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Sample records for cell cleavage

  1. Failure of cell cleavage induces senescence in tetraploid primary cells.

    Science.gov (United States)

    Panopoulos, Andreas; Pacios-Bras, Cristina; Choi, Justin; Yenjerla, Mythili; Sussman, Mark A; Fotedar, Rati; Margolis, Robert L

    2014-10-15

    Tetraploidy can arise from various mitotic or cleavage defects in mammalian cells, and inheritance of multiple centrosomes induces aneuploidy when tetraploid cells continue to cycle. Arrest of the tetraploid cell cycle is therefore potentially a critical cellular control. We report here that primary rat embryo fibroblasts (REF52) and human foreskin fibroblasts become senescent in tetraploid G1 after drug- or small interfering RNA (siRNA)-induced failure of cell cleavage. In contrast, T-antigen-transformed REF52 and p53+/+ HCT116 tumor cells rapidly become aneuploid by continuing to cycle after cleavage failure. Tetraploid primary cells quickly become quiescent, as determined by loss of the Ki-67 proliferation marker and of the fluorescent ubiquitination-based cell cycle indicator/late cell cycle marker geminin. Arrest is not due to DNA damage, as the γ-H2AX DNA damage marker remains at control levels after tetraploidy induction. Arrested tetraploid cells finally become senescent, as determined by SA-β-galactosidase activity. Tetraploid arrest is dependent on p16INK4a expression, as siRNA suppression of p16INK4a bypasses tetraploid arrest, permitting primary cells to become aneuploid. We conclude that tetraploid primary cells can become senescent without DNA damage and that induction of senescence is critical to tetraploidy arrest.

  2. Cell-surface acceleration of urokinase-catalyzed receptor cleavage

    DEFF Research Database (Denmark)

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

    1997-01-01

    937 cell lysates, had the same amino termini as uPAR(2+3), generated by uPA in a purified system. In both cases cleavage had occurred at two positions in the hinge region connecting domain 1 and 2, between Arg83-Ala84 and Arg89-Ser90, respectively. The uPA-catalyzed cleavage of uPAR is a new negative...

  3. Glycoprotein B cleavage is important for murid herpesvirus 4 to infect myeloid cells.

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    Glauser, Daniel L; Milho, Ricardo; Frederico, Bruno; May, Janet S; Kratz, Anne-Sophie; Gillet, Laurent; Stevenson, Philip G

    2013-10-01

    Glycoprotein B (gB) is a conserved herpesvirus virion component implicated in membrane fusion. As with many-but not all-herpesviruses, the gB of murid herpesvirus 4 (MuHV-4) is cleaved into disulfide-linked subunits, apparently by furin. Preventing gB cleavage for some herpesviruses causes minor infection deficits in vitro, but what the cleavage contributes to host colonization has been unclear. To address this, we mutated the furin cleavage site (R-R-K-R) of the MuHV-4 gB. Abolishing gB cleavage did not affect its expression levels, glycosylation, or antigenic conformation. In vitro, mutant viruses entered fibroblasts and epithelial cells normally but had a significant entry deficit in myeloid cells such as macrophages and bone marrow-derived dendritic cells. The deficit in myeloid cells was not due to reduced virion binding or endocytosis, suggesting that gB cleavage promotes infection at a postendocytic entry step, presumably viral membrane fusion. In vivo, viruses lacking gB cleavage showed reduced lytic spread in the lungs. Alveolar epithelial cell infection was normal, but alveolar macrophage infection was significantly reduced. Normal long-term latency in lymphoid tissue was established nonetheless.

  4. Global identification of target recognition and cleavage by the Microprocessor in human ES cells.

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    Seong, Youngmo; Lim, Do-Hwan; Kim, Augustine; Seo, Jae Hong; Lee, Young Sik; Song, Hoseok; Kwon, Young-Soo

    2014-11-10

    The Microprocessor plays an essential role in canonical miRNA biogenesis by facilitating cleavage of stem-loop structures in primary transcripts to yield pre-miRNAs. Although miRNA biogenesis has been extensively studied through biochemical and molecular genetic approaches, it has yet to be addressed to what extent the current miRNA biogenesis models hold true in intact cells. To address the issues of in vivo recognition and cleavage by the Microprocessor, we investigate RNAs that are associated with DGCR8 and Drosha by using immunoprecipitation coupled with next-generation sequencing. Here, we present global protein-RNA interactions with unprecedented sensitivity and specificity. Our data indicate that precursors of canonical miRNAs and miRNA-like hairpins are the major substrates of the Microprocessor. As a result of specific enrichment of nascent cleavage products, we are able to pinpoint the Microprocessor-mediated cleavage sites per se at single-nucleotide resolution. Unexpectedly, a 2-nt 3' overhang invariably exists at the ends of cleaved bases instead of nascent pre-miRNAs. Besides canonical miRNA precursors, we find that two novel miRNA-like structures embedded in mRNAs are cleaved to yield pre-miRNA-like hairpins, uncoupled from miRNA maturation. Our data provide a framework for in vivo Microprocessor-mediated cleavage and a foundation for experimental and computational studies on miRNA biogenesis in living cells.

  5. Electrochemical Protein Cleavage in a Microfluidic Cell with Integrated Boron Doped Diamond Electrodes

    NARCIS (Netherlands)

    van den Brink, Floris T G; Zhang, Tao; Ma, Liwei; Bomer, Johan; Odijk, Mathieu; Olthuis, Wouter; Permentier, Hjalmar P; Bischoff, Rainer; van den Berg, Albert

    2016-01-01

    Specific electrochemical cleavage of peptide bonds at the C-terminal side of tyrosine and tryptophan generates peptides amenable to liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis for protein identification. To this end we developed a microfluidic electrochemical cell of 160 nL vo

  6. Polarity and cell division orientation in the cleavage embryo: from worm to human

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    Ajduk, Anna; Zernicka-Goetz, Magdalena

    2016-01-01

    Cleavage is a period after fertilization, when a 1-cell embryo starts developing into a multicellular organism. Due to a series of mitotic divisions, the large volume of a fertilized egg is divided into numerous smaller, nucleated cells—blastomeres. Embryos of different phyla divide according to different patterns, but molecular mechanism of these early divisions remains surprisingly conserved. In the present paper, we describe how polarity cues, cytoskeleton and cell-to-cell communication interact with each other to regulate orientation of the early embryonic division planes in model animals such as Caenorhabditis elegans, Drosophila and mouse. We focus particularly on the Par pathway and the actin-driven cytoplasmic flows that accompany it. We also describe a unique interplay between Par proteins and the Hippo pathway in cleavage mammalian embryos. Moreover, we discuss the potential meaning of polarity, cytoplasmic dynamics and cell-to-cell communication as quality biomarkers of human embryos. PMID:26660321

  7. Trichomonas vaginalis metalloproteinase induces mTOR cleavage of SiHa cells.

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

    2014-12-01

    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.

  8. Structural basis of cell wall cleavage by a staphylococcal autolysin.

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    Sebastian Zoll

    2010-03-01

    Full Text Available The major autolysins (Atl of Staphylococcus epidermidis and S. aureus play an important role in cell separation, and their mutants are also attenuated in virulence. Therefore, autolysins represent a promising target for the development of new types of antibiotics. Here, we report the high-resolution structure of the catalytically active amidase domain AmiE (amidase S. epidermidis from the major autolysin of S. epidermidis. This is the first protein structure with an amidase-like fold from a bacterium with a gram-positive cell wall architecture. AmiE adopts a globular fold, with several alpha-helices surrounding a central beta-sheet. Sequence comparison reveals a cluster of conserved amino acids that define a putative binding site with a buried zinc ion. Mutations of key residues in the putative active site result in loss of activity, enabling us to propose a catalytic mechanism. We also identified and synthesized muramyltripeptide, the minimal peptidoglycan fragment that can be used as a substrate by the enzyme. Molecular docking and digestion assays with muramyltripeptide derivatives allow us to identify key determinants of ligand binding. This results in a plausible model of interaction of this ligand not only for AmiE, but also for other PGN-hydrolases that share the same fold. As AmiE active-site mutations also show a severe growth defect, our findings provide an excellent platform for the design of specific inhibitors that target staphylococcal cell separation and can thereby prevent growth of this pathogen.

  9. Differential cleavage of IRES trans-acting factors (ITAFs) in cells infected by human rhinovirus.

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    Chase, Amanda J; Semler, Bert L

    2014-01-20

    Human rhinovirus (HRV) is a major causative agent of the common cold, and thus has several important health implications. As a member of the picornavirus family, HRV has a small genomic RNA that utilizes several host cell proteins for RNA replication. Host proteins poly(rC) binding protein 2 (PCBP2) and polypyrimidine tract binding protein (PTB) are cleaved by a viral proteinase during the course of infection by the related picornavirus, poliovirus. The cleavage of PCBP2 and PTB inhibits poliovirus translation and has been proposed to mediate a switch in poliovirus template usage from translation to RNA replication. HRV RNA replication also requires a switch in template usage from translation to RNA replication; however, the mechanism is not yet known. We demonstrate that PCBP2 and PTB are differentially cleaved during HRV infection in different cell lines, suggesting that HRV utilizes a mechanism distinct from PCBP2 or PTB cleavage to mediate a switch in template usage.

  10. Site of cleavage in infected cells and polypeptides of representative paramyxoviruses grown in cultured cells of the chorioallantoic membrane

    Energy Technology Data Exchange (ETDEWEB)

    Seto, J.T.; Garten, W.; Rott, R. (Giessen Univ. (Germany, F.R.))

    1981-01-01

    Cultured cells of the chorioallantoic membrane (CAM) fulfilled the need of using the same cell system that was permissive for representative paramyxoviruses to carry out studies on the biosynthesis of their glycoproteins in infected cells. The polypeptide composition of the respective paramyxoviruses (Newcastle disease virus (NDV), paramyxovirus Yucaipa (PMY), and Sendai virus), grown in eggs and CAM-cells, was essentially identical. In egg-grown PMY a large glycoprotein (LGP) was present but only in some CAM-grown preparations of virus labeled with (/sup 3/H)-glucosamine and rarely in (/sup 35/S)-methionine or (/sup 3/H)-amino acids (valine, leucine, and tyrosine) labeled viruses. The site of cleavage of precursor F/sub 0/ to Fsub(1,2) was not the same. In contrast to the cleavage of Sendai virus glycoprotein, cleavage was intracellular in NDV and PMY infected cells. Homologous antisera against the glycoproteins failed to inhibit cleavage of HN/sub 0/ or F/sub 0/ in cells infected with the representative paramyxoviruses.

  11. 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) and penetratin(43-58), was through Fmoc (fluoren-9-ylmethoxycarbonyl) chemistry. Metabolic degradation kinetics of the tested CPP in contact with three cell-cultured epithelial models, MDCK (Madin-Darby canine kidney), Calu-3 and TR146, was evaluated by reversed-phase HPLC. Identification of the resulting...

  12. A Single-Cell Platform for Monitoring Viral Proteolytic Cleavage in Different Cellular Compartments

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    Abbadessa, Darin; Smurthwaite, Cameron A.; Reed, Connor W.; Wolkowicz, Roland

    2015-01-01

    Infectious diseases affect human health despite advances in biomedical research and drug discovery. Among these, viruses are especially difficult to tackle due to the sudden transfer from animals to humans, high mutational rates, resistance to current treatments, and the intricacies of their molecular interactions with the host. As an example of these interactions, we describe a cell-based approach to monitor specific proteolytic events executed by either the viral-encoded protease or by host proteins on the virus. We then emphasize the significance of examining proteolysis within the subcellular compartment where cleavage occurs naturally. We show the power of stable expression, highlighting the usefulness of the cell-based multiplexed approach, which we have adapted to two independent assays previously developed to monitor (a) the activity of the HIV-1-encoded protease or (b) the cleavage of the HIV-1-encoded envelope protein by the host. Multiplexing was achieved by mixing cells each carrying a different assay or, alternatively, by engineering cells expressing two assays. Multiplexing relies on the robustness of the individual assays and their clear discrimination, further enhancing screening capabilities in an attempt to block proteolytic events required for viral infectivity and spread. PMID:27688710

  13. Proteolytic cleavage of protein kinase Cmu upon induction of apoptosis in U937 cells.

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    Häussermann, S; Kittstein, W; Rincke, G; Johannes, F J; Marks, F; Gschwendt, M

    1999-12-03

    Treatment of U937 cells with various apoptosis-inducing agents, such as TNFalpha and beta-D-arabinofuranosylcytosine (ara-C) alone or in combination with the phorbol ester 12-O-tetradecanoylphorbol-13-acetate (TPA), bryostatin 1 or cycloheximide, causes proteolytic cleavage of protein kinase Cmu (PKCmu) between the regulatory and catalytic domain, generating a 62 kDa catalytic fragment of the kinase. The formation of this fragment is effectively suppressed by the caspase-3 inhibitor Z-DEVD-FMK. In accordance with these in vivo data, treatment of recombinant PKCmu with caspase-3 in vitro results also in the generation of a 62 kDa fragment (p62). Treatment of several aspartic acid to alanine mutants of PKCmu with caspase-3 resulted in an unexpected finding. PKCmu is not cleaved at one of the typical cleavage sites containing the motif DXXD but at the atypical site CQND378/S379. The respective fragment (amino acids 379-912) was expressed in bacteria as a GST fusion protein (GST-p62) and partially purified. In contrast to the intact kinase, the fragment does not respond to the activating cofactors TPA and phosphatidylserine and is thus unable to phosphorylate substrates effectively.

  14. Cleavage of Armadillo/beta-catenin by the caspase DrICE in Drosophila apoptotic epithelial cells

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    Kessler Thomas

    2009-02-01

    Full Text Available Abstract Background During apoptosis cells become profoundly restructured through concerted cleavage of cellular proteins by caspases. In epithelial tissues, apoptotic cells loose their apical/basal polarity and are extruded from the epithelium. We used the Drosophila embryo as a system to investigate the regulation of components of the zonula adherens during apoptosis. Since Armadillo/beta-catenin (Arm is a major regulator of cadherin-mediated adhesion, we analyzed the mechanisms of Arm proteolysis in apoptosis. Results We define early and late apoptotic stages and find that early in apoptosis Dα-catenin remains relatively stable, while Arm and DE-cadherin protein levels are strongly reduced. Arm is cleaved by caspases in embryo extracts and we provide evidence that the caspase-3 homolog drICE cleaves Arm in vitro and in vivo. Cleavage by drICE creates a stable protein fragment that remains associated with the plasma membrane early in apoptosis. To further understand the role of caspase-mediated cleavage of Arm, we examined potential caspase cleavage sites and found that drICE cleaves Arm at a unique DQVD motif in the N-terminal domain of the protein. Mutation of the drICE cleavage site in Arm results in a protein that is not cleaved in vitro and in vivo. Furthermore we provide evidence that cleavage of Arm plays a role in the removal of DE-cadherin from the plasma membrane during apoptosis. Conclusion This study defines the specificity of caspase cleavage of Arm in Drosophila apoptotic cells. Our data suggest that N-terminal truncation of Arm by caspases is evolutionarily conserved and thus might provide a principal mechanism involved in the disassembly of adherens junctions during apoptosis.

  15. Pelargonium quercetorum Agnew induces apoptosis without PARP or cytokeratin 18 cleavage in non-small cell lung cancer cell lines

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    Aztopal, Nazlihan; Cevatemre, Buse; Sarimahmut, Mehmet; Ari, Ferda; Dere, Egemen; Ozel, Mustafa Zafer; Firat, Mehmet; Ulukaya, Engin

    2016-01-01

    Pelargonium species have various uses in folk medicine as traditional remedies, and several of them have been screened for their biological activity, including anticancer. Pelargonium quercetorum Agnew (P. quercetorum) is traditionally used for its anthelminthic activity. However, little is known about its biological activity or its effect on cancer cells. The aim of the present study was to determine the cytotoxic activity of P. quercetorum extract on lung cancer cell lines with varying properties. Following the analyses of its chemical composition, the cytotoxic activity was screened by the adenosine triphosphate viability test. M30-Apoptosense® and M65 EpiDeath® enzyme-linked immunosorbent assays were used to determine the cell death mode (apoptosis vs. necrosis). For apoptosis, additional methods, including Annexin-V-fluorescein isothiocyanate (FITC) and Hoechst 33342 staining, were employed. The cleavage of poly (adenosine diphosphate-ribose) polymerase (PARP) was assayed by western blotting to further dissect the apoptosis mechanism. The methanol extract of P. quercetorum caused cytotoxic activity in a dose-dependent manner. The mode of cell death was apoptosis, as evidenced by the positive staining of the cells for Annexin-V-FITC and the presence of pyknotic nuclei. Notably, neither PARP cleavage nor cytokeratin 18 fragmentation were observed. P.quercetorum caused cell death by an apoptosis mechanism that is slightly different from classical apoptosis. Therefore, future in vivo experiments are required for further understanding of the effect of this plant on cancer cells. PMID:27446448

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

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    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.

  17. TACE cleavage of proamphiregulin regulates GPCR-induced proliferation and motility of cancer cells.

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    Gschwind, Andreas; Hart, Stefan; Fischer, Oliver M; Ullrich, Axel

    2003-05-15

    Communication between G protein-coupled receptor (GPCR) and epidermal growth factor receptor (EGFR) signalling systems involves cell surface proteolysis of EGF-like precursors. The underlying mechanisms of EGFR signal transactivation pathways, however, are largely unknown. We demonstrate that in squamous cell carcinoma cells, stimulation with the GPCR agonists LPA or carbachol specifically results in metalloprotease cleavage and release of amphiregulin (AR). Moreover, AR gene silencing by siRNA or inhibition of AR biological activity by neutralizing antibodies and heparin prevents GPCR-induced EGFR tyrosine phosphorylation, downstream mitogenic signalling events, cell proliferation, migration and activation of the survival mediator Akt/PKB. Therefore, despite some functional redundancy among EGF family ligands, the present study reveals a distinct and essential role for AR in GPCR-triggered cellular responses. Furthermore, we present evidence that blockade of the metalloprotease-disintegrin tumour necrosis factor-alpha-converting enzyme (TACE) by the tissue inhibitor of metalloprotease-3, a dominant-negative TACE mutant or RNA interference suppresses GPCR-stimulated AR release, EGFR activation and downstream events. Thus, TACE can function as an effector of GPCR-mediated signalling and represents a key element of the cellular receptor cross-talk network.

  18. Autocrine motility factor promotes HER2 cleavage and signaling in breast cancer cells

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    Kho, Dhong Hyo; Nangia-Makker, Pratima; Balan, Vitaly; Hogan, Victor; Tait, Larry; Wang, Yi; Raz, Avraham

    2013-01-01

    Trastuzumab (Herceptin®) is an effective targeted therapy in HER2 overexpressing human breast carcinoma. However, many HER2-positive patients initially or eventually become resistant to this treatment, so elucidating mechanisms of trastuzumab resistance that emerge in breast carcinoma cells is clinically important. Here we show that autocrine motility factor (AMF) binds to HER2 and induces cleavage to the ectodomain-deleted and constitutively active form p95HER2. Mechanistic investigations indicated that interaction of AMF with HER2 triggers HER2 phosphorylation and metalloprotease-mediated ectodomain shedding, activating PI3K and MAPK signaling and ablating the ability of trastuzumab to inhibit breast carcinoma cell growth. Further, we found that HER2 expression and AMF secretion were inversely related in breast carcinoma cells. Based on this evidence that AMF may contribute to HER2-mediated breast cancer progression, our findings suggest that AMF-HER2 interaction might be a novel target for therapeutic management of breast cancer patients whose disease is resistant to trastuzumab. PMID:23248119

  19. BAP31 and its caspase cleavage product regulate cell surface expression of tetraspanins and integrin-mediated cell survival.

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    Stojanovic, Marina; Germain, Marc; Nguyen, Mai; Shore, Gordon C

    2005-08-26

    BAP31, a resident integral protein of the endoplasmic reticulum membrane, regulates the export of other integral membrane proteins to the downstream secretory pathway. Here we show that cell surface expression of the tetraspanins CD9 and CD81 is compromised in mouse cells from which the Bap31 gene has been deleted. CD9 and CD81 facilitate the function of multiprotein complexes at the plasma membrane, including integrins. Of note, BAP31 does not appear to influence the egress of alpha5beta1 or alpha(v)beta3 integrins to the cell surface, but in Bap31-null mouse cells, these integrins are not able to maintain cellular adhesion to the extracellular matrix in the presence of reduced serum. Consequently, Bap31-null cells are sensitive to serum starvation-induced apoptosis. Reconstitution of wild-type BAP31 into these Bap31-null cells restores integrin-mediated cell attachment and cell survival after serum stress, whereas interference with the functions of CD9, alpha5beta1, or alpha(v)beta3 by antagonizing antibodies makes BAP31 cells act similar to Bap31-null cells in these respects. Finally, in human KB epithelial cells protected from apoptosis by BCL-2, the caspase-8 cleavage product, p20 BAP31, inhibits egress of tetraspanin and integrin-mediated cell attachment. Thus, p20 BAP31 can operate upstream of BCL-2 in living cells to influence cell surface properties due to its effects on protein egress from the endoplasmic reticulum.

  20. Malt1-dependent RelB cleavage promotes canonical NF-kappaB activation in lymphocytes and lymphoma cell lines.

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    Hailfinger, Stephan; Nogai, Hendrik; Pelzer, Christiane; Jaworski, Maike; Cabalzar, Katrin; Charton, Jean-Enno; Guzzardi, Montserrat; Décaillet, Chantal; Grau, Michael; Dörken, Bernd; Lenz, Peter; Lenz, Georg; Thome, Margot

    2011-08-30

    The protease activity of the paracaspase Malt1 contributes to antigen receptor-mediated lymphocyte activation and lymphomagenesis. Malt1 activity is required for optimal NF-κB activation, but little is known about the responsible substrate(s). Here we report that Malt1 cleaved the NF-κB family member RelB after Arg-85. RelB cleavage induced its proteasomal degradation and specifically controlled DNA binding of RelA- or c-Rel-containing NF-κB complexes. Overexpression of RelB inhibited expression of canonical NF-κB target genes and led to impaired survival of diffuse large B-cell lymphoma cell lines characterized by constitutive Malt1 activity. These findings identify a central role for Malt1-dependent RelB cleavage in canonical NF-κB activation and thereby provide a rationale for the targeting of Malt1 in immunomodulation and cancer treatment.

  1. Efficient cleavage of p220 by poliovirus 2Apro expression in mammalian cells: effects on vaccinia virus.

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    Aldabe, R; Feduchi, E; Novoa, I; Carrasco, L

    1995-10-24

    Poliovirus protease 2A cleaves p220, a component of initiation factor eIF-4F. Polyclonal antibodies that recognize p220 and the cleaved products from different species have been raised. Transfection of several cell lines with poliovirus 2Apro cloned in different plasmids leads to efficient cleavage of p220 upon infection with VT7, a recombinant vaccinia virus that expresses the T7 RNA polymerase. Under these conditions vaccinia virus protein synthesis is severely inhibited, while expression of poliovirus protein 2C from a similar plasmid has no effect. These results show by the first time the effects of p220 cleavage on vaccinia virus translation in the infected cells.

  2. Synthesis, characterization, plasmid cleavage and cytotoxicity of cancer cells by a copper(II) complex of anthracenyl-terpyridine.

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    Kumar, Amit; Chinta, Jugun Prakash; Ajay, Amrendra Kumar; Bhat, Manoj Kumar; Rao, Chebrolu P

    2011-11-01

    Metallo-organic compounds are interesting to study for their antitumor activity and related applications. This paper deals with the syntheses, characterization, structure determination of a copper complex of anthracenyl terpyridine (1) and its plasmid cleavage and cytotoxicity towards different cancer cell lines. The complex binds CT-DNA through partial intercalation mode. The plasmid cleavage studies carried out using pBR322 and pUC18 resulted in the formation of all the three forms of the plasmid DNA. Plasmid cleavage studies carried out with a non-redoxable Zn(2+) complex (2) supported the role of the redox activity of copper in 1. The complex 1 showed remarkable antiproliferative activity against cancer cell lines, viz., cervical (HeLa, SiHa, CaSki), breast (MCF-7), liver (HepG2) and lung (H1299). A considerable lowering was observed in the IC(50) values of HPV-infected (viz., HeLa, SiHa, CaSki) vs. non-HPV-infected cell lines (MCF-7, HepG2, H1299). Antiproliferative activity of 1 was found to be much higher than the carboplatin when treated with the same cell lines. Incubation of the cells with 1 results in granular structures only with the HPV-infected cells and not with others as studied by phase contrast and fluorescence microscopy. The lower IC(50) value observed in case of 1 with HPV-infected cell lines may be correlated with the involvement of HPV oncoprotein. The role of HPV has been further augmented by transfecting the MCF-7 cells (originally not possessing HPV copy) with e6 oncoprotein cDNA. To our knowledge this is the first copper complex that causes cell death by interacting with HPV oncoprotein followed by exhibition of remarkable antiproliferative activity.

  3. Proteolytic cleavage of pertussis toxin S1 subunit is not essential for its activity in mammalian cells

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    Plaut Roger D

    2005-02-01

    Full Text Available Abstract Background Pertussis toxin (PT is an exotoxin virulence factor produced by Bordetella pertussis, the causative agent of whooping cough. PT consists of an active subunit (S1 that ADP-ribosylates the alpha subunit of several mammalian G proteins, and a B oligomer (S2–S5 that binds glycoconjugate receptors on cells. PT appears to enter cells by endocytosis, and retrograde transport through the Golgi apparatus may be important for its cytotoxicity. A previous study demonstrated that proteolytic processing of S1 occurs after PT enters mammalian cells. We sought to determine whether this proteolytic processing of S1 is necessary for PT cytotoxicity. Results Protease inhibitor studies suggested that S1 processing may involve a metalloprotease, and processing does not involve furin, a mammalian cell protease that cleaves several other bacterial toxins. However, inhibitor studies showed a general lack of correlation of S1 processing with PT cellular activity. A combination of replacement, insertion and deletion mutations in the C-terminal region of S1, as well as mass spectrometry data, suggested that the cleavage site is located around residue 203–204, but that cleavage is not strongly sequence-dependent. Processing of S1 was abolished by each of 3 overlapping 8 residue deletions just downstream of the putative cleavage site, but not by smaller deletions in the same region. Processing of the various mutant forms of PT did not correlate with cellular activity of the toxin, nor with the ability of the bacteria producing them to infect the mouse respiratory tract. In addition, S1 processing was not detected in transfected cells expressing S1, even though S1 was fully active in these cells. Conclusions S1 processing is not essential for the cellular activity of PT. This distinguishes it from the processing of various other bacterial toxins, which has been shown to be important for their cytotoxicity. S1 processing may be mediated primarily by a

  4. Thrombin Cleavage of Osteopontin Modulates Its Activities in Human Cells In Vitro and Mouse Experimental Autoimmune Encephalomyelitis In Vivo.

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    Boggio, Elena; Dianzani, Chiara; Gigliotti, Casimiro Luca; Soluri, Maria Felicia; Clemente, Nausicaa; Cappellano, Giuseppe; Toth, Erika; Raineri, Davide; Ferrara, Benedetta; Comi, Cristoforo; Dianzani, Umberto; Chiocchetti, Annalisa

    2016-01-01

    Osteopontin is a proinflammatory cytokine and plays a pathogenetic role in multiple sclerosis and its animal model, experimental autoimmune encephalomyelitis (EAE), by recruiting autoreactive T cells into the central nervous system. Osteopontin functions are modulated by thrombin cleavage generating N- and C-terminal fragment, whose individual roles are only partly known. Published data are difficult to compare since they have been obtained with heterogeneous approaches. Interestingly, thrombin cleavage of osteopontin unmasks a cryptic domain of interaction with α 4 β 1 integrin that is the main adhesion molecule involved in lymphocyte transmigration to the brain and is the target for natalizumab, the most potent drug preventing relapses. We produced recombinant osteopontin and its N- and C-terminal fragments in an eukaryotic system in order to allow their posttranslational modifications. We investigated, in vitro, their effect on human cells and in vivo in EAE. We found that the osteopontin cleavage plays a key role in the function of this cytokine and that the two fragments exert distinct effects both in vitro and in vivo. These findings suggest that drugs targeting each fragment may be used to fine-tune the pathological effects of osteopontin in several diseases.

  5. Comparison of non-canonical PAMs for CRISPR/Cas9-mediated DNA cleavage in human cells.

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    Zhang, Yilan; Ge, Xianglian; Yang, Fayu; Zhang, Liping; Zheng, Jiayong; Tan, Xuefang; Jin, Zi-Bing; Qu, Jia; Gu, Feng

    2014-06-23

    CRISPR/Cas9-mediated DNA cleavage (CCMDC) is becoming increasingly used for efficient genome engineering. Proto-spacer adjacent motif (PAM) adjacent to target sequence is one of the key components in the design of CCMDC strategies. It has been reported that NAG sequences are the predominant non-canonical PAM for CCMDC at the human EMX locus, but it is not clear whether it is universal at other loci. In the present study, we attempted to use a GFP-reporter system to comprehensively and quantitatively test the efficiency of CCMDC with non-canonical PAMs in human cells. The initial results indicated that the effectiveness of NGA PAM for CCMDC is much higher than that of other 14 PAMs including NAG. Then we further designed another three pairs of NGG, NGA and NAG PAMs at different locations in the GFP gene and investigated the corresponding DNA cleavage efficiency. We observed that one group of NGA PAMs have a relatively higher DNA cleavage efficiency, while the other groups have lower efficiency, compared with the corresponding NAG PAMs. Our study clearly demonstrates that NAG may not be the universally predominant non-canonical PAM for CCMDC in human cells. These findings raise more concerns over off-target effects in CRISPR/Cas9-mediated genome engineering.

  6. Carboxypeptidase D is the only enzyme responsible for antibody C-terminal lysine cleavage in Chinese hamster ovary (CHO) cells.

    Science.gov (United States)

    Hu, Zhilan; Zhang, Henry; Haley, Benjamin; Macchi, Frank; Yang, Feng; Misaghi, Shahram; Elich, Joseph; Yang, Renee; Tang, Yun; Joly, John C; Snedecor, Bradley R; Shen, Amy

    2016-10-01

    Heterogeneity of C-terminal lysine levels often observed in therapeutic monoclonal antibodies is believed to result from the proteolysis by endogenous carboxypeptidase(s) during cell culture production. Identifying the responsible carboxypeptidase(s) for C-terminal lysine cleavage in CHO cells would provide valuable insights for antibody production cell culture processes development and optimization. In this study, five carboxypeptidases, CpD, CpM, CpN, CpB, and CpE, were studied for message RNA (mRNA) expression by qRT-PCR analysis in two most commonly used blank hosts (DUXB-11 derived DHFR-deficient DP12 host and DHFR-positive CHOK1 host), used for therapeutic antibody production, as well an antibody-expressing cell line derived from each host. Our results showed that CpD had the highest mRNA expression. When CpD mRNA levels were reduced by RNAi (RNA interference) technology, C-terminal lysine levels increased, whereas there was no obvious change in C-terminal lysine levels when a different carboxypeptidase mRNA level was knocked down suggesting that carboxypeptidase D is the main contributor for C-terminal lysine processing. Most importantly, when CpD expression was knocked out by CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats) technology, C-terminal lysine cleavage was completely abolished in CpD knockout cells based on mass spectrometry analysis, demonstrating that CpD is the only endogenous carboxypeptidase that cleaves antibody heavy chain C-terminal lysine in CHO cells. Hence, our work showed for the first time that the cleavage of antibody heavy chain C-terminal lysine is solely mediated by the carboxypeptidase D in CHO cells and our finding provides one solution to eliminating C-terminal lysine heterogeneity for therapeutic antibody production by knocking out CpD gene expression. Biotechnol. Bioeng. 2016;113: 2100-2106. © 2016 Wiley Periodicals, Inc.

  7. Efficient Nuclear DNA Cleavage in Human Cancer Cells by Synthetic Bleomycin Mimics

    NARCIS (Netherlands)

    Li, Qian; van der Wijst, Monique G. P.; Kazemier, Hinke G.; Rots, Marianne G.; Roelfes, Gerard

    2014-01-01

    Iron complexes of N,N-bis(2-Pyridylmethyl)-N-bis(2-pyridyl)-methylamine (N4Py) have proven to be excellent synthetic mimics of the Bleomycins (BLMs), which are a family of natural antibiotics used clinically in the treatment of certain cancers. However, most investigations of DNA cleavage activity o

  8. 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.

  9. Sea urchin akt activity is Runx-dependent and required for post-cleavage stage cell division

    KAUST Repository

    Robertson, Anthony J.

    2013-03-25

    In animal development following the initial cleavage stage of embryogenesis, the cell cycle becomes dependent on intercellular signaling and controlled by the genomically encoded ontogenetic program. Runx transcription factors are critical regulators of metazoan developmental signaling, and we have shown that the sea urchin Runx gene runt-1, which is globally expressed during early embryogenesis, functions in support of blastula stage cell proliferation and expression of the mitogenic genes pkc1, cyclinD, and several wnts. To obtain a more comprehensive list of early runt-1 regulatory targets, we screened a Strongylocentrotus purpuratus microarray to identify genes mis-expressed in mid-blastula stage runt-1 morphants. This analysis showed that loss of Runx function perturbs the expression of multiple genes involved in cell division, including the pro-growth and survival kinase Akt (PKB), which is significantly underexpressed in runt-1 morphants. Further genomic analysis revealed that Akt is encoded by two genes in the S. purpuratus genome, akt-1 and akt-2, both of which contain numerous canonical Runx target sequences. The transcripts of both genes accumulate several fold during blastula stage, contingent on runt-1 expression. Inhibiting Akt expression or activity causes blastula stage cell cycle arrest, whereas overexpression of akt-1 mRNA rescues cell proliferation in runt-1 morphants. These results indicate that post-cleavage stage cell division requires Runx-dependent expression of akt.

  10. Sea urchin akt activity is Runx-dependent and required for post-cleavage stage cell division

    Directory of Open Access Journals (Sweden)

    Anthony J. Robertson

    2013-03-01

    In animal development following the initial cleavage stage of embryogenesis, the cell cycle becomes dependent on intercellular signaling and controlled by the genomically encoded ontogenetic program. Runx transcription factors are critical regulators of metazoan developmental signaling, and we have shown that the sea urchin Runx gene runt-1, which is globally expressed during early embryogenesis, functions in support of blastula stage cell proliferation and expression of the mitogenic genes pkc1, cyclinD, and several wnts. To obtain a more comprehensive list of early runt-1 regulatory targets, we screened a Strongylocentrotus purpuratus microarray to identify genes mis-expressed in mid-blastula stage runt-1 morphants. This analysis showed that loss of Runx function perturbs the expression of multiple genes involved in cell division, including the pro-growth and survival kinase Akt (PKB, which is significantly underexpressed in runt-1 morphants. Further genomic analysis revealed that Akt is encoded by two genes in the S. purpuratus genome, akt-1 and akt-2, both of which contain numerous canonical Runx target sequences. The transcripts of both genes accumulate several fold during blastula stage, contingent on runt-1 expression. Inhibiting Akt expression or activity causes blastula stage cell cycle arrest, whereas overexpression of akt-1 mRNA rescues cell proliferation in runt-1 morphants. These results indicate that post-cleavage stage cell division requires Runx-dependent expression of akt.

  11. A novel COX-independent mechanism of sulindac sulfide involves cleavage of epithelial cell adhesion molecule protein.

    Science.gov (United States)

    Liggett, Jason L; Min, Kyung-Won; Smolensky, Dmitriy; Baek, Seung Joon

    2014-08-01

    Non-steroidal anti-inflammatory drugs (NSAIDs) are extensively used over the counter to treat headaches and inflammation as well as clinically to prevent cancer among high-risk groups. The inhibition of cyclooxygenase (COX) activity by NSAIDs plays a role in their anti-tumorigenic properties. NSAIDs also have COX-independent activity which is not fully understood. In this study, we report a novel COX-independent mechanism of sulindac sulfide (SS), which facilitates a previously uncharacterized cleavage of epithelial cell adhesion molecule (EpCAM) protein. EpCAM is a type I transmembrane glycoprotein that has been implemented as an over-expressed oncogene in many cancers including colon, breast, pancreas, and prostate. We found EpCAM to be down-regulated by SS in a manner that is independent of COX activity, transcription regulation, de novo protein synthesis, and proteasomal degradation pathway. Our findings clearly demonstrate that SS drives cleavage of the extracellular portion of EpCAM near the N-terminus. This SS driven cleavage is blocked by a deleting amino acids 55-81 as well as simply mutating arginine residues at positions 80 and 81 to alanine of EpCAM. Proteolysis of EpCAM by SS may provide a novel mechanism by which NSAIDs affect anti-tumorigenesis at the post-translational level.

  12. Cathepsin K cleavage of SDF-1α inhibits its chemotactic activity towards glioblastoma stem-like cells.

    Science.gov (United States)

    Hira, Vashendriya V V; Verbovšek, Urška; Breznik, Barbara; Srdič, Matic; Novinec, Marko; Kakar, Hala; Wormer, Jill; der Swaan, Britt Van; Lenarčič, Brigita; Juliano, Luiz; Mehta, Shwetal; Van Noorden, Cornelis J F; Lah, Tamara T

    2017-03-01

    Glioblastoma (GBM) is the most aggressive primary brain tumor with poor patient survival that is at least partly caused by malignant and therapy-resistant glioma stem-like cells (GSLCs) that are protected in GSLC niches. Previously, we have shown that the chemo-attractant stromal-derived factor-1α (SDF-1α), its C-X-C receptor type 4 (CXCR4) and the cysteine protease cathepsin K (CatK) are localized in GSLC niches in glioblastoma. Here, we investigated whether SDF-1α is a niche factor that through its interactions with CXCR4 and/or its second receptor CXCR7 on GSLCs facilitates their homing to niches. Furthermore, we aimed to prove that SDF-1α cleavage by CatK inactivates SDF-1α and inhibits the invasion of GSLCs. We performed mass spectrometric analysis of cleavage products of SDF-1α after proteolysis by CatK. We demonstrated that CatK cleaves SDF-1α at 3 sites in the N-terminus, which is the region of SDF-1α that binds to its receptors. Confocal imaging of human GBM tissue sections confirmed co-localization of SDF-1α and CatK in GSLC niches. In accordance, 2D and 3D invasion experiments using CXCR4/CXCR7-expressing GSLCs and GBM cells showed that SDF-1α had chemotactic activity whereas CatK cleavage products of SDF-1α did not. Besides, CXCR4 inhibitor plerixafor inhibited invasion of CXCR4/CXCR7-expressing GSLCs. In conclusion, CatK can cleave and inactivate SDF-1α. This implies that CatK activity facilitates migration of GSLCs out of niches. We propose that activation of CatK may be a promising strategy to prevent homing of GSLCs in niches and thus render these cells sensitive to chemotherapy and radiation.

  13. The Relationship between Cell Number, Division Behavior and Developmental Potential of Cleavage Stage Human Embryos: A Time-Lapse Study.

    Science.gov (United States)

    Kong, Xiangyi; Yang, Shuting; Gong, Fei; Lu, Changfu; Zhang, Shuoping; Lu, Guangxiu; Lin, Ge

    2016-01-01

    Day 3 cleavage embryo transfer is routine in many assisted reproductive technology centers today. Embryos are usually selected according to cell number, cell symmetry and fragmentation for transfer. Many studies have showed the relationship between cell number and embryo developmental potential. However, there is limited understanding of embryo division behavior and their association with embryo cell number and developmental potential. A retrospective and observational study was conducted to investigate how different division behaviors affect cell number and developmental potential of day 3 embryos by time-lapse imaging. Based on cell number at day 3, the embryos (from 104 IVF/intracytoplasmic sperm injection (ICSI) treatment cycles, n = 799) were classified as follows: less than 5 cells (10C; n = 42). Division behavior, morphokinetic parameters and blastocyst formation rate were analyzed in 5 groups of day 3 embryos with different cell numbers. In 10C embryos increased compared to 7-8C embryos (45.8%, 33.3% vs. 11.1%, respectively). In ≥5C embryos, FR and DC significantly reduced developmental potential, whereas division behaviors. In NB embryos, the blastocyst formation rate increased with cell number from 7.4% (10C). In NB embryos, the cell cycle elongation or shortening was the main cause for abnormally low or high cell number, respectively. After excluding embryos with abnormal division behaviors, the developmental potential, implantation rate and live birth rate of day 3 embryos increased with cell number.

  14. Bifunctional alkylating agent-mediated MGMT-DNA cross-linking and its proteolytic cleavage in 16HBE cells.

    Science.gov (United States)

    Cheng, Jin; Ye, Feng; Dan, Guorong; Zhao, Yuanpeng; Wang, Bin; Zhao, Jiqing; Sai, Yan; Zou, Zhongmin

    2016-08-15

    Nitrogen mustard (NM), a bifunctional alkylating agent (BAA), contains two alkyl arms and can act as a cross-linking bridge between DNA and protein to form a DNA-protein cross-link (DPC). O(6)-methylguanine-DNA methyltransferase (MGMT), a DNA repair enzyme for alkyl adducts removal, is found to enhance cell sensitivity to BAAs and to promote damage, possibly due to its stable covalent cross-linking with DNA mediated by BAAs. To investigate MGMT-DNA cross-link (mDPC) formation and its possible dual roles in NM exposure, human bronchial epithelial cell line 16HBE was subjected to different concentrations of HN2, a kind of NM, and we found mDPC was induced by HN2 in a concentration-dependent manner, but the mRNA and total protein of MGMT were suppressed. As early as 1h after HN2 treatment, high mDPC was achieved and the level maintained for up to 24h. Quick total DPC (tDPC) and γ-H2AX accumulation were observed. To evaluate the effect of newly predicted protease DVC1 on DPC cleavage, we applied siRNA of MGMT and DVC1, MG132 (proteasome inhibitor), and NMS-873 (p97 inhibitor) and found that proteolysis plays a role. DVC1 was proven to be more important in the cleavage of mDPC than tDPC in a p97-dependent manner. HN2 exposure induced DVC1 upregulation, which was at least partially contributed to MGMT cleavage by proteolysis because HN2-induced mDPC level and DNA damage was closely related with DVC1 expression. Homologous recombination (HR) was also activated. Our findings demonstrated that MGMT might turn into a DNA damage promoter by forming DPC when exposed to HN2. Proteolysis, especially DVC1, plays a crucial role in mDPC repair.

  15. Cu(II) complexes of glyco-imino-aromatic conjugates in DNA binding, plasmid cleavage and cell cytotoxicity

    Indian Academy of Sciences (India)

    Amit Kumar; Atanu Mitra; Amrendra Kumar Ajay; Manoj Kumar Bhat; Chebrolu P Rao

    2012-11-01

    Binding of metal complexes of C2-glucosyl conjugates with DNA has been established by absorption and fluorescence studies. Conformational changes occurred in DNA upon binding have been studied by circular dichroism. All these studies are suggestive that the metal complexes bind to DNA through intercalation. Binding of di-nuclear copper complex 5 was found to be stronger when compared to the other complexes studied. Copper complexes were found to cleave the plasmid DNA in the absence of oxidizing or reducing agent, whereas, zinc complexes do not cleave. Metal complexes have shown toxicity to the HeLa and MCF-7 cell lines.Morphological studies, western blot and FACS analysis are suggestive of apoptotic cell death induced by the metal complexes. Di-nuclear copper complexes were found to be better as compared to the mononuclear ones in binding, plasmid cleavage and also in causing more cell death.

  16. Implications of caspase-dependent proteolytic cleavage of cyclin A1 in DNA damage-induced cell death

    Energy Technology Data Exchange (ETDEWEB)

    Woo, Sang Hyeok; Seo, Sung-Keum [Division of Radiation Cancer Research, Korea Institute of Radiological and Medical Sciences, 215-4 Gongneung-dong, Nowon-gu, Seoul (Korea, Republic of); An, Sungkwan; Choe, Tae-Boo [Department of Microbiological Engineering, Kon-Kuk University, Gwangjin-gu, Seoul (Korea, Republic of); Hong, Seok-Il [Department of Laboratory Medicine, Korea Cancer Center Hospital, Korea Institute of Radiological and Medical Sciences, 215-4 Gongneung-dong, Nowon-gu, Seoul (Korea, Republic of); Lee, Yun-Han, E-mail: yhlee87@yuhs.ac [Department of Radiation Oncology, College of Medicine, Yonsei University, 250 Seongsan-no, Seodaemun-gu, Seoul (Korea, Republic of); Park, In-Chul, E-mail: parkic@kcch.re.kr [Division of Radiation Cancer Research, Korea Institute of Radiological and Medical Sciences, 215-4 Gongneung-dong, Nowon-gu, Seoul (Korea, Republic of)

    2014-10-24

    Highlights: • Caspase-1 mediates doxorubicin-induced downregulation of cyclin A1. • Active caspase-1 effectively cleaved cyclin A1 at D165. • Cyclin A1 expression is involved in DNA damage-induced cell death. - Abstract: Cyclin A1 is an A-type cyclin that directly binds to CDK2 to regulate cell-cycle progression. In the present study, we found that doxorubicin decreased the expression of cyclin A1 at the protein level in A549 lung cancer cells, while markedly downregulating its mRNA levels. Interestingly, doxorubicin upregulated caspase-1 in a concentration-dependent manner, and z-YAVD-fmk, a specific inhibitor of caspase-1, reversed the doxorubicin-induced decrease in cyclin A1 in A549 lung cancer and MCF7 breast cancer cells. Active caspase-1 effectively cleaved cyclin A1 at D165 into two fragments, which in vitro cleavage assays showed were further cleaved by caspase-3. Finally, we found that overexpression of cyclin A1 significantly reduced the cytotoxicity of doxorubicin, and knockdown of cyclin A1 by RNA interference enhanced the sensitivity of cells to ionizing radiation. Our data suggest a new mechanism for the downregulation of cyclin A1 by DNA-damaging stimuli that could be intimately involved in the cell death induced by DNA damage-inducing stimuli, including doxorubicin and ionizing radiation.

  17. NALP3 inflammasome upregulation and CASP1 cleavage of the glucocorticoid receptor cause glucocorticoid resistance in leukemia cells.

    Science.gov (United States)

    Paugh, Steven W; Bonten, Erik J; Savic, Daniel; Ramsey, Laura B; Thierfelder, William E; Gurung, Prajwal; Malireddi, R K Subbarao; Actis, Marcelo; Mayasundari, Anand; Min, Jaeki; Coss, David R; Laudermilk, Lucas T; Panetta, John C; McCorkle, J Robert; Fan, Yiping; Crews, Kristine R; Stocco, Gabriele; Wilkinson, Mark R; Ferreira, Antonio M; Cheng, Cheng; Yang, Wenjian; Karol, Seth E; Fernandez, Christian A; Diouf, Barthelemy; Smith, Colton; Hicks, J Kevin; Zanut, Alessandra; Giordanengo, Audrey; Crona, Daniel; Bianchi, Joy J; Holmfeldt, Linda; Mullighan, Charles G; den Boer, Monique L; Pieters, Rob; Jeha, Sima; Dunwell, Thomas L; Latif, Farida; Bhojwani, Deepa; Carroll, William L; Pui, Ching-Hon; Myers, Richard M; Guy, R Kiplin; Kanneganti, Thirumala-Devi; Relling, Mary V; Evans, William E

    2015-06-01

    Glucocorticoids are universally used in the treatment of acute lymphoblastic leukemia (ALL), and resistance to glucocorticoids in leukemia cells confers poor prognosis. To elucidate mechanisms of glucocorticoid resistance, we determined the prednisolone sensitivity of primary leukemia cells from 444 patients newly diagnosed with ALL and found significantly higher expression of CASP1 (encoding caspase 1) and its activator NLRP3 in glucocorticoid-resistant leukemia cells, resulting from significantly lower somatic methylation of the CASP1 and NLRP3 promoters. Overexpression of CASP1 resulted in cleavage of the glucocorticoid receptor, diminished the glucocorticoid-induced transcriptional response and increased glucocorticoid resistance. Knockdown or inhibition of CASP1 significantly increased glucocorticoid receptor levels and mitigated glucocorticoid resistance in CASP1-overexpressing ALL. Our findings establish a new mechanism by which the NLRP3-CASP1 inflammasome modulates cellular levels of the glucocorticoid receptor and diminishes cell sensitivity to glucocorticoids. The broad impact on the glucocorticoid transcriptional response suggests that this mechanism could also modify glucocorticoid effects in other diseases.

  18. NALP3 inflammasome up-regulation and CASP1 cleavage of the glucocorticoid receptor causes glucocorticoid resistance in leukemia cells

    Science.gov (United States)

    Paugh, Steven W.; Bonten, Erik J.; Savic, Daniel; Ramsey, Laura B.; Thierfelder, William E.; Gurung, Prajwal; Malireddi, R. K. Subbarao; Actis, Marcelo; Mayasundari, Anand; Min, Jaeki; Coss, David R.; Laudermilk, Lucas T.; Panetta, John C.; McCorkle, J. Robert; Fan, Yiping; Crews, Kristine R.; Stocco, Gabriele; Wilkinson, Mark R.; Ferreira, Antonio M.; Cheng, Cheng; Yang, Wenjian; Karol, Seth E.; Fernandez, Christian A.; Diouf, Barthelemy; Smith, Colton; Hicks, J. Kevin; Zanut, Alessandra; Giordanengo, Audrey; Crona, Daniel; Bianchi, Joy J.; Holmfeldt, Linda; Mullighan, Charles G.; den Boer, Monique L.; Pieters, Rob; Jeha, Sima; Dunwell, Thomas L.; Latif, Farida; Bhojwani, Deepa; Carroll, William L.; Pui, Ching-Hon; Myers, Richard M.; Guy, R. Kiplin; Kanneganti, Thirumala-Devi; Relling, Mary V.; Evans, William E.

    2015-01-01

    Glucocorticoids are universally used in the treatment of acute lymphoblastic leukemia (ALL), and leukemia cell resistant to glucocorticoids confers a poor prognosis. To elucidate mechanisms of glucocorticoid resistance, we determined the sensitivity to prednisolone of primary leukemia cells from 444 newly diagnosed ALL patients, revealing significantly higher expression of caspase 1 (CASP1) and its activator NLRP3 in glucocorticoid resistant leukemia cells, due to significantly lower somatic methylation of CASP1 and NLRP3 promoters. Over-expression of CASP1 resulted in cleavage of the glucocorticoid receptor, diminished glucocorticoid-induced transcriptional response and increased glucocorticoid resistance. Knockdown or inhibition of CASP1 significantly increased glucocorticoid receptor levels and mitigated glucocorticoid resistance in CASP1 overexpressing ALL. Our findings establish a new mechanism by which the NLRP3/CASP1 inflammasome modulates cellular levels of the glucocorticoid receptor and diminishes cell sensitivity to glucocorticoids. The broad impact on glucocorticoid transcriptional response suggests this mechanism could also modify glucocorticoid effects in other diseases. PMID:25938942

  19. Miniaturized electrochemical cells for applications in drug screening and protein cleavage

    NARCIS (Netherlands)

    Odijk, Mathieu

    2011-01-01

    This thesis describes the design of a small flow-through electrochemical cell in a lab-on-chip format. This cell contains a complete, integrated 3-electrode setup with platinum working and counter electrodes and a palladium or iridiumoxide pseudo-reference electrode. The cell has sub-microliter inte

  20. A c-di-GMP effector system controls cell adhesion by inside-out signaling and surface protein cleavage.

    Directory of Open Access Journals (Sweden)

    Peter D Newell

    Full Text Available In Pseudomonas fluorescens Pf0-1 the availability of inorganic phosphate (Pi is an environmental signal that controls biofilm formation through a cyclic dimeric GMP (c-di-GMP signaling pathway. In low Pi conditions, a c-di-GMP phosphodiesterase (PDE RapA is expressed, depleting cellular c-di-GMP and causing the loss of a critical outer-membrane adhesin LapA from the cell surface. This response involves an inner membrane protein LapD, which binds c-di-GMP in the cytoplasm and exerts a periplasmic output promoting LapA maintenance on the cell surface. Here we report how LapD differentially controls maintenance and release of LapA: c-di-GMP binding to LapD promotes interaction with and inhibition of the periplasmic protease LapG, which targets the N-terminus of LapA. We identify conserved amino acids in LapA required for cleavage by LapG. Mutating these residues in chromosomal lapA inhibits LapG activity in vivo, leading to retention of the adhesin on the cell surface. Mutations with defined effects on LapD's ability to control LapA localization in vivo show concomitant effects on c-di-GMP-dependent LapG inhibition in vitro. To establish the physiological importance of the LapD-LapG effector system, we track cell attachment and LapA protein localization during Pi starvation. Under this condition, the LapA adhesin is released from the surface of cells and biofilms detach from the substratum. This response requires c-di-GMP depletion by RapA, signaling through LapD, and proteolytic cleavage of LapA by LapG. These data, in combination with the companion study by Navarro et al. presenting a structural analysis of LapD's signaling mechanism, give a detailed description of a complete c-di-GMP control circuit--from environmental signal to molecular output. They describe a novel paradigm in bacterial signal transduction: regulation of a periplasmic enzyme by an inner membrane signaling protein that binds a cytoplasmic second messenger.

  1. High cell density and latent membrane protein 1 expression induce cleavage of the mixed lineage leukemia gene at 11q23 in nasopharyngeal carcinoma cell line

    Directory of Open Access Journals (Sweden)

    Sim Sai-Peng

    2010-09-01

    Full Text Available Abstract Background Nasopharyngeal carcinoma (NPC is commonly found in Southern China and South East Asia. Epstein-Barr virus (EBV infection is well associated with NPC and has been implicated in its pathogenesis. Moreover, various chromosome rearrangements were reported in NPC. However, the underlying mechanism of chromosome rearrangement remains unclear. Furthermore, the relationship between EBV and chromosome rearrangement with respect to the pathogenesis of NPC has not been established. We hypothesize that during virus- or stress-induced apoptosis, chromosomes are initially cleaved at the base of the chromatin loop domain structure. Upon DNA repair, cell may survive with rearranged chromosomes. Methods In this study, cells were seeded at various densities to induce apoptosis. Genomic DNA extracted was processed for Southern hybridization. In order to investigate the role of EBV, especially the latent membrane protein 1 (LMP1, LMP1 gene was overexpressed in NPC cells and chromosome breaks were analyzed by inverse polymerase chain (IPCR reaction. Results Southern analysis revealed that high cell density resulted in cleavage of the mixed lineage leukemia (MLL gene within the breakpoint cluster region (bcr. This high cell density-induced cleavage was significantly reduced by caspase inhibitor, Z-DEVD-FMK. Similarly, IPCR analysis showed that LMP1 expression enhanced cleavage of the MLL bcr. Breakpoint analysis revealed that these breaks occurred within the matrix attachment region/scaffold attachment region (MAR/SAR. Conclusions Since MLL locates at 11q23, a common deletion site in NPC, our results suggest a possibility of stress- or virus-induced apoptosis in the initiation of chromosome rearrangements at 11q23. The breakpoint analysis results also support the role of chromatin structure in defining the site of chromosome rearrangement.

  2. Cleavage of SNAP-25 and VAMP-2 impairs store-operated Ca2+ entry in mouse pancreatic acinar cells.

    Science.gov (United States)

    Rosado, Juan A; Redondo, Pedro C; Salido, Ginés M; Sage, Stewart O; Pariente, Jose A

    2005-01-01

    We recently reported that store-operated Ca(2+) entry (SOCE) in nonexcitable cells is likely to be mediated by a reversible interaction between Ca(2+) channels in the plasma membrane and the endoplasmic reticulum, a mechanism known as "secretion-like coupling." As for secretion, in this model the actin cytoskeleton plays a key regulatory role. In the present study we have explored the involvement of the secretory proteins synaptosome-associated protein (SNAP-25) and vesicle-associated membrane protein (VAMP) in SOCE in pancreatic acinar cells. Cleavage of SNAP-25 and VAMPs by treatment with botulinum toxin A (BoNT A) and tetanus toxin (TeTx), respectively, effectively inhibited amylase secretion stimulated by the physiological agonist CCK-8. BoNT A significantly reduced Ca(2+) entry induced by store depletion using thapsigargin or CCK-8. In addition, treatment with BoNT A once SOCE had been activated reduced Ca(2+) influx, indicating that SNAP-25 is needed for both the activation and maintenance of SOCE in pancreatic acinar cells. VAMP-2 and VAMP-3 are expressed in mouse pancreatic acinar cells. Both proteins associate with the cytoskeleton upon Ca(2+) store depletion, although only VAMP-2 seems to be sensitive to TeTx. Treatment of pancreatic acinar cells with TeTx reduced the activation of SOCE without affecting its maintenance. These findings support a role for SNAP-25 and VAMP-2 in the activation of SOCE in pancreatic acinar cells and show parallels between this process and secretion in a specialized secretory cell type.

  3. Lipopolysaccharide-enhanced early proliferation of insulin secreting NIT-1 cell is associated with nuclear factor-kappaBmediated inhibition of caspase 3 cleavage

    Institute of Scientific and Technical Information of China (English)

    LIU Shan-ying; LIANG Qi-jun; LIN Tian-xin; FAN Xin-lan; LIANG Ying; Uwe Heemann; LI Yan

    2011-01-01

    Background Increased levels of plasma lipopolysaccharide (LPS) have been found in obesity and diabetes patients.This study was to investigate the effect of LPS on pancreatic beta-cell viability and the involvement of caspase 3 in NIT-1 cell line.Methods Mouse insulinoma NIT-1 cells were treated with LPS for the indicated time and dose.Cell viability was measured by cell counting kit-8 reagent.Toll-like receptor 4 (TLR4),caspase 3 and cleaved caspase 3 were detected by Western blotting.Insulin was determined by radioimmunoassay (RIA).Results LPS promoted NIT-1 cell proliferation at 1 μg/ml,peaked at 72 hours of incubation.A reduction in cleavage of caspase 3 was observed upon LPS treatment.Bay11-7082,a specific inhibitor of nuclear factor (NF)-κB,blunted LPS-induced inhibition of caspase 3 cleavage.Reduction in chronic insulin secretion was observed after treatment with LPS at 1 μg/ml for 48 and 72 hours,not for 24 hours.TLR4 protein was upregulated when NIT-1 cells were treated with LPS at 1 pg/ml for 24 hours.Conclusions LPS promotes early NIT-1 cell proliferation in association with NF-κB-mediated inhibition of caspase 3 cleavage.LPS exerts a time-dependent inhibitory effect on chronic insulin secretion from NIT-1 cells.

  4. Resolution of telomere associations by TRF1 cleavage in mouse embryonic stem cells

    NARCIS (Netherlands)

    Lisaingo, Kathleen; Uringa, Evert-Jan; Lansdorp, Peter M.

    2014-01-01

    Telomere associations have been observed during key cellular processes such as mitosis, meiosis, and carcinogenesis and must be resolved before cell division to prevent genome instability. Here we establish that telomeric repeat-binding factor 1 (TRF1), a core component of the telomere protein compl

  5. hnRNP F Influences Binding of a 64-Kilodalton Subunit of Cleavage Stimulation Factor to mRNA Precursors in Mouse B Cells

    OpenAIRE

    Veraldi, Kristen L.; Arhin, George K.; Martincic, Kathleen; Chung-Ganster, Ling-Hsiu; Wilusz, Jeffrey; Milcarek, Christine

    2001-01-01

    Previous studies on the regulation of polyadenylation of the immunoglobulin (Ig) heavy-chain pre-mRNA argued for trans-acting modifiers of the cleavage-polyadenylation reaction operating differentially during B-cell developmental stages. Using four complementary approaches, we demonstrate that a change in the level of hnRNP F is an important determinant in the regulated use of alternative polyadenylation sites between memory and plasma stage B cells. First, by Western analyses of cellular pro...

  6. Cathepsin S Cleavage of Protease-Activated Receptor-2 on Endothelial Cells Promotes Microvascular Diabetes Complications.

    Science.gov (United States)

    Kumar Vr, Santhosh; Darisipudi, Murthy N; Steiger, Stefanie; Devarapu, Satish Kumar; Tato, Maia; Kukarni, Onkar P; Mulay, Shrikant R; Thomasova, Dana; Popper, Bastian; Demleitner, Jana; Zuchtriegel, Gabriele; Reichel, Christoph; Cohen, Clemens D; Lindenmeyer, Maja T; Liapis, Helen; Moll, Solange; Reid, Emma; Stitt, Alan W; Schott, Brigitte; Gruner, Sabine; Haap, Wolfgang; Ebeling, Martin; Hartmann, Guido; Anders, Hans-Joachim

    2016-06-01

    Endothelial dysfunction is a central pathomechanism in diabetes-associated complications. We hypothesized a pathogenic role in this dysfunction of cathepsin S (Cat-S), a cysteine protease that degrades elastic fibers and activates the protease-activated receptor-2 (PAR2) on endothelial cells. We found that injection of mice with recombinant Cat-S induced albuminuria and glomerular endothelial cell injury in a PAR2-dependent manner. In vivo microscopy confirmed a role for intrinsic Cat-S/PAR2 in ischemia-induced microvascular permeability. In vitro transcriptome analysis and experiments using siRNA or specific Cat-S and PAR2 antagonists revealed that Cat-S specifically impaired the integrity and barrier function of glomerular endothelial cells selectively through PAR2. In human and mouse type 2 diabetic nephropathy, only CD68(+) intrarenal monocytes expressed Cat-S mRNA, whereas Cat-S protein was present along endothelial cells and inside proximal tubular epithelial cells also. In contrast, the cysteine protease inhibitor cystatin C was expressed only in tubules. Delayed treatment of type 2 diabetic db/db mice with Cat-S or PAR2 inhibitors attenuated albuminuria and glomerulosclerosis (indicators of diabetic nephropathy) and attenuated albumin leakage into the retina and other structural markers of diabetic retinopathy. These data identify Cat-S as a monocyte/macrophage-derived circulating PAR2 agonist and mediator of endothelial dysfunction-related microvascular diabetes complications. Thus, Cat-S or PAR2 inhibition might be a novel strategy to prevent microvascular disease in diabetes and other diseases.

