate hydrazo compound and 5-amino salicylic acid quinoneimine. The mechanism is proposed in which activated complex of 5-amino salicylic acid quinoneimine and intermediate hydrazo compound is formed with the simultaneous loss of one proton

  20. 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

  1. 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

  2. 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.

  3. 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.

  4. Dinitrogen binding and cleavage by multinuclear iron complexes.

    Science.gov (United States)

    McWilliams, Sean F; Holland, Patrick L

    2015-07-21

    The iron-molybdenum cofactor of nitrogenase has unprecedented coordination chemistry, including a high-spin iron cluster called the iron-molybdenum cofactor (FeMoco). Thus, understanding the mechanism of nitrogenase challenges coordination chemists to understand the fundamental N2 chemistry of high-spin iron sites. This Account summarizes a series of studies in which we have synthesized a number of new compounds with multiple iron atoms, characterized them using crystallography and spectroscopy, and studied their reactions in detail. These studies show that formally iron(I) and iron(0) complexes with three- and four-coordinate metal atoms have the ability to weaken and break the triple bond of N2. These reactions occur at or below room temperature, indicating that they are kinetically facile. This in turn implies that iron sites in the FeMoco are chemically reasonable locations for N2 binding and reduction. The careful evaluation of these compounds and their reaction pathways has taught important lessons about what characteristics make iron more effective for N2 activation. Cooperation of two iron atoms can lengthen and weaken the N-N bond, while three working together enables iron atoms to completely cleave the N-N bond to nitrides. Alkali metals (typically introduced into the reaction as part of the reducing agent) are thermodynamically useful because the alkali metal cations stabilize highly reduced complexes, pull electron density into the N2 unit, and make reduced nitride products more stable. Alkali metals can also play a kinetic role, because cation-π interactions with the supporting ligands can hold iron atoms near enough to one another to facilitate the cooperation of multiple iron atoms. Many of these principles may also be relevant to the iron-catalyzed Haber-Bosch process, at which collections of iron atoms (often promoted by the addition of alkali metals) break the N-N bond of N2. The results of these studies teach more general lessons as well. They

  5. 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

  6. Protein cleavage strategies for an improved analysis of the membrane proteome

    Directory of Open Access Journals (Sweden)

    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.

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

    Science.gov (United States)

    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

  8. 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

  9. 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.

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

    Directory of Open Access Journals (Sweden)

    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

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

    Directory of Open Access Journals (Sweden)

    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.

  12. 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

  13. 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...

  14. 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.

  15. 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.

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

    Science.gov (United States)

    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

  17. 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

  18. 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

  19. 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.

  20. 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.

  1. 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.

  2. 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.

  3. 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.)

  4. 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)

  5. 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

  6. 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...

  7. 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

  8. [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.

  9. 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

  10. Irreversible processes kinetic theory

    CERN Document Server

    Brush, Stephen G

    2013-01-01

    Kinetic Theory, Volume 2: Irreversible Processes deals with the kinetic theory of gases and the irreversible processes they undergo. It includes the two papers by James Clerk Maxwell and Ludwig Boltzmann in which the basic equations for transport processes in gases are formulated, together with the first derivation of Boltzmann's ""H-theorem"" and a discussion of this theorem, along with the problem of irreversibility.Comprised of 10 chapters, this volume begins with an introduction to the fundamental nature of heat and of gases, along with Boltzmann's work on the kinetic theory of gases and s

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

    Science.gov (United States)

    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.

  12. SHORT COMMUNICATION CATALYTIC KINETIC ...

    African Journals Online (AJOL)

    IV) catalyzes the discoloring reaction of DBS-arsenazo oxidized by potassium bromate, a new catalytic kinetic spectrophotometric method for the determination of trace titanium (IV) was developed. The linear range of the determination of ...

  13. 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.

  14. 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.

  15. 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.

  16. 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.

  17. 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...

  18. 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-.

  19. 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.

  20. 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)

  1. 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.

  2. 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...

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

    NARCIS (Netherlands)

    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

  4. 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...

  5. 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...

  6. 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

  7. 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

  8. 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.

  9. 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

  10. 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)

  11. 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

  12. 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

  13. 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.

  14. 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...

  15. 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.

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

    Directory of Open Access Journals (Sweden)

    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.

  17. 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

  18. 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.

  19. 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

  20. 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.

  1. 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.

  2. 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

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

    Directory of Open Access Journals (Sweden)

    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

  4. 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.

  5. 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.

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

    Science.gov (United States)

    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.

  7. 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

  8. 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.

  9. Relativistic Chiral Kinetic Theory

    International Nuclear Information System (INIS)

    Stephanov, Mikhail

    2016-01-01

    This very brief review of the recent progress in chiral kinetic theory is based on the results of Refs. [J.-Y. Chen, D. T. Son, M. A. Stephanov, H.-U. Yee, Y. Yin, Lorentz Invariance in Chiral Kinetic Theory, Phys. Rev. Lett. 113 (18) (2014) 182302. doi: (10.1103/PhysRevLett.113.182302); J.-Y. Chen, D. T. Son, M. A. Stephanov, Collisions in Chiral Kinetic Theory, Phys. Rev. Lett. 115 (2) (2015) 021601. doi: (10.1103/PhysRevLett.115.021601); M. A. Stephanov, H.-U. Yee, The no-drag frame for anomalous chiral fluid, Phys. Rev. Lett. 116 (12) (2016) 122302. doi: (10.1103/PhysRevLett.116.122302)].

  10. Relativistic Chiral Kinetic Theory

    Energy Technology Data Exchange (ETDEWEB)

    Stephanov, Mikhail

    2016-12-15

    This very brief review of the recent progress in chiral kinetic theory is based on the results of Refs. [J.-Y. Chen, D. T. Son, M. A. Stephanov, H.-U. Yee, Y. Yin, Lorentz Invariance in Chiral Kinetic Theory, Phys. Rev. Lett. 113 (18) (2014) 182302. doi: (10.1103/PhysRevLett.113.182302); J.-Y. Chen, D. T. Son, M. A. Stephanov, Collisions in Chiral Kinetic Theory, Phys. Rev. Lett. 115 (2) (2015) 021601. doi: (10.1103/PhysRevLett.115.021601); M. A. Stephanov, H.-U. Yee, The no-drag frame for anomalous chiral fluid, Phys. Rev. Lett. 116 (12) (2016) 122302. doi: (10.1103/PhysRevLett.116.122302)].

  11. Erbium hydride decomposition kinetics.

    Energy Technology Data Exchange (ETDEWEB)

    Ferrizz, Robert Matthew

    2006-11-01

    Thermal desorption spectroscopy (TDS) is used to study the decomposition kinetics of erbium hydride thin films. The TDS results presented in this report are analyzed quantitatively using Redhead's method to yield kinetic parameters (E{sub A} {approx} 54.2 kcal/mol), which are then utilized to predict hydrogen outgassing in vacuum for a variety of thermal treatments. Interestingly, it was found that the activation energy for desorption can vary by more than 7 kcal/mol (0.30 eV) for seemingly similar samples. In addition, small amounts of less-stable hydrogen were observed for all erbium dihydride films. A detailed explanation of several approaches for analyzing thermal desorption spectra to obtain kinetic information is included as an appendix.

  12. Relativistic Kinetic Theory

    Science.gov (United States)

    Vereshchagin, Gregory V.; Aksenov, Alexey G.

    2017-02-01

    Preface; Acknowledgements; Acronyms and definitions; Introduction; Part I. Theoretical Foundations: 1. Basic concepts; 2. Kinetic equation; 3. Averaging; 4. Conservation laws and equilibrium; 5. Relativistic BBGKY hierarchy; 6. Basic parameters in gases and plasmas; Part II. Numerical Methods: 7. The basics of computational physics; 8. Direct integration of Boltzmann equations; 9. Multidimensional hydrodynamics; Part III. Applications: 10. Wave dispersion in relativistic plasma; 11. Thermalization in relativistic plasma; 12. Kinetics of particles in strong fields; 13. Compton scattering in astrophysics and cosmology; 14. Self-gravitating systems; 15. Neutrinos, gravitational collapse and supernovae; Appendices; Bibliography; Index.

  13. Quantum kinetic theory

    CERN Document Server

    Bonitz, Michael

    2016-01-01

    This book presents quantum kinetic theory in a comprehensive way. The focus is on density operator methods and on non-equilibrium Green functions. The theory allows to rigorously treat nonequilibrium dynamics in quantum many-body systems. Of particular interest are ultrafast processes in plasmas, condensed matter and trapped atoms that are stimulated by rapidly developing experiments with short pulse lasers and free electron lasers. To describe these experiments theoretically, the most powerful approach is given by non-Markovian quantum kinetic equations that are discussed in detail, including computational aspects.

  14. 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)

  15. 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

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

    Directory of Open Access Journals (Sweden)

    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.

  17. 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

  18. 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...

  19. 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.

  20. 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

  1. 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.

  2. Oxidative desulfurization: kinetic modelling.

    Science.gov (United States)

    Dhir, S; Uppaluri, R; Purkait, M K

    2009-01-30

    Increasing environmental legislations coupled with enhanced production of petroleum products demand, the deployment of novel technologies to remove organic sulfur efficiently. This work represents the kinetic modeling of ODS using H(2)O(2) over tungsten-containing layered double hydroxide (LDH) using the experimental data provided by Hulea et al. [V. Hulea, A.L. Maciuca, F. Fajula, E. Dumitriu, Catalytic oxidation of thiophenes and thioethers with hydrogen peroxide in the presence of W-containing layered double hydroxides, Appl. Catal. A: Gen. 313 (2) (2006) 200-207]. The kinetic modeling approach in this work initially targets the scope of the generation of a superstructure of micro-kinetic reaction schemes and models assuming Langmuir-Hinshelwood (LH) and Eley-Rideal (ER) mechanisms. Subsequently, the screening and selection of above models is initially based on profile-based elimination of incompetent schemes followed by non-linear regression search performed using the Levenberg-Marquardt algorithm (LMA) for the chosen models. The above analysis inferred that Eley-Rideal mechanism describes the kinetic behavior of ODS process using tungsten-containing LDH, with adsorption of reactant and intermediate product only taking place on the catalyst surface. Finally, an economic index is presented that scopes the economic aspects of the novel catalytic technology with the parameters obtained during regression analysis to conclude that the cost factor for the catalyst is 0.0062-0.04759 US $ per barrel.

  3. Oxidative desulfurization: Kinetic modelling

    International Nuclear Information System (INIS)

    Dhir, S.; Uppaluri, R.; Purkait, M.K.

    2009-01-01

    Increasing environmental legislations coupled with enhanced production of petroleum products demand, the deployment of novel technologies to remove organic sulfur efficiently. This work represents the kinetic modeling of ODS using H 2 O 2 over tungsten-containing layered double hydroxide (LDH) using the experimental data provided by Hulea et al. [V. Hulea, A.L. Maciuca, F. Fajula, E. Dumitriu, Catalytic oxidation of thiophenes and thioethers with hydrogen peroxide in the presence of W-containing layered double hydroxides, Appl. Catal. A: Gen. 313 (2) (2006) 200-207]. The kinetic modeling approach in this work initially targets the scope of the generation of a superstructure of micro-kinetic reaction schemes and models assuming Langmuir-Hinshelwood (LH) and Eley-Rideal (ER) mechanisms. Subsequently, the screening and selection of above models is initially based on profile-based elimination of incompetent schemes followed by non-linear regression search performed using the Levenberg-Marquardt algorithm (LMA) for the chosen models. The above analysis inferred that Eley-Rideal mechanism describes the kinetic behavior of ODS process using tungsten-containing LDH, with adsorption of reactant and intermediate product only taking place on the catalyst surface. Finally, an economic index is presented that scopes the economic aspects of the novel catalytic technology with the parameters obtained during regression analysis to conclude that the cost factor for the catalyst is 0.0062-0.04759 US $ per barrel

  4. Modeling chemical kinetics graphically

    NARCIS (Netherlands)

    Heck, A.

    2012-01-01

    In literature on chemistry education it has often been suggested that students, at high school level and beyond, can benefit in their studies of chemical kinetics from computer supported activities. Use of system dynamics modeling software is one of the suggested quantitative approaches that could

  5. CATALYTIC KINETIC SPECTROPHOTOMETRIC DETERMINATION ...

    African Journals Online (AJOL)

    Preferred Customer

    acetylchlorophosphonazo(CPApA) by hydrogen peroxide in 0.10 M phosphoric acid. A novel catalytic kinetic-spectrophotometric method is proposed for the determination of copper based on this principle. Copper(II) can be determined spectrophotometrically ...

  6. Kinetic energy budget details

    Indian Academy of Sciences (India)

    Abstract. This paper presents the detailed turbulent kinetic energy budget and higher order statistics of flow behind a surface-mounted rib with and without superimposed acoustic excitation. Pattern recognition technique is used to determine the large-scale structure magnitude. It is observed that most of the turbulence ...

  7. Point kinetics modeling

    International Nuclear Information System (INIS)

    Kimpland, R.H.

    1996-01-01

    A normalized form of the point kinetics equations, a prompt jump approximation, and the Nordheim-Fuchs model are used to model nuclear systems. Reactivity feedback mechanisms considered include volumetric expansion, thermal neutron temperature effect, Doppler effect and void formation. A sample problem of an excursion occurring in a plutonium solution accidentally formed in a glovebox is presented

  8. 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.

  9. 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.

  10. 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.

  11. 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

  12. 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.

  13. 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

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

    Science.gov (United States)

    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

  15. 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

  16. 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.

  17. 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.

  18. 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

  19. 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.

  20. 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

  1. 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

  2. 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.

  3. 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

  4. Analysis of Network Topologies Underlying Ethylene Growth Response Kinetics

    Directory of Open Access Journals (Sweden)

    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

  5. 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.

  6. 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.

  7. 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.

  8. 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

  9. 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.

  10. 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

  11. 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

  12. 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

  13. 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.

  14. 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.

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

    Directory of Open Access Journals (Sweden)

    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.

  16. LLNL Chemical Kinetics Modeling Group

    Energy Technology Data Exchange (ETDEWEB)

    Pitz, W J; Westbrook, C K; Mehl, M; Herbinet, O; Curran, H J; Silke, E J

    2008-09-24

    The LLNL chemical kinetics modeling group has been responsible for much progress in the development of chemical kinetic models for practical fuels. The group began its work in the early 1970s, developing chemical kinetic models for methane, ethane, ethanol and halogenated inhibitors. Most recently, it has been developing chemical kinetic models for large n-alkanes, cycloalkanes, hexenes, and large methyl esters. These component models are needed to represent gasoline, diesel, jet, and oil-sand-derived fuels.

  17. 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.

  18. 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

  19. 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.

  20. 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.

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

    Institute of Scientific and Technical Information of China (English)

    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.

  2. 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...

  3. 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...

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

    Directory of Open Access Journals (Sweden)

    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.

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

    Science.gov (United States)

    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.

  6. Kinetic energy absorbing pad

    International Nuclear Information System (INIS)

    Bricmont, R.J.; Hamilton, P.A.; Ming Long Ting, R.

    1981-01-01

    Reactors, fuel processing plants etc incorporate pipes and conduits for fluids under high pressure. Fractures, particularly adjacent to conduit elbows, produce a jet of liquid which whips the broken conduit at an extremely high velocity. An enormous impact load would be applied to any stationary object in the conduit's path. The design of cellular, corrugated metal impact pads to absorb the kinetic energy of the high velocity conduits is given. (U.K.)

  7. 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

  8. 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.

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

    Science.gov (United States)

    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.

  10. Calcite Dissolution Kinetics

    Science.gov (United States)

    Berelson, W.; Subhas, A.; Dong, S.; Naviaux, J.; Adkins, J. F.

    2016-12-01

    A geological buffer for high atmospheric CO2 concentrations is neutralization via reaction with CaCO3. We have been studying the dissolution kinetics of carbonate minerals using labeled 13C calcite and Picarro-based measurements of 13C enrichments in solution DIC. This methodology has greatly facilitated our investigation of dissolution kinetics as a function of water carbonate chemistry, temperature and pressure. One can adjust the saturation state Omega by changing the ion activity product (e.g. adjusting carbonate ion concentration), or by changing the solubility product (e.g. adjusting temperature or pressure). The canonical formulation of dissolution rate vs. omega has been refined (Subhas et al. 2015) and shows distinct non-linear behavior near equilibrium and rates in sea water of 1-3 e-6 g/cm2day at omega = 0.8. Carbonic anhydrase (CA), an enzyme that catalyzes the hydration of dissolved CO2 to carbonic acid, was shown (in concentrations 500x. This result points to the importance of carbonic acid in enhancing dissolution at low degrees of undersaturation. CA activity and abundance in nature must be considered regarding the role it plays in catalyzing dissolution. We also have been investigating the role of temperature on dissolution kinetics. An increase of 16C yields an order of magnitude increase in dissolution rate. Temperature (and P) also change Omega critical, the saturation state where dissolution rates change substantially. Increasing pressure (achieved in a pressure reaction chamber we built) also shifts Omega critical closer to equilibrium and small pressure increases have large impact on dissolution kinetics. Dissolution rates are enhanced by an order of magnitude for a change in pressure of 1500 psi relative to the dissolution rate achieved by water chemistry effects alone for an omega of 0.8. We've shown that the thermodynamic determination of saturation state does not adequately describe the kinetics of dissolution. The interplay of mineral

  11. 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.

  12. 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.

  13. 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

  14. 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.

  15. 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.

  16. 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.

  17. 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.

  18. 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.

  19. Cleavage and formation of molecular dinitrogen in a single system assisted by molybdenum complexes bearing ferrocenyldiphosphine.

    Science.gov (United States)

    Miyazaki, Takamasa; Tanaka, Hiromasa; Tanabe, Yoshiaki; Yuki, Masahiro; Nakajima, Kazunari; Yoshizawa, Kazunari; Nishibayashi, Yoshiaki

    2014-10-20

    The N≡N bond of molecular dinitrogen bridging two molybdenum atoms in the pentamethylcyclopentadienyl molybdenum complexes that bear ferrocenyldiphosphine as an auxiliary ligand is homolytically cleaved under visible light irradiation at room temperature to afford two molar molybdenum nitride complexes. Conversely, the bridging molecular dinitrogen is reformed by the oxidation of the molybdenum nitride complex at room temperature. This result provides a successful example of the cleavage and formation of molecular dinitrogen induced by a pair of two different external stimuli using a single system assisted by molybdenum complexes bearing ferrocenyldiphosphine under ambient conditions. © 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  20. 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...