  7. Deciphering the Mechanism of Alternative Cleavage and Polyadenylation in Mantle Cell Lymphoma (MCL)

    Science.gov (United States)

    2014-10-01

    express- ion in glioblastoma cells enhances their tumorigenic properties and increases tumour size,whereasCFIm25overexpression reduces these...inCFIm25knockdowncells haveoverall higher express- ion levels (Fig. 2d).Weobserved that 64%of transcriptswith shortened 39UTRs exhibited significantly increased...lines used (HeLa, U251 and LN229) were cultured in DMEM supplemented with 10% FBS (11% penicillin and streptomy- cin) in a 5% CO2 incubator at 37 uC

  8. Deciphering the Mechanism of Alternative Cleavage and Polyadenylation in Mantle Cell Lymphoma (MCL)

    Science.gov (United States)

    2015-12-01

    Like other hematological malignancies, MCL is characterized by numerous chromosomal translocations. RNA-Sequencing is limited by the relatively...Attend class at CSHL on Deep Sequencing Technology) • Attended CSHL training course in Advanced Sequencing Technology and Applications, November 12-24...226-231. 9. Bertoni F, Zucca E, Cavalli F. Mantle cell lymphoma. Current Opinion in Hematology 2004;11:411-418. 10. Wiestner A, Tehrani M, Chiorazzi

  9. Cometabolic degradation of chloramphenicol via a meta-cleavage pathway in a microbial fuel cell and its microbial community.

    Science.gov (United States)

    Zhang, Qinghua; Zhang, Yanyan; Li, Daping

    2017-04-01

    The performance of a microbial fuel cell (MFC) in terms of degradation of chloramphenicol (CAP) was investigated. Approximately 84% of 50mg/L CAP was degraded within 12h in the MFC. A significant interaction of pH, temperature, and initial CAP concentration was found on removal of CAP, and a maximum degradation rate of 96.53% could theoretically be achieved at 31.48°C, a pH of 7.12, and an initial CAP concentration of 106.37mg/L. Moreover, CAP was further degraded through a ring-cleavage pathway. The antibacterial activity of CAP towards Escherichia coli ATCC 25922 and Shewanella oneidensis MR-1 was largely eliminated by MFC treatment. High-throughput sequencing analysis indicated that Azonexus, Comamonas, Nitrososphaera, Chryseobacterium, Azoarcus, Rhodococcus, and Dysgonomonas were the predominant genera in the MFC anode biofilm. In conclusion, the MFC shows potential for the treatment of antibiotic residue-containing wastewater due to its high rates of CAP removal and energy recovery.

  10. MMP2-cleavage of DMP1 generates a bioactive peptide promoting differentiation of dental pulp stem/progenitor cell

    Directory of Open Access Journals (Sweden)

    C Chaussain

    2009-11-01

    Full Text Available Dentin Matrix Protein 1 (DMP1 plays a regulatory role in dentin mineralization and can also function as a signaling molecule. MMP-2 (matrix metalloproteinase-2 is a predominant protease in the dentin matrix that plays a prominent role in tooth formation and a potential role during the carious process. The possibility that MMP-2 can cleave DMP1 to release biologically active peptides was investigated in this study. DMP1, both in the recombinant form and in its native state within the dentin matrix, was shown to be a substrate for MMP-2. Proteolytic processing of DMP1 by MMP-2 produced two major peptides, one that contains the C-terminal region of the protein known to carry both the ASARM (aspartic acid and serine rich domain domain involved in biomineralization and the DNA binding site of DMP1. In vitro experiments with recombinant N- and C-terminal polypeptides mimicking the MMP-2 cleavage products of DMP1 demonstrated an effect of the C-polypeptide on the differentiation of dental pulp stem/progenitor cells to a putative odontoblast phenotype. In vivo implantation of this peptide in a rat injured pulp model induced a rapid formation of a homogeneous dentin bridge covered by a palisade of orientated cells expressing dentin sialoprotein (DSP and DMP1, attesting an efficient repair process. These data suggest that a peptide generated through the proteolytic processing of DMP1 by MMP-2 can regulate the differentiation of mesenchymal cells during dentinogenesis and thus sustain reparative dentin formation in pathological situations such as carious decay. In addition, these data open a new therapeutic possibility of using this peptide to regenerate dentin after an injury.

  11. In vitro evaluation of triazenes: DNA cleavage, antibacterial activity and cytotoxicity against acute myeloid leukemia cells

    Energy Technology Data Exchange (ETDEWEB)

    Domingues, Vanessa O.; Hoerner, Rosmari; Reetz, Luiz G.B.; Kuhn, Fabio, E-mail: rosmari.ufsm@gmail.co [Universidade Federal de Santa Maria (UFSM), RS (Brazil). Dept. de Analises Clinicas e Toxicologicas; Coser, Virginia M.; Rodrigues, Jacqueline N.; Bauchspiess, Rita; Pereira, Waldir V. [Hospital Universitario de Santa Maria, RS (Brazil). Dept. de Hematologia-Oncologia; Paraginski, Gustavo L.; Locatelli, Aline; Fank, Juliana de O.; Giglio, Vinicius F.; Hoerner, Manfredo, E-mail: hoerner.manfredo@gmail.co [Universidade Federal de Santa Maria (UFSM), RS (Brazil). Dept. de Quimica

    2010-07-01

    The asymmetric diazoamines 1-(2-chlorophenyl)-3-(4-carboxyphenyl)triazene (1), 1-(2-fluorophenyl)-3-(4-carboxyphenyl)triazene (2) and 1-(2-fluorophenyl)-3-(4-amidophenyl) triazene (3) were evaluated for their ability to cleave pUC18 and pBSKII plasmid DNA, antibacterial activity and in vitro cytotoxicity against acute myeloid leukemia cells and normal leukocytes using the bioassay of reduction of 3-(4,5-dimethylthiazole-2-yl)-2,5-diphenyltetrazolium bromide (MTT). The triazenes showed ability to cleave the two types of plasmid DNA: triazene 1 at pH 8.0 and 50 deg C; triazene 2 at pH 6.5 and 37 and 50 deg C; triazene 3 at pH 6.5 and 37 deg C. The compounds presented cytotoxic activity against myeloid leukemia cells. Compound 1 showed high activity against B. cereus (MIC = 32 {mu}g mL{sup -1}). The observation of intermolecular hydrogen bonding in the solid state of compound 3, based on the structural analysis by X-ray crystallography, as well as the results of IR and UV-Vis spectroscopic analyses of compounds 1, 2 and 3 are discussed in the present work. (author)

  12. Efficient hammerhead ribozyme-mediated cleavage of the structured hepatitis B virus encapsidation signal in vitro and in cell extracts, but not in intact cells.

    Science.gov (United States)

    Beck, J; Nassal, M

    1995-01-01

    Hepatitis B virus (HBV), the causative agent of B-type hepatitis in man, is a small enveloped DNA virus that replicates through reverse transcription of an RNA intermediate, the terminally redundant RNA pregenome. An essential highly conserved cis-element present twice on this RNA is the encapsidation signal epsilon, a stem-loop structure that is critical for pregenome packaging and reverse transcription. Epsilon is hence an attractive target for antiviral therapy. Its structure, however, is a potential obstacle to antivirals whose action depends on hybridization, e.g. ribozymes. Here we demonstrate effective in vitro cleavage inside epsilon by hammerhead ribozymes containing flanking sequences complementary to an adjacent less structured region. Upon co-transfection with a HBV expression construct corresponding ribozymes embedded in a U6 snRNA context led to a significant, though modest, reduction in the steady-state level of HBV pregenomes. Inactive ribozyme mutants revealed that antisense effects contributed substantially to this reduction, however, efficient epsilon cleavage by the intracellularly expressed ribozymes was observed in Mg(2+)-supplemented cell lysates. Artificial HBV pregenomes carrying the ribozymes in cis and model RNAs lacking all HBV sequences except epsilon exhibited essentially the same behaviour. Hence, neither the absence of co-localization of ribozyme and target nor a viral component, but rather a cellular factor(s), is responsible for the strikingly different ribozyme activities inside cells and in cellular extracts. Images PMID:8559651

  13. The Cell Death Pathway Regulates Synapse Elimination through Cleavage of Gelsolin in Caenorhabditis elegans Neurons

    Directory of Open Access Journals (Sweden)

    Lingfeng Meng

    2015-06-01

    Full Text Available Synapse elimination occurs in development, plasticity, and disease. Although the importance of synapse elimination has been documented in many studies, the molecular mechanisms underlying this process are unclear. Here, using the development of C. elegans RME neurons as a model, we have uncovered a function for the apoptosis pathway in synapse elimination. We find that the conserved apoptotic cell death (CED pathway and axonal mitochondria are required for the elimination of transiently formed clusters of presynaptic components in RME neurons. This function of the CED pathway involves the activation of the actin-filament-severing protein, GSNL-1. Furthermore, we show that caspase CED-3 cleaves GSNL-1 at a conserved C-terminal region and that the cleaved active form of GSNL-1 promotes its actin-severing ability. Our data suggest that activation of the CED pathway contributes to selective elimination of synapses through disassembly of the actin filament network.

  14. Rapid cytochrome c release, activation of caspases 3, 6, 7 and 8 followed by Bap31 cleavage in HeLa cells treated with photodynamic therapy.

    Science.gov (United States)

    Granville, D J; Carthy, C M; Jiang, H; Shore, G C; McManus, B M; Hunt, D W

    1998-10-16

    Photodynamic therapy (PDT) is a clinical approach that utilizes light-activated drugs for the treatment of a variety of pathologic conditions. The initiating events of PDT-induced apoptosis are poorly defined. It has been shown for other proapoptotic stimuli that the integral endoplasmic reticulum protein Bap31 is cleaved by caspases 1 and 8, but not by caspase-3. Further, a 20 kDa Bap31 cleavage fragment is generated which can induce apoptosis. In the current report, we sought to determine whether Bap31 cleavage and generation of p20 is an early event in PDT-induced apoptosis. The mitochondrial release of cytochrome c, involvement of caspases 1, 2, 3, 4, 6, 7, 8, and 10 and the status of several known caspase substrates, including Bap31, were evaluated in PDT-treated HeLa cells. Cytochrome c appeared in the cytosol immediately following light activation of the photosensitizer benzoporphyrin derivative monoacid ring A. Activation of caspases 3, 6, 7, and 8 was evident within 1-2 h post PDT. Processing of caspases 1, 2, 4, and 10 was not observed. Cleavage of Bap31 was observed at 2-3 h post PDT. The caspase-3 inhibitor DEVD-fmk blocked caspase-8 and Bap31 cleavage suggesting that caspase-8 and Bap31 processing occur downstream of caspase-3 activation in PDT-induced apoptosis. These results demonstrate that release of mitochondrial cytochrome c into the cytoplasm is a primary event following PDT, preceding caspase activation and cleavage of Bap31. To our knowledge, this is the first example of a chemotherapeutic agent inducing caspase-8 activation and demonstrates that caspase-8 activation can occur after cytochrome c release.

  15. Centralspindlin in Rappaport's cleavage signaling.

    Science.gov (United States)

    Mishima, Masanori

    2016-05-01

    Cleavage furrow in animal cell cytokinesis is formed by cortical constriction driven by contraction of an actomyosin network activated by Rho GTPase. Although the role of the mitotic apparatus in furrow induction has been well established, there remain discussions about the detailed molecular mechanisms of the cleavage signaling. While experiments in large echinoderm embryos highlighted the role of astral microtubules, data in smaller cells indicate the role of central spindle. Centralspindlin is a constitutive heterotetramer of MKLP1 kinesin and the non-motor CYK4 subunit and plays crucial roles in formation of the central spindle and recruitment of the downstream cytokinesis factors including ECT2, the major activator of Rho during cytokinesis, to the site of division. Recent reports have revealed a role of this centralspindlin-ECT2 pathway in furrow induction both by the central spindle and by the astral microtubules. Here, a unified view of the stimulation of cortical contractility by this pathway is discussed. Cytokinesis, the division of the whole cytoplasm, is an essential process for cell proliferation and embryonic development. In animal cells, cytokinesis is executed using a contractile network of actin filaments driven by a myosin-II motor that constricts the cell cortex (cleavage furrow ingression) into a narrow channel between the two daughter cells, which is resolved by scission (abscission) [1-3]. The anaphase-specific organization of the mitotic apparatus (MA, spindle with chromosomes plus asters) positions the cleavage furrow and plays a major role in spatial coupling between mitosis and cytokinesis [4-6]. The nucleus and chromosomes are dispensable for furrow specification [7-10], although they contribute to persistent furrowing and robust completion in some cell types [11,12]. Likewise, centrosomes are not essential for cytokinesis, but they contribute to the general fidelity of cell division [10,13-15]. Here, classical models of cleavage furrow

  16. Methionine catabolism in Arabidopsis cells is initiated by a gamma-cleavage process and leads to S-methylcysteine and isoleucine syntheses.

    Science.gov (United States)

    Rébeillé, Fabrice; Jabrin, Samuel; Bligny, Richard; Loizeau, Karen; Gambonnet, Bernadette; Van Wilder, Valérie; Douce, Roland; Ravanel, Stéphane

    2006-10-17

    Despite recent progress in elucidating the regulation of methionine (Met) synthesis, little is known about the catabolism of this amino acid in plants. In this article, we present several lines of evidence indicating that the cleavage of Met catalyzed by Met gamma-lyase is the first step in this process. First, we cloned an Arabidopsis cDNA coding a functional Met gamma-lyase (AtMGL), a cytosolic enzyme catalyzing the conversion of Met into methanethiol, alpha-ketobutyrate, and ammonia. AtMGL is present in all of the Arabidopsis organs and tissues analyzed, except in quiescent dry mature seeds, thus suggesting that AtMGL is involved in the regulation of Met homeostasis in various situations. Also, we demonstrated that the expression of AtMGL was induced in Arabidopsis cells in response to high Met levels, probably to bypass the elevated Km of the enzyme for Met. Second, [13C]-NMR profiling of Arabidopsis cells fed with [13C]Met allowed us to identify labeled S-adenosylmethionine, S-methylmethionine, S-methylcysteine (SMC), and isoleucine (Ile). The unexpected production of SMC and Ile was directly associated to the function of Met gamma-lyase. Indeed, we showed that part of the methanethiol produced during Met cleavage could react with an activated form of serine to produce SMC. The second product of Met cleavage, alpha-ketobutyrate, entered the pathway of Ile synthesis in plastids. Together, these data indicate that Met catabolism in Arabidopsis cells is initiated by a gamma-cleavage process and can result in the formation of the essential amino acid Ile and a potential storage form for sulfide or methyl groups, SMC.

  17. Caspase-mediated cleavage of Beclin1 inhibits autophagy and promotes apoptosis induced by S1 in human ovarian cancer SKOV3 cells.

    Science.gov (United States)

    Li, Xiaoning; Su, Jing; Xia, Meihui; Li, Hongyan; Xu, Ye; Ma, Chunhui; Ma, Liwei; Kang, Jingsong; Yu, Huimei; Zhang, Zhichao; Sun, Liankun

    2016-02-01

    S1, a novel BH3 mimetic, can induce apoptosis dependent on Bax/Bak through inhibition of Bcl-2 in various tumors. S1 also induces autophagy through interrupting the interaction of Bcl-2 and Beclin1. Our results showed that S1 induces apoptosis in human ovarian cancer SKOV3 cells in a time- and dose-dependent manner. Autophagy precedes apoptosis, in SKOV3 cells treated with S1 (6 μmol/L), autophagy reached the maximum peak at 12 h after treatment and decreased to 24 h. In SKOV3 cells treated with different concentrations of S1 for 24 h, the highest level of autophagy was observed with 5 μmol/L and decreased to 10 μmol/L. Autophagy inhibitors 3-MA and CQ enhanced apoptosis induced by S1 in SKOV3 cells. However, overactivation of caspases in apoptosis induced by S1 may inhibit the autophagy-inducing function of Beclin1. Because the pan-caspase inhibitor Z-VAD recovered the autophagy-inducing function of Beclin1 through reduction of activated caspase-mediated cleavage of Beclin1. Furthermore, the Beclin1 cleavage products could further increase apoptosis induced by S1 in SKOV3 cells. This indicates that apoptosis induced by high doses and long exposure of S1 causes the overactivation of caspases and subsequent cleavage of Beclin1, and inhibits the protection of autophagy. Moreover, the cleaved product of Beclin1 further promotes apoptosis induced by S1 in SKOV3 cells. Our results suggest this may be a molecular mechanism for enhancing the sensitivity of cancer cells to apoptosis induced by small molecular compound targeting Bcl-2.

  18. CD40 ligand induced cytotoxicity in carcinoma cells is enhanced by inhibition of metalloproteinase cleavage and delivery via a conditionally-replicating adenovirus

    Directory of Open Access Journals (Sweden)

    Young Lawrence S

    2010-03-01

    Full Text Available Abstract Background CD40 and its ligand (CD40L play a critical role in co-ordinating immune responses. CD40 is also expressed in lymphoid malignancies and a number of carcinomas. In carcinoma cells the physiological outcome of CD40 ligation depends on the level of receptor engagement with low levels promoting cell survival and high levels inducing cell death. The most profound induction of cell death in carcinoma cells is induced by membrane-bound rather than recombinant soluble CD40L, but like other TNF family ligands, it is cleaved from the membrane by matrix metalloproteinases. Results We have generated a replication-deficient adenovirus expressing a mutant CD40L that is resistant to metalloproteinase cleavage such that ligand expression is retained at the cell membrane. Here we show that the mutated, cleavage-resistant form of CD40L is a more potent inducer of apoptosis than wild-type ligand in CD40-positive carcinoma cell lines. Since transgene expression via replication-deficient adenovirus vectors in vivo is low, we have also engineered a conditionally replicating E1A-CR2 deleted adenovirus to express mutant CD40L, resulting in significant amplification of ligand expression and consequent enhancement of its therapeutic effect. Conclusions Combined with numerous studies demonstrating its immunotherapeutic potential, these data provide a strong rationale for the exploitation of the CD40-CD40L pathway for the treatment of solid tumours.

  19. Comparison of non-canonical PAMs for CRISPR/Cas9-mediated DNA cleavage in human cells

    OpenAIRE

    Yilan Zhang; Xianglian Ge; Fayu Yang; Liping Zhang; Jiayong Zheng; Xuefang Tan; Zi-Bing Jin; Jia Qu; Feng Gu

    2014-01-01

    CRISPR/Cas9 -mediated DNA cleavage (CCMDC) is becoming increasingly used for efficient genome engineering. Proto-spacer adjacent motif (PAM) adjacent to target sequence is one of the key components in the design of CCMDC strategies. It has been reported that NAG sequences are the predominant non-canonical PAM for CCMDC at the human EMX locus, but it is not clear whether it is universal at other loci. In the present study, we attempted to use a GFP-reporter system to comprehensively and quanti...

  20. Grb7 and Hax1 may colocalize partially to mitochondria in EGF treated SKBR3 cells and their interaction can affect Caspase3 cleavage of Hax1

    Science.gov (United States)

    Qian, Lei; Bradford, Andrew M.; Cooke, Peter H.; Lyons, Barbara A.

    2017-01-01

    Growth factor receptor bound protein 7 (Grb7) is a signal transducing adaptor protein that mediates specific protein-protein interactions in multiple signaling pathways. Grb7, with Grb10 and Grb14, are members of the Grb7 protein family. The topology of the Grb7 family members contains several protein-binding domains that facilitate the formation of protein complexes and high signal transduction efficiency. Grb7 has been found overexpressed in several types of cancers and cancer cell lines, and is presumed involved in cancer progression through promotion of cell proliferation and migration via interactions with the ErbB2 (HER2) receptor, FAK (focal adhesion kinase), Ras-GTPases, and other signaling partners. We previously reported Grb7 binds to Hax1 (HS1 associated protein X1) isoform 1, an anti-apoptotic protein also involved in cell proliferation and calcium homeostasis. In this study, we confirm the in vitro Grb7/Hax1 interaction is exclusive to these two proteins and their interaction does not depend on Grb7 dimerization state. In addition, we report Grb7 and Hax1 isoform 1 may colocalize partially to mitochondria in EGF treated SKBR3 cells and growth conditions can affect this colocalization. Moreover, Grb7 can affect Caspase3 cleavage of the Hax1 isoform 1 in vitro, and Grb7 expression may slow the Caspase3 cleavage of Hax1 isoform 1 in apoptotic HeLa cells. Finally, Grb7 is shown to increase cell viability in apoptotic HeLa cells in a time dependent manner. Taken together, these discoveries provide clues for the role of a Grb7/Hax1 protein interaction in apoptosis pathways involving Hax1. PMID:26869103

  1. Presenilin 1/gamma-secretase is associated with cadmium-induced E-cadherin cleavage and COX-2 gene expression in T47D breast cancer cells.

    Science.gov (United States)

    Park, Chang Seok; Kim, Ohn Soon; Yun, Sang-Moon; Jo, Sangmee A; Jo, Inho; Koh, Young Ho

    2008-12-01

    Cadmium is a heavy metal that has multiple toxic effects on human health and has been classified as a human carcinogen. E-cadherin is a major target of cadmium; however, the roles of E-cadherin and cadmium and the mechanisms of tumor progression remain to be defined. Here, we demonstrate that cadmium increases E-cadherin processing via a gamma-secretase in the T47D breast cancer cell lines. This presenilin 1 (PS1)/gamma-secretase-dependent cleavage of E-cadherin was accompanied by changes in reactive oxygen species or calcium. E-cadherin cleavage was blocked by a PS1 dominant-negative mutant, gamma-secretase inhibitors [N-[N-(3,5-Difluorophenacetyl-L-alanyl)]-S-phenylglycine t-butyl ester (DAPT) and L-685,486], antioxidants (N-acetylcysteine and Mn(III)tetrakis(1-methyl-4-pyridyl)porphyrin pentachloride), or a calcium chelating drug 1,2-bis(o-Aminophenoxy)ethane-N,N,N',N'-tetraacetic acid tetra(acetoxymethyl) ester. Immunofluorescence analysis confirmed the disappearance of E-cadherin staining at the cell surface. Those inhibitors attenuated cadmium-induced cytotoxicity. Additionally, cadmium treatment increased cell motility and invasion ability, which was abated by DAPT. Interestingly, cyclooxygenase-2 (COX-2) expression induced by cadmium was also inhibited by DAPT. The cadmium-induced cell motility and invasion ability were inhibited by a COX-2 inhibitor, NS398. Our data indicate a novel molecular mechanism that links cytotoxicity of cadmium and disrupted E-cadherin processing to adherens junctions; cadmium induces COX-2 expression via gamma-secretase, which increases cell motility and invasion ability. Understanding the downstream signaling cascades of cadmium that promote tumor progression might be a key to the development of novel therapeutic strategies.

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

    Directory of Open Access Journals (Sweden)

    Robert J Lake

    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.

  3. The position of DNA cleavage by TALENs and cell synchronization influences the frequency of gene editing directed by single-stranded oligonucleotides.

    Directory of Open Access Journals (Sweden)

    Natalia Rivera-Torres

    Full Text Available With recent technological advances that enable DNA cleavage at specific sites in the human genome, it may now be possible to reverse inborn errors, thereby correcting a mutation, at levels that could have an impact in a clinical setting. We have been developing gene editing, using single-stranded DNA oligonucleotides (ssODNs, as a tool to direct site specific single base changes. Successful application of this technique has been demonstrated in many systems ranging from bacteria to human (ES and somatic cells. While the frequency of gene editing can vary widely, it is often at a level that does not enable clinical application. As such, a number of stimulatory factors such as double-stranded breaks are known to elevate the frequency significantly. The majority of these results have been discovered using a validated HCT116 mammalian cell model system where credible genetic and biochemical readouts are available. Here, we couple TAL-Effector Nucleases (TALENs that execute specific ds DNA breaks with ssODNs, designed specifically to repair a missense mutation, in an integrated single copy eGFP gene. We find that proximal cleavage, relative to the mutant base, is key for enabling high frequencies of editing. A directionality of correction is also observed with TALEN activity upstream from the target base being more effective in promoting gene editing than activity downstream. We also find that cells progressing through S phase are more amenable to combinatorial gene editing activity. Thus, we identify novel aspects of gene editing that will help in the design of more effective protocols for genome modification and gene therapy in natural genes.

  4. Altered catalytic activity of and DNA cleavage by DNA topoisomerase II from human leukemic cells selected for resistance to VM-26.

    Science.gov (United States)

    Danks, M K; Schmidt, C A; Cirtain, M C; Suttle, D P; Beck, W T

    1988-11-29

    The simultaneous development of resistance to the cytotoxic effects of several classes of natural product anticancer drugs, after exposure to only one of these agents, is referred to as multiple drug resistance (MDR). At least two distinct mechanisms for MDR have been postulated: that associated with P-glycoprotein and that thought to be due to an alteration in DNA topoisomerase II activity (at-MDR). We describe studies with two sublines of human leukemic CCRF-CEM cells approximately 50-fold resistant (CEM/VM-1) and approximately 140-fold resistant (CEM/VM-1-5) to VM-26, a drug known to interfere with DNA topoisomerase II activity. Each of these lines is cross-resistant to other drugs known to affect topoisomerase II but not cross-resistant to vinblastine, an inhibitor of mitotic spindle formation. We found little difference in the amount of immunoreactive DNA topoisomerase II in 1.0 M NaCl nuclear extracts of the two resistant and parental cell lines. However, topoisomerase II in nuclear extracts of the resistant sublines is altered in both catalytic activity (unknotting) of and DNA cleavage by this enzyme. Also, the rate at which catenation occurs is 20-30-fold slower with the CEM/VM-1-5 preparations. The effect of VM-26 on both strand passing and DNA cleavage is inversely related to the degree of primary resistance of each cell line. Our data support the hypothesis that at-MDR is due to an alteration in topoisomerase II or in a factor modulating its activity.

  5. T cell antigen receptor stimulation induces MALT1 paracaspase-mediated cleavage of the NF-kappaB inhibitor A20.

    Science.gov (United States)

    Coornaert, Beatrice; Baens, Mathijs; Heyninck, Karen; Bekaert, Tine; Haegman, Mira; Staal, Jens; Sun, Lijun; Chen, Zhijian J; Marynen, Peter; Beyaert, Rudi

    2008-03-01

    The paracaspase MALT1 mediates T cell antigen receptor-induced signaling to the transcription factor NF-kappaB and is indispensable for T cell activation and proliferation. Enhanced expression of MALT1 or aberrant expression of a fusion protein of the apoptosis inhibitor API2 and MALT1 has been linked to mucosa-associated lymphoid tissue lymphoma. Despite the presence of a caspase-like domain, MALT1 proteolytic activity has not yet been demonstrated. Here we show that T cell antigen receptor stimulation induced recruitment of the NF-kappaB inhibitor A20 into a complex of MALT1 and the adaptor protein Bcl-10, leading to MALT1-mediated processing of A20. API2-MALT1 expression likewise resulted in cleavage of A20. MALT1 cleaved human A20 after arginine 439 and impaired its NF-kappaB-inhibitory function. Our studies identify A20 as a substrate of MALT1 and emphasize the importance of MALT1 proteolytic activity in the 'fine tuning' of T cell antigen receptor signaling.

  6. Induction of cell death by ternary copper(II) complexes of L-tyrosine and diimines: role of coligands on DNA binding and cleavage and anticancer activity.

    Science.gov (United States)

    Ramakrishnan, Sethu; Rajendiran, Venugopal; Palaniandavar, Mallayan; Periasamy, Vaiyapuri Subbarayan; Srinag, Bangalore Suresh; Krishnamurthy, Hanumanthappa; Akbarsha, Mohammad Abdulkader

    2009-02-16

    viscosity of DNA bound to 1 decreases, indicating the shortening of the DNA chain length by means of the formation of kinks or bends. All complexes exhibit effective DNA (pUC19 DNA) cleavage at 100 microM complex concentrations, and the order of DNA cleavage ability varies as 3 > 2 > 4 > 1. Interestingly, 3 exhibits a DNA cleavage rate constant that is higher than that of the other complexes only at 100 microM concentration, whereas 4 exhibits the highest cleavage rate constant at 80 microM complex concentration. The oxidative DNA cleavage follows the order 4 > 3 > 2 > 1. Mechanistic studies reveal that the DNA cleavage pathway involves hydroxyl radicals. Interestingly, only 4 displays efficient photonuclease activity upon irradiation with 365 nm light, which occurs through double-strand DNA breaks involving hydroxyl radicals. Furthermore, cytotoxicity studies on the nonsmall lung cancer (H-460) cell line show that the IC(50) values of 2-4 are more or less equal to cisplatin for the same cell line, indicating that they have the potential to act as very effective anticancer drugs in a time-dependent manner. The study of cytological changes reveals the higher induction of apoptosis and mitotic catastrophe for 4 and 3, respectively. The alkaline single-cell gel electrophoresis (comet assay), DNA laddering, and AO/EB and Hoechst 33258 staining assays have also been employed in finding the extent of DNA damage. Flow cytometry analysis shows an increase in the percentage of cells with apoptotic morphological features in the sub-G(0)/G(1) phase for 4, whereas it shows mitotic catastrophe for 3.

  7. Extended cleavage specificity of the mast cell chymase from the crab-eating macaque (Macaca fascicularis): an interesting animal model for the analysis of the function of the human mast cell chymase.

    Science.gov (United States)

    Thorpe, Michael; Yu, Jing; Boinapally, Vamsi; Ahooghalandari, Parvin; Kervinen, Jukka; Garavilla, Lawrence de; Hellman, Lars

    2012-12-01

    Serine proteases are the major protein constituents within mast cell secretory granules. These proteases are subdivided into chymases and tryptases depending on their primary cleavage specificity. Here, we present the extended cleavage specificity of the macaque mast cell chymase and compare the specificity with human chymase (HC) and dog chymase (DC) that were produced in the same insect cell expression host. The macaque chymase (MC) shows almost identical characteristics as the HC, including both primary and extended cleavage specificities as well as sensitivity to protease inhibitors, whereas the DC differs in several of these characteristics. Although previous studies have shown that mouse mast cell protease-4 (mMCP-4) is similar in its hydrolytic specificity to the HC, mouse mast cells contain several related enzymes. Thus mice may not be the most appropriate model organism for studying HC activity and inhibition. Importantly, macaques express only one chymase and, as primates, are closely related to human general physiology. In addition, the human and macaque enzymes both cleave angiotensin I (Ang I) in the same way, generating primarily angiotensin II (Ang II) and they do not further degrade the peptide like most rodent enzymes do. Both enzymes also cleave two additional potential in vivo substrates, fibronectin and secretory leukocyte protease inhibitor (SLPI) in a similar way. Given the fact that both HC and MC are encoded by a single gene with high sequence homology and that many physiological processes are similar between these species, the macaque may be a very interesting model to study the physiological role of the chymase and to determine the potency and potential side-effects of various chymase inhibitors designed for therapeutic human use.

  8. NALP3 inflammasome upregulation and CASP1 cleavage of the glucocorticoid receptor cause glucocorticoid resistance in leukemia cells

    NARCIS (Netherlands)

    S.W. Paugh (Steven); E.J. Bonten (Erik J.); D. Savic (Daniel); L.B. Ramsey (Laura B.); W.E. Thierfelder (William E.); P. Gurung (Prajwal); R.K.S. Malireddi (R. K. Subbarao); M. Actis (Marcelo); A. Mayasundari (Anand); J. Min (Jaeki); D.R. Coss (David R.); L.T. Laudermilk (Lucas T.); J.C. Panetta (John); J.R. McCorkle (J. Robert); Y. Fan (Yiping); K.R. Crews (Kristine R.); G. Stocco (Gabriele); M.R. Wilkinson (Mark R.); A.M. Ferreira (Antonio M.); C. Cheng (Cheng); W. Yang (Wenjian); S.E. Karol (Seth E.); C.A. Fernandez (Christian A.); B. Diouf (Barthelemy); C. Smith (Colton); J.K. Hicks (J Kevin); A. Zanut (Alessandra); A. Giordanengo (Audrey); D.J. Crona; J.J. Bianchi (Joy J.); L. Holmfeldt (Linda); C.G. Mullighan (Charles); M.L. den Boer (Monique); R. Pieters (Rob); S. Jeha (Sima); T.L. Dunwell (Thomas L.); F. Latif (Farida); D. Bhojwani (Deepa); W.L. Carroll (William L.); C.-H. Pui (Ching-Hon); R.M. Myers (Richard M.); R.K. Guy (R Kiplin); T.-D. Kanneganti (Thirumala-Devi); M.V. Relling (Mary); W.E. Evans (William)

    2015-01-01

    textabstractGlucocorticoids are universally used in the treatment of acute lymphoblastic leukemia (ALL), and resistance to glucocorticoids in leukemia cells confers poor prognosis. To elucidate mechanisms of glucocorticoid resistance, we determined the prednisolone sensitivity of primary leukemia ce

  9. Limited caspase cleavage of human BAP31.

    Science.gov (United States)

    Määttä, J; Hallikas, O; Welti, S; Hildén, P; Schröder, J; Kuismanen, E

    2000-11-10

    Human BAP31 was cleaved at both of its two identical caspase cleavage sites in two previously reported models of apoptosis. We show here that only the most carboxy-terminal site is cleaved during apoptosis induced in HeLa cells by tunicamycin, tumor necrosis factor and cycloheximide, or staurosporine. Similar results were obtained in HL-60 cells using Fas/APO-1 antibodies, or cycloheximide. This limited cleavage, which is inhibited by several caspase inhibitors, removes eight amino acids from human BAP31 including the KKXX coat protein I binding motif. Ectopic expression of the resulting cleavage product induces redistribution of mannosidase II from the Golgi and prevents endoplasmic reticulum to Golgi transport of virus glycoproteins.

  10. Peptide bond cleavage site determination of novel proteolytic enzymes found in ROS 17/2.8 cell lysates.

    Science.gov (United States)

    Guidon, P T; Perrin, D; Harrison, P

    1996-02-01

    We have identified proteolytic activities in the rat osteoblastic osteosarcoma cell line ROS 17/2.8 which are capable of cleaving a peptide substrate for protein kinase C-mediated phosphorylation (PSPKC, Pro-Leu-Ser-Arg-Thr-Leu-Ser-Val-Ala-Ala-Lys). Using polyacrylamide gel electrophoresis conditions similar to those used to resolve small molecular weight proteins, the peptide bonds of PSPKC which are cleaved by the proteolytic activities present in ROS 17/2.8 cell lysates have been determined. These activities cleave the Ser-Arg, Thr-Leu, and Ser-Val peptide bonds. To date, no proteolytic activities present in osteoblast cell lysates have been described with the aforementioned peptide bond specificities, suggesting that these activities are novel. The PSPKC-cleaved peptide fragment pattern generated was similar for several different osteoblast cell lysates. Lysates generated from different rat tissues were also able to cleave PSPKC, but the peptide fragment pattern generated by ROS 17/2.8 cell lysates appeared to be unique amongst these tissues.

  11. In vivo exposure of young adult male rats to methoxychlor reduces serum testosterone levels and ex vivo Leydig cell testosterone formation and cholesterol side-chain cleavage activity.

    Science.gov (United States)

    Murono, Eisuke P; Derk, Raymond C; Akgul, Yucel

    2006-02-01

    Methoxychlor (MC) was developed as a replacement for the banned pesticide DDT. After in vivo administration, it is metabolized in the liver to 2,2-bis(p-hydroxyphenyl)-1,1,1-trichloroethane (HPTE), which is proposed to be the active agent. Both MC and HPTE have been shown to exhibit weak estrogenic and antiandrogenic activities, and they are thought to exert their effects through estrogen and androgen receptors, respectively. Although in vitro studies using cultured rat Leydig cells have reported that HPTE inhibits both basal and hCG-stimulated testosterone formation, the response of circulating testosterone levels to in vivo MC has been more variable. Therefore, the current studies evaluated whether the daily in vivo administration of MC (0, 5, 40 and 200 mg/kg body weight) for a short duration (days 54-60 of age) by gavage altered serum testosterone levels and ex vivo Leydig cell testosterone formation in young adult male rats. These results demonstrate that both fluid-retained and fluid-expressed seminal vesicle weights declined to 44 and 60% of control, respectively, in the 200 mg/kg MC-exposed animals. Similarly, serum testosterone and dehydroepiandrosterone levels declined to 41 and 45% of control, respectively, in the 200 mg/kg MC-exposed animals; however, serum LH and FSH levels were unaffected. Ex vivo Leydig cell basal testosterone formation over 4h declined to 49% of control in animals exposed to 200 mg/kg MC, and ex vivo Leydig cell P450 cholesterol side-chain cleavage activity declined to 79 and 50% of control in animals exposed to 40 and 200 mg/kg of MC, respectively, supporting previous in vitro studies which demonstrated the sensitivity of this step to MC.

  12. Vasoactive intestinal peptide-induced expression of cytochrome P450 cholesterol side-chain cleavage and 17 alpha-hydroxylase enzyme activity in hen granulosa cells.

    Science.gov (United States)

    Johnson, A L; Li, Z; Gibney, J A; Malamed, S

    1994-08-01

    Experiments were conducted to determine whether vasoactive intestinal peptide (VIP) can regulate expression of cytochrome P450 side-chain cleavage (P450scc) and P450 17 alpha-hydroxylase (P450 17 alpha-OH) mRNA levels and enzyme activity in granulosa cells from nonhierarchal (6-8-mm) follicles. Initial studies demonstrated that immunoreactive VIP is localized within the theca (but not granulosa) layer of both resting (< 0.5-mm follicles) and 6-8-mm follicles, thus providing a potential paracrine mechanism of action for VIP. While short-term (3 h) incubation of granulosa cells with VIP (0.001-1.0 microM) failed to stimulate progesterone production from 6-8-mm follicle granulosa cells, a 4-h culture period in the presence of VIP resulted in increased cyclic AMP (cAMP) accumulation, and a 24-h culture period resulted in progesterone synthesis and increased P450scc mRNA levels; control levels of each endpoint measurement were not altered within the period observed. By contrast, culture with the growth factor transforming growth factor alpha (TGF alpha) in the presence of VIP (1 microM) prevented increases in P450scc mRNA levels and progesterone production. Similar effects of VIP and TGF alpha in the presence of VIP were demonstrated for P450 17 alpha-OH mRNA levels and enzyme activity. Finally, there was an additive effect of VIP (0.1 microM) plus recombinant human (rh) FSH (100 mIU) on the initiation of progesterone production in cultured 6-8-mm follicle granulosa cells compared to the addition of VIP or rhFSH alone.(ABSTRACT TRUNCATED AT 250 WORDS)

  13. The polyadenylation factor subunit CLEAVAGE AND POLYADENYLATION SPECIFICITY FACTOR30: A key factor of programmed cell death and a regulator of immunity in arabidopsis

    KAUST Repository

    Bruggeman, Quentin

    2014-04-04

    Programmed cell death (PCD) is essential for several aspects of plant life, including development and stress responses. Indeed, incompatible plant-pathogen interactions are well known to induce the hypersensitive response, a localized cell death. Mutational analyses have identified several key PCD components, and we recently identified the mips1 mutant of Arabidopsis (Arabidopsis thaliana), which is deficient for the key enzyme catalyzing the limiting step of myoinositol synthesis. One of the most striking features of mips1 is the light-dependent formation of lesions on leaves due to salicylic acid (SA)-dependent PCD, revealing roles for myoinositol or inositol derivatives in the regulation of PCD. Here, we identified a regulator of plant PCD by screening for mutants that display transcriptomic profiles opposing that of the mips1 mutant. Our screen identified the oxt6 mutant, which has been described previously as being tolerant to oxidative stress. In the oxt6 mutant, a transfer DNA is inserted in the CLEAVAGE AND POLYADENYLATION SPECIFICITY FACTOR30 (CPSF30) gene, which encodes a polyadenylation factor subunit homolog. We show that CPSF30 is required for lesion formation in mips1 via SA-dependent signaling, that the prodeath function of CPSF30 is not mediated by changes in the glutathione status, and that CPSF30 activity is required for Pseudomonas syringae resistance. We also show that the oxt6 mutation suppresses cell death in other lesion-mimic mutants, including lesion-simulating disease1, mitogen-activated protein kinase4, constitutive expressor of pathogenesis-related genes5, and catalase2, suggesting that CPSF30 and, thus, the control of messenger RNA 3′ end processing, through the regulation of SA production, is a key component of plant immune responses. © 2014 American Society of Plant Biologists. All rights reserved.

  14. The methoxychlor metabolite, HPTE, directly inhibits the catalytic activity of cholesterol side-chain cleavage (P450scc) in cultured rat ovarian cells.

    Science.gov (United States)

    Akgul, Yucel; Derk, Raymond C; Meighan, Terence; Rao, K Murali Krishna; Murono, Eisuke P

    2008-01-01

    Exposure to the pesticide methoxychlor in rodents is linked to impaired steroid production, ovarian atrophy and reduced fertility. Following in vivo administration, it is rapidly converted by the liver to 2,2-bis-(p-hydroxyphenyl)-1,1,1-trichloroethane (HPTE), the reported active metabolite. Both methoxychlor and HPTE have weak estrogenic and antiandrogenic activities, and these effects are thought to be mediated through the estrogen and androgen receptors, respectively. Previous in vivo studies on methoxychlor exposure to female animals have demonstrated decreased progesterone production but no change in serum estrogen levels. We recently showed that HPTE specifically inhibits the P450 cholesterol side-chain cleavage (P450scc, CYP11A1) step resulting in decreased androgen production by cultured rat testicular Leydig cells. The current studies examined the mechanism of action of HPTE on progesterone production by cultured ovarian cells (granulosa and theca-interstitial) from pregnant mare serum gonadotropin-primed immature rats. In addition, we evaluated whether the effects of HPTE on rat ovarian cell progesterone biosynthesis were mediated through the estrogen or androgen receptors. Exposure to HPTE (0, 10, 50 or 100nM) alone progressively inhibited progesterone formation in cultured theca-interstitial and granulosa cells and the P450scc catalytic activity in theca-interstitial cells in a dose-dependent manner with significant declines starting at 50nM. However, HPTE did not change mRNA levels of the P450scc system (P450scc, adrenodoxin reductase and adrenodoxin) as well as P450scc protein levels. Of interest, estradiol, xenoestrogens (bisphenol-A or 4-tert-octylphenol), a pure antiestrogen (ICI 182,780), or antiandrogens (4-hydroxyflutamide or the vinclozolin metabolite M-2), had no effect on progesterone production even at 1000nM. Co-treatment of HPTE with ICI 182,780 did not block the effect of HPTE on progesterone formation. These studies suggest that the

  15. A small molecule PAI-1 functional inhibitor attenuates neointimal hyperplasia and vascular smooth muscle cell survival by promoting PAI-1 cleavage.

    Science.gov (United States)

    Simone, Tessa M; Higgins, Stephen P; Archambeault, Jaclyn; Higgins, Craig E; Ginnan, Roman G; Singer, Harold; Higgins, Paul J

    2015-05-01

    Plasminogen activator inhibitor-1 (PAI-1), the primary inhibitor of urokinase-and tissue-type plasminogen activators (uPA and tPA), is an injury-response gene implicated in the development of tissue fibrosis and cardiovascular disease. PAI-1 mRNA and protein levels were elevated in the balloon catheter-injured carotid and in the vascular smooth muscle cell (VSMC)-enriched neointima of ligated arteries. PAI-1/uPA complex formation and PAI-1 antiproteolytic activity can be inhibited, via proteolytic cleavage, by the small molecule antagonist tiplaxtinin which effectively increased the VSMC apoptotic index in vitro and attenuated carotid artery neointimal formation in vivo. In contrast to the active full-length serine protease inhibitor (SERPIN), elastase-cleaved PAI-1 (similar to tiplaxtinin) also promoted VSMC apoptosis in vitro and similarly reduced neointimal formation in vivo. The mechanism through which cleaved PAI-1 (CL-PAI-1) stimulates apoptosis appears to involve the TNF-α family member TWEAK (TNF-α weak inducer of apoptosis) and it's cognate receptor, fibroblast growth factor (FGF)-inducible 14 (FN14). CL-PAI-1 sensitizes cells to TWEAK-stimulated apoptosis while full-length PAI-1 did not, presumably due to its ability to down-regulate FN14 in a low density lipoprotein receptor-related protein 1 (LRP1)-dependent mechanism. It appears that prolonged exposure of VSMCs to CL-PAI-1 induces apoptosis by augmenting TWEAK/FN14 pro-apoptotic signaling. This work identifies a critical, anti-stenotic, role for a functionally-inactive (at least with regard to its protease inhibitory function) cleaved SERPIN. Therapies that promote the conversion of full-length to cleaved PAI-1 may have translational implications.

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

    DEFF Research Database (Denmark)

    Kristensen, Thea; Normann, Preben; Gullberg, Maria

    2016-01-01

    The foot-and-mouth disease virus (FMDV) capsid precursor, P1-2A, is cleaved by FMDV 3C protease to yield VP0, VP3, VP1 and 2A. Cleavage of the VP1/2A junction is the slowest. Serotype O FMDVs with uncleaved VP1-2A (having a K210E substitution in VP1; at position P2 in cleavage site) have been des...... have implications for the testing of potential antiviral agents targeting the FMDV 3C protease....

  17. Mutation in cpsf6/CFIm68 (Cleavage and Polyadenylation Specificity Factor Subunit 6) causes short 3'UTRs and disturbs gene expression in developing embryos, as revealed by an analysis of primordial germ cell migration using the medaka mutant naruto.

    Science.gov (United States)

    Sasado, Takao; Kondoh, Hisato; Furutani-Seiki, Makoto; Naruse, Kiyoshi

    2017-01-01

    Our previous studies analyzing medaka mutants defective in primordial germ cell (PGC) migration identified cxcr4b and cxcr7, which are both receptors of the chemokine sdf1/cxcl12, as key regulators of PGC migration. Among PGC migration mutants, naruto (nar) is unique in that the mutant phenotype includes gross morphological abnormalities of embryos, suggesting that the mutation affects a broader range of processes. A fine genetic linkage mapping and genome sequencing showed the nar gene encodes Cleavage and Polyadenylation Specificity Factor subunit 6 (CPSF6/CFIm68). CPSF6 is a component of the Cleavage Factor Im complex (CFIm) which plays a key role in pre-mRNA 3'-cleavage and polyadenylation. 3'RACE of sdf1a/b and cxcr7 transcripts in the mutant embryos indicated shorter 3'UTRs with poly A additions occurring at more upstream positions than wild-type embryos, suggesting CPSF6 functions to prevent premature 3'UTR cleavage. In addition, expression of the coding region sequences of sdf1a/b in nar mutants was more anteriorly extended in somites than wild-type embryos, accounting for the abnormally extended distribution of PGCs in nar mutants. An expected consequence of shortening 3'UTR is the escape from the degradation mechanism mediated by microRNAs interacting with distal 3'UTR sequence. The abnormal expression pattern of sdf1a coding sequence may be at least partially accounted for by this mechanism. Given the pleiotropic effects of nar mutation, further analysis using the nar mutant will reveal processes in which CPSF6 plays essential regulatory roles in poly A site selection and involvement of 3'UTRs in posttranscriptional gene regulation in various genes in vivo.

  18. Mutation in cpsf6/CFIm68 (Cleavage and Polyadenylation Specificity Factor Subunit 6) causes short 3'UTRs and disturbs gene expression in developing embryos, as revealed by an analysis of primordial germ cell migration using the medaka mutant naruto

    Science.gov (United States)

    Kondoh, Hisato; Furutani-Seiki, Makoto; Naruse, Kiyoshi

    2017-01-01

    Our previous studies analyzing medaka mutants defective in primordial germ cell (PGC) migration identified cxcr4b and cxcr7, which are both receptors of the chemokine sdf1/cxcl12, as key regulators of PGC migration. Among PGC migration mutants, naruto (nar) is unique in that the mutant phenotype includes gross morphological abnormalities of embryos, suggesting that the mutation affects a broader range of processes. A fine genetic linkage mapping and genome sequencing showed the nar gene encodes Cleavage and Polyadenylation Specificity Factor subunit 6 (CPSF6/CFIm68). CPSF6 is a component of the Cleavage Factor Im complex (CFIm) which plays a key role in pre-mRNA 3'-cleavage and polyadenylation. 3'RACE of sdf1a/b and cxcr7 transcripts in the mutant embryos indicated shorter 3’UTRs with poly A additions occurring at more upstream positions than wild-type embryos, suggesting CPSF6 functions to prevent premature 3’UTR cleavage. In addition, expression of the coding region sequences of sdf1a/b in nar mutants was more anteriorly extended in somites than wild-type embryos, accounting for the abnormally extended distribution of PGCs in nar mutants. An expected consequence of shortening 3'UTR is the escape from the degradation mechanism mediated by microRNAs interacting with distal 3’UTR sequence. The abnormal expression pattern of sdf1a coding sequence may be at least partially accounted for by this mechanism. Given the pleiotropic effects of nar mutation, further analysis using the nar mutant will reveal processes in which CPSF6 plays essential regulatory roles in poly A site selection and involvement of 3'UTRs in posttranscriptional gene regulation in various genes in vivo. PMID:28253363

  19. Site specificity of DSP-PP cleavage by BMP1.

    Science.gov (United States)

    Yang, Robert T; Lim, Glendale L; Yee, Colin T; Fuller, Robert S; Ritchie, Helena H

    2014-08-01

    Bone morphogenic protein 1 (BMP1), a metalloproteinase, is known to cleave a wide variety of extracellular matrix proteins, suggesting that a consensus substrate cleavage amino acid sequence might exist. However, while such a consensus sequence has been proposed based on P4 to P4' (i.e. the four amino acids flanking either side of the BMP1 cleavage site; P4P3P2P1|P1'P2'P3'P4') sequence homologies between two BMP1 substrates, dentin matrix protein 1 and dentin sialoprotein phosphophoryn (DSP-PP) (i.e. xMQx|DDP), no direct testing has so far been attempted. Using an Sf9 cell expression system, we have been able to produce large amounts of uncleaved DSP-PP. Point mutations introduced into this recombinant DSP-PP were then tested for their effects on DSP-PP cleavage by either Sf9 endogenous tolloid-related protein 1 (TLR-1) or by its human homolog, BMP1. Here, we have measured DSP-PP cleavage efficiencies after modifications based on P4-P4' sequence comparisons with dentin matrix protein 1, as well as for prolysyl oxidase and chordin, two other BMP1 substrates. Our results demonstrate that any mutations within or outside of the DSP-PP P4 to P4' cleavage site can block, impair or accelerate DSP-PP cleavage, and suggest that its BMP1 cleavage site is highly conserved in order to regulate its cleavage efficiency, possibly with additional assistance from its conserved exosites. Thus, BMP1 cleavage cannot be based on a consensus substrate cleavage site.

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

    DEFF Research Database (Denmark)

    Gullberg, Maria; Polacek, Charlotta; Belsham, Graham

    2014-01-01

    The foot-and-mouth disease virus (FMDV) capsid protein precursor P1-2A is cleaved by the virus-encoded 3C protease to VP0, VP3, VP1 and 2A. It was shown previously that modification of a single amino acid residue (K210E) within the VP1 protein and close to the VP1/2A cleavage site, inhibited clea...

  1. Reduced DNA topoisomerase II activity and drug-induced DNA cleavage activity in an adriamycin-resistant human small cell lung carcinoma cell line

    NARCIS (Netherlands)

    de Jong, Steven; Zijlstra, J G; de Vries, Liesbeth; Mulder, Nanno

    1990-01-01

    In a previous study we suggested that, in addition to the reduced Adriamycin accumulation, part of the resistance in an Adriamycin-resistant human small cell lung carcinoma cell line (GLC4/ADR) could be explained by supposing a changed Adriamycin-DNA-topoisomerase II (Topo II) interaction. The prese

  2. PI-PLCβ1b affects Akt activation, cyclin E expression, and caspase cleavage, promoting cell survival in pro-B-lymphoblastic cells exposed to oxidative stress.

    Science.gov (United States)

    Piazzi, Manuela; Blalock, William L; Bavelloni, Alberto; Faenza, Irene; Raffini, Mirco; Tagliavini, Francesca; Manzoli, Lucia; Cocco, Lucio

    2015-04-01

    The phosphoinositide-dependent signal transduction pathway has been implicated in the control of a variety of biologic processes, such as the regulation of cellular metabolism and homeostasis, cell proliferation and differentiation, and apoptosis. One of the key players in the regulation of inositol lipid signaling is the phospholipase Cβ1 (PI-PLCβ1), that hydrolyzes phosphatidylinositol 4,5-bisphosphate [PtIns(4,5)P2], giving rise to the second messengers inositol triphosphate and diacylglicerol. PI-PLCβ1 has been associated with the regulation of several cellular functions, some of which have not yet been fully understood. In particular, it has been reported that PI-PLCβ1 protects murine fibroblasts from oxidative stress-induced cell death. The mediators of oxidative stress, reactive oxygen species (ROS), have been shown to regulate major epigenetic processes, causing the silencing of tumor suppressors and enhancing the proliferation of leukemic cells under oxidative stress. Investigation of the interplay between ROS, PI-PLCβ1, and their signaling mediators in leukemia might therefore reveal innovative targets of pharmacological therapy in the treatment for leukemia. In this work, we demonstrate that in pro-B-lymphoblastic cells (Ba/F3), treated with H2O2, PI-PLCβ1b conferred resistance to cell death, promoting cell cycle progression and cell proliferation and influencing the expression of cyclin A and E. Interestingly, we found that, expression of PI-PLCβ1b affects the activity of caspase-3, caspase-7, and of several protein kinases induced by oxidative stress. In particular, PI-PLCβ1b expression completely abolished the phosphorylation of Erk1/2 MAP kinases, down-regulated phosphatase and tensin homolog (PTEN), and up-regulated the phosphorylation of Akt, thereby sustaining cellular proliferation.

  3. Quantification of DNA cleavage specificity in Hi-C experiments.

    Science.gov (United States)

    Meluzzi, Dario; Arya, Gaurav

    2016-01-01

    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.

  4. 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.

  5. Mechanisms for ribotoxin-induced ribosomal RNA cleavage

    Energy Technology Data Exchange (ETDEWEB)

    He, Kaiyu [Department of Microbiology and Molecular Genetics (United States); Center for Integrative Toxicology, Michigan State University, East Lansing, MI 48824 (United States); Zhou, Hui-Ren [Food Science and Human Nutrition (United States); Pestka, James J., E-mail: pestka@msu.edu [Department of Microbiology and Molecular Genetics (United States); Food Science and Human Nutrition (United States); Center for Integrative Toxicology, Michigan State University, East Lansing, MI 48824 (United States)

    2012-11-15

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

  6. The antioxidative properties of S-allyl cysteine not only influence somatic cells but also improve early embryo cleavage in pigs

    Directory of Open Access Journals (Sweden)

    Markéta Dvořáková

    2016-08-01

    Full Text Available In vitro cultivation systems for oocytes and embryos are characterised by increased levels of reactive oxygen species (ROS, which can be balanced by the addition of suitable antioxidants. S-allyl cysteine (SAC is a sulfur compound naturally occurring in garlic (Allium sativum, which is responsible for its high antioxidant properties. In this study, we demonstrated the capacity of SAC (0.1, 0.5 and 1.0 mM to reduce levels of ROS in maturing oocytes significantly after 24 (reduced by 90.33, 82.87 and 91.62%, respectively and 48 h (reduced by 86.35, 94.42 and 99.05%, respectively cultivation, without leading to a disturbance of the standard course of meiotic maturation. Oocytes matured in the presence of SAC furthermore maintained reduced levels of ROS even 22 h after parthenogenic activation (reduced by 66.33, 61.64 and 57.80%, respectively. In these oocytes we also demonstrated a growth of early embryo cleavage rate (increased by 33.34, 35.00 and 35.00%, respectively. SAC may be a valuable supplement to cultivation media.

  7. 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 cell...... stage (p 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...

  8. Cleavage of E-Cadherin by Matrix Metalloproteinase-7 Promotes Cellular Proliferation in Nontransformed Cell Lines via Activation of RhoA

    Directory of Open Access Journals (Sweden)

    Conor C. Lynch

    2010-01-01

    Full Text Available Perturbations in cell-cell contact machinery occur frequently in epithelial cancers and result in increased cancer cell migration and invasion. Previously, we demonstrated that MMP-7, a protease implicated in mammary and intestinal tumor growth, can process the adherens junction component E-cadherin. This observation leads us to test whether MMP-7 processing of E-cadherin could directly impact cell proliferation in nontransformed epithelial cell lines (MDCK and C57MG. Our goal was to investigate the possibility that MMP-7 produced by cancer cells may have effects on adjacent normal epithelium. Here, we show that MMP-7 processing of E-cadherin mediates, (1 loss of cell-cell contact, (2 increased cell migration, (3 a loss of epithelial cell polarization and (4 increased cell proliferation via RhoA activation. These data demonstrate that MMP-7 promotes epithelial cell proliferation via the processing of E-cadherin and provide insights into the molecular mechanisms that govern epithelial cell growth.

  9. Fe3O4 nanoparticle loaded paclitaxel induce multiple myeloma apoptosis by cell cycle arrest and increase cleavage of caspases in vitro

    Science.gov (United States)

    Yang, Cuiping; He, Xiangfeng; Chen, Junsong; Chen, Dengyu; Liu, Yunjing; Xiong, Fei; Shi, Fangfang; Dou, Jun; Gu, Ning

    2013-08-01

    Multiple myeloma (MM) still remains an incurable disease in spite of extending the patient survival by new therapies. The hypothesis of cancer stem cells (CSCs) states that although chemotherapy kills most tumor cells, it is believed to leave a reservoir of CSCs that allows the tumor cell propagation. The objective of this research was to evaluate the therapeutic effect of new paclitaxel-Fe3O4 nanoparticles (PTX-NPs) with an average size range of 7.17 ± 1.31 nm on MM CSCs in vitro. The characteristics of CD138-CD34- cells, isolated from human MM RPMI 8226 and NCI-H929 cell lines by the magnetic associated cell sorting method, were identified by the assays of colony formation, cell proliferation, drug resistance, cell migration, and tumorigenicity in non-obese diabetic/severe combined immunodeficiency (NOD/SCID) mice, respectively. Inhibitory effects of PTX-NPs on CD138-CD34- cells were evaluated by a variety of assays in vitro. The results showed that the CD138-CD34- cells were capable of forming colonies, exhibited high proliferative and migratory ability, possessed a strong drug resistance, and had powerful tumorigenicity in NOD/SCID mice compared to non-CD138-CD34- cells. PTX-NPs significantly inhibited CD138- CD34- cell viability and invasive ability, and resulted in G0/G1 cell cycle arrest and apoptosis compared with PTX alone. We concluded that the CD138-CD34- phenotype cells might be CSCs in RPMI 8226 and NCI-H929 cell lines. PTX-NPs had an obvious inhibitory effect on MM CD138-CD34- CSCs. The findings may provide a guideline for PTX-NPs' treatment of MM CSCs in preclinical investigation.

  10. Fe{sub 3}O{sub 4} nanoparticle loaded paclitaxel induce multiple myeloma apoptosis by cell cycle arrest and increase cleavage of caspases in vitro

    Energy Technology Data Exchange (ETDEWEB)

    Yang, Cuiping [Medical School, Southeast University, Department of Pathogenic Biology and Immunology (China); He, Xiangfeng [Affiliated Tumor Hospital of Nantong University, Department of Medical Oncology (China); Chen, Junsong; Chen, Dengyu; Liu, Yunjing [Medical School, Southeast University, Department of Pathogenic Biology and Immunology (China); Xiong, Fei [Southeast University, School of Biological Science and Medical Engineering (China); Shi, Fangfang [Zhongda Hospital, Southeast University, Department of Oncology (China); Dou, Jun, E-mail: njdoujun@yahoo.com.cn [Medical School, Southeast University, Department of Pathogenic Biology and Immunology (China); Gu, Ning, E-mail: guning@seu.edu.cn [Southeast University, School of Biological Science and Medical Engineering (China)

    2013-08-15

    Multiple myeloma (MM) still remains an incurable disease in spite of extending the patient survival by new therapies. The hypothesis of cancer stem cells (CSCs) states that although chemotherapy kills most tumor cells, it is believed to leave a reservoir of CSCs that allows the tumor cell propagation. The objective of this research was to evaluate the therapeutic effect of new paclitaxel-Fe{sub 3}O{sub 4} nanoparticles (PTX-NPs) with an average size range of 7.17 {+-} 1.31 nm on MM CSCs in vitro. The characteristics of CD138{sup -}CD34{sup -} cells, isolated from human MM RPMI 8226 and NCI-H929 cell lines by the magnetic associated cell sorting method, were identified by the assays of colony formation, cell proliferation, drug resistance, cell migration, and tumorigenicity in non-obese diabetic/severe combined immunodeficiency (NOD/SCID) mice, respectively. Inhibitory effects of PTX-NPs on CD138{sup -}CD34{sup -} cells were evaluated by a variety of assays in vitro. The results showed that the CD138{sup -}CD34{sup -} cells were capable of forming colonies, exhibited high proliferative and migratory ability, possessed a strong drug resistance, and had powerful tumorigenicity in NOD/SCID mice compared to non-CD138{sup -}CD34{sup -} cells. PTX-NPs significantly inhibited CD138{sup -} CD34{sup -} cell viability and invasive ability, and resulted in G0/G1 cell cycle arrest and apoptosis compared with PTX alone. We concluded that the CD138{sup -}CD34{sup -} phenotype cells might be CSCs in RPMI 8226 and NCI-H929 cell lines. PTX-NPs had an obvious inhibitory effect on MM CD138{sup -}CD34{sup -} CSCs. The findings may provide a guideline for PTX-NPs' treatment of MM CSCs in preclinical investigation.

  11. Mitochondria localize to the cleavage furrow in mammalian cytokinesis.

    Science.gov (United States)

    Lawrence, Elizabeth J; Mandato, Craig A

    2013-01-01

    Mitochondria are dynamic organelles with multiple cellular functions, including ATP production, calcium buffering, and lipid biosynthesis. Several studies have shown that mitochondrial positioning is regulated by the cytoskeleton during cell division in several eukaryotic systems. However, the distribution of mitochondria during mammalian cytokinesis and whether the distribution is regulated by the cytoskeleton has not been examined. Using live spinning disk confocal microscopy and quantitative analysis of mitochondrial fluorescence intensity, we demonstrate that mitochondria are recruited to the cleavage furrow during cytokinesis in HeLa cells. After anaphase onset, the mitochondria are recruited towards the site of cleavage furrow formation, where they remain enriched as the furrow ingresses and until cytokinesis completion. Furthermore, we show that recruitment of mitochondria to the furrow occurs in multiple mammalian cells lines as well as in monopolar, bipolar, and multipolar divisions, suggesting that the mechanism of recruitment is conserved and robust. Using inhibitors of cytoskeleton dynamics, we show that the microtubule cytoskeleton, but not actin, is required to transport mitochondria to the cleavage furrow. Thus, mitochondria are specifically recruited to the cleavage furrow in a microtubule-dependent manner during mammalian cytokinesis. Two possible reasons for this could be to localize mitochondrial function to the furrow to facilitate cytokinesis and / or ensure accurate mitochondrial inheritance.

  12. Apoptosis of the teratocarcinoma cell line Tera-1 leads to the cleavage of HERV-K10gag proteins by caspases and/or granzyme B.

    Science.gov (United States)

    Beyer, T D; Kolowos, W; Dumitriu, I E; Voll, R E; Heyder, P; Gaipl, U S; Kalden, J R; Herrmann, M

    2002-09-01

    Redistribution, post-translational modifications and coclustering with viral antigens contribute to the immunogenicity of apoptotic cell-derived autoantigens. Almost all known targets of the humoral autoimmune response in systemic lupus erythematosus (SLE) are cleaved by caspases or granzyme B during apoptosis. Antibodies against retroviral proteins can frequently be detected in the sera of SLE patients without overt retroviral infections. These antibodies may represent cross-reactive antibodies or may have been induced by proteins encoded by endogenous retroviral sequences. We used Tera-1 cells that abundantly express a group-specific antigen of human endogenous retroviruses, HERV-K10gag polyprotein, to investigate its processing during apoptosis. Tera-1 cells induced to undergo apoptosis showed an altered HERV-K10gag processing compared with viable cells. In addition, granzyme B was able to cleave HERV-K10gag isolated from viable Tera-1 cells. Similar to nuclear autoantigens, endogenous retroviral proteins are cleaved during the execution phase of apoptosis. These post-translational modifications may result in the generation of T-cell neoepitopes or a changed epitope hierarchy of retroviral proteins. Therefore, immunogenicity of retroviral antigens in SLE patients may result from a similar mechanism as described for nuclear autoantigens.

  13. Viability of corneal epithelial cells and cleavage of corneal basement after ethanol effect%乙醇对角膜上皮瓣活性及基底膜分离定位研究

    Institute of Scientific and Technical Information of China (English)

    陈颖欣; 蓝平; 高明宏; 范忠义; 宋福林; 徐旭; 于静

    2008-01-01

    Objective To evaluate the viability of corneal epithelial cells and to determine the anatomic cleavage on the epithelial basement membrane after various exposure times to 20% ethanol during epithelial flap preparation in laser-assisted subepithelial keratectomy(LASEK)in cadaver eyes.Methods Six human cadaver eyes were exposed to 20% ethanol for 20,30 and 40 seconds(2 eyes for each group),and another one eye was used as the control.PCNA staining was performed to determine the viability of corneal epithelial cells.Immunofluorescence staining using monoclonal antibodies against collagen Ⅶ,and immunohistological staining using monoclonal antibodies against laminin were performed to detect the anatomic location of the cleavage plane on the corneal epithelial flaps created by 20 seconds exposure to 20% ethanol in cadaver eyes.Results Hematoxylin and eosin staining of epithelial flaps revealed a coherent stratified epithelium.The PCNA positive rates of the epithelial cells in the flap decreased in the 20-second group,30-seconcl group and 40-second group successively.Immunohistological staining to laminin was patchy in the lifted flap and the remaining corneal basement membrane.Immunofluorescence to collagen Ⅶ,the main component of anchoring fibrils remained exclusively in the corneal bed.Conclusions Viability of the epithelial flap decreased with longer time exposure to ethanol.The cleavage plane of the ethanol-treated corneal epithelial flap is located between the lamina lucida and the lamina densa of the basement membrane where laminin forms hemidesmesome.%目的 探讨不同乙醇作用时间对角膜上皮瓣活性的影响,并确定角膜上皮瓣的解剖分离层面.方法 按标准准分子激光角膜上皮瓣下磨镶术(LASEK)方法制备7只尸体眼角膜上皮瓣,其中对照组1只眼,其余6只分为A、B、C 3个组,每组2只眼,乙醇浸润时间分别为20、30和40 S.对7只眼进行HE、增殖细胞核抗原(PCNA)、层黏连蛋白免疫组化

  14. Signal peptide cleavage is essential for surface expression of a regulatory T cell surface protein, leucine rich repeat containing 32 (LRRC32

    Directory of Open Access Journals (Sweden)

    Sugiyama Hideaki

    2011-05-01

    Full Text Available Abstract Background Elevated numbers of regulatory T cells (Tregs have been implicated in certain cancers. Depletion of Tregs has been shown to increase anti-tumor immunity. Tregs also play a critical role in the suppression of autoimmune responses. The study of Tregs has been hampered by a lack of adequate surface markers. Leucine Rich Repeat Containing 32 (LRRC32, also known as Glycoprotein A Repetitions Predominant (GARP, has been postulated as a novel surface marker of activated Tregs. However, there is limited information regarding the processing of LRRC32 or the regulatory phenotype and functional activity of Tregs expressing LRRC32. Results Using naturally-occurring freshly isolated Tregs, we demonstrate that low levels of LRRC32 are present intracellularly prior to activation and that freshly isolated LRRC32+ Tregs are distinct from LRRC32- Tregs with respect to the expression of surface CD62L. Using LRRC32 transfectants of HEK cells, we demonstrate that the N-terminus of LRRC32 is cleaved prior to expression of the protein at the cell surface. Furthermore, we demonstrate using a construct containing a deleted putative signal peptide region that the presence of a signal peptide region is critical to cell surface expression of LRRC32. Finally, mixed lymphocyte assays demonstrate that LRRC32+ Tregs are more potent suppressors than LRRC32- Tregs. Conclusions A cleaved signal peptide site in LRRC32 is necessary for surface localization of native LRRC32 following activation of naturally-occurring freshly-isolated regulatory T cells. LRRC32 expression appears to alter the surface expression of activation markers of T cells such as CD62L. LRRC32 surface expression may be useful as a marker that selects for more potent Treg populations. In summary, understanding the processing and expression of LRRC32 may provide insight into the mechanism of action of Tregs and the refinement of immunotherapeutic strategies aimed at targeting these cells.

  15. 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...

  16. 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.

  17. APP cleavage in live cells guided by C99%C99引导淀粉样蛋白前体裂解过程的活体细胞研究

    Institute of Scientific and Technical Information of China (English)

    李晓晴; 梁燕玲; 常丽英; 张苏明; 骆清铭; 张旻; 张智红; 邢变枝; 杨华静; 郭守刚; 黎逢光

    2008-01-01

    Objective To construct recombinant eukaryotic expression plasmid encoding Swedish and Flemish mutations of amyloid precursor protein (APP) fused with fluorescent protein and to investigate the APP cleavage progress. Methods The last 300 bases of APP (named as C99 containing Flemish mutation), together with cyan and yellow fluorescence sequence (named as CFP and YFP,respectively) were obtained by polymerase chain reaction (PCR). The 54 bases in the middle of APP sequence were synthesized (named as 54 bp containing Swedish mutation). The 4 fragments mentioned above (CFP, YFP, C99 as well as 54 bp) were inserted into the vector pcDNA3.0. By genetic engineering, the recombinant plasmid pcDNA3.0-CFP-54bp-YFP-C99 was constructed and identified by enzyme digestion, PCR and sequencing. Then the plasmid was transfected into SH-SY5Y cells. Its expression was examined by fluorescence confocal microscopy and the fluorescence resonance energy transfer (FRET) signal was collected. The amyloid beta (A) deposition was detected by immunocytochemistry. Results (1) DNA sequencing showed the sequence of the constructed recombinant plasmid was correct. (2) FRET and two types of fluorescence could be seen by the spectrum confocal fluorescence microscopy. (3) The expression product of fusion gene was correct and cleaved by and secretases. (4) The A deposition was detected in the cell membrane, cytoplasma and intercellular space. Conclusion (1) The fusion protein can generate A by and γproteolytic processing. (2) It is for the first time to observe the APP cleavage by FRET. (3) It is also the first time to find that APP may be cleaved during its transportation from cell plasma to cell membrane. (4) C99 is very important for the correct cleavage of APP. Our test data strongly suggest that C99 may function as the signal peptide. It might guide and direct the APP to the right location for the cleavage.%目的 构建含有Swedish突变和Flemish突变的荧光真核表达系统,研究淀粉

  18. Cleavage of INDOLE-3-ACETIC ACID INDUCIBLE28 mRNA by microRNA847 upregulates auxin signaling to modulate cell proliferation and lateral organ growth in Arabidopsis.

    Science.gov (United States)

    Wang, Jing-Jing; Guo, Hui-Shan

    2015-03-01

    MicroRNAs function in a range of developmental processes. Here, we demonstrate that miR847 targets the mRNA of the auxin/indole acetic acid (Aux/IAA) repressor-encoding gene IAA28 for cleavage. The rapidly increased accumulation of miR847 in Arabidopsis thaliana coincided with reduced IAA28 mRNA levels upon auxin treatment. This induction of miR847 by auxin was abolished in auxin receptor tir1-1 and auxin-resistant axr1-3 mutants. Further analysis demonstrates that miR847 functions as a positive regulator of auxin-mediated lateral organ development by cleaving IAA28 mRNA. Importantly, the ectopic expression of miR847 increases the expression of cell cycle genes as well as the neoplastic activity of leaf cells, prolonging later-stage rosette leaf growth and producing leaves with serrated margins. Moreover, both miR847 and IAA28 mRNAs are specifically expressed in marginal meristems of rosette leaves and lateral root initiation sites. Our data indicate that auxin-dependent induction of miR847 positively regulates meristematic competence by clearing IAA28 mRNA to upregulate auxin signaling, thereby determining the duration of cell proliferation and lateral organ growth in Arabidopsis. IAA28 mRNA encodes an Aux/IAA repressor protein, which is degraded through the proteasome in response to auxin. Altered signal sensitization to IAA28 mRNA levels, together with targeted IAA28 degradation, ensures a robust signal derepression.

  19. Fracto—emissions in Catastrophic Cleavage Process

    Institute of Scientific and Technical Information of China (English)

    HonglaiTAN; WeiYANG

    1996-01-01

    Fracto-emissions accompanying crack propagation are observed in the recent experiments.The energy impulses during and after fracture stimulate the fracto-emissions.Model concerning atomic scale cleavage processes is proposed to formulate a catastrophic fracure theory relevant to these phenomena.A criterion for catastrophic jump of the cleavage potential is applied to representative crystals.

  20. Microstructure and cleavage in lath martensitic steels

    Directory of Open Access Journals (Sweden)

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

    2013-01-01

    Full Text Available 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.

  1. Neutrophil elastase cleavage of the gC1q domain impairs the EMILIN1-α4β1 integrin interaction, cell adhesion and anti-proliferative activity

    Science.gov (United States)

    Maiorani, Orlando; Pivetta, Eliana; Capuano, Alessandra; Modica, Teresa Maria Elisa; Wassermann, Bruna; Bucciotti, Francesco; Colombatti, Alfonso; Doliana, Roberto; Spessotto, Paola

    2017-01-01

    The extracellular matrix glycoprotein EMILIN1 exerts a wide range of functions mainly associated with its gC1q domain. Besides providing functional significance for adhesion and migration, the direct interaction between α4β1 integrin and EMILIN1-gC1q regulates cell proliferation, transducing net anti-proliferative effects. We have previously demonstrated that EMILIN1 degradation by neutrophil elastase (NE) is a specific mechanism leading to the loss of functions disabling its regulatory properties. In this study we further analysed the proteolytic activity of NE, MMP-3, MMP-9, and MT1-MMP on EMILIN1 and found that MMP-3 and MT1-MMP partially cleaved EMILIN1 but without affecting the functional properties associated with the gC1q domain, whereas NE was able to fully impair the interaction of gC1q with the α4β1 integrin by cleaving this domain outside of the E933 integrin binding site. By a site direct mutagenesis approach we mapped the bond between S913 and R914 residues and selected the NE-resistant R914W mutant still able to interact with the α4β1 integrin after NE treatment. Functional studies showed that NE impaired the EMILIN1-α4β1 integrin interaction by cleaving the gC1q domain in a region crucial for its proper structural conformation, paving the way to better understand NE effects on EMILIN1-cell interaction in pathological context. PMID:28074935

  2. 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.

  3. Hyperphosphorylation and cleavage at D421 enhance tau secretion.

    Directory of Open Access Journals (Sweden)

    Vanessa Plouffe

    Full Text Available It is well established that tau pathology propagates in a predictable manner in Alzheimer's disease (AD. Moreover, tau accumulates in the cerebrospinal fluid (CSF of AD's patients. The mechanisms underlying the propagation of tau pathology and its accumulation in the CSF remain to be elucidated. Recent studies have reported that human tau was secreted by neurons and non-neuronal cells when it was overexpressed indicating that tau secretion could contribute to the spreading of tau pathology in the brain and could lead to its accumulation in the CSF. In the present study, we showed that the overexpression of human tau resulted in its secretion by Hela cells. The main form of tau secreted by these cells was cleaved at the C-terminal. Surprisingly, secreted tau was dephosphorylated at several sites in comparison to intracellular tau which presented a strong immunoreactivity to all phospho-dependent antibodies tested. Our data also revealed that phosphorylation and cleavage of tau favored its secretion by Hela cells. Indeed, the mimicking of phosphorylation at 12 sites known to be phosphorylated in AD enhanced tau secretion. A mutant form of tau truncated at D421, the preferential cleavage site of caspase-3, was also significantly more secreted than wild-type tau. Taken together, our results indicate that hyperphosphorylation and cleavage of tau by favoring its secretion could contribute to the propagation of tau pathology in the brain and its accumulation in the CSF.

  4. Site Specificity of Cleavage of DSP-PP by BMP1

    Science.gov (United States)

    Yang, Robert T.; Lim, Glendale L.; Yee, Colin T.; Fuller, Robert S.; Ritchie, Helena H.

    2015-01-01

    Bone morphogenic protein 1 (BMP1), a metalloproteinase, is known to cleave a wide variety of extracellular matrix proteins, suggesting that a consensus substrate cleavage amino acid sequence might exist. However, while such a consensus sequence has been proposed based on P4 to P4′ (i.e., the four amino acids flanking either side of the BMP1 cleavage site; P4P3P2P1|P1′P2′P3′P4′) sequence homologies between two BMP1 substrates, dentin matrix protein 1 and dentin sialoprotein phosphophoryn (DSP-PP) (i.e., xMQx | DDP), no direct testing has so far been attempted. Using an Sf9 cell expression system, we have been able to produce large amounts of uncleaved DSP-PP,. Point mutations introduced into this recombinant DSP-PP were then tested for their affects on DSP-PP cleavage by either Sf9 endogenous tolloid-related protein 1 (TLR-1) or by its human homolog, BMP1. Here we have measured DSP-PP cleavage efficiencies after modifications based on P4-P4′ sequence comparisons with dentin matrix protein 1, as well as for prolysyl oxidase and chordin, two other BMP1 substrates. Our results demonstrate that any mutations within or outside of the DSP-PP P4 to P4′ cleavage site can block, impair or accelerate DSP-PP cleavage, and suggest that its BMP1 cleavage site is highly conserved in order to regulate its cleavage efficiency, possibly with additional assistance from its conserved exosites. Thus, BMP1 cleavage cannot be based on a consensus substrate cleavage site. PMID:25158199

  5. Development and application of bond cleavage reactions in bioorthogonal chemistry.

    Science.gov (United States)

    Li, Jie; Chen, Peng R

    2016-03-01

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

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

    Science.gov (United States)

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

    2015-12-01

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

  7. Observation of Early Cleavage in Animal Development: A Simple Technique for Obtaining the Eggs of Rhabditis (Nematoda)

    Science.gov (United States)

    Hinchliffe, J. R.

    1973-01-01

    Outlines the advantages of using the readily available eggs of the nematode Rhabditis in studying the early cleavage stages of animal development. Discusses the identification and life history of Rhabditis, how to culture and examine the organism, the cleavage stages and cell lineage, and sources of visual aids. (JR)

  8. Identification of BACE1 cleavage sites in human voltage-gated sodium channel beta 2 subunit

    Directory of Open Access Journals (Sweden)

    Kovacs Dora M

    2010-12-01

    Full Text Available Abstract Background The voltage-gated sodium channel β2 subunit (Navβ2 is a physiological substrate of BACE1 (β-site APP cleaving enzyme and γ-secretase, two proteolytic enzymes central to Alzheimer's disease pathogenesis. Previously, we have found that the processing of Navβ2 by BACE1 and γ-secretase regulates sodium channel metabolism in neuronal cells. In the current study we identified the BACE1 cleavage sites in human Navβ2. Results We found a major (147-148 L↓M, where ↓ indicates the cleavage site and a minor (144145 L↓Q BACE1 cleavage site in the extracellular domain of human Navβ2 using a cell-free BACE1 cleavage assay followed by mass spectrometry. Next, we introduced two different double mutations into the identified major BACE1 cleavage site in human Navβ2: 147LM/VI and 147LM/AA. Both mutations dramatically decreased the cleavage of human Navβ2 by endogenous BACE1 in cell-free BACE1 cleavage assays. Neither of the two mutations affected subcellular localization of Navβ2 as confirmed by confocal fluorescence microscopy and subcellular fractionation of cholesterol-rich domains. Finally, wildtype and mutated Navβ2 were expressed along BACE1 in B104 rat neuroblastoma cells. In spite of α-secretase still actively cleaving the mutant proteins, Navβ2 cleavage products decreased by ~50% in cells expressing Navβ2 (147LM/VI and ~75% in cells expressing Navβ2 (147LM/AA as compared to cells expressing wildtype Navβ2. Conclusion We identified a major (147-148 L↓M and a minor (144-145 L↓Q BACE1 cleavage site in human Navβ2. Our in vitro and cell-based results clearly show that the 147-148 L↓M is the major BACE1 cleavage site in human Navβ2. These findings expand our understanding of the role of BACE1 in voltage-gated sodium channel metabolism.

  9. Ectodomain cleavage of the EGF ligands HB-EGF, neuregulin1-beta, and TGF-alpha is specifically triggered by different stimuli and involves different PKC isoenzymes.

    Science.gov (United States)

    Herrlich, Andreas; Klinman, Eva; Fu, Jonathan; Sadegh, Cameron; Lodish, Harvey

    2008-12-01

    Metalloproteinase cleavage of transmembrane proteins (ectodomain cleavage), including the epidermal growth factor (EGF) ligands heparin-binding EGF-like growth factor (HB-EGF), neuregulin (NRG), and transforming growth factor-alpha (TGF-alpha), is important in many cellular signaling pathways and is disregulated in many diseases. It is largely unknown how physiological stimuli of ectodomain cleavage--hypertonic stress, phorbol ester, or activation of G-protein-coupled receptors [e.g., by lysophosphatidic acid (LPA)]--are molecularly connected to metalloproteinase activation. To study this question, we developed a fluorescence-activated cell sorting (FACS)- based assay that measures cleavage of EGF ligands in single living cells. EGF ligands expressed in mouse lung epithelial cells are differentially and specifically cleaved depending on the stimulus. Inhibition of protein kinase C (PKC) isoenzymes or metalloproteinase inhibition by batimastat (BB94) showed that different regulatory signals are used by different stimuli and EGF substrates, suggesting differential effects that act on the substrate, the metalloproteinase, or both. For example, hypertonic stress led to strong cleavage of HB-EGF and NRG but only moderate cleavage of TGF-alpha. HB-EGF, NRG, and TGF-alpha cleavage was not dependent on PKC, and only HB-EGF and NRG cleavage were inhibited by BB94. In contrast, phorbol 12-myristate-13-acetate (TPA) -induced cleavage of HB-EGF, NRG, and TGF-alpha was dependent on PKC and sensitive to BB94 inhibition. LPA led to significant cleavage of only NRG and TGF-alpha and was inhibited by BB94; only LPA-induced NRG cleavage required PKC. Surprisingly, specific inhibition of atypical PKCs zeta and iota [not activated by diacylglycerol (DAG) and calcium] significantly enhanced TPA-induced NRG cleavage. Employed in a high-throughput cloning strategy, our cleavage assay should allow the identification of candidate proteins involved in signal transduction of different

  10. Synthesis and enzymatic cleavage of dual-ligand quantum dots

    Energy Technology Data Exchange (ETDEWEB)

    Sewell, Sarah L. [Department of Biomedical Engineering, Vanderbilt University, Nashville, TN (United States); Giorgio, Todd D., E-mail: todd.d.giorgio@vanderbilt.edu [Department of Biomedical Engineering, Vanderbilt University, Nashville, TN (United States); Department of Chemical and Biomolecular Engineering, Vanderbilt University, Nashville, TN (United States)

    2009-05-05

    Site directed therapy promises to minimize treatment-limiting systemic effects associated with cytotoxic agents that have no specificity for pathologic tissues. One general strategy is to target cell surface receptors uniquely presented on particular tissues. Highly specific in vivo targeting of an emerging neoplasm through a single molecular recognition mechanism has not generally been successful. Nonspecific binding and specific binding to non-target cells compromise the therapeutic index of small molecule, ubiquitous cancer targeting ligands. In this work, we have designed and fabricated a nanoparticle (NP) construct that could potentially overcome the current limitations of targeted in vivo delivery. Quantum dots (QDs) were functionalized with a poly(ethylene glycol) (PEG) modified to enable specific cleavage by matrix metalloprotease-7 (MMP-7). The QDs were further functionalized with folic acid, a ligand for a cell surface receptor that is overexpressed in many tumors, but also expressed in some normal tissues. The nanomolecular construct is designed so that the PEG initially conceals the folate ligand and construct binding to cells is inhibited. MMP-7 activated peptide cleavage and subsequent unmasking of the folate ligand occurs only near tumor tissue, resulting in a proximity activated (PA) targeting system. QDs functionalized with both the MMP-7 cleavable substrate and folic acid were successfully synthesized and characterized. The proteolytic capability of the dual ligand QD construct was quantitatively assessed by fluorometric analysis and compared to a QD construct functionalized with only the PA ligand. The dual ligand PA nanoparticles studied here exhibit significant susceptibility to cleavage by MMP-7 at physiologically relevant conditions. The capacity to autonomously convert a biopassivated nanostructure to a tissue-specific targeted delivery agent in vivo represents a paradigm change for site-directed therapies.

  11. 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.

  12. Recycling of protein subunits during DNA translocation and cleavage by Type I restriction-modification enzymes.

    Science.gov (United States)

    Simons, Michelle; Szczelkun, Mark D

    2011-09-01

    The Type I restriction-modification enzymes comprise three protein subunits; HsdS and HsdM that form a methyltransferase (MTase) and HsdR that associates with the MTase and catalyses Adenosine-5'-triphosphate (ATP)-dependent DNA translocation and cleavage. Here, we examine whether the MTase and HsdR components can 'turnover' in vitro, i.e. whether they can catalyse translocation and cleavage events on one DNA molecule, dissociate and then re-bind a second DNA molecule. Translocation termination by both EcoKI and EcoR124I leads to HsdR dissociation from linear DNA but not from circular DNA. Following DNA cleavage, the HsdR subunits appear unable to dissociate even though the DNA is linear, suggesting a tight interaction with the cleaved product. The MTases of EcoKI and EcoAI can dissociate from DNA following either translocation or cleavage and can initiate reactions on new DNA molecules as long as free HsdR molecules are available. In contrast, the MTase of EcoR124I does not turnover and additional cleavage of circular DNA is not observed by inclusion of RecBCD, a helicase-nuclease that degrades the linear DNA product resulting from Type I cleavage. Roles for Type I restriction endonuclease subunit dynamics in restriction alleviation in the cell are discussed.

  13. Potential-modulated DNA cleavage by (N-salicylideneglycinato)copper(II) complex.

    Science.gov (United States)

    Yang, Zhou-Sheng; Wang, Yan-Ling; Liu, Yun-Chun; Zhao, Guang-Chao

    2005-11-01

    The interaction of aqua (N-salicylideneglycinato)copper(II) (Cu(salgly)2+) complex with calf thymus DNA has been investigated by cyclic voltammetry. Potential-modulated DNA cleavage in the presence of Cu(salgly)2+ complex was performed at a gold electrode in a thin layer cell. DNA can be efficiently cleaved by electrochemically reducing Cu(salgly)2+ complex to Cu(salgly)+ complex at -0.7 V (vs. Ag/AgCl). When the solution was aerated with a small flow of O2 during electrolysis, the extent of DNA cleavage was dramatically enhanced, and hydroxyl radical scavengers inhibited DNA cleavage. These results suggested that O2 and hydroxyl radical were involved in potential-modulated DNA cleavage reaction. The percentage of DNA cleavage was enhanced as the working potential was shifted to more negative values and the electrolysis time was increased. It was also dependent on the ratio of Cu(salgly)2+ complex to DNA concentration. The cleaved DNA fragments were separated by high performance liquid chromatography (HPLC). The experimental results indicated that the method for potential-modulated DNA cleavage by Cu(salgly)2+ complex was simple and efficient.

  14. Cleavage site analysis in picornaviral polyproteins

    DEFF Research Database (Denmark)

    Blom, Nikolaj; Hansen, Jan; Blaas, Dieter;

    1996-01-01

    are indeed cleaved awaits experimental verification. Additionally, we report several errors detected in the protein databases. A computer server for prediction of cleavage sites by picornaviral proteinases is publicly available at the e-mail address NetPicoRNA@cbs.dtu.dk or via WWW at http://www.cbs.dtu.dk/services/NetPicoRNA...

  15. Regulation of DNA Replication in Early Embryonic Cleavages

    Directory of Open Access Journals (Sweden)

    Chames Kermi

    2017-01-01

    Full Text Available Early embryonic cleavages are characterized by short and highly synchronous cell cycles made of alternating S- and M-phases with virtually absent gap phases. In this contracted cell cycle, the duration of DNA synthesis can be extraordinarily short. Depending on the organism, the whole genome of an embryo is replicated at a speed that is between 20 to 60 times faster than that of a somatic cell. Because transcription in the early embryo is repressed, DNA synthesis relies on a large stockpile of maternally supplied proteins stored in the egg representing most, if not all, cellular genes. In addition, in early embryonic cell cycles, both replication and DNA damage checkpoints are inefficient. In this article, we will review current knowledge on how DNA synthesis is regulated in early embryos and discuss possible consequences of replicating chromosomes with little or no quality control.

  16. piRNA-directed cleavage of meiotic transcripts regulates spermatogenesis.

    Science.gov (United States)

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

    2015-05-15

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

  17. Dynamic nature of cleavage bodies and their spatial relationship to DDX1 bodies, Cajal bodies, and gems.

    Science.gov (United States)

    Li, Lei; Roy, Ken; Katyal, Sachin; Sun, Xuejun; Bléoo, Stacey; Godbout, Roseline

    2006-03-01

    DDX1 bodies, cleavage bodies, Cajal bodies (CBs), and gems are nuclear suborganelles that contain factors involved in RNA transcription and/or processing. Although all four nuclear bodies can exist as distinct entities, they often colocalize or overlap with each other. To better understand the relationship between these four nuclear bodies, we examined their spatial distribution as a function of the cell cycle. Here, we report that whereas DDX1 bodies, CBs and gems are present throughout interphase, CPSF-100-containing cleavage bodies are predominantly found during S and G2 phases, whereas CstF-64-containing cleavage bodies are primarily observed during S phase. All four nuclear bodies associate with each other during S phase, with cleavage bodies colocalizing with DDX1 bodies, and cleavage bodies/DDX1 bodies residing adjacent to gems and CBs. Although inhibitors of RNA transcription had no effect on DDX1 bodies or cleavage bodies, inhibitors of DNA replication resulted in loss of CstF-64-containing cleavage bodies. A striking effect on nuclear structures was observed with latrunculin B, an inhibitor of actin polymerization, resulting in the formation of needlelike nuclear spicules made up of CstF-64, CPSF-100, RNA, and RNA polymerase II. Our results suggest that cleavage body components are highly dynamic in nature.

  18. Dynamic Nature of Cleavage Bodies and Their Spatial Relationship to DDX1 Bodies, Cajal Bodies, and Gems

    Science.gov (United States)

    Li, Lei; Roy, Ken; Katyal, Sachin; Sun, Xuejun; Bléoo, Stacey; Godbout, Roseline

    2006-01-01

    DDX1 bodies, cleavage bodies, Cajal bodies (CBs), and gems are nuclear suborganelles that contain factors involved in RNA transcription and/or processing. Although all four nuclear bodies can exist as distinct entities, they often colocalize or overlap with each other. To better understand the relationship between these four nuclear bodies, we examined their spatial distribution as a function of the cell cycle. Here, we report that whereas DDX1 bodies, CBs and gems are present throughout interphase, CPSF-100-containing cleavage bodies are predominantly found during S and G2 phases, whereas CstF-64-containing cleavage bodies are primarily observed during S phase. All four nuclear bodies associate with each other during S phase, with cleavage bodies colocalizing with DDX1 bodies, and cleavage bodies/DDX1 bodies residing adjacent to gems and CBs. Although inhibitors of RNA transcription had no effect on DDX1 bodies or cleavage bodies, inhibitors of DNA replication resulted in loss of CstF-64-containing cleavage bodies. A striking effect on nuclear structures was observed with latrunculin B, an inhibitor of actin polymerization, resulting in the formation of needlelike nuclear spicules made up of CstF-64, CPSF-100, RNA, and RNA polymerase II. Our results suggest that cleavage body components are highly dynamic in nature. PMID:16371507

  19. Extra Copper-mediated Enhancement of the DNA Cleavage Activity Supported with Wild-type Cu, Zn Superoxide Dismutase

    Institute of Scientific and Technical Information of China (English)

    ZHOU Ruo-Yu; JIANG Wei; ZHANG Li-Na; WANG Li; LIU Chang-Lin

    2008-01-01

    It is well known that the primary function of wild type Cu, Zn superoxide dismutase (holo SOD) is to catalyze the conversion of the superoxide anion to H2O2 and O2 as an antioxidant enzyme. However, the aberrant copper-mediated oxidation chemistry in the enzyme (including its mutation forms) that damages nucleic acids, proteins including itself and cell membrane has attracted extensive attention in the past decade. The present study examined the hydrogen peroxide-dependent DNA cleavage activity supported with the combinations between holo SOD and extra copper (holo SOD+nCu(Ⅱ)). The results indicate that the presence of extra copper can enhance the DNA cleavage activity and a cooperative effect between holo SOD and the extra Cu(Ⅱ) occurs in DNA cleavage. The relative activity and kinetic assay showed that the DNA cleavage activity of holo SOD+nCu(Ⅱ) was enhanced upon addition of extra Cu(Ⅱ). The favorable pH regions for the DNA cleavage were observed to be 3.6-5.6 and 9.0-10, suggesting the species responsible for the DNA cleavage are different in different pH regions. In addition,to obtain an insight into DNA cleavage pathways, the effect of free radical scavengers and inhibitors on the DNA cleavage activity was probed.

  20. Cleavage and survival of Xenopus embryos exposed to 8 T static magnetic fields in a rotating clinostat.

    Science.gov (United States)

    Eguchi, Yawara; Ueno, Shoogo; Kaito, Chikara; Sekimizu, Kazuhisa; Shiokawa, Koichiro

    2006-05-01

    In this study, we examined cleavage and survival of fertilized Xenopus embryos exposed to 8 T static magnetic fields (SMFs). We investigated fertilized Xenopus embryos exposed to magnetic field either in static chamber or in a rotating culture system. Our results showed that the exposure to the strong magnetic field of 8 T changed the third cleavage furrow from the usual horizontal one to a perpendicular one; however, when the direction of gravity was randomized by exposing embryos to magnetic field in a rotating culture system, the third cleavage furrow were formed horizontally, a finding which suggests that the observed distortion of the third cleavage furrow in magnetism-exposed embryos was accomplished by altering gravity effects which were elicited by diamagnetic force due to high gradient magnetic field. Our results also showed that the exposure to the strong magnetic field did not damage survival. These results demonstrate that SMF and altering gravity cause distortion of the third cleavage furrow and show that effects of exposing cleavage embryos to magnetic field were transient and did not affect the post-cleavage development. We also showed that strong magnetic field is not hazardous to the cleavage and blastula-gastrula transition of developing embryonic cells.

  1. Differential modulation of prM cleavage, extracellular particle distribution, and virus infectivity by conserved residues at nonfurin consensus positions of the dengue virus pr-M junction.

    Science.gov (United States)

    Junjhon, Jiraphan; Lausumpao, Matthawee; Supasa, Sunpetchuda; Noisakran, Sansanee; Songjaeng, Adisak; Saraithong, Prakaimuk; Chaichoun, Kridsada; Utaipat, Utaiwan; Keelapang, Poonsook; Kanjanahaluethai, Amornrat; Puttikhunt, Chunya; Kasinrerk, Watchara; Malasit, Prida; Sittisombut, Nopporn

    2008-11-01

    In the generation of flavivirus particles, an internal cleavage of the envelope glycoprotein prM by furin is required for the acquisition of infectivity. Unlike cleavage of the prM of other flaviviruses, cleavage of dengue virus prM is incomplete in many cell lines; the partial cleavage reflects the influence of residues at furin nonconsensus positions of the pr-M junction, as flaviviruses share basic residues at positions P1, P2, and P4, recognized by furin. In this study, viruses harboring the alanine-scanning and other multiple-point mutations of the pr-M junction were generated, employing a dengue virus background that exhibited 60 to 70% prM cleavage and a preponderance of virion-sized extracellular particles. Analysis of prM and its cleavage products in viable mutants revealed a cleavage-suppressive effect at the conserved P3 Glu residue, as well as the cleavage-augmenting effects at the P5 Arg and P6 His residues, indicating an interplay between opposing modulatory influences mediated by these residues on the cleavage of the pr-M junction. Changes in the prM cleavage level were associated with altered proportions of extracellular virions and subviral particles; mutants with reduced cleavage were enriched with subviral particles and prM-containing virions, whereas the mutant with enhanced cleavage was deprived of these particles. Alterations of virus multiplication were detected in mutants with reduced prM cleavage and were correlated with their low specific infectivities. These findings define the functional roles of charged residues located adjacent to the furin consensus sequence in the cleavage of dengue virus prM and provide plausible mechanisms by which the reduction in the pr-M junction cleavability may affect virus replication.

  2. 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

  3. Cleavage of galectin-3 by matrix metalloproteases induces angiogenesis in breast cancer

    Science.gov (United States)

    Nangia-Makker, Pratima; Wang, Yi; Raz, Tirza; Tait, Larry; Balan, Vitaly; Hogan, Victor; Raz, Avraham

    2012-01-01

    Galectin-3 cleavage is related to progression of human breast and prostate cancer and is partly responsible for tumor growth, angiogenesis and apoptosis resistance in mouse models. A functional polymorphism in galectin-3 gene, determining its susceptibility to cleavage by matrix metalloproteinases (MMPs)-2/-9 is related to racial disparity in breast cancer incidence in Asian and Caucasian women. The purpose of our study is to evaluate (i) if cleavage of galectin-3 could be related to angiogenesis during the progression of human breast cancer, (ii) the role of cleaved galectin-3 in induction of angiogenesis and (iii) determination of the galectin-3 domain responsible for induction of angiogenic response. Galectin-3 null breast cancer cells BT-459 were transfected with either cleavable full-length galectin-3 or its fragmented peptides. Chemotaxis, chemoinvasion, heterotypic aggregation, epithelial-endothelial cell interactions and angiogenesis were compared to noncleavable galectin-3. BT-549-H64 cells harboring cleavable galectin-3 exhibited increased chemotaxis, invasion and interactions with endothelial cells resulting in angiogenesis and 3D morphogenesis compared to BT-549-P64 cells harboring noncleavable galectin-3. BT-549-H64 cells induced increased migration and phosphorylation of focal adhesion kinase in migrating endothelial cells. Endothelial cells cocultured with BT-549 cells transfected with galectin-3 peptides indicate that amino acids 1–62 and 33–250 stimulate migration and morphogenesis of endothelial cells. Immunohistochemical analysis of blood vessel density and galectin-3 cleavage in a breast cancer progression tissue array support the in vitro findings. We conclude that the cleavage of the N terminus of galectin-3 followed by its release in the tumor microenvironment in part leads to breast cancer angiogenesis and progression. PMID:20162566

  4. Surveillance and Cleavage of Eukaryotic tRNAs

    Directory of Open Access Journals (Sweden)

    Cyrille Megel

    2015-01-01

    Full Text Available Beyond their central role in protein synthesis, transfer RNAs (tRNAs have many other crucial functions. This includes various roles in the regulation of gene expression, stress responses, metabolic processes and priming reverse transcription. In the RNA world, tRNAs are, with ribosomal RNAs, among the most stable molecules. Nevertheless, they are not eternal. As key elements of cell function, tRNAs need to be continuously quality-controlled. Two tRNA surveillance pathways have been identified. They act on hypo-modified or mis-processed pre-tRNAs and on mature tRNAs lacking modifications. A short overview of these two pathways will be presented here. Furthermore, while the exoribonucleases acting in these pathways ultimately lead to complete tRNA degradation, numerous tRNA-derived fragments (tRFs are present within a cell. These cleavage products of tRNAs now potentially emerge as a new class of small non-coding RNAs (sncRNAs and are suspected to have important regulatory functions. The tRFs are evolutionarily widespread and created by cleavage at different positions by various endonucleases. Here, we review our present knowledge on the biogenesis and function of tRFs in various organisms.

  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

    This investigation was stimulated by reports that one-electron reductions of monoaryldialkylsulfonium salts never give aryl bond cleavage whereas reductions of diarylmonoalkylsulfonium salts preferentially give aryl bond cleavage. We studied the product ratios from the reductive cleavage of di-4-...

  6. Establishment of cell lines stably co-expressing Japanese encephalitis virus prM and E protein with a mutant in prM furin cleavage site%流行性乙型脑炎prM蛋白剪切位点突变的prM-E蛋白表达细胞系的建立

    Institute of Scientific and Technical Information of China (English)

    李业南; 陈振师; 步志高; 华荣虹

    2013-01-01

    In order to establish the BHK-21 cell lines stably co-expressing prM-E fusion protein of Japanese encephalitis virus (JEV) with a mutant furin site to disable the pre-peptide cleavage of prM, BHK-21 cells was transfected with the recombinant plasmid pCAG-JEV-prM(R89A)E, which was constructed by introducing a point mutation at the cleavage site of prM gene by PCR and cloned into eukaryotic vector pCAG-neo with E gene. Cells stably co-expressing prM-E fusion protein were selected in the present of G418 and identified by indirect immunofluorescence assay and western blot, In addition, the cells displaying high-level protein expression were purified through limiting dilution cloning. Eventfully, the results showed that the cell line was able to express the intact prM of prM-E fusion protein. The established cell line would provide a basis for further study the effect of the cleavage event on mature mechanism of JEV and the development of JEV subunit vaccine.%为建立稳定共表达乙型脑炎病-毒(JEV)完整M前体蛋白(prM)和E蛋白的BHK-21细胞系,本研究在prM蛋白furin蛋白酶剪切位点编码基因中引入突变,将突变后的prM基因克隆于质粒中构建重组表达质粒pCAG-JEV-prM(R89A)E.将重组质粒转染BHK-21细胞,转染48 h后细胞用含G418的选择性培养基选择培养,进一步经细胞克隆纯化制备表达剪切位点突变的JEV prM-E蛋白的稳定细胞系.经IFA和western blot鉴定表明,该细胞系能表达JEV prM与E蛋白,所表达的prM蛋白未发生剪切;细胞经多次传代后仍能够稳定地共表达prM与E蛋白.该细胞系的建立为研究prM蛋白剪切对JEV粒子形成的影响以及亚单位疫苗的制备奠定了基础.

  7. The cleavage of phosphoenolpyruvate by vanadate.

    Science.gov (United States)

    Aureliano, M; Leta, J; Madeira, V M; de Meis, L

    1994-05-30

    Vanadate rapidly promotes the cleavage of phosphoenolpyruvate with phosphate liberation. This was not observed when ATP, glucose-6-phosphate and acetyl phosphate were incubated with vanadate. 51V NMR spectra shows that phosphoenolpyruvate and acetyl phosphate broadened and shifted upfield the monomeric vanadate signal at -561 ppm, indicative of vanadate/phosphate interactions. Comparatively, smaller changes were detected when glucose-6-phosphate was added to the vanadate solution. The shift behavior was not observed in the presence of ATP, ADP or pyruvate.

  8. 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.

  9. Differential Modulation of prM Cleavage, Extracellular Particle Distribution, and Virus Infectivity by Conserved Residues at Nonfurin Consensus Positions of the Dengue Virus pr-M Junction▿

    OpenAIRE

    Junjhon, Jiraphan; Lausumpao, Matthawee; Supasa, Sunpetchuda; Noisakran, Sansanee; Songjaeng, Adisak; Saraithong, Prakaimuk; Chaichoun, Kridsada; Utaipat, Utaiwan; Keelapang, Poonsook; Kanjanahaluethai, Amornrat; Puttikhunt, Chunya; Kasinrerk, Watchara; Malasit, Prida; Sittisombut, Nopporn

    2008-01-01

    In the generation of flavivirus particles, an internal cleavage of the envelope glycoprotein prM by furin is required for the acquisition of infectivity. Unlike cleavage of the prM of other flaviviruses, cleavage of dengue virus prM is incomplete in many cell lines; the partial cleavage reflects the influence of residues at furin nonconsensus positions of the pr-M junction, as flaviviruses share basic residues at positions P1, P2, and P4, recognized by furin. In this study, viruses harboring ...

  10. SVM-based prediction of caspase substrate cleavage sites

    Directory of Open Access Journals (Sweden)

    Ranganathan Shoba

    2006-12-01

    Full Text Available Abstract Background Caspases belong to a class of cysteine proteases which function as critical effectors in apoptosis and inflammation by cleaving substrates immediately after unique sites. Prediction of such cleavage sites will complement structural and functional studies on substrates cleavage as well as discovery of new substrates. Recently, different computational methods have been developed to predict the cleavage sites of caspase substrates with varying degrees of success. As the support vector machines (SVM algorithm has been shown to be useful in several biological classification problems, we have implemented an SVM-based method to investigate its applicability to this domain. Results A set of unique caspase substrates cleavage sites were obtained from literature and used for evaluating the SVM method. Datasets containing (i the tetrapeptide cleavage sites, (ii the tetrapeptide cleavage sites, augmented by two adjacent residues, P1' and P2' amino acids and (iii the tetrapeptide cleavage sites with ten additional upstream and downstream flanking sequences (where available were tested. The SVM method achieved an accuracy ranging from 81.25% to 97.92% on independent test sets. The SVM method successfully predicted the cleavage of a novel caspase substrate and its mutants. Conclusion This study presents an SVM approach for predicting caspase substrate cleavage sites based on the cleavage sites and the downstream and upstream flanking sequences. The method shows an improvement over existing methods and may be useful for predicting hitherto undiscovered cleavage sites.

  11. Cleavage crystallography of liquid metal embrittled aluminum alloys

    Science.gov (United States)

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

    1991-01-01

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

  12. Cleavage by MALT1 induces cytosolic release of A20.

    Science.gov (United States)

    Malinverni, Claire; Unterreiner, Adeline; Staal, Jens; Demeyer, Annelies; Galaup, Marion; Luyten, Marcel; Beyaert, Rudi; Bornancin, Frédéric

    2010-10-01

    The MALT1 paracaspase has arginine-directed proteolytic activity. A20 is a dual ubiquitin-editing enzyme involved in termination of NF-κB signaling. Upon T- or B-cell receptor engagement human (h) A20 is cleaved by MALT1 after arginine 439, yielding an N-terminal fragment (hA20p50) and a C-terminal one (hA20p37). The hA20p50 fragment has never been detected directly, thus limiting insight into the functional consequences of MALT1-mediated cleavage of A20. Here, various antibodies were tested, including newly generated hA20p50 and hA20p37 specific antibodies, leading to detection of the hA20p50 fragment produced after MALT1-mediated cleavage of ectopically expressed as well as endogenous A20 proteins. The properties of both A20 fragments, generated upon co-expression with a constitutively active MALT1 protein, were further studied by sub-cellular fractionation and fluorescence microscopy. In contrast to full-length A20 which is particulate and insoluble, we found hA20p50 to be soluble and readily released into the cytosol whereas hA20p37 was partially soluble, thus suggesting loss of compartmentalization as a possible mechanism for MALT1-mediated dampening of A20 function.

  13. CLEAVAGE OF SOFTWOOD KRAFT PULP FIBRES BY HCL AND CELLULASES

    Directory of Open Access Journals (Sweden)

    Paul Ander

    2008-05-01

    Full Text Available A new pulp fibre testing procedure called the HCl method was used to compare different spruce and pine fibres and mixtures of these fibres to calculate number of fibre cleavages in dislocations and other weak points. This method was compared with treatment of softwood kraft pulp fibres using different cellulase mixtures. The HCl method can distinguish between mill- and laboratory-made softwood kraft pulp fibres from the same wood batch. The sugar release is characterized by xylose and other hemicellulose sugars and little glucose. This is in contrast to cellulases, which despite strong fibre cleavage, did not distinguish between mill- and laboratory-made pulp fibres and released large amounts of glucose from the fibres. Hemicellulose degradation by HCl and deep penetration of the acid into the primary and secondary fibre cell walls at 80°C seems to be of major importance for the differentiation between mill and laboratory pulp fibres. Cellulases, in contrast, act mostly on the fibre surfaces, and deep penetration only takes place in amorphous regions of dislocations.

  14. Apoptosis Mediated by HIV Protease is Preceded by Cleavage of Bcl-2

    Science.gov (United States)

    Strack, Peter R.; West Frey, Michelle; Rizzo, Christopher J.; Cordova, Beverly; George, Henry J.; Meade, Raymond; Ho, Siew Peng; Corman, Jeanne; Tritch, Radonna; Korant, Bruce D.

    1996-09-01

    Expression of the human immunodeficiency virus type 1 (HIV) protease in cultured cells leads to apoptosis, preceded by cleavage of bcl-2, a key negative regulator of cell death. In contrast, a high level of bcl-2 protects cells in vitro and in vivo from the viral protease and prevents cell death following HIV infection of human lymphocytes, while reducing the yields of viral structural proteins, infectivity, and tumor necrosis factor α . We present a model for HIV replication in which the viral protease depletes the infected cells of bcl-2, leading to oxidative stress-dependent activation of NFkappa B, a cellular factor required for HIV transcription, and ultimately to cell death. Purified bcl-2 is cleaved by HIV protease between phenylalanine 112 and alanine 113. The results suggest a new option for HIV gene therapy; bcl-2 muteins that have noncleavable alterations surrounding the HIV protease cleavage site.

  15. 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.

  16. The dorsoventral axis is specified prior to first cleavage in the direct developing sea urchin Heliocidaris erythrogramma.

    Science.gov (United States)

    Henry, J J; Wray, G A; Raff, R A

    1990-11-01

    Previous fate mapping studies as well as the culture of isolated blastomeres have revealed that the dorsoventral axis is specified as early as the 2-cell stage in the embryos of the direct developing echinoid, Heliocidaris erythrogramma. Normally, the first cleavage plane includes the animal-vegetal axis and bisects the embryo between future dorsal and ventral halves. Experiments were performed to establish whether the dorsoventral axis is set up prior to the first cleavage division in H. erythrogramma. Eggs were elongated and fertilized in silicone tubes of a small diameter in order to orient the cleavage spindle and thus the first plane of cell division. Following first cleavage, one of the two resulting blastomeres was then microinjected with a fluorescent cell lineage tracer dye. Fate maps were made after culturing these embryos to larval stages. The results indicate that the first cleavage division can be made to occur at virtually any angle relative to the animal-vegetal and dorsoventral axes. Therefore, the dorsoventral axis is specified prior to first cleavage. We argue that this axis resides in the unfertilized oocyte rather than being set up as a consequence of fertilization.

  17. 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

  18. Endoproteolytic cleavage of TUG protein regulates GLUT4 glucose transporter translocation.

    Science.gov (United States)

    Bogan, Jonathan S; Rubin, Bradley R; Yu, Chenfei; Löffler, Michael G; Orme, Charisse M; Belman, Jonathan P; McNally, Leah J; Hao, Mingming; Cresswell, James A

    2012-07-06

    To promote glucose uptake into fat and muscle cells, insulin causes the translocation of GLUT4 glucose transporters from intracellular vesicles to the cell surface. Previous data support a model in which TUG traps GLUT4-containing vesicles and tethers them intracellularly in unstimulated cells and in which insulin mobilizes this pool of vesicles by releasing this tether. Here we show that TUG undergoes site-specific endoproteolytic cleavage, which separates a GLUT4-binding, N-terminal region of TUG from a C-terminal region previously suggested to bind an intracellular anchor. Cleavage is accelerated by insulin stimulation in 3T3-L1 adipocytes and is highly dependent upon adipocyte differentiation. The N-terminal TUG cleavage product has properties of a novel 18-kDa ubiquitin-like modifier, which we call TUGUL. The C-terminal product is observed at the expected size of 42 kDa and also as a 54-kDa form that is released from membranes into the cytosol. In transfected cells, intact TUG links GLUT4 to PIST and also binds Golgin-160 through its C-terminal region. PIST is an effector of TC10α, a GTPase previously shown to transmit an insulin signal required for GLUT4 translocation, and we show using RNAi that TC10α is required for TUG proteolytic processing. Finally, we demonstrate that a cleavage-resistant form of TUG does not support highly insulin-responsive GLUT4 translocation or glucose uptake in 3T3-L1 adipocytes. Together with previous results, these data support a model whereby insulin stimulates TUG cleavage to liberate GLUT4 storage vesicles from the Golgi matrix, which promotes GLUT4 translocation to the cell surface and enhances glucose uptake.

  19. 2ʹ-O-methyl nucleotide modified DNA substrates influence the cleavage efficiencies of BamHI and BglII

    Indian Academy of Sciences (India)

    Zhaoxue Tong; Bin Zhao; Guojie Zhao; Hong Shang; Yifu Guan

    2014-09-01

    Induction of endonucleolytic DNA cleavage is an essential event that links the initiating stimuli to the final effects of cells. The cleavage efficiency and thus the final yield could be affected by many factors, including structures of DNA substrates, composite structures of enzymes–substrates or enzymes–nucleic analogs and so on. However, it is not clear whether a nucleotide derivative-substituted in DNA substrates can influence the efficiency of enzymatic cleavage. To investigate the effect of sugar pucker conformation on DNA–protein interactions, we used 2′--methyl modified nucleotides (OMeN) to modify DNA substrates of isocaudemers BamHI and BglII in this study, and used FRET assay as an efficient method for analysis of enzyme cleavage. Experimental results demonstrated that OMeN-substituted recognition sequences influenced the cleavage rates significantly in a position-dependent manner. OMeN substitutions can reduce the cleavage as expected. Surprisingly, OMeN substitutions can also enhance the cleavage rates. The kinetics parameters of max and m have been obtained by fitting the Michaelis-Menten kinetic equation. These 2′-OMe nucleotides could behave as a regulatory element to modulate the enzymatic activity in vitro, and this property could enrich our understanding about the endonuclease cleavage mechanism and enhance our ability to regulate the enzymatic cleavage efficiency for applications in synthetic biology.

  20. Intratree Variability of Cleavage Resistance of Chinese Fir from Plantation

    Institute of Scientific and Technical Information of China (English)

    XU Ming; REN Haiqing; LUO Xiuqin; YIN Yafang

    2006-01-01

    This paper studied the variation of cleavage resistance of Chinese fir wood from plantation.Six trees of 36 years old were investigated,and the cleavage resistance properties for 672 samples made of the trees were tested.The samples were cut from the sapwood and heartwood at different directions (south and north) and heights (1.3,3.3,5.3 and 7.3 m) of the trees.The result showed that:tangential cleavage resistance was higher than radial one, and cleavage resistance of sapwood was higher than that of heartwood,but there was no significant difference in cleavage resistances between sections of the north and the south of the trees.There was a little variation in cleavage resistance between the radial and tangential from butt to top log,which shows alittle decrease with the height from 1.3 to 5.3 m,but a rise in the top of the trees.

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

    Directory of Open Access Journals (Sweden)

    Chenyu Zhang

    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

  2. A Python analytical pipeline to identify prohormone precursors and predict prohormone cleavage sites

    Directory of Open Access Journals (Sweden)

    Bruce Southey

    2008-12-01

    Full Text Available Neuropeptides and hormones are signaling molecules that support cell-cell communication in the central nervous system. Experimentally characterizing neuropeptides requires significant efforts because of the complex and variable processing of prohormone precursor proteins into neuropeptides and hormones. We demonstrate the power and flexibility of the Python language to develop components of an bioinformatic analytical pipeline to identify precursors from genomic data and to predict cleavage as these precursors are en route to the final bioactive peptides. We identified 75 precursors in the rhesus genome, predicted cleavage sites using support vector machines and compared the rhesus predictions to putative assignments based on homology to human sequences. The correct classification rate of cleavage using the support vector machines was over 97% for both human and rhesus data sets. The functionality of Python has been important to develop and maintain NeuroPred (http://neuroproteomics.scs.uiuc.edu/neuropred.html, a user-centered web application for the neuroscience community that provides cleavage site prediction from a wide range of models, precision and accuracy statistics, post-translational modifications, and the molecular mass of potential peptides. The combined results illustrate the suitability of the Python language to implement an all-inclusive bioinformatics approach to predict neuropeptides that encompasses a large number of interdependent steps, from scanning genomes for precursor genes to identification of potential bioactive neuropeptides.

  3. A python analytical pipeline to identify prohormone precursors and predict prohormone cleavage sites.

    Science.gov (United States)

    Southey, Bruce R; Sweedler, Jonathan V; Rodriguez-Zas, Sandra L

    2008-01-01

    Neuropeptides and hormones are signaling molecules that support cell-cell communication in the central nervous system. Experimentally characterizing neuropeptides requires significant efforts because of the complex and variable processing of prohormone precursor proteins into neuropeptides and hormones. We demonstrate the power and flexibility of the Python language to develop components of an bioinformatic analytical pipeline to identify precursors from genomic data and to predict cleavage as these precursors are en route to the final bioactive peptides. We identified 75 precursors in the rhesus genome, predicted cleavage sites using support vector machines and compared the rhesus predictions to putative assignments based on homology to human sequences. The correct classification rate of cleavage using the support vector machines was over 97% for both human and rhesus data sets. The functionality of Python has been important to develop and maintain NeuroPred (http://neuroproteomics.scs.uiuc.edu/neuropred.html), a user-centered web application for the neuroscience community that provides cleavage site prediction from a wide range of models, precision and accuracy statistics, post-translational modifications, and the molecular mass of potential peptides. The combined results illustrate the suitability of the Python language to implement an all-inclusive bioinformatics approach to predict neuropeptides that encompasses a large number of interdependent steps, from scanning genomes for precursor genes to identification of potential bioactive neuropeptides.

  4. Proteolytic cleavage of cadherins: Functional role of the cleaved extracellular and cytoplasmic domains

    OpenAIRE

    2006-01-01

    Dynamic regulation of cadherin mediated cell-cell adhesion is crucial for morphogenesis and tissue homeostasis. Cadherin adhesive function can be regulated by distinct proteolytic cleavage events, resulting in release of either the ectodomain or cytoplasmic domain. However, it is unclear if the released fragments have biological activity by themselves. This thesis analyses the functional significance of the generated cadherin fragments. Using Xenopus laevis development as model system, it was...

  5. Regulation by retinoids of luteinizing hormone/chorionic gonadotropin receptor, cholesterol side-chain cleavage cytochrome P-450, 3 beta-hydroxysteroid dehydrogenase/delta (5-4)-isomerase and 17 alpha-hydroxylase/C17-20 lyase cytochrome P-450 messenger ribonucleic acid levels in the K9 mouse Leydig cell line.

    Science.gov (United States)

    Lefèvre, A; Rogier, E; Astraudo, C; Duquenne, C; Finaz, C

    1994-12-01

    Vitamin A is a potent regulator of testicular function. We have reported that retinol (R) and retinoic acid (RA) induced a down regulation of luteinizing hormone/human chorionic gonadotropin (LH/CG) binding sites in K9 Leydig cells. In the present study we evaluated the effect of R and RA on LH/CG receptors, cholesterol side-chain cleavage cytochrome P-450 (P-450 scc), 17 alpha-hydroxylase/C17-20 lyase (P-450 17 alpha) and 3 beta-hydroxysteroid dehydrogenase (3 beta HSD) mRNA levels in K9 mouse Leydig cells. To validate K9 cells as a model for studying Leydig cell steroidogenesis at the molecular level, we first investigated the effect of hCG on mRNA levels of the steroidogenic enzymes. P-450 scc, 3 beta HSD and P-450 17 alpha were expressed constitutively. The addition of 10 ng/ml hCG enhanced mRNA levels for the three genes within 2 h. Maximal accumulation of P-450 scc, P-450 17 alpha and 3 beta HSD mRNA in treated cells represents a 2.5-, 8.5- and 4-fold increase over control values, respectively. P-450 17 alpha expression reached a maximum by 4 h and then declined rapidly to return to control value by 24 h. The pattern of LH/CG receptor mRNAs in K9 cells was very similar to that of MA10 Leydig cells and showed six transcripts of 1.1, 1.6, 1.9, 2.6, 4.2 and 7.0 kb. Treatment of cells with R or RA resulted in a time- and dose-dependent decrease in all six species.(ABSTRACT TRUNCATED AT 250 WORDS)

  6. Resistance to topoisomerase cleavage complex induced lethality in Escherichia coli via titration of transcription regulators PurR and FNR

    Directory of Open Access Journals (Sweden)

    Liu I-Fen

    2011-12-01

    Full Text Available Abstract Background Accumulation of gyrase cleavage complex in Escherichia coli from the action of quinolone antibiotics induces an oxidative damage cell death pathway. The oxidative cell death pathway has also been shown to be involved in the lethality following accumulation of cleavage complex formed by bacterial topoisomerase I with mutations that result in defective DNA religation. Methods A high copy number plasmid clone spanning the upp-purMN region was isolated from screening of an E. coli genomic library and analyzed for conferring increased survival rates following accumulation of mutant topoisomerase I proteins as well as treatment with the gyrase inhibitor norfloxacin. Results Analysis of the intergenic region upstream of purM demonstrated a novel mechanism of resistance to the covalent protein-DNA cleavage complex through titration of the cellular transcription regulators FNR and PurR responsible for oxygen sensing and repression of purine nucleotide synthesis respectively. Addition of adenine to defined growth medium had similar protective effect for survival following accumulation of topoisomerase cleavage complex, suggesting that increase in purine level can protect against cell death. Conclusions Perturbation of the global regulator FNR and PurR functions as well as increase in purine nucleotide availability could affect the oxidative damage cell death pathway initiated by topoisomerase cleavage complex.