  1. 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.)

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

    Science.gov (United States)

    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

  3. Temperature dependence of the fracture toughness and the cleavage fracture strength of a pressure vessel steel

    International Nuclear Information System (INIS)

    Kotilainen, H.

    1980-01-01

    A new model for the temperature dependence of the fracture toughness has been sought. It is based on the yielding processes at the crack tip, which are thought to be competitive with fracture. Using this method a good correlation between measured and calculated values of fracture toughness has been found for a Cr-Mo-V pressure vessel steel as well as for A533B. It has been thought that the application of this method can reduce the number of surveillance specimens in nuclear reactors. A method for the determination of the cleavage fracture strength has been proposed. 28 refs

  4. Structure of the Cpf1 endonuclease R-loop complex after target DNA cleavage

    DEFF Research Database (Denmark)

    Stella, Stefano; Alcón, Pablo; Montoya, Guillermo

    2017-01-01

    involved in DNA unwinding to form a CRISPR RNA (crRNA)-DNA hybrid and a displaced DNA strand. The protospacer adjacent motif (PAM) is recognized by the PAM-interacting domain. The loop-lysine helix-loop motif in this domain contains three conserved lysine residues that are inserted in a dentate manner...... and the crRNA-DNA hybrid, avoiding DNA re-annealing. Mutations in key residues reveal a mechanism linking the PAM and DNA nuclease sites. Analysis of the Cpf1 structures proposes a singular working model of RNA-guided DNA cleavage, suggesting new avenues for redesign of Cpf1....

  5. Kinetics of tetrataenite disordering

    International Nuclear Information System (INIS)

    Dos Santos, E.; Gattacceca, J.; Rochette, P.; Fillion, G.; Scorzelli, R.B.

    2015-01-01

    Tetrataenite is a chemically ordered L1 0 -type Fe 50 Ni 50 alloy detected for the first time in 1977 by 57 Fe Mössbauer spectroscopy studies in iron meteorites. The thermal history of meteorites, in particular short thermal events like those associated to hypervelocity impacts, can be constrained by tracing the presence of tetrataenite or its disordering into taenite. The knowledge of the disordering kinetics of tetrataenite, that is associated with changes in its magnetic properties, is still very fragmentary so that the time–temperature history of these meteorites cannot be constrained in details. Furthermore, knowledge of disordering kinetics is important due to potential technological application of tetrataenite as a rare-earth free strong magnet. Thus, this work provides the first time–temperature data for disordering reaction of tetrataenite. We have shown that disordering is not an instantaneous process but is a kinetic limited reaction. It was shown that disordering may take place at any temperature above the order–disorder transition for L 10 superstructure phase (∼320 °C) when the appropriate time-scale is considered. This result means that the apparent Curie point for tetrataenite is not an absolute property in the sense that any estimate of this parameter should be referred to a given time-scale. - Highlights: • The first time–temperature data for tetrataenite disordering reaction is provided. • Previous works does not give a complete picture of tetrataenite disordering. • Apparent Curie temperature of tetrataenite should be referred to a time-scale. • Tetrataenite can be used as a probe to detect thermal/shock events recorded in meteorites

  6. 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

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

    Directory of Open Access Journals (Sweden)

    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

  8. Quantum kinetic Ising models

    International Nuclear Information System (INIS)

    Augusiak, R; Cucchietti, F M; Lewenstein, M; Haake, F

    2010-01-01

    In this paper, we introduce a quantum generalization of classical kinetic Ising models (KIM), described by a certain class of quantum many-body master equations. Similarly to KIMs with detailed balance that are equivalent to certain Hamiltonian systems, our models reduce to a set of Hamiltonian systems determining the dynamics of the elements of the many-body density matrix. The ground states of these Hamiltonians are well described by the matrix product, or pair entangled projected states. We discuss critical properties of such Hamiltonians, as well as entanglement properties of their low-energy states.

  9. 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.

  10. 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.

  11. 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.

  12. 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.

  13. Kinetics of coal pyrolysis

    Energy Technology Data Exchange (ETDEWEB)

    Seery, D.J.; Freihaut, J.D.; Proscia, W.M. (United Technologies Research Center, East Hartford, CT (USA)); Howard, J.B.; Peters, W.; Hsu, J.; Hajaligol, M.; Sarofim, A. (Massachusetts Inst. of Tech., Cambridge, MA (USA)); Jenkins, R.; Mallin, J.; Espindola-Merin, B. (Pennsylvania State Univ., University Park, PA (USA)); Essenhigh, R.; Misra, M.K. (Ohio State Univ., Columbus, OH (USA))

    1989-07-01

    This report contains results of a coordinated, multi-laboratory investigation of coal devolatilization. Data is reported pertaining to the devolatilization for bituminous coals over three orders of magnitude in apparent heating rate (100 to 100,000 + {degree}C/sec), over two orders of magnitude in particle size (20 to 700 microns), final particle temperatures from 400 to 1600{degree}C, heat transfer modes ranging from convection to radiative, ambient pressure ranging from near vacuum to one atmosphere pressure. The heat transfer characteristics of the reactors are reported in detail. It is assumed the experimental results are to form the basis of a devolatilization data base. Empirical rate expressions are developed for each phase of devolatilization which, when coupled to an awareness of the heat transfer rate potential of a particular devolatilization reactor, indicate the kinetics emphasized by a particular system reactor plus coal sample. The analysis indicates the particular phase of devolatilization that will be emphasized by a particular reactor type and, thereby, the kinetic expressions appropriate to that devolatilization system. Engineering rate expressions are developed from the empirical rate expressions in the context of a fundamental understanding of coal devolatilization developed in the course of the investigation. 164 refs., 223 figs., 44 tabs.

  14. Repair kinetics in tissues

    International Nuclear Information System (INIS)

    Thames, H.D.

    1989-01-01

    Monoexponential repair kinetics is based on the assumption of a single, dose-independent rate of repair of sublethal injury in the target cells for tissue injury after exposure to ionizing radiation. Descriptions of the available data based on this assumption have proved fairly successful for both acutely responding (skin, lip mucosa, gut) and late-responding (lung, spinal cord) normal tissues. There are indications of biphasic exponential repair in both categories, however. Unfortunately, the data usually lack sufficient resolution to permit unambiguous determination of the repair rates. There are also indications that repair kinetics may depend on the size of the dose. The data are conflicting on this account, however, with suggestions of both faster and slower repair after larger doses. Indeed, experiments that have been explicitly designed to test this hypothesis show either no effect (gut, spinal cord), faster repair after higher doses (lung, kidney), or slower repair after higher doses (skin). Monoexponential repair appears to be a fairly accurate description that provides an approximation to a more complicated picture, the elucidation of whose details will, however, require very careful and extensive experimental study. (author). 30 refs.; 1 fig

  15. Diffusion Influenced Adsorption Kinetics.

    Science.gov (United States)

    Miura, Toshiaki; Seki, Kazuhiko

    2015-08-27

    When the kinetics of adsorption is influenced by the diffusive flow of solutes, the solute concentration at the surface is influenced by the surface coverage of solutes, which is given by the Langmuir-Hinshelwood adsorption equation. The diffusion equation with the boundary condition given by the Langmuir-Hinshelwood adsorption equation leads to the nonlinear integro-differential equation for the surface coverage. In this paper, we solved the nonlinear integro-differential equation using the Grünwald-Letnikov formula developed to solve fractional kinetics. Guided by the numerical results, analytical expressions for the upper and lower bounds of the exact numerical results were obtained. The upper and lower bounds were close to the exact numerical results in the diffusion- and reaction-controlled limits, respectively. We examined the validity of the two simple analytical expressions obtained in the diffusion-controlled limit. The results were generalized to include the effect of dispersive diffusion. We also investigated the effect of molecular rearrangement of anisotropic molecules on surface coverage.

  16. Chemical kinetics and reaction mechanism

    International Nuclear Information System (INIS)

    Jung, Ou Sik; Park, Youn Yeol

    1996-12-01

    This book is about chemical kinetics and reaction mechanism. It consists of eleven chapters, which deal with reaction and reaction speed on reaction mechanism, simple reaction by rate expression, reversible reaction and simultaneous reaction, successive reaction, complicated reaction mechanism, assumption for reaction mechanism, transition state theory, successive reaction and oscillating reaction, reaction by solution, research method high except kinetics on reaction mechanism, high reaction of kinetics like pulsed radiolysis.

  17. Chemical kinetics of gas reactions

    CERN Document Server

    Kondrat'Ev, V N

    2013-01-01

    Chemical Kinetics of Gas Reactions explores the advances in gas kinetics and thermal, photochemical, electrical discharge, and radiation chemical reactions. This book is composed of 10 chapters, and begins with the presentation of general kinetic rules for simple and complex chemical reactions. The next chapters deal with the experimental methods for evaluating chemical reaction mechanisms and some theories of elementary chemical processes. These topics are followed by discussions on certain class of chemical reactions, including unimolecular, bimolecular, and termolecular reactions. The rema

  18. 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.

  19. 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.

  20. Single-stranded DNA cleavage by divergent CRISPR-Cas9 enzymes

    Science.gov (United States)

    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

  1. 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.

  2. 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

  3. 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.

  4. 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.

  5. 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.

  6. 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.

  7. Live births after polar body biopsy and frozen-thawed cleavage stage embryo transfer: case report.

    Science.gov (United States)

    Guimarães, Fernando; Roque, Matheus; Valle, Marcello; Kostolias, Alessandra; Azevedo, Rodrigo A de; Martinhago, Ciro D; Sampaio, Marcos; Geber, Selmo

    2016-12-01

    Pre-implantation genetic diagnosis (PGD) or screening (PGS) technology, has emerged and developed in the past few years, benefiting couples as it allows the selection and transfer of healthy embryos during IVF treatments. These techniques can be performed in oocytes (polar-body biopsy) or embryos (blastomere or trophectoderm biopsy). In this case report, we describe the first two live births to be published in Brazil after a polar-body (PB) biopsy. In case 1, a 42-year-old was submitted to PB biopsy with PGS due to advanced maternal age and poor ovarian reserve. Five MII oocytes underwent first and second polar body biopsy and four cleavage embryos were cryopreserved. The PGS analysis resulted in two euploid embryos (next generation sequence). A frozen-thawed embryo transfer (FET) was performed after endometrial priming and a healthy baby was delivered after a cesarean section (37 weeks, female, 3390g, 47.5 cm). In case 2, a 40-year old patient with balanced translocation and poor ovarian response was submitted to PB biopsy. Two MII oocytes underwent first and second polar body biopsy and two embryos were cryopreserved in cleavage stage. The analysis resulted in one euploid embryo that was transferred after endometrial priming. A preterm healthy baby (34 weeks, female, 2100g, 40 cm) was delivered via cesarean section. In conclusion, although the blastocyst biopsy is the norm when performing PGS/PGD during IVF treatments, other alternatives (as PB biopsy) should be considered in some specific situations.

  8. 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

  9. 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 Caliphatic-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.

  10. 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.

  11. 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.

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

    Science.gov (United States)

    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.

  13. 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)

  14. 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.

  15. 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.

  16. 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.

  17. 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.

  18. 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.

  19. 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

  20. Oxidative ring cleavage of 2,3-dihydrophthalazine-1,4-dione in ...

    Indian Academy of Sciences (India)

    and non-aqueous solutions: Electrochemical and kinetic studies. D NEMATOLLAHIa,∗ ... as anti-convulsant,2 anti-microbial,3 .... of tetrabutylammonium perchlorate (0.1 M), in methanol, ethanol and acetic acid, at a glassy carbon electrode.

  1. 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.

  2. Adsorption analysis equilibria and kinetics

    CERN Document Server

    Do, Duong D

    1998-01-01

    This book covers topics of equilibria and kinetics of adsorption in porous media. Fundamental equilibria and kinetics are dealt with for homogeneous as well as heterogeneous particles. Five chapters of the book deal with equilibria and eight chapters deal with kinetics. Single component as well as multicomponent systems are discussed. In kinetics analysis, we deal with the various mass transport processes and their interactions inside a porous particle. Conventional approaches as well as the new approach using Maxwell-Stefan equations are presented. Various methods to measure diffusivity, such

  3. 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.

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

    Science.gov (United States)

    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.

  5. 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.

  6. 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.

  7. Kinetics of Social Contagion

    Science.gov (United States)

    Ruan, Zhongyuan; Iñiguez, Gerardo; Karsai, Márton; Kertész, János

    2015-11-01

    Diffusion of information, behavioral patterns or innovations follows diverse pathways depending on a number of conditions, including the structure of the underlying social network, the sensitivity to peer pressure and the influence of media. Here we study analytically and by simulations a general model that incorporates threshold mechanism capturing sensitivity to peer pressure, the effect of "immune" nodes who never adopt, and a perpetual flow of external information. While any constant, nonzero rate of dynamically introduced spontaneous adopters leads to global spreading, the kinetics by which the asymptotic state is approached shows rich behavior. In particular, we find that, as a function of the immune node density, there is a transition from fast to slow spreading governed by entirely different mechanisms. This transition happens below the percolation threshold of network fragmentation, and has its origin in the competition between cascading behavior induced by adopters and blocking due to immune nodes. This change is accompanied by a percolation transition of the induced clusters.

  8. The temperature hydration kinetics

    Directory of Open Access Journals (Sweden)

    Mircea Oroian

    2017-07-01

    Full Text Available The aim of this study is to evaluate the hydration kinetics of lentil seeds (Lens culinaris in water at different temperatures (25, 32.5, 40, 55, 70 and 80 °C for assessing the adequacy of models for describing the absorption phenomena during soaking. The diffusion coefficient values were calculated using Fick’s model for spherical and hemispherical geometries and the values were in the range of 10−6 m2/s. The experimental data were fitted to Peleg, Sigmoidal, Weibull and Exponential models. The models adequacy was determined using regression coefficients (R2, root mean square error (RMSE and reduced chi-square (χ2. The Peleg model is the suitable one for predicting the experimental data. Temperature had a positive and significant effect on the water absorption capacities and absorption was an endothermic process.

  9. 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.

  10. Kinetic theory of Jeans instability

    NARCIS (Netherlands)

    Trigger, S.A.; Ershkovic, A.I.; Heijst, van G.J.F.; Schram, P.P.J.M.

    2004-01-01

    Kinetic treatment of the Jeans gravitational instability, with collisions taken into account, is presented. The initial-value problem for the distribution function which obeys the kinetic equation, with the collision integral conserving the number of particles, is solved. Dispersion relation is

  11. 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

  12. 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...

  13. 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)

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

    Science.gov (United States)

    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.

  15. PLK1 regulation of PCNT cleavage ensures fidelity of centriole separation during mitotic exit.

    Science.gov (United States)

    Kim, Jaeyoun; Lee, Kwanwoo; Rhee, Kunsoo

    2015-12-09

    Centrioles are duplicated and segregated in close link to the cell cycle. During mitosis, daughter centrioles are disengaged and eventually separated from mother centrioles. New daughter centrioles may be generated only after centriole separation. Therefore, centriole separation is considered a licensing step for centriole duplication. It was previously known that separase specifically cleaves pericentrin (PCNT) during mitotic exit. Here we report that PCNT has to be phosphorylated by PLK1 to be a suitable substrate of separase. Phospho-resistant mutants of PCNT are not cleaved by separase and eventually inhibit centriole separation. Furthermore, phospho-mimetic PCNT mutants rescue centriole separation even in the presence of a PLK1 inhibitor. On the basis on these results, we propose that PLK1 phosphorylation is a priming step for separase-mediated cleavage of PCNT and eventually for centriole separation. PLK1 phosphorylation of PCNT provides an additional layer of regulatory mechanism to ensure the fidelity of centriole separation during mitotic exit.

  16. AAU-Specific RNA Cleavage Mediated by MazF Toxin Endoribonuclease Conserved in Nitrosomonas europaea

    Directory of Open Access Journals (Sweden)

    Tatsuki Miyamoto

    2016-06-01

    Full Text Available Nitrosomonas europaea carries numerous toxin-antitoxin systems. However, despite the abundant representation in its chromosome, studies have not surveyed the underlying molecular functions in detail, and their biological roles remain enigmatic. In the present study, we found that a chromosomally-encoded MazF family member, predicted at the locus NE1181, is a functional toxin endoribonuclease, and constitutes a toxin-antitoxin system, together with its cognate antitoxin, MazE. Massive parallel sequencing provided strong evidence that this toxin endoribonuclease exhibits RNA cleavage activity, primarily against the AAU triplet. This sequence-specificity was supported by the results of fluorometric assays. Our results indicate that N. europaea alters the translation profile and regulates its growth using the MazF family of endoribonuclease under certain stressful conditions.

  17. Synthesis, characterization, DNA binding and cleavage studies of mixed-ligand copper (II complexes

    Directory of Open Access Journals (Sweden)

    M. Sunita

    2017-05-01

    Full Text Available New two copper complexes of type [Cu(Bzimpy(LH2O]SO4 (where L = 2,2′ bipyridine (bpy, and ethylene diamine (en, Bzimpy = 2,6-bis(benzimidazole-2ylpyridine have been synthesized and characterized by elemental analyses, molar conductance measurements, magnetic susceptibility measurements, mass, IR, electronic and EPR spectral studies. Based on elemental and spectral studies six coordinated geometries were assigned to the two complexes. DNA-binding properties of these metal complexes were investigated using absorption spectroscopy, fluorescence spectroscopy, viscosity measurements and thermal denaturation methods. Experimental studies suggest that the complexes bind to DNA through intercalation. These complexes also promote the cleavage of plasmid pBR322, in the presence of H2O2.