  7. Synthesis, photochemistry, DNA cleavage/binding and cytotoxic properties of fluorescent quinoxaline and quinoline hydroperoxides.

    Science.gov (United States)

    Chowdhury, Nilanjana; Gangopadhyay, Moumita; Karthik, S; Pradeep Singh, N D; Baidya, Mithu; Ghosh, S K

    2014-01-01

    Novel fluorescent quinoxaline and quinoline hydroperoxides were shown to perform dual role as both fluorophores for cell imaging and photoinduced DNA cleaving agents. Photophysical studies of newly synthesized quinoxaline and quinoline hydroperoxides showed that they all exhibited moderate to good fluorescence. Photolysis of quinoxaline and quinoline hydroperoxides in acetonitrile using UV light above 350nm resulted in the formation of corresponding ester compounds via γ-hydrogen abstraction by excited carbonyl chromophore. Single strand DNA cleavage was achieved on irradiation of newly synthesized hydroperoxides by UV light (⩾350nm). Both hydroxyl radicals and singlet oxygen were identified as reactive oxygen species (ROS) responsible for the DNA cleavage. Further, we showed quinoline hydroperoxide binds to ct-DNA via intercalative mode. In vitro biological studies revealed that quinoline hydroperoxide has good biocompatibility, cellular uptake property and cell imaging ability. Finally, we showed that quinoline hydroperoxide can permeate into cells efficiently and may cause cytotoxicity upon irradiation by UV light.

  8. prpC-related signal transduction is influenced by copper, membrane integrity and the alpha cleavage site

    Institute of Scientific and Technical Information of China (English)

    Cathryn L Haigh; Victoria A Lewis; Laura J Vella; Colin L Masters; Andrew F Hill; Victoria A Lawson; Steven J Collins

    2009-01-01

    The copper-binding, membrane-anchored, cellular prion protein (PrPC) has two constitutive cleavage sites pro-ducing distinct N- and C-terminal fragments (N1/C1 and N2/C2). Using RKI3 cells expressing either human PrPC, mouse PrPC or mouse PrPC carrying the 3F4 epitope, this study explored the influence of the PrPC primary sequence on endoproteolytic cleavage and one putative PrPC function, MAP kinase signal transduction, in response to exoge-nous copper with or without a perturbed membrane environment. PrPC primary sequence, especially that around the N1/C1 cleavage site, appeared to influence basal levels of proteolysis at this location and extracellular signal-regulat-ed kinase 1/2 (ERK1/2) phosphorylation, with increased processing demonstrating an inverse relationship with basal ERK1/2 activation. Human PrPC showed increased N1/C1 cleavage in response to copper alone, accompanied by spe-cific p38 and JNK/SAPK phosphorylation. Combined exposure to copper plus the cholesterol-sequestering antibiotic filipin resulted in a mouse PrPC-specific substantial increase in signal protein phosphorylation, accompanied by an increase in N1/C1 cleavage. Mouse PrPC harboring the human N1/C1 cleavage site assumed more human-like profiles basally and in response to copper and altered membrane environments. Our results demonstrate that the PrPC pri-mary sequence around the N1/C1 cleavage site influences endoproteolytic processing at this location, which appears linked to MAP kinase signal transduction both basally and in response to copper. Further, the primary sequence ap-pears to confer a mutual dependence of N1/C1 cleavage and membrane integrity on the fidelity of prpC-related signal transduction in response to exogenous stimuli.

  9. 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 BACKGROUND: 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. METHODOLOGY/PRINCIPAL FINDING: 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. CONCLUSIONS: 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.

  10. Presence of Meiotic Spindles Indicates Early Cleavage of Embryos

    Institute of Scientific and Technical Information of China (English)

    2007-01-01

    Objective To assess whether the detection of the meiotic spindle could anticipate the appearance of early cleavage.Methods Oocytes were obtained from stimulated ovaries of consenting patients undergoing oocytes retrieval for ICSI.Spindles were imaged with the Polscope.After ICSI,oocytes with or without spindles were cultured for examination of early cleavage and embryo development.A total of 328 oocytes from 50 cycles were examined with the Polscope and inseminated by ICSI.Results Spindles were imaged in 81.7% of oocytes.After ICSI,more oocytes with spindles (78.4%) fertilized normally than oocytes without spindles (53.3%)(P<0.001).At 25-27 h post ICSI.more fertilized oocytes developed from oocytes with spindles (81.9%) were detected early cleavage than those from oocytes without spindles(28.1%)(P<0.001).Significantly more embryos with early cleavage (82.2%) developed to high quality embryos at d 3 compared with the embryos without early cleavage(48.3%)(P=0.001).The value of rs related to the relationship between spindles and early cleavage was 0.420(P<0.0001).Conclusion The existing of the early cleavage may have a predictive value on the opportunity of high quality embryos and the existing of the spindle may have a predictive value in the appearance of early cleavage.

  11. Control of cleavage spindle orientation in Caenorhabditis elegans: The role of the genes par-2 and par-3

    Energy Technology Data Exchange (ETDEWEB)

    Cheng, N.N.; Kirby, C.M.; Kemphues, K.J. [Cornell Univ., Ithaca, NY (United States)

    1995-02-01

    Polarized asymmetric divisions play important roles in the development of plants and animals. The first two embryonic cleavages of Caenorhabditis elegans provide an opportunity to study the mechanisms controlling polarized asymmetric divisions. The first cleavage is unequal, producing daughters with different sizes and fates. The daughter blastomeres divide with different orientations at the second cleavage; the anterior blastomere divides equally across the long axis of the egg, whereas the posterior blastomere divides unequally along the long axis. We report here the results of our analysis of the genes par-2 and par-3 with respect to their contribution to the polarity of these divisions. Strong loss-of-function mutations in both genes lead to an equal first cleavage and an altered second cleavage. Interestingly, the mutations exhibit striking gene-specific differences at the second cleavage. The par-2 mutations lead to transverse spindle orientations in both blastomeres, whereas par-3 mutations lead to longitudinal spindle orientations in both blastomeres. The spindle orientation defects correlate with defects in centrosome movements during both the first and the second cell cycle. Temperature shift experiments with par-2 (it5ts) indicate that the par-2(+) activity is not required after the two-cell stage. Analysis of double mutants shows that par-3 is epistatic to par-2. We propose a model wherein par-2(+) and par-3(+) act in concert during the first cell cycle to affect asymmetric modification of the cytoskeleton. This polar modification leads to different behaviors of centrosomes in the anterior and posterior and leads ultimately to blastomere-specific spindle orientations at the second cleavage. 44 refs., 5 figs., 5 tabs.

  12. ADAM10 overexpression shifts lympho- and myelopoiesis by dysregulating site 2/site 3 cleavage products of Notch.

    Science.gov (United States)

    Gibb, David R; Saleem, Sheinei J; Kang, Dae-Joong; Subler, Mark A; Conrad, Daniel H

    2011-04-01

    Although the physiological consequences of Notch signaling in hematopoiesis have been extensively studied, the differential effects of individual notch cleavage products remain to be elucidated. Given that ADAM10 is a critical regulator of Notch and that its deletion is embryonically lethal, we generated mice that overexpress ADAM10 (ADAM10 transgenic [A10Tg]) at early stages of lympho- and myeloid development. Transgene expression resulted in abrogated B cell development, delayed T cell development in the thymus, and unexpected systemic expansion of CD11b(+)Gr-1(+) cells, also known as myeloid-derived suppressor cells. Mixed bone marrow reconstitution assays demonstrated that transgene expression altered hematopoiesis via a cell-intrinsic mechanism. Consistent with previously reported observations, we hypothesized that ADAM10 overexpression dysregulated Notch by uncoupling the highly regulated proteolysis of Notch receptors. This was confirmed using an in vitro model of hematopoiesis via culturing A10Tg hematopoietic Lineage(-)Sca-1(+)c-Kit(+) cells with OP-9 stromal cells in the presence or absence of Delta-like 1, a primary ligand for Notch. Blockade of the site 2 (S2) and site 3 (S3) cleavage of the Notch receptor demonstrated differential effects on hematopoiesis. OP9-DL1 cultures containing the ADAM10 inhibitor (S2 cleavage site) enhanced and rescued B cell development from wild-type and A10Tg Lineage(-)Sca-1(+)c-Kit(+) cells, respectively. In contrast, blockade of γ-secretase at the S3 cleavage site induced accumulation of the S2 product and consequently prevented B cell development and resulted in myeloid cell accumulation. Collectively, these findings indicate that the differential cleavage of Notch into S2 and S3 products regulated by ADAM10 is critical to hematopoietic cell-fate determination.

  13. 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.

  14. The N-terminal domain allosterically regulates cleavage and activation of the epithelial sodium channel.

    Science.gov (United States)

    Kota, Pradeep; Buchner, Ginka; Chakraborty, Hirak; Dang, Yan L; He, Hong; Garcia, Guilherme J M; Kubelka, Jan; Gentzsch, Martina; Stutts, M Jackson; Dokholyan, Nikolay V

    2014-08-15

    The epithelial sodium channel (ENaC) is activated upon endoproteolytic cleavage of specific segments in the extracellular domains of the α- and γ-subunits. Cleavage is accomplished by intracellular proteases prior to membrane insertion and by surface-expressed or extracellular soluble proteases once ENaC resides at the cell surface. These cleavage events are partially regulated by intracellular signaling through an unknown allosteric mechanism. Here, using a combination of computational and experimental techniques, we show that the intracellular N terminus of γ-ENaC undergoes secondary structural transitions upon interaction with phosphoinositides. From ab initio folding simulations of the N termini in the presence and absence of phosphatidylinositol 4,5-bisphosphate (PIP2), we found that PIP2 increases α-helical propensity in the N terminus of γ-ENaC. Electrophysiology and mutation experiments revealed that a highly conserved cluster of lysines in the γ-ENaC N terminus regulates accessibility of extracellular cleavage sites in γ-ENaC. We also show that conditions that decrease PIP2 or enhance ubiquitination sharply limit access of the γ-ENaC extracellular domain to proteases. Further, the efficiency of allosteric control of ENaC proteolysis is dependent on Tyr(370) in γ-ENaC. Our findings provide an allosteric mechanism for ENaC activation regulated by the N termini and sheds light on a potential general mechanism of channel and receptor activation.

  15. The N-terminal Domain Allosterically Regulates Cleavage and Activation of the Epithelial Sodium Channel*

    Science.gov (United States)

    Kota, Pradeep; Buchner, Ginka; Chakraborty, Hirak; Dang, Yan L.; He, Hong; Garcia, Guilherme J. M.; Kubelka, Jan; Gentzsch, Martina; Stutts, M. Jackson; Dokholyan, Nikolay V.

    2014-01-01

    The epithelial sodium channel (ENaC) is activated upon endoproteolytic cleavage of specific segments in the extracellular domains of the α- and γ-subunits. Cleavage is accomplished by intracellular proteases prior to membrane insertion and by surface-expressed or extracellular soluble proteases once ENaC resides at the cell surface. These cleavage events are partially regulated by intracellular signaling through an unknown allosteric mechanism. Here, using a combination of computational and experimental techniques, we show that the intracellular N terminus of γ-ENaC undergoes secondary structural transitions upon interaction with phosphoinositides. From ab initio folding simulations of the N termini in the presence and absence of phosphatidylinositol 4,5-bisphosphate (PIP2), we found that PIP2 increases α-helical propensity in the N terminus of γ-ENaC. Electrophysiology and mutation experiments revealed that a highly conserved cluster of lysines in the γ-ENaC N terminus regulates accessibility of extracellular cleavage sites in γ-ENaC. We also show that conditions that decrease PIP2 or enhance ubiquitination sharply limit access of the γ-ENaC extracellular domain to proteases. Further, the efficiency of allosteric control of ENaC proteolysis is dependent on Tyr370 in γ-ENaC. Our findings provide an allosteric mechanism for ENaC activation regulated by the N termini and sheds light on a potential general mechanism of channel and receptor activation. PMID:24973914

  16. Cleavage of resveratrol in fungi: characterization of the enzyme Rco1 from Ustilago maydis.

    Science.gov (United States)

    Brefort, Thomas; Scherzinger, Daniel; Limón, M Carmen; Estrada, Alejandro F; Trautmann, Danika; Mengel, Carina; Avalos, Javier; Al-Babili, Salim

    2011-02-01

    Ustilago maydis, the causative agent of corn smut disease, contains two genes encoding members of the carotenoid cleavage oxygenase family, a group of enzymes that cleave double bonds in different substrates. One of them, Cco1, was formerly identified as a β-carotene cleaving enzyme. Here we elucidate the function of the protein encoded by the second gene, termed here as Ustilago maydis Resveratrol cleavage oxygenase 1 (Um Rco1). In vitro incubations of heterologously expressed and purified UM Rco1 with different carotenoid and stilbene substrates demonstrate that it cleaves the interphenyl Cα-Cβ double bond of the phytoalexin resveratrol and its derivative piceatannol. Um Rco1 exhibits a high degree of substrate specificity, as suggested by the lack of activity on carotenoids and the other resveratrol-related compounds tested. The activity of Um Rco1 was confirmed by incubation of U. maydis rco1 deletion and over-expression strains with resveratrol. Furthermore, treatment with resveratrol resulted in striking alterations of cell morphology. However, pathogenicity assays indicated that Um rco1 is largely dispensable for biotrophic development. Our work reveals Um Rco1 as the first eukaryotic resveratrol cleavage enzyme identified so far. Moreover, Um Rco1 represents a subfamily of fungal enzymes likely involved in the degradation of stilbene compounds, as suggested by the cleavage of resveratrol by homologs from Aspergillus fumigatus, Chaetomium globosum and Botryotinia fuckeliana.

  17. Characterization of the prohormone complement in cattle using genomic libraries and cleavage prediction approaches

    Directory of Open Access Journals (Sweden)

    Rodriguez-Zas Sandra L

    2009-05-01

    Full Text Available Abstract Background Neuropeptides are cell to cell signalling molecules that regulate many critical biological processes including development, growth and reproduction. These peptides result from the complex processing of prohormone proteins, making their characterization both challenging and resource demanding. In fact, only 42 neuropeptide genes have been empirically confirmed in cattle. Neuropeptide research using high-throughput technologies such as microarray and mass spectrometry require accurate annotation of prohormone genes and products. However, the annotation and associated prediction efforts, when based solely on sequence homology to species with known neuropeptides, can be problematic. Results Complementary bioinformatic resources were integrated in the first survey of the cattle neuropeptide complement. Functional neuropeptide characterization was based on gene expression profiles from microarray experiments. Once a gene is identified, knowledge of the enzymatic processing allows determination of the final products. Prohormone cleavage sites were predicted using several complementary cleavage prediction models and validated against known cleavage sites in cattle and other species. Our bioinformatics approach identified 92 cattle prohormone genes, with 84 of these supported by expressed sequence tags. Notable findings included an absence of evidence for a cattle relaxin 1 gene and evidence for a cattle galanin-like peptide pseudogene. The prohormone processing predictions are likely accurate as the mammalian proprotein convertase enzymes, except for proprotein convertase subtilisin/kexin type 9, were also identified. Microarray analysis revealed the differential expression of 21 prohormone genes in the liver associated with nutritional status and 8 prohormone genes in the placentome of embryos generated using different reproductive techniques. The neuropeptide cleavage prediction models had an exceptional performance, correctly

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

    Science.gov (United States)

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

    2016-11-04

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

  19. Label-free Quantification of Proteins in Single Embryonic Cells with Neural Fate in the Cleavage-Stage Frog (Xenopus laevis) Embryo using Capillary Electrophoresis Electrospray Ionization High-Resolution Mass Spectrometry (CE-ESI-HRMS).

    Science.gov (United States)

    Lombard-Banek, Camille; Reddy, Sushma; Moody, Sally A; Nemes, Peter

    2016-08-01

    Quantification of protein expression in single cells promises to advance a systems-level understanding of normal development. Using a bottom-up proteomic workflow and multiplexing quantification by tandem mass tags, we recently demonstrated relative quantification between single embryonic cells (blastomeres) in the frog (Xenopus laevis) embryo. In this study, we minimize derivatization steps to enhance analytical sensitivity and use label-free quantification (LFQ) for single Xenopus cells. The technology builds on a custom-designed capillary electrophoresis microflow-electrospray ionization high-resolution mass spectrometry platform and LFQ by MaxLFQ (MaxQuant). By judiciously tailoring performance to peptide separation, ionization, and data-dependent acquisition, we demonstrate an ∼75-amol (∼11 nm) lower limit of detection and quantification for proteins in complex cell digests. The platform enabled the identification of 438 nonredundant protein groups by measuring 16 ng of protein digest, or embryo. LFQ intensity was validated as a quantitative proxy for protein abundance. Correlation analysis was performed to compare protein quantities between the embryo and n = 3 different single D11 blastomeres, which are fated to develop into the nervous system. A total of 335 nonredundant protein groups were quantified in union between the single D11 cells spanning a 4 log-order concentration range. LFQ and correlation analysis detected expected proteomic differences between the whole embryo and blastomeres, and also found translational differences between individual D11 cells. LFQ on single cells raises exciting possibilities to study gene expression in other cells and models to help better understand cell processes on a systems biology level.

  20. Ostensible enzyme promiscuity: alkene cleavage by peroxidases.

    Science.gov (United States)

    Mutti, Francesco G; Lara, Miguel; Kroutil, Markus; Kroutil, Wolfgang

    2010-12-17

    Enzyme promiscuity is generally accepted as the ability of an enzyme to catalyse alternate chemical reactions besides the 'natural' one. In this paper peroxidases were shown to catalyse the cleavage of a C=C double bond adjacent to an aromatic moiety for selected substrates at the expense of molecular oxygen at an acidic pH. It was clearly shown that the reaction occurs due to the presence of the enzyme; furthermore, the reactivity was clearly linked to the hemin moiety of the peroxidase. Comparison of the transformations catalysed by peroxidase and by hemin chloride revealed that these two reactions proceed equally fast; additional experiments confirmed that the peptide backbone was not obligatory for the reaction and only a single functional group of the enzyme was required, namely in this case the prosthetic group (hemin). Consequently, we propose to define such a promiscuous activity as 'ostensible enzyme promiscuity'. Thus, we call an activity that is catalysed by an enzyme 'ostensible enzyme promiscuity' if the reactivity can be tracked back to a single catalytic site, which on its own can already perform the reaction equally well in the absence of the peptide backbone.

  1. A cleavage toughness master curve model

    Science.gov (United States)

    Odette, G. R.; He, M. Y.

    2000-12-01

    Development of fusion power will require a fracture toughness database, derived largely from small specimen tests, closely integrated with methods to assess first wall and blanket structural integrities. A master curve-shift (MC-ΔT) method has been proposed as an engineering expedient to treat the effects of structural geometry, irradiation, loading rates and safety margins. However, a number of issues related to the MC-ΔT method remain to be resolved, including the universality of MC shapes. A new micromechanical model of fracture toughness in the cleavage transition regime is proposed that combines analytical representations of finite element analysis simulations of crack-tip stress fields with a local critical stress-critical stressed area (σ∗-A∗) fracture criterion. This model, has been successful in predicting geometry effects, as well as high loading rate and irradiation hardening-induced Charpy shifts. By incorporating a modest temperature dependence in σ∗(T), an inconsistency between model predictions and an observed universal-type MC shape is resolved.

  2. Secologanin synthase which catalyzes the oxidative cleavage of loganin into secologanin is a cytochrome P450.

    Science.gov (United States)

    Yamamoto, H; Katano, N; Ooi, A; Inoue, K

    2000-01-01

    Secologanin synthase, an enzyme catalyzing the oxidative cleavage of the cyclopentane ring in loganin to form secologanin, was detected in microsomal preparations from cell suspension cultures of Lonicera japonica. The reaction required NADPH and molecular oxygen, and was blocked by carbon monoxide as well as by several other cytochrome P450 inhibitors, indicating that the reaction was mediated by cytochrome P450. Of the substrates examined, only specificity for loganin was demonstrated. A possible reaction mechanism is described.

  3. 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.

  4. Berberine inhibits growth, induces G1 arrest and apoptosis in human epidermoid carcinoma A431 cells by regulating Cdki-Cdk-cyclin cascade, disruption of mitochondrial membrane potential and cleavage of caspase 3 and PARP.

    Science.gov (United States)

    Mantena, Sudheer K; Sharma, Som D; Katiyar, Santosh K

    2006-10-01

    Chemotherapeutic approach using non-toxic botanicals may be one of the strategies for the management of the skin cancers. Here we report that in vitro treatment of human epidermoid carcinoma A431 cells with berberine, a naturally occurring isoquinoline alkaloid, decreased cell viability (3-77%, P berberine-induced G(1) cell cycle arrest was mediated through the increased expression of Cdki proteins (Cip1/p21 and Kip1/p27), a simultaneous decrease in Cdk2, Cdk4, Cdk6 and cyclins D1, D2 and E and enhanced binding of Cdki-Cdk. In additional studies, treatment of A431 cells with berberine (15-75 microM) for 72 h resulted in a significant dose-dependent increase in apoptosis (31-60%, P berberine-treated control (11.7%), which was associated with an increased expression of pro-apoptotic protein Bax, decreased expression of anti-apoptotic proteins Bcl-2 and Bcl-xl, disruption of mitochondrial membrane potential, and activation of caspases 9, 3 and poly (ADP-ribose) polymerase. Pretreatment of A431 cells with the pan-caspase inhibitor (z-VAD-fmk) significantly blocked the berberine-induced apoptosis in A431 cells confirmed that berberine-induced apoptosis is mediated through activation of caspase 3-dependent pathway. Together, this study for the first time identified berberine as a chemotherapeutic agent against human epidermoid carcinoma A431 cells in vitro, further in vivo studies are required to determine whether berberine could be an effective chemotherapeutic agent for the management of non-melanoma skin cancers.

  5. Effects of silver ions (Ag+) on contractile ring function and microtubule dynamics during first cleavage in Ilyanassa obsoleta

    Science.gov (United States)

    Conrad, A. H.; Stephens, A. P.; Paulsen, A. Q.; Schwarting, S. S.; Conrad, G. W.; Spooner, B. S. (Principal Investigator)

    1994-01-01

    The terminal phase of cell division involves tight constriction of the cleavage furrow contractile ring, stabilization/elongation of the intercellular bridge, and final separation of the daughter cells. At first cleavage, the fertilized eggs of the mollusk, Ilyanassa obsoleta, form two contractile rings at right angles to each other in the same cytoplasm that constrict to tight necks and partition the egg into a trefoil shape. The cleavage furrow contractile ring (CF) normally constricts around many midbody microtubules (MTs) and results in cleavage; the polar lobe constriction contractile ring (PLC) normally constricts around very few MTs and subsequently relaxes without cleavage. In the presence of Ag+ ions, the PLC 1) begins MT-dependent rapid constriction sooner than controls, 2) encircles more MTs than control egg PLCs, 3) elongates much more than control PLCs, and 4) remains tightly constricted and effectively cleaves the polar lobe from the egg. If Ag(+)-incubated eggs are returned to normal seawater at trefoil, tubulin fluorescence disappears from the PLC neck and the neck relaxes. If nocodazole, a drug that depolymerizes MTs, is added to Ag(+)-incubated eggs during early PLC constriction, the PLC is not stabilized and eventually relaxes. However, if nocodazole is added to Ag(+)-incubated eggs at trefoil, tubulin fluorescence disappears from the PLC neck but the neck remains constricted. These results suggest that Ag+ accelerates and gradually stabilizes the PLC constriction by a mechanism that is initially MT-dependent, but that progressively becomes MT-independent.

  6. 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.

  7. 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

  8. Specificity of the proteasome cleavage to the antigen protein

    Institute of Scientific and Technical Information of China (English)

    2009-01-01

    In the MHC classⅠmolecule binding antigenic peptides processing and presentation pathway,the ubiquitin-proteasome system plays a key role in degrading the protein substrate.For the purpose of studying the specificities of proteasomal cleavage sites,partial least squares method is used to predict the proteasomal cleavage sites,and the predictive accuracy of the model is 82.8%.The specificities of the cleavage sites and the adjacent positions come from the contribution of the amino acids of the samples to the cleavage sites,showing the information of proteasome interacting with antigen protein.It demonstrates that the proteasome cleaving to target protein is selective,but not random.

  9. Synthesis and Cleavage Activity of Artifical Minic Polypeptides

    Institute of Scientific and Technical Information of China (English)

    Yong YE; Xiao Lian HU; Ping LI; Ming Yu NIU; Li Feng CAO; Yu Fen ZHAO

    2006-01-01

    Two artificial minic polypeptides which are synthetic analogues of natural products with DNA affinity were synthesized, and theirs cleavage activity with DNA were examined. The structures of these compounds was confirmed by 1H NMR, MS and IR.

  10. 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...

  11. Observation of amyloid precursor protein cleavage and Aβ generation in living cells by using multiphoton laser scanning microscopy%多光子激光扫描成像技术对活细胞内淀粉样前体蛋白裂解和β-淀粉样蛋白生成的观察

    Institute of Scientific and Technical Information of China (English)

    李晓晴; 张苏明; 杨华静; 张智红

    2007-01-01

    Objective To investigate the proteolytic mechanism of amyloid precursor protein (APP) and to explore amyloidbeta (Aβ) generation in living neurons. Methods DNA fragments were amplified by PCR or synthesized. The four fragments- CFP- 54bp- YFP and C99 were ligated into pcDNAS.O vector to construct the recombinant plasmids pcDNA3.0-CFP-54bp-YFP and pcDNA3.0-CFP-54bp-YFP-C99. The SH-SY5Y cells were transiently transfected with pcDNA3.0-CFP-54bp-YFP or pcDNA3.0-CFP-54bp-YFP-C99.The SH-SY5Y cells were transiently transfected with pcDNA3.0-CFP-54bp-YFP or pcDNA3.0-CFP-54bp-YFP-C99.The expression of fusion gene was examined under a multiphoton laser scanning microscope.Fluorescence resonance energy transfer (FRET) was used to measure the p cleavage and y cleavage of APP.Aβ generation was confirmed by immunocytochemistry and multiphoton laser scanning microscopy.Cell viability was tested by MTT assay at different time points.Results (1) The double restriction endonuclease digestion and sequencing analysis confirmed the authenticity of the recombinant plasmids pcDNA3.0-CFP-54bp-YFP and pcDNA3.0-CFP-54bp-YFP-C99.(2) Blue and yellow fluorescences were detected in the transfected cells.(3) FRET occurred in pcDNA3.0-CFP-54bp-YFP-transfected cells but not in pcDNA3.0-CFP-54bp-YFP-C99-transfected cells.(4) Aβ was produced in the pcDNA3.0-CFP-54bp-YFP-C99 transfected cells.(5) Aβ-deposition was widespread in the cell.(6) Cell viability decreased along with the intracellular Aβ deposition.Conclusion C99 is important for the APP β cleavage.Aβ may be generated and deposited in cells at the early stage of Alzheimer's disease.Intracellular Aβ accumulation brings deleterious effects on cells.%目的 在活细胞内探究淀粉样前体蛋白(amyloid precursor protein,APP)的裂解和β-淀粉样蛋白(amyloid beta,Aβ)的生成机制.方法 利用PCR扩增CFP(编码蓝色荧光蛋白),YFP(编码黄色荧光蛋白)和C99(编码APP最后99个氨基酸)三片段.含有54

  12. Cleavage of a specific bond in troponin C by thrombin.

    Science.gov (United States)

    Leavis, P C; Rosenfeld, S; Lu, R C

    1978-08-21

    Limited proteolysis of rabbit skeletal troponin C with bovine thrombin yielded two fragments, TH1 (Mr = 11000) containing Ca2+ binding regions I--III and TH2 (Mr = 6000) containing region IV. Determination of the partial sequences of the fragments established the site of cleavage at Arg120-Ala121. Secondary cleavage by thrombin at other arginyl or lysyl residues in troponin C was ruled out by the sequence data and by the amino acid compositions of the two fragments.

  13. 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.

  14. 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

  15. The effects of proteasome inhibitor lactacystin on mouse oocyte meiosis and first cleavage

    Institute of Scientific and Technical Information of China (English)

    TAN Xin; PENG An; WANG Yongchao; TANG Zuoqing

    2005-01-01

    In order to study the effects of ubiquitin-proteasome pathway (UPP) on mouse oocyte meiosis and cleavage, oocytes undergoing maturation and parthenogenetic activation and 1-cell embryos were treated with lactacystin, a specific inhibitor of proteasome. The results indicared that the rate of GVBD was not influenced by the treatment, but polar body extrusion, parthenogenesis and first cleavage were inhibited. Immunofluorescent staining using anti β-tubulin antibody indicated that the continuous treatment of lactacystin from GV stage disorganized microtubules and spindle assembly. When metaphase stage oocytes were treated with the drug,the already formed spindle structure was not affected, but the oocytes were arrested at metaphases. The 1-cell embryos were arrested at interphase or metaphase of first mitosis when they were incubated in the drug. Proteasome regulatory subunit PA700 was located in the spindle region, as indicated by immunofluorescence. These results suggest that UPP has effects on the process of oocyte meiosis and early cleavage in many aspects, including normal organization of spindle at prophase and segregation of chromosomes at anaphase for normal meiosis.

  16. Active site specificity profiling of the matrix metalloproteinase family: Proteomic identification of 4300 cleavage sites by nine MMPs explored with structural and synthetic peptide cleavage analyses.

    Science.gov (United States)

    Eckhard, Ulrich; Huesgen, Pitter F; Schilling, Oliver; Bellac, Caroline L; Butler, Georgina S; Cox, Jennifer H; Dufour, Antoine; Goebeler, Verena; Kappelhoff, Reinhild; Keller, Ulrich Auf dem; Klein, Theo; Lange, Philipp F; Marino, Giada; Morrison, Charlotte J; Prudova, Anna; Rodriguez, David; Starr, Amanda E; Wang, Yili; Overall, Christopher M

    2016-01-01

    Secreted and membrane tethered matrix metalloproteinases (MMPs) are key homeostatic proteases regulating the extracellular signaling and structural matrix environment of cells and tissues. For drug targeting of proteases, selectivity for individual molecules is highly desired and can be met by high yield active site specificity profiling. Using the high throughput Proteomic Identification of protease Cleavage Sites (PICS) method to simultaneously profile both the prime and non-prime sides of the cleavage sites of nine human MMPs, we identified more than 4300 cleavages from P6 to P6' in biologically diverse human peptide libraries. MMP specificity and kinetic efficiency were mainly guided by aliphatic and aromatic residues in P1' (with a ~32-93% preference for leucine depending on the MMP), and basic and small residues in P2' and P3', respectively. A wide differential preference for the hallmark P3 proline was found between MMPs ranging from 15 to 46%, yet when combined in the same peptide with the universally preferred P1' leucine, an unexpected negative cooperativity emerged. This was not observed in previous studies, probably due to the paucity of approaches that profile both the prime and non-prime sides together, and the masking of subsite cooperativity effects by global heat maps and iceLogos. These caveats make it critical to check for these biologically highly important effects by fixing all 20 amino acids one-by-one in the respective subsites and thorough assessing of the inferred specificity logo changes. Indeed an analysis of bona fide MEROPS physiological substrate cleavage data revealed that of the 37 natural substrates with either a P3-Pro or a P1'-Leu only 5 shared both features, confirming the PICS data. Upon probing with several new quenched-fluorescent peptides, rationally designed on our specificity data, the negative cooperativity was explained by reduced non-prime side flexibility constraining accommodation of the rigidifying P3 proline with

  17. Identification of off-target cleavage sites of zinc finger nucleases and TAL effector nucleases using predictive models.

    Science.gov (United States)

    Fine, Eli J; Cradick, Thomas J; Bao, Gang

    2014-01-01

    Using engineered nucleases, such as Zinc Finger Nucleases (ZFNs) or Transcription Activator-Like Effector Nucleases (TALENs), to make targeted genomic modifications has become a common technique to create new model organisms and custom cell lines, and has shown great promise for disease treatment. However, these nucleases have the potential for off-target cleavage that could confound interpretation of experimental results and be detrimental for therapeutic use. Here, we describe a method to test for nuclease cleavage at potential off-target sites predicted by bioinformatics models.

  18. 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...

  19. 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.

  20. Foot-and-mouth disease virus leader proteinase: structural insights into the mechanism of intermolecular cleavage.

    Science.gov (United States)

    Steinberger, Jutta; Grishkovskaya, Irina; Cencic, Regina; Juliano, Luiz; Juliano, Maria A; Skern, Tim

    2014-11-01

    Translation of foot-and-mouth disease virus RNA initiates at one of two start codons leading to the synthesis of two forms of leader proteinase L(pro) (Lab(pro) and Lb(pro)). These forms free themselves from the viral polyprotein by intra- and intermolecular self-processing and subsequently cleave the cellular eukaryotic initiation factor (eIF) 4 G. During infection, Lb(pro) removes six residues from its own C-terminus, generating sLb(pro). We present the structure of sLb(pro) bound to the inhibitor E64-R-P-NH2, illustrating how sLb(pro) can cleave between Lys/Gly and Gly/Arg pairs. In intermolecular cleavage on polyprotein substrates, Lb(pro) was unaffected by P1 or P1' substitutions and processed a substrate containing nine eIF4GI cleavage site residues whereas sLb(pro) failed to cleave the eIF4GI containing substrate and cleaved appreciably more slowly on mutated substrates. Introduction of 70 eIF4GI residues bearing the Lb(pro) binding site restored cleavage. These data imply that Lb(pro) and sLb(pro) may have different functions in infected cells.

  1. In vitro proteolytic cleavage of Gazdar murine sarcoma virus p65gag.

    Science.gov (United States)

    Maxwell, S; Arlinghaus, R B

    1981-09-01

    Moloney murine leukemia virus, disrupted in concentrations of 0.1 to 0.5% Nonidet P-40, catalyzed the cleavage of p65, the gag gene polyprotein of the Gazdar strain of murine sarcoma virus, into polypeptides with sizes and antigenic determinants of murine leukemia virus-specified p30, p15, pp12, and p10. Cleavage performed in the presence of 0.15% Nonidet P-40 in water yielded polypeptides of approximately 40,000 (P40) and 25,000 (P25) Mr. In vitro cleavage performed in a buffered solution containing dithiothreitol in addition to 0.1% Nonidet P-40 allowed the efficient processing of P40 to p30 and a band migrating with p10. Immunoprecipitation with monospecific sera indicated that P40 contained p30 and p10, whereas P25 contained p15 and pp12 determinants. P40 and P25 are similar in size and antigenic properties to Pr40gag and Pr25gag observed in infected cells (Naso et al, J. Virol. 32:187-198, 1979).

  2. Cleavage of CXCR1 on neutrophils disables bacterial killing in cystic fibrosis lung disease.

    Science.gov (United States)

    Hartl, Dominik; Latzin, Philipp; Hordijk, Peter; Marcos, Veronica; Rudolph, Carsten; Woischnik, Markus; Krauss-Etschmann, Susanne; Koller, Barbara; Reinhardt, Dietrich; Roscher, Adelbert A; Roos, Dirk; Griese, Matthias

    2007-12-01

    Interleukin-8 (IL-8) activates neutrophils via the chemokine receptors CXCR1 and CXCR2. However, the airways of individuals with cystic fibrosis are frequently colonized by bacterial pathogens, despite the presence of large numbers of neutrophils and IL-8. Here we show that IL-8 promotes bacterial killing by neutrophils through CXCR1 but not CXCR2. Unopposed proteolytic activity in the airways of individuals with cystic fibrosis cleaved CXCR1 on neutrophils and disabled their bacterial-killing capacity. These effects were protease concentration-dependent and also occurred to a lesser extent in individuals with chronic obstructive pulmonary disease. Receptor cleavage induced the release of glycosylated CXCR1 fragments that were capable of stimulating IL-8 production in bronchial epithelial cells via Toll-like receptor 2. In vivo inhibition of proteases by inhalation of alpha1-antitrypsin restored CXCR1 expression and improved bacterial killing in individuals with cystic fibrosis. The cleavage of CXCR1, the functional consequences of its cleavage, and the identification of soluble CXCR1 fragments that behave as bioactive components represent a new pathophysiologic mechanism in cystic fibrosis and other chronic lung diseases.

  3. The DNA cleavage reaction of topoisomerase II: wolf in sheep's clothing.

    Science.gov (United States)

    Deweese, Joseph E; Osheroff, Neil

    2009-02-01

    Topoisomerase II is an essential enzyme that is required for virtually every process that requires movement of DNA within the nucleus or the opening of the double helix. This enzyme helps to regulate DNA under- and overwinding and removes knots and tangles from the genetic material. In order to carry out its critical physiological functions, topoisomerase II generates transient double-stranded breaks in DNA. Consequently, while necessary for cell survival, the enzyme also has the capacity to fragment the genome. The DNA cleavage/ligation reaction of topoisomerase II is the target for some of the most successful anticancer drugs currently in clinical use. However, this same reaction also is believed to trigger chromosomal translocations that are associated with specific types of leukemia. This article will familiarize the reader with the DNA cleavage/ligation reaction of topoisomerase II and other aspects of its catalytic cycle. In addition, it will discuss the interaction of the enzyme with anticancer drugs and the mechanisms by which these agents increase levels of topoisomerase II-generated DNA strand breaks. Finally, it will describe dietary and environmental agents that enhance DNA cleavage mediated by the enzyme.

  4. Cleavage entropy as quantitative measure of protease specificity.

    Directory of Open Access Journals (Sweden)

    Julian E Fuchs

    2013-04-01

    Full Text Available A purely information theory-guided approach to quantitatively characterize protease specificity is established. We calculate an entropy value for each protease subpocket based on sequences of cleaved substrates extracted from the MEROPS database. We compare our results with known subpocket specificity profiles for individual proteases and protease groups (e.g. serine proteases, metallo proteases and reflect them quantitatively. Summation of subpocket-wise cleavage entropy contributions yields a measure for overall protease substrate specificity. This total cleavage entropy allows ranking of different proteases with respect to their specificity, separating unspecific digestive enzymes showing high total cleavage entropy from specific proteases involved in signaling cascades. The development of a quantitative cleavage entropy score allows an unbiased comparison of subpocket-wise and overall protease specificity. Thus, it enables assessment of relative importance of physicochemical and structural descriptors in protease recognition. We present an exemplary application of cleavage entropy in tracing substrate specificity in protease evolution. This highlights the wide range of substrate promiscuity within homologue proteases and hence the heavy impact of a limited number of mutations on individual substrate specificity.

  5. Cleavage entropy as quantitative measure of protease specificity.

    Science.gov (United States)

    Fuchs, Julian E; von Grafenstein, Susanne; Huber, Roland G; Margreiter, Michael A; Spitzer, Gudrun M; Wallnoefer, Hannes G; Liedl, Klaus R

    2013-04-01

    A purely information theory-guided approach to quantitatively characterize protease specificity is established. We calculate an entropy value for each protease subpocket based on sequences of cleaved substrates extracted from the MEROPS database. We compare our results with known subpocket specificity profiles for individual proteases and protease groups (e.g. serine proteases, metallo proteases) and reflect them quantitatively. Summation of subpocket-wise cleavage entropy contributions yields a measure for overall protease substrate specificity. This total cleavage entropy allows ranking of different proteases with respect to their specificity, separating unspecific digestive enzymes showing high total cleavage entropy from specific proteases involved in signaling cascades. The development of a quantitative cleavage entropy score allows an unbiased comparison of subpocket-wise and overall protease specificity. Thus, it enables assessment of relative importance of physicochemical and structural descriptors in protease recognition. We present an exemplary application of cleavage entropy in tracing substrate specificity in protease evolution. This highlights the wide range of substrate promiscuity within homologue proteases and hence the heavy impact of a limited number of mutations on individual substrate specificity.

  6. A strategy for developing a hammerhead ribozyme for selective RNA cleavage depending on substitutional RNA editing.

    Science.gov (United States)

    Fukuda, Masatora; Kurihara, Kei; Tanaka, Yasuyoshi; Deshimaru, Masanobu

    2012-09-01

    Substitutional RNA editing plays a crucial role in the regulation of biological processes. Cleavage of target RNA that depends on the specific site of substitutional RNA editing is a useful tool for analyzing and regulating intracellular processes related to RNA editing. Hammerhead ribozymes have been utilized as small catalytic RNAs for cleaving target RNA at a specific site and may be used for RNA-editing-specific RNA cleavage. Here we reveal a design strategy for a hammerhead ribozyme that specifically recognizes adenosine to inosine (A-to-I) and cytosine to uracil (C-to-U) substitutional RNA-editing sites and cleaves target RNA. Because the hammerhead ribozyme cleaves one base upstream of the target-editing site, the base that pairs with the target-editing site was utilized for recognition. RNA-editing-specific ribozymes were designed such that the recognition base paired only with the edited base. These ribozymes showed A-to-I and C-to-U editing-specific cleavage activity against synthetic serotonin receptor 2C and apolipoprotein B mRNA fragments in vitro, respectively. Additionally, the ribozyme designed for recognizing A-to-I RNA editing at the Q/R site on filamin A (FLNA) showed editing-specific cleavage activity against physiologically edited FLNA mRNA extracted from cells. We demonstrated that our strategy is effective for cleaving target RNA in an editing-dependent manner. The data in this study provided an experimental basis for the RNA-editing-dependent degradation of specific target RNA in vivo.

  7. 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.

  8. Copper-obatoclax derivative complexes mediate DNA cleavage and exhibit anti-cancer effects in hepatocellular carcinoma.

    Science.gov (United States)

    Su, Jung-Chen; Chang, Jung-Hua; Huang, Jui-Wen; Chen, Peter P-Y; Chen, Kuen-Feng; Tseng, Ping-Hui; Shiau, Chung-Wai

    2015-02-25

    Obatoclax is an indole-pyrrole compound that induces cancer cell apoptosis through targeting the anti-apoptotic Bcl-2 protein family. Previously, we developed a series of obatoclax derivatives and studied their STAT3 inhibition-dependent activity against cancer cell lines. The obatoclax analog, prodigiosin, has been reported to mediate DNA cleavage in cancer cells by coordinating with copper complexes. To gain an understanding of copper-obatoclax complex activity, we applied obatoclax derivatives to examine their copper-mediated nuclease activity as a means to establish a basis for structure activity relationship. Replacement of the indole ring of obatoclax with furanyl, thiophenyl or Boc-indolyl rings reduced the DNA cleavage ability. The same effect was achieved through the replacement of the obatoclax pyrrolyl ring with thiazolidinedione and thioacetal. Among the compounds tested, we demonstrated that the complex of obatoclax or compound 7 with copper exhibited potent DNA strand scission which correlated with HCC cell growth inhibition.

  9. 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.

  10. 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.

  11. Cleavage of Poly(A)-binding protein by coxsackievirus 2A protease in vitro and in vivo: another mechanism for host protein synthesis shutoff?

    Science.gov (United States)

    Kerekatte, V; Keiper, B D; Badorff, C; Cai, A; Knowlton, K U; Rhoads, R E

    1999-01-01

    Infection of cells by picornaviruses of the rhinovirus, aphthovirus, and enterovirus groups results in the shutoff of host protein synthesis but allows viral protein synthesis to proceed. Although considerable evidence suggests that this shutoff is mediated by the cleavage of eukaryotic translation initiation factor eIF4G by sequence-specific viral proteases (2A protease in the case of coxsackievirus), several experimental observations are at variance with this view. Thus, the cleavage of other cellular proteins could contribute to the shutoff of host protein synthesis and stimulation of viral protein synthesis. Recent evidence indicates that the highly conserved 70-kDa cytoplasmic poly(A)-binding protein (PABP) participates directly in translation initiation. We have now found that PABP is also proteolytically cleaved during coxsackievirus infection of HeLa cells. The cleavage of PABP correlated better over time with the host translational shutoff and onset of viral protein synthesis than did the cleavage of eIF4G. In vitro experiments with purified rabbit PABP and recombinant human PABP as well as in vivo experiments with Xenopus oocytes and recombinant Xenopus PABP demonstrate that the cleavage is catalyzed by 2A protease directly. N- and C-terminal sequencing indicates that cleavage occurs uniquely in human PABP at 482VANTSTQTM downward arrowGPRPAAAAAA500, separating the four N-terminal RNA recognition motifs (80%) from the C-terminal homodimerization domain (20%). The N-terminal cleavage product of PABP is less efficient than full-length PABP in restoring translation to a PABP-dependent rabbit reticulocyte lysate translation system. These results suggest that the cleavage of PABP may be another mechanism by which picornaviruses alter the rate and spectrum of protein synthesis.

  12. 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; Lee, Pyung Cheon

    2013-06-01

    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-13 C-14, C-15 C-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.

  13. 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...

  14. Meganuclease-mediated virus self-cleavage facilitates tumor-specific virus replication.

    Science.gov (United States)

    Gürlevik, Engin; Schache, Peter; Goez, Anneliese; Kloos, Arnold; Woller, Norman; Armbrecht, Nina; Manns, Michael P; Kubicka, Stefan; Kühnel, Florian

    2013-09-01

    Meganucleases can specifically cleave long DNA sequence motifs, a feature that makes them an ideal tool for gene engineering in living cells. In a proof-of-concept study, we investigated the use of the meganuclease I-Sce I for targeted virus self-disruption to generate high-specific oncolytic viruses. For this purpose, we provided oncolytic adenoviruses with a molecular circuit that selectively responds to p53 activation by expression of I-Sce I subsequently leading to self-disruption of the viral DNA via heterologous I-Sce I recognition sites within the virus genome. We observed that virus replication and cell lysis was effectively impaired in p53-normal cells, but not in p53-dysfunctional tumor cells. I-Sce I activity led to effective intracellular processing of viral DNA as confirmed by detection of specific cleavage products. Virus disruption did not interfere with E1A levels indicating that reduction of functional virus genomes was the predominant cause for conditional replication. Consequently, tumor-specific replication was further enhanced when E1A expression was additionally inhibited by targeted transcriptional repression. Finally, we demonstrated p53-dependent oncolysis by I-Sce I-expressing viruses in vitro and in vivo, and demonstrated effective inhibition of tumor growth. In summary, meganuclease-mediated virus cleavage represents a promising approach to provide oncolytic viruses with attractive safety profiles.

  15. Effects of S1 Cleavage on the Structure, Surface Export, and Signaling Activity of Human Notch1 and Notch2

    Science.gov (United States)

    Gordon, Wendy R.; Vardar-Ulu, Didem; L'Heureux, Sarah; Ashworth, Todd; Malecki, Michael J.; Sanchez-Irizarry, Cheryll; McArthur, Debbie G.; Histen, Gavin; Mitchell, Jennifer L.; Aster, Jon C.; Blacklow, Stephen C.

    2009-01-01

    Background Notch receptors are normally cleaved during maturation by a furin-like protease at an extracellular site termed S1, creating a heterodimer of non-covalently associated subunits. The S1 site lies within a key negative regulatory region (NRR) of the receptor, which contains three highly conserved Lin12/Notch repeats and a heterodimerization domain (HD) that interact to prevent premature signaling in the absence of ligands. Because the role of S1 cleavage in Notch signaling remains unresolved, we investigated the effect of S1 cleavage on the structure, surface trafficking and ligand-mediated activation of human Notch1 and Notch2, as well as on ligand-independent activation of Notch1 by mutations found in human leukemia. Principal Findings The X-ray structure of the Notch1 NRR after furin cleavage shows little change when compared with that of an engineered Notch1 NRR lacking the S1-cleavage loop. Likewise, NMR studies of the Notch2 HD domain show that the loop containing the S1 site can be removed or cleaved without causing a substantial change in its structure. However, Notch1 and Notch2 receptors engineered to resist S1 cleavage exhibit unexpected differences in surface delivery and signaling competence: S1-resistant Notch1 receptors exhibit decreased, but detectable, surface expression and ligand-mediated receptor activation, whereas S1-resistant Notch2 receptors are fully competent for cell surface delivery and for activation by ligands. Variable dependence on S1 cleavage also extends to T-ALL-associated NRR mutations, as common class 1 mutations display variable decrements in ligand-independent activation when introduced into furin-resistant receptors, whereas a class 2 mutation exhibits increased signaling activity. Conclusions/Significance S1 cleavage has distinct effects on the surface expression of Notch1 and Notch2, but is not generally required for physiologic or pathophysiologic activation of Notch proteins. These findings are consistent with

  16. Effects of S1 cleavage on the structure, surface export, and signaling activity of human Notch1 and Notch2.

    Directory of Open Access Journals (Sweden)

    Wendy R Gordon

    Full Text Available Notch receptors are normally cleaved during maturation by a furin-like protease at an extracellular site termed S1, creating a heterodimer of non-covalently associated subunits. The S1 site lies within a key negative regulatory region (NRR of the receptor, which contains three highly conserved Lin12/Notch repeats and a heterodimerization domain (HD that interact to prevent premature signaling in the absence of ligands. Because the role of S1 cleavage in Notch signaling remains unresolved, we investigated the effect of S1 cleavage on the structure, surface trafficking and ligand-mediated activation of human Notch1 and Notch2, as well as on ligand-independent activation of Notch1 by mutations found in human leukemia.The X-ray structure of the Notch1 NRR after furin cleavage shows little change when compared with that of an engineered Notch1 NRR lacking the S1-cleavage loop. Likewise, NMR studies of the Notch2 HD domain show that the loop containing the S1 site can be removed or cleaved without causing a substantial change in its structure. However, Notch1 and Notch2 receptors engineered to resist S1 cleavage exhibit unexpected differences in surface delivery and signaling competence: S1-resistant Notch1 receptors exhibit decreased, but detectable, surface expression and ligand-mediated receptor activation, whereas S1-resistant Notch2 receptors are fully competent for cell surface delivery and for activation by ligands. Variable dependence on S1 cleavage also extends to T-ALL-associated NRR mutations, as common class 1 mutations display variable decrements in ligand-independent activation when introduced into furin-resistant receptors, whereas a class 2 mutation exhibits increased signaling activity.S1 cleavage has distinct effects on the surface expression of Notch1 and Notch2, but is not generally required for physiologic or pathophysiologic activation of Notch proteins. These findings are consistent with models for receptor activation in which

  17. Effects of S1 Cleavage on the Structure, Surface Export, and Signaling Activity of Human Notch1 and Notch2

    Energy Technology Data Exchange (ETDEWEB)

    Gordon, Wendy R.; Vardar-Ulu, Didem; L' Heureux, Sarah; Ashworth, Todd; Malecki, Michael J.; Sanchez-Irizarry, Cheryll; McArthur, Debbie G.; Histen, Gavin; Mitchell, Jennifer L.; Aster, Jon C.; Blacklow, Stephen C.; (BWH); (Wellesley)

    2009-09-25

    Notch receptors are normally cleaved during maturation by a furin-like protease at an extracellular site termed S1, creating a heterodimer of non-covalently associated subunits. The S1 site lies within a key negative regulatory region (NRR) of the receptor, which contains three highly conserved Lin12/Notch repeats and a heterodimerization domain (HD) that interact to prevent premature signaling in the absence of ligands. Because the role of S1 cleavage in Notch signaling remains unresolved, we investigated the effect of S1 cleavage on the structure, surface trafficking and ligand-mediated activation of human Notch1 and Notch2, as well as on ligand-independent activation of Notch1 by mutations found in human leukemia. The X-ray structure of the Notch1 NRR after furin cleavage shows little change when compared with that of an engineered Notch1 NRR lacking the S1-cleavage loop. Likewise, NMR studies of the Notch2 HD domain show that the loop containing the S1 site can be removed or cleaved without causing a substantial change in its structure. However, Notch1 and Notch2 receptors engineered to resist S1 cleavage exhibit unexpected differences in surface delivery and signaling competence: S1-resistant Notch1 receptors exhibit decreased, but detectable, surface expression and ligand-mediated receptor activation, whereas S1-resistant Notch2 receptors are fully competent for cell surface delivery and for activation by ligands. Variable dependence on S1 cleavage also extends to T-ALL-associated NRR mutations, as common class 1 mutations display variable decrements in ligand-independent activation when introduced into furin-resistant receptors, whereas a class 2 mutation exhibits increased signaling activity. S1 cleavage has distinct effects on the surface expression of Notch1 and Notch2, but is not generally required for physiologic or pathophysiologic activation of Notch proteins. These findings are consistent with models for receptor activation in which ligand-binding or

  18. 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...

  19. The syntheses, characterization, antimicrobial, DNA cleavage and cytotoxic activities of novel terephthalato complexes

    Science.gov (United States)

    Yıldız, Özge; Çolak, Alper Tolga; Yılmaz, Murat; İça, Tuba; Oztopcu-Vatan, Pinar; Topaloǧlu, Emel; Çolak, Ferdaǧ

    2017-01-01

    [Cu(tp)(dmpd)2] (1), [Cu(tp)(pen)2] (2), [Cu(tp)(dmen)2] (3), [Cu(tp)(deen)2]·4H2O (4), [Cu(tp)(mpen)2]·2H2O (5) and [Cu(tp)(amp)2]·3H2O (6) (H2tp = Benzene-1,4-dicarboxylic acid or terephthalic acid, dmpd = 2,2-dimethyl-1,3-propanediamine, pen = 1,3-propanediamine, dmen = N,N-dimethylethylenediamine, deen = N,N-diethylethylenediamine, mpen = N-methyl-1,3-propanediamine and amp = 2-aminomethylpyridine) were synthesised and characterised by elemental analysis, spectroscopic measurements (UV-vis. and FT-IR spectra) and thermal analysis technique. These complexes have been screened for antimicrobial activities and DNA cleavage. Antimicrobial activity of compounds 1-6 were evaluated by the agar diffusion method. The DNA cleavage activities of the complexes were evaluated by agarose gel electrophoresis. In addition, cytotoxic activities of all complexes were performed prostate carcinoma cells LNCaP and DU145 by MTT assay for 24 and 48 h. Especially after 24 h treatment, 6 complex increased the apoptotic and necrotic cell death in both cell lines in a concentration dependent manner. Particularly, 6 complex good show antimicrobial, nuclease and cytotoxic activity.

  20. Tyrosine-phosphorylated Galectin-3 Protein Is Resistant to Prostate-specific Antigen (PSA) Cleavage*

    Science.gov (United States)

    Balan, Vitaly; Nangia-Makker, Pratima; Kho, Dhong Hyo; Wang, Yi; Raz, Avraham

    2012-01-01

    Galectin-3 is a chimeric carbohydrate-binding protein, which interacts with cell surface carbohydrate-containing molecules and extracellular matrix glycoproteins and has been implicated in various biological processes such as cell growth, angiogenesis, motility, and metastasis. It is expressed in a wide range of tumor cells and is associated with tumor progression. The functions of galectin-3 are dependent on its localization and post-translational modifications such as cleavage and phosphorylation. Recently, we showed that galectin-3 Tyr-107 is phosphorylated by c-Abl; concomitantly, it was also shown that galectin-3 can be cleaved at this site by prostate-specific antigen (PSA), a chymotrypsin-like serine protease, after Tyr-107, resulting in loss of galectin-3 multivalency while preserving its carbohydrate binding activity. Galectin-3 is largely a monomer in solution but may form a homodimer by self-association through its carbohydrate recognition domain, whereas, in the presence of a ligand, galectin-3 polymerizes up to pentamers utilizing its N-terminal domain. Oligomerization is a unique feature of secreted galectin-3, which allows its function by forming ordered galectin-glycan structures, i.e. lattices, on the cell surface or through direct engagement of specific cell surface glycoconjugates by traditional ligand-receptor binding. We questioned whether Tyr-107 phosphorylation by c-Abl affects galectin-3 cleavage by PSA. The data suggest a role for galectin-3 in prostate cells associated with increased activity of c-Abl kinase and loss of phosphatase and tensin homologue deleted on chromosome 10 (PTEN) activity. In addition, the ratio of phosphorylated/dephosphorylated galectin-3 might be used as a complementary value to that of PSA for prognosis of prostate cancer and a novel therapeutic target for the treatment of prostate cancer. PMID:22232548

  1. Specificity of human rhinovirus 2A(pro) is determined by combined spatial properties of four cleavage site residues.

    Science.gov (United States)

    Neubauer, David; Aumayr, Martina; Gösler, Irene; Skern, Tim

    2013-07-01

    The 2A proteinase (2A(pro)) of human rhinoviruses cleaves the virally encoded polyprotein between the C terminus of VP1 and its own N terminus. Poor understanding of the 2A(pro) substrate specificity of this enzyme has hampered progress in developing inhibitors that may serve as antiviral agents. We show here that the 2A(pro) of human rhinovirus (HRV) 1A and 2 (rhinoviruses from genetic group A) cannot self-process at the HRV14 (a genetic group B rhinovirus) cleavage site. When the amino acids in the cleavage site of HRV2 2A(pro) (Ile-Ile-Thr-Thr-Ala*Gly-Pro-Ser-Asp) were singly or doubly replaced with the corresponding HRV14 residues (Asp-Ile-Lys-Ser-Tyr*Gly-Leu-Gly-Pro) at positions from P3 to P2', HRV1A and HRV2 2A(pro) cleavage took place at WT levels. However, when three or more positions of the HRV1A or 2 2A(pro) were substituted (e.g. at P2, P1 and P2'), cleavage in vitro was essentially eliminated. Introduction of the full HRV14 cleavage site into a full-length clone of the HRV1A and transfection of HeLa cells with a transcribed RNA did not give rise to viable virus. In contrast, revertant viruses bearing cysteine at the P1 position or proline at P2' were obtained when an RNA bearing the three inhibitory amino acids was transfected. Reversions in the enzyme affecting substrate specificity were not found in any of the in vivo experiments. Modelling of oligopeptide substrates onto the structure of HRV2 2A(pro) revealed no appreciable differences in residues of HRV2 and HRV14 in the respective substrate binding sites, suggesting that the overall shape of the substrate is important in determining binding efficiency.

  2. Effect of multifunctional protein YB-1 on the AP site cleavage by AP endonuclease 1 and tyrosyl phosphodiesterase 1

    Directory of Open Access Journals (Sweden)

    Ovchinnikov L. P.

    2012-07-01

    Full Text Available Apurinic/apyrimidinic sites (AP sites which represent one of the most abundantly generated DNA lesions in the cell are generally repaired by base excision repair (BER pathway. Multifunctional protein YB-1 is known to participate in cellular response to genotoxic stress and was shown to interact with several components of BER – DNA glycosylases NTH1, NEIL2, DNA polymerase and DNA ligase III. Therefore, it is of great interest to investigate the influence of YB-1 on one of the major BER enzymes, responsible for AP site cleavage, AP endonuclease APE1, and on tyrosyl phosphodiesterase Tdp1, participating in APE1 independent pathway of AP site repair. Aim. Effect of multifunctional protein YB-1 on the AP site cleavage by the activities of APE1 and Tdp1 was studied. Methods. Gel-mobility shift assays and enzyme activity tests. Results. YB-1 was shown to inhibit the cleavage of AP site located in single-stranded DNA by both APE1 and Tdp1. Stimulation of APE1 activity on protruding double-stranded DNA in the presence of YB-1 was observed, whereas no effect on Tdp1-mediated cleavage of AP site in double-stranded DNA was found. Conclusions. YB-1 can modulate the repair of AP sites in DNA by both positively stimulating APE1 during the classic BER of AP sites and avoiding a possible generation of doublestrand breaks, arising from the cleavage of single-stranded portion of DNA substrate already used by different DNA-processing pathway

  3. 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 ...

  4. Two Antagonistic MALT1 Auto-Cleavage Mechanisms Reveal a Role for TRAF6 to Unleash MALT1 Activation.

    Science.gov (United States)

    Ginster, Stefanie; Bardet, Maureen; Unterreiner, Adeline; Malinverni, Claire; Renner, Florian; Lam, Stephen; Freuler, Felix; Gerrits, Bertran; Voshol, Johannes; Calzascia, Thomas; Régnier, Catherine H; Renatus, Martin; Nikolay, Rainer; Israël, Laura; Bornancin, Frédéric

    2017-01-01

    The paracaspase MALT1 has arginine-directed proteolytic activity triggered by engagement of immune receptors. Recruitment of MALT1 into activation complexes is required for MALT1 proteolytic function. Here, co-expression of MALT1 in HEK293 cells, either with activated CARD11 and BCL10 or with TRAF6, was used to explore the mechanism of MALT1 activation at the molecular level. This work identified a prominent self-cleavage site of MALT1 isoform A (MALT1A) at R781 (R770 in MALT1B) and revealed that TRAF6 can activate MALT1 independently of the CBM. Intramolecular cleavage at R781/R770 removes a C-terminal TRAF6-binding site in both MALT1 isoforms, leaving MALT1B devoid of the two key interaction sites with TRAF6. A previously identified auto-proteolysis site of MALT1 at R149 leads to deletion of the death-domain, thereby abolishing interaction with BCL10. By using MALT1 isoforms and cleaved fragments thereof, as well as TRAF6 WT and mutant forms, this work shows that TRAF6 induces N-terminal auto-proteolytic cleavage of MALT1 at R149 and accelerates MALT1 protein turnover. The MALT1 fragment generated by N-terminal self-cleavage at R149 was labile and displayed enhanced signaling properties that required an intact K644 residue, previously shown to be a site for mono-ubiquitination of MALT1. Conversely, C-terminal self-cleavage at R781/R770 hampered the ability for self-cleavage at R149 and stabilized MALT1 by hindering interaction with TRAF6. C-terminal self-cleavage had limited impact on MALT1A but severely reduced MALT1B proteolytic and signaling functions. It also abrogated NF-κB activation by N-terminally cleaved MALT1A. Altogether, this study provides further insights into mechanisms that regulate the scaffolding and activation cycle of MALT1. It also emphasizes the reduced functional capacity of MALT1B as compared to MALT1A.

  5. Differential neuregulin 1 cleavage in the prefrontal cortex and hippocampus in schizophrenia and bipolar disorder: preliminary findings.

    Directory of Open Access Journals (Sweden)

    Ketan Marballi

    Full Text Available BACKGROUND: Neuregulin 1 (NRG1 is a key candidate susceptibility gene for both schizophrenia (SCZ and bipolar disorder (BPD. The function of the NRG1 transmembrane proteins is regulated by cleavage. Alteration of membrane bound-NRG1 cleavage has been previously shown to be associated with behavioral impairments in mouse models lacking expression of NRG1-cleavage enzymes such as BACE1 and gamma secretase. We sought to determine whether alterations in NRG1 cleavage and associated enzymes occur in patients with SCZ and BPD. METHODOLOGY/PRINCIPAL FINDINGS: Using human postmortem brain, we evaluated protein expression of NRG1 cleavage products and enzymes that cleave at the external (BACE1, ADAM17, ADAM19 and internal (PS1-gamma secretase sides of the cell membrane. We used three different cohorts (Controls, SCZ and BPD and two distinct brain regions: BA9-prefrontal cortex (Controls (n = 6, SCZ (n = 6 and BPD (n = 6 and hippocampus (Controls (n = 5, SCZ (n = 6 and BPD (n = 6. In BA9, the ratio of the NRG1 N-terminal fragment relative to full length was significantly upregulated in the SCZ cohort (Bonferroni test, p = 0.011. ADAM17 was negatively correlated with full length NRG1 levels in the SCZ cohort (r = -0.926, p = 0.008. In the hippocampus we found significantly lower levels of a soluble 50 kDa NRG1 fragment in the two affected groups compared the control cohort (Bonferroni test, p = 0.0018. We also examined the relationship of specific symptomatology criteria with measures of NRG1 cleavage using the Bipolar Inventory of Signs and Symptoms Scale (BISS and the Montgomery Åsberg Depression Rating Scale (MADRS. Our results showed a positive correlation between ADAM19 and psychosis (r = 0.595 p = 0.019; PS1 and mania (r = 0.535, p = 0.040; PS1 and depression (r = 0.567, p = 0.027 in BA9, and BACE1 with anxiety (r = 0.608, p = 0.03 in the hippocampus. CONCLUSION/SIGNIFICANCE: Our preliminary findings suggest region-specific alterations in NRG1

  6. 4-Dimethylaminoazobenzenes: carcinogenicities and reductive cleavage by microsomal azo reductase.

    Science.gov (United States)

    Lambooy, J P; Koffman, B M

    1985-01-01

    Twenty-four 4-dimethylaminoazobenzenes (DABs) in which systematic structural modifications have been made in the prime ring have been studied for substrate specificity for microsomal azo reductase. The DABs were also evaluated for carcinogenicity and it was found that there was no correlation between carcinogenicity and extent of azo bond cleavage by azo reductase. While any substituent in the prime ring reduces the rate of cleavage of the azo bond relative to the unsubstituted dye, there is a correlation between substituent size and susceptibility to the enzyme. Substituent size was also found to be a significant factor in the induction of hepatomas by the dyes. Preliminary studies have shown that there appears to be a positive correlation between microsomal riboflavin content and the activity of the azo reductase.

  7. 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 assayed. The following PNAs were used: T10-LysNH2, T5CT4-LysNH2 and T2CT2CT4-LysNH2 and the corresponding targets cloned into pUC 19 were flanked by BamH1, Sal1 or Pstl sites, respectively. In all cases it was found that complete inhibition of restriction enzyme cleavage was obtained...

  8. MerTK cleavage limits proresolving mediator biosynthesis and exacerbates tissue inflammation.

    Science.gov (United States)

    Cai, Bishuang; Thorp, Edward B; Doran, Amanda C; Subramanian, Manikandan; Sansbury, Brian E; Lin, Chyuan-Sheng; Spite, Matthew; Fredman, Gabrielle; Tabas, Ira

    2016-06-07

    The acute inflammatory response requires a coordinated resolution program to prevent excessive inflammation, repair collateral damage, and restore tissue homeostasis, and failure of this response contributes to the pathology of numerous chronic inflammatory diseases. Resolution is mediated in part by long-chain fatty acid-derived lipid mediators called specialized proresolving mediators (SPMs). However, how SPMs are regulated during the inflammatory response, and how this process goes awry in inflammatory diseases, are poorly understood. We now show that signaling through the Mer proto-oncogene tyrosine kinase (MerTK) receptor in cultured macrophages and in sterile inflammation in vivo promotes SPM biosynthesis by a mechanism involving an increase in the cytoplasmic:nuclear ratio of a key SPM biosynthetic enzyme, 5-lipoxygenase. This action of MerTK is linked to the resolution of sterile peritonitis and, after ischemia-reperfusion (I/R) injury, to increased circulating SPMs and decreased remote organ inflammation. MerTK is susceptible to ADAM metallopeptidase domain 17 (ADAM17)-mediated cell-surface cleavage under inflammatory conditions, but the functional significance is not known. We show here that SPM biosynthesis is increased and inflammation resolution is improved in a new mouse model in which endogenous MerTK was replaced with a genetically engineered variant that is cleavage-resistant (Mertk(CR)). Mertk(CR) mice also have increased circulating levels of SPMs and less lung injury after I/R. Thus, MerTK cleavage during inflammation limits SPM biosynthesis and the resolution response. These findings contribute to our understanding of how SPM synthesis is regulated during the inflammatory response and suggest new therapeutic avenues to boost resolution in settings where defective resolution promotes disease progression.

  9. Raman characterization of Avocado Sunblotch viroid and its response to external perturbations and self-cleavage

    Science.gov (United States)

    2014-01-01

    Background Viroids are the smallest pathogens of plants. To date the structural and conformational details of the cleavage of Avocado sunblotch viroid (ASBVd) and the catalytic role of Mg2+ ions in efficient self-cleavage are of crucial interest. Results We report the first Raman characterization of the structure and activity of ASBVd, for plus and minus viroid strands. Both strands exhibit a typical A-type RNA conformation with an ordered double-helical content and a C3′-endo/anti sugar pucker configuration, although small but specific differences are found in the sugar puckering and base-stacking regions. The ASBVd(-) is shown to self-cleave 3.5 times more actively than ASBVd(+). Deuteration and temperature increase perturb differently the double-helical content and the phosphodiester conformation, as revealed by corresponding characteristic Raman spectral changes. Our data suggest that the structure rigidity and stability are higher and the D2O accessibility to H-bonding network is lower for ASBVd(+) than for ASBVd(-). Remarkably, the Mg2+-activated self-cleavage of the viroid does not induce any significant alterations of the secondary viroid structure, as evidenced from the absence of intensity changes of Raman marker bands that, however exhibit small but noticeable frequency downshifts suggesting several minor changes in phosphodioxy, internal loops and hairpins of the cleaved viroids. Conclusions Our results demonstrate the sensitivity of Raman spectroscopy in monitoring structural and conformational changes of the viroid and constitute the basis for further studies of its interactions with therapeutic agents and cell membranes. PMID:24655924

  10. 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.

  11. Cleavage Mapping the Topology of Protein Folding Intermediates

    Science.gov (United States)

    2007-11-02

    investigate the changes that occur in two of these mutants. V66L has a greatly lowered m value while that of A90S is substantially increased (5...stability of the folded state of nuclease. The cleavage technique will be used to investigate the changes that occur in two of these mutants. V66L...Connecticut, 06520 3Instituto de Qufmica y Fisicoquimica Biolögicas, Facultad de Farmacia y Bioqufmica (UBA-CONICET), Buenos Aires, Argentina 4

  12. Recent cleavages in the religious right in Turkey

    OpenAIRE

    1993-01-01

    Ankara : Department of Political Science and Public Administration, Bilkent Univ., 1993. Thesis (Master's) -- Bilkent University, 1993. Includes bibliographical references. In this study,recent cleavages within the Prosperity Party is described. For this purpose, first the ideologies of the National Order Party and the National Salvation Party are taken up. For the PP is the continuation of the NOP and NSP, the PP cannot be understood as distinct from these two parties...

  13. 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.

  14. Deformation-dependent enzyme mechanokinetic cleavage of type I collagen.

    Science.gov (United States)

    Wyatt, Karla E-K; Bourne, Jonathan W; Torzilli, Peter A

    2009-05-01

    Collagen is a key structural protein in the extracellular matrix of many tissues. It provides biological tissues with tensile mechanical strength and is enzymatically cleaved by a class of matrix metalloproteinases known as collagenases. Collagen enzymatic kinetics has been well characterized in solubilized, gel, and reconstituted forms. However, limited information exists on enzyme degradation of structurally intact collagen fibers and, more importantly, on the effect of mechanical deformation on collagen cleavage. We studied the degradation of native rat tail tendon fibers by collagenase after the fibers were mechanically elongated to strains of epsilon=1-10%. After the fibers were elongated and the stress was allowed to relax, the fiber was immersed in Clostridium histolyticum collagenase and the decrease in stress (sigma) was monitored as a means of calculating the rate of enzyme cleavage of the fiber. An enzyme mechanokinetic (EMK) relaxation function T(E)(epsilon) in s(-1) was calculated from the linear stress-time response during fiber cleavage, where T(E)(epsilon) corresponds to the zero order Michaelis-Menten enzyme-substrate kinetic response. The EMK relaxation function T(E)(epsilon) was found to decrease with applied strain at a rate of approximately 9% per percent strain, with complete inhibition of collagen cleavage predicted to occur at a strain of approximately 11%. However, comparison of the EMK response (T(E) versus epsilon) to collagen's stress-strain response (sigma versus epsilon) suggested the possibility of three different EMK responses: (1) constant T(E)(epsilon) within the toe region (epsiloncollagen triple helix may be by a conformational change in the triple helix since the decrease in T(E)(epsilon) appeared concomitant with stretching of the collagen molecule.

  15. Elasto-viscoplastic phase field modelling of anisotropic cleavage fracture

    Science.gov (United States)

    Shanthraj, P.; Svendsen, B.; Sharma, L.; Roters, F.; Raabe, D.

    2017-02-01

    A finite-strain anisotropic phase field method is developed to model the localisation of damage on a defined family of crystallographic planes, characteristic of cleavage fracture in metals. The approach is based on the introduction of an undamaged configuration, and the inelastic deformation gradient mapping this configuration to a damaged configuration is microstructurally represented by the opening of a set of cleavage planes in the three fracture modes. Crack opening is modelled as a dissipative process, and its evolution is thermodynamically derived. To couple this approach with a physically-based phase field method for brittle fracture, a scalar measure of the overall local damage is introduced, whose evolution is determined by the crack opening rates, and weakly coupled with the non-local phase field energy representing the crack opening resistance in the classical sense of Griffith. A finite-element implementation of the proposed model is employed to simulate the crack propagation path in a laminate and a polycrystalline microstructure. As shown in this work, it is able to predict the localisation of damage on the set of pre-defined cleavage planes, as well as the kinking and branching of the crack resulting from the crystallographic misorientation across the laminate boundary and the grain boundaries respectively.

  16. Examination of Early Cleavage an its Importance in IVF Treatment

    Directory of Open Access Journals (Sweden)

    Fancsovits P

    2006-01-01

    Full Text Available Since the introduction of assisted reproduction, the number of multiple pregnancies has increased due to the high number of transferred embryos. There is an urgent need for IVF specialists to reduce the number of embryos transferred without the risk of decreasing pregnancy rates. Embryos are selected for transfer on the basis of their developmental stage and morphology. The number of blastomeres of the embryo indicates the speed of early embryo development which correlates to the viability of the embryo. Examination of early embryo development, especially the timing of the first cleavage, can be recommended as a tool for predicting embryo viability. Observation of timing of the first cleavage and its different stages helps us to identify fast- and slow-developing embryos. Early pronuclear breakdown and early cleavage of the zygote are indicators of fast embryo development and good embryo viability. Thereby, they can lead to high implantation and pregnancy rates. The aim of this paper is to provide an overview of the timing of early embryo development and to show its significance in IVF treatment.

  17. 人M-07e白血病细胞凋亡过程中激活Caspase-3引起的Bcl-2 蛋白酶解与Lynp53/56激酶失活相关%Cleavage of Bcl-2 Protein by Activated Caspase-3 Is Associated with Inactivation of Lynp53/56 Kinase Activity in Human M-07e Leukemic Cells during Apoptosis

    Institute of Scientific and Technical Information of China (English)

    张学敏; 胡美茹; 兰雨; 于鸣; Ben D-M Chen

    2000-01-01

    The growth of M-07e human megakaryocytie leukemia cells is strictly dependent on GM-CSF. In M-07e cells, the GM-CSF receptor (GM-CSF R) is composed of two subunits: a low affinity α subunit and a phosphorylated β subunit, which is constitutively linked to lyn53/56 protein tyrosine kinase. In this study, The role of lyn kinase in regulating TGF-β 1-induced apoptosis in M-07e cells was examined. The removal of rhGM-CSF from the culture medium resulted in down-regulation of lyn kinase activity, followed by growth inhibition and programmed cell death. Apoptosis of M-07e cells was accompanied with a massive cleavage of Bcl-2 and Bax proteins into shortened fragments with molecular mass of 22 kD and 18 kD, respectively. Using specific inhibitors, the cleavage of Bcl-2, but not Bax, was found to be processed through activated caspase-3 (CPP32), which is abundantly expressed in M-07e cells. TGF-β 1 inhibited rhGM-CSF-stimulated cell growth and promoted apoptosis in M-07e cells with a pattern identical to that induced by rhGM-CSF depletion, which included massive cleavage of both Bcl-2 and Bax proteins and inactivation of lyn kinase activity. TGF-β 1 did not affect the levels of lyn protein or the β-subunit, neither did it block the interaction between these two components. Also, TGF-β 1 treatment did not diminish the expression of the α subunit in M-07e cells. Our results showed that TGF-β 1 inhibits cell proliferation and promotes apoptosis in M-07e cells by inactivating the GM-CSF R-associated lyn kinase activity. Further, This study showed that Bcl-2 cleavage by activated CPP32 is a naturally occurring event associated with apoptosis, which is under the regulation of lyn kinase activation.%人巨核白血病细胞系M-07e的生长严格依赖于GM-CSF.在M-07e细胞,GM-CSF受体(GM-CSF R)由两个亚基所组成:低亲合力的配体特异的α亚基和一个磷酸化的β亚基,后者与lynp53/56酪氨酸蛋白激酶固定相连.本研究

  18. Cleavage of model substrates by archaeal RNase P: role of protein cofactors in cleavage-site selection.

    Science.gov (United States)

    Sinapah, Sylvie; Wu, Shiying; Chen, Yu; Pettersson, B M Fredrik; Gopalan, Venkat; Kirsebom, Leif A

    2011-02-01

    RNase P is a catalytic ribonucleoprotein primarily involved in tRNA biogenesis. Archaeal RNase P comprises a catalytic RNase P RNA (RPR) and at least four protein cofactors (RPPs), which function as two binary complexes (POP5•RPP30 and RPP21• RPP29). Exploiting the ability to assemble a functional Pyrococcus furiosus (Pfu) RNase P in vitro, we examined the role of RPPs in influencing substrate recognition by the RPR. We first demonstrate that Pfu RPR, like its bacterial and eukaryal counterparts, cleaves model hairpin loop substrates albeit at rates 90- to 200-fold lower when compared with cleavage by bacterial RPR, highlighting the functionally comparable catalytic cores in bacterial and archaeal RPRs. By investigating cleavage-site selection exhibited by Pfu RPR (±RPPs) with various model substrates missing consensus-recognition elements, we determined substrate features whose recognition is facilitated by either POP5•RPP30 or RPP21•RPP29 (directly or indirectly via the RPR). Our results also revealed that Pfu RPR + RPP21•RPP29 displays substrate-recognition properties coinciding with those of the bacterial RPR-alone reaction rather than the Pfu RPR, and that this behaviour is attributable to structural differences in the substrate-specificity domains of bacterial and archaeal RPRs. Moreover, our data reveal a hierarchy in recognition elements that dictates cleavage-site selection by archaeal RNase P.

  19. 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

  20. Zinc overload enhances APP cleavage and Aβ deposition in the Alzheimer mouse brain.

    Directory of Open Access Journals (Sweden)

    Chun-Yan Wang

    Full Text Available BACKGROUND: Abnormal zinc homeostasis is involved in β-amyloid (Aβ plaque formation and, therefore, the zinc load is a contributing factor in Alzheimer's disease (AD. However, the involvement of zinc in amyloid precursor protein (APP processing and Aβ deposition has not been well established in AD animal models in vivo. METHODOLOGY/PRINCIPAL FINDINGS: In the present study, APP and presenilin 1 (PS1 double transgenic mice were treated with a high dose of zinc (20 mg/ml ZnSO4 in drinking water. This zinc treatment increased APP expression, enhanced amyloidogenic APP cleavage and Aβ deposition, and impaired spatial learning and memory in the transgenic mice. We further examined the effects of zinc overload on APP processing in SHSY-5Y cells overexpressing human APPsw. The zinc enhancement of APP expression and cleavage was further confirmed in vitro. CONCLUSIONS/SIGNIFICANCE: The present data indicate that excess zinc exposure could be a risk factor for AD pathological processes, and alteration of zinc homeostasis is a potential strategy for the prevention and treatment of AD.

  1. 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

    2012-04-01

    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.

  2. 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.

  3. A new take on V(DJ recombination: transcription driven nuclear and chromatin reorganization in RAG–mediated cleavage.

    Directory of Open Access Journals (Sweden)

    Julie eChaumeil

    2013-12-01

    Full Text Available It is nearly thirty years since the Alt lab first put forward the accessibility model, which proposes that cleavage of the various loci is controlled by lineage and stage specific factors that regulate RAG access to the different loci. Numerous labs have since demonstrated that locus opening is regulated at multiple levels that include sterile transcription, changes in chromatin packaging and alterations in locus conformation. Here we focus on the interplay between transcription and RAG binding in facilitating targeted cleavage. We discuss the results of recent studies that implicate transcription in regulating nuclear organization and altering the composition of resident nucleosomes to promote regional access to the recombinase machinery. Additionally we include new data that provide insight into the role of the RAG proteins in defining nuclear organization in recombining T cells.

  4. Expression of a naturally occurring angiotensin AT(1) receptor cleavage fragment elicits caspase-activation and apoptosis.

    Science.gov (United States)

    Cook, Julia L; Singh, Akannsha; DeHaro, Dawn; Alam, Jawed; Re, Richard N

    2011-11-01

    Several transmembrane receptors are documented to accumulate in nuclei, some as holoreceptors and others as cleaved receptor products. Our prior studies indicate that a population of the 7-transmembrane angiotensin type-1 receptor (AT(1)R) is cleaved in a ligand-augmented manner after which the cytoplasmic, carboxy-terminal cleavage fragment (CF) traffics to the nucleus. In the present report, we determine the precise cleavage site within the AT(1)R by mass spectrometry and Edman sequencing. Cleavage occurs between Leu(305) and Gly(306) at the junction of the seventh transmembrane domain and the intracellular cytoplasmic carboxy-terminal domain. To evaluate the function of the CF distinct from the holoreceptor, we generated a construct encoding the CF as an in-frame yellow fluorescent protein fusion. The CF accumulates in nuclei and induces apoptosis in CHO-K1 cells, rat aortic smooth muscle cells (RASMCs), MCF-7 human breast adenocarcinoma cells, and H9c2 rat cardiomyoblasts. All cell types show nuclear fragmentation and disintegration, as well as evidence for phosphotidylserine displacement in the plasma membrane and activated caspases. RASMCs specifically showed a 5.2-fold increase (P < 0.001) in CF-induced active caspases compared with control and a 7.2-fold increase (P < 0.001) in cleaved caspase-3 (Asp174). Poly(ADP-ribose)polymerase was upregulated 4.8-fold (P < 0.001) in CF expressing cardiomyoblasts and colocalized with terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL). CF expression also induces DNA laddering, the gold-standard for apoptosis in all cell types studied. CF-induced apoptosis, therefore, appears to be a general phenomenon as it is observed in multiple cell types including smooth muscle cells and cardiomyoblasts.

  5. Cytotoxicity and DNA cleavage with core-shell nanocomposites functionalized by a KH domain DNA binding peptide

    Science.gov (United States)

    Bazak, Remon; Ressl, Jan; Raha, Sumita; Doty, Caroline; Liu, William; Wanzer, Beau; Salam, Seddik Abdel; Elwany, Samy; Paunesku, Tatjana; Woloschak, Gayle E.

    2013-11-01

    A nanoconjugate was composed of metal oxide nanoparticles decorated with peptides and fluorescent dye and tested for DNA cleavage following UV light activation. The peptide design was based on a DNA binding domain, the so called KH domain of the hnRNPK protein. This ``KH peptide'' enabled cellular uptake of nanoconjugates and their entry into cell nuclei. The control nanoconjugate carried no peptide; it consisted only of the metal oxide nanoparticle prepared as Fe3O4@TiO2 nanocomposite and the fluorescent dye alizarin red S. These components of either construct are responsible for nanoconjugate activation by UV light and the resultant production of reactive oxygen species (ROS). Production of ROS at different subcellular locations causes damage to different components of cells: only nanoconjugates inside cell nuclei can be expected to cause DNA cleavage. Degradation of cellular DNA with KH peptide decorated nanoconjugates exceeded the DNA damage obtained from control, no-peptide nanoconjugate counterparts. Moreover, caspase activation and cell death were more extensive in the same cells.A nanoconjugate was composed of metal oxide nanoparticles decorated with peptides and fluorescent dye and tested for DNA cleavage following UV light activation. The peptide design was based on a DNA binding domain, the so called KH domain of the hnRNPK protein. This ``KH peptide'' enabled cellular uptake of nanoconjugates and their entry into cell nuclei. The control nanoconjugate carried no peptide; it consisted only of the metal oxide nanoparticle prepared as Fe3O4@TiO2 nanocomposite and the fluorescent dye alizarin red S. These components of either construct are responsible for nanoconjugate activation by UV light and the resultant production of reactive oxygen species (ROS). Production of ROS at different subcellular locations causes damage to different components of cells: only nanoconjugates inside cell nuclei can be expected to cause DNA cleavage. Degradation of cellular DNA

  6. NH2-terminal cleavage of xenopus fibroblast growth factor 3 is necessary for optimal biological activity and receptor binding.

    Science.gov (United States)

    Antoine, M; Daum, M; Köhl, R; Blecken, V; Close, M J; Peters, G; Kiefer, P

    2000-11-01

    Fibroblast growth factor 3 (FGF3) was originally identified as the mouse proto-oncogene Int-2, which is activated by proviral insertion in tumors induced by mouse mammary tumor virus. To facilitate the biological characterization of the ligand, we have analyzed its homologue in Xenopus laevis, XFGF3. Here we confirm that the X. laevis genome contains two distinct FGF3 alleles, neither of which is capable of encoding the NH2-terminally extended forms specified by the mouse and human FGF3 genes. Unlike the mammalian proteins, XFGF3 is efficiently secreted as a Mr 31,000 glycoprotein, gp31, which undergoes proteolytic cleavage to produce an NH2-terminally truncated product, gp27. Processing removes a segment of 18 amino acids immediately distal to the signal peptide that is not present in the mammalian homologues. By inserting an epitope-tag adjacent to the cleavage site, we show that a substantial amount of the gp27 is generated intracellularly, although processing can also occur in the extracellular matrix. Two residues are also removed from the COOH terminus. To compare the biological properties of the different forms, cDNAs were constructed that selectively give rise to the larger, gp31, or smaller, gp27, forms of XFGF3. As judged by their ability to cause morphological transformation of NIH3T3 cells, their mitogenicity on specific cell types, and their affinity for the IIIb and IIIc isoforms of Xenopus FGF receptors, gp27 has a much higher biological activity than gp31. Sequence comparison revealed an intriguing similar cleavage motif immediately downstream of the signal peptide cleavage site in the NH2-terminus of mouse and human FGF3. Analysis of secreted mutant mouse FGF3 confirmed an additional NH2-terminal processing at the corresponding sequence motif. NH2-terminal trimming of Xenopus and mammalian FGF3s may therefore be a prerequisite of optimal biological activity.

  7. Oxidative stress up-regulates the expression of β-Amyloid precursor protein cleavage enzyme 1 in SH-SY5Y human neuroblastoma cells%氧化应激上调人神经母细胞瘤细胞内β-裂解酶的表达

    Institute of Scientific and Technical Information of China (English)

    谷心灵; 孟斐; 李良

    2012-01-01

    Objective To investigate the role of oxidative stress in the expression of p-Amyloid precursor protein cleavage enzyme 1 ( BACE1) and the changes DNA methylation and histone acetylation. Methods Cultured SH-SY5Y cells treated with H2O2 were used to test the expressions of BACE1, DNA methyltransferases 1, 3A (DN-MT1,DNMT3A) and histone deacetyltranferase (HDAC) by were examined by Western blot. The level of mRNA of BACE1 was assessed by RT-PCR. Acetylation level of histone H3 and H4 was examined by optical density assay. Results Both BACE1 mRNA and protein levels were up-regulated significantly after H2O2 treatment for 1 and 72 h; DNMT1 and DNMT3A expressions were decreased to 75% and 65% of control respectively after H2O2 treatment for 72 h; HD AC3 level was increased by 1.6 folds as compared with control; While the level of histone H3 acetylation was decreased and there was no change with histone H4 acetylation. Conclusions Oxidative stress may regulate BACE1 expression in SH-SY5 Y through alteration of DNA methylation and histone acetylation which play a role in Alzheimer's disease (AD) pathogenesis.%目的 研究氧化应激对人神经母细胞瘤细胞(SH-SY5Y)β-裂解酶(BACE1)表达的影响及组蛋白乙酰化、DNA甲基化的改变.方法 采用H2O2处理体外培养的SH-SY5Y,Westem blot法检测细胞的BACE1表达及DNA甲基转移酶(DNMTs)和组蛋白去乙酰化酶(HDAC)的表达;实时定量PCR检测BACE1 mRNA的表达;吸光度值法检测组蛋白3(H3)和组蛋白4(H4)整体乙酰化水平.结果 SH-SY5Y细胞经H2O2处理1和72 h后BACE1 mRNA和蛋白表达均明显增多;H2O2处理72 h后DNMT1、DNMT3A表达均下降,分别是对照组的75%和65%(P<0.01);而组蛋白去乙酰化酶HDAC3的表达增高至对照组的1.6倍(P<0.01);同时,组蛋白H3整体乙酰化水平下降,但H4乙酰化水平无明显改变.结论 氧化应激可能通过改变SH-SY5Y细胞内DNA甲基化水平及组蛋白乙酰化状态调节BACE1的

  8. 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.

  9. Role of the proteasome in excitotoxicity-induced cleavage of glutamic acid decarboxylase in cultured hippocampal neurons.

    Directory of Open Access Journals (Sweden)

    Márcio S Baptista

    Full Text Available Glutamic acid decarboxylase is responsible for synthesizing GABA, the major inhibitory neurotransmitter, and exists in two isoforms--GAD65 and GAD67. The enzyme is cleaved under excitotoxic conditions, but the mechanisms involved and the functional consequences are not fully elucidated. We found that excitotoxic stimulation of cultured hippocampal neurons with glutamate leads to a time-dependent cleavage of GAD65 and GAD67 in the N-terminal region of the proteins, and decrease the corresponding mRNAs. The cleavage of GAD67 was sensitive to the proteasome inhibitors MG132, YU102 and lactacystin, and was also abrogated by the E1 ubiquitin ligase inhibitor UBEI-41. In contrast, MG132 and UBEI-41 were the only inhibitors tested that showed an effect on GAD65 cleavage. Excitotoxic stimulation with glutamate also increased the amount of GAD captured in experiments where ubiquitinated proteins and their binding partners were isolated. However, no evidences were found for direct GADs ubiquitination in cultured hippocampal neurons, and recombinant GAD65 was not cleaved by purified 20S or 26S proteasome preparations. Since calpains, a group of calcium activated proteases, play a key role in GAD65/67 cleavage under excitotoxic conditions the results suggest that GADs are cleaved after ubiquitination and degradation of an unknown binding partner by the proteasome. The characteristic punctate distribution of GAD65 along neurites of differentiated cultured hippocampal neurons was significantly reduced after excitotoxic injury, and the total GAD activity measured in extracts from the cerebellum or cerebral cortex at 24h postmortem (when there is a partial cleavage of GADs was also decreased. The results show a role of the UPS in the cleavage of GAD65/67 and point out the deregulation of GADs under excitotoxic conditions, which is likely to affect GABAergic neurotransmission. This is the first time that the UPS has been implicated in the events triggered during

  10. Kinetics of acid-catalyzed cleavage of cumene hydroperoxide.

    Science.gov (United States)

    Levin, M E; Gonzales, N O; Zimmerman, L W; Yang, J

    2006-03-17

    The cleavage of cumene hydroperoxide, in the presence of sulfuric acid, to form phenol and acetone has been examined by adiabatic calorimetry. As expected, acid can catalyze cumene hydroperoxide reaction at temperatures below that of thermally-induced decomposition. At elevated acid concentrations, reactivity is also observed at or below room temperature. The exhibited reactivity behavior is complex and is significantly affected by the presence of other species (including the products). Several reaction models have been explored to explain the behavior and these are discussed.

  11. 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...... a sequential C-terminal degradation pattern characteristic of this dipeptylpeptidase. The substrate primary structure was not found to be essential for regulation of the proteolytic activity of the cysteine peptidases studied. However, the degradation patterns obtained imply that calpains are involved...

  12. Airway protease/antiprotease imbalance in atopic asthmatics contributes to increased Influenza A virus cleavage and replication

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    Kesic Matthew J

    2012-09-01

    Full Text Available Abstract Asthmatics are more susceptible to influenza infections, yet mechanisms mediating this enhanced susceptibility are unknown. Influenza virus hemagglutinin (HA protein binds to sialic acid residues on the host cells. HA requires cleavage to allow fusion of the viral HA with host cell membrane, which is mediated by host trypsin-like serine protease. We show data here demonstrating that the protease:antiprotease ratio is increased in the nasal mucosa of asthmatics and that these changes were associated with increased proteolytic activation of influenza. These data suggest that disruption of the protease balance in asthmatics enhances activation and infection of influenza virus.

  13. Nucleoside modifications in RNA limit activation of 2'-5'-oligoadenylate synthetase and increase resistance to cleavage by RNase L.

    Science.gov (United States)

    Anderson, Bart R; Muramatsu, Hiromi; Jha, Babal K; Silverman, Robert H; Weissman, Drew; Karikó, Katalin

    2011-11-01

    The interferon-induced enzymes 2'-5'-oligoadenylate synthetase (OAS) and RNase L are key components of innate immunity involved in sensory and effector functions following viral infections. Upon binding target RNA, OAS is activated to produce 2'-5'-linked oligoadenylates (2-5A) that activate RNase L, which then cleaves single-stranded self and non-self RNA. Modified nucleosides that are present in cellular transcripts have been shown to suppress activation of several RNA sensors. Here, we demonstrate that in vitro transcribed, unmodified RNA activates OAS, induces RNase L-mediated ribosomal RNA (rRNA) cleavage and is rapidly cleaved by RNase L. In contrast, RNA containing modified nucleosides activates OAS less efficiently and induces limited rRNA cleavage. Nucleoside modifications also make RNA resistant to cleavage by RNase L. Examining translation in RNase L(-/-) cells and mice confirmed that RNase L activity reduces translation of unmodified mRNA, which is not observed with modified mRNA. Additionally, mRNA containing the nucleoside modification pseudouridine is translated longer and has an extended half-life. The observation that modified nucleosides in RNA reduce 2-5A pathway activation joins OAS and RNase L to the list of RNA sensors and effectors whose functions are limited when RNA is modified, confirming the role of nucleoside modifications in suppressing immune recognition of RNA.

  14. Vesicle-associated membrane protein (VAMP) cleavage by a new metalloprotease from the Brazilian scorpion Tityus serrulatus.

    Science.gov (United States)

    Fletcher, Paul L; Fletcher, Maryann D; Weninger, Keith; Anderson, Trevor E; Martin, Brian M

    2010-03-01

    We present evidence that venom from the Brazilian scorpion Tityus serrulatus and a purified fraction selectively cleave essential SNARE proteins within exocrine pancreatic tissue. Western blotting for vesicle-associated membrane protein type v-SNARE proteins (or synaptobrevins) reveals characteristic alterations to venom-treated excised pancreatic lobules in vitro. Immunocytochemistry by electron microscopy confirms both the SNARE identity as VAMP2 and the proteolysis of VAMP2 as a marked decrease in secondary antibody-conjugated colloidal gold particles that are predominantly associated with mature zymogen granules. Studies with recombinant SNARE proteins were used to determine the specific cleavage site in VAMP2 and the susceptibility of VAMP8 (endobrevin). The VAMP2 cleavage site is between the transmembrane anchor and the SNARE motif that assembles into the ternary SNARE complex. Inclusion of divalent chelating agents (EDTA) with fraction nu, an otherwise active purified component from venom, eliminates SNARE proteolysis, suggesting the active protein is a metalloprotease. The unique cleavages of VAMP2 and VAMP8 may be linked to pancreatitis that develops following scorpion envenomation as both of these v-SNARE proteins are associated with zymogen granule membranes in pancreatic acinar cells. We have isolated antarease, a metalloprotease from fraction nu that cleaves VAMP2, and report its amino acid sequence.

  15. Vesicle-associated Membrane Protein (VAMP) Cleavage by a New Metalloprotease from the Brazilian Scorpion Tityus serrulatus*

    Science.gov (United States)

    Fletcher, Paul L.; Fletcher, Maryann D.; Weninger, Keith; Anderson, Trevor E.; Martin, Brian M.

    2010-01-01

    We present evidence that venom from the Brazilian scorpion Tityus serrulatus and a purified fraction selectively cleave essential SNARE proteins within exocrine pancreatic tissue. Western blotting for vesicle-associated membrane protein type v-SNARE proteins (or synaptobrevins) reveals characteristic alterations to venom-treated excised pancreatic lobules in vitro. Immunocytochemistry by electron microscopy confirms both the SNARE identity as VAMP2 and the proteolysis of VAMP2 as a marked decrease in secondary antibody-conjugated colloidal gold particles that are predominantly associated with mature zymogen granules. Studies with recombinant SNARE proteins were used to determine the specific cleavage site in VAMP2 and the susceptibility of VAMP8 (endobrevin). The VAMP2 cleavage site is between the transmembrane anchor and the SNARE motif that assembles into the ternary SNARE complex. Inclusion of divalent chelating agents (EDTA) with fraction ν, an otherwise active purified component from venom, eliminates SNARE proteolysis, suggesting the active protein is a metalloprotease. The unique cleavages of VAMP2 and VAMP8 may be linked to pancreatitis that develops following scorpion envenomation as both of these v-SNARE proteins are associated with zymogen granule membranes in pancreatic acinar cells. We have isolated antarease, a metalloprotease from fraction ν that cleaves VAMP2, and report its amino acid sequence. PMID:20026600

  16. Decreased linear ubiquitination of NEMO and FADD on apoptosis with caspase-mediated cleavage of HOIP.

    Science.gov (United States)

    Goto, Eiji; Tokunaga, Fuminori

    2017-02-09

    NF-κB is crucial to regulate immune and inflammatory responses and cell survival. LUBAC generates a linear ubiquitin chain and activates NF-κB through ubiquitin ligase (E3) activity in the HOIP subunit. Here, we show that HOIP is predominantly cleaved by caspase at Asp390 upon apoptosis, and that is subjected to proteasomal degradation. We identified that FADD, as well as NEMO, is a substrate for LUBAC. Although the C-terminal fragment of HOIP retains NF-κB activity, linear ubiquitination of NEMO and FADD decreases upon apoptosis. Moreover, the N-terminal fragment of HOIP binds with deubiquitinases, such as OTULIN and CYLD-SPATA2. These results indicate that caspase-mediated cleavage of HOIP divides critical functional regions of HOIP, and that this regulates linear (de)ubiquitination of substrates upon apoptosis.

  17. Anthrax lethal factor cleavage of Nlrp1 is required for activation of the inflammasome.

    Directory of Open Access Journals (Sweden)

    Jonathan L Levinsohn

    Full Text Available NOD-like receptor (NLR proteins (Nlrps are cytosolic sensors responsible for detection of pathogen and danger-associated molecular patterns through unknown mechanisms. Their activation in response to a wide range of intracellular danger signals leads to formation of the inflammasome, caspase-1 activation, rapid programmed cell death (pyroptosis and maturation of IL-1β and IL-18. Anthrax lethal toxin (LT induces the caspase-1-dependent pyroptosis of mouse and rat macrophages isolated from certain inbred rodent strains through activation of the NOD-like receptor (NLR Nlrp1 inflammasome. Here we show that LT cleaves rat Nlrp1 and this cleavage is required for toxin-induced inflammasome activation, IL-1 β release, and macrophage pyroptosis. These results identify both a previously unrecognized mechanism of activation of an NLR and a new, physiologically relevant protein substrate of LT.

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

    Science.gov (United States)

    Tati, Ramesh; Kristoffersson, Ann-Charlotte; Manea Hedström, Minola; Mörgelin, Matthias; Wieslander, Jörgen; van Kooten, Cees; Karpman, Diana

    2017-02-01

    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.

  19. The Mycobacterium tuberculosis ORF Rv0654 encodes a carotenoid oxygenase mediating central and excentric cleavage of conventional and aromatic carotenoids.

    Science.gov (United States)

    Scherzinger, Daniel; Scheffer, Erdmann; Bär, Cornelia; Ernst, Hansgeorg; Al-Babili, Salim

    2010-11-01

    Mycobacterium tuberculosis, the causative agent of tuberculosis, is assumed to lack carotenoids, which are widespread pigments fulfilling important functions as radical scavengers and as a source of apocarotenoids. In mammals, the synthesis of apocarotenoids, including retinoic acid, is initiated by the β-carotene cleavage oxygenases I and II catalyzing either a central or an excentric cleavage of β-carotene, respectively. The M. tuberculosis ORF Rv0654 codes for a putative carotenoid oxygenase conserved in other mycobacteria. In the present study, we investigated the corresponding enzyme, here named M. tuberculosis carotenoid cleavage oxygenase (MtCCO). Using heterologously expressed and purified protein, we show that MtCCO converts several carotenoids and apocarotenoids in vitro. Moreover, the identification of the products suggests that, in contrast to other carotenoid oxygenases, MtCCO cleaves the central C15-C15' and an excentric double bond at the C13-C14 position, leading to retinal (C(20)), β-apo-14'-carotenal (C(22)) and β-apo-13-carotenone (C(18)) from β-carotene, as well as the corresponding hydroxylated products from zeaxanthin and lutein. Moreover, the enzyme cleaves also 3,3'-dihydroxy-isorenieratene representing aromatic carotenoids synthesized by other mycobacteria. Quantification of the products from different substrates indicates that the preference for each of the cleavage positions is determined by the hydroxylation and the nature of the ionone ring. The data obtained in the present study reveal MtCCO to be a novel carotenoid oxygenase and indicate that M. tuberculosis may utilize carotenoids from host cells and interfere with their retinoid metabolism.

  20. Cleavage of kininogen and subsequent bradykinin release by the complement component: mannose-binding lectin-associated serine protease (MASP-1.

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    József Dobó

    Full Text Available Bradykinin (BK, generated from high-molecular-weight kininogen (HK is the major mediator of swelling attacks in hereditary angioedema (HAE, a disease associated with C1-inhibitor deficiency. Plasma kallikrein, activated by factor XIIa, is responsible for most of HK cleavage. However other proteases, which activate during episodes of angioedema, might also contribute to BK production. The lectin pathway of the complement system activates after infection and oxidative stress on endothelial cells generating active serine proteases: MASP-1 and MASP-2. Our aim was to study whether activated MASPs are able to digest HK to release BK. Initially we were trying to find potential new substrates of MASP-1 in human plasma by differential gel electrophoresis, and we identified kininogen cleavage products by this proteomic approach. As a control, MASP-2 was included in the study in addition to MASP-1 and kallikrein. The proteolytic cleavage of HK by MASPs was followed by SDS-PAGE, and BK release was detected by HPLC. We showed that MASP-1 was able to cleave HK resulting in BK production. MASP-2 could also cleave HK but could not release BK. The cleavage pattern of MASPs is similar but not strictly identical to that of kallikrein. The catalytic efficiency of HK cleavage by a recombinant version of MASP-1 and MASP-2 was about 4.0×10(2 and 2.7×10(2 M(-1 s(-1, respectively. C1-inhibitor, the major inhibitor of factor XIIa and kallikrein, also prevented the cleavage of HK by MASPs. In all, a new factor XII- and kallikrein-independent mechanism of bradykinin production by MASP-1 was demonstrated, which may contribute to the pro-inflammatory effect of the lectin pathway of complement and to the elevated bradykinin levels in HAE patients.

  1. A single molecule assay for measuring site-specific DNA cleavage.

    Science.gov (United States)

    Gambino, Stefano; Mousley, Briana; Cathcart, Lindsay; Winship, Janelle; Loparo, Joseph J; Price, Allen C

    2016-02-15

    Sequence-specific DNA cleavage is a key step in a number of genomic transactions. Here, we report a single-molecule technique that allows the simultaneous measurement of hundreds of DNAs, thereby collecting significant statistics in a single experiment. Microbeads are tethered with single DNA molecules in a microfluidic channel. After the DNA cleavage reaction is initiated, the time of cleavage of each DNA is recorded using video microscopy. We demonstrate the utility of our method by measuring the cleavage kinetics of NdeI, a type II restriction endonuclease.

  2. Huntingtin cleavage product A forms in neurons and is reduced by gamma-secretase inhibitors

    Directory of Open Access Journals (Sweden)

    Betschart Claudia

    2010-12-01

    Full Text Available Abstract Background The mutation in Huntington's disease is a polyglutamine expansion near the N-terminus of huntingtin. Huntingtin expressed in immortalized neurons is cleaved near the N-terminus to form N-terminal polypeptides known as cleavage products A and B (cpA and cpB. CpA and cpB with polyglutamine expansion form inclusions in the nucleus and cytoplasm, respectively. The formation of cpA and cpB in primary neurons has not been established and the proteases involved in the formation of these fragments are unknown. Results Delivery of htt cDNA into the mouse striatum using adeno-associated virus or into primary cortical neurons using lentivirus generated cpA and cpB, indicating that neurons in brain and in vitro can form these fragments. A screen of small molecule protease inhibitors introduced to clonal striatal X57 cells and HeLa cells identified compounds that reduced levels of cpA and are inhibitors of the aspartyl proteases cathepsin D and cathepsin E. The most effective compound, P1-N031, is a transition state mimetic for aspartyl proteases. By western blot analysis, cathepsin D was easily detected in clonal striatal X57 cells, mouse brain and primary neurons, whereas cathepsin E was only detectible in clonal striatal X57 cells. In primary neurons, levels of cleavage product A were not changed by the same compounds that were effective in clonal striatal cells or by mRNA silencing to partially reduce levels of cathepsin D. Instead, treating primary neurons with compounds that are known to inhibit gamma secretase activity either indirectly (Imatinib mesylate, Gleevec or selectively (LY-411,575 or DAPT reduced levels of cpA. LY-411,575 or DAPT also increased survival of primary neurons expressing endogenous full-length mutant huntingtin. Conclusion We show that cpA and cpB are produced from a larger huntingtin fragment in vivo in mouse brain and in primary neuron cultures. The aspartyl protease involved in forming cpA has cathepsin

  3. Enzymic Pathways for Formation of Carotenoid Cleavage Products

    Science.gov (United States)

    Fleischmann, Peter; Zorn, Holger

    Degraded carotenoids (apocarotenoids, norisoprenoids) have been a subject of intensive research for several decades. From the perspective of human physiology and nutrition, the retinoids, acting as vitamins, signalling molecules, and visual pigments, attracted the greatest attention (Chapters 15 and 16). Plant scientists, however, detected a wealth of different apocarotenoids, presumably derived by the excentric cleavage of carotenoids in various species, the plant hormone abscisic acid (1, Scheme 6) being the best-investigated example. With the onset of fruit ripening, flower opening or senescence of green tissues, carotenoids are degraded oxidatively to smaller, volatile compounds. The natural biological functions of the reaction products are outlined in Chapter 15. As many of these apocarotenoids act as potent flavour compounds, food chemists and flavourists worldwide have investigated meticulously their structural and sensory properties. Many aspects of carotenoid metabolites and breakdown products as aroma compounds are presented in a comprehensive book [1].

  4. Dinitrogen cleavage and hydrogenation by a trinuclear titanium polyhydride complex.

    Science.gov (United States)

    Shima, Takanori; Hu, Shaowei; Luo, Gen; Kang, Xiaohui; Luo, Yi; Hou, Zhaomin

    2013-06-28

    Both the Haber-Bosch and biological ammonia syntheses are thought to rely on the cooperation of multiple metals in breaking the strong N≡N triple bond and forming an N-H bond. This has spurred investigations of the reactivity of molecular multimetallic hydrides with dinitrogen. We report here the reaction of a trinuclear titanium polyhydride complex with dinitrogen, which induces dinitrogen cleavage and partial hydrogenation at ambient temperature and pressure. By (1)H and (15)N nuclear magnetic resonance, x-ray crystallographic, and computational studies of some key reaction steps and products, we have determined that the dinitrogen (N2) reduction proceeds sequentially through scission of a N2 molecule bonded to three Ti atoms in a μ-η(1):η(2):η(2)-end-on-side-on fashion to give a μ2-N/μ3-N dinitrido species, followed by intramolecular hydrogen migration from Ti to the μ2-N nitrido unit.

  5. Cleavage of an amide bond by a ribozyme

    Science.gov (United States)

    Dai, X.; De Mesmaeker, A.; Joyce, G. F.; Miller, S. L. (Principal Investigator)

    1995-01-01

    A variant form of a group I ribozyme, optimized by in vitro evolution for its ability to catalyze magnesium-dependent phosphoester transfer reactions involving DNA substrates, also catalyzes the cleavage of an unactivated alkyl amide when that linkage is presented in the context of an oligodeoxynucleotide analog. Substrates containing an amide bond that joins either two DNA oligos, or a DNA oligo and a short peptide, are cleaved in a magnesium-dependent fashion to generate the expected products. The first-order rate constant, kcat, is 0.1 x 10(-5) min-1 to 1 x 10(-5) min-1 for the DNA-flanked substrates, which corresponds to a rate acceleration of more than 10(3) as compared with the uncatalyzed reaction.

  6. A designer bleomycin with significantly improved DNA cleavage activity.

    Science.gov (United States)

    Huang, Sheng-Xiong; Feng, Zhiyang; Wang, Liyan; Galm, Ute; Wendt-Pienkowski, Evelyn; Yang, Dong; Tao, Meifeng; Coughlin, Jane M; Duan, Yanwen; Shen, Ben

    2012-08-15

    The bleomycins (BLMs) are used clinically in combination with a number of other agents for the treatment of several types of tumors, and the BLM, etoposide, and cisplatin treatment regimen cures 90-95% of metastatic testicular cancer patients. BLM-induced pneumonitis is the most feared, dose-limiting side effect of BLM in chemotherapy, which can progress into lung fibrosis and affect up to 46% of the total patient population. There have been continued efforts to develop new BLM analogues in the search for anticancer drugs with better clinical efficacy and lower lung toxicity. We have previously cloned and characterized the biosynthetic gene clusters for BLMs from Streptomyces verticillus ATCC15003, tallysomycins from Streptoalloteichus hindustanus E465-94 ATCC31158, and zorbamycin (ZBM) from Streptomyces flavoviridis SB9001. Comparative analysis of the three biosynthetic machineries provided the molecular basis for the formulation of hypotheses to engineer novel analogues. We now report engineered production of three new analogues, 6'-hydroxy-ZBM, BLM Z, and 6'-deoxy-BLM Z and the evaluation of their DNA cleavage activities as a measurement for their potential anticancer activity. Our findings unveiled: (i) the disaccharide moiety plays an important role in the DNA cleavage activity of BLMs and ZBMs, (ii) the ZBM disaccharide significantly enhances the potency of BLM, and (iii) 6'-deoxy-BLM Z represents the most potent BLM analogue known to date. The fact that 6'-deoxy-BLM Z can be produced in reasonable quantities by microbial fermentation should greatly facilitate follow-up mechanistic and preclinical studies to potentially advance this analogue into a clinical drug.

  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. C-S bond cleavage by a polyketide synthase domain.

    Science.gov (United States)

    Ma, Ming; Lohman, Jeremy R; Liu, Tao; Shen, Ben

    2015-08-18

    Leinamycin (LNM) is a sulfur-containing antitumor antibiotic featuring an unusual 1,3-dioxo-1,2-dithiolane moiety that is spiro-fused to a thiazole-containing 18-membered lactam ring. The 1,3-dioxo-1,2-dithiolane moiety is essential for LNM's antitumor activity, by virtue of its ability to generate an episulfonium ion intermediate capable of alkylating DNA. We have previously cloned and sequenced the lnm gene cluster from Streptomyces atroolivaceus S-140. In vivo and in vitro characterizations of the LNM biosynthetic machinery have since established that: (i) the 18-membered macrolactam backbone is synthesized by LnmP, LnmQ, LnmJ, LnmI, and LnmG, (ii) the alkyl branch at C-3 of LNM is installed by LnmK, LnmL, LnmM, and LnmF, and (iii) leinamycin E1 (LNM E1), bearing a thiol moiety at C-3, is the nascent product of the LNM hybrid nonribosomal peptide synthetase (NRPS)-acyltransferase (AT)-less type I polyketide synthase (PKS). Sulfur incorporation at C-3 of LNM E1, however, has not been addressed. Here we report that: (i) the bioinformatics analysis reveals a pyridoxal phosphate (PLP)-dependent domain, we termed cysteine lyase (SH) domain (LnmJ-SH), within PKS module-8 of LnmJ; (ii) the LnmJ-SH domain catalyzes C-S bond cleavage by using l-cysteine and l-cysteine S-modified analogs as substrates through a PLP-dependent β-elimination reaction, establishing l-cysteine as the origin of sulfur at C-3 of LNM; and (iii) the LnmJ-SH domain, sharing no sequence homology with any other enzymes catalyzing C-S bond cleavage, represents a new family of PKS domains that expands the chemistry and enzymology of PKSs and might be exploited to incorporate sulfur into polyketide natural products by PKS engineering.

  9. 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.

  10. Two Antagonistic MALT1 Auto-Cleavage Mechanisms Reveal a Role for TRAF6 to Unleash MALT1 Activation

    OpenAIRE

    Ginster, Stefanie; Bardet, Maureen; Unterreiner, Adeline; Malinverni, Claire; Renner, Florian; Lam, Stephen; Freuler, Felix; Gerrits, Bertran; Voshol, Johannes; Calzascia, Thomas; Régnier, Catherine H.; Renatus, Martin; Nikolay, Rainer; Israël, Laura; Bornancin, Frédéric

    2017-01-01

    The paracaspase MALT1 has arginine-directed proteolytic activity triggered by engagement of immune receptors. Recruitment of MALT1 into activation complexes is required for MALT1 proteolytic function. Here, co-expression of MALT1 in HEK293 cells, either with activated CARD11 and BCL10 or with TRAF6, was used to explore the mechanism of MALT1 activation at the molecular level. This work identified a prominent self-cleavage site of MALT1 isoform A (MALT1A) at R781 (R770 in MALT1B) and revealed ...

  11. Knockdown of pre-mRNA cleavage factor Im 25 kDa promotes neurite outgrowth

    Energy Technology Data Exchange (ETDEWEB)

    Fukumitsu, Hidefumi, E-mail: hfukumi@gifu-pu.ac.jp [Laboratory of Molecular Biology, Department of Biofunctional Analysis, Gifu Pharmaceutical University, Daigakunishi 1-25-4, Gifu 501 1196 (Japan); Soumiya, Hitomi; Furukawa, Shoei [Laboratory of Molecular Biology, Department of Biofunctional Analysis, Gifu Pharmaceutical University, Daigakunishi 1-25-4, Gifu 501 1196 (Japan)

    2012-09-07

    Highlights: Black-Right-Pointing-Pointer CFIm25 knockdown promoted NGF-induced neurite out growth from PC12 cells. Black-Right-Pointing-Pointer Depletion of CFIm25 did not influence the morphology of proliferating PC12 cells. Black-Right-Pointing-Pointer CFIm regulated NGF-induced neurite outgrowth via coordinating RhoA activity. Black-Right-Pointing-Pointer CFIm25 knockdown increase the number of primary dendrites of hippocampal neurons. -- Abstract: Mammalian precursor mRNA (pre-mRNA) cleavage factor I (CFIm) plays important roles in the selection of poly(A) sites in a 3 Prime -untranslated region (3 Prime -UTR), producing mRNAs with variable 3 Prime ends. Because 3 Prime -UTRs often contain cis elements that impact stability or localization of mRNA or translation, alternative polyadenylation diversifies utilization of primary transcripts in mammalian cells. However, the physiological role of CFIm remains unclear. CFIm acts as a heterodimer comprising a 25 kDa subunit (CFIm25) and one of the three large subunits-CFIm59, CFIm68, or CFIm72. CFIm25 binds directly to RNA and introduces and anchors the larger subunit. To examine the physiological roles of CFIm, we knocked down the CFIm25 gene in neuronal cells using RNA interference. Knockdown of CFIm25 increased the number of primary dendrites of developing hippocampal neurons and promoted nerve growth factor (NGF)-induced neurite extension from rat pheochromocytoma PC12 cells without affecting the morphology of proliferating PC12 cells. On the other hand, CFIm25 knockdown did not influence constitutively active or dominantly negative RhoA suppression or promotion of NGF-induced neurite extension from PC12 cells, respectively. Taken together, our results indicate that endogenous CFIm may promote neuritogenesis in developing neurons by coordinating events upstream of NGF-induced RhoA inactivation.

  12. Carbon-carbon bond cleavage in activation of the prodrug nabumetone

    DEFF Research Database (Denmark)

    Varfaj, Fatbardha; Zulkifli, Siti N A; Park, Hyoung-Goo;

    2014-01-01

    Carbon-carbon bond cleavage reactions are catalyzed by, among others, lanosterol 14-demethylase (CYP51), cholesterol side-chain cleavage enzyme (CYP11), sterol 17β-lyase (CYP17), and aromatase (CYP19). Because of the high substrate specificities of these enzymes and the complex nature of their su...

  13. Oxidative cleavage of benzylic C-N bonds under metal-free conditions.

    Science.gov (United States)

    Gong, Jin-Long; Qi, Xinxin; Wei, Duo; Feng, Jian-Bo; Wu, Xiao-Feng

    2014-10-14

    An interesting procedure for the oxidative cleavage of benzylic C-N bonds has been developed. Using TBAI as the catalyst and H2O2 as the oxidant, various benzylamines were transformed into their corresponding aromatic aldehydes in moderate to good yields. Notably, this is the first example of an oxidative cleavage of benzylic C-N bonds under metal-free conditions.

  14. Chromium(VI) reduction by catechol(amine)s results in DNA cleavage in vitro

    DEFF Research Database (Denmark)

    Pattison, D I; Davies, Michael Jonathan; Levina, A;

    2001-01-01

    ) or 4-tert-butylcatechol (5) do not damage DNA. The Cr(VI)/catechol(amine) reactions have been studied at low added H(2)O(2) concentrations, which lead to enhanced DNA cleavage with 1 and induce DNA cleavage with 4. The Cr(V) and organic intermediates generated by the reactions of Cr(VI) with 1 or 4...

  15. 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

    such as the cystic fibrosis transmembrane conductance regulator ( CFTR), transcription factors CREB-RP and OCT-I, 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...

  16. 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.

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

    Science.gov (United States)

    Shiju, C; Arish, D; Kumaresan, S

    2013-03-15

    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 H(2)O(2). 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.

  18. Broad specificity profiling of TALENs results in engineered nucleases with improved DNA-cleavage specificity.

    Science.gov (United States)

    Guilinger, John P; Pattanayak, Vikram; Reyon, Deepak; Tsai, Shengdar Q; Sander, Jeffry D; Joung, J Keith; Liu, David R

    2014-04-01

    Although transcription activator-like effector nucleases (TALENs) can be designed to cleave chosen DNA sequences, TALENs have activity against related off-target sequences. To better understand TALEN specificity, we profiled 30 unique TALENs with different target sites, array length and domain sequences for their abilities to cleave any of 10(12) potential off-target DNA sequences using in vitro selection and high-throughput sequencing. Computational analysis of the selection results predicted 76 off-target substrates in the human genome, 16 of which were accessible and modified by TALENs in human cells. The results suggest that (i) TALE repeats bind DNA relatively independently; (ii) longer TALENs are more tolerant of mismatches yet are more specific in a genomic context; and (iii) excessive DNA-binding energy can lead to reduced TALEN specificity in cells. Based on these findings, we engineered a TALEN variant that exhibits equal on-target cleavage activity but tenfold lower average off-target activity in human cells.

  19. Synthesis, characterization, cytotoxicity, DNA cleavage and antimicrobial activity of homodinuclear lanthanide complexes of phenylthioacetic acid

    Institute of Scientific and Technical Information of China (English)

    T. F. Abbs Fen Reji; A. Jeena Pearl; Bojaxa A. Rosy

    2013-01-01

    Lanthanide complexes of Eu(III), Gd(III), Nd(III), Sm(III), and Tb(III) with phenylthioacetic acid were synthesized and characterized by elemental analysis, mass, infrared radiation (IR), electronic spectra, molar conductance, thermogravimetric analysis (TGA), and powder X-ray diffraction (XRD). The results showed that the lanthanide complexes were homodinuclear in nature. The two lanthanide ions were bridged by eight oxygen atoms from four carboxylate groups. Thermal decomposition profiles were consis-tent with the proposed formulations. Powder XRD studies showed that all the complexes were amorphous in nature. Antimicrobial studies indicated that these complexes exhibited more activity than the ligand itself. The DNA cleavage activity of the ligand and its complexes were assayed on CT DNA using gel electrophoresis in the presence of H2O2. The result showed that the Eu(III) and Nd(III) complexes completely cleaved the DNA. The anticancer activities of the complexes were also studied towards human cervical cancer cell line (HeLa) and colon cancer cells (HCT116) and it was found that the Eu(III) and Nd(III) complexes were more active than the corresponding Gd(III), Sm(III), Tb(III) complexes and the free ligand on both the cancer cells.

  20. Soluble Axl is generated by ADAM10-dependent cleavage and associates with Gas6 in mouse serum.

    Science.gov (United States)

    Budagian, Vadim; Bulanova, Elena; Orinska, Zane; Duitman, Erwin; Brandt, Katja; Ludwig, Andreas; Hartmann, Dieter; Lemke, Greg; Saftig, Paul; Bulfone-Paus, Silvia

    2005-11-01

    Axl receptor tyrosine kinase exists as a transmembrane protein and as a soluble molecule. We show that constitutive and phorbol 12-myristate 13-acetate-induced generation of soluble Axl (sAxl) involves the activity of disintegrin-like metalloproteinase 10 (ADAM10). Spontaneous and inducible Axl cleavage was inhibited by the broad-spectrum metalloproteinase inhibitor GM6001 and by hydroxamate GW280264X, which is capable of blocking ADAM10 and ADAM17. Furthermore, murine fibroblasts deficient in ADAM10 expression exhibited a significant reduction in constitutive and inducible Axl shedding, whereas reconstitution of ADAM10 restored sAxl production, suggesting that ADAM10-mediated proteolysis constitutes a major mechanism for sAxl generation in mice. Partially overlapping 14-amino-acid stretch deletions in the membrane-proximal region of Axl dramatically affected sAxl generation, indicating that these regions are involved in regulating the access of the protease to the cleavage site. Importantly, relatively high circulating levels of sAxl are present in mouse sera in a heterocomplex with Axl ligand Gas6. Conversely, two other family members, Tyro3 and Mer, were not detected in mouse sera and conditioned medium. sAxl is constitutively released by murine primary cells such as dendritic and transformed cell lines. Upon immobilization, sAxl promoted cell migration and induced the phosphorylation of Axl and phosphatidylinositol 3-kinase. Thus, ADAM10-mediated generation of sAxl might play an important role in diverse biological processes.