  18. A beta2-microglobulin cleavage variant fibrillates at near-physiological pH

    DEFF Research Database (Denmark)

    Corlin, Dorthe B; Johnsen, Christina K; Nissen, Mogens H

    2009-01-01

    Beta2-microglobulin (beta2m) deposits as amyloid in dialysis-related amyloidosis (DRA), predominantly in joints. The molecular mechanisms underlying the amyloidogenicity of beta2m are still largely unknown. In vitro, acidic conditions, pH ... several days. Here, we show that amyloid fibrils are generated in less than an hour when a cleavage variant of beta2m--found in the circulation of many dialysis patients--is exposed to pH levels (pH 6.6) occurring in joints during inflammation. Aggregation and fibrillation, including seeding effects...... with intact, native beta2m were studied by Thioflavin T fluorescence spectroscopy, turbidimetry, capillary electrophoresis, and electron microscopy. We conclude that a biologically relevant variant of beta2m is amyloidogenic at slightly acidic pH. Also, only a very small amount of preformed fibrils...

  19. Graves' Disease Mechanisms: The Role of Stimulating, Blocking, and Cleavage Region TSH Receptor Antibodies

    Science.gov (United States)

    Morshed, S. A.; Davies, T. F.

    2016-01-01

    The immunologic processes involved in Graves' disease (GD) have one unique characteristic – the autoantibodies to the TSH receptor (TSHR) – which have both linear and conformational epitopes. Three types of TSHR antibodies (stimulating, blocking, and cleavage) with different functional capabilities have been described in GD patients, which induce different signaling effects varying from thyroid cell proliferation to thyroid cell death. The establishment of animal models of GD by TSHR antibody transfer or by immunization with TSHR antigen has confirmed its pathogenic role and, therefore, GD is the result of a breakdown in TSHR tolerance. Here we review some of the characteristics of TSHR antibodies with a special emphasis on new developments in our understanding of what were previously called “neutral” antibodies and which we now characterize as autoantibodies to the “cleavage” region of the TSHR ectodomain. PMID:26361259

  20. Monozygotic Triplets and Dizygotic Twins following Transfer of Three Poor-Quality Cleavage Stage Embryos

    Directory of Open Access Journals (Sweden)

    Reshef Tal

    2012-01-01

    Full Text Available Background. Assisted reproductive technology has been linked to the increased incidence of monozygotic twinning. It is of clinical importance due to the increased risk of complications in multiple pregnancies in general and in monozygotic twins in particular. Case. A 29-year-old female, nulligravida underwent her first IVF cycle. Three poor-quality cleavage stage embryos were transferred resulting in monochorionic triamniotic triplets and dichorionic diamniotic twins. Selective embryo reduction was performed at 12 weeks leaving dichorionic twins. The patient underwent emergency cesarean section due to preterm labor and nonreassuring fetal heart tracing at 30 weeks of gestation. Conclusion. Our case emphasizes that even embryos with significant morphological abnormalities should be considered viable and the possibility of simultaneous spontaneous embryo splitting must be factored into determining number of embryos to transfer.

  1. Effects of caffeine on cleavage delay of sea urchin eggs induced by ethidium bromide or puromycin

    International Nuclear Information System (INIS)

    Kimura, Hiroshi

    1975-01-01

    The mitotic delay was induced when sea urchin eggs were treated with either ethidium bromide or puromycin, as observed with eggs fertilized with x- or UV-irradiated sperm. Treatment of these eggs with caffeine during the period of early prophase block resulted in the reduction of the mitotic delay. Protein synthesis of these eggs was not affected by x-irradiation but inhibited by ethidium bromide or puromycin. Caffeine was almost ineffective in changing the protein synthesis of eggs inseminated with x-irradiated sperm or treated with ethidium bromide. These facts mean that additive synthesis of protein is not required for the reduction by caffeine of the mitotic delay. Some role of protein synthesis in the reduction by caffeine of the cleavage delay is not excluded for puromycin treated eggs, since caffeine counteracted the inhibitory effect of puromycin on protein synthesis. (author)

  2. Oxidative C-C bond cleavage of 1,2-diols by silver(II)

    International Nuclear Information System (INIS)

    Kumar, A.

    1981-01-01

    Oxidation of ethylene glycol and related compounds by Ag(II) has been investigated. Complexation of these substrates by Ag(II) precedes their oxidation. Oxidation occurs through electron transfer from an OH group to the Ag(II) within the complex resulting in the formation of alkoxyl-type radicals. The radicals thus formed undergo β-scission to give cleavage products. For ethylene glycol a complexation rate 1.3 x 10 6 M -1 s -1 and oxidation rate approx. 3 x 10 3 s -1 were observed. A general trend for the type of the substrates which would undergo C-C bond scission by Ag(II) is discussed

  3. Synthesis, characterization and DNA cleavage activity of nickel(II adducts with aromatic heterocyclic bases

    Directory of Open Access Journals (Sweden)

    G. H. PHILIP

    2010-01-01

    Full Text Available Mixed ligand complexes of nickel(II with 2,4-dihydroxyaceto-phenone oxime (DAPO and 2,4-dihydroxybenzophenone oxime (DBPO as primary ligands, and pyridine (Py and imidazole (Im as secondary ligands were synthesized and characterized by molar conductivity, magnetic moments measurements, as well as by electronic, IR, and 1H-NMR spectroscopy. Electrochemical studies were performed by cyclic voltammetry. The active signals are assignable to the NiIII/II and NiII/I redox couples. The binding interactions between the metal complexes and calf thymus DNA were investigated by absorption and thermal denaturation. The cleavage activity of the complexes was determined using double-stranded pBR322 circular plasmid DNA by gel electrophoresis. All complexes showed increased nuclease activity in the presence of the oxidant H2O2. The nuclease activities of mixed ligand complexes were compared with those of the parent copper(II complexes.

  4. Chemical cleavage reactions of DNA on solid support: application in mutation detection

    Directory of Open Access Journals (Sweden)

    Cotton Richard GH

    2003-05-01

    Full Text Available Abstract Background The conventional solution-phase Chemical Cleavage of Mismatch (CCM method is time-consuming, as the protocol requires purification of DNA after each reaction step. This paper describes a new version of CCM to overcome this problem by immobilizing DNA on silica solid supports. Results DNA test samples were loaded on to silica beads and the DNA bound to the solid supports underwent chemical modification reactions with KMnO4 (potassium permanganate and hydroxylamine in 3M TEAC (tetraethylammonium chloride solution. The resulting modified DNA was then simultaneously cleaved by piperidine and removed from the solid supports to afford DNA fragments without the requirement of DNA purification between reaction steps. Conclusions The new solid-phase version of CCM is a fast, cost-effective and sensitive method for detection of mismatches and mutations.

  5. Ratiometric Signaling of Hypochlorite by the Oxidative Cleavage of Sulfonhydrazide-Based Rhodamine-Dansyl Dyad.

    Science.gov (United States)

    Lee, Hyo Jin; Cho, Min Jeoung; Chang, Suk-Kyu

    2015-09-08

    A reaction-based probe 1 for hypochlorite signaling was designed by the conjugation of two fluorophores, rhodamine and dansyl moieties, by the reaction of rhodamine B base with dansylhydrazine. Probe 1 exhibited pronounced hypochlorite-selective chromogenic and fluorescent signaling behavior over other oxidants used in practical applications, such as hydrogen peroxide, peracetic acid, and ammonium persulfate, as well as commonly encountered metal ions and anions. Signaling was attributed to the hypochlorite-induced oxidative cleavage of the sulfonhydrazide linkage of the probe. In particular, favorable ratiometric fluorescence signaling was possible by utilizing the emissions of the two fluorophores. A detection limit of 1.13 × 10(-6) M (0.058 ppm) was estimated for the determination of hypochlorite. A paper-based test strip was prepared and was used as a semiquantitative indicator for the presence of hypochlorite in aqueous solutions. The probe was also successfully applied for the determination of hypochlorite in practical tap water samples.

  6. Size effects and strain localization in atomic-scale cleavage modeling

    International Nuclear Information System (INIS)

    Elsner, B A M; Müller, S

    2015-01-01

    In this work, we study the adhesion and decohesion of Cu(1 0 0) surfaces using density functional theory (DFT) calculations. An upper stress to surface decohesion is obtained via the universal binding energy relation (UBER), but the model is limited to rigid separation of bulk-terminated surfaces. When structural relaxations are included, an unphysical size effect arises if decohesion is considered to occur as soon as the strain energy equals the energy of the newly formed surfaces. We employ the nudged elastic band (NEB) method to show that this size effect is opposed by a size-dependency of the energy barriers involved in the transition. Further, we find that the transition occurs via a localization of bond strain in the vicinity of the cleavage plane, which resembles the strain localization at the tip of a sharp crack that is predicted by linear elastic fracture mechanics. (paper)

  7. Frustration across the periodic table: heterolytic cleavage of dihydrogen by metal complexes.

    Science.gov (United States)

    Bullock, R Morris; Chambers, Geoffrey M

    2017-08-28

    This perspective examines frustrated Lewis pairs (FLPs) in the context of heterolytic cleavage of H 2 by transition metal complexes, with an emphasis on molecular complexes bearing an intramolecular Lewis base. FLPs have traditionally been associated with main group compounds, yet many reactions of transition metal complexes support a broader classification of FLPs that includes certain types of transition metal complexes with reactivity resembling main group-based FLPs. This article surveys transition metal complexes that heterolytically cleave H 2 , which vary in the degree that the Lewis pairs within these systems interact. Many of the examples include complexes bearing a pendant amine functioning as the base with the metal functioning as the hydride acceptor. Consideration of transition metal compounds in the context of FLPs can inspire new innovations and improvements in transition metal catalysis.This article is part of the themed issue 'Frustrated Lewis pair chemistry'. © 2017 The Author(s).

  8. No Country For Old Cleavages: Political attitudes and beliefs amidst the Greek debt crisis

    Directory of Open Access Journals (Sweden)

    Elias Dinas

    2012-08-01

    Full Text Available The Greek youth enters in their formative period amidst a period of a severe debt crisis that has been having unforeseeable implications to the established party system. How do these new political developments affect the attitudes, beliefs and the repertoire of political actions of this generation? What is the role of old cleavages and traditional division lines in this ever-changing political setting? Drawing on a novel sample from university students, the paper assesses the impact of the crisis on young people’s political beliefs. The findings suggest that the classic left-right division is not adequate to represent the much more nuanced and complex divisions generated as a result of the crisis. Some of the information provided in this survey helps to explore the role of new seemingly important division lines in helping us understand the dynamics of party competition and public opinion.

  9. Radioimmunoelectrophoresis, a sensitive method for detecting cleavage of the fifth component of human complement (C5)

    International Nuclear Information System (INIS)

    Perez, H.D.; Ong, R.; Banda, D.; Goldstein, I.M.

    1983-01-01

    A method has been developed for detecting cleavage of human C5 in serum and whole blood as a consequence of complement activation. Standard, single-dimension immunoelectrophoresis was performed using as antibody a radioiodinated IgG fraction prepared from a commercially available antiserum to human C5. Autoradiographs developed after radioimmunoelectrophoresis of either normal human serum or functionally pure human C5 revealed only one precipitin band. In contrast, when either zymosan-treated serum or trypsin-treated human C5 were examined with this technique, two additional precipitin bands were detected. One migrated more anodally than native C5 while the other remained at the origin (cathode). Radioimmunoelectrophoresis was significantly more sensitive as an indicator of complement activation in human serum than either measurements of total hemolytic complement or a standard assay for complement (C5)-derived chemotactic activity. (Auth.)

  10. Cleavage of nicotinamide adenine dinucleotide by the ribosome-inactivating protein from Momordica charantia.

    Science.gov (United States)

    Vinkovic, M; Dunn, G; Wood, G E; Husain, J; Wood, S P; Gill, R

    2015-09-01

    The interaction of momordin, a type 1 ribosome-inactivating protein from Momordica charantia, with NADP(+) and NADPH has been investigated by X-ray diffraction analysis of complexes generated by co-crystallization and crystal soaking. It is known that the proteins of this family readily cleave the adenine-ribose bond of adenosine and related nucleotides in the crystal, leaving the product, adenine, bound to the enzyme active site. Surprisingly, the nicotinamide-ribose bond of oxidized NADP(+) is cleaved, leaving nicotinamide bound in the active site in the same position but in a slightly different orientation to that of the five-membered ring of adenine. No binding or cleavage of NADPH was observed at pH 7.4 in these experiments. These observations are in accord with current views of the enzyme mechanism and may contribute to ongoing searches for effective inhibitors.

  11. 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

  12. 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.

  13. 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.

  14. Preventing cleavage of Mer promotes efferocytosis and suppresses acute lung injury in bleomycin treated mice

    International Nuclear Information System (INIS)

    Lee, Ye-Ji; Lee, Seung-Hae; Youn, Young-So; Choi, Ji-Yeon; Song, Keung-Sub; Cho, Min-Sun; Kang, Jihee Lee

    2012-01-01

    Mer receptor tyrosine kinase (Mer) regulates macrophage activation and promotes apoptotic cell clearance. Mer activation is regulated through proteolytic cleavage of the extracellular domain. To determine if membrane-bound Mer is cleaved during bleomycin-induced lung injury, and, if so, how preventing the cleavage of Mer enhances apoptotic cell uptake and down-regulates pulmonary immune responses. During bleomycin-induced acute lung injury in mice, membrane-bound Mer expression decreased, but production of soluble Mer and activity as well as expression of disintegrin and metalloproteinase 17 (ADAM17) were enhanced . Treatment with the ADAM inhibitor TAPI-0 restored Mer expression and diminished soluble Mer production. Furthermore, TAPI-0 increased Mer activation in alveolar macrophages and lung tissue resulting in enhanced apoptotic cell clearance in vivo and ex vivo by alveolar macrophages. Suppression of bleomycin-induced pro-inflammatory mediators, but enhancement of hepatocyte growth factor induction were seen after TAPI-0 treatment. Additional bleomycin-induced inflammatory responses reduced by TAPI-0 treatment included inflammatory cell recruitment into the lungs, levels of total protein and lactate dehydrogenase activity in bronchoalveolar lavage fluid, as well as caspase-3 and caspase-9 activity and alveolar epithelial cell apoptosis in lung tissue. Importantly, the effects of TAPI-0 on bleomycin-induced inflammation and apoptosis were reversed by coadministration of specific Mer-neutralizing antibodies. These findings suggest that restored membrane-bound Mer expression by TAPI-0 treatment may help resolve lung inflammation and apoptosis after bleomycin treatment. -- Highlights: ►Mer expression is restored by TAPI-0 treatment in bleomycin-stimulated lung. ►Mer signaling is enhanced by TAPI-0 treatment in bleomycin-stimulated lung. ►TAPI-0 enhances efferocytosis and promotes resolution of lung injury.

  15. Evolutionarily distinct carotenoid cleavage dioxygenases are responsible for crocetin production in Buddleja davidii.

    Science.gov (United States)

    Ahrazem, Oussama; Diretto, Gianfranco; Argandoña, Javier; Rubio-Moraga, Ángela; Julve, José Manuel; Orzáez, Diego; Granell, Antonio; Gómez-Gómez, Lourdes

    2017-07-20

    Crocetin, one of the few colored apocarotenoids known in nature, is present in flowers and fruits and has long been used medicinally and as a colorant. Saffron is the main source of crocetin, although a few other plants produce lower amounts of this apocarotenoid. Notably, Buddleja davidii accumulates crocetin in its flowers. Recently, a carotenoid dioxygenase cleavage enzyme, CCD2, has been characterized as responsible for crocetin production in Crocus species. We searched for CCD2 homologues in B. davidii and identified several CCD enzymes from the CCD1 and CCD4 subfamilies. Unexpectedly, two out of the three CCD4 enzymes, namely BdCCD4.1 and BdCCD4.3, showed 7,8;7',8' activity in vitro and in vivo over zeaxanthin. In silico analyses of these enzymes and CCD2 allowed the determination of key residues for this activity. Both BdCCD4 genes are highly expressed during flower development and transcripts levels parallel the accumulation of crocins in the petals. Phylogenetic analysis showed that BdCCD4.2 grouped with almost all the characterized CCD4 enzymes, while BdCCD4.1 and BdCCD4.3 form a new sub-cluster together with CCD4 enzymes from certain Lamiales species. The present study indicates that convergent evolution led to the acquisition of 7,8;7',8' apocarotenoid cleavage activity in two separate CCD enzyme families. © The Author 2017. Published by Oxford University Press on behalf of the Society for Experimental Biology. All rights reserved. For permissions, please email: journals.permissions@oup.com.

  16. The Conformational Dynamics of Cas9 Governing DNA Cleavage Are Revealed by Single-Molecule FRET

    Directory of Open Access Journals (Sweden)

    Mengyi Yang

    2018-01-01

    Full Text Available Summary: 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. : Yang et al. revealed significant conformational dynamics of Cas9 at global and local scales using single-molecule FRET. They uncovered surprising long-range allosteric communication between the HNH nuclease domain and the RNA/DNA heteroduplex at the PAM-distal end that serves as a proofreading checkpoint to govern the nuclease activity and specificity of Cas9. Keywords: CRISPR, Cas9, single-molecule, FRET, conformational dynamics, proofreading, off-target, allosteric communication, genome editing

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

    Directory of Open Access Journals (Sweden)

    Zuzana Zubáčová

    Full Text Available All eukaryotic organisms contain mitochondria or organelles that evolved from the same endosymbiotic event like classical mitochondria. Organisms inhabiting low oxygen environments often contain mitochondrial derivates known as hydrogenosomes, mitosomes or neutrally as mitochondrion-like organelles. The detailed investigation has shown unexpected evolutionary plasticity in the biochemistry and protein composition of these organelles in various protists. We investigated the mitochondrion-like organelle in Trimastix pyriformis, a free-living member of one of the three lineages of anaerobic group Metamonada. Using 454 sequencing we have obtained 7 037 contigs from its transcriptome and on the basis of sequence homology and presence of N-terminal extensions we have selected contigs coding for proteins that putatively function in the organelle. Together with the results of a previous transcriptome survey, the list now consists of 23 proteins - mostly enzymes involved in amino acid metabolism, transporters and maturases of proteins and transporters of metabolites. We have no evidence of the production of ATP in the mitochondrion-like organelle of Trimastix but we have obtained experimental evidence for the presence of enzymes of the glycine cleavage system (GCS, which is part of amino acid metabolism. Using homologous antibody we have shown that H-protein of GCS localizes into vesicles in the cell of Trimastix. When overexpressed in yeast, H- and P-protein of GCS and cpn60 were transported into mitochondrion. In case of H-protein we have demonstrated that the first 16 amino acids are necessary for this transport. Glycine cleavage system is at the moment the only experimentally localized pathway in the mitochondrial derivate of Trimastix pyriformis.