  1. Soluble Axl Is Generated by ADAM10-Dependent Cleavage and Associates with Gas6 in Mouse Serum†

    Science.gov (United States)

    Budagian, Vadim; Bulanova, Elena; Orinska, Zane; Duitman, Erwin; Brandt, Katja; Ludwig, Andreas; Hartmann, Dieter; Lemke, Greg; Saftig, Paul; Bulfone-Paus, Silvia

    2005-01-01

    Axl receptor tyrosine kinase exists as a transmembrane protein and as a soluble molecule. We show that constitutive and phorbol 12-myristate 13-acetate-induced generation of soluble Axl (sAxl) involves the activity of disintegrin-like metalloproteinase 10 (ADAM10). Spontaneous and inducible Axl cleavage was inhibited by the broad-spectrum metalloproteinase inhibitor GM6001 and by hydroxamate GW280264X, which is capable of blocking ADAM10 and ADAM17. Furthermore, murine fibroblasts deficient in ADAM10 expression exhibited a significant reduction in constitutive and inducible Axl shedding, whereas reconstitution of ADAM10 restored sAxl production, suggesting that ADAM10-mediated proteolysis constitutes a major mechanism for sAxl generation in mice. Partially overlapping 14-amino-acid stretch deletions in the membrane-proximal region of Axl dramatically affected sAxl generation, indicating that these regions are involved in regulating the access of the protease to the cleavage site. Importantly, relatively high circulating levels of sAxl are present in mouse sera in a heterocomplex with Axl ligand Gas6. Conversely, two other family members, Tyro3 and Mer, were not detected in mouse sera and conditioned medium. sAxl is constitutively released by murine primary cells such as dendritic and transformed cell lines. Upon immobilization, sAxl promoted cell migration and induced the phosphorylation of Axl and phosphatidylinositol 3-kinase. Thus, ADAM10-mediated generation of sAxl might play an important role in diverse biological processes. PMID:16227584

  2. Mitochondrial tRNA cleavage by tRNA-targeting ribonuclease causes mitochondrial dysfunction observed in mitochondrial disease

    Energy Technology Data Exchange (ETDEWEB)

    Ogawa, Tetsuhiro, E-mail: atetsu@mail.ecc.u-tokyo.ac.jp; Shimizu, Ayano; Takahashi, Kazutoshi; Hidaka, Makoto; Masaki, Haruhiko, E-mail: amasaki@mail.ecc.u-tokyo.ac.jp

    2014-08-15

    Highlights: • MTS-tagged ribonuclease was translocated successfully to the mitochondrial matrix. • MTS-tagged ribonuclease cleaved mt tRNA and reduced COX activity. • Easy and reproducible method of inducing mt tRNA dysfunction. - Abstract: Mitochondrial DNA (mtDNA) is a genome possessed by mitochondria. Since reactive oxygen species (ROS) are generated during aerobic respiration in mitochondria, mtDNA is commonly exposed to the risk of DNA damage. Mitochondrial disease is caused by mitochondrial dysfunction, and mutations or deletions on mitochondrial tRNA (mt tRNA) genes are often observed in mtDNA of patients with the disease. Hence, the correlation between mt tRNA activity and mitochondrial dysfunction has been assessed. Then, cybrid cells, which are constructed by the fusion of an enucleated cell harboring altered mtDNA with a ρ{sup 0} cell, have long been used for the analysis due to difficulty in mtDNA manipulation. Here, we propose a new method that involves mt tRNA cleavage by a bacterial tRNA-specific ribonuclease. The ribonuclease tagged with a mitochondrial-targeting sequence (MTS) was successfully translocated to the mitochondrial matrix. Additionally, mt tRNA cleavage, which resulted in the decrease of cytochrome c oxidase (COX) activity, was observed.

  3. Real-time monitoring of DNAzyme cleavage process using fluorescent assay

    Institute of Scientific and Technical Information of China (English)

    Xiang Xian Meng; Xiao Hai Yang; Ke Min Wang; Wei Hong Tan; Qiu Ping Guo

    2009-01-01

    Detection of deoxyribozyme (DNAzyme) cleavage process usually needs complex and time-consuming radial labeling, gel electrophoresis and autoradiography. This paper reported an approach to detect DNAzyme cleavage process in real time using a fluorescence probe. The probe was employed as DNAzyme substrate to convert directly the cleavage information into fluorescence signal in real time. Compared with traditional approach, this non-isotope method not only brought a convenient means to monitor the DNAzyme cleavage reaction, but also offered abundant dynamic data for choosing potential gene therapeutic agents. It provides a new tool for DNAzyme research, as well as a new insight into research on human disease diagnosis. Based on this method, 8-17deoxyribozyme (8-17DNAzyme) against hepatitis C virus RNA (HCV-RNA) was designed and the cleavage process was studied in real time.

  4. Ultrasensitive monitoring of ribozyme cleavage product using molecular-beacon-ligation system

    Institute of Scientific and Technical Information of China (English)

    MENG XiangXian; TANG ZhiWen; WANG KeMin; TAN WeiHong; YANG XiaoHai; LI Jun; GUO QiuPing

    2007-01-01

    This paper reports a new approach to detect ribozyme cleavage product based on the molecular- beacon-ligation system. The molecular beacon, designed in such a way that one-half of its loop is complementary to ribozyme cleavage product, is used to monitor ligation process of RNA/DNA complex in a homogeneous solution and to convert directly cleavage product information into fluorescence signal. The method need not label ribozyme and ribozyme substrate, which is fast, simple and ultrasensitive for detection of cleavage product. Detection limit of the assay is 0.05 nmol/L. The cleavage product of hammerhead ribozyme against hepatitis C virus RNA (HCV-RNA) was detected perfectly based on this assay. Owing to its ultrasensitivity, excellent specificity, convenience and fidelity, this method might hold out great promise in ribozyme reaction and ribozyme gene therapy.

  5. DSP-PP precursor protein cleavage by tolloid-related-1 protein and by bone morphogenetic protein-1.

    Science.gov (United States)

    Ritchie, Helena H; Yee, Colin T; Tang, Xu-Na; Dong, Zhihong; Fuller, Robert S

    2012-01-01

    Dentin sialoprotein (DSP) and phosphophoryn (PP), acidic proteins critical to dentin mineralization, are translated from a single transcript as a DSP-PP precursor that undergoes specific proteolytic processing to generate DSP and PP. The cleavage mechanism continues to be controversial, in part because of the difficulty of obtaining DSP-PP from mammalian cells and dentin matrix. We have infected Sf9 cells with a recombinant baculovirus to produce large amounts of secreted DSP-PP(240), a variant form of rat DSP-PP. Mass spectrometric analysis shows that DSP-PP(240) secreted by Sf9 cells undergoes specific cleavage at the site predicted from the N-terminal sequence of PP extracted from dentin matrix: SMQG(447)↓D(448)DPN. DSP-PP(240) is cleaved after secretion by a zinc-dependent activity secreted by Sf9 cells, generating DSP(430) and PP(240) products that are stable in the medium. DSP-PP processing activity is constitutively secreted by Sf9 cells, but secretion is diminished 3 days after infection. Using primers corresponding to the highly conserved catalytic domain of Drosophila melanogaster tolloid (a mammalian BMP1 homolog), we isolated a partial cDNA for a Spodopotera frugiperda tolloid-related-1 protein (TLR1) that is 78% identical to Drosophila TLR1 but only 65% identical to Drosophila tolloid. Tlr1 mRNA decreased rapidly in Sf9 cells after baculovirus infection and was undetectable 4d after infection, paralleling the observed decrease in secretion of the DSP-PP(240) processing activity after infection. Human BMP1 is more similar to Sf9 and Drosophila TLR1 than to tolloid, and Sf9 TLR1 is more similar to BMP1 than to other mammalian homologs. Recombinant human BMP1 correctly processed baculovirus-expressed DSP-PP(240) in a dose-dependent manner. Together, these data suggest that the physiologically accurate cleavage of mammalian DSP-PP(240) in the Sf9 cell system represents the action of a conserved processing enzyme and support the proposed role of BMP1 in

  6. DSP-PP precursor protein cleavage by tolloid-related-1 protein and by bone morphogenetic protein-1.

    Directory of Open Access Journals (Sweden)

    Helena H Ritchie

    Full Text Available Dentin sialoprotein (DSP and phosphophoryn (PP, acidic proteins critical to dentin mineralization, are translated from a single transcript as a DSP-PP precursor that undergoes specific proteolytic processing to generate DSP and PP. The cleavage mechanism continues to be controversial, in part because of the difficulty of obtaining DSP-PP from mammalian cells and dentin matrix. We have infected Sf9 cells with a recombinant baculovirus to produce large amounts of secreted DSP-PP(240, a variant form of rat DSP-PP. Mass spectrometric analysis shows that DSP-PP(240 secreted by Sf9 cells undergoes specific cleavage at the site predicted from the N-terminal sequence of PP extracted from dentin matrix: SMQG(447↓D(448DPN. DSP-PP(240 is cleaved after secretion by a zinc-dependent activity secreted by Sf9 cells, generating DSP(430 and PP(240 products that are stable in the medium. DSP-PP processing activity is constitutively secreted by Sf9 cells, but secretion is diminished 3 days after infection. Using primers corresponding to the highly conserved catalytic domain of Drosophila melanogaster tolloid (a mammalian BMP1 homolog, we isolated a partial cDNA for a Spodopotera frugiperda tolloid-related-1 protein (TLR1 that is 78% identical to Drosophila TLR1 but only 65% identical to Drosophila tolloid. Tlr1 mRNA decreased rapidly in Sf9 cells after baculovirus infection and was undetectable 4d after infection, paralleling the observed decrease in secretion of the DSP-PP(240 processing activity after infection. Human BMP1 is more similar to Sf9 and Drosophila TLR1 than to tolloid, and Sf9 TLR1 is more similar to BMP1 than to other mammalian homologs. Recombinant human BMP1 correctly processed baculovirus-expressed DSP-PP(240 in a dose-dependent manner. Together, these data suggest that the physiologically accurate cleavage of mammalian DSP-PP(240 in the Sf9 cell system represents the action of a conserved processing enzyme and support the proposed role of BMP

  7. A METABOLITE OF METHOXYCHLOR,2,2-BIS(P-HYDOXYPHENYL)-1,1,1- TRICHLOROETHANE REDUCES TESTOSTERONE BIOSYNTHESIS IN RAT LEYDIG CELLS THROUGH SUPPRESSION OF STEADY-STATE MESSENGER RIBONUCLEIC ACID LEVELS OF THE CHOLESTEROL SIDE-CHAIN CLEAVAGE ENZYME

    Science.gov (United States)

    Postnatal development of Leydig cells involves transformation through three stages: progenitor, immature, and adult Leydig cells. The process of differentiation is accompanied by a progressive increase in the capacity of Leydig cells to produce testosterone (T). T promotes the ma...

  8. Computational redesign of endonuclease DNA binding and cleavage specificity

    Science.gov (United States)

    Ashworth, Justin; Havranek, James J.; Duarte, Carlos M.; Sussman, Django; Monnat, Raymond J.; Stoddard, Barry L.; Baker, David

    2006-06-01

    The reprogramming of DNA-binding specificity is an important challenge for computational protein design that tests current understanding of protein-DNA recognition, and has considerable practical relevance for biotechnology and medicine. Here we describe the computational redesign of the cleavage specificity of the intron-encoded homing endonuclease I-MsoI using a physically realistic atomic-level forcefield. Using an in silico screen, we identified single base-pair substitutions predicted to disrupt binding by the wild-type enzyme, and then optimized the identities and conformations of clusters of amino acids around each of these unfavourable substitutions using Monte Carlo sampling. A redesigned enzyme that was predicted to display altered target site specificity, while maintaining wild-type binding affinity, was experimentally characterized. The redesigned enzyme binds and cleaves the redesigned recognition site ~10,000 times more effectively than does the wild-type enzyme, with a level of target discrimination comparable to the original endonuclease. Determination of the structure of the redesigned nuclease-recognition site complex by X-ray crystallography confirms the accuracy of the computationally predicted interface. These results suggest that computational protein design methods can have an important role in the creation of novel highly specific endonucleases for gene therapy and other applications.

  9. DNA targeting and cleavage by an engineered metalloprotein dimer.

    Science.gov (United States)

    Wong-Deyrup, Siu Wah; Prasannan, Charulata; Dupureur, Cynthia M; Franklin, Sonya J

    2012-03-01

    Nature has illustrated through numerous examples that protein dimerization has structural and functional advantages. We previously reported the design and characterization of an engineered "metallohomeodomain" protein (C2) based on a chimera of the EF-hand Ca-binding motif and the helix-turn-helix motif of homeodomains (Lim and Franklin in Protein Sci. 15:2159-2165, 2004). This small metalloprotein binds the hard metal ions Ca(II) and Ln(III) and interacts with DNA with modest sequence preference and affinity, yet exhibits only residual DNA cleavage activity. Here we have achieved substantial improvement in function by constructing a covalent dimer of this C2 module (F2) to create a larger multidomain protein. As assayed via fluorescence spectroscopy, this F2 protein binds Ca(II) more avidly (25-fold) than C2 on a per-domain basis; in gel shift selection experiments, metallated F2 exhibits a specificity toward 5'-TAATTA-3' sequences. Finally, Ca(2)F2 cleaves plasmid DNA and generates a linear product in a Ca(II)-dependent way, unlike the CaC2 monomer. To the best of our knowledge this activation of Ca(II) in the context of an EF-hand binding motif is unique and represents a significant step forward in the design of artificial metallonucleases by utilizing biologically significant metal ions.

  10. Sequence-specific interactions of drugs interfering with the topoisomerase-DNA cleavage complex.

    Science.gov (United States)

    Palumbo, Manlio; Gatto, Barbara; Moro, Stefano; Sissi, Claudia; Zagotto, Giuseppe

    2002-07-18

    DNA-processing enzymes, such as the topoisomerases (tops), represent major targets for potent anticancer (and antibacterial) agents. The drugs kill cells by poisoning the enzymes' catalytic cycle. Understanding the molecular details of top poisoning is a fundamental requisite for the rational development of novel, more effective antineoplastic drugs. In this connection, sequence-specific recognition of the top-DNA complex is a key step to preferentially direct the action of the drugs onto selected genomic sequences. In fact, the (reversible) interference of drugs with the top-DNA complex exhibits well-defined preferences for DNA bases in the proximity of the cleavage site, each drug showing peculiarities connected to its structural features. A second level of selectivity can be observed when chemically reactive groups are present in the structure of the top-directed drug. In this case, the enzyme recognizes or generates a unique site for covalent drug-DNA binding. This will further subtly modulate the drug's efficiency in stimulating DNA damage at selected sites. Finally, drugs can discriminate not only among different types of tops, but also among different isoenzymes, providing an additional level of specific selection. Once the molecular basis for DNA sequence-dependent recognition has been established, the above-mentioned modes to generate selectivity in drug poisoning can be rationally exploited, alone or in combination, to develop tailor-made drugs targeted at defined loci in cancer cells.

  11. Synthesis, characterization, DNA binding, DNA cleavage, protein binding and cytotoxic activities of Ru(II) complexes.

    Science.gov (United States)

    Thota, Sreekanth; Vallala, Srujana; Yerra, Rajeshwar; Rodrigues, Daniel Alencar; Raghavendra, Nulgumnalli Manjunathaiah; Barreiro, Eliezer J

    2016-01-01

    We report on the synthesis of novel Ru(II) compounds (Ru-1 to Ru-8) bearing R-pdc, 4-Cl-pbinh ligands (where R=4-CF3, 4-F, 4-OH pdc=3-phenyl-5-(1H-pyrrol-2-yl)-4,5-dihydro-1H-pyrazole-1-carbothioamide, pbinh=phenoxybenzylidene isonicotinyl hydrazides) and their in vitro antitumor activity toward the cell lines murine leukemia L1210, human lymphocyte CEM, human epithelial cervical carcinoma HeLa, BEL-7402 and Molt4/C8. Some of the complexes exhibited more potent antiproliferative activity against cell lines than the standard drug cisplatin. Ruthenium complex Ru-2 displayed potent cytotoxicity with than that of cisplatin. DNA-binding, DNA cleavage and protein binding properties of ruthenium complexes with these ligands are reported. Interactions of these ruthenium complexes with DNA revealed an intercalative mode of binding between them. Synchronous fluorescence spectra proved that the interaction of ruthenium complexes with bovine serum albumin (BSA) resulted in a conformational change of the latter.

  12. 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.

  13. Synthesis, characterization, biological activity and DNA cleavage studies of tridentate Schiff bases and their Co(II complexes

    Directory of Open Access Journals (Sweden)

    P. Kavitha

    2016-01-01

    Full Text Available In the present study a series of Co(II complexes of formyl chromone Schiff bases have been synthesized characterized by analytical, molar conductance, IR, electronic, magnetic susceptibility, thermal, fluorescence and powder XRD measurements and screened for various biological activities (antimicrobial, antioxidant, nematicidal, DNA cleavage and cytotoxicity. In all the Co(II complexes 1:2 metal to ligand molar ratio was obtained from analytical data. The molar conductance data confirm that all complexes are non-electrolytic in nature. Based on the electronic and magnetic data, an octahedral geometry is ascribed for all the Co(II complexes. Thermal behaviour of the synthesized complexes illustrates the general decomposition patterns of the complexes. The X-ray analysis data show that all the Co(II complexes have triclinic crystal system with different unit cell parameters. Metal complexes have greater antimicrobial activity than ligands. Antioxidant and nematicidal activities indicate that the ligands exhibit greater activity when compared to their respective Co(II complexes. All ligands and Co(II complexes of HL1 and HL2 showed considerable anticancer activity against Raw, MCF-7 and COLO 205 cell lines. All ligands and their Co(II complexes showed more pronounced DNA cleavage activity in the presence of H2O2.

  14. Nuclear Localization and Cleavage of STAT6 Is Induced by Kaposi’s Sarcoma-Associated Herpesvirus for Viral Latency

    Science.gov (United States)

    Zhang, Liming; Li, Yuhong; Feng, Yanling; Xu, Jianqing; Wang, Bin; Yuan, Zhenghong; Robertson, Erle S.; Cai, Qiliang

    2017-01-01

    Emerging evidence implies that STAT6 plays an important role in both the adaptive and innate immune responses to virus infection. Kaposi’s sarcoma-associated herpesvirus (KSHV) is an oncogenic γ-herpesvirus agent associated with several human malignancies, including Kaposi’s sarcoma (KS) and primary effusion lymphomas (PELs). Previously, we demonstrated that KSHV blocks IL-4-induced STAT6 phosphorylation and retains a basal IL-13/STAT6 constitutive activation for cell survival and proliferation. However, the mechanism by which KSHV regulates STAT6 remains largely unknown. Here, we found that KSHV-encoded LANA interacts with STAT6 and promotes nuclear localization of STAT6 independent of the tyrosine 641-phosphorylation state. Moreover, nuclear localization of STAT6 is also dramatically increased in KS tissue. The latent antigen LANA induces serine protease-mediated cleavage of STAT6 in the nucleus, where the cleaved STAT6 lacking transactivation domain functions as a dominant-negative regulator to repress transcription of Replication and Transcription Activator (RTA) and in turn shut off viral lytic replication. Blockade of STAT6 by small interference RNA dramatically enhances expression of RTA, and in turn reduces KSHV-infected endothelial cell growth and colony formation. Taken together, these results suggest that nuclear localization and cleavage of STAT6 is important for modulating the viral latency and pathogenesis of KSHV. PMID:28099521

  15. Cleavage of the angiotensin II type 1 receptor and nuclear accumulation of the cytoplasmic carboxy-terminal fragment.

    Science.gov (United States)

    Cook, Julia L; Mills, Sarah J; Naquin, Ryan T; Alam, Jawed; Re, Richard N

    2007-04-01

    Our published studies show that the distribution of the ANG II type 1 (AT(1)) receptor (AT(1)R), expressed as a enhanced yellow fluorescent fusion (YFP) protein (AT(1)R/EYFP), is altered upon cellular treatment with ANG II or coexpression with intracellular ANG II. AT(1)R accumulates in nuclei of cells only in the presence of ANG II. Several transmembrane receptors are known to accumulate in nuclei, some as holoreceptors and others as cleaved receptor products. The present study was designed to determine whether the AT(1)R is cleaved before nuclear transport. A plasmid encoding a rat AT(1)R labeled at the amino terminus with enhanced cyan fluorescent protein (CFP) and at the carboxy terminus with EYFP was employed. Image analyses of this protein in COS-7 cells, CCF-STTG1 glial cells, and A10 vascular smooth muscle cells show the two fluorescent moieties to be largely spatially colocalized in untreated cells. ANG II treatment, however, leads to a separation of the fluorescent moieties with yellow fluorescence accumulating in more than 30% of cellular nuclei. Immunoblot analyses of extracts and conditioned media from transfected cells indicate that the CFP domain fused to the extracellular amino-terminal AT(1)R domain is cleaved from the membrane and that the YFP domain, together with the intracellular cytoplasmic carboxy terminus of the AT(1)R, is also cleaved from the membrane-bound receptor. The carboxy terminus of the AT(1)R is essential for cleavage; cleavage does not occur in protein deleted with respect to this region. Overexpressed native AT(1)R (nonfusion) is also cleaved; the intracellular 6-kDa cytoplasmic domain product accumulates to a significantly higher level with ANG II treatment.

  16. Binding of factor VIII to von willebrand factor is enabled by cleavage of the von Willebrand factor propeptide and enhanced by formation of disulfide-linked multimers.

    Science.gov (United States)

    Bendetowicz, A V; Morris, J A; Wise, R J; Gilbert, G E; Kaufman, R J

    1998-07-15

    von Willebrand factor (vWF) is a multimeric adhesive glycoprotein with one factor VIII binding site/subunit. Prior reports suggest that posttranslational modifications of vWF, including formation of N-terminal intersubunit disulfide bonds and subsequent cleavage of the propeptide, influence availability and/or affinity of factor VIII binding sites. We found that deletion of the vWF propeptide produced a dimeric vWF molecule lacking N-terminal intersubunit disulfide bonds. This molecule bound fluorescein-labeled factor VIII with sixfold lower affinity than multimeric vWF in an equilibrium flow cytometry assay (approximate KDs, 5 nmol/L v 0.9 nmol/L). Coexpression of propeptide-deleted vWF with the vWF propeptide in trans yielded multimeric vWF that displayed increased affinity for factor VIII. Insertion of an alanine residue at the N-terminus of the mature vWF subunit destroyed binding to factor VIII, indicating that the native mature N-terminus is required for factor VIII binding. The requirement for vWF propeptide cleavage was shown by (1) a point mutation of the vWF propeptide cleavage site yielding pro-vWF that was defective in factor VIII binding and (2) correlation between efficiency of intracellular propeptide cleavage and factor VIII binding. Furthermore, in a cell-free system, addition of the propeptide-cleaving enzyme PACE/furin enabled factor VIII binding in parallel with propeptide cleavage. Our results indicate that high-affinity factor VIII binding sites are located on N-terminal disulfide-linked vWF subunits from which the propeptide has been cleaved.

  17. BCL10GFP fusion protein as a substrate for analysis of determinants required for Mucosa-Associated Lymphoid Tissue 1 (MALT1-mediated cleavage

    Directory of Open Access Journals (Sweden)

    Jou Shin-Yi

    2012-10-01

    Full Text Available Abstract Background MALT1 belongs to a family of paracaspase and modulates NF-κB signaling pathways through its scaffolding function and proteolytic activity. MALT1 cleaves protein substrates after a positively charged Arginine residue. BCL10, a 233 amino acids polypeptide, is identified as one of the MALT1 proteolytic substrates. MALT1 cleaves BCL10 at the C-terminal end of Arg228. A mere 5 amino acids difference between the substrate and the proteolytic product made it difficult to tell whether the cleavage event took place by using a simple western blot analysis. Here, BCL10GFP was constructed and utilized to examine the specificity and domain determinants for MALT1 cleavage in cells. Methods Various BCL10GFP constructs were transfected into HEK293T cell with MALT1 construct by using calcium phosphate-DNA precipitation method. Lysates of transfectants were resolved by SDS/PAGE and analyzed by western blot analysis. Results BCL10GFP was proteolytically processed by MALT1 as BCL10. The integrity of caspase recruitment domain (CARD and MALT1-interacting domain on BCL10 were required for MALT1 proteolytic activity. Besides the invariant P1 cleavage site Arg228, P4 Leu225 played a role in defining BCL10 as a good substrate for MALT1. Conclusions We offered a way of monitoring the catalytic activity of MALT1 in HEK293T cells using BCL10GFP as a substrate. BCL10GFP can be utilized as a convenient tool for studying the determinants for efficient MALT1 cleavage in HEK293T cells

  18. Molecular determinants of survival motor neuron (SMN protein cleavage by the calcium-activated protease, calpain.

    Directory of Open Access Journals (Sweden)

    Jennifer L Fuentes

    Full Text Available Spinal muscular atrophy (SMA is a leading genetic cause of childhood mortality, caused by reduced levels of survival motor neuron (SMN protein. SMN functions as part of a large complex in the biogenesis of small nuclear ribonucleoproteins (snRNPs. It is not clear if defects in snRNP biogenesis cause SMA or if loss of some tissue-specific function causes disease. We recently demonstrated that the SMN complex localizes to the Z-discs of skeletal and cardiac muscle sarcomeres, and that SMN is a proteolytic target of calpain. Calpains are implicated in muscle and neurodegenerative disorders, although their relationship to SMA is unclear. Using mass spectrometry, we identified two adjacent calpain cleavage sites in SMN, S192 and F193. Deletion of small motifs in the region surrounding these sites inhibited cleavage. Patient-derived SMA mutations within SMN reduced calpain cleavage. SMN(D44V, reported to impair Gemin2 binding and amino-terminal SMN association, drastically inhibited cleavage, suggesting a role for these interactions in regulating calpain cleavage. Deletion of A188, a residue mutated in SMA type I (A188S, abrogated calpain cleavage, highlighting the importance of this region. Conversely, SMA mutations that interfere with self-oligomerization of SMN, Y272C and SMNΔ7, had no effect on cleavage. Removal of the recently-identified SMN degron (Δ268-294 resulted in increased calpain sensitivity, suggesting that the C-terminus of SMN is important in dictating availability of the cleavage site. Investigation into the spatial determinants of SMN cleavage revealed that endogenous calpains can cleave cytosolic, but not nuclear, SMN. Collectively, the results provide insight into a novel aspect of the post-translation regulation of SMN.

  19. Direct proteolytic cleavage of NLRP1B is necessary and sufficient for inflammasome activation by anthrax lethal factor.

    Directory of Open Access Journals (Sweden)

    Joseph Chavarría-Smith

    Full Text Available Inflammasomes are multimeric protein complexes that respond to infection by recruitment and activation of the Caspase-1 (CASP1 protease. Activated CASP1 initiates immune defense by processing inflammatory cytokines and by causing a rapid and lytic cell death called pyroptosis. Inflammasome formation is orchestrated by members of the nucleotide-binding domain and leucine-rich repeat (NLR or AIM2-like receptor (ALR protein families. Certain NLRs and ALRs have been shown to function as direct receptors for specific microbial ligands, such as flagellin or DNA, but the molecular mechanism responsible for activation of most NLRs is still poorly understood. Here we determine the mechanism of activation of the NLRP1B inflammasome in mice. NLRP1B, and its ortholog in rats, is activated by the lethal factor (LF protease that is a key virulence factor secreted by Bacillus anthracis, the causative agent of anthrax. LF was recently shown to cleave mouse and rat NLRP1 directly. However, it is unclear if cleavage is sufficient for NLRP1 activation. Indeed, other LF-induced cellular events have been suggested to play a role in NLRP1B activation. Surprisingly, we show that direct cleavage of NLRP1B is sufficient to induce inflammasome activation in the absence of LF. Our results therefore rule out the need for other LF-dependent cellular effects in activation of NLRP1B. We therefore propose that NLRP1 functions primarily as a sensor of protease activity and thus could conceivably detect a broader spectrum of pathogens than just B. anthracis. By adding proteolytic cleavage to the previously established ligand-receptor mechanism of NLR activation, our results illustrate the remarkable flexibility with which the NLR architecture can be deployed for the purpose of pathogen-detection and host defense.

  20. In vitro proteolytic cleavage of Gazdar murine sarcoma virus p65gag.

    OpenAIRE

    Maxwell, S.; Arlinghaus, R B

    1981-01-01

    Moloney murine leukemia virus, disrupted in concentrations of 0.1 to 0.5% Nonidet P-40, catalyzed the cleavage of p65, the gag gene polyprotein of the Gazdar strain of murine sarcoma virus, into polypeptides with sizes and antigenic determinants of murine leukemia virus-specified p30, p15, pp12, and p10. Cleavage performed in the presence of 0.15% Nonidet P-40 in water yielded polypeptides of approximately 40,000 (P40) and 25,000 (P25) Mr. In vitro cleavage performed in a buffered solution co...

  1. Enzymology of the carotenoid cleavage dioxygenases: reaction mechanisms, inhibition and biochemical roles.

    Science.gov (United States)

    Harrison, Peter J; Bugg, Timothy D H

    2014-02-15

    Carotenoid cleavage dioxygenases (CCDs) are a large family of non-heme iron (II) dependent enzymes. CCDs catalyse the selective oxidative cleavage of carotenoids to produce apocarotenoids. Apocarotenoid derived molecules form important signalling molecules in plants in the form of abscisic acid and strigolactone and in mammals in the form of retinal. Very little is known biochemically about the CCDs and only a handful of CCDs have been biochemically characterised. Mechanistically, debate surrounds whether CCDs utilise a mono or dioxygenase mechanism. Here, we review the biochemical roles of CCDs, discuss the mechanisms by which CCD cleavage is proposed to occur, and discuss recent reports of selective CCD enzyme inhibitors.

  2. Guest-host interactions in the cleavage of phenylphenyl acetates by -cyclodextrin in alkaline medium

    Indian Academy of Sciences (India)

    V Raj; T Chandrakala; K Rajasekaran

    2008-05-01

    Kinetics of cleavage of phenylphenyl acetates (PPA) and several para-substituted PPAs in basic aqueous sodium carbonate-bicarbonate buffer containing -cyclodextrin (CD) have been studied. The reaction exhibits saturation type kinetics and CD accelerates the rate of cleavage by the formation of 1G : 1H inclusion complex. The kinetic results indicate that aryloxy moiety of PPA is included in the hydrophobic cavity of CD. The overall rate constants for the cleavage of the [CD-ester] complex correlate with the Hammett -constants and Hansch hydrophobicity parameters . At higher concentrations of CD, there is an additional catalysis due to the formation of weak 1G : 2H complex.

  3. Up-regulation of c-Jun inhibits proliferation and induces apoptosis via caspase-triggered c-Abl cleavage in human multiple myeloma.

    Science.gov (United States)

    Podar, Klaus; Raab, Marc S; Tonon, Giovanni; Sattler, Martin; Barilà, Daniela; Zhang, Jing; Tai, Yu-Tzu; Yasui, Hiroshi; Raje, Noopur; DePinho, Ronald A; Hideshima, Teru; Chauhan, Dharminder; Anderson, Kenneth C

    2007-02-15

    Here we show the antimyeloma cytotoxicity of adaphostin and carried out expression profiling of adaphostin-treated multiple myeloma (MM) cells to identify its molecular targets. Surprisingly, c-Jun was the most up-regulated gene even at the earliest point of analysis (2 h). We also observed adaphostin-induced c-Abl cleavage in immunoblot analysis. Proteasome inhibitor bortezomib, but not melphalan or dexamethasone, induced similar effects, indicating unique agent-dependent mechanisms. Using caspase inhibitors, as well as caspase-resistant mutants of c-Abl (TM-c-Abl and D565A-Abl), we then showed that c-Abl cleavage in MM cells requires caspase activity. Importantly, both overexpression of the c-Abl fragment or c-Jun and knockdown of c-Abl and c-Jun expression by small interfering RNA confirmed that adaphostin-induced c-Jun up-regulation triggers downstream caspase-mediated c-Abl cleavage, inhibition of MM cell growth, and induction of apoptosis. Finally, our data suggest that this mechanism may not only be restricted to MM but may also be important in a broad range of malignancies including erythroleukemia and solid tumors.

  4. Mechanistic Investigation of Acid-Catalyzed Cleavage of Aryl-Ether Linkages: Implications for Lignin Depolymerization

    Energy Technology Data Exchange (ETDEWEB)

    Sturgeon, M. R.; Kim, S.; Chmely, S. C.; Foust, T. D.; Beckham, G. T.

    2013-01-01

    Carbon-oxygen bonds are the primary inter-monomer linkages lignin polymers in plant cell walls, and as such, catalyst development to cleave these linkages is of paramount importance to deconstruct biomass to its constituent monomers for the production of renewable fuels and chemicals. For many decades, acid catalysis has been used to depolymerize lignin. Lignin is a primary component of plant cell walls, which is connected primarily by aryl-ether linkages, and the mechanism of its deconstruction by acid is not well understood, likely due to its heterogeneous and complex nature compared to cellulose. For effective biomass conversion strategies, utilization of lignin is of significant relevance and as such understanding the mechanisms of catalytic lignin deconstruction to constituent monomers and oligomers is of keen interest. Here, we present a comprehensive experimental and theoretical study of the acid catalysis of a range of dimeric species exhibiting the b-O-4 linkage, the most common inter-monomer linkage in lignin. We demonstrate that the presence of a phenolic species dramatically increases the rate of cleavage in acid at 150 degrees C. Quantum mechanical calculations on dimers with the para-hydroxyl group demonstrate that this acid-catalyzed pathway differs from the nonphenolic dimmers. Importantly, this result implies that depolymerization of native lignin in the plant cell wall will proceed via an unzipping mechanism wherein b-O-4 linkages will be cleaved from the ends of the branched, polymer chains inwards toward the center of the polymer. To test this hypothesis further, we synthesized a homopolymer of b-O-4 with a phenolic hydroxyl group, and demonstrate that it is cleaved in acid from the end containing the phenolic hydroxyl group. This result suggests that genetic modifications to lignin biosynthesis pathways in plants that will enable lower severity processes to fractionate lignin for upgrading and for easier access to the carbohydrate fraction of

  5. Ku-mediated coupling of DNA cleavage and repair during programmed genome rearrangements in the ciliate Paramecium tetraurelia.

    Directory of Open Access Journals (Sweden)

    Antoine Marmignon

    2014-08-01

    Full Text Available During somatic differentiation, physiological DNA double-strand breaks (DSB can drive programmed genome rearrangements (PGR, during which DSB repair pathways are mobilized to safeguard genome integrity. Because of their unique nuclear dimorphism, ciliates are powerful unicellular eukaryotic models to study the mechanisms involved in PGR. At each sexual cycle, the germline nucleus is transmitted to the progeny, but the somatic nucleus, essential for gene expression, is destroyed and a new somatic nucleus differentiates from a copy of the germline nucleus. In Paramecium tetraurelia, the development of the somatic nucleus involves massive PGR, including the precise elimination of at least 45,000 germline sequences (Internal Eliminated Sequences, IES. IES excision proceeds through a cut-and-close mechanism: a domesticated transposase, PiggyMac, is essential for DNA cleavage, and DSB repair at excision sites involves the Ligase IV, a specific component of the non-homologous end-joining (NHEJ pathway. At the genome-wide level, a huge number of programmed DSBs must be repaired during this process to allow the assembly of functional somatic chromosomes. To understand how DNA cleavage and DSB repair are coordinated during PGR, we have focused on Ku, the earliest actor of NHEJ-mediated repair. Two Ku70 and three Ku80 paralogs are encoded in the genome of P. tetraurelia: Ku70a and Ku80c are produced during sexual processes and localize specifically in the developing new somatic nucleus. Using RNA interference, we show that the development-specific Ku70/Ku80c heterodimer is essential for the recovery of a functional somatic nucleus. Strikingly, at the molecular level, PiggyMac-dependent DNA cleavage is abolished at IES boundaries in cells depleted for Ku80c, resulting in IES retention in the somatic genome. PiggyMac and Ku70a/Ku80c co-purify as a complex when overproduced in a heterologous system. We conclude that Ku has been integrated in the Paramecium

  6. Ku-mediated coupling of DNA cleavage and repair during programmed genome rearrangements in the ciliate Paramecium tetraurelia.

    Science.gov (United States)

    Marmignon, Antoine; Bischerour, Julien; Silve, Aude; Fojcik, Clémentine; Dubois, Emeline; Arnaiz, Olivier; Kapusta, Aurélie; Malinsky, Sophie; Bétermier, Mireille

    2014-08-01

    During somatic differentiation, physiological DNA double-strand breaks (DSB) can drive programmed genome rearrangements (PGR), during which DSB repair pathways are mobilized to safeguard genome integrity. Because of their unique nuclear dimorphism, ciliates are powerful unicellular eukaryotic models to study the mechanisms involved in PGR. At each sexual cycle, the germline nucleus is transmitted to the progeny, but the somatic nucleus, essential for gene expression, is destroyed and a new somatic nucleus differentiates from a copy of the germline nucleus. In Paramecium tetraurelia, the development of the somatic nucleus involves massive PGR, including the precise elimination of at least 45,000 germline sequences (Internal Eliminated Sequences, IES). IES excision proceeds through a cut-and-close mechanism: a domesticated transposase, PiggyMac, is essential for DNA cleavage, and DSB repair at excision sites involves the Ligase IV, a specific component of the non-homologous end-joining (NHEJ) pathway. At the genome-wide level, a huge number of programmed DSBs must be repaired during this process to allow the assembly of functional somatic chromosomes. To understand how DNA cleavage and DSB repair are coordinated during PGR, we have focused on Ku, the earliest actor of NHEJ-mediated repair. Two Ku70 and three Ku80 paralogs are encoded in the genome of P. tetraurelia: Ku70a and Ku80c are produced during sexual processes and localize specifically in the developing new somatic nucleus. Using RNA interference, we show that the development-specific Ku70/Ku80c heterodimer is essential for the recovery of a functional somatic nucleus. Strikingly, at the molecular level, PiggyMac-dependent DNA cleavage is abolished at IES boundaries in cells depleted for Ku80c, resulting in IES retention in the somatic genome. PiggyMac and Ku70a/Ku80c co-purify as a complex when overproduced in a heterologous system. We conclude that Ku has been integrated in the Paramecium DNA cleavage

  7. Lanthanide-Mediated Dephosphorylation Used for Peptide Cleavage during Solid Phase Peptide Synthesis

    Directory of Open Access Journals (Sweden)

    Byunghee Yoo

    2013-04-01

    Full Text Available Lanthanide(III ions can accelerate the hydrolysis of phosphomonoesters and phosphodiesters in neutral aqueous solution. In this paper, lanthanide-mediated dephosphorylation has been applied in aqueous media as an orthogonal cleavage condition that can be employed in conventional solid phase peptide synthesis (SPPS. A phosphorylated polymeric support for SPPS was developed using Boc chemistry. The cleavage of resin-bound phosphates was investigated with the addition of Eu(III, Yb(III, acid or base, a mixture of solvents or different temperatures. To demonstrate the utility of this approach for SPPS, a peptide sequence was synthesized on a phosphorylated polymeric support and quantitatively cleaved with lanthanide ions in neutral aqueous media. The protecting groups for side chains were retained during peptide cleavage using lanthanide ions. This new methodology provides a mild orthogonal cleavage condition of phosphoester as a linker during SPPS.

  8. Facile P-C/C-H Bond-Cleavage Reactivity of Nickel Bis(diphosphine) Complexes.

    Science.gov (United States)

    Zhang, Shaoguang; Li, Haixia; Appel, Aaron M; Hall, Michael B; Bullock, R Morris

    2016-07-04

    Unusual cleavage of P-C and C-H bonds of the P2 N2 ligand, in heteroleptic [Ni(P2 N2 )(diphosphine)](2+) complexes under mild conditions, results in the formation of an iminium formyl nickelate featuring a C,P,P-tridentate coordination mode. The structures of both the heteroleptic [Ni(P2 N2 )(diphosphine)](2+) complexes and the resulting iminium formyl nickelate have been characterized by NMR spectroscopy and single-crystal X-ray diffraction analysis. Density functional theory (DFT) calculations were employed to investigate the mechanism of the P-C/C-H bond cleavage, which involves C-H bond cleavage, hydride rotation, Ni-C/P-H bond formation, and P-C bond cleavage.

  9. Photolytic Cleavage and Condensation Reactions of Cyclohexa-2,4-dienones with Diamines

    Directory of Open Access Journals (Sweden)

    Sung Kee Chung

    2000-07-01

    Full Text Available Cyclohexa-2,4-diene-1-one sulfone derivate undergoes ring cleavage to afford bis-amides containing a diene moiety on irradiation with visible light in the presence of various diamines.

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

    Science.gov (United States)

    Zhang, Shaofeng; Basile, Franco

    2011-01-01

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

  11. Biomarkers derived from heterolytic and homolytic cleavage of allylic hydroperoxides resulting from alkenone autoxidation

    Digital Repository Service at National Institute of Oceanography (India)

    Rontania, J.F.; Harji, R.; Volkmanc, J.K.

    , hydroxyacids and alkyldiols resulted from the reduction during the NaBH4 treatment of the corresponding aldehydes, ketoxyacids and ketoxyaldehydes formed from heterolytic or hemolytic cleavages of allylic hydroperoxyl groups resulting from the oxidation...

  12. From Bedding To Cleavage: The Evolution Of Clay Fabric Near A Thrust.

    Science.gov (United States)

    Boiron, T.; Aubourg, C.; Valier, P., Sr.

    2015-12-01

    In foothills, the propagation of faults within incompetent bed is potentially accompanied by the development of oblique cleavage. This is particularly demonstrated in the Southern Pyrenees where the out-of-sequence propagation of a flat thrust imposed the development of oblique cleavage within the flat-lying Pamplona marls. Over hundreds of meters, it is possible to trace step by step the cleavage development. The horizontal bedding is gradually superimposed by oblique cleavage (dip ~60°N). At the end of the studied outcrop, a pervasive ~mm spaced cleavage is observed. The anisotropy of magnetic susceptibility (AMS) is a classic tool for study the deformation in shales. In the Pamplona marls, AMS is essentially controlled by clays fabric. AMS shows that the degree of anisotropy Pj is not the best parameter to highlight the degree of deformation of marls. This parameter is positively correlated to the bulk magnetic susceptibility Km (~10-4 SI). On the contrary, the shape parameter T is more consistent with the degree of deformation: higher is the degree of deformation observed in outcrop (occurrence of pervasive cleavage), lower is the T parameter. As m-spaced cleavage starts to develop, the shape parameter T decreases linearly from ~0.8 to ~0.2, reflecting a gradual disorganization of clay particles. Despite the development of mm-spaced cleavage, the magnetic fabric remains oblate and is still dominated by the sedimentary fabric. This means that the bulk fabric of clay particles remains parallel to the bedding plane. Our study demonstrates that AMS is a powerful tool to trace the deformation of clay rocks and that the study of the shape parameter T is a robust and fast gauge of clays fabric. .

  13. 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...... LNA-modified DNA oligonucleotides. Furthermore, introduction of LNA nucleotides protects against cleavage by the restriction endonucleases PvuII, PstI, SacI, KpnI and EcoRI....

  14. Photocatalytic C-C Bond Cleavage and Amination of Cycloalkanols by Cerium(III) Chloride Complex.

    Science.gov (United States)

    Guo, Jing-Jing; Hu, Anhua; Chen, Yilin; Sun, Jianfeng; Tang, Haoming; Zuo, Zhiwei

    2016-12-05

    A general strategy for the cleavage and amination of C-C bonds of cycloalkanols has been achieved through visible-light-induced photoredox catalysis utilizing a cerium(III) chloride complex. This operationally simple methodology has been successfully applied to a wide array of unstrained cyclic alcohols, and represents the first example of catalytic C-C bond cleavage and functionalization of unstrained secondary cycloalkanols.

  15. A strategy for developing a hammerhead ribozyme for selective RNA cleavage depending on substitutional RNA editing

    OpenAIRE

    Fukuda, Masatora; Kurihara, Kei; Tanaka, Yasuyoshi; Deshimaru, Masanobu

    2012-01-01

    Engineered site-specific RNA cleavage is widely used for gene regulation, RNA mapping, and synthetic RNA production. Here the authors extend the range of engineered recognition selectivity to include cleavage of sequence motifs containing naturally occurring base modifications. They describe and implement a designer hammerhead ribozyme that cleaves a target sequence 1 nt from a site of adenosine to inosine (A-to-I) or cytosine to uracil (C-to-U) editing in synthetic or physiological mRNA cont...

  16. 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...

  17. 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.

  18. DNA binding, DNA cleavage, and cytotoxicity studies of two new copper (II) complexes.

    Science.gov (United States)

    Kashanian, Soheila; Khodaei, Mohammad Mehdi; Roshanfekr, Hamideh; Shahabadi, Nahid; Rezvani, Alireza; Mansouri, Ghobad

    2011-05-01

    The DNA binding behavior of [Cu(phen)(phen-dione)Cl]Cl (1) and [Cu(bpy)(phen-dione)Cl]Cl (2) was studied with a series of techniques including UV-vis absorption, circular dichroism spectroscopy, and viscometric methods. Cytotoxicity effect and DNA unwinding properties were also investigated. The results indicate that the Cu(II) complexes interact with calf-thymus DNA by both partially intercalative and hydrogen binding. These findings have been further substantiated by the determination of intrinsic binding constants spectrophotometrically, 12.5 × 10(5) and 5 × 10(5) for 1 and 2, respectively. Our findings suggest that the type of ligands and structure of complexes have marked effect on the binding affinity of complexes involving CT-DNA. Circular dichroism results show that complex 1 causes considerable increase in base stacking of DNA, whereas 2 decreases the base stacking, which is related to more extended aromatic area of 1,10-phenanthroline in 1 rather than bipyridine in 2. Slow decrease in DNA viscosity indicates partially intercalative binding in addition to hydrogen binding on the surface of DNA. The second binding mode was also confirmed by additional tests: interaction in denaturation condition and acidic pH. Also, these new complexes induced cleavage in pUC18 plasmid DNA as indicated in gel electrophoresis and showed excellent antitumor activity against K562 (human chronic myeloid leukemia) cells.

  19. Synthesis, characterization, DNA binding, cleavage activity and cytotoxicity of copper(II) complexes.

    Science.gov (United States)

    Li, Mei-Jin; Lan, Tao-Yu; Cao, Xiu-Hui; Yang, Huang-Hao; Shi, Yupeng; Yi, Changqing; Chen, Guo-Nan

    2014-02-21

    Three new mononuclear copper(II) complexes, [Cu(L2)](2+) (1), [Cu(acac)(L)](+) (2), and [Cu(acac-Cl)(L)](+) (3) (L = 2-(4-pyridine)oxazo[4,5-f]1,10-phenanthroline (4-PDOP); acac = acetylacetone; acac-Cl = 3-chloroacetylacetone), have been synthesized and characterized by elemental analysis, high resolution mass spectrometry (Q-TOF), and IR spectroscopy. Two of the complexes were structurally characterized by single-crystal X-ray diffraction techniques. Their interactions with DNA were studied by UV-vis absorption and emission spectra, viscosity, thermal melting, DNA unwinding assay and CD spectroscopy. The nucleolytic cleavage activity of the compounds was carried out on double stranded pBR322 circular plasmid DNA by using a gel electrophoresis experiment in the presence and absence of an oxidant (H2O2). Active oxygen intermediates such as hydroxyl radicals and hydrogen peroxide generated in the presence of L and complexes 1-3 may act as active species for the DNA scission. The cytotoxicity of the complexes against HepG2 cancer cells was also studied.

  20. Shear-induced unfolding and enzymatic cleavage of full-length VWF multimers

    CERN Document Server

    Lippok, Svenja; Obser, Tobias; Kleemeier, Lars; Schneppenheim, Reinhard; Budde, Ulrich; Netz, Roland R; Rädler, Joachim O

    2015-01-01

    Proteolysis of the multimeric blood coagulation protein von Willebrand Factor (VWF) by ADAMTS13 is crucial for prevention of microvascular thrombosis. ADAMTS13 cleaves VWF within the mechanosensitive A2 domain, which is believed to open under shear flow. Here, we combine Fluorescence Correlation Spectroscopy (FCS) and a microfluidic shear cell to monitor real-time kinetics of full-length VWF proteolysis as a function of shear stress. For comparison, we also measure the Michaelis-Menten kinetics of ADAMTS13 cleavage of wild-type VWF in the absence of shear but partially denaturing conditions. Under shear, ADAMTS13 activity on full-length VWF arises without denaturing agent as evidenced by FCS and gel-based multimer analysis. In agreement with Brownian hydrodynamics simulations, we find a sigmoidal increase of the enzymatic rate as a function of shear at a threshold shear rate 5522/s. The same flow-rate dependence of ADAMTS13 activity we also observe in blood plasma, which is relevant to predict hemostatic dysf...

  1. Transcriptome profiles of embryos before and after cleavage in Eriocheir sinensis: identification of developmental genes at the earliest stages

    Science.gov (United States)

    Hui, Min; Cui, Zhaoxia; Liu, Yuan; Song, Chengwen

    2016-09-01

    In crab, embryogenesis is a complicated developmental program marked by a series of critical events. RNA-Sequencing technology offers developmental biologists a way to identify many more developmental genes than ever before. Here, we present a comprehensive analysis of the transcriptomes of Eriocheir sinensis oosperms (Os) and embryos at the 2-4 cell stage (Cs), which are separated by a cleavage event. A total of 18 923 unigenes were identified, and 403 genes matched with gene ontology (GO) terms related to developmental processes. In total, 432 differentially expressed genes (DEGs) were detected between the two stages. Nine DEGs were specifically expressed at only one stage. These DEGs may be relevant to stage-specific molecular events during development. A number of DEGs related to `hedgehog signaling pathway', `wnt signaling pathway' `germplasm', `nervous system', `sensory perception' and `segment polarity' were identified as being up-regulated at the Cs stage. The results suggest that these embryonic developmental events begin before the early cleavage event in crabs, and that many of the genes expressed in the two transcriptomes might be maternal genes. Our study provides ample information for further research on the molecular mechanisms underlying crab development.

  2. Nuclear substructure reorganization during late stageerythropoiesis is selective and does not involve caspase cleavage ofmajor nuclear substructural proteins

    Energy Technology Data Exchange (ETDEWEB)

    Krauss, Sharon Wald; Lo, Annie J.; Short, Sarah A.; Koury, MarkJ.; Mohandas, Narla; Chasis, Joel Anne

    2005-04-06

    Enucleation, a rare feature of mammalian differentiation, occurs in three cell types: erythroblasts, lens epithelium and keratinocytes. Previous investigations suggest that caspase activation functions in lens epithelial and keratinocyte enucleation, as well as in early erythropoiesis encompassing BFU-E differentiation to proerythroblast. To determine whether caspase activation contributes to later erythropoiesis and whether nuclear substructures other than chromatin reorganize, we analyzed distributions of nuclear subcompartment proteins and assayed for caspase-induced cleavage of subcompartmental target proteins in mouse erythroblasts. We found that patterns of lamin B in the filamentous network interacting with both the nuclear envelope and DNA, nuclear matrix protein NuMA, and splicing factors Sm and SC35 persisted during nuclear condensation, consistent with effective transcription of genes expressed late in differentiation. Thus nuclear reorganization prior to enucleation is selective, allowing maintenance of critical transcriptional processes independent of extensive chromosomal reorganization. Consistent with these data, we found no evidence for caspase-induced cleavage of major nuclear subcompartment proteins during late erythropoiesis, in contrast to what has been observed in early erythropoiesis and in lens epithelial and keratinocyte differentiation. These findings imply that nuclear condensation and extrusion during terminal erythroid differentiation involve novel mechanisms that do not entail major activation of apoptotic machinery.

  3. The democratization of gene editing: Insights from site-specific cleavage and double-strand break repair.

    Science.gov (United States)

    Jasin, Maria; Haber, James E

    2016-08-01

    DNA double-strand breaks (DSBs) are dangerous lesions that if not properly repaired can lead to genomic change or cell death. Organisms have developed several pathways and have many factors devoted to repairing DSBs, which broadly occurs by homologous recombination, which relies on an identical or homologous sequence to template repair, or nonhomologous end-joining. Much of our understanding of these repair mechanisms has come from the study of induced DNA cleavage by site-specific endonucleases. In addition to their biological role, these cellular pathways can be co-opted for gene editing to study gene function or for gene therapy or other applications. While the first gene editing experiments were done more than 20 years ago, the recent discovery of RNA-guided endonucleases has simplified approaches developed over the years to make gene editing an approach that is available to the entire biomedical research community. Here, we review DSB repair mechanisms and site-specific cleavage systems that have provided insight into these mechanisms and led to the current gene editing revolution.

  4. The peptide antibiotic microcin B17 induces double-strand cleavage of DNA mediated by E. coli DNA gyrase.

    Science.gov (United States)

    Vizán, J L; Hernández-Chico, C; del Castillo, I; Moreno, F

    1991-02-01

    Microcin B17 (MccB17) is a bactericidal peptide antibiotic which inhibits DNA replication. Two Escherichia coli MccB17 resistant mutants were isolated and the mutations were shown to map to 83 min of the genetic map. Cloning of the mutations and Tn5 insertional analysis demonstrated that they were located inside gyrB. The approximate location of the mutations within gyrB was determined by constructing hybrid genes, as a previous step to sequencing. Both mutations were shown to consist of a single AT----GC transition at position 2251 of the gene, which produces a Trp751----Arg substitution in the amino acid sequence of the GyrB polypeptide. The inhibitory effect of MccB17 on replicative cell-free extracts was assayed. In this in vitro system, interaction of MccB17 with a component of the extracts induced double-strand cleavage of plasmid DNA. In vivo treatment with MccB17 also induced a well-defined cleavage pattern on chromosomal DNA. These effects were not observed with a MccB17-resistant, gyrB mutant. Altogether, our results indicate that MccB17 blocks DNA gyrase by trapping an enzyme-DNA cleavable complex. Thus, the mode of action of this peptide antibiotic resembles that of quinolones and a variety of antitumour drugs currently used in cancer chemotherapy. MccB17 is the first peptide shown to inhibit a type II DNA topoisomerase.

  5. The polycomb group protein EED varies in its ability to access the nucleus in porcine oocytes and cleavage stage embryos.

    Science.gov (United States)

    Foust, Kallie B; Li, Yanfang; Park, Kieun; Wang, Xin; Liu, Shihong; Cabot, Ryan A

    2012-08-01

    Chromatin-modifying complexes serve essential functions during mammalian embryonic development. Polycomb group proteins EED, SUZ12, and EZH2 have been shown to mediate methylation of the lysine 27 residue of histone protein H3 (H3K27), an epigenetic mark that is linked with transcriptional repression. H3K27 trimethylation has been shown to be present on chromatin in mature porcine oocytes, pronuclear and 2-cell stage embryos, with H3K27 trimethylation decreasing at the 4-cell stage and not detectable in blastocyst stage embryos. The goals of this study were to determine the intracellular localization of the polycomb group protein EED in porcine oocytes and cleavage stage porcine embryos produced by in vitro fertilization and to determine the binding abilities of karyopherin α subtypes toward EED. Our results revealed that EED had a strong nuclear localization in 4-cell and blastocyst stage embryos and a strong perinuclear staining in GV-stage oocytes; EED was not detectable in the nuclei of pronuclear or 2-cell stage embryos. An in vitro binding assay was performed to assess the ability of EED to interact with a series of karyopherin α subtypes; results from this experiment revealed that EED can interact with several karyopherin α subtypes, but with varying degrees of affinity. Together these data indicate that EED displays a dynamic change in intracellular localization in progression from immature oocyte to cleavage stage embryo and that EED possess differing in vitro binding affinities toward individual karyopherin α subtypes, which may in part regulate the nuclear access of EED during this window of development.

  6. Cleavage pattern and fate map of the mesentoblast, 4d, in the gastropod Crepidula: a hallmark of spiralian development

    Directory of Open Access Journals (Sweden)

    Lyons Deirdre C

    2012-09-01

    Full Text Available Abstract Background Animals with a spiral cleavage program, such as mollusks and annelids, make up the majority of the superphylum Lophotrochozoa. The great diversity of larval and adult body plans in this group emerges from this highly conserved developmental program. The 4d micromere is one of the most conserved aspects of spiralian development. Unlike the preceding pattern of spiral divisions, cleavages within the 4d teloblastic sublineages are bilateral, representing a critical transition towards constructing the bilaterian body plan. These cells give rise to the visceral mesoderm in virtually all spiralians examined and in many species they also contribute to the endodermal intestine. Hence, the 4d lineage is an ideal one for studying the evolution and diversification of the bipotential endomesodermal germ layer in protostomes at the level of individual cells. Little is known of how division patterns are controlled or how mesodermal and endodermal sublineages diverge in spiralians. Detailed modern fate maps for 4d exist in only a few species of clitellate annelids, specifically in glossiphoniid leeches and the sludge worm Tubifex. We investigated the 4d lineage in the gastropod Crepidula fornicata, an established model system for spiralian biology, and in a closely related direct-developing species, C. convexa. Results High-resolution cell lineage tracing techniques were used to study the 4d lineage of C. fornicata and C. convexa. We present a new nomenclature to name the progeny of 4d, and report the fate map for the sublineages up through the birth of the first five pairs of teloblast daughter cells (when 28 cells are present in the 4d sublineage, and describe each clone’s behavior during gastrulation and later stages as these undergo differentiation. We identify the precise origin of the intestine, two cells of the larval kidney complex, the larval retractor muscles and the presumptive germ cells, among others. Other tissues that arise

  7. Carbon-carbon bond cleavage in activation of the prodrug nabumetone.

    Science.gov (United States)

    Varfaj, Fatbardha; Zulkifli, Siti N A; Park, Hyoung-Goo; Challinor, Victoria L; De Voss, James J; Ortiz de Montellano, Paul R

    2014-05-01

    Carbon-carbon bond cleavage reactions are catalyzed by, among others, lanosterol 14-demethylase (CYP51), cholesterol side-chain cleavage enzyme (CYP11), sterol 17β-lyase (CYP17), and aromatase (CYP19). Because of the high substrate specificities of these enzymes and the complex nature of their substrates, these reactions have been difficult to characterize. A CYP1A2-catalyzed carbon-carbon bond cleavage reaction is required for conversion of the prodrug nabumetone to its active form, 6-methoxy-2-naphthylacetic acid (6-MNA). Despite worldwide use of nabumetone as an anti-inflammatory agent, the mechanism of its carbon-carbon bond cleavage reaction remains obscure. With the help of authentic synthetic standards, we report here that the reaction involves 3-hydroxylation, carbon-carbon cleavage to the aldehyde, and oxidation of the aldehyde to the acid, all catalyzed by CYP1A2 or, less effectively, by other P450 enzymes. The data indicate that the carbon-carbon bond cleavage is mediated by the ferric peroxo anion rather than the ferryl species in the P450 catalytic cycle. CYP1A2 also catalyzes O-demethylation and alcohol to ketone transformations of nabumetone and its analogs.

  8. Expression and in vitro cleavage activity of anti-caspase-7 hammerhead ribozymes

    Institute of Scientific and Technical Information of China (English)

    Wei Zhang; Qing Xie; Xia-Qiu Zhou; Shan Jiang; You-Xin Jin

    2004-01-01

    AIM: To prepare hammerhead ribozymes against mouse caspase-7 and identify their cleavage activityin vitro, in order to select a ribozyme with specific cleavage activity against mouse caspase-7 as a potential gene therapy for apoptosis-related diseases.METHODS: Anti-caspase-7 ribozymes targeting sites 333and 394 (named Rz333 and Rz394) were designed by computer software, and their DNA sequences encoding ribozymes were synthesized. Caspase-7 DNA sequence was acquired by RT-PCR. Ribozymes and caspase-7 DNA obtained byin vitro transcription were cloned into pBSKneo U6' and pGEM-T vectors, respectively. The cleavage activity of ribozymes against mouse caspase-7 was identified by cleavage experimentsin vitro.RESULTS: Rz333 and Rz394 were designed and their DNA sequences were synthesized respectively. The expression vector of caspase-7 and plasmids containing Rz333 and Rz394 were reconstructed successfully. Ribozymes and caspase-7 mRNA were expressed byin vitro transcription.In vitro cleavage experiment showed that 243-nt and 744-nt segments were produced after caspase-7 mRNA was mixed with Rz333 in equivalent, and the cleavage efficiency was 67.98%. No cleaved segment was observed when caspase-7 mRNA was mixed with Rz394.CONCLUSION: Rz333 can site-specific cleave mouse caspase-7 mRNA, and it shows a potential for gene therapy of apoptosis-related diseases by down-regulating gene expression of caspase-7.

  9. 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.