  18. Kinetics of thyroid hormones

    International Nuclear Information System (INIS)

    Inada, Mitsuo; Nishikawa, Mitsushige; Naito, Kimikazu; Ishii, Hitoshi; Tanaka, Kiyoshi

    1980-01-01

    Kinetics of thyroid hormones were outlined, and recent progress in metabolism of these hormones was also described. Recently, not only T 4 and T 3 but also rT 3 , 3,3'-T 2 , 3',5'-T 2 , and 3,5-T 2 can be measured by RIA. To clarify metabolic pathways of these hormones, metabolic clearance rate and production rate of these hormones were calculated. As single-compartment analysis was insufficient to clarify disappearance curves of thyroid hormones in blood such as T 3 and T 2 of which metabolic speed was so fast, multi-compartment analysis or non-compartment analysis were also performed. Thyroid hormones seemed to be measured more precisely by constant infusion method. At the first step of T 4 metabolism, T 3 was formed by 5'-monodeiodination of T 4 , and rT 3 was formed by 5-monodeiodination of T 4 . As metabolic pathways of T 3 and rT 3 , conversion of them to 3,3'-T 2 or to 3',5'-T 2 and 3,5-T 2 was supposed. This subject will be an interesting research theme in future. (Tsunoda, M.)

  19. 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

  20. Kinetic effects on magnetohydrodynamic phenomena

    International Nuclear Information System (INIS)

    Naito, Hiroshi; Matsumoto, Taro

    2001-01-01

    Resistive and ideal magnetohydrodynamic (MHD) theories are insufficient to adequately explain MHD phenomena in the high-temperature plasma. Recent progress in numerical simulations concerning kinetic effects on magnetohydrodynamic phenomena is summarized. The following three topics are studied using various models treating extended-MHD phenomena. (1) Kinetic modifications of internal kink modes in tokamaks with normal and reversed magnetic shear configurations. (2) Temporal evolution of the toroidal Alfven eigenmode and fishbone mode in tokamaks with energetic ions. (3) Kinetic stabilization of a title mode in field-reversed configurations by means of anchoring ions and beam ions. (author)

  1. Capillary gas chromatographic analysis of mycolic acid cleavage products, cellular fatty acids, and alcohols of Mycobacterium xenopi.

    OpenAIRE

    Luquin, M; Lopez, F; Ausina, V

    1989-01-01

    The fatty acids, alcohols, and mycolic acids of 26 strains of Mycobacterium xenopi were studied by capillary gas chromatography and thin-layer chromatography. All strains contained alpha-, keto-, and omega-carboxymycolates. The primary mycolic acid cleavage product was hexacosanoic acid. The fatty acid patterns and, especially, the presence of 2-docosanol are characteristic markers of M. xenopi.

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

    Directory of Open Access Journals (Sweden)

    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.

  3. 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...

  4. 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

  5. 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.

  6. 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...

  7. Successive heterolytic cleavages of H2 achieve N2 splitting on silica-supported tantalum hydrides: A DFT proposed mechanism

    KAUST Repository

    Solá ns, Xavier Luis; Chow, Catherine; Gouré , Eric; Kaya, Yasemin; Basset, Jean-Marie; Taoufik, Mostafa; Quadrelli, Elsje Alessandra; Eisenstein, Odile

    2012-01-01

    DFT(B3PW91) calculations have been carried out to propose a pathway for the N2 cleavage by H2 in the presence of silica-supported tantalum hydride complexes [(≡ SiO)2TaHx] that forms [(≡SiO)2Ta(NH)(NH2)] (Science2007, 317, 1056). The calculations

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

    Directory of Open Access Journals (Sweden)

    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.

  9. 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...

  10. Synthesis, spectral, crystal structure, thermal behavior, antimicrobial and DNA cleavage potential of two octahedral cadmium complexes: A supramolecular structure

    Czech Academy of Sciences Publication Activity Database

    Montazerozohori, M.; Musavi, S.A.; Masoudiasl, A.; Naghiha, A.; Dušek, Michal; Kučeráková, Monika

    2015-01-01

    Roč. 137, FEB (2015), s. 389-396 ISSN 1386-1425 R&D Projects: GA ČR(CZ) GA14-03276S Institutional support: RVO:68378271 Keywords : Schiff base * Cd(II) * DNA cleavage * TG/DTG analysis * X-ray structure analysis Subject RIV: BM - Solid Matter Physics ; Magnetism Impact factor: 2.653, year: 2015

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

    Directory of Open Access Journals (Sweden)

    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.

  12. 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.

  13. 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

  14. 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

  15. Bicarbonate kinetics in Indian males

    Indian Academy of Sciences (India)

    Madhu

    ized kinetics of bicarbonate using a three-compartment model, to assess which compartmental fluxes changed dur- .... total VCO2 was < 3 % and the average respiratory quotient ..... a part of the nonrespiratory losses of 13CO2 occur to this.

  16. Kinetic equations in dirty superconductors

    International Nuclear Information System (INIS)

    Kraehenbuehl, Y.

    1981-01-01

    Kinetic equations for superconductors in the dirty limit are derived using a method developed for superfluid systems, which allows a systematic expansion in small parameters; exact charge conservation is obeyed. (orig.)

  17. 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.

  18. 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

  19. 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.

  20. 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.

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

    Directory of Open Access Journals (Sweden)

    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.

  2. 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.

  3. Cell kinetics and therapeutic efficiency

    International Nuclear Information System (INIS)

    Andreeff, M.; Abenhardt, W.; Gruner, B.; Stoffner, D.; Mainz Univ.

    1976-01-01

    The study shows that cell kinetics effects correlate with the effects of cytostatic drugs in the tumour model investigated here. It should, however, be noted that even genetically related tumour cell types may react differently to the same cytostatic drug, and that the cell kinetics effects, due to the changes in the cell cycle, cannot be predicted but should be followed with a very fast method, e.g. sequential flan fluorescence cytophotometry, for optimal therapeutic results. (orig./GSE) [de

  4. Kinetic parameters from thermogravimetric analysis

    Science.gov (United States)

    Kiefer, Richard L.

    1993-01-01

    High performance polymeric materials are finding increased use in aerospace applications. Proposed high speed aircraft will require materials to withstand high temperatures in an oxidative atmosphere for long periods of time. It is essential that accurate estimates be made of the performance of these materials at the given conditions of temperature and time. Temperatures of 350 F (177 C) and times of 60,000 to 100,000 hours are anticipated. In order to survey a large number of high performance polymeric materials on a reasonable time scale, some form of accelerated testing must be performed. A knowledge of the rate of a process can be used to predict the lifetime of that process. Thermogravimetric analysis (TGA) has frequently been used to determine kinetic information for degradation reactions in polymeric materials. Flynn and Wall studied a number of methods for using TGA experiments to determine kinetic information in polymer reactions. Kinetic parameters, such as the apparent activation energy and the frequency factor, can be determined in such experiments. Recently, researchers at the McDonnell Douglas Research Laboratory suggested that a graph of the logarithm of the frequency factor against the apparent activation energy can be used to predict long-term thermo-oxidative stability for polymeric materials. Such a graph has been called a kinetic map. In this study, thermogravimetric analyses were performed in air to study the thermo-oxidative degradation of several high performance polymers and to plot their kinetic parameters on a kinetic map.

  5. METABOLIC ENGINEERING TO DEVELOP A PATHWAY FOR THE SELECTIVE CLEAVAGE OF CARBON-NITROGEN BONDS

    Energy Technology Data Exchange (ETDEWEB)

    John J. Kilbane II

    2004-10-01

    The objective of the project is to develop biochemical pathways for the selective cleavage of C-N bonds in molecules found in petroleum. The initial phase of the project was focused on the isolation or development of an enzyme capable of cleaving the C-N bond in aromatic amides, specifically 2-aminobiphenyl. The objective of the second phase of the research will be to construct a biochemical pathway for the selective removal of nitrogen from carbazole by combining the carA genes from Sphingomonas sp. GTIN11 with the gene(s) encoding an appropriate deaminase. The objective of the final phase of the project will be to develop derivative C-N bond cleaving enzymes that have broader substrate ranges and to demonstrate the use of such strains to selectively remove nitrogen from petroleum. During the first year of the project (October, 2002-September, 2003) enrichment culture experiments resulted in the isolation of microbial cultures that utilize aromatic amides as sole nitrogen sources, several amidase genes were cloned and were included in directed evolution experiments to obtain derivatives that can cleave C-N bonds in aromatic amides, and the carA genes from Sphingomonas sp. GTIN11, and Pseudomonas resinovorans CA10 were cloned in vectors capable of replicating in Escherichia coli. During the second year of the project (October, 2003-September, 2004) enrichment culture experiments succeeded in isolating a mixed bacterial culture that can utilize 2-aminobiphenyl as a sole nitrogen source, directed evolution experiments were focused on the aniline dioxygenase enzyme that is capable of deaminating aniline, and expression vectors were constructed to enable the expression of genes encoding C-N bond cleaving enzymes in Rhodococcus hosts. The construction of a new metabolic pathway to selectively remove nitrogen from carbazole and other molecules typically found in petroleum should lead to the development of a process to improve oil refinery efficiency by reducing the

  6. Development of Synthetic Catalysts for Peptide Bond Cleavage (Synthesis and Complete Kinetic Analysis of Compounds 6A, 7A, 8A).

    Science.gov (United States)

    1987-08-05

    03824 Dr. Ronald Breslow Columbia University Departmont of Cemistry New York, NY 10027 Dr. James P. Colmen Department of Cmistry Stanford University...of Massachusetts Amherst, MA 01003 Dr. Harden M. McConnell Stanford Univesity Department of Cemistry Stanford, CA 94305 Dr. Kristin lowtmn Mertes...Institute of Technology Cambridge, MA 02139 Dr. J. H. Richards California Institute of Technology Division of Cemistry and Ch e.cal Engineering

  7. METABOLIC ENGINEERING TO DEVELOP A PATHWAY FOR THE SELECTIVE CLEAVAGE OF CARBON-NITROGEN BONDS

    Energy Technology Data Exchange (ETDEWEB)

    John J. Kilbane III

    2003-12-01

    The objective of the project is to develop biochemical pathways for the selective cleavage of C-N bonds in molecules found in petroleum. The initial phase of the project will focus on the isolation or development of an enzyme capable of cleaving the C-N bond in aromatic amides, specifically 2-aminobiphenyl. The objective of the second phase of the research will be to construct a biochemical pathway for the selective removal of nitrogen from carbazole by combining the carA genes from Sphingomonas sp. GTIN11 with the gene(s) encoding an appropriate amidase. The objective of the final phase of the project will be to develop derivative CN bond cleaving enzymes that have broader substrate ranges and to demonstrate the use of such strains to selectively remove nitrogen from petroleum. The project is on schedule and no major difficulties have been encountered. During the first year of the project (October, 2002-September, 2003) enrichment culture experiments have resulted in the isolation of promising cultures that may be capable of cleaving C-N bonds in aromatic amides, several amidase genes have been cloned and are currently undergoing directed evolution to obtain derivatives that can cleave C-N bonds in aromatic amides, and the carA genes from Sphingomonas sp. GTIN11, and Pseudomonas resinovorans CA10 were cloned in vectors capable of replicating in Escherichia coli. Future research will address expression of these genes in Rhodococcus erythropolis. Enrichment culture experiments and directed evolution experiments continue to be a main focus of research activity and further work is required to obtain an appropriate amidase that will selectively cleave C-N bonds in aromatic substrates. Once an appropriate amidase gene is obtained it must be combined with genes encoding an enzyme capable of converting carbazole to 2'aminobiphenyl-2,3-diol: specifically carA genes. The carA genes from two sources have been cloned and are ready for construction of C-N bond cleavage

  8. A ginseng saponin metabolite-induced apoptosis in HepG2 cells involves a mitochondria-mediated pathway and its downstream caspase-8 activation and Bid cleavage

    International Nuclear Information System (INIS)

    Oh, Seon-Hee; Lee, Byung-Hoon

    2004-01-01

    20-O-(β-D-Glucopyranosyl)-20(S)-protopanaxadiol (IH901), an intestinal bacterial metabolite of ginseng saponin formed from ginsenosides Rb1, Rb2, and Rc, is suggested to be a potential chemopreventive agent. Here, we show that IH901 induces apoptosis in human hepatoblastoma HepG2 cells. IH901 led to an early activation of procaspase-3 (12 h posttreatment), and the activation of caspase-8 became evident only later (18 h posttreatment). Caspase activation was a necessary requirement for apoptosis because caspase inhibitors significantly inhibited cell death by IH901. Treatment of HepG2 cells with IH901 also induced the cleavage of cytosolic factors such as Bid and Bax and translocation of truncated Bid (tBid) to mitochondria. A time-dependent release of cytochrome c from mitochondria was observed, which was accompanied by activation of caspase-9. A broad-spectrum caspase inhibitor, N-benzyloxycarbonyl-Val-Ala-Asp-fluoromethylketone (zVAD-fmk), and a specific inhibitor for caspase-8, N-benzyloxycarbonyl-Ile-Glu-Thr-Asp-fluoromethylketone (zIETD-fmk), abrogated Bid processing and translocation, and caspase-3 activation. Cytochrome c release was inhibited by zVAD-fmk, however, the inhibition by zIETD-fmk was not complete. The activation of caspase-8 was inhibited not only by zIETD-fmk but also by zVAD-fmk. The results, together with the kinetic change of caspase activation, indicate that activation of caspase-8 occurred downstream of caspase-3 and -9. Our data suggest that the activation of caspase-8 after early caspase-3 activation might act as an amplification loop necessary for successful apoptosis. Primary hepatocytes isolated from normal Sprague-Dawley rats were not affected by IH901 (0-60 μM). The very low toxicity in normal hepatocytes and high activity in hepatoblastoma HepG2 cells suggest that IH901 is a promising experimental cancer chemopreventive agent

  9. Gyrocenter-gauge kinetic theory

    International Nuclear Information System (INIS)

    Qin, H.; Tang, W.M.; Lee, W.W.

    2000-01-01

    Gyrocenter-gauge kinetic theory is developed as an extension of the existing gyrokinetic theories. In essence, the formalism introduced here is a kinetic description of magnetized plasmas in the gyrocenter coordinates which is fully equivalent to the Vlasov-Maxwell system in the particle coordinates. In particular, provided the gyroradius is smaller than the scale-length of the magnetic field, it can treat high frequency range as well as the usual low frequency range normally associated with gyrokinetic approaches. A significant advantage of this formalism is that it enables the direct particle-in-cell simulations of compressional Alfven waves for MHD applications and of RF waves relevant to plasma heating in space and laboratory plasmas. The gyrocenter-gauge kinetic susceptibility for arbitrary wavelength and arbitrary frequency electromagnetic perturbations in a homogeneous magnetized plasma is shown to recover exactly the classical result obtained by integrating the Vlasov-Maxwell system in the particle coordinates. This demonstrates that all the waves supported by the Vlasov-Maxwell system can be studied using the gyrocenter-gauge kinetic model in the gyrocenter coordinates. This theoretical approach is so named to distinguish it from the existing gyrokinetic theory, which has been successfully developed and applied to many important low-frequency and long parallel wavelength problems, where the conventional meaning of gyrokinetic has been standardized. Besides the usual gyrokinetic distribution function, the gyrocenter-gauge kinetic theory emphasizes as well the gyrocenter-gauge distribution function, which sometimes contains all the physics of the problems being studied, and whose importance has not been realized previously. The gyrocenter-gauge distribution function enters Maxwell's equations through the pull-back transformation of the gyrocenter transformation, which depends on the perturbed fields. The efficacy of the gyrocenter-gauge kinetic approach is

  10. Crystallization Kinetics within a Generic Modelling Framework

    DEFF Research Database (Denmark)

    Meisler, Kresten Troelstrup; von Solms, Nicolas; Gernaey, Krist

    2013-01-01

    An existing generic modelling framework has been expanded with tools for kinetic model analysis. The analysis of kinetics is carried out within the framework where kinetic constitutive models are collected, analysed and utilized for the simulation of crystallization operations. A modelling...... procedure is proposed to gain the information of crystallization operation kinetic model analysis and utilize this for faster evaluation of crystallization operations....

  11. Localization of self-interacting domains within betaretrovirus Gag polyproteins

    Czech Academy of Sciences Publication Activity Database

    Zábranský, Aleš; Sakalian, M.; Pichová, Iva

    2005-01-01

    Roč. 332, - (2005), s. 659-666 ISSN 0042-6822 R&D Projects: GA ČR(CZ) GP203/02/P020 Grant - others:NIH(US) R01AI43230 Institutional research plan: CEZ:AV0Z4055905 Keywords : MMTV * M-PMV * betaretrovirus Subject RIV: CE - Biochemistry Impact factor: 3.080, year: 2005

  12. Isolation of recombinant phage antibodies targeting the hemagglutinin cleavage site of highly pathogenic avian influenza virus.

    Directory of Open Access Journals (Sweden)

    Jinhua Dong

    Full Text Available Highly pathogenic avian influenza (HPAI H5N1 viruses, which have emerged in poultry and other wildlife worldwide, contain a characteristic multi-basic cleavage site (CS in the hemagglutinin protein (HA. Because this arginine-rich CS is unique among influenza virus subtypes, antibodies against this site have the potential to specifically diagnose pathogenic H5N1. By immunizing mice with the CS peptide and screening a phage display library, we isolated four antibody Fab fragment clones that specifically bind the antigen peptide and several HPAI H5N1 HA proteins in different clades. The soluble Fab fragments expressed in Escherichia coli bound the CS peptide and the H5N1 HA protein with nanomolar affinity. In an immunofluorescence assay, these Fab fragments stained cells infected with HPAI H5N1 but not those infected with a less virulent strain. Lastly, all the Fab clones could detect the CS peptide and H5N1 HA protein by open sandwich ELISA. Thus, these recombinant Fab fragments will be useful novel reagents for the rapid and specific detection of HPAI H5N1 virus.

  13. Synthesis and DNA cleavage activity of Bis-3-chloropiperidines as alkylating agents.

    Science.gov (United States)

    Zuravka, Ivonne; Roesmann, Rolf; Sosic, Alice; Wende, Wolfgang; Pingoud, Alfred; Gatto, Barbara; Göttlich, Richard

    2014-09-01

    Nitrogen mustards are an important class of bifunctional alkylating agents routinely used in chemotherapy. They react with DNA as electrophiles through the formation of highly reactive aziridinium ion intermediates. The antibiotic 593A, with potential antitumor activity, can be considered a naturally occurring piperidine mustard containing a unique 3-chloropiperidine ring. However, the total synthesis of this antibiotic proved to be rather challenging. With the aim of designing simplified analogues of this natural product, we developed an efficient bidirectional synthetic route to bis-3-chloropiperidines joined by flexible, conformationally restricted, or rigid diamine linkers. The key step involves an iodide-catalyzed double cyclization of unsaturated bis-N-chloroamines to simultaneously generate both piperidine rings. Herein we describe the synthesis and subsequent evaluation of a series of novel nitrogen-bridged bis-3-chloropiperidines, enabling the study of the impact of the linker structure on DNA alkylation properties. Our studies reveal that the synthesized compounds possess DNA alkylating abilities and induce strand cleavage, with a strong preference for guanine residues. © 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  14. Expression Profile of Carotenoid Cleavage Dioxygenase Genes in Summer Squash (Cucurbita pepo L.).

    Science.gov (United States)

    González-Verdejo, Clara I; Obrero, Ángeles; Román, Belén; Gómez, Pedro

    2015-06-01

    Carotenoids are important dietary components that can be found in vegetable crops. The accumulation of these compounds in fruit and vegetables is altered by the activity of carotenoid cleavage dioxygenases (CCDs) enzymes that produce their degradation. The aim of this work was to study the possible implication of CCD genes in preventing carotenoid storage in the horticultural crop summer squash (Cucurbita pepo L.). The relationship between the presence of these compounds and gene expression for CCDs was studied in three varieties showing different peel and flesh colour. Expression analysis for the CCD genes CpNCED1, CpNCED2, CpNCED3, CpNCED9, CpCCD1, CpCCD4a, CpCCD4b and CpCCD8 was carried out on different organs and at several fruit developmental stages. The results showed that the CpCCD4a and CpCCD4b genes were highly expressed in the variety with lowest carotenoid content suggesting a putative role in carotenoid accumulation pattern in summer squash fruit.

  15. Caspase cleavage of viral proteins, another way for viruses to make the best of apoptosis.

    Science.gov (United States)

    Richard, A; Tulasne, D

    2012-03-08

    Viral infection constitutes an unwanted intrusion that needs to be eradicated by host cells. On one hand, one of the first protective barriers set up to prevent viral replication, spread or persistence involves the induction of apoptotic cell death that aims to limit the availability of the cellular components for viral amplification. On the other hand, while they completely depend on the host molecular machinery, viruses also need to evade the cellular responses that are meant to destroy them. The existence of numerous antiapoptotic products within the viral kingdom proves that apoptosis constitutes a major threat that should better be bypassed. Among the different strategies developed to deal with apoptosis, one is based on what viruses do best: backfiring the cell on itself. Several unrelated viruses have been described to take advantage of apoptosis induction by expressing proteins targeted by caspases, the key effectors of apoptotic cell death. Caspase cleavage of these proteins results in various consequences, from logical apoptosis inhibition to more surprising enhancement or attenuation of viral replication. The present review aims at discussing the characterization and relevance of this post-translational modification that adds a new complexity in the already intricate host-apoptosis-virus triangle.

  16. A β2-microglobulin cleavage variant fibrillates at near-physiological pH

    International Nuclear Information System (INIS)

    Corlin, Dorthe B.; Johnsen, Christina K.; Nissen, Mogens H.; Heegaard, Niels H.H.

    2009-01-01

    β 2 -microglobulin (β 2 m) deposits as amyloid in dialysis-related amyloidosis (DRA), predominantly in joints. The molecular mechanisms underlying the amyloidogenicity of β 2 m are still largely unknown. In vitro, acidic conditions, pH 2 m within several days. Here, we show that amyloid fibrils are generated in less than an hour when a cleavage variant of β 2 m-found in the circulation of many dialysis patients-is exposed to pH levels (pH 6.6) occurring in joints during inflammation. Aggregation and fibrillation, including seeding effects with intact, native β 2 m were studied by Thioflavin T fluorescence spectroscopy, turbidimetry, capillary electrophoresis, and electron microscopy. We conclude that a biologically relevant variant of β 2 m is amyloidogenic at slightly acidic pH. Also, only a very small amount of preformed fibrils of this variant is required to induce fibrillation of native β 2 m. This may explain the apparent lack of detectable amounts of the variant β 2 m in extracts of amyloid from DRA patients.

  17. On the substrate- and stereospecificity of the plant carotenoid cleavage dioxygenase 7

    KAUST Repository

    Bruno, Mark; Hofmann, Manuel; Vermathen, Martina; Alder, Adrian; Beyer, Peter D.; Al-Babili, Salim

    2014-01-01

    Strigolactones are phytohormones synthesized from carotenoids via a stereospecific pathway involving the carotenoid cleavage dioxygenases 7 (CCD7) and 8. CCD7 cleaves 9-cis-β-carotene to form a supposedly 9-cis-configured β-apo-10′-carotenal. CCD8 converts this intermediate through a combination of yet undetermined reactions into the strigolactone-like compound carlactone. Here, we investigated the substrate and stereo-specificity of the Arabidopsis and pea CCD7 and determined the stereo-configuration of the β-apo-10′-carotenal intermediate by using Nuclear Magnetic Resonance Spectroscopy. Our data unequivocally demonstrate the 9-cis-configuration of the intermediate. Both CCD7s cleave different 9-cis-carotenoids, yielding hydroxylated 9-cis-apo-10′-carotenals that may lead to hydroxylated carlactones, but show highest affinity for 9-cis-β-carotene. © 2014 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.

  18. Casein Kinase 1 Coordinates Cohesin Cleavage, Gametogenesis, and Exit from M Phase in Meiosis II.

    Science.gov (United States)

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

    2017-01-09

    Meiosis consists of DNA replication followed by two consecutive nuclear divisions and gametogenesis or spore formation. While meiosis I has been studied extensively, less is known about the regulation of meiosis II. Here we show that Hrr25, the conserved casein kinase 1δ of budding yeast, links three mutually independent key processes of meiosis II. First, Hrr25 induces nuclear division by priming centromeric cohesin for cleavage by separase. Hrr25 simultaneously phosphorylates Rec8, the cleavable subunit of cohesin, and removes from centromeres the cohesin protector composed of shugoshin and the phosphatase PP2A. Second, Hrr25 initiates the sporulation program by inducing the synthesis of membranes that engulf the emerging nuclei at anaphase II. Third, Hrr25 mediates exit from meiosis II by activating pathways that trigger the destruction of M-phase-promoting kinases. Thus, Hrr25 synchronizes formation of the single-copy genome with gamete differentiation and termination of meiosis. Copyright © 2017 Elsevier Inc. All rights reserved.

  19. Cadmium but not lead exposure affects Xenopus laevis fertilization and embryo cleavage

    Energy Technology Data Exchange (ETDEWEB)

    Slaby, Sylvain [Univ. Lille Nord de France, EA 4515 – LGCgE – Laboratoire Génie Civil et géo-Environnement, Université de Lille 1, Cité scientifique, SN3, F-59655 Villeneuve d’Ascq (France); Univ. Lille, CNRS, INRA, UMR 8576 – UGSF – Unité de Glycobiologie Structurale et Fonctionnelle, F-59000 Lille (France); Lemière, Sébastien [Univ. Lille Nord de France, EA 4515 – LGCgE – Laboratoire Génie Civil et géo-Environnement, Université de Lille 1, Cité scientifique, SN3, F-59655 Villeneuve d’Ascq (France); Hanotel, Julie; Lescuyer, Arlette [Univ. Lille, CNRS, INRA, UMR 8576 – UGSF – Unité de Glycobiologie Structurale et Fonctionnelle, F-59000 Lille (France); Demuynck, Sylvain [Univ. Lille Nord de France, EA 4515 – LGCgE – Laboratoire Génie Civil et géo-Environnement, Université de Lille 1, Cité scientifique, SN3, F-59655 Villeneuve d’Ascq (France); Bodart, Jean-François [Univ. Lille, CNRS, INRA, UMR 8576 – UGSF – Unité de Glycobiologie Structurale et Fonctionnelle, F-59000 Lille (France); and others

    2016-08-15

    Highlights: • First embryonic steps were studied. • Fertilization success was impacted by cadmium exposures. • Oocytes were most affected instead of spermatozoa by cadmium exposures. • First embryonic cleavages were slown down or stopped by cadmium exposures. • Lead exposures did not affected fertilization and segmentation. - Abstract: Among the toxicological and ecotoxicological studies, few have investigated the effects on germ cells, gametes or embryos, while an impact at these stages will result in serious damage at a population level. Thus, it appeared essential to characterize consequences of environmental contaminant exposures at these stages. Therefore, we proposed to assess the effects of exposure to cadmium and lead ions, alone or in a binary mixture, on early stages of Xenopus laevis life cycle. Fertilization and cell division during segmentation were the studied endpoints. Cadmium ion exposures decreased in the fertilization rates in a concentration-dependent manner, targeting mainly the oocytes. Exposure to this metal ions induced also delays or blockages in the embryonic development. For lead ion exposure, no such effect was observed. For the exposure to the mixture of the two metal ions, concerning the fertilization success, we observed results similar to those obtained with the highest cadmium ion concentration.

  20. Rapid polyether cleavage via extracellular one-electron oxidation by a brown-rot basidiomycete.

    Science.gov (United States)

    Kerem, Z; Bao, W; Hammel, K E

    1998-09-01

    Fungi that cause brown rot of wood are essential biomass recyclers and also the principal agents of decay in wooden structures, but the extracellular mechanisms by which they degrade lignocellulose remain unknown. To test the hypothesis that brown-rot fungi use extracellular free radical oxidants as biodegradative tools, Gloeophyllum trabeum was examined for its ability to depolymerize an environmentally recalcitrant polyether, poly(ethylene oxide) (PEO), that cannot penetrate cell membranes. Analyses of degraded PEOs by gel permeation chromatography showed that the fungus cleaved PEO rapidly by an endo route. 13C NMR analyses of unlabeled and perdeuterated PEOs recovered from G. trabeum cultures showed that a major route for depolymerization was oxidative C---C bond cleavage, a reaction diagnostic for hydrogen abstraction from a PEO methylene group by a radical oxidant. Fenton reagent (Fe(II)/H2O2) oxidized PEO by the same route in vitro and therefore might account for PEO biodegradation if it is produced by the fungus, but the data do not rule out involvement of less reactive radicals. The reactivity and extrahyphal location of this PEO-degrading system suggest that its natural function is to participate in the brown rot of wood and that it may enable brown-rot fungi to degrade recalcitrant organopollutants.

  1. Subcellular RNA profiling links splicing and nuclear DICER1 to alternative cleavage and polyadenylation.

    Science.gov (United States)

    Neve, Jonathan; Burger, Kaspar; Li, Wencheng; Hoque, Mainul; Patel, Radhika; Tian, Bin; Gullerova, Monika; Furger, Andre

    2016-01-01

    Alternative cleavage and polyadenylation (APA) plays a crucial role in the regulation of gene expression across eukaryotes. Although APA is extensively studied, its regulation within cellular compartments and its physiological impact remains largely enigmatic. Here, we used a rigorous subcellular fractionation approach to compare APA profiles of cytoplasmic and nuclear RNA fractions from human cell lines. This approach allowed us to extract APA isoforms that are subjected to differential regulation and provided us with a platform to interrogate the molecular regulatory pathways that shape APA profiles in different subcellular locations. Here, we show that APA isoforms with shorter 3' UTRs tend to be overrepresented in the cytoplasm and appear to be cell-type-specific events. Nuclear retention of longer APA isoforms occurs and is partly a result of incomplete splicing contributing to the observed cytoplasmic bias of transcripts with shorter 3' UTRs. We demonstrate that the endoribonuclease III, DICER1, contributes to the establishment of subcellular APA profiles not only by expected cytoplasmic miRNA-mediated destabilization of APA mRNA isoforms, but also by affecting polyadenylation site choice. © 2016 Neve et al.; Published by Cold Spring Harbor Laboratory Press.

  2. Approximate techniques for predicting size effects on cleavage fracture toughness (Jc)

    International Nuclear Information System (INIS)

    Kirk, M.T.; Dodds, R.H. Jr.

    1993-07-01

    This investigation examines the ability of an elastic T-stress analysis coupled with modified boundary layer (MBL) solution to predict stresses ahead of a crack tip in a variety of planar geometries. The approximate stresses are used as input to estimate the effective driving force for cleavage fracture (J 0 ) using the micromechanically based approach introduced by Dodds and Anderson. Finite element analyses for a wide variety of planar cracked geometries are conducted which have elastic biaxiality parameters (β) ranging from -0.99 (very low constraint) to +2.96 (very high constraint). The magnitude and sign of β indicate the rate at which crack-tip constraint changes with increasing applied load. All results pertain to a moderately strain hardening material (strain hardening exponent (η) of 10). These analyses suggest that β is an effective indicator of both the accuracy of T-MBL estimates of J 0 and of applicability limits on evolving fracture analysis methodologies (i.e. T-MBL, J-Q, and J/J 0 ). Specifically, when 1β1>0.4 these analyses show that the T-MBL approximation of J 0 is accurate to within 20% of a detailed finite-element analysis. As ''structural type'' configurations, i.e. shallow cracks in tension, generally have 1β1>0.4, it appears that only an elastic analysis may be needed to determine reasonably accurate J 0 values for structural conditions

  3. PARP-1 cleavage fragments: signatures of cell-death proteases in neurodegeneration

    Directory of Open Access Journals (Sweden)

    Alexander Jonathan S

    2010-12-01

    Full Text Available Abstract The normal function of poly (ADP-ribose polymerase-1 (PARP-1 is the routine repair of DNA damage by adding poly (ADP ribose polymers in response to a variety of cellular stresses. Recently, it has become widely appreciated that PARP-1 also participates in diverse physiological and pathological functions from cell survival to several forms of cell death and has been implicated in gene transcription, immune responses, inflammation, learning, memory, synaptic functions, angiogenesis and aging. In the CNS, PARP inhibition attenuates injury in pathologies like cerebral ischemia, trauma and excitotoxicity demonstrating a central role of PARP-1 in these pathologies. PARP-1 is also a preferred substrate for several 'suicidal' proteases and the proteolytic action of suicidal proteases (caspases, calpains, cathepsins, granzymes and matrix metalloproteinases (MMPs on PARP-1 produces several specific proteolytic cleavage fragments with different molecular weights. These PARP-1 signature fragments are recognized biomarkers for specific patterns of protease activity in unique cell death programs. This review focuses on specific suicidal proteases active towards PARP-1 to generate signature PARP-1 fragments that can identify key proteases and particular forms of cell death involved in pathophysiology. The roles played by some of the PARP-1 fragments and their associated binding partners in the control of different forms of cell death are also discussed.

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

    Science.gov (United States)

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

    2016-11-23

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

  5. On the substrate- and stereospecificity of the plant carotenoid cleavage dioxygenase 7

    KAUST Repository

    Bruno, Mark

    2014-05-01

    Strigolactones are phytohormones synthesized from carotenoids via a stereospecific pathway involving the carotenoid cleavage dioxygenases 7 (CCD7) and 8. CCD7 cleaves 9-cis-β-carotene to form a supposedly 9-cis-configured β-apo-10′-carotenal. CCD8 converts this intermediate through a combination of yet undetermined reactions into the strigolactone-like compound carlactone. Here, we investigated the substrate and stereo-specificity of the Arabidopsis and pea CCD7 and determined the stereo-configuration of the β-apo-10′-carotenal intermediate by using Nuclear Magnetic Resonance Spectroscopy. Our data unequivocally demonstrate the 9-cis-configuration of the intermediate. Both CCD7s cleave different 9-cis-carotenoids, yielding hydroxylated 9-cis-apo-10′-carotenals that may lead to hydroxylated carlactones, but show highest affinity for 9-cis-β-carotene. © 2014 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.

  6. Theoretical perspectives on electron transfer and charge separation events in photochemical water cleavage systems

    International Nuclear Information System (INIS)

    Kozak, J.J.; Lenoir, P.M.; Musho, M.K.; Tembe, B.L.

    1984-01-01

    We study in this paper the dynamics induced by models for photochemical water cleavage systems, focusing on the spatial and temporal factors influencing electron transfer and charge separation processes in such systems. The reaction-diffusion theory is formulated in full generality and the consequences explored in a number of spatio-temporal regimes, viz. the spatially homogeneous system in the long-time limit (i.e. the steady state for a well-stirred system), the spatially homogeneous system in evolution, and the spatially inhomogeneous system in evolution (where, in the latter study, we consider electron transfer at the cluster surface to be governed by a rate constant that reflects the localized nature of such processes). The results of numerical simulations are presented for all three cases and used to highlight the importance of heterogeneous environments in enhancing the cage escape yield of charge separated species, and to demonstrate the dependence of the hydrogen yield on the localization of electron-transfer processes in the vicinity of the microcatalyst surface

  7. Matrix metalloproteinase-1 facilitates MSC migration via cleavage of IGF-2/IGFBP2 complex.

    Science.gov (United States)

    Guan, Shou P; Lam, Alan T L; Newman, Jennifer P; Chua, Kevin L M; Kok, Catherine Y L; Chong, Siao T; Chua, Melvin L K; Lam, Paula Y P

    2018-01-01

    The specific mechanism underlying the tumor tropism of human mesenchymal stem cells (MSCs) for cancer is not well defined. We previously showed that the migration potential of MSCs correlated with the expression and protease activity of matrix metalloproteinase (MMP)-1. Furthermore, highly tumor-tropic MSCs expressed higher levels of MMP-1 and insulin-like growth factor (IGF)-2 than poorly migrating MSCs. In this study, we examined the functional roles of IGF-2 and MMP-1 in mediating the tumor tropism of MSCs. Exogenous addition of either recombinant IGF-2 or MMP-1 could stimulate MSC migration. The correlation between IGF-2, MMP-1 expression, and MSC migration suggests that MMP-1 may play a role in regulating MSC migration via the IGF-2 signaling cascade. High concentrations of IGF binding proteins (IGFBPs) can inhibit IGF-stimulated functions by blocking its binding to its receptors and proteolysis of IGFBP is an important mechanism for the regulation of IGF signaling. We thus hypothesized that MMP-1 acts as an IGFBP2 proteinase, resulting in the cleavage of IGF-2/IGFBP2 complex and extracellular release of free IGF-2. Indeed, our results showed that conditioned media from highly migrating MSCs, which expressed high levels of MMP-1, cleaved the IGF-2/IGFBP2 complex. Taken together, these results showed that the MMP-1 secreted by highly tumor-tropic MSCs cleaved IGF-2/IGFBP2 complex. Free IGF-2 released from the complex may facilitate MSC migration toward tumor.