  10. 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

  11. The role of the methyltransferase domain of bifunctional restriction enzyme RM.BpuSI in cleavage activity.

    Directory of Open Access Journals (Sweden)

    Arthur Sarrade-Loucheur

    Full Text Available Restriction enzyme (REase RM.BpuSI can be described as a Type IIS/C/G REase for its cleavage site outside of the recognition sequence (Type IIS, bifunctional polypeptide possessing both methyltransferase (MTase and endonuclease activities (Type IIC and endonuclease activity stimulated by S-adenosyl-L-methionine (SAM (Type IIG. The stimulatory effect of SAM on cleavage activity presents a major paradox: a co-factor of the MTase activity that renders the substrate unsusceptible to cleavage enhances the cleavage activity. Here we show that the RM.BpuSI MTase activity modifies both cleavage substrate and product only when they are unmethylated. The MTase activity is, however, much lower than that of M1.BpuSI and is thought not to be the major MTase for host DNA protection. SAM and sinefungin (SIN increase the Vmax of the RM.BpuSI cleavage activity with a proportional change in Km, suggesting the presence of an energetically more favorable pathway is taken. We further showed that RM.BpuSI undergoes substantial conformational changes in the presence of Ca(2+, SIN, cleavage substrate and/or product. Distinct conformers are inferred as the pre-cleavage/cleavage state (in the presence of Ca(2+, substrate or both and MTase state (in the presence of SIN and substrate, SIN and product or product alone. Interestingly, RM.BpuSI adopts a unique conformation when only SIN is present. This SIN-bound state is inferred as a branch point for cleavage and MTase activity and an intermediate to an energetically favorable pathway for cleavage, probably through increasing the binding affinity of the substrate to the enzyme under cleavage conditions. Mutation of a SAM-binding residue resulted in altered conformational changes in the presence of substrate or Ca(2+ and eliminated cleavage activity. The present study underscores the role of the MTase domain as facilitator of efficient cleavage activity for RM.BpuSI.

  12. Carbon-Oxygen Bond Cleavage by Bis(imino)pyridine Iron Compounds : Catalyst Deactivation Pathways and Observation of Acyl C-O Bond Cleavage in Esters

    NARCIS (Netherlands)

    Trovitch, Ryan J.; Lobkovsky, Emil; Bouwkamp, Marco W.; Chirik, Paul J.

    2008-01-01

    Investigations into the substrate scope of bis(imino)pyridine iron-catalyzed hydrogenation and [2 pi + 2 pi]. diene cyclization reactions identified C-O bond cleavage as a principal deactivation pathway. Addition of diallyl or allyl ethyl ether to the bis(imino)pyridine iron dinitrogen complex, ((iP

  13. Increased cleavage and blastocyst rate in ewes treated with bovine somatotropin 5 days before the end of progestin-based estrous synchronization.

    Science.gov (United States)

    Montero-Pardo, A; Hernández-Cerón, J; Rojas-Maya, S; Valencia, J; Rodríguez-Cortez, A; Gutiérrez, C G

    2011-05-01

    Treatment with bovine somatotropin (bST) during estrous synchronization increased fertility and prolificacy in sheep. In the present study, a single dose of bST 5 days before the end of progestin treatment improved cleavage and embryo development. Stage of estrous cycle was synchronized in ewes (n=32) with progestin and superovulation was induced by use of FSH. Five days before the end of progestin treatment, ewes were randomly assigned to two groups: bST group (n=16) received a depot injection of 125 mg of bST sc (Lactotropina, Elanco, México) and the control group (n=16) received saline solution. Estrous was detected with rams fitted with an apron every 2 h and estrous sheep were mated every 8 h whilst in estrous. Embryos were recovered on Day 7 post mating, assessed microscopically and fixed in 4% paraformaldehyde. Cell number in blastocysts was counted after Hoechst 33342 staining. Plasma concentrations of IGF-I, insulin and progesterone were determined in eight sheep per group from the day of bST treatment to the day of embryo recovery. Cleavage rate, percentage of transferable embryos (transferable embryos/cleaved) and percentage of embryos reaching the blastocyst stage (blastocyst/cleaved) were compared between groups by logistic regression. IGF-I, insulin and progesterone plasma concentrations were analyzed by ANOVA for repeated measurements and cell number by ANOVA. Cleavage rate was greater (Psheep. Plasma concentrations of IGF-I and insulin were greater (P<0.01) in the bST-treated group. No changes were observed in progesterone concentrations (P=0.5). It is concluded that bST injection 5 days before progestin removal increases cleavage rate and the proportion of embryos that reach the blastocyst stage. These effects are associated with an increase in IGF-I and insulin concentrations.

  14. Requirements for efficient proteolytic cleavage of prelamin A by ZMPSTE24.

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    Jemima Barrowman

    Full Text Available BACKGROUND: The proteolytic maturation of the nuclear protein lamin A by the zinc metalloprotease ZMPSTE24 is critical for human health. The lamin A precursor, prelamin A, undergoes a multi-step maturation process that includes CAAX processing (farnesylation, proteolysis and carboxylmethylation of the C-terminal CAAX motif, followed by ZMPSTE24-mediated cleavage of the last 15 amino acids, including the modified C-terminus. Failure to cleave the prelamin A "tail", due to mutations in either prelamin A or ZMPSTE24, results in a permanently prenylated form of prelamin A that underlies the premature aging disease Hutchinson-Gilford Progeria Syndrome (HGPS and related progeroid disorders. METHODOLOGY/PRINCIPAL FINDINGS: Here we have investigated the features of the prelamin A substrate that are required for efficient cleavage by ZMPSTE24. We find that the C-terminal 41 amino acids of prelamin A contain sufficient context to allow cleavage of the tail by ZMPSTE24. We have identified several mutations in amino acids immediately surrounding the cleavage site (between Y646 and L647 that interfere with efficient cleavage of the prelamin A tail; these mutations include R644C, L648A and N650A, in addition to the previously reported L647R. Our data suggests that 9 of the 15 residues within the cleaved tail that lie immediately upstream of the CAAX motif are not critical for ZMPSTE24-mediated cleavage, as they can be replaced by the 9 amino acid HA epitope. However, duplication of the same 9 amino acids (to increase the distance between the prenyl group and the cleavage site impairs the ability of ZMPSTE24 to cleave prelamin A. CONCLUSIONS/SIGNIFICANCE: Our data reveals amino acid preferences flanking the ZMPSTE24 cleavage site of prelamin A and suggests that spacing from the farnesyl-cysteine to the cleavage site is important for optimal ZMPSTE24 cleavage. These studies begin to elucidate the substrate requirements of an enzyme activity critical to human

  15. Simian sarcoma virus-encoded gag-related protein: in vitro cleavage by Friend leukemia virus-associated proteolytic activity.

    Science.gov (United States)

    Hafenrichter, R; Thiel, H J

    1985-05-01

    The simian sarcoma virus (SSV) encodes a gag-related 65,000-Da protein (SSV p65) which is not processed in SSV nonproducer cells (SSV-NP cells) (H.-J. Thiel, T. J. Matthews, E. M. Broughton, K. J. Weinhold, D. P. Bolognesi, T. Graf, and H. Beug (1981a), Virology 114, 124-131). In order to cleave SSV p65, retroviral particles containing this antigen were incubated with extracts from the heterologous helper virus Friend leukemia virus (FLV). Superinfection of SSV-NP cells by FLV has been previously shown to result in processing of SSV p65 in vivo (H.-J. Thiel, F. Weiland, R. Hafenrichter, T. J. Matthews, and K. J. Weinhold (1982), Virology 123, 229-234). In vitro cleavage was most efficient in the presence of a nonionic detergent (greater than 0.1% Nonidet-P40) and a reducing agent (greater than 5 mM dithiothreitol) at a pH of 7.0. The products, termed SSV p55 (p15, p12, p30), SSV p30, SSV p25 (p15, p12), and SSV p10, were characterized by (1) molecular weight, (2) kinetics experiments, (3) incorporation of different radiolabeled amino acids, and (4) comparison with SSAV structural proteins. Kinetics experiments with two amino acids ([3H]leucine, [35S]cysteine) revealed that initial processing of SSV p65 produced SSV p55 and SSV p10, with subsequent processing of SSV p55 occurring thereafter. In contrast to the Moloney system, the major intermediate p40 (p30, p10) could not be clearly demonstrated. A direct comparison of SSAV p10 and the cleavage product SSV p10 by SDS-PAGE suggests that SSAV pr65gag and SSV p65 differ slightly by molecular weight.

  16. 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.

  17. Megakaryocytes Regulate Expression of Pyk2 Isoforms and Caspase-mediated Cleavage of Actin in Osteoblasts*

    Science.gov (United States)

    Kacena, Melissa A.; Eleniste, Pierre P.; Cheng, Ying-Hua; Huang, Su; Shivanna, Mahesh; Meijome, Tomas E.; Mayo, Lindsey D.; Bruzzaniti, Angela

    2012-01-01

    The proliferation and differentiation of osteoblast (OB) precursors are essential for elaborating the bone-forming activity of mature OBs. However, the mechanisms regulating OB proliferation and function are largely unknown. We reported that OB proliferation is enhanced by megakaryocytes (MKs) via a process that is regulated in part by integrin signaling. The tyrosine kinase Pyk2 has been shown to regulate cell proliferation and survival in a variety of cells. Pyk2 is also activated by integrin signaling and regulates actin remodeling in bone-resorbing osteoclasts. In this study, we examined the role of Pyk2 and actin in the MK-mediated increase in OB proliferation. Calvarial OBs were cultured in the presence of MKs for various times, and Pyk2 signaling cascades in OBs were examined by Western blotting, subcellular fractionation, and microscopy. We found that MKs regulate the temporal expression of Pyk2 and its subcellular localization. We also found that MKs regulate the expression of two alternatively spliced isoforms of Pyk2 in OBs, which may regulate OB differentiation and proliferation. MKs also induced cytoskeletal reorganization in OBs, which was associated with the caspase-mediated cleavage of actin, an increase in focal adhesions, and the formation of apical membrane ruffles. Moreover, BrdU incorporation in MK-stimulated OBs was blocked by the actin-polymerizing agent, jasplakinolide. Collectively, our studies reveal that Pyk2 and actin play an important role in MK-regulated signaling cascades that control OB proliferation and may be important for therapeutic interventions aimed at increasing bone formation in metabolic diseases of the skeleton. PMID:22447931

  18. Megakaryocytes regulate expression of Pyk2 isoforms and caspase-mediated cleavage of actin in osteoblasts.

    Science.gov (United States)

    Kacena, Melissa A; Eleniste, Pierre P; Cheng, Ying-Hua; Huang, Su; Shivanna, Mahesh; Meijome, Tomas E; Mayo, Lindsey D; Bruzzaniti, Angela

    2012-05-18

    The proliferation and differentiation of osteoblast (OB) precursors are essential for elaborating the bone-forming activity of mature OBs. However, the mechanisms regulating OB proliferation and function are largely unknown. We reported that OB proliferation is enhanced by megakaryocytes (MKs) via a process that is regulated in part by integrin signaling. The tyrosine kinase Pyk2 has been shown to regulate cell proliferation and survival in a variety of cells. Pyk2 is also activated by integrin signaling and regulates actin remodeling in bone-resorbing osteoclasts. In this study, we examined the role of Pyk2 and actin in the MK-mediated increase in OB proliferation. Calvarial OBs were cultured in the presence of MKs for various times, and Pyk2 signaling cascades in OBs were examined by Western blotting, subcellular fractionation, and microscopy. We found that MKs regulate the temporal expression of Pyk2 and its subcellular localization. We also found that MKs regulate the expression of two alternatively spliced isoforms of Pyk2 in OBs, which may regulate OB differentiation and proliferation. MKs also induced cytoskeletal reorganization in OBs, which was associated with the caspase-mediated cleavage of actin, an increase in focal adhesions, and the formation of apical membrane ruffles. Moreover, BrdU incorporation in MK-stimulated OBs was blocked by the actin-polymerizing agent, jasplakinolide. Collectively, our studies reveal that Pyk2 and actin play an important role in MK-regulated signaling cascades that control OB proliferation and may be important for therapeutic interventions aimed at increasing bone formation in metabolic diseases of the skeleton.

  19. Orbit/CLASP is required for myosin accumulation at the cleavage furrow in Drosophila male meiosis.

    Directory of Open Access Journals (Sweden)

    Daishi Kitazawa

    Full Text Available Peripheral microtubules (MTs near the cell cortex are essential for the positioning and continuous constriction of the contractile ring (CR in cytokinesis. Time-lapse observations of Drosophila male meiosis showed that myosin II was first recruited along the cell cortex independent of MTs. Then, shortly after peripheral MTs made contact with the equatorial cortex, myosin II was concentrated there in a narrow band. After MT contact, anillin and F-actin abruptly appeared on the equatorial cortex, simultaneously with myosin accumulation. We found that the accumulation of myosin did not require centralspindlin, but was instead dependent on Orbit, a Drosophila ortholog of the MT plus-end tracking protein CLASP. This protein is required for stabilization of central spindle MTs, which are essential for cytokinesis. Orbit was also localized in a mid-zone of peripheral MTs, and was concentrated in a ring at the equatorial cortex during late anaphase. Fluorescence resonance energy transfer experiments indicated that Orbit is closely associated with F-actin in the CR. We also showed that the myosin heavy chain was in close proximity with Orbit in the cleavage furrow region. Centralspindlin was dispensable in Orbit ring formation. Instead, the Polo-KLP3A/Feo complex was required for the Orbit accumulation independently of the Orbit MT-binding domain. However, orbit mutations of consensus sites for the phosphorylation of Cdk1 or Polo did not influence the Orbit accumulation, suggesting an indirect regulatory role of these protein kinases in Orbit localization. Orbit was also necessary for the maintenance of the CR. Our data suggest that Orbit plays an essential role as a connector between MTs and the CR in Drosophila male meiosis.

  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. Topography and Atomic Structure Investigations Of (100 Cleavage Surface of In4Se3 Layered Crystals

    Directory of Open Access Journals (Sweden)

    P.V. Galiy

    2014-06-01

    Full Text Available The atomic microstructure and crystallography of (100 surfaces of In4Se3 layered crystals obtained by cleavage in situ were studied by the methods of scanning tunneling and atomic force microscopies (STM, AFM and low energy electron diffraction (LEED for reflection. The obtained results indicate the existence of periodic corrugated structures on the cleavage surface. It is shown that (100 In4Se3 cleavage surface is structurally stable and doesn't undergo reconstruction in a wide temperature range of 77-295 K. The anisotropy of thermal expansion along the main crystallography directions in the (100 In4Se3 cleavage plane has been shown. The evaluation of the two-dimensional lattice constant in the cleavage (100 surface plane of orthorhombic In4Se3 layered crystal was done. The calculated values of the lattice constants in consequence of LEED study, such as b  11,475 Å and c  3,734 Å, coincide well with those obtained by the AFM and STM (b  13-14 Å and c  4 Å, and correlate, within the errors limits, with the corresponding values obtained by X-ray diffraction (b  12,308(1 Å and c  4,0810(5 Å. Besides, the obtained results of cleavage surface structure studies show the correctness of filtering application concerning topography images and indicate the adequacy of the model used for calculations of the cleavage (100 surfaces lattice constants of In4Se3 in accordance with the LEED results. The influence of the LEED experimental module structure on the results has been considered.

  2. The eIF4G–homolog p97 can activate translation independent of caspase cleavage

    Science.gov (United States)

    Nousch, Marco; Reed, Victoria; Bryson-Richardson, Robert J.; Currie, Peter D.; Preiss, Thomas

    2007-01-01

    The eukaryotic initiation factor (eIF) 4G family plays a central role during translation initiation, bridging between the 5′ and 3′ ends of the mRNA via its N-terminal third while recruiting other factors and ribosomes through its central and C-terminal third. The protein p97/NAT1/DAP5 is homologous to the central and C-terminal thirds of eIF4G. p97 has long been considered to be a translational repressor under normal cellular conditions. Further, caspase cleavage liberates a p86 fragment that is thought to mediate cap-independent translation in apoptotic cells. We report here that, surprisingly, human p97 is polysome associated in proliferating cells and moves to stress granules in stressed, nonapoptotic cells. Tethered-function studies in living cells show that human p97 and p86 both can activate translation; however, we were unable to detect polysome association of p86 in apoptotic cells. We further characterized the zebrafish orthologs of p97, and found both to be expressed throughout embryonic development. Their simultaneous knockdown by morpholino injection led to impaired mesoderm formation and early embryonic lethality, indicating conservation of embryonic p97 function from fish to mammals. These data indicate that full-length p97 is a translational activator with essential role(s) in unstressed cells, suggesting a reassessment of current models of p97 function. PMID:17237356

  3. The eIF4G-homolog p97 can activate translation independent of caspase cleavage.

    Science.gov (United States)

    Nousch, Marco; Reed, Victoria; Bryson-Richardson, Robert J; Currie, Peter D; Preiss, Thomas

    2007-03-01

    The eukaryotic initiation factor (eIF) 4G family plays a central role during translation initiation, bridging between the 5' and 3' ends of the mRNA via its N-terminal third while recruiting other factors and ribosomes through its central and C-terminal third. The protein p97/NAT1/DAP5 is homologous to the central and C-terminal thirds of eIF4G. p97 has long been considered to be a translational repressor under normal cellular conditions. Further, caspase cleavage liberates a p86 fragment that is thought to mediate cap-independent translation in apoptotic cells. We report here that, surprisingly, human p97 is polysome associated in proliferating cells and moves to stress granules in stressed, nonapoptotic cells. Tethered-function studies in living cells show that human p97 and p86 both can activate translation; however, we were unable to detect polysome association of p86 in apoptotic cells. We further characterized the zebrafish orthologs of p97, and found both to be expressed throughout embryonic development. Their simultaneous knockdown by morpholino injection led to impaired mesoderm formation and early embryonic lethality, indicating conservation of embryonic p97 function from fish to mammals. These data indicate that full-length p97 is a translational activator with essential role(s) in unstressed cells, suggesting a reassessment of current models of p97 function.

  4. Resolution of joint molecules by RuvABC and RecG following cleavage of the Escherichia coli chromosome by EcoKI.

    Directory of Open Access Journals (Sweden)

    Laura Wardrope

    Full Text Available DNA double-strand breaks can be repaired by homologous recombination involving the formation and resolution of Holliday junctions. In Escherichia coli, the RuvABC resolvasome and the RecG branch-migration enzyme have been proposed to act in alternative pathways for the resolution of Holliday junctions. Here, we have studied the requirements for RuvABC and RecG in DNA double-strand break repair after cleavage of the E. coli chromosome by the EcoKI restriction enzyme. We show an asymmetry in the ability of RuvABC and RecG to deal with joint molecules in vivo. We detect linear DNA products compatible with the cleavage-ligation of Holliday junctions by the RuvABC pathway but not by the RecG pathway. Nevertheless we show that the XerCD-mediated pathway of chromosome dimer resolution is required for survival regardless of whether the RuvABC or the RecG pathway is active, suggesting that crossing-over is a common outcome irrespective of the pathway utilised. This poses a problem. How can cells resolve joint molecules, such as Holliday junctions, to generate crossover products without cleavage-ligation? We suggest that the mechanism of bacterial DNA replication provides an answer to this question and that RecG can facilitate replication through Holliday junctions.

  5. Genetic Predisposition To Acquire a Polybasic Cleavage Site for Highly Pathogenic Avian Influenza Virus Hemagglutinin

    Science.gov (United States)

    Nao, Naganori; Yamagishi, Junya; Miyamoto, Hiroko; Igarashi, Manabu; Manzoor, Rashid; Ohnuma, Aiko; Tsuda, Yoshimi; Furuyama, Wakako; Shigeno, Asako; Kajihara, Masahiro; Kishida, Noriko; Yoshida, Reiko

    2017-01-01

    ABSTRACT Highly pathogenic avian influenza viruses with H5 and H7 hemagglutinin (HA) subtypes evolve from low-pathogenic precursors through the acquisition of multiple basic amino acid residues at the HA cleavage site. Although this mechanism has been observed to occur naturally only in these HA subtypes, little is known about the genetic basis for the acquisition of the polybasic HA cleavage site. Here we show that consecutive adenine residues and a stem-loop structure, which are frequently found in the viral RNA region encoding amino acids around the cleavage site of low-pathogenic H5 and H7 viruses isolated from waterfowl reservoirs, are important for nucleotide insertions into this RNA region. A reporter assay to detect nontemplated nucleotide insertions and deep-sequencing analysis of viral RNAs revealed that an increased number of adenine residues and enlarged stem-loop structure in the RNA region accelerated the multiple adenine and/or guanine insertions required to create codons for basic amino acids. Interestingly, nucleotide insertions associated with the HA cleavage site motif were not observed principally in the viral RNA of other subtypes tested (H1, H2, H3, and H4). Our findings suggest that the RNA editing-like activity is the key mechanism for nucleotide insertions, providing a clue as to why the acquisition of the polybasic HA cleavage site is restricted to the particular HA subtypes. PMID:28196963

  6. Characteristics of the fast electron emission produced during the cleavage of crystals

    Indian Academy of Sciences (India)

    B P Chandra; N L Patel; S S Rahangdale; R P Patel; V K Patle

    2003-01-01

    The present paper reports the fast electron emission produced during the cleavage of alkali halide crystals and models the dynamics of the process. The mechano-emission arises as a result of the ionization of surface traps at the expense of the energy which is released in the annihilation of the defects which are formed during cleavage. The slow electrons which appear upon the ionization of surface traps are subsequently accelerated in the field of negatively charged segment of the freshly cleaved surface. Considering the basic mechanism of fast electron emission, expressions are derived which are able to explain satisfactorily the temporal, thermal, charge, surface, coloration, water adsorption and other characteristics of the fast electron emission produced during the cleavage of crystals. The decay time of the charges on the newly created surfaces, and the velocity of cracks can be determined from the measurements of fast electron emission produced during the cleavage of crystals. It is shown that two types of diffusing centres are responsible for the charge relaxation and thereby for the emission of fast electrons produced during the cleavage of alkali halide crystals.

  7. Caspase cleavage sites in the human proteome: CaspDB, a database of predicted substrates.

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    Sonu Kumar

    Full Text Available Caspases are enzymes belonging to a conserved family of cysteine-dependent aspartic-specific proteases that are involved in vital cellular processes and play a prominent role in apoptosis and inflammation. Determining all relevant protein substrates of caspases remains a challenging task. Over 1500 caspase substrates have been discovered in the human proteome according to published data and new substrates are discovered on a daily basis. To aid the discovery process we developed a caspase cleavage prediction method using the recently published curated MerCASBA database of experimentally determined caspase substrates and a Random Forest classification method. On both internal and external test sets, the ranking of predicted cleavage positions is superior to all previously developed prediction methods. The in silico predicted caspase cleavage positions in human proteins are available from a relational database: CaspDB. Our database provides information about potential cleavage sites in a verified set of all human proteins collected in Uniprot and their orthologs, allowing for tracing of cleavage motif conservation. It also provides information about the positions of disease-annotated single nucleotide polymorphisms, and posttranslational modifications that may modulate the caspase cleaving efficiency.

  8. Two-step cleavage of hairpin RNA with 5' overhangs by human DICER

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    Suzuki Harukazu

    2011-02-01

    Full Text Available Abstract Background DICER is an RNase III family endoribonuclease that processes precursor microRNAs (pre-miRNAs and long double-stranded RNAs, generating microRNA (miRNA duplexes and short interfering RNA duplexes with 20~23 nucleotides (nts in length. The typical form of pre-miRNA processed by the Drosha protein is a hairpin RNA with 2-nt 3' overhangs. On the other hand, production of mature miRNA from an endogenous hairpin RNA with 5' overhangs has also been reported, although the mechanism for this process is unknown. Results In this study, we show that human recombinant DICER protein (rDICER processes a hairpin RNA with 5' overhangs in vitro and generates an intermediate duplex with a 29 nt-5' strand and a 23 nt-3' strand, which was eventually cleaved into a canonical miRNA duplex via a two-step cleavage. The previously identified endogenous pre-miRNA with 5' overhangs, pre-mmu-mir-1982 RNA, is also determined to be a substrate of rDICER through the same two-step cleavage. Conclusions The two-step cleavage of a hairpin RNA with 5' overhangs shows that DICER releases double-stranded RNAs after the first cleavage and binds them again in the inverse direction for a second cleavage. These findings have implications for how DICER may be able to interact with or process differing precursor structures.

  9. Distinct mechanisms for DNA cleavage by myoglobin with a designed heme active center.

    Science.gov (United States)

    Zhao, Yuan; Du, Ke-Jie; Gao, Shu-Qin; He, Bo; Wen, Ge-Bo; Tan, Xiangshi; Lin, Ying-Wu

    2016-03-01

    Heme proteins perform diverse biological functions, of which myoglobin (Mb) is a representative protein. In this study, the O2 carrier Mb was shown to cleave double stranded DNA upon aerobic dithiothreitol-induced reduction, which is fine-tuned by an additional distal histidine, His29 or His43, engineered in the heme active center. Spectroscopic (UV-vis and EPR) and inhibition studies suggested that free radicals including singlet oxygen and hydroxyl radical are responsible for efficient DNA cleavage via an oxidative cleavage mechanism. On the other hand, L29E Mb, with a distinct heme active center involving three water molecules in the met form, was found to exhibit an excellent DNA cleavage activity that was not depending on O2. Inhibition and ligation studies demonstrated for the first time that L29E Mb cleaves double stranded DNA into both the nicked circular and linear forms via a hydrolytic cleavage mechanism, which resembles native endonucleases. This study provides valuable insights into the distinct mechanisms for DNA cleavage by heme proteins, and lays down a base for creating artificial DNA endonucleases by rational design of heme proteins. Moreover, this study suggests that the diverse functions of heme proteins can be fine-tuned by rational design of the heme active center with a hydrogen-bonding network.

  10. Mycoplasma hyopneumoniae Surface proteins Mhp385 and Mhp384 bind host cilia and glycosaminoglycans and are endoproteolytically processed by proteases that recognize different cleavage motifs.

    Science.gov (United States)

    Deutscher, Ania T; Tacchi, Jessica L; Minion, F Chris; Padula, Matthew P; Crossett, Ben; Bogema, Daniel R; Jenkins, Cheryl; Kuit, Tracey A; Walker, Mark J; Djordjevic, Steven P

    2012-03-02

    P97 and P102 paralogues occur as endoproteolytic cleavage fragments on the surface of Mycoplasma hyopneumoniae that bind glycosaminoglycans, plasminogen, and fibronectin and perform essential roles in colonization of ciliated epithelia. We show that the P102 paralogue Mhp384 is efficiently cleaved at an S/T-X-F↓X-D/E-like site, creating P60(384) and P50(384). The P97 paralogue Mhp385 is inefficiently cleaved, with tryptic peptides from a 115 kDa protein (P115(385)) and 88 kDa (P88(385)) and 27 kDa (P27(385)) cleavage fragments identified by LC-MS/MS. This is the first time a preprotein belonging to the P97 and P102 paralogue families has been identified by mass spectrometry. The semitryptic peptide (752)IQFELEPISLNV(763) denotes the C-terminus of P88(385) and defines the novel cleavage site (761)L-N-V↓A-V-S(766) in Mhp385. P115(385), P88(385), P27(385), P60(384), and P50(384) were shown to reside extracellularly, though it is unknown how the fragments remain attached to the cell surface. Heparin- and cilium-binding sites were identified within P60(384), P50(384), and P88(385). No primary function was attributed to P27(385); however, this molecule contains four tandem R1 repeats with similarity to porcine collagen type VI (α3 chain). P97 and P102 paralogue families are adhesins targeted by several proteases with different cleavage efficiencies, and this process generates combinatorial complexity on the surface of M. hyopneumoniae.

  11. Subunit architecture of the Golgi Dsc E3 ligase required for sterol regulatory element-binding protein (SREBP) cleavage in fission yeast.

    Science.gov (United States)

    Lloyd, S Julie-Ann; Raychaudhuri, Sumana; Espenshade, Peter J

    2013-07-19

    The membrane-bound sterol regulatory element-binding protein (SREBP) transcription factors regulate lipogenesis in mammalian cells and are activated through sequential cleavage by the Golgi-localized Site-1 and Site-2 proteases. The mechanism of fission yeast SREBP cleavage is less well defined and, in contrast, requires the Golgi-localized Dsc E3 ligase complex. The Dsc E3 ligase consists of five integral membrane subunits, Dsc1 through Dsc5, and resembles membrane E3 ligases that function in endoplasmic reticulum-associated degradation. Using immunoprecipitation assays and blue native electrophoresis, we determined the subunit architecture for the complex of Dsc1 through Dsc5, showing that the Dsc proteins form subcomplexes and display defined connectivity. Dsc2 is a rhomboid pseudoprotease family member homologous to mammalian UBAC2 and a central component of the Dsc E3 ligase. We identified conservation in the architecture of the Dsc E3 ligase and the multisubunit E3 ligase gp78 in mammals. Specifically, Dsc1-Dsc2-Dsc5 forms a complex resembling gp78-UBAC2-UBXD8. Further characterization of Dsc2 revealed that its C-terminal UBA domain can bind to ubiquitin chains but that the Dsc2 UBA domain is not essential for yeast SREBP cleavage. Based on the ability of rhomboid superfamily members to bind transmembrane proteins, we speculate that Dsc2 functions in SREBP recognition and binding. Homologs of Dsc1 through Dsc4 are required for SREBP cleavage and virulence in the human opportunistic pathogen Aspergillus fumigatus. Thus, these studies advance our organizational understanding of multisubunit E3 ligases involved in endoplasmic reticulum-associated degradation and fungal pathogenesis.

  12. Mutation G805R in the transmembrane domain of the LDL receptor gene causes familial hypercholesterolemia by inducing ectodomain cleavage of the LDL receptor in the endoplasmic reticulum

    Directory of Open Access Journals (Sweden)

    Thea Bismo Strøm

    2014-01-01

    Full Text Available More than 1700 mutations in the low density lipoprotein receptor (LDLR gene have been found to cause familial hypercholesterolemia (FH. These are commonly divided into five classes based upon their effects on the structure and function of the LDLR. However, little is known about the mechanism by which mutations in the transmembrane domain of the LDLR gene cause FH. We have studied how the transmembrane mutation G805R affects the function of the LDLR. Based upon Western blot analyses of transfected HepG2 cells, mutation G805R reduced the amounts of the 120 kDa precursor LDLR in the endoplasmic reticulum. This led to reduced amounts of the mature 160 kDa LDLR at the cell surface. However, significant amounts of a secreted 140 kDa G805R-LDLR ectodomain fragment was observed in the culture media. Treatment of the cells with the metalloproteinase inhibitor batimastat largely restored the amounts of the 120 and 160 kDa forms in cell lysates, and prevented secretion of the 140 kDa ectodomain fragment. Together, these data indicate that a metalloproteinase cleaved the ectodomain of the 120 kDa precursor G805R-LDLR in the endoplasmic reticulum. It was the presence of the polar Arg805 and not the lack of Gly805 which led to ectodomain cleavage. Arg805 also prevented γ-secretase cleavage within the transmembrane domain. It is conceivable that introducing a charged residue within the hydrophobic membrane lipid bilayer, results in less efficient incorporation of the 120 kDa G805R-LDLR in the endoplasmic reticulum membrane and makes it a substrate for metalloproteinase cleavage.

  13. IRE1α nucleotide sequence cleavage specificity in the unfolded protein response.

    Science.gov (United States)

    Poothong, Juthakorn; Sopha, Pattarawut; Kaufman, Randal J; Tirasophon, Witoon

    2017-01-01

    Inositol-requiring enzyme 1 (IRE1) is a conserved sensor of the unfolded protein response that has protein kinase and endoribonuclease (RNase) enzymatic activities and thereby initiates HAC1/XBP1 splicing. Previous studies demonstrated that human IRE1α (hIRE1α) does not cleave Saccharomyces cerevisiae HAC1 mRNA. Using an in vitro cleavage assay, we show that adenine to cytosine nucleotide substitution at the +1 position in the 3' splice site of HAC1 RNA is required for specific cleavage by hIRE1α. A similar restricted nucleotide specificity in the RNA substrate was observed for XBP1 splicing in vivo. Together these findings underscore the essential role of cytosine nucleotide at +1 in the 3' splice site for determining cleavage specificity of hIRE1α.

  14. Alkene cleavage catalysed by heme and nonheme enzymes: reaction mechanisms and biocatalytic applications.

    Science.gov (United States)

    Mutti, Francesco G

    2012-01-01

    The oxidative cleavage of alkenes is classically performed by chemical methods, although they display several drawbacks. Ozonolysis requires harsh conditions (-78°C, for a safe process) and reducing reagents in a molar amount, whereas the use of poisonous heavy metals such as Cr, Os, or Ru as catalysts is additionally plagued by low yield and selectivity. Conversely, heme and nonheme enzymes can catalyse the oxidative alkene cleavage at ambient temperature and atmospheric pressure in an aqueous buffer, showing excellent chemo- and regioselectivities in certain cases. This paper focuses on the alkene cleavage catalysed by iron cofactor-dependent enzymes encompassing the reaction mechanisms (in case where it is known) and the application of these enzymes in biocatalysis.

  15. MAGNETIC STRIPS TO SIMULATE LAYERED BRITTLE SOLIDS IN CLEAVAGE AND FRACTURE EXPERIMENTS

    Institute of Scientific and Technical Information of China (English)

    Francisco G.Emmerich; Alfredo G.Cunha; Carlos M.A.Girelli; Arnobio I.Vassem

    2008-01-01

    A characteristic of the fracture and cleavage experiments is that they are usually intrinsically destructive.Cracks do not completely heal in an unstressed system,even in crystals such as mica.Here,we used magnetic solids composed of magnetic strips for the non-destructive cleavage and brittle fracture experiments.Between the magnetic strips materials with different mechanical characteristics can be inserted,such as Teflon or foam strips,to change the mechanical properties of the solid.For the cleavage experiments,we developed an apparatus where parameters such as the main involved force can be measured easily.By inserting flaws,the magnetic solid can be used in dynamic fracture experiments,with the advantages of simulating macroscopically a non-destructive experiment in an easier way,that happen in real materials with much higher velocities.The apparatus and the used magnetic solid may be useful for demonstrations of fractures in classes.

  16. Multiple-turnover cleavage of double-stranded DNA by sandwiched zinc-finger nuclease.

    Science.gov (United States)

    Mineta, Yusuke; Okamoto, Tomoyuki; Takenaka, Kosuke; Doi, Norio; Aoyama, Yasuhiro; Sera, Takashi

    2009-01-01

    To refine zinc-finger nuclease (ZFN) technology, we constructed a sandwiched ZFN, in which a DNA cleavage enzyme was sandwiched with two artificial zinc-finger proteins (AZPs). Because the sandwiched ZFN is designed to cleave the DNA between the two AZP-binding sites, the sandwiched ZFN is expected to bind preferentially to a DNA substrate rather than to cleavage products and thereby cleave it with multiple turnovers. To prove the concept, we sandwiched a staphylococcal nuclease (SNase), which cleaves DNA as a monomer, between two 3-finger AZPs. The AZP-sandwiched SNase cleaved large amounts of dsDNA site-specifically. Such multiple-turnover cleavage was not observed with control nucleases that possess a single AZP.

  17. Cleavage-induced termination in U2 snRNA gene expression

    Energy Technology Data Exchange (ETDEWEB)

    Nabavi, Sadeq [Department of Molecular and Cellular Biology, University of Guelph, Guelph, Ontario, Canada N1G 2W1 (Canada); Nazar, Ross N., E-mail: rnnazar@uoguelph.ca [Department of Molecular and Cellular Biology, University of Guelph, Guelph, Ontario, Canada N1G 2W1 (Canada)

    2010-03-12

    The maturation of many small nuclear RNAs is dependent on RNase III-like endonuclease mediated cleavage, which generates a loading site for the exosome complex that trims the precursor at its 3' end. Using a temperature sensitive Pac1 nuclease, here we show that the endonuclease cleavage is equally important in terminating the transcription of the U2 snRNA in Schizosaccharomyces pombe. Using a temperature sensitive Dhp1p 5' {yields} 3' exonuclease, we demonstrate that it also is an essential component of the termination pathway. Taken together the results support a 'reversed torpedoes' model for the termination and maturation of the U2 snRNA; the Pac1 endonuclease cleavage provides entry sites for the 3' and 5' exonuclease activities, leading to RNA maturation in one direction and transcript termination in the other.

  18. Scandium(iii) triflate-promoted serine/threonine-selective peptide bond cleavage.

    Science.gov (United States)

    Ni, Jizhi; Sohma, Youhei; Kanai, Motomu

    2017-02-01

    The site-selective cleavage of peptide bonds is an important chemical modification that is useful not only for the structural determination of peptides, but also as an artificial modulator of peptide/protein function and properties. Here we report site-selective hydrolysis of peptide bonds at the Ser and Thr positions with a high conversion yield. This chemical cleavage relies on Sc(iii)-promoted N,O-acyl rearrangement and subsequent hydrolysis. The method is applicable to a broad scope of polypeptides with various functional groups, including a post-translationally modified peptide that is unsuitable for enzymatic hydrolysis. The system was further extended to site-selective cleavage of a native protein, Aβ1-42, which is closely related to the onset of Alzheimer's disease.

  19. Indirect DNA Sequence Recognition and Its Impact on Nuclease Cleavage Activity.

    Science.gov (United States)

    Lambert, Abigail R; Hallinan, Jazmine P; Shen, Betty W; Chik, Jennifer K; Bolduc, Jill M; Kulshina, Nadia; Robins, Lori I; Kaiser, Brett K; Jarjour, Jordan; Havens, Kyle; Scharenberg, Andrew M; Stoddard, Barry L

    2016-06-07

    LAGLIDADG meganucleases are DNA cleaving enzymes used for genome engineering. While their cleavage specificity can be altered using several protein engineering and selection strategies, their overall targetability is limited by highly specific indirect recognition of the central four base pairs within their recognition sites. In order to examine the physical basis of indirect sequence recognition and to expand the number of such nucleases available for genome engineering, we have determined the target sites, DNA-bound structures, and central four cleavage fidelities of nine related enzymes. Subsequent crystallographic analyses of a meganuclease bound to two noncleavable target sites, each containing a single inactivating base pair substitution at its center, indicates that a localized slip of the mutated base pair causes a small change in the DNA backbone conformation that results in a loss of metal occupancy at one binding site, eliminating cleavage activity.

  20. Computational prediction of cleavage using proteasomal in vitro digestion and MHC I ligand data

    Institute of Scientific and Technical Information of China (English)

    Yu-feng LU; Hao SHENG; Yi ZHANG; Zhi-yang LI

    2013-01-01

    Proteasomes are responsible for the production of the majority of cytotoxic T lymphocyte (CTL) epitopes.Hence,it is important to identify correctly which peptides will be generated by proteasomes from an unknown protein.However,the pool of proteasome cleavage data used in the prediction algorithms,whether from major histocompatibility complex (MHC) I ligand or in vitro digestion data,is not identical to in vivo proteasomal digestion products.Therefore,the accuracy and reliability of these models still need to be improved.In this paper,three types of proteasomal cleavage data,constitutive proteasome (cCP),immunoproteasome (iCP) in vitro cleavage,and MHC I ligandv data,were used for training cleave-site predictive methods based on the kernel-function stabilized matrix method (KSMM).The predictive accuracies of the KSMM+pair coefficients were 75.0%,72.3%,and 83.1% for cCP,iCP,and MHC I ligand data,respectively,which were comparable to the results from support vector machine (SVM).The three proteasomal cleavage methods were combined in turn with MHC I-peptide binding predictions to model MHC I-peptide processing and the presentation pathway.These integrations markedly improved MHC I peptide identification,increasing area under the receiver operator characteristics (ROC) curve (AUC) values from 0.82 to 0.91.The results suggested that both MHC I ligand and proteasomal in vitro degradation data can give an exact simulation of in vivo processed digestion.The information extracted from cCP and iCP in vitro cleavage data demonstrated that both cCP and iCP are selective in their usage of peptide bonds for cleavage.

  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. Shutoff of RNA polymerase II transcription by poliovirus involves 3C protease-mediated cleavage of the TATA-binding protein at an alternative site: incomplete shutoff of transcription interferes with efficient viral replication.

    Science.gov (United States)

    Kundu, Pallob; Raychaudhuri, Santanu; Tsai, Weimin; Dasgupta, Asim

    2005-08-01

    The TATA-binding protein (TBP) plays a crucial role in cellular transcription catalyzed by all three DNA-dependent RNA polymerases. Previous studies have shown that TBP is targeted by the poliovirus (PV)-encoded protease 3C(pro) to bring about shutoff of cellular RNA polymerase II-mediated transcription in PV-infected cells. The processing of the majority of viral precursor proteins by 3C(pro) involves cleavages at glutamine-glycine (Q-G) sites. We present evidence that suggests that the transcriptional inactivation of TBP by 3C(pro) involves cleavage at the glutamine 104-serine 105 (Q104-S105) site of TBP and not at the Q18-G19 site as previously thought. The TBP Q104-S105 cleavage by 3C(pro) is greatly influenced by the presence of an aliphatic amino acid at the P4 position, a hallmark of 3C(pro)-mediated proteolysis. To examine the importance of host cell transcription shutoff in the PV life cycle, stable HeLa cell lines were created that express recombinant TBP resistant to cleavage by the viral proteases, called GG rTBP. Transcription shutoff was significantly impaired and delayed in GG rTBP cells upon infection with poliovirus compared with the cells that express wild-type recombinant TBP (wt rTBP). Infection of GG rTBP cells with poliovirus resulted in small plaques, significantly reduced viral RNA synthesis, and lower viral yields compared to the wt rTBP cell line. These results suggest that a defect in transcription shutoff can lead to inefficient replication of poliovirus in cultured cells.

  3. Reductive cleavage of nitrite to form terminal uranium mono-oxo complexes.

    Science.gov (United States)

    Lewis, Andrew J; Carroll, Patrick J; Schelter, Eric J

    2013-01-09

    Uranium terminal mono-oxo complexes are prepared with a unique activation of nitrite following reductive cleavage of an N-O bond with loss of nitric oxide. The thermodynamic driving force of U═O bond formation differentiates this reactivity from known mechanisms of nitrite reduction, which are typically mediated by proton transfer. Mechanistic details are explored by DFT supporting a simple homolytic cleavage pathway from a κ(1)-ONO bound intermediate. Complexes of the formula U(VI)OX[N(SiMe(3))(2)](3) are formed providing a trigonal bipyramidal framework into which ligands trans to the U═O bond may be installed.

  4. Characterization of insulin-degrading enzyme-mediated cleavage of Aβ in distinct aggregation states.

    Science.gov (United States)

    Hubin, Ellen; Cioffi, Federica; Rozenski, Jef; van Nuland, Nico A J; Broersen, Kerensa

    2016-06-01

    To enhance our understanding of the potential therapeutic utility of insulin-degrading enzyme (IDE) in Alzheimer's disease (AD), we studied in vitro IDE-mediated degradation of different amyloid-beta (Aβ) peptide aggregation states. Our findings show that IDE activity is driven by the dynamic equilibrium between Aβ monomers and higher ordered aggregates. We identify Met(35)-Val(36) as a novel IDE cleavage site in the Aβ sequence and show that Aβ fragments resulting from IDE cleavage form non-toxic amorphous aggregates. These findings need to be taken into account in therapeutic strategies designed to increase Aβ clearance in AD patients by modulating IDE activity.

  5. DNA Cleavage of the Copper(Ⅱ) Complexes with a Polyamine Ditopic Ligand

    Institute of Scientific and Technical Information of China (English)

    Yi Bin WEI; Pin YANG

    2005-01-01

    A new binuclear complex [Cu2L(OH)](ClO4)3·2H2O has been synthesized and characterized, where L=2,6-bis{[bis-(2-aminoethyl)amino]methyl}-benzene. In the presence of0.5 mmol/L complex at pH 8.10 and 37℃, the complex can efficiently cleavage pBR322 DNA with a rate constant kobs of 1.35 × 10-4 s-1. The cleavage occurred by a non-oxidative mechanism showing activity to be dependent on pH.

  6. Near-IR Light-Mediated Cleavage of Antibody-Drug Conjugates Using Cyanine Photocages.

    Science.gov (United States)

    Nani, Roger R; Gorka, Alexander P; Nagaya, Tadanobu; Kobayashi, Hisataka; Schnermann, Martin J

    2015-11-09

    Despite significant progress in the clinical application of antibody drug conjugates (ADCs), novel cleavage strategies that provide improved selectivity are still needed. Herein is reported the first approach that uses near-IR light to cleave a small molecule from a biomacromolecule, and its application to the problem of ADC linkage. The preparation of cyanine antibody conjugates, drug cleavage mediated by 690 nm light, and initial in vitro and in vivo evaluation is described. These studies provide the critical chemical underpinning from which to develop this near-IR light cleavable linker strategy.

  7. The Cleavage and Polyadenylation Specificity Factor 6 (CPSF6) Subunit of the Capsid-recruited Pre-messenger RNA Cleavage Factor I (CFIm) Complex Mediates HIV-1 Integration into Genes.

    Science.gov (United States)

    Rasheedi, Sheeba; Shun, Ming-Chieh; Serrao, Erik; Sowd, Gregory A; Qian, Juan; Hao, Caili; Dasgupta, Twishasri; Engelman, Alan N; Skowronski, Jacek

    2016-05-27

    HIV-1 favors integration into active genes and gene-enriched regions of host cell chromosomes, thus maximizing the probability of provirus expression immediately after integration. This requires cleavage and polyadenylation specificity factor 6 (CPSF6), a cellular protein involved in pre-mRNA 3' end processing that binds HIV-1 capsid and connects HIV-1 preintegration complexes to intranuclear trafficking pathways that link integration to transcriptionally active chromatin. CPSF6 together with CPSF5 and CPSF7 are known subunits of the cleavage factor I (CFIm) 3' end processing complex; however, CPSF6 could participate in additional protein complexes. The molecular mechanisms underpinning the role of CPSF6 in HIV-1 infection remain to be defined. Here, we show that a majority of cellular CPSF6 is incorporated into the CFIm complex. HIV-1 capsid recruits CFIm in a CPSF6-dependent manner, which suggests that the CFIm complex mediates the known effects of CPSF6 in HIV-1 infection. To dissect the roles of CPSF6 and other CFIm complex subunits in HIV-1 infection, we analyzed virologic and integration site targeting properties of a CPSF6 variant with mutations that prevent its incorporation into CFIm We show, somewhat surprisingly, that CPSF6 incorporation into CFIm is not required for its ability to direct preferential HIV-1 integration into genes. The CPSF5 and CPSF7 subunits appear to have only a minor, if any, role in this process even though they appear to facilitate CPSF6 binding to capsid. Thus, CPSF6 alone controls the key molecular interactions that specify HIV-1 preintegration complex trafficking to active chromatin.

  8. The Role of Trop2 Cleavage Products in Prostate Tumorigenesis

    Science.gov (United States)

    2014-10-01

    Kuboshima M, Kuroiwa N, Okazumi S, Matsubara H, Nomura F, Takiguchi M, Hiwasa T. 2004. Serological identification of TROP2 by recombi - nant cDNA expression...transduced with lentivirus, resulting in the integration of viral DNA into the genome of the target cell and all of its progeny. If both...region X) and DNA was isolated separately from Fig. 1. Myc and myrAKT are coexpressed in advanced prostate cancer and synergize to initiate human

  9. Natural soluble interleukin-15Ralpha is generated by cleavage that involves the tumor necrosis factor-alpha-converting enzyme (TACE/ADAM17).

    Science.gov (United States)

    Budagian, Vadim; Bulanova, Elena; Orinska, Zane; Ludwig, Andreas; Rose-John, Stefan; Saftig, Paul; Borden, Ernest C; Bulfone-Paus, Silvia

    2004-09-24

    This study shows that the high affinity alpha-chain of the interleukin (IL)-15 receptor exists not only in membrane-anchored but also in soluble form. Soluble IL-15Ralpha (sIL-15Ralpha) can be detected in mouse sera and cell-conditioned media by enzyme-linked immunosorbent assay and by immunoprecipitation and Western blotting. This protein has a molecular mass of about 30 kDa because of the presence of a single N-glycosylation site, which is reduced to 26 kDa after N-glycosidase treatment. Transmembrane IL-15Ralpha is constitutively converted into its soluble form by proteolytic cleavage that involves tumor necrosis factor-alpha-converting enzyme (TACE), and this process is further enhanced by phorbol 12-myristate 13-acetate (PMA) stimulation. The hydroxamate GW280264X, which is capable of blocking TACE and the closely related disintegrin-like metalloproteinase 10 (ADAM10), effectively inhibited both spontaneous and PMA-inducible cleavage of IL-15Ralpha, whereas GI254023X, which preferentially blocks ADAM10, was ineffective. Overexpression of TACE but not ADAM10 in COS-7 cells enhanced the constitutive and PMA-inducible cleavage of IL-15Ralpha. Moreover, murine fibroblasts deficient in TACE but not ADAM10 expression exhibited a significant reduction in the spontaneous and inducible IL-15Ralpha shedding, whereas a reconstitution of TACE in these cells restored the release of sIL-15Ralpha, thereby suggesting that TACE-mediated proteolysis may represent a major mechanism for sIL-15Ralpha generation in mice. The existence of natural sIL-15Ralpha offers novel insights into the complex biology of IL-15 and envisages a new level for therapeutic intervention.

  10. Heightened cleavage of Axl receptor tyrosine kinase by ADAM metalloproteases may contribute to disease pathogenesis in SLE.

    Science.gov (United States)

    Orme, Jacob J; Du, Yong; Vanarsa, Kamala; Mayeux, Jessica; Li, Li; Mutwally, Azza; Arriens, Cristina; Min, Soyoun; Hutcheson, Jack; Davis, Laurie S; Chong, Benjamin F; Satterthwaite, Anne B; Wu, Tianfu; Mohan, Chandra

    2016-08-01

    Systemic lupus erythematosus (SLE) is characterized by antibody-mediated chronic inflammation in the kidney, lung, skin, and other organs to cause inflammation and damage. Several inflammatory pathways are dysregulated in SLE, and understanding these pathways may improve diagnosis and treatment. In one such pathway, Axl tyrosine kinase receptor responds to Gas6 ligand to block inflammation in leukocytes. A soluble form of the Axl receptor ectodomain (sAxl) is elevated in serum from patients with SLE and lupus-prone mice. We hypothesized that sAxl in SLE serum originates from the surface of leukocytes and that the loss of leukocyte Axl contributes to the disease. We determined that macrophages and B cells are a source of sAxl in SLE and in lupus-prone mice. Shedding of the Axl ectodomain from the leukocytes of lupus-prone mice is mediated by the matrix metalloproteases ADAM10 and TACE (ADAM17). Loss of Axl from lupus-prone macrophages renders them unresponsive to Gas6-induced anti-inflammatory signaling in vitro. This phenotype is rescued by combined ADAM10/TACE inhibition. Mice with Axl-deficient macrophages develop worse disease than controls when challenged with anti-glomerular basement membrane (anti-GBM) sera in an induced model of nephritis. ADAM10 and TACE also mediate human SLE PBMC Axl cleavage. Collectively, these studies indicate that increased metalloprotease-mediated cleavage of leukocyte Axl may contribute to end organ disease in lupus. They further suggest dual ADAM10/TACE inhibition as a potential therapeutic modality in SLE.

  11. Identification of protein SUMOylation sites by mass spectrometry using combined microwave-assisted aspartic acid cleavage and tryptic digestion

    Science.gov (United States)

    Osula, Omoruyi; Swatkoski, Stephen; Cotter, Robert J.

    2012-01-01

    SUMO (Small-Ubiquitin-like MOdifier) is a post-translational modifier of protein substrates at lysine residues that conjugates to proteins in response to various changes in the cell. As a result of SUMO modification, marked changes in transcription regulation, DNA repair, subcellular localization, and mitosis, among other cellular processes, are known to occur. However, while the identification of ubiquitylation sites by mass spectrometry is aided in part by the presence of a small di-amino acid GlyGly “tag” that remains on lysine residues following tryptic digestion, SUMOylation poses a particular challenge as the absence of a basic residue near to the SUMO C-terminus results in a significant 27 or 32 amino acid sequence branch conjugated to the substrate peptide. MS/MS analyses of these branch peptides generally reveal abundant fragment ions resulting from cleavage of the SUMO tail, but which obscure those needed for characterizing the target peptide sequence. Other approaches for identifying SUMO substrates exist and include overexpression of the SUMO isoforms using an N-terminal histidine tag, as well as site-directed mutagenesis of the C-terminal end of the SUMO sequence. Here, we employ combined enzymatic/chemical approaches which serve to shorten the SUMO tag, and thus help to simplify SUMO spectra, making interpretation of mass spectra and location of the SUMOylation site easier. As described in this report, we demonstrate a method for identifying SUMOylation sites using three commercially available SUMO- modified isoforms, and by employing acid-only and acid/trypsin cleavage strategies. These approaches were carried out using MALDI-TOF and LC/MS instrumentation, along with CID and ETD fragmentation. PMID:22576878

  12. Site-specifically Hydrolytic Cleavage of Oxidized Insulin B Chain With Cu(II) Ion

    Institute of Scientific and Technical Information of China (English)

    2000-01-01

    Electrospray mass spectrometry investigation shows that denatured oxidized insulin B chain can be selectively cleaved by simple Cu(II) ion and the site of cleavage is at Gly8-Ser9 bond which is second amide bond left from His 10 in the sequence of oxidized insulin B chain.

  13. Facile P-C/C-H Bond-Cleavage Reactivity of Nickel Bis(diphosphine) Complexes

    Energy Technology Data Exchange (ETDEWEB)

    Zhang, Shaoguang [Pacific Northwest National Lab. (PNNL), Richland, WA (United States); Li, Haixia [Texas A & M Univ., College Station, TX (United States). Dept. of Chemistry; Appel, Aaron M. [Pacific Northwest National Lab. (PNNL), Richland, WA (United States); Hall, Michael B. [Texas A & M Univ., College Station, TX (United States). Dept. of Chemistry; Bullock, R. Morris [Pacific Northwest National Lab. (PNNL), Richland, WA (United States)

    2016-06-07

    Unusual cleavage of P-C and C-H bonds of the P2N2 ligand in heteroleptic [Ni(P2N2)(diphosphine)]2+ complexes results in the formation of an iminium formyl nickelate featuring a C,P,P-tridentate coordination mode.

  14. 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

    specifically designed oligonucleotide targets that are attached to DNA origami templates. In this way, we use a highly selective approach to compare the efficiency of the electron-induced dissociation of a single disulfide bond with the more complex cleavage of the DNA backbone within a TT dinucleotide...

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

    Directory of Open Access Journals (Sweden)

    R Adam Smith

    2008-03-01

    Full Text Available R Adam Smith, Sarah L Sewell, Todd D GiorgioDepartment of Biomedical Engineering, Vanderbilt University, Nashville, TN, USAAbstract: 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.Keywords: quantum dot, MMP-7, protease, proximity activated nanoparticle

  16. Unusual nickel-mediated C-S cleavage of alkyl and aryl sulfoxides.

    Science.gov (United States)

    Schaub, Thomas; Backes, Marc; Radius, Udo

    2007-05-28

    The first examples of transition metal mediated C-S cleavage of sulfoxides containing sp2- and sp3-hybridized carbon bonds attached to the sulfur atom and the first example of a structurally characterized complex featuring an oxygen-bound sulfinyl ligand are presented.

  17. Antisense oligonucleotide-mediated exon skipping as a strategy to reduce proteolytic cleavage of ataxin-3.

    Science.gov (United States)

    Toonen, Lodewijk J A; Schmidt, Iris; Luijsterburg, Martijn S; van Attikum, Haico; van Roon-Mom, Willeke M C

    2016-10-12

    Spinocerebellar ataxia type-3 (SCA3) is a neurodegenerative disorder caused by a polyglutamine repeat expansion in the ataxin-3 protein. Cleavage of mutant ataxin-3 by proteolytic enzymes yields ataxin-3 fragments containing the polyglutamine stretch. These shorter ataxin-3 fragments are thought to be involved in SCA3 pathogenesis due to their increased cellular toxicity and their involvement in formation of the characteristic neuronal aggregates. As a strategy to prevent formation of toxic cleavage fragments, we investigated an antisense oligonucleotide-mediated modification of the ataxin-3 pre-mRNA through exon skipping of exon 8 and 9, resulting in the removal of a central 88 amino acid region of the ataxin-3 protein. This removed protein region contains several predicted cleavage sites and two ubiquitin-interacting motifs. In contrast to unmodified mutant ataxin-3, the internally truncated ataxin-3 protein did not give rise to potentially toxic cleavage fragments when incubated with caspases. In vitro experiments did not show cellular toxicity of the modified ataxin-3 protein. However, the modified protein was incapable of binding poly-ubiquitin chains, which may interfere with its normal deubiquitinating function. Low exon skipping efficiencies combined with reduction in important ataxin-3 protein functions suggest that skipping of exon 8 and 9 is not a viable therapeutic option for SCA3.

  18. 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.

  19. Quercetin-Iron Complex: Synthesis, Characterization, Antioxidant, DNA Binding, DNA Cleavage, and Antibacterial Activity Studies.

    Science.gov (United States)

    Raza, Aun; Xu, Xiuquan; Xia, Li; Xia, Changkun; Tang, Jian; Ouyang, Zhen

    2016-11-01

    Quercetin-iron (II) complex was synthesized and characterized by elemental analysis, ultraviolet-visible spectrophotometry, fourier transform infrared spectroscopy, mass spectrometry, proton nuclear magnetic resonance spectroscopy, thermogravimetry and differential scanning calorimetry, scanning electron micrography and molar conductivity. The low molar conductivity value investigates the non-electrolyte nature of the complex. The elemental analysis and other physical and spectroscopic methods reveal the 1:2 stoichiometric ratio (metal:ligand) of the complex. Antioxidant study of the quercetin and its metal complex against 2, 2-di-phenyl-1-picryl hydrazyl radical showed that the complex has much more radical scavenging activity than free quercetin. The interaction of quercetin-iron (II) complex with DNA was determined using ultraviolet visible spectra, fluorescence spectra and agarose gel electrophoresis. The results showed that quercetin-iron (II) complex can intercalate moderately with DNA, quench a strong intercalator ethidium bromide and compete for the intercalative binding sites. The complex showed significant cleavage of pBR 322 DNA from supercoiled form to nicked circular form and these cleavage effects were dose-dependent. Moreover, the mechanism of DNA cleavage indicated that it was an oxidative cleavage pathway. These results revealed the potential nuclease activity of complex to cleave DNA. In addition, antibacterial activity of complex on E.coli and S. aureus was also investigated. The results showed that complex has higher antibacterial activity than ligand.

  20. Rutin-Nickel Complex: Synthesis, Characterization, Antioxidant, DNA Binding, and DNA Cleavage Activities.

    Science.gov (United States)

    Raza, Aun; Bano, Shumaila; Xu, Xiuquan; Zhang, Rong Xian; Khalid, Haider; Iqbal, Furqan Muhammad; Xia, Changkun; Tang, Jian; Ouyang, Zhen

    2016-12-17

    The rutin-nickel (II) complex (RN) was synthesized and characterized by elemental analysis, UV-visible spectroscopy, IR, mass spectrometry, (1)H NMR, TG-DSC, SEM, and molar conductivity. The low molar conductivity value investigates the non-electrolyte nature of the complex. The elemental analysis and other physical and spectroscopic methods reveal the 1:2 stoichiometric ratio (metal/ligand) of the complex. An antioxidant study of rutin and its metal complex against DPPH radical showed that the complex has more radical scavenging activity than free rutin. The interaction of complex RN with DNA was determined using fluorescence spectra and agarose gel electrophoresis. The results showed that RN can intercalate moderately with DNA, quench a strong intercalator ethidium bromide (EB), and compete for the intercalative binding sites. The complex showed significant cleavage of pBR 322 DNA from supercoiled form (SC) to nicked circular form (NC), and these cleavage effects were dose-dependent. Moreover, the mechanism of DNA cleavage indicated that it was a hydrolytic cleavage pathway. These results revealed the potential nuclease activity of the complex to cleave DNA.

  1. A novel purification method for histidine-tagged proteins containing a thrombin cleavage site

    NARCIS (Netherlands)

    Hefti, M.H.; Vugt-Toorn, van der C.J.; Dixon, R.; Vervoort, J.J.M.

    2001-01-01

    A general procedure for the purification of histidine-tagged proteins has been developed using immobilized metal-ion affinity chromatography. This two-step purification method can be used for proteins containing a hexahistidine tag and a thrombin cleavage site, yielding high amounts of purified prot

  2. Cleavage mediated by the catalytic domain of bacterial RNase P RNA.

    Science.gov (United States)

    Wu, Shiying; Kikovska, Ema; Lindell, Magnus; Kirsebom, Leif A

    2012-09-14

    Like other RNA molecules, RNase P RNA (RPR) is composed of domains, and these have different functions. Here, we provide data demonstrating that the catalytic (C) domain of Escherichia coli (Eco) RPR when separated from the specificity (S) domain mediates cleavage using various model RNA hairpin loop substrates. Compared to full-length Eco RPR, the rate constant, k(obs), of cleavage for the truncated RPR (CP RPR) was reduced 30- to 13,000-fold depending on substrate. Specifically, the structural architecture of the -1/+73 played a significant role where a C(-1)/G(+73) pair had the most dramatic effect on k(obs). Substitution of A(248) (E. coli numbering), positioned near the cleavage site in the RNase P-substrate complex, with G in the CP RPR resulted in 30-fold improvement in rate. In contrast, strengthening the interaction between the RPR and the 3' end of the substrate only had a modest effect. Interestingly, although deleting the S-domain gave a reduction in the rate, it resulted in a less erroneous RPR with respect to cleavage site selection. These data support and extend our understanding of the coupling between the distal interaction between the S-domain and events at the active site. Our findings will also be discussed with respect to the structure of RPR derived from different organisms.