  8. Synthesis of isatin thiosemicarbazones derivatives: in vitro anti-cancer, DNA binding and cleavage activities.

    Science.gov (United States)

    Ali, Amna Qasem; Teoh, Siang Guan; Salhin, Abdussalam; Eltayeb, Naser Eltaher; Khadeer Ahamed, Mohamed B; Abdul Majid, A M S

    2014-05-05

    New derivatives of thiosemicarbazone Schiff base with isatin moiety were synthesized L1-L6. The structures of these compounds were characterized based on the spectroscopic techniques. Compound L6 was further characterized by XRD single crystal. The interaction of these compounds with calf thymus (CT-DNA) exhibited high intrinsic binding constant (k(b)=5.03-33.00×10(5) M(-1)) for L1-L3 and L5 and (6.14-9.47×10(4) M(-1)) for L4 and L6 which reflect intercalative activity of these compounds toward CT-DNA. This result was also confirmed by the viscosity data. The electrophoresis studies reveal the higher cleavage activity of L1-L3 than L4-L6. The in vitro anti-proliferative activity of these compounds against human colon cancer cell line (HCT 116) revealed that the synthesized compounds (L3, L6 and L2) exhibited good anticancer potency. Copyright © 2014 Elsevier B.V. All rights reserved.

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

    Science.gov (United States)

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

    2014-02-04

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

  10. Cell Death-Associated Ribosomal RNA Cleavage in Postmortem Tissues and Its Forensic Applications.

    Science.gov (United States)

    Kim, Ji Yeon; Kim, Yunmi; Cha, Hyo Kyeong; Lim, Hye Young; Kim, Hyungsub; Chung, Sooyoung; Hwang, Juck-Joon; Park, Seong Hwan; Son, Gi Hoon

    2017-06-30

    Estimation of postmortem interval (PMI) is a key issue in the field of forensic pathology. With the availability of quantitative analysis of RNA levels in postmortem tissues, several studies have assessed the postmortem degradation of constitutively expressed RNA species to estimate PMI. However, conventional RNA quantification as well as biochemical and physiological changes employed thus far have limitations related to standardization or normalization. The present study focuses on an interesting feature of the subdomains of certain RNA species, in which they are site-specifically cleaved during apoptotic cell death. We found that the D8 divergent domain of ribosomal RNA (rRNA) bearing cell death-related cleavage sites was rapidly removed during postmortem RNA degradation. In contrast to the fragile domain, the 5' terminal region of 28S rRNA was remarkably stable during the postmortem period. Importantly, the differences in the degradation rates between the two domains in mammalian 28S rRNA were highly proportional to increasing PMI with a significant linear correlation observed in mice as well as human autopsy tissues. In conclusion, we demonstrate that comparison of the degradation rates between domains of a single RNA species provides quantitative information on postmortem degradation states, which can be applied for the estimation of PMI.

  11. Covalent bond force profile and cleavage in a single polymer chain

    Science.gov (United States)

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

    2000-08-01

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

  12. Cleavage of rRNA ensures translational cessation in sperm at fertilization

    Science.gov (United States)

    Johnson, G.D.; Sendler, E.; Lalancette, C.; Hauser, R.; Diamond, M.P.; Krawetz, S.A.

    2011-01-01

    Intact ribosomal RNAs (rRNAs) comprise the majority of somatic transcripts, yet appear conspicuously absent in spermatozoa, perhaps reflecting cytoplasmic expulsion during spermatogenesis. To discern their fate, total RNA retained in mature spermatozoa from three fertile donors was characterized by Next Generation Sequencing. In all samples, >75% of total sequence reads aligned to rRNAs. The distribution of reads along the length of these transcripts exhibited a high degree of non-uniformity that was reiterated between donors. The coverage of sequencing reads was inversely correlated with guanine-cytosine (GC)-richness such that sequences greater than ∼70% GC were virtually absent in all sperm RNA samples. To confirm the loss of sequence, the relative abundance of specific regions of the 28S transcripts in sperm was established by 7-Deaza-2′-deoxy-guanosine-5′-triphosphate RT–PCR. The inability to amplify specific regions of the 28S sequence from sperm despite the abundant representation of this transcript in the sequencing libraries demonstrates that approximately three-quarters of RNA retained in the mature male gamete are products of rRNA fragmentation. Hence, cleavage (not expulsion of the RNA component of the translational machinery) is responsible for preventing spurious translation following spermiogenesis. These results highlight the potential importance of those transcripts, including many mRNAs, which evade fragmentation and remain intact when sperm are delivered at fertilization. Sequencing data are deposited in GEO as: GSE29160. PMID:21831882

  13. Homodinuclear lanthanide complexes of phenylthiopropionic acid: Synthesis, characterization, cytotoxicity, DNA cleavage, and antimicrobial activity

    Science.gov (United States)

    Shiju, C.; Arish, D.; Kumaresan, S.

    2013-03-01

    Lanthanide complexes of La(III), Pr(III), Nd(III), Sm(III), and Ho(III) with phenylthiopropionic acid were synthesized and characterized by elemental analysis, mass, IR, electronic spectra, molar conductance, TGA, and powder XRD. The results show that the lanthanide complexes are homodinuclear in nature. The two lanthanide ions are bridged by eight oxygen atoms from four carboxylate groups. Thermal decomposition profiles are consistent with the proposed formulations. Powder XRD studies show that all the complexes are amorphous in nature. Antimicrobial studies indicate that these complexes exhibit more activity than the ligand itself. The DNA cleavage activity of the ligand and its complexes were assayed on Escherichia coli DNA using gel electrophoresis in the presence of H2O2. The result shows that the Pr(III) and Nd(III) complexes have completely cleaved the DNA. The anticancer activities of the complexes have also been studied towards human cervical cancer cell line (HeLa) and colon cancer cells (HCT116) and it was found that the La(III) and Nd(III) complexes are more active than the corresponding Pr(III), Sm(III), Ho(III) complexes, and the free ligand on both the cancer cells.

  14. Anodic carbon-boron bond cleavage through the intermediacy of electrogenerated bromonium ion

    International Nuclear Information System (INIS)

    Shi Deqing; Gitkis, Anna; Becker, James Y.

    2007-01-01

    The electrochemical properties of a series of cyclic arylboronic esters, XC 6 H 4 B(OR) 2 [RR = CH 2 CH 2 ; X = H (1a); p-Me (1b); p-OMe (1c); p-Cl (1d); p-Ph (1e); m-Cl (1f); m-OMe (1g); CF 3 (1h); OMe (1i); 2,6-dimethyl (1j); 1b with RR = (CH 2 ) 3 , (1k); 1b with RR = CMe 2 CMe 2 , (1m)] has been studied in acetonitrile by cyclic voltammetry (CV) and controlled-potential electrolysis (CPE). The CV of representative examples of aryl borates with different substituents show one irreversible oxidation wave on a Pt cathode, at 1.8-1.9 V (vs. Ag/AgCl), with a negligible substituent effect. The cathodic CPE process led to small amounts of biaryls only, whereas the direct anodic CPE could not be carried out practically due to low currents. However, in the presence of electrogenerated bromonium (or iodonium) ions a C-B bond cleavage does take place to yield the corresponding bromoaryls, brominated phenols, and arylboronic acids as the major products

  15. Anodic carbon-boron bond cleavage through the intermediacy of electrogenerated bromonium ion

    Energy Technology Data Exchange (ETDEWEB)

    Shi Deqing; Gitkis, Anna [Department of Chemistry, Ben-Gurion University of the Negev, Beer Sheva 84105 (Israel); Becker, James Y. [Department of Chemistry, Ben-Gurion University of the Negev, Beer Sheva 84105 (Israel)], E-mail: becker@bgu.ac.il

    2007-12-31

    The electrochemical properties of a series of cyclic arylboronic esters, XC{sub 6}H{sub 4}B(OR){sub 2} [RR = CH{sub 2}CH{sub 2}; X = H (1a); p-Me (1b); p-OMe (1c); p-Cl (1d); p-Ph (1e); m-Cl (1f); m-OMe (1g); CF{sub 3} (1h); OMe (1i); 2,6-dimethyl (1j); 1b with RR = (CH{sub 2}){sub 3}, (1k); 1b with RR = CMe{sub 2}CMe{sub 2}, (1m)] has been studied in acetonitrile by cyclic voltammetry (CV) and controlled-potential electrolysis (CPE). The CV of representative examples of aryl borates with different substituents show one irreversible oxidation wave on a Pt cathode, at 1.8-1.9 V (vs. Ag/AgCl), with a negligible substituent effect. The cathodic CPE process led to small amounts of biaryls only, whereas the direct anodic CPE could not be carried out practically due to low currents. However, in the presence of electrogenerated bromonium (or iodonium) ions a C-B bond cleavage does take place to yield the corresponding bromoaryls, brominated phenols, and arylboronic acids as the major products.

  16. Synthesis, characterization, anti-microbial, DNA binding and cleavage studies of Schiff base metal complexes

    Directory of Open Access Journals (Sweden)

    Poomalai Jayaseelan

    2016-09-01

    Full Text Available A novel Schiff base ligand has been prepared by the condensation between butanedione monoxime with 3,3′-diaminobenzidine. The ligand and metal complexes have been characterized by elemental analysis, UV, IR, 1H NMR, conductivity measurements, EPR and magnetic studies. The molar conductance studies of Cu(II, Ni(II, Co(II and Mn(II complexes showed non-electrolyte in nature. The ligand acts as dibasic with two N4-tetradentate sites and can coordinate with two metal ions to form binuclear complexes. The spectroscopic data of metal complexes indicated that the metal ions are complexed with azomethine nitrogen and oxyimino nitrogen atoms. The binuclear metal complexes exhibit octahedral arrangements. DNA binding properties of copper(II metal complex have been investigated by electronic absorption spectroscopy. Results suggest that the copper(II complex bind to DNA via an intercalation binding mode. The nucleolytic cleavage activities of the ligand and their complexes were assayed on CT-DNA using gel electrophoresis in the presence and absence of H2O2. The ligand showed increased nuclease activity when administered as copper complex and copper(II complex behave as efficient chemical nucleases with hydrogen peroxide activation. The anti-microbial activities and thermal studies have also been studied. In anti-microbial activity all complexes showed good anti-microbial activity higher than ligand against gram positive, gram negative bacteria and fungi.

  17. Differential spatio-temporal expression of carotenoid cleavage dioxygenases regulates apocarotenoid fluxes during AM symbiosis.

    Science.gov (United States)

    López-Ráez, Juan A; Fernández, Iván; García, Juan M; Berrio, Estefanía; Bonfante, Paola; Walter, Michael H; Pozo, María J

    2015-01-01

    Apocarotenoids are a class of compounds that play important roles in nature. In recent years, a prominent role for these compounds in arbuscular mycorrhizal (AM) symbiosis has been shown. They are derived from carotenoids by the action of the carotenoid cleavage dioxygenase (CCD) enzyme family. In the present study, using tomato as a model, the spatio-temporal expression pattern of the CCD genes during AM symbiosis establishment and functioning was investigated. In addition, the levels of the apocarotenoids strigolactones (SLs), C13 α-ionol and C14 mycorradicin (C13/C14) derivatives were analyzed. The results suggest an increase in SLs promoted by the presence of the AM fungus at the early stages of the interaction, which correlated with an induction of the SL biosynthesis gene SlCCD7. At later stages, induction of SlCCD7 and SlCCD1 expression in arbusculated cells promoted the production of C13/C14 apocarotenoid derivatives. We show here that the biosynthesis of apocarotenoids during AM symbiosis is finely regulated throughout the entire process at the gene expression level, and that CCD7 constitutes a key player in this regulation. Once the symbiosis is established, apocarotenoid flux would be turned towards the production of C13/C14 derivatives, thus reducing SL biosynthesis and maintaining a functional symbiosis. Copyright © 2014 Elsevier Ireland Ltd. All rights reserved.

  18. Chromosome fragility at FRAXA in human cleavage stage embryos at risk for fragile X syndrome.

    Science.gov (United States)

    Verdyck, Pieter; Berckmoes, Veerle; De Vos, Anick; Verpoest, Willem; Liebaers, Inge; Bonduelle, Maryse; De Rycke, Martine

    2015-10-01

    Fragile X syndrome (FXS), the most common inherited intellectual disability syndrome, is caused by expansion and hypermethylation of the CGG repeat in the 5' UTR of the FMR1 gene. This expanded repeat, also known as the rare fragile site FRAXA, causes X chromosome fragility in cultured cells from patients but only when induced by perturbing pyrimidine synthesis. We performed preimplantation genetic diagnosis (PGD) on 595 blastomeres biopsied from 442 cleavage stage embryos at risk for FXS using short tandem repeat (STR) markers. In six blastomeres, from five embryos an incomplete haplotype was observed with loss of all alleles telomeric to the CGG repeat. In all five embryos, the incomplete haplotype corresponded to the haplotype carrying the CGG repeat expansion. Subsequent analysis of additional blastomeres from three embryos by array comparative genomic hybridization (aCGH) confirmed the presence of a terminal deletion with a breakpoint close to the CGG repeat in two blastomeres from one embryo. A blastomere from another embryo showed the complementary duplication. We conclude that a CGG repeat expansion at FRAXA causes X chromosome fragility in early human IVF embryos at risk for FXS. © 2015 Wiley Periodicals, Inc.

  19. 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

  20. 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

  1. 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

  2. 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.

  3. 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.

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

    Directory of Open Access Journals (Sweden)

    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.

  5. Transformation kinetics for nucleus clusters

    International Nuclear Information System (INIS)

    Villa, Elena; Rios, Paulo R.

    2009-01-01

    A rigorous mathematical approach based on stochastic geometry concepts is presented to extend previous Johnson-Mehl, Avrami, Kolmogorov treatment of transformation kinetics to situations in which nuclei are not homogeneously located in space but are located in clusters. An exact analytical solution is presented here for the first time assuming that nucleation sites follow a Matern cluster process. The influence of Matern cluster process parameters on subsequent growth kinetics and the microstructural path are illustrated by means of numerical examples. Moreover, using the superposition principle, exact analytical solutions are also obtained when nucleation takes place by a combination of a Matern cluster process and an inhomogeneous Poisson point process. The new solutions presented here significantly increase the number of exactly solvable cases available to formal kinetics.

  6. Chapter 22. Cell population kinetics

    International Nuclear Information System (INIS)

    Tubiana, M.

    1975-01-01

    The main contribution of radioisotopes to the development of a new discipline, cell population kinetics, was shown. The aim of this science is to establish, for each tissue of the organism, the life span of its component cells and the mechanisms governing its growth, its differentiation and its homeostasis with respect to outside attacks. Labelling techniques have been used to follow the cells during these various processes. The case of non-dividing cells was considered first, taking as example, the red blood cells of which the lifetime was studied, after which the case of proliferating cells was examined using 14 C- or tritium-labelled thymidine. The methods used to measure the cell cycle parameters were described: labelled-mitosis curve method, double-labelling and continuous labelling methods, proliferation coefficient measurement. Cell kinetics were shown to allow an interpretation of radiobiological data. Finally the practical value of cell kinetics research was shown [fr

  7. Kinetic theory and transport phenomena

    CERN Document Server

    Soto, Rodrigo

    2016-01-01

    This textbook presents kinetic theory, which is a systematic approach to describing nonequilibrium systems. The text is balanced between the fundamental concepts of kinetic theory (irreversibility, transport processes, separation of time scales, conservations, coarse graining, distribution functions, etc.) and the results and predictions of the theory, where the relevant properties of different systems are computed. The book is organised in thematic chapters where different paradigmatic systems are studied. The specific features of these systems are described, building and analysing the appropriate kinetic equations. Specifically, the book considers the classical transport of charges, the dynamics of classical gases, Brownian motion, plasmas, and self-gravitating systems, quantum gases, the electronic transport in solids and, finally, semiconductors. Besides these systems that are studied in detail, concepts are applied to some modern examples including the quark–gluon plasma, the motion of bacterial suspen...

  8. 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)

  9. A multicenter prospective study to assess the effect of early cleavage on embryo quality, implantation, and live-birth rate.

    Science.gov (United States)

    de los Santos, Maria José; Arroyo, Gemma; Busquet, Ana; Calderón, Gloria; Cuadros, Jorge; Hurtado de Mendoza, Maria Victoria; Moragas, Marta; Herrer, Raquel; Ortiz, Agueda; Pons, Carme; Ten, Jorge; Vilches, Miguel Angel; Figueroa, Maria José

    2014-04-01

    To investigate the impact of early cleavage (EC) on embryo quality, implantation, and live-birth rates. Prospective cross-sectional study. Multicenter study. Seven hundred embryo transfers and 1,028 early-stage human embryos. None. Implantation according to the presence of EC and embryo quality. The presence of EC is associated with embryo quality, especially in cycles with autologous oocytes. However, the use of EC as an additional criterion for selecting an embryo for transfer does not appear to significantly improve likelihood of implantation. Furthermore, embryos that presented EC had live-birth rates per implanted embryo similar to those that did not show any sign of cleavage. At least for conventional embryo culture and morphologic evaluations, the additional evaluation of EC in embryos may not be valuable to improve embryo implantation. Copyright © 2014 American Society for Reproductive Medicine. Published by Elsevier Inc. All rights reserved.

  10. 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

  11. 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.

  12. 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...

  13. 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.

  14. 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

  15. 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.

  16. Photo-assisted cyanation of transition metal nitrates coupled with room temperature C-C bond cleavage of acetonitrile.

    Science.gov (United States)

    Zou, Shihui; Li, Renhong; Kobayashi, Hisayoshi; Liu, Juanjuan; Fan, Jie

    2013-03-07

    It is a challenge to use acetonitrile as a cyanating agent because of the difficulty in cleaving its C-CN bond. Herein, we report a mild photo-assisted route to conduct the cyanation of transition metal nitrates using acetonitrile as the cyanating agent coupled with room-temperature C-C bond cleavage. DFT calculations and experimental observations suggest a radical-involved reaction mechanism, which excludes toxicity from free cyanide ions.

  17. Injection of an antibody against a p21 c-Ha-ras protein inhibits cleavage in axolotl eggs.

    OpenAIRE

    Baltus, E; Hanocq-Quertier, J; Hanocq, F; Brachet, J

    1988-01-01

    The presence of a ras protein was demonstrated in cleaving axolotl eggs by selective immunoprecipitation with a polyclonal antibody against a peptide encoded by the c-Ha-ras oncogene, cellular homolog of the v-Ha-ras oncogene of Harvey rat sarcoma virus. Injection of this antibody into axolotl oocytes subjected to progesterone treatment does not prevent meiotic maturation. Injection of the same antibody into a blastomere of axolotl eggs at the 2- or 4-cell stage causes cleavage arrest in the ...

  18. 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

  19. Modulation of HIV-1 Gag NC/p1 cleavage efficiency affects protease inhibitor resistance and viral replicative capacity

    Czech Academy of Sciences Publication Activity Database

    Maarseveen van, N. M.; Andersson, Dan; Lepšík, Martin; Fun, A.; Schipper, P. J.; Jong de, D.; Boucher, Ch. A. B.; Nijhuis, M.

    2012-01-01

    Roč. 9, č. 29 (2012), s. 1-7 ISSN 1742-4690 EU Projects: European Commission(XE) 37693 - HIV PI RESISTANCE Grant - others:Dutch AIDS Fund(XE) 2006028; (NWO) VIDI(XE) 91796349 Institutional research plan: CEZ:AV0Z40550506 Keywords : HIV-1 * protease * Gag * resistance * cleavage Subject RIV: CE - Biochemistry Impact factor: 5.657, year: 2012

  20. Selected readings in chemical kinetics

    CERN Document Server

    Back, Margaret H

    2013-01-01

    Selected Readings in Chemical Kinetics covers excerpts from 12 papers in the field of general and gas-phase kinetics. The book discusses papers on the laws of connexion between the conditions of a chemical change and its amount; on the reaction velocity of the inversion of the cane sugar by acids; and the calculation in absolute measure of velocity constants and equilibrium constants in gaseous systems. The text then tackles papers on simple gas reactions; on the absolute rate of reactions in condensed phases; on the radiation theory of chemical action; and on the theory of unimolecular reacti

  1. Chemical kinetics and reaction dynamics

    CERN Document Server

    Houston, Paul L

    2006-01-01

    This text teaches the principles underlying modern chemical kinetics in a clear, direct fashion, using several examples to enhance basic understanding. It features solutions to selected problems, with separate sections and appendices that cover more technical applications.Each chapter is self-contained and features an introduction that identifies its basic goals, their significance, and a general plan for their achievement. This text's important aims are to demonstrate that the basic kinetic principles are essential to the solution of modern chemical problems, and to show how the underlying qu

  2. Chemical kinetics and combustion modeling

    Energy Technology Data Exchange (ETDEWEB)

    Miller, J.A. [Sandia National Laboratories, Livermore, CA (United States)

    1993-12-01

    The goal of this program is to gain qualitative insight into how pollutants are formed in combustion systems and to develop quantitative mathematical models to predict their formation rates. The approach is an integrated one, combining low-pressure flame experiments, chemical kinetics modeling, theory, and kinetics experiments to gain as clear a picture as possible of the process in question. These efforts are focused on problems involved with the nitrogen chemistry of combustion systems and on the formation of soot and PAH in flames.

  3. Deuteration kinetics of the graphene

    Energy Technology Data Exchange (ETDEWEB)

    Nefedov, Alexei; Woell, Christof [KIT, Leopoldshafen (Germany); Paris, Alessio; Calliari, Lucia [FBK-CMM, Trento (Italy); Verbitskiy, Nikolay [MSU, Moscow (Russian Federation); University of Vienna, Vienna (Austria); Wang, Ying; Irle, Stephan [Nagoya University, Nagoya (Japan); Fedorov, Alexander [IFW Dresden, Dresden (Germany); St. Petersburg University, St. Petersburg (Russian Federation); Haberer, Danny; Knupfer, Martin; Buechner, Bernd [IFW Dresden, Dresden (Germany); Oetzelt, Martin [BESSY II, Berlin (Germany); Petaccia, Luca [Elettra, Trieste (Italy); Usachov, Dmitry [St. Petersburg University, St. Petersburg (Russian Federation); Vyalikh, Denis [St. Petersburg University, St. Petersburg (Russian Federation); TU Dresden, Dresden (Germany); Sagdev, Hermann [MPI fuer Polymerforschung, Mainz (Germany); Yashina, Lada [MSU, Moscow (Russian Federation); Grueneis, Alexander [IFW Dresden, Dresden (Germany); University of Vienna, Vienna (Austria)

    2013-07-01

    The kinetics of the hydrogenation/deuteration reaction of graphene was studied by time-dependent x-ray photoemission spectroscopy (XPS). The graphene layer was then exposed to hydrogen or deuterium atomic gas beams, obtained by thermal cracking in a tungsten capillary at T=3000 K. After each step XPS of the C1s line was performed in order to measure H/C and D/C ratios. We have observed a strong kinetic isotope effect for the hydrogenation/deuteration reaction leading to substantially faster adsorption and higher maximum D/C ratios as compared to H/C (D/C 35% vs. H/C 25%).

  4. Vlasov simulations of Kinetic Alfven Waves at proton kinetic scales

    NARCIS (Netherlands)

    C.L. Vasconez; F. Valentini (Francesco); E. Camporeale (Enrico); P. Veltri

    2014-01-01

    htmlabstractKinetic Alfv ́en waves represent an important subject in space plasma physics, since they are thought to play a crucial role in the development of the turbulent energy cascade in the solar wind plasma at short wavelengths (of the order of the proton inertial length d p and beyond). A

  5. 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.

  6. 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.

  7. 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

  8. 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.

  9. 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.

  10. 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.

  11. 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

  12. 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.

  13. 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

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

    Directory of Open Access Journals (Sweden)

    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 

  15. 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

  16. 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"

  17. Cleavages and co-operation in the UK alcohol industry: A qualitative study

    Directory of Open Access Journals (Sweden)

    Holden Chris

    2012-06-01

    Full Text Available Abstract Background It is widely believed that corporate actors exert substantial influence on the making of public health policy, including in the alcohol field. However, the industry is far from being monolithic, comprising a range of producers and retailers with varying and diverse interests. With a focus on contemporary debates concerning the minimum pricing of alcohol in the UK, this study examined the differing interests of actors within the alcohol industry, the cleavages which emerged between them on this issue and how this impacted on their ability to organise themselves collectively to influence the policy process. We conducted 35 semi-structured interviews between June and November 2010 with respondents from all sectors of the industry as well as a range of non-industry actors who had knowledge of the alcohol policy process, including former Ministers, Members of the UK Parliament and the Scottish Parliament, civil servants, members of civil society organisations and professionals. Methods The paper draws on an analysis of publicly available documents and 35 semi-structured interviews with respondents from the alcohol industry (on- and off-trade including retailers, producers of wines, spirits and beers and trade associations and a range of non-industry actors with knowledge of the alcohol policy process (including former Ministers, Members of Parliament and of the Scottish Parliament, civil servants, members of civil society organisations and professional groups. Interviews were recorded, transcribed and analysed using Nvivo qualitative analysis software. Processes of triangulation between data sources and different types of respondent sought to ensure we gained as accurate a picture as possible of industry participation in the policy process. Results Divergences of interest were evident between producers and retailers and within the retail sector between the on and off trade. Divisions within the alcohol industry, however, existed

  18. Hydrogen production through microheterogeneous photocatalysis of hydrogen sulfide cleavage. The thiosulfate cycle

    Energy Technology Data Exchange (ETDEWEB)

    Borgarello, E; Serpone, N; Graetzel, M; Pelizzetti, E

    1985-01-01

    Cleavage of hydrogen sulfide into hydrogen and sulfur occurs in alkaline aqueous CdS dispersions under visible light illumination. Small quantities of a noble metal catalyst (RuO/sub 2/) loaded onto 'naked' CdS particles markedly improve the yield of hydrogen formation. The effect of RuO/sub 2/ is ascribed to catalysis of electron transfer to proton. Simultaneous and efficient photogeneration of hydrogen and thiosulfate occurs in CdS dispersions containing both sulfite and bisulfide (or sulfide) ions. Electron transfer from the conduction band of CdS to that of TiO/sub 2/ particles occurs in alkaline suspensions containing these HS/sup -/ ions and has been exploited to improve the performance of a system achieving decomposition of H/sub 2/S by visible light. Equally important is a recent finding that the performance of a system containing 'naked' CdS in combination with RuO/sub 2/-loaded TiO/sub 2/ particles is far better than that of CdS/RuO/sub 2/ alone. Additionally, conduction band electrons produced by bandgap excitation of TiO/sub 2/ particles efficiently reduce thiosulfate to sulfide and sulfite. The valence band process in alklaine TiO/sub 2/ dispersions is thought to involve oxidation of S/sub 2/O/sub 3//sup 2 -/ to tetrathionate, S/sub 4/O/sub 6//sup 2 -/, which quantitatively dismutates into sulfite and thiosulfate. The photodriven disproportionation of thiosulfate into sulfide and sulfate is of great interest in systems that photochemically cleave hydrogen sulfide into hydrogen and sulfur. (author).

  19. Drosophila acetylcholinesterase: demonstration of a glycoinositol phospholipid anchor and an endogenous proteolytic cleavage

    International Nuclear Information System (INIS)

    Haas, R.; Marshall, T.L.; Rosenberry, T.L.

    1988-01-01

    The presence of a glycoinositol phospholipid anchor Drosophila acetylcholinesterase (AChE) was shown by several criteria. Chemical analysis of highly purified Drosophila AChE demonstrated approximately one residue of inositol per enzyme subunit. Selective cleavage by Staphylococcus aureus phosphatidylinositol-specific phospholipase C (PI-PLC) was tested with Drosophila AChE radiolabeled by the photoactivatable affinity probe 3-(trifluoromethyl)-3-(m-[ 125 I]iodophenyl)diazirine ([ 125 I]TID), a reagent that specifically labels the lipid moiety of glycoinositol phospholipid-anchored proteins. Digestion with PI-PLC released 75% of this radiolabel from the protein. Gel electrophoresis of Drosophila AChE in sodium dodecyl sulfate indicated prominent 55- and 16-kDa bands and a faint 70-kDa band. The [ 125 ]I]TID label was localized on the 55-kDa fragment, suggesting that this fragment is the C-terminal portion of the protein. In support of this conclusion, a sensitive microsequencing procedure that involved manual Edman degradation combined with radiomethylation was used to determine residues 2-5 of the 16-kDa fragment. Comparison with the Drosophila AChE cDNA sequence confirmed that the 16-kDa fragment includes the N-terminus of AChE. Furthermore, the position of the N-terminal amino acid of the mature Drosophila AChE is closely homologous to that of Torpedo AChE. The presence of radiomethylatable ethanolamine in both 16- and 55-kDa fragments was also confirmed. Thus, Drosophila AChE may include a second posttranslational modification involving ethanolamine

  20. Atomistic Details of the Associative Phosphodiester Cleavage in Human Ribonuclease H

    International Nuclear Information System (INIS)

    Elsasser, Brigitta M.; Fels, Gregor

    2010-01-01

    During translation of the genetic information of DNA into proteins, mRNA is synthesized by RNA polymerase and after the transcription process degraded by RNase H. The endoribonuclease RNase H is a member of the nucleotidyl-transferase (NT) superfamily and is known to hydrolyze the phosphodiester bonds of RNA which is hybridized to DNA. Retroviral RNase H is part of the viral reverse transcriptase enzyme that is indispensable for the proliferation of retroviruses, such as HIV. Inhibitors of this enzyme could therefore provide new drugs against diseases like AIDS. In our study we investigated the molecular mechanism of RNA cleavage by human RNase H using a comprehensive high level DFT/B3LYP QM/MM theoretical method for the calculation of the stationary points and nudged elastic band (NEB) and free energy calculations to identify the transition state structures, the rate limiting step and the reaction barrier. Our calculations reveal that the catalytic mechanism proceeds in two steps and that the nature of the nucleophile is a water molecule. In the first step, the water attack on the scissile phosphorous is followed by a proton transfer from the water to the O2P oxygen and a trigonal bipyramidal pentacoordinated phosphorane is formed. Subsequently, in the second step the proton is shuttled to the O30 oxygen to generate the product state. During the reaction mechanism two Mg2+ ions support the formation of a stable associated in-line SN2-type phosphorane intermediate. Our calculated energy barrier of 19.3 kcal mol*1 is in excellent agreement with experimental findings (20.5 kcal mol*1). These results may contribute to the clarification and understanding of the RNase H reaction mechanism and of further enzymes from the RNase family.

  1. Controllable synthesis of silver and silver sulfide nanocrystals via selective cleavage of chemical bonds

    International Nuclear Information System (INIS)

    Tang Aiwei; Wang Yu; Ye Haihang; Zhou Chao; Yang Chunhe; Li Xu; Peng Hongshang; Zhang Fujun; Hou Yanbing; Teng Feng

    2013-01-01

    A one-step colloidal process has been adopted to prepare silver (Ag) and silver sulfide (Ag 2 S) nanocrystals, thus avoiding presynthesis of an organometallic precursor and the injection of a toxic phosphine agent. During the reaction, a layered intermediate compound is first formed, which then acts as a precursor, decomposing into the nanocrystals. The composition of the as-obtained products can be controlled by selective cleavage of S–C bonds or Ag–S bonds. Pure Ag 2 S nanocrystals can be obtained by directly heating silver acetate (Ag(OAc)) and n-dodecanethiol (DDT) at 200 ° C without any surfactant, and pure Ag nanocrystals can be synthesized successfully if the reaction temperature is reduced to 190 ° C and the amount of DDT is decreased to 1 ml in the presence of a non-coordinating organic solvent (1-octadecene, ODE). Otherwise, the mixture of Ag and Ag 2 S is obtained by directly heating Ag(OAc) in DDT by increasing the reaction temperature or in a mixture of DDT and ODE at 200 ° C. The formation mechanism has been discussed in detail in terms of selective S–C and Ag–S bond dissociation due to the nucleophilic attack of DDT and the lower bonding energy of Ag–S. Interestingly, some products can easily self-assemble into two- or three-dimensional (2D or 3D) highly ordered superlattice structures on a copper grid without any additional steps. The excess DDT plays a key role in the superlattice structure due to the bundling and interdigitation of the thiolate molecules adsorbed on the as-obtained nanocrystals. (paper)

  2. Structure-function analysis of Staphylococcus aureus amidase reveals the determinants of peptidoglycan recognition and cleavage.

    Science.gov (United States)

    Büttner, Felix Michael; Zoll, Sebastian; Nega, Mulugeta; Götz, Friedrich; Stehle, Thilo

    2014-04-18

    The bifunctional major autolysin AtlA of Staphylococcus aureus cleaves the bacterium's peptidoglycan network (PGN) at two distinct sites during cell division. Deletion of the enzyme results in large cell clusters with disordered division patterns, indicating that AtlA could be a promising target for the development of new antibiotics. One of the two functions of AtlA is performed by the N-acetylmuramyl-l-alanine amidase AmiA, which cleaves the bond between the carbohydrate and the peptide moieties of PGN. To establish the structural requirements of PGN recognition and the enzymatic mechanism of cleavage, we solved the crystal structure of the catalytic domain of AmiA (AmiA-cat) in complex with a peptidoglycan-derived ligand at 1.55 Å resolution. The peptide stem is clearly visible in the structure, forming extensive contacts with protein residues by docking into an elongated groove. Less well defined electron density and the analysis of surface features indicate likely positions of the carbohydrate backbone and the pentaglycine bridge. Substrate specificity analysis supports the importance of the pentaglycine bridge for fitting into the binding cleft of AmiA-cat. PGN of S. aureus with l-lysine tethered with d-alanine via a pentaglycine bridge is completely hydrolyzed, whereas PGN of Bacillus subtilis with meso-diaminopimelic acid directly tethered with d-alanine is not hydrolyzed. An active site mutant, H370A, of AmiA-cat was completely inactive, providing further support for the proposed catalytic mechanism of AmiA. The structure reported here is not only the first of any bacterial amidase in which both the PGN component and the water molecule that carries out the nucleophilic attack on the carbonyl carbon of the scissile bond are present; it is also the first peptidoglycan amidase complex structure of an important human pathogen.

  3. STM observation of a box-shaped graphene nanostructure appeared after mechanical cleavage of pyrolytic graphite

    Energy Technology Data Exchange (ETDEWEB)

    Lapshin, Rostislav V., E-mail: rlapshin@gmail.com [Solid Nanotechnology Laboratory, Institute of Physical Problems, Zelenograd, Moscow 124460 (Russian Federation); Department of Photosensitive Nano and Microsystems, Moscow Institute of Electronic Technology, Zelenograd, Moscow 124498 (Russian Federation)

    2016-01-01

    Graphical abstract: - Highlights: • A previously unknown 3D box-shaped graphene (BSG) nanostructure has been detected. • The nanostructure is a multilayer system of parallel nanochannels having quadrangular cross-section. • Typical width of a nanochannel facet makes 25 nm, typical wall/facet thickness is 1 nm. • A mechanism qualitatively explaining the nanostructure formation has been proposed. • Possible applications of the BSG nanostructure are briefly discussed. - Abstract: A description is given of a three-dimensional box-shaped graphene (BSG) nanostructure formed/uncovered by mechanical cleavage of highly oriented pyrolytic graphite (HOPG). The discovered nanostructure is a multilayer system of parallel hollow channels located along the surface and having quadrangular cross-section. The thickness of the channel walls/facets is approximately equal to 1 nm. The typical width of channel facets makes about 25 nm, the channel length is 390 nm and more. The investigation of the found nanostructure by means of a scanning tunneling microscope (STM) allows us to draw a conclusion that it is possible to make spatial constructions of graphene similar to the discovered one by mechanical compression, bending, splitting, and shifting graphite surface layers. The distinctive features of such constructions are the following: simplicity of the preparation method, small contact area between graphene planes and a substrate, large surface area, nanometer cross-sectional sizes of the channels, large aspect ratio. Potential fields of application include: ultra-sensitive detectors, high-performance catalytic cells, nanochannels for DNA manipulation, nanomechanical resonators, electron multiplication channels, high-capacity sorbents for hydrogen storage.

  4. Patterns of proteolytic cleavage and carbodiimide derivatization in sarcoplasmic reticulum adenosinetriphosphatase

    International Nuclear Information System (INIS)

    de Ancos, J.G.; Inesi, G.

    1988-01-01

    Two series of experiments were carried out to characterize (a) peptide fragments of sarcoplasmic reticulum (SR) ATPase, based on proteolysis with different enzymes and distribution of known labels, and (b) specific labeling and functional inactivation patterns, following ATPase derivatization with dicyclohexylcarbodiimide (DCCD) under various conditions. Digestion with trypsin or chymotrypsin results in the initial cleavage of the SR ATPase in two fragments of similar size and then into smaller fragments, while subtilisin and thermolysin immediately yield smaller fragments. Peptide fragments were assigned to segments of the protein primary structure and to functionally relevant domains, such as those containing the 32 P at the active site and the fluorescein isothiocyanate at the nucleotide site. ATPase derivatization with [ 14 C]DCCD under mild conditions produced selective inhibition of ATPase hydrolytic catalysis without significant incorporation of the 14 C radioactive label. This effect is attributed to blockage of catalytically active residues by reaction of the initial DCCD adduct with endogenous or exogenous nucleophiles. ATPase derivatization with [ 14 C]DCCD under more drastic conditions produced inhibition of calcium binding, 14 C radioactive labeling of tryptic fragments A 1 and A 2 (but not of B), and extensive cross-linking. The presence of calcium during derivatization prevented functional inactivation, radioactive labeling of fragment A 2 , and internal cross-linking of fragment A 1 . It is proposed that both A 1 and A 2 fragments participate in formation of the calcium binding domain and that the labeled residues of fragment A 2 are directly involved in calcium complexation. A diagram is constructed, representing the relative positions of labels and functional domains within the ATPase protein

  5. Oxidative cleavage of a phenolic diarylpropane lignin model dimer by manganese peroxidase from Phanerochaete chrysosporium

    International Nuclear Information System (INIS)

    Wariishi, Hiroyuki; Valli, K.; Gold, M.H.

    1989-01-01

    In the presence of Mn II and H 2 O 2 , homogeneous manganese peroxidase oxidized 1-(3,5-dimethoxy-4-hydroxyphenyl)-2-(4-methoxyphenyl)-1,3-dihydroxypropane (I) to yield 1-(3,5-dimethoxy-4-hydroxyphenyl)-2-(4-methoxyphenyl)-1-oxo-3-hydroxypropane (II), 2,6-dimethoxy-1,4-benzoquinone (III), 2,6-dimethoxy-1,4-dihydroxybenzene (IV), 1-(4-methoxyphenyl)-1-oxo-2-hydroxyethane (V), 1-(4-methoxyphenyl)-1,2-dihydroxyethane (VI), syringaldehyde (VIII), and 2-(4-methoxyphenyl)-3-hydroxypropanal (IX). Chemically prepared manganese(III) malonate catalyzed the same reactions. Oxidation of I in H 2 18 O under argon resulted in >80% incorporation of 18 O into the phenylglycol VI, the hydroquinone IV, and the quinone III. Oxidation of I in H 2 18 O under aerobic conditions resulted in 40% incorporation of 18 O into VI but no 18 O incorporation into V. Finally, oxidation of I under 18 O 2 resulted in 89% and 28% incorporation of 18 O into V and VI, respectively. These results are explained by mechanisms involving the one-electron oxidation of the substrate I by enzyme-generated Mn III to produce a phenoxy radical intermediate I'. Subsequent C α -C β bond cleavage of the radical intermediate yields syringaldehyde (VIII) and a C 6 -C 2 benzylic radical. Syringaldehyde is oxidized by Mn III in several steps to a cyclohexadiene cation intermediate I double-prime, which is attacked by water to yield the benzoquinone III. The C 6 -C 2 radical is scavenged by O 2 to form a peroxy radical that decomposes to V and VI. In these reactions, Mn III generated by manganese peroxidase catalyzes both formation of the substrate phenoxy radical and oxidation of carbon-centered radical intermediates, to yield reactive cations

  6. Can intermuscular cleavage planes provide proper transverse screw angle? Comparison of two paraspinal approaches.

    Science.gov (United States)

    Cheng, Xiaofei; Ni, Bin; Liu, Qi; Chen, Jinshui; Guan, Huapeng

    2013-01-01

    The goal of this study was to determine which paraspinal approach provided a better transverse screw angle (TSA) for each vertebral level in lower lumbar surgery. Axial computed tomography (CT) images of 100 patients, from L3 to S1, were used to measure the angulation parameters, including transverse pedicle angle (TPA) and transverse cleavage plane angle (TCPA) of entry from the two approaches. The difference value between TCPA and TPA, defined as difference angle (DA), was calculated. Statistical differences of DA obtained by the two approaches and the angulation parameters between sexes, and the correlation between each angulation parameter and age or body mass index (BMI) were analyzed. TPA ranged from about 16° at L3 to 30° at S1. TCPA through the Wiltse's and Weaver's approach ranged from about -10° and 25° at L3 to 12° and 32° at S1, respectively. The absolute values of DA through the Weaver's approach were significantly lower than those through the Wiltse's approach at each level. The angulation parameters showed no significant difference with sex and no significant correlation with age or BMI. In the lower lumbar vertebrae (L3-L5) and S1, pedicle screw placement through the Weaver's approach may more easily yield the preferred TSA consistent with TPA than that through the Wiltse's approach. The reference values obtained in this paper may be applied regardless of sex, age or BMI and the descriptive statistical results may be used as references for applying the two paraspinal approaches.

  7. Structure-Function Analysis of Staphylococcus aureus Amidase Reveals the Determinants of Peptidoglycan Recognition and Cleavage*

    Science.gov (United States)

    Büttner, Felix Michael; Zoll, Sebastian; Nega, Mulugeta; Götz, Friedrich; Stehle, Thilo

    2014-01-01

    The bifunctional major autolysin AtlA of Staphylococcus aureus cleaves the bacterium's peptidoglycan network (PGN) at two distinct sites during cell division. Deletion of the enzyme results in large cell clusters with disordered division patterns, indicating that AtlA could be a promising target for the development of new antibiotics. One of the two functions of AtlA is performed by the N-acetylmuramyl-l-alanine amidase AmiA, which cleaves the bond between the carbohydrate and the peptide moieties of PGN. To establish the structural requirements of PGN recognition and the enzymatic mechanism of cleavage, we solved the crystal structure of the catalytic domain of AmiA (AmiA-cat) in complex with a peptidoglycan-derived ligand at 1.55 Å resolution. The peptide stem is clearly visible in the structure, forming extensive contacts with protein residues by docking into an elongated groove. Less well defined electron density and the analysis of surface features indicate likely positions of the carbohydrate backbone and the pentaglycine bridge. Substrate specificity analysis supports the importance of the pentaglycine bridge for fitting into the binding cleft of AmiA-cat. PGN of S. aureus with l-lysine tethered with d-alanine via a pentaglycine bridge is completely hydrolyzed, whereas PGN of Bacillus subtilis with meso-diaminopimelic acid directly tethered with d-alanine is not hydrolyzed. An active site mutant, H370A, of AmiA-cat was completely inactive, providing further support for the proposed catalytic mechanism of AmiA. The structure reported here is not only the first of any bacterial amidase in which both the PGN component and the water molecule that carries out the nucleophilic attack on the carbonyl carbon of the scissile bond are present; it is also the first peptidoglycan amidase complex structure of an important human pathogen. PMID:24599952

  8. Transfer of human frozen-thawed embryos with further cleavage during culture increases pregnancy rates

    Directory of Open Access Journals (Sweden)

    Bharat V Joshi

    2010-01-01

    Full Text Available Aim: To compare the pregnancy rate following transfer of frozen-thawed embryos with or without overnight culture after thawing. Settings and Design: This is a retrospective analysis of frozen-thawed embryo transfer (FET cycles performed between January 2006 and December 2008. Materials and Methods: Out of 518 thaw cycles, 504 resulted in embryo transfers (ETs. Of the total FET cycles, 415 were performed after an overnight culture of embryos (group A; and in 89 cycles, ET was performed within 2 hours of embryo thawing (group B. Statistical Analysis: The data were statistically analyzed using chi-square test. Results: We observed that with FET, women ≤30 years of age had a significantly higher (P=0.003 pregnancy rate (PR=28.9% as compared to women >30 years of age (17.5%. A significantly higher (P<0.001FNx08 pregnancy rate was also observed in women receiving 3 frozen-thawed embryos (29% as compared to those who received less than 3 embryos (10.7%. The difference in PR between group A (PR=24.3% and group B (PR=20.3% was not statistically significant. However, within group A, ET with cleaved embryos showed significantly ( P≤0.01 higher pregnancy rate compared to the uncleaved embryos, depending on the number of cleaved embryos transferred. Conclusion: No significant difference was noticed between FETs made with transfer of embryos with overnight culture and those without culture. However, within the cultured group, transfer of embryos cleaved during overnight culture gave significantly higher PR than transfers without any cleavage.

  9. Interactions of tetracationic porphyrins with DNA and their effects on DNA cleavage

    Science.gov (United States)

    Lebedeva, Natalya Sh.; Yurina, Elena S.; Gubarev, Yury A.; Syrbu, Sergey A.

    2018-06-01

    The interaction of tetracationic porphyrins with DNA was studied using UV-Vis absorption, fluorescence spectroscopy and viscometry, and the particle sizes were determined. Аs cationic porphyrins, two isomer porphyrins, 3,3‧,3″,3‴-(5,10,15,20-Porphyrintetrayl)tetrakis(1-methylpyridinium) (TMPyP3) and 4,4‧,4″,4‴-(5,10,15,20-Porphyrintetrayl)tetrakis(1-methylpyridinium) (TMPyP4), were studied. They differ in the position of NCH3+ group in phenyl ring of the porphyrins and hence, in degree of freedom of rotation of the phenyl rings about the central macrocycle. It was found that intercalated complexes are formed at DNA/porphyrin molar ratios (R) of 2.2 and 3.9 for TMPyP3 и TMPyP4, respectively. Decreasing R up to 0.4 and 0.8 for TMPyP3 и TMPyP4, respectively, leads mainly to formation of outside complexes due to π-π stacking between the porphyrin chromophores interacting electrostatically with phosphate framework of DNA. Each type of the obtained complexes was characterized using Scatchard approach. It was ascertained that the affinity of TMPyP4 to DNA is stronger than TMPyP3, meanwhile the wedge effect of the latter is higher. The differences between the porphyrin isomers become more evident at irradiation of their complexes with DNA. It was established that irradiation of the intercalated complexes results in DNA fragmentation. In the case of TMPyP4, DNA fragments of different size are formed. The irradiation of the outside DNA/porphyrin complexes leads to cleavage of DNA (TMPyP3 and TMPyP4) and partial destruction of the complex due to photolysis of the porphyrin (TMPyP3).

  10. Cleavage of Hyaluronan and CD44 Adhesion Molecule Regulate Astrocyte Morphology via Rac1 Signalling.

    Directory of Open Access Journals (Sweden)

    Anna Konopka

    Full Text Available Communication of cells with their extracellular environment is crucial to fulfill their function in physiological and pathophysiological conditions. The literature data provide evidence that such a communication is also important in case of astrocytes. Mechanisms that contribute to the interaction between astrocytes and extracellular matrix (ECM proteins are still poorly understood. Hyaluronan is the main component of ECM in the brain, where its major receptor protein CD44 is expressed by a subset of astrocytes. Considering the fact that functions of astrocytes are tightly coupled with changes in their morphology (e.g.: glutamate clearance in the synaptic cleft, migration, astrogliosis, we investigated the influence of hyaluronan cleavage by hyaluronidase, knockdown of CD44 by specific shRNA and CD44 overexpression on astrocyte morphology. Our results show that hyaluronidase treatment, as well as knockdown of CD44, in astrocytes result in a "stellate"-like morphology, whereas overexpression of CD44 causes an increase in cell body size and changes the shape of astrocytes into flattened cells. Moreover, as a dynamic reorganization of the actin cytoskeleton is supposed to be responsible for morphological changes of cells, and this reorganization is controlled by small GTPases of the Rho family, we hypothesized that GTPase Rac1 acts as a downstream effector for hyaluronan and CD44 in astrocytes. We used FRET-based biosensor and a dominant negative mutant of Rac1 to investigate the involvement of Rac1 activity in hyaluronidase- and CD44-dependent morphological changes of astrocytes. Both, hyaluronidase treatment and knockdown of CD44, enhances Rac1 activity while overexpression of CD44 reduces the activity state in astrocytes. Furthermore, morphological changes were blocked by specific inhibition of Rac1 activity. These findings indicate for the first time that regulation of Rac1 activity is responsible for hyaluronidase and CD44-driven morphological

  11. Cleavage of Hyaluronan and CD44 Adhesion Molecule Regulate Astrocyte Morphology via Rac1 Signalling.

    Science.gov (United States)

    Konopka, Anna; Zeug, Andre; Skupien, Anna; Kaza, Beata; Mueller, Franziska; Chwedorowicz, Agnieszka; Ponimaskin, Evgeni; Wilczynski, Grzegorz M; Dzwonek, Joanna

    2016-01-01

    Communication of cells with their extracellular environment is crucial to fulfill their function in physiological and pathophysiological conditions. The literature data provide evidence that such a communication is also important in case of astrocytes. Mechanisms that contribute to the interaction between astrocytes and extracellular matrix (ECM) proteins are still poorly understood. Hyaluronan is the main component of ECM in the brain, where its major receptor protein CD44 is expressed by a subset of astrocytes. Considering the fact that functions of astrocytes are tightly coupled with changes in their morphology (e.g.: glutamate clearance in the synaptic cleft, migration, astrogliosis), we investigated the influence of hyaluronan cleavage by hyaluronidase, knockdown of CD44 by specific shRNA and CD44 overexpression on astrocyte morphology. Our results show that hyaluronidase treatment, as well as knockdown of CD44, in astrocytes result in a "stellate"-like morphology, whereas overexpression of CD44 causes an increase in cell body size and changes the shape of astrocytes into flattened cells. Moreover, as a dynamic reorganization of the actin cytoskeleton is supposed to be responsible for morphological changes of cells, and this reorganization is controlled by small GTPases of the Rho family, we hypothesized that GTPase Rac1 acts as a downstream effector for hyaluronan and CD44 in astrocytes. We used FRET-based biosensor and a dominant negative mutant of Rac1 to investigate the involvement of Rac1 activity in hyaluronidase- and CD44-dependent morphological changes of astrocytes. Both, hyaluronidase treatment and knockdown of CD44, enhances Rac1 activity while overexpression of CD44 reduces the activity state in astrocytes. Furthermore, morphological changes were blocked by specific inhibition of Rac1 activity. These findings indicate for the first time that regulation of Rac1 activity is responsible for hyaluronidase and CD44-driven morphological changes of

  12. 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.

  13. 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.

  14. 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.

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

    Science.gov (United States)

    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.

  16. Elements of plasma kinetic theory

    International Nuclear Information System (INIS)

    Guasp, J.

    1976-01-01

    The physical foundations of plasma kinetic equations are exposed inside a series of seminars on plasma and fusion physics. The Vlasov and collisional equations with its application range have been discussed. The momenta equations for the macroscopic magnitudes and the more usual approximations have been obtained: two fluid equations for cold and warm plasmas, magnetohydrodynamic equations and the double-adiabatic theory. (author)

  17. Kinetic studies on leucite precursors

    Czech Academy of Sciences Publication Activity Database

    Mrázová, M.; Kloužková, A.; Kohoutková, Martina

    2009-01-01

    Roč. 7, č. 2 (2009), s. 205-210 ISSN 1895-1066 R&D Projects: GA MPO 2A-1TP1/063 Institutional research plan: CEZ:AV0Z40320502 Keywords : leucite * crystallization kinetics * hydrothermal Subject RIV: JH - Ceramics, Fire-Resistant Materials and Glass Impact factor: 1.065, year: 2009

  18. Some parameters of radionuclide kinetics

    International Nuclear Information System (INIS)

    Prokof'ev, O.N.; Smirnov, V.A.; Belen'kij, E.I.

    1978-01-01

    Numerical values of the rates of radionuclide absorption into, and elimination from, bovine organs were determined. Kinetic rate constants of radionuclides such as 89 Sr, 99 Mo, 131 I, 132 Tl, and 140 Be were calculated. The calculations were done for muscle, liver, and kidney

  19. Solving Simple Kinetics without Integrals

    Science.gov (United States)

    de la Pen~a, Lisandro Herna´ndez

    2016-01-01

    The solution of simple kinetic equations is analyzed without referencing any topic from differential equations or integral calculus. Guided by the physical meaning of the rate equation, a systematic procedure is used to generate an approximate solution that converges uniformly to the exact solution in the case of zero, first, and second order…

  20. Robustness Analysis of Kinetic Structures

    DEFF Research Database (Denmark)

    Kirkegaard, Poul Henning; Sørensen, John Dalsgaard

    2009-01-01

    Kinetic structures in architecture follows a new trend which is emerging in responsive architecture coined by Nicholas Negroponte when he proposed that architecture may benefit from the integration of computing power into built spaces and structures, and that better performing, more rational...