  3. 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.

  4. Arthrobacter luteus restriction endonuclease cleavage map of X174 RF DNA

    NARCIS (Netherlands)

    Vereijken, J.M.; Mansfeld, A.D.M. van; Baas, P.D.; Jansz, H.S.

    1975-01-01

    Cleavage of X174 RF DNA with the restriction endonuclease from Arthrobacter luteus (Alu I) produces 23 fragments of approximately 24–1100 base pairs in length. The order of most of these fragments has been established by digestion of Haemophilus influenzae Rd (Hind II) and Haemophilus aegyptius (Hae

  5. Carbon-Carbon Bond Cleavage Reaction: Synthesis of Multisubstituted Pyrazolo[1,5-a]pyrimidines.

    Science.gov (United States)

    Saikia, Pallabi; Gogoi, Sanjib; Boruah, Romesh C

    2015-07-02

    A new carbon-carbon bond cleavage reaction was developed for the efficient synthesis of multisubstituted pyrazolo[1,5-a]pyrimidines. This base induced reaction of 1,3,5-trisubstituted pentane-1,5-diones and substituted pyrazoles afforded good yields of the pyrazolo[1,5-a]pyrimidines.

  6. The party politics of economic reform: Public opinion, party positions and partisan cleavages

    NARCIS (Netherlands)

    Padgett, Stephen

    2005-01-01

    This article focuses on the capacity of parties to cultivate public opinion to accept welfare state reform. 'Preference shaping', it is argued, depends on the intensity of party 'messages', which will be at their strongest where there are sharply defined partisan cleavages in opinion. The aversion o

  7. Prediction of Signal Peptide Cleavage Sites with Subsite-Coupled and Template Matching Fusion Algorithm.

    Science.gov (United States)

    Zhang, Shao-Wu; Zhang, Ting-He; Zhang, Jun-Nan; Huang, Yufei

    2014-03-01

    Fast and effective prediction of signal peptides (SP) and their cleavage sites is of great importance in computational biology. The approaches developed to predict signal peptide can be roughly divided into machine learning based, and sliding windows based. In order to further increase the prediction accuracy and coverage of organism for SP cleavage sites, we propose a novel method for predicting SP cleavage sites called Signal-CTF that utilizes machine learning and sliding windows, and is designed for N-termial secretory proteins in a large variety of organisms including human, animal, plant, virus, bacteria, fungi and archaea. Signal-CTF consists of three distinct elements: (1) a subsite-coupled and regularization function with a scaled window of fixed width that selects a set of candidates of possible secretion-cleavable segment for a query secretory protein; (2) a sum fusion system that integrates the outcomes from aligning the cleavage site template sequence with each of the aforementioned candidates in a scaled window of fixed width to determine the best candidate cleavage sites for the query secretory protein; (3) a voting system that identifies the ultimate signal peptide cleavage site among all possible results derived from using scaled windows of different width. When compared with Signal-3L and SignalP 4.0 predictors, the prediction accuracy of Signal-CTF is 4-12 %, 10-25 % higher than that of Signal-3L for human, animal and eukaryote, and SignalP 4.0 for eukaryota, Gram-positive bacteria and Gram-negative bacteria, respectively. Comparing with PRED-SIGNAL and SignalP 4.0 predictors on the 32 archaea secretory proteins of used in Bagos's paper, the prediction accuracy of Signal-CTF is 12.5 %, 25 % higher than that of PRED-SIGNAL and SignalP 4.0, respectively. The predicting results of several long signal peptides show that the Signal-CTF can better predict cleavage sites for long signal peptides than SignalP, Phobius, Philius, SPOCTOPUS, Signal

  8. Thermodynamic Strategies for C-O Bond Formation and Cleavage via Tandem Catalysis.

    Science.gov (United States)

    Lohr, Tracy L; Li, Zhi; Marks, Tobin J

    2016-05-17

    To reduce global reliance on fossil fuels, new renewable sources of energy that can be used with the current infrastructure are required. Biomass represents a major source of renewable carbon based fuel; however, the high oxygen content (∼40%) limits its use as a conventional fuel. To utilize biomass as an energy source, not only with current infrastructure, but for maximum energy return, the oxygen content must be reduced. One method to achieve this is to develop selective catalytic methods to cleave C-O bonds commonly found in biomass (aliphatic and aromatic ethers and esters) for the eventual removal of oxygen in the form of volatile H2O or carboxylic acids. Once selective methods of C-O cleavage are understood and perfected, application to processing real biomass feedstocks such as lignin can be undertaken. This Laboratory previously reported that recyclable "green" lanthanide triflates are excellent catalysts for C-O bond-forming hydroalkoxylation reactions. Based on the virtues of microscopic reversibility, the same lanthanide triflate catalyst should catalyze the reverse C-O cleavage process, retrohydroalkoxylation, to yield an alcohol and an alkene. However, ether C-O bond-forming (retrohydroalkoxylation) to form an alcohol and alkene is endothermic. Guided by quantum chemical analysis, our strategy is to couple endothermic, in tandem, ether C-O bond cleavage with exothermic alkene hydrogenation, thereby leveraging the combined catalytic cycles thermodynamically to form an overall energetically favorable C-O cleavage reaction. This Account reviews recent developments on thermodynamically leveraged tandem catalysis for ether and more recently, ester C-O bond cleavage undertaken at Northwestern University. First, the fundamentals of lanthanide-catalyzed hydroelementation are reviewed, with particular focus on ether C-O bond formation (hydroalkoxylation). Next, the reverse C-O cleavage/retrohydroalkoxylation processes enabled by tandem catalysis are

  9. Cleavage Factor I Links Transcription Termination to DNA Damage Response and Genome Integrity Maintenance in Saccharomyces cerevisiae

    Science.gov (United States)

    Gaillard, Hélène; Aguilera, Andrés

    2014-01-01

    During transcription, the nascent pre-mRNA undergoes a series of processing steps before being exported to the cytoplasm. The 3′-end processing machinery involves different proteins, this function being crucial to cell growth and viability in eukaryotes. Here, we found that the rna14-1, rna15-1, and hrp1-5 alleles of the cleavage factor I (CFI) cause sensitivity to UV-light in the absence of global genome repair in Saccharomyces cerevisiae. Unexpectedly, CFI mutants were proficient in UV-lesion repair in a transcribed gene. DNA damage checkpoint activation and RNA polymerase II (RNAPII) degradation in response to UV were delayed in CFI-deficient cells, indicating that CFI participates in the DNA damage response (DDR). This is further sustained by the synthetic growth defects observed between rna14-1 and mutants of different repair pathways. Additionally, we found that rna14-1 suffers severe replication progression defects and that a functional G1/S checkpoint becomes essential in avoiding genetic instability in those cells. Thus, CFI function is required to maintain genome integrity and to prevent replication hindrance. These findings reveal a new function for CFI in the DDR and underscore the importance of coordinating transcription termination with replication in the maintenance of genomic stability. PMID:24603480

  10. High-throughput profiling of off-target DNA cleavage reveals RNA-programmed Cas9 nuclease specificity.

    Science.gov (United States)

    Pattanayak, Vikram; Lin, Steven; Guilinger, John P; Ma, Enbo; Doudna, Jennifer A; Liu, David R

    2013-09-01

    The RNA-programmable Cas9 endonuclease cleaves double-stranded DNA at sites complementary to a 20-base-pair guide RNA. The Cas9 system has been used to modify genomes in multiple cells and organisms, demonstrating its potential as a facile genome-engineering tool. We used in vitro selection and high-throughput sequencing to determine the propensity of eight guide-RNA:Cas9 complexes to cleave each of 10(12) potential off-target DNA sequences. The selection results predicted five off-target sites in the human genome that were confirmed to undergo genome cleavage in HEK293T cells upon expression of one of two guide-RNA:Cas9 complexes. In contrast to previous models, our results show that guide-RNA:Cas9 specificity extends past a 7- to 12-base-pair seed sequence. Our results also suggest a tradeoff between activity and specificity both in vitro and in cells as a shorter, less-active guide RNA is more specific than a longer, more-active guide RNA. High concentrations of guide-RNA:Cas9 complexes can cleave off-target sites containing mutations near or within the PAM that are not cleaved when enzyme concentrations are limiting.

  11. Cleavage factor I links transcription termination to DNA damage response and genome integrity maintenance in Saccharomyces cerevisiae.

    Directory of Open Access Journals (Sweden)

    Hélène Gaillard

    2014-03-01

    Full Text Available During transcription, the nascent pre-mRNA undergoes a series of processing steps before being exported to the cytoplasm. The 3'-end processing machinery involves different proteins, this function being crucial to cell growth and viability in eukaryotes. Here, we found that the rna14-1, rna15-1, and hrp1-5 alleles of the cleavage factor I (CFI cause sensitivity to UV-light in the absence of global genome repair in Saccharomyces cerevisiae. Unexpectedly, CFI mutants were proficient in UV-lesion repair in a transcribed gene. DNA damage checkpoint activation and RNA polymerase II (RNAPII degradation in response to UV were delayed in CFI-deficient cells, indicating that CFI participates in the DNA damage response (DDR. This is further sustained by the synthetic growth defects observed between rna14-1 and mutants of different repair pathways. Additionally, we found that rna14-1 suffers severe replication progression defects and that a functional G1/S checkpoint becomes essential in avoiding genetic instability in those cells. Thus, CFI function is required to maintain genome integrity and to prevent replication hindrance. These findings reveal a new function for CFI in the DDR and underscore the importance of coordinating transcription termination with replication in the maintenance of genomic stability.

  12. p75NTR-dependent Rac1 activation requires receptor cleavage and activation of an NRAGE and NEDD9 signaling cascade.

    Science.gov (United States)

    Zeinieh, Michele; Salehi, Amir; Rajkumar, Vijidha; Barker, Philip A

    2015-02-01

    The p75 neurotrophin receptor (p75NTR, also known as tumor necrosis factor receptor superfamily member 16) is implicated in diverse cellular events, but fundamental aspects of its signaling mechanisms remain unclear. To address this, we have established a novel bioassay to characterize signaling cascades activated by p75NTR. We show that in COS7 cells, p75NTR expression causes a large increase in cell surface area that relies on the activation of Rac1, and we demonstrate that the p75NTR-dependent COS7 phenotype is dependent on ADAM17- and c-secretase-dependent cleavage of p75NTR and generation of the p75NTR intracellular domain (p75NTRICD). We show that the p75NTR adaptor protein NRAGE (also known as MAGED1) acts downstream of the p75NTRICD in this cascade and, through a yeast two-hybrid screen, identify NEDD9, a Cas family adaptor protein, as a novel NRAGE-binding partner that mediates p75NTR-dependent Rac1 activation and cell spreading. Our results demonstrate a crucial role for p75NTR cleavage in small GTPase activation and define a novel Rac1 activation pathway involving the p75NTRICD, NRAGE andNEDD9.

  13. Efficient and specific internal cleavage of a retroviral palindromic DNA sequence by tetrameric HIV-1 integrase.

    Directory of Open Access Journals (Sweden)

    Olivier Delelis

    Full Text Available BACKGROUND: HIV-1 integrase (IN catalyses the retroviral integration process, removing two nucleotides from each long terminal repeat and inserting the processed viral DNA into the target DNA. It is widely assumed that the strand transfer step has no sequence specificity. However, recently, it has been reported by several groups that integration sites display a preference for palindromic sequences, suggesting that a symmetry in the target DNA may stabilise the tetrameric organisation of IN in the synaptic complex. METHODOLOGY/PRINCIPAL FINDINGS: We assessed the ability of several palindrome-containing sequences to organise tetrameric IN and investigated the ability of IN to catalyse DNA cleavage at internal positions. Only one palindromic sequence was successfully cleaved by IN. Interestingly, this symmetrical sequence corresponded to the 2-LTR junction of retroviral DNA circles-a palindrome similar but not identical to the consensus sequence found at integration sites. This reaction depended strictly on the cognate retroviral sequence of IN and required a full-length wild-type IN. Furthermore, the oligomeric state of IN responsible for this cleavage differed from that involved in the 3'-processing reaction. Palindromic cleavage strictly required the tetrameric form, whereas 3'-processing was efficiently catalysed by a dimer. CONCLUSIONS/SIGNIFICANCE: Our findings suggest that the restriction-like cleavage of palindromic sequences may be a general physiological activity of retroviral INs and that IN tetramerisation is strongly favoured by DNA symmetry, either at the target site for the concerted integration or when the DNA contains the 2-LTR junction in the case of the palindromic internal cleavage.

  14. 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

  15. Anaerobic DNA cleavage in red light by dicopper(II) complexes on disulphide bond activation

    Indian Academy of Sciences (India)

    Debojyoti Lahiri; Ritankar Majumdar; Ashis K Patra; Akhil R Chakravarty

    2010-05-01

    Binuclear complexes [Cu(-RSSR)]2 (1) and [M2(-PDS)(H2O)]2 (M = Cu(II), 2; Fe(II), 3), where H2RSSR is a reduced Schiff base derived from 2-(thioethyl)salicylaldimine having a disulphide moiety and H2PDS is derived from dimerization of D-penicillamine, have been prepared, structurally characterized, and their photo-induced DNA cleavage activity studied. The crystal structure of 1 shows the complex as a discrete binuclear species with each metal in a CuN2O2 square-planar geometry (Cu…Cu, 6.420 Å). The tetradentate RSSR2- acts as a bridging ligand. The sulphur atoms in the disulphide unit do not interact with the metal ions. Complexes 1-3 do not show any DNA cleavage activity in darkness. The copper(II) complexes exhibit chemical nuclease activity in the presence of 3-mercaptopropionic acid. Cleavage of supercoiled DNA has been observed in UV-A light of 365 nm for 1 and red light of 647.1 nm for both 1 and 2 in air. Mechanistic data reveal the involvement of the disulphide unit as photosensitizer generating hydroxyl radicals ($^{\\bullet}$OH) as the reactive species. Photo-induced DNA cleavage in red light seems to involve sulphide radicals in a type-I process and hydroxyl radicals. The dicopper(II) complexes show significant anaerobic photo-induced DNA cleavage activity in red light under argon following type-I pathway without involving any reactive oxygen species.

  16. Effect of Presenilin Mutations on APP Cleavage; Insights into the Pathogenesis of FAD.

    Science.gov (United States)

    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-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, six 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, six 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.

  17. Serine-selective aerobic cleavage of peptides and a protein using a water-soluble copper-organoradical conjugate.

    Science.gov (United States)

    Seki, Yohei; Tanabe, Kana; Sasaki, Daisuke; Sohma, Youhei; Oisaki, Kounosuke; Kanai, Motomu

    2014-06-16

    The site-specific cleavage of peptide bonds is an important chemical modification of biologically relevant macromolecules. The reaction is not only used for routine structural determination of peptides, but is also a potential artificial modulator of protein function. Realizing the substrate scope beyond the conventional chemical or enzymatic cleavage of peptide bonds is, however, a formidable challenge. Here we report a serine-selective peptide-cleavage protocol that proceeds at room temperature and near neutral pH value, through mild aerobic oxidation promoted by a water-soluble copper-organoradical conjugate. The method is applicable to the site-selective cleavage of polypeptides that possess various functional groups. Peptides comprising D-amino acids or sensitive disulfide pairs are competent substrates. The system is extendable to the site-selective cleavage of a native protein, ubiquitin, which comprises more than 70 amino acid residues.

  18. Investigations into the mechanisms of pyridine ring cleavage in vismodegib.

    Science.gov (United States)

    Khojasteh, S Cyrus; Yue, Qin; Ma, Shuguang; Castanedo, Georgette; Chen, Jacob Z; Lyssikatos, Joseph; Mulder, Teresa; Takahashi, Ryan; Ly, Justin; Messick, Kirsten; Jia, Wei; Liu, Lichuan; Hop, Cornelis E C A; Wong, Harvey

    2014-03-01

    Vismodegib (Erivedge, GDC-0449) is a first-in-class, orally administered small-molecule Hedgehog pathway inhibitor that is approved for the treatment of advanced basal cell carcinoma. Previously, we reported results from preclinical and clinical radiolabeled mass balance studies in which we determined that metabolism is the main route of vismodegib elimination. The metabolites of vismodegib are primarily the result of oxidation followed by glucuronidation. The focus of the current work is to probe the mechanisms of formation of three pyridine ring-cleaved metabolites of vismodegib, mainly M9, M13, and M18, using in vitro, ex vivo liver perfusion and in vivo rat studies. The use of stable-labeled ((13)C2,(15)N)vismodegib on the pyridine ring exhibited that the loss of carbon observed in both M9 and M13 was from the C-6 position of pyridine. Interestingly, the source of the nitrogen atom in the amide of M9 was from the pyridine. Evidence for the formation of aldehyde intermediates was observed using trapping agents as well as (18)O-water. Finally, we conclude that cytochrome P450 is involved in the formation of M9, M13, and M18 and that M3 (the major mono-oxidative metabolite) is not the precursor for the formation of these cleaved products; rather, M18 is the primary cleaved metabolite.

  19. Initiation of V(D)J recombination in a cell-free system

    NARCIS (Netherlands)

    D.C. van Gent (Dik); J.F. McBlane; D.A. Ramsden; M.J. Sadokfsky; J.E. Hesse (Joanne); M. Gellert

    1995-01-01

    textabstractCells performing V(D)J recombination make specific cuts in DNA at recombination signal sequences. Here, we show that nuclear extracts of pre-B cell lines carry out this specific cleavage. The products of cleavage are the same as found previously in thymocytes: full-leng

  20. Identification and characterization of a cleavage site in the proteolysis of orf virus 086 protein

    Directory of Open Access Journals (Sweden)

    Xiaoping eWang

    2016-04-01

    Full Text Available The ORF virus (ORFV is among the parapoxvirus genus of the poxviridae family, but little is known about the proteolytic pathways of ORFV encoding proteins. By contrast, the proteolysis mechanism of the vaccinia virus has been extensively explored. Vaccinia virus core protein P4a undergoes a proteolytic process that takes place at a conserved cleavage site Ala-Gly-X (where X is any amino acid and participates in virus assembly. Bioinformatics analysis revealed that an ORFV encoding protein, ORFV086, has a similar structure to the Vaccinia virus P4a core protein. In this study, we focus on the kinetic analysis and proteolysis mechanism of ORFV086. We found, via kinetic analysis, that ORFV086 is a late gene that starts to express at 8 hours post infection at mRNA level and 12 to 24 hours post infection at the protein level. The ORFV086 precursor and a 21kDa fragment can be observed in mature ORFV virions. The same bands were detected at only 3 hours post infection, suggesting that both the ORFV086 precursor and the 21kDa fragment are viral structural proteins. ORFV086 was cleaved from 12 to 24 hours post infection. The cleavage took place at different sites,resulting in seven bands with differing molecular weights. Sequence alignment revealed that five putative cleavage sites were predicted at C-terminal and internal regions of ORFV086. To investigate whether those cleavage sites are involved in proteolytic processing, full length and several deletion mutant ORFV086 recombinant proteins were expressed and probed. The GGS site that produced a 21kDa cleavage fragment was confirmed by identification of N/C-terminal FLAG epitope recombinant proteins, site-directed mutagenesis and Pulse-chase analysis. Interestingly, chase results demonstrated that, at late times, ORFV086 is partially cleaved. Taken together, we concluded that GGS is a cleavage site in ORFV086 and produces a 21kDa fragment post infection. Both ORFV086 precursor and the 21kDa fragment

  1. Inhibition of Lassa virus glycoprotein cleavage and multicycle replication by site 1 protease-adapted alpha(1-antitrypsin variants.

    Directory of Open Access Journals (Sweden)

    Anna Maisa

    Full Text Available BACKGROUND: Proteolytic processing of the Lassa virus envelope glycoprotein precursor GP-C by the host proprotein convertase site 1 protease (S1P is a prerequisite for the incorporation of the subunits GP-1 and GP-2 into viral particles and, hence, essential for infectivity and virus spread. Therefore, we tested in this study the concept of using S1P as a target to block efficient virus replication. METHODOLOGY/PRINCIPAL FINDING: We demonstrate that stable cell lines inducibly expressing S1P-adapted alpha(1-antitrypsin variants inhibit the proteolytic maturation of GP-C. Introduction of the S1P recognition motifs RRIL and RRLL into the reactive center loop of alpha(1-antitrypsin resulted in abrogation of GP-C processing by endogenous S1P to a similar level observed in S1P-deficient cells. Moreover, S1P-specific alpha(1-antitrypsins significantly inhibited replication and spread of a replication-competent recombinant vesicular stomatitis virus expressing the Lassa virus glycoprotein GP as well as authentic Lassa virus. Inhibition of viral replication correlated with the ability of the different alpha(1-antitrypsin variants to inhibit the processing of the Lassa virus glycoprotein precursor. CONCLUSIONS/SIGNIFICANCE: Our data suggest that glycoprotein cleavage by S1P is a promising target for the development of novel anti-arenaviral strategies.

  2. Crystallization and preliminary X-ray diffraction analysis of two N-terminal fragments of the DNA-cleavage domain of topoisomerase IV from Staphylococcus aureus

    Energy Technology Data Exchange (ETDEWEB)

    Carr, Stephen B., E-mail: bmbsbc@bmb.leeds.ac.uk [Astbury Centre for Structural Molecular Biology, University of Leeds, Leeds LS2 9JT (United Kingdom); Makris, George [Omega Mediation Hellas Ltd, Clinical and Pharma Consulting, 11525 N. Psychiko, Athens (Greece); Phillips, Simon E. V.; Thomas, Christopher D. [Astbury Centre for Structural Molecular Biology, University of Leeds, Leeds LS2 9JT (United Kingdom)

    2006-11-01

    The crystallization and data collection of topoisomerase IV from S. aureus is described. Phasing by molecular replacement proved difficult owing to the presence of translational NCS and strategies used to overcome this are discussed. DNA topoisomerase IV removes undesirable topological features from DNA molecules in order to help maintain chromosome stability. Two constructs of 56 and 59 kDa spanning the DNA-cleavage domain of the A subunit of topoisomerase IV from Staphylococcus aureus (termed GrlA56 and GrlA59) have been crystallized. Crystals were grown at 291 K using the sitting-drop vapour-diffusion technique with PEG 3350 as a precipitant. Preliminary X-ray analysis revealed that GrlA56 crystals belong to space group P2{sub 1}, diffract to a resolution of 2.9 Å and possess unit-cell parameters a = 83.6, b = 171.5, c = 87.8 Å, β = 90.1°, while crystals of GrlA59 belong to space group P2{sub 1}2{sub 1}2, with unit-cell parameters a = 41.5, b = 171.89, c = 87.9 Å. These crystals diffract to a resolution of 2.8 Å. This is the first report of the crystallization and preliminary X-ray analysis of the DNA-cleavage domain of a topoisomerase IV from a Gram-positive organism.

  3. Crystallization and preliminary X-ray diffraction analysis of two N-terminal fragments of the DNA-cleavage domain of topoisomerase IV from Staphylococcus aureus.

    Science.gov (United States)

    Carr, Stephen B; Makris, George; Phillips, Simon E V; Thomas, Christopher D

    2006-11-01

    DNA topoisomerase IV removes undesirable topological features from DNA molecules in order to help maintain chromosome stability. Two constructs of 56 and 59 kDa spanning the DNA-cleavage domain of the A subunit of topoisomerase IV from Staphylococcus aureus (termed GrlA56 and GrlA59) have been crystallized. Crystals were grown at 291 K using the sitting-drop vapour-diffusion technique with PEG 3350 as a precipitant. Preliminary X-ray analysis revealed that GrlA56 crystals belong to space group P2(1), diffract to a resolution of 2.9 A and possess unit-cell parameters a = 83.6, b = 171.5, c = 87.8 A, beta = 90.1 degrees, while crystals of GrlA59 belong to space group P2(1)2(1)2, with unit-cell parameters a = 41.5, b = 171.89, c = 87.9 A. These crystals diffract to a resolution of 2.8 A. This is the first report of the crystallization and preliminary X-ray analysis of the DNA-cleavage domain of a topoisomerase IV from a Gram-positive organism.

  4. DNA of a circular minichromosome linearized by restriction enzymes or other reagents is resistant to further cleavage: an influence of chromatin topology on the accessibility of DNA.

    Science.gov (United States)

    Kumala, Sławomir; Hadj-Sahraoui, Yasmina; Rzeszowska-Wolny, Joanna; Hancock, Ronald

    2012-10-01

    The accessibility of DNA in chromatin is an essential factor in regulating its activities. We studied the accessibility of the DNA in a ∼170 kb circular minichromosome to DNA-cleaving reagents using pulsed-field gel electrophoresis and fibre-fluorescence in situ hybridization on combed DNA molecules. Only one of several potential sites in the minichromosome DNA was accessible to restriction enzymes in permeabilized cells, and in growing cells only a single site at an essentially random position was cut by poisoned topoisomerase II, neocarzinostatin and γ-radiation, which have multiple potential cleavage sites; further sites were then inaccessible in the linearized minichromosomes. Sequential exposure to combinations of these reagents also resulted in cleavage at only a single site. Minichromosome DNA containing single-strand breaks created by a nicking endonuclease to relax any unconstrained superhelicity was also cut at only a single position by a restriction enzyme. Further sites became accessible after ≥95% of histones H2A, H2B and H1, and most non-histone proteins were extracted. These observations suggest that a global rearrangement of the three-dimensional packing and interactions of nucleosomes occurs when a circular minichromosome is linearized and results in its DNA becoming inaccessible to probes.

  5. Caspase cleavage product of BAP31 induces mitochondrial fission through endoplasmic reticulum calcium signals, enhancing cytochrome c release to the cytosol.

    Science.gov (United States)

    Breckenridge, David G; Stojanovic, Marina; Marcellus, Richard C; Shore, Gordon C

    2003-03-31

    Stimulation of cell surface death receptors activates caspase-8, which targets a limited number of substrates including BAP31, an integral membrane protein of the endoplasmic reticulum (ER). Recently, we reported that a caspase-resistant BAP31 mutant inhibited several features of Fas-induced apoptosis, including the release of cytochrome c (cyt.c) from mitochondria (Nguyen, M., D.G. Breckenridge, A. Ducret, and G.C. Shore. 2000. Mol. Cell. Biol. 20:6731-6740), implicating ER-mitochondria crosstalk in this pathway. Here, we report that the p20 caspase cleavage fragment of BAP31 can direct pro-apoptotic signals between the ER and mitochondria. Adenoviral expression of p20 caused an early release of Ca2+ from the ER, concomitant uptake of Ca2+ into mitochondria, and mitochondrial recruitment of Drp1, a dynamin-related protein that mediates scission of the outer mitochondrial membrane, resulting in dramatic fragmentation and fission of the mitochondrial network. Inhibition of Drp1 or ER-mitochondrial Ca2+ signaling prevented p20-induced fission of mitochondria. p20 strongly sensitized mitochondria to caspase-8-induced cyt.c release, whereas prolonged expression of p20 on its own ultimately induced caspase activation and apoptosis through the mitochondrial apoptosome stress pathway. Therefore, caspase-8 cleavage of BAP31 at the ER stimulates Ca2+-dependent mitochondrial fission, enhancing the release of cyt.c in response to this initiator caspase.

  6. Evaluation of DNA-binding, DNA cleavage, antioxidant and cytotoxic activity of mononuclear ruthenium(II) carbonyl complexes of benzaldehyde 4-phenyl-3-thiosemicarbazones

    Science.gov (United States)

    Sampath, Krishnan; Sathiyaraj, Subbaiyan; Jayabalakrishnan, Chinnasamy

    2013-11-01

    Two 4-phenyl-3-thiosemicarbazone ligands, (E)-2-(2-chlorobenzylidene)-N-phenylhydrazinecarbothioamide (HL1) and (E)-2-(2-nitrobenzylidene)-N-phenylhydrazinecarbothioamide (HL2), and its ruthenium(II) complexes were synthesized and characterized by physico-chemical and spectroscopic methods. The Schiff bases act as bidentate, monobasic chelating ligands with S and N as the donor sites and are preferably found in the thiol form in all the complexes studied. The molecular structure of HL1 and HL2 were determined by single crystal X-ray diffraction method. DNA binding of the compounds was investigated by absorption spectroscopy which indicated that the compounds bind to DNA via intercalation. The oxidative cleavage of the complexes with CT-DNA inferred that the effects of cleavage are dose dependent. Antioxidant study of the ligands and complexes showed significant antioxidant activity against DPPH radical. In addition, the in vitro cytotoxicity of the ligands and complexes assayed against HeLa and MCF-7 cell lines showed higher cytotoxic activity with the lower IC50 values indicating their efficiency in killing the cancer cells even at low concentrations.

  7. Mixed ligand ruthenium(III) complexes of benzaldehyde 4-methyl-3-thiosemicarbazones with triphenylphosphine/triphenylarsine co-ligands: Synthesis, DNA binding, DNA cleavage, antioxidative and cytotoxic activity

    Science.gov (United States)

    Sampath, K.; Sathiyaraj, S.; Raja, G.; Jayabalakrishnan, C.

    2013-08-01

    The new ruthenium(III) complexes with 4-methyl-3-thiosemicarbazone ligands, (E)-2-(2-chlorobenzylidene)-N-methylhydrazinecarbothioamide (HL1) and (E)-2-(2-nitrobenzylidene)-N-methylhydrazinecarbothioamide (HL2), were prepared and characterized by various physico-chemical and spectroscopic methods. The title compounds act as bidentate, monobasic chelating ligands with S and N as the donor sites and are preferably found in the thiol form in all the complexes studied. The molecular structure of HL1 and HL2 were determined by single crystal X-ray diffraction method. DNA binding of the ligands and complexes were investigated by absorption spectroscopy and IR spectroscopy. It reveals that the compounds bind to nitrogenous bases of DNA via intercalation. The oxidative cleavage of the complexes with CT-DNA inferred that the effects of cleavage are dose dependent. Antioxidant study of the ligands and complexes showed the significant antioxidant activity against DPPH radical. In addition, the in vitro cytotoxicity of the ligands and complexes against MCF-7 cell line was assayed which showed higher cytotoxic activity with the lower IC50 values indicating their efficiency in killing the cancer cells even at low concentrations.

  8. A 20 Residues Motif Delineates the Furin Cleavage Site and its Physical Properties May Influence Viral Fusion

    Directory of Open Access Journals (Sweden)

    Sun Tian

    2009-01-01

    Full Text Available Furin is a proprotein convertase that proteolytically cleaves protein precursors to yield functional proteins. Efficient cleavage depends on the presence of a specific sequence motif on the substrate. Currently, the cleavage site motif is described as a four amino acid pattern: R-X-[K/R]-R↓. However, not all furin cleavage recognition sites can be described by this pattern and not all R-X-[K/R]-R↓ sites are cleaved by furin. Since many furin substrates are involved in the pathogenesis of viral infection and human diseases, it is important to accurately characterize the furin cleavage site motif. In this study, the furin cleavage site motif was characterized using statistical analysis. The data were interpreted within the 3D crystal structure of the furin catalytic domain. The results indicate that the furin cleavage site motif is comprised of about 20 residues, P14–P6´. Specific physical properties such as volume, charge, and hydrophilicity are required at specific positions. The furin cleavage site motif is divided into two parts: 1 one core region (8 amino acids, positions P6–P2´ packed inside the furin binding pocket; 2 two polar regions (8 amino acids, positions P7–P14; and 4 amino acids, positions P3´–P6´ located outside the furin binding pocket. The physical properties of the core region contribute to the binding strength of the furin substrate, while the polar regions provide a solvent accessible environment and facilitate the accessibility of the core region to the furin binding pocket. This furin cleavage site motif also revealed a dynamic relationship linking the evolution of physical properties in region P1´–P6´ of viral fusion peptides, furin cleavage efficacy, and viral infectivity.

  9. Entropic origin of cobalt-carbon bond cleavage catalysis in adenosylcobalamin-dependent ethanolamine ammonia-lyase.

    Science.gov (United States)

    Wang, Miao; Warncke, Kurt

    2013-10-09

    Adenosylcobalamin-dependent enzymes accelerate the cleavage of the cobalt-carbon (Co-C) bond of the bound coenzyme by >10(10)-fold. The cleavage-generated 5'-deoxyadenosyl radical initiates the catalytic cycle by abstracting a hydrogen atom from substrate. Kinetic coupling of the Co-C bond cleavage and hydrogen-atom-transfer steps at ambient temperatures has interfered with past experimental attempts to directly address the factors that govern Co-C bond cleavage catalysis. Here, we use time-resolved, full-spectrum electron paramagnetic resonance spectroscopy, with temperature-step reaction initiation, starting from the enzyme-coenzyme-substrate ternary complex and (2)H-labeled substrate, to study radical pair generation in ethanolamine ammonia-lyase from Salmonella typhimurium at 234-248 K in a dimethylsulfoxide/water cryosolvent system. The monoexponential kinetics of formation of the (2)H- and (1)H-substituted substrate radicals are the same, indicating that Co-C bond cleavage rate-limits radical pair formation. Analysis of the kinetics by using a linear, three-state model allows extraction of the microscopic rate constant for Co-C bond cleavage. Eyring analysis reveals that the activation enthalpy for Co-C bond cleavage is 32 ± 1 kcal/mol, which is the same as for the cleavage reaction in solution. The origin of Co-C bond cleavage catalysis in the enzyme is, therefore, the large, favorable activation entropy of 61 ± 6 cal/(mol·K) (relative to 7 ± 1 cal/(mol·K) in solution). This represents a paradigm shift from traditional, enthalpy-based mechanisms that have been proposed for Co-C bond-breaking in B12 enzymes. The catalysis is proposed to arise from an increase in protein configurational entropy along the reaction coordinate.

  10. Peptides whose uptake by cells is controllable

    Energy Technology Data Exchange (ETDEWEB)

    Jiang, Tao; Tsien, Roger Y

    2014-02-04

    A generic structure for the peptides of the present invention includes A-X-B-C, where C is a cargo moiety, the B portion includes basic amino acids, X is a cleavable linker sequence, and the A portion includes acidic amino acids. The intact structure is not significantly taken up by cells; however, upon extracellular cleavage of X, the B-C portion is taken up, delivering the cargo to targeted cells. Cargo may be, for example, a contrast agent for diagnostic imaging, a chemotherapeutic drug, or a radiation-sensitizer for therapy. Cleavage of X allows separation of A from B, unmasking the normal ability of the basic amino acids in B to drag cargo C into cells near the cleavage event. X is cleaved extracellularly, preferably under physiological conditions. D-amino acids are preferred for the A and B portions, to minimize immunogenicity and nonspecific cleavage by background peptidases or proteases.

  11. Peptides whose uptake by cells is controllable

    Energy Technology Data Exchange (ETDEWEB)

    Jiang, Tao [San Diego, CA; Tsien, Roger Y [La Jolla, CA

    2012-02-07

    A generic structure for the peptides of the present invention includes A-X-B-C, where C is a cargo moiety, the B portion includes basic amino acids, X is a cleavable linker sequence, and the A portion includes acidic amino acids. The intact structure is not significantly taken up by cells; however, upon extracellular cleavage of X, the B-C portion is taken up, delivering the cargo to targeted cells. Cargo may be, for example, a contrast agent for diagnostic imaging, a chemotherapeutic drug, or a radiation-sensitizer for therapy. Cleavage of X allows separation of A from B, unmasking the normal ability of the basic amino acids in B to drag cargo C into cells near the cleavage event. X is cleaved extracellularly, preferably under physiological conditions. D-amino acids are preferred for the A and B portions, to minimize immunogenicity and nonspecific cleavage by background peptidases or proteases.

  12. Peptides whose uptake by cells is controllable

    Science.gov (United States)

    Jiang, Tao; Tsien, Roger Y.

    2008-10-07

    A generic structure for the peptides of the present invention includes A-X-B-C, where C is a cargo moiety, the B portion includes basic amino acids, X is a cleavable linker sequence, and the A portion includes acidic amino acids. The intact structure is not significantly taken up by cells; however, upon extracellular cleavage of X, the B-C portion is taken up, delivering the cargo to targeted cells. Cargo may be, for example, a contrast agent for diagnostic imaging, a chemotherapeutic drug, or a radiation-sensitizer for therapy. Cleavage of X allows separation of A from B, unmasking the normal ability of the basic amino acids in B to drag cargo C into cells near the cleavage event. X is cleaved extracellularly, preferably under physiological conditions. D-amino acids are preferred for the A and B portions, to minimize immunogenicity and nonspecific cleavage by background peptidases or proteases.

  13. Structural basis for duplex RNA recognition and cleavage by Archaeoglobus fulgidus C3PO

    Science.gov (United States)

    Parizotto, Eneida A; Lowe, Edward D; Parker, James S

    2013-01-01

    Oligomeric complexes of Trax and Translin proteins, known as C3POs, participate in a variety of eukaryotic nucleic acid metabolism pathways including RNAi and tRNA processing. In RNAi in humans and Drosophila, C3PO activates pre-RISC by removing the passenger strand of the siRNA precursor duplex using nuclease activity present in Trax. It is not known how C3POs engage with nucleic acid substrates. Here we identify a single protein from Archaeoglobus fulgidus that assembles into an octamer with striking similarity to human C3PO. The structure in complex with duplex RNA reveals that the octamer entirely encapsulates a single thirteen base-pair RNA duplex inside a large inner cavity. Trax-like subunit catalytic sites target opposite strands of the duplex for cleavage, separated by seven base pairs. The structure provides insight into the mechanism of RNA recognition and cleavage by an archaeal C3PO-like complex. PMID:23353787

  14. Structures of a CRISPR-Cas9 R-loop complex primed for DNA cleavage.

    Science.gov (United States)

    Jiang, Fuguo; Taylor, David W; Chen, Janice S; Kornfeld, Jack E; Zhou, Kaihong; Thompson, Aubri J; Nogales, Eva; Doudna, Jennifer A

    2016-02-19

    Bacterial adaptive immunity and genome engineering involving the CRISPR (clustered regularly interspaced short palindromic repeats)-associated (Cas) protein Cas9 begin with RNA-guided DNA unwinding to form an RNA-DNA hybrid and a displaced DNA strand inside the protein. The role of this R-loop structure in positioning each DNA strand for cleavage by the two Cas9 nuclease domains is unknown. We determine molecular structures of the catalytically active Streptococcus pyogenes Cas9 R-loop that show the displaced DNA strand located near the RuvC nuclease domain active site. These protein-DNA interactions, in turn, position the HNH nuclease domain adjacent to the target DNA strand cleavage site in a conformation essential for concerted DNA cutting. Cas9 bends the DNA helix by 30°, providing the structural distortion needed for R-loop formation.

  15. Mercury Detoxification by Bacteria: Simulations of Transcription Activation and Mercury-Carbon Bond Cleavage

    Energy Technology Data Exchange (ETDEWEB)

    Guo, Hao-Bo [ORNL; Parks, Jerry M [ORNL; Johs, Alexander [ORNL; Smith, Jeremy C [ORNL

    2011-01-01

    In this chapter, we summarize recent work from our laboratory and provide new perspective on two important aspects of bacterial mercury resistance: the molecular mechanism of transcriptional regulation by MerR, and the enzymatic cleavage of the Hg-C bond in methylmercury by the organomercurial lyase, MerB. Molecular dynamics (MD) simulations of MerR reveal an opening-and-closing dynamics, which may be involved in initiating transcription of mercury resistance genes upon Hg(II) binding. Density functional theory (DFT) calculations on an active-site model of the enzyme reveal how MerB catalyzes the Hg-C bond cleavage using cysteine coordination and acid-base chemistry. These studies provide insight into the detailed mechanisms of microbial gene regulation and defense against mercury toxicity.

  16. VAMP/synaptobrevin cleavage by tetanus and botulinum neurotoxins is strongly enhanced by acidic liposomes.

    Science.gov (United States)

    Caccin, Paola; Rossetto, Ornella; Rigoni, Michela; Johnson, Eric; Schiavo, Giampietro; Montecucco, Cesare

    2003-05-01

    Tetanus and botulinum neurotoxins (TeNT and BoNTs) block neuroexocytosis via specific cleavage and inactivation of SNARE proteins. Such activity is exerted by the N-terminal 50 kDa light chain (L) domain, which is a zinc-dependent endopeptidase. TeNT, BoNT/B, /D, /F and /G cleave vesicle associated membrane protein (VAMP), a protein of the neurotransmitter-containing small synaptic vesicles, at different single peptide bonds. Since the proteolytic activity of these metalloproteases is higher on native VAMP inserted in synaptic vesicles than on recombinant VAMP, we have investigated the influence of liposomes of different lipid composition on this activity. We found that the rate of VAMP cleavage with all neurotoxins tested here is strongly enhanced by negatively charged lipid mixtures. This effect is at least partially due to the binding of the metalloprotease to the lipid membranes, with electrostatic interactions playing an important role.

  17. Histidine-Based Lipopeptides Enhance Cleavage of Nucleic Acids: Interactions with DNA and Hydrolytic Properties.

    Science.gov (United States)

    Bélières, M; Déjugnat, C; Chouini-Lalanne, N

    2015-12-16

    Interaction studies and cleavage activity experiments were carried out between plasmid DNA and a series of histidine-based lipopeptides. Specific fluorescent probes (ethidium bromide, Hoechst 33342, and pyrene) were used to monitor intercalation, minor groove binding, and self-assembly of lipopeptides, respectively. Association between DNA and lipopeptides was thus evidenced, highlighting the importance of both histidine and hydrophobic tail in the interaction process. DNA cleavage in the presence of lipopeptides was then detected by gel electrophoresis and quantified, showing the importance of histidine and the involvement of its side-chain imidazole in the hydrolysis mechanism. These systems could then be developed as synthetic nucleases while raising concern of introducing histidine in the design of lipopeptide-based transfection vectors.

  18. Evidence for cleavage of the metalloprotease Vsm from Vibrio splendidus strain JZ6 by an M20 peptidase (PepT-like protein at low temperature

    Directory of Open Access Journals (Sweden)

    Rui Liu

    2016-10-01

    Full Text Available Metalloprotease Vsm is a major extracellular virulence factor of Vibrio splendidus. The toxicity of Vsm from V. splendidus strain JZ6 has been characterized, and production of this virulence factor proved to be temperature-regulated. The present study provides evidence that two forms (JZE1 and JZE2 of Vsm protein exist in extracellular products (ECPs of strain JZ6, and a significant conversion of these two forms was detected by SDS-PAGE and immunoblotting analyses of samples obtained from cells grown at 4, 10, 16, 20, 24 and 28 ℃. Mass spectroscopy confirmed that JZE1 was composed only of the peptidase_M4 domain of Vsm, and JZE2 contained both the PepSY domain and the peptidase_M4 domain. An M20 peptidase T-like protein (PepTL was screened from the transcriptome data of strain JZ6, which was considered as a crucial molecule to produce the active Vsm (JZE1 by cleavage of the propeptide. Similar to that of Vsm, PepTL mRNA accumulation was highest at 4 ℃ (836.82-fold of that at 28 ℃, decreased with increasing of temperature and reached its lowest level at 28 ℃. Deletion of the gene encoding the PepTL resulted in a mutant strain that did not produce the JZE1 cleavage product. The peptidase activity of PepTL recombinant protein (rPepTL was confirmed by cleaving the Vsm in ECPs with an in vitro degradation reaction. These results demonstrate that PepTL participates in activating Vsm in strain JZ6 by proteolytic cleavage at low temperature.

  19. Exchanging Alkyl Groups through Unstrained C-C Bond Cleavage in the Presence of a Copper Catalyst.

    Science.gov (United States)

    Wada, Masaru; Noda, Yushi; Nishikata, Takashi

    2017-04-05

    Although numerous reports exist on strained C-C bond cleavage reactions in aryl substitutions, the cleavage methodology for unstrained C-C bonds in alkylation reactions has not yet been established. We found that unstrained allylic C-C bonds can be cleaved using α-radicals to form C(sp(3) )-C(sp(3) ) bonds in the presence of a copper catalyst. In this reaction, the property of leaving and loading radicals is very important for radical fragmentations. In this paper, we investigated the effects of these properties in cleavage reactions for unstrained C-C bonds.

  20. 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.

  1. 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

    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...... is well above that of the publicly available chloroplast localization predictor PSORT. Cleavage sites are predicted using a scoring matrix derived by an automatic motif-finding algorithm. Approximately 60% of the known cleavage sites in our sequence collection were predicted to within +/-2 residues from...

  2. Experimental study about nano-deformation field near quasi-cleavage crack tip

    Institute of Scientific and Technical Information of China (English)

    邢永明; 戴福隆; 杨卫FML

    2000-01-01

    Using the nano-moire method, we measure the near tip nanoscopic deformation on the {111 } plane of single crystal silicon with a loaded quasi-cleavage crack running in the [110] direction. The measured strain distribution ahead of the crack tip agrees with the linear elastic fracture mechanics prediction up to 10 nm from the crack tip. Dislocations of Peierls type are detected and they extend from the crack tip over a length of hundreds of Burgers vectors.

  3. Unexpected cleavage of 2-azido-2-(hydroxymethyl)oxetanes: conformation determines reaction pathway?

    Science.gov (United States)

    Farber, Elisa; Herget, Jackson; Gascón, José A; Howell, Amy R

    2010-11-19

    An unanticipated cleavage of 2-azido-2-(hydroxymethyl)oxetanes is reported. In attempts to oxidize the title oxetanyl alcohols to the corresponding carboxylic acids with RuO4, cleaved nitriles were formed as the sole isolable products, while a closely related tetrahydrofuran gave solely the expected carboxylic acid. Quantum chemical calculations suggest that the divergent outcomes are governed by conformational differences in the azidoalcohols.

  4. 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.

  5. Manipulations of Amyloid Precursor Protein Cleavage Disrupt the Circadian Clock in Aging Drosophila

    OpenAIRE

    Blake, Matthew R.; Holbrook, Scott D.; Kotwica-Rolinska, Joanna; Chow, Eileen; Kretzschmar, Doris; Giebultowicz, Jadwiga M.

    2015-01-01

    Alzheimer’s disease (AD) is a neurodegenerative disease characterized by severe cognitive deterioration. While causes of AD pathology are debated, a large body of evidence suggests that increased cleavage of Amyloid Precursor Protein (APP) producing the neurotoxic Amyloid-β (Aβ) peptide plays a fundamental role in AD pathogenesis. One of the detrimental behavioral symptoms commonly associated with AD is the fragmentation of sleep-activity cycles with increased nighttime activity and daytime n...

  6. Aerobic dehydrogenative α-diarylation of benzyl ketones with aromatics through carbon-carbon bond cleavage.

    Science.gov (United States)

    More, Nagnath Yadav; Jeganmohan, Masilamani

    2014-02-01

    Substituted benzyl ketones reacted with aromatics in the presence of K2S2O8 in CF3COOH at room temperature, yielding α-diaryl benzyl ketones through a carbon-carbon bond cleavage. In the reaction, two new carbon-carbon bonds were formed and one carbon-carbon bond was cleaved. It is very interesting that two different nucleophiles such as benzyl ketones and aromatics were coupled together without metal, which is unusual in organic synthesis.

  7. Cleavage of serum response factor mediated by enteroviral protease 2A contributes to impaired cardiac function

    Institute of Scientific and Technical Information of China (English)

    Jerry Wong; Jingchun Zhang; Bobby Yanagawa; Zongshu Luo; Xiangsheng Yang; Jiang Chang; Bruce McManus; Honglin Luo

    2012-01-01

    Enteroviral infection can lead to dilated cardiomyopathy (DCM),which is a major cause of cardiovascular mortality worldwide.However,the pathogenetic mechanisms have not been fully elucidated.Serum response factor (SRF) is a cardiac-enriched transcription regulator controlling the expression of a variety of target genes,including those involved in the contractile apparatus and immediate early response,as well as microRNAs that silence the expression of cardiac regulatory factors.Knockout of SRF in the heart results in downregulation of cardiac contractile gene expression and development of DCM.The goal of this study is to understand the role of SRF in enterovirus-induced cardiac dysfunction and progression to DCM.Here we report that SRF is cleaved following enteroviral infection of mouse heart and cultured cardiomyocytes.This cleavage is accompanied by impaired cardiac function and downregulation of cardiac-specific contractile and regulatory genes.Further investigation by antibody epitope mapping and site-directed mutagenesis demonstrates that SRF cleavage occurs at the region of its transactivation domain through the action of virus-encoded protease 2A.Moreover,we demonstrate that cleavage of SRF dissociates its transactivation domain from DNA-binding domain,resulting in the disruption of SRF-mediated gene transactivation.In addition to loss of functional SRF,finally we report that the N-terminal fragment of SRF cleavage products can also act as a dominant-negative transcription factor,which likely competes with the native SRF for DNA binding.Our results suggest a mechanism by which virus infection impairs heart function and may offer a new therapeutic strategy to ameliorate myocardial damage and progression to DCM.

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

    Science.gov (United States)

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

    2016-09-14

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

  9. 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.

  10. Reaction Pathways and Energetics of Etheric C–O Bond Cleavage Catalyzed by Lanthanide Triflates

    Energy Technology Data Exchange (ETDEWEB)

    Assary, Rajeev S.; Atesin, Abdurrahman C.; Li, Zhi; Curtiss, Larry A.; Marks, Tobin J.

    2013-09-06

    Efficient and selective cleavage of etheric C-O bonds is crucial for converting biomass into platform chemicals and liquid transportation fuels. In this contribution, computational methods at the DFT B3LYP level of theory are employed to understand the efficacy of lanthanide triflate catalysts (Ln(OTf)3, Ln = La, Ce, Sm, Gd, Yb, and Lu) in cleaving etheric C-O bonds. In agreement with experiment, the calculations indicate that the reaction pathway for C-O cleavage occurs via a C-H → O-H proton transfer in concert with weakening of the C-O bond of the coordinated ether substrate to ultimately yield a coordinated alkenol. The activation energy for this process falls as the lanthanide ionic radius decreases, reflecting enhanced metal ion electrophilicity. Details of the reaction mechanism for Yb(OTf)3-catalyzed ring opening are explored in depth, and for 1-methyl-d3-butyl phenyl ether, the computed primary kinetic isotope effect of 2.4 is in excellent agreement with experiment (2.7), confirming that etheric ring-opening pathway involves proton transfer from the methyl group alpha to the etheric oxygen atom, which is activated by the electrophilic lanthanide ion. Calculations of the catalytic pathway using eight different ether substrates indicate that the more rapid cleavage of acyclic versus cyclic ethers is largely due to entropic effects, with the former C-O bond scission processes increasing the degrees of freedom/particles as the transition state is approached.

  11. Reaction between radicals and N-alkoxyamines As coordinated cleavage with fragmentation

    Science.gov (United States)

    Denisov, E. T.; Shestakov, A. F.

    2015-08-01

    Quantum chemical calculations of the enthalpy and activation energy of two reactions with MeO{2/⊙} attacking the CH- and CH2-groups of 2,2,6,6-tetramethylpiperidineoxy-2'-butane are performed. It is shown that the cleavage of hydrogen atoms is accompanied by coordinated breaking of N-O-bonds in the former case and C-O-bonds in the latter. Based on the obtained results, a new scheme is proposed for the cyclic mechanism behind the cleavage of chains on nitroxyl radicals in oxidizing hydrocarbons and polymers that agrees with experimental data. At the center of this cyclic mechanism lies the fast exothermic reaction between peroxyl radicals and N-alkoxyamine with the cleavage of H atoms and the coordinated fragmentation of molecules. Using the model of intersecting parabolas, an algorithm for calculating the enthalpies, activation energies, and rate constants of these reactions with the participation of alkyl, alkoxy, aminyl, peroxyl, phenoxyl, thiyl, and hydroxyl radicals is proposed.

  12. 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.

  13. Effects of 2'-O-methyl nucleotide substitution on EcoRI endonuclease cleavage activities.

    Directory of Open Access Journals (Sweden)

    Guojie Zhao

    Full Text Available To investigate the effect of sugar pucker conformation on DNA-protein interactions, we used 2'-O-methyl nucleotide (2'-OMeN to modify the EcoRI recognition sequence -TGAATTCT-, and monitored the enzymatic cleavage process using FRET method. The 2'-O-methyl nucleotide has a C3'-endo sugar pucker conformation different from the C2'-endo sugar pucker conformation of native DNA nucleotides. The initial reaction velocities were measured and the kinetic parameters, Km and Vmax were derived using Michaelis-Menten equation. Experimental results showed that 2'-OMeN substitutions for the EcoRI recognition sequence decreased the cleavage efficiency for A2, A3 and T4 substitutions significantly, and 2'-OMeN substitution for T5 residue inhibited the enzymatic activity completely. In contrast, substitutions for G1 and C6 could maintain the original activity. 2'-fluoro nucleic acid (2'-FNA and locked nucleic acid (LNA having similar C3'-endo sugar pucker conformation also demonstrated similar enzymatic results. This position-dependent enzymatic cleavage property might be attributed to the phosphate backbone distortion caused by the switch from C2'-endo to C3'-endo sugar pucker conformation, and was interpreted on the basis of the DNA-EcoRI structure. These 2'-modified nucleotides could behave as a regulatory element to modulate the enzymatic activity in vitro, and this property will have potential applications in genetic engineering and biomedicine.

  14. Synthesis, characterization, and photoactivated DNA cleavage by copper (II)/cobalt (II) mediated macrocyclic complexes.

    Science.gov (United States)

    Naik, H R Prakash; Naik, H S Bhojya; Aravinda, T; Lamani, D S

    2010-01-01

    We report the synthesis of new photonuclease consisting of two Co(II)/Cu(II) complexes of macrocyclic fused quinoline. Metal complexes are [MLX(2)], type where M = Co(II) (5), Cu(II) (6), and X = Cl, and are well characterized by elemental analysis, Fourier transform infrared spectroscopy, (1)H-NMR and electronic spectra. We have shown that photocleavage of plasmid DNA is markedly enhanced when this ligand is irradiated in the presence of Cu(II), and more so than that of cobalt. The chemistry of ternary and binary Co(II) complexes showing efficient light induced (360 nm) DNA cleavage activity is summarized. The role of the metal in photoinduced DNA cleavage reactions is explored by designing complex molecules having macrocyclic structure. The mechanistic pathways are found to be concentration dependent on Co(II)/Cu(II) complexes and the photoexcitation energy photoredox chemistry. Highly effective DNA cleavage ability of 6 is attributed to the effective cooperation of the metal moiety.

  15. Yeast SREBP cleavage activation requires the Golgi Dsc E3 ligase complex.

    Science.gov (United States)

    Stewart, Emerson V; Nwosu, Christine C; Tong, Zongtian; Roguev, Assen; Cummins, Timothy D; Kim, Dong-Uk; Hayles, Jacqueline; Park, Han-Oh; Hoe, Kwang-Lae; Powell, David W; Krogan, Nevan J; Espenshade, Peter J

    2011-04-22

    Mammalian lipid homeostasis requires proteolytic activation of membrane-bound sterol regulatory element binding protein (SREBP) transcription factors through sequential action of the Golgi Site-1 and Site-2 proteases. Here we report that while SREBP function is conserved in fungi, fission yeast employs a different mechanism for SREBP cleavage. Using genetics and biochemistry, we identified four genes defective for SREBP cleavage, dsc1-4, encoding components of a transmembrane Golgi E3 ligase complex with structural homology to the Hrd1 E3 ligase complex involved in endoplasmic reticulum-associated degradation. The Dsc complex binds SREBP and cleavage requires components of the ubiquitin-proteasome pathway: the E2-conjugating enzyme Ubc4, the Dsc1 RING E3 ligase, and the proteasome. dsc mutants display conserved aggravating genetic interactions with components of the multivesicular body pathway in fission yeast and budding yeast, which lacks SREBP. Together, these data suggest that the Golgi Dsc E3 ligase complex functions in a post-ER pathway for protein degradation.

  16. Enantioselective epoxidation and carbon-carbon bond cleavage catalyzed by Coprinus cinereus peroxidase and myeloperoxidase.

    Science.gov (United States)

    Tuynman, A; Spelberg, J L; Kooter, I M; Schoemaker, H E; Wever, R

    2000-02-01

    We demonstrate that myeloperoxidase (MPO) and Coprinus cinereus peroxidase (CiP) catalyze the enantioselective epoxidation of styrene and a number of substituted derivatives with a reasonable enantiomeric excess (up to 80%) and in a moderate yield. Three major differences with respect to the chloroperoxidase from Caldariomyces fumago (CPO) are observed in the reactivity of MPO and CiP toward styrene derivatives. First, in contrast to CPO, MPO and CiP produced the (S)-isomers of the epoxides in enantiomeric excess. Second, for MPO and CiP the H(2)O(2) had to be added very slowly (10 eq in 16 h) to prevent accumulation of catalytically inactive enzyme intermediates. Under these conditions, CPO hardly showed any epoxidizing activity; only with a high influx of H(2)O(2) (300 eq in 1.6 h) was epoxidation observed. Third, both MPO and CiP formed significant amounts of (substituted) benzaldehydes as side products as a consequence of C-alpha-C-beta bond cleavage of the styrene derivatives, whereas for CPO and cytochrome c peroxidase this activity is not observed. C-alpha-C-beta cleavage was the most prominent reaction catalyzed by CiP, whereas with MPO the relative amount of epoxide formed was higher. This is the first report of peroxidases catalyzing both epoxidation reactions and carbon-carbon bond cleavage. The results are discussed in terms of mechanisms involving ferryl oxygen transfer and electron transfer, respectively.

  17. Exploring Regioselective Bond Cleavage and Cross-Coupling Reactions using a Low-Valent Nickel Complex.

    Science.gov (United States)

    Desnoyer, Addison N; Friese, Florian W; Chiu, Weiling; Drover, Marcus W; Patrick, Brian O; Love, Jennifer A

    2016-03-14

    Recently, esters have received much attention as transmetalation partners for cross-coupling reactions. Herein, we report a systematic study of the reactivity of a series of esters and thioesters with [{(dtbpe)Ni}2(μ-η(2):η(2)-C6H6)] (dtbpe=1,2-bis(di-tert-butyl)phosphinoethane), which is a source of (dtbpe)nickel(0). Trifluoromethylthioesters were found to form η(2)-carbonyl complexes. In contrast, acetylthioesters underwent rapid Cacyl-S bond cleavage followed by decarbonylation to generate methylnickel complexes. This decarbonylation could be pushed backwards by the addition of CO, allowing for regeneration of the thioester. Most of the thioester complexes were found to undergo stoichiometric cross-coupling with phenylboronic acid to yield sulfides. While ethyl trifluoroacetate was also found to form an η(2)-carbonyl complex, phenyl esters were found to predominantly undergo Caryl-O bond cleavage to yield arylnickel complexes. These could also undergo transmetalation to yield biaryls. Attempts to render the reactions catalytic were hindered by ligand scrambling to yield nickel bis(acetate) complexes, the formation of which was supported by independent syntheses. Finally, 2-naphthyl acetate was also found to undergo clean Caryl-O bond cleavage, and although stoichiometric cross-coupling with phenylboronic acid proceeded with good yield, catalytic turnover has so far proven elusive.

  18. Genetic insight of the H5N1 hemagglutinin cleavage site

    Institute of Scientific and Technical Information of China (English)

    2007-01-01

    The cleavability of the hemagglutinin (HA) plays a major role in virulence of avian influenza viruses. Detailed analyses of the cleavage sequences and their evolution would give insights into the high pathogenicity of the H5N1 virus. HA segments were visually identifiable in the cellular automata (CA) image, and a feature gene segment (FGS) was only found in H5N1 rather than any other subtype. This FGS is a 30-bp gene segment mainly consisting of 'A' and 'G'. When translated into amino acids the FGS converted into a sequence of mainly basic amino acids with positive charges. This feature amino acid segment (FAAS) was located in the cleavage site loop of HA which was potentially cleavable by various proteases. The 3D structure of H5N1 HA was reconstructed using homology modelling. It was found that the cleavage site loop was well exposed to potential proteases. The molecular surfaces were reconstructed to study how mutation and deletion of some amino acids in the FAAS affected the charge distribution. It was found that some mutations had severely changed the landscape of the charge distribution. Statistical analyses of FAAS were made with respect to when and where the H5N1 viruses were found. In 2005, there were less un-mutated FAAS than the other years according to temporal evolution, and more mutated FAAS appeared in China than other regions according to geographic distribution. These results are helpful for exploring the evolution of virus high pathogenicity.

  19. 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; Whittaker, Gary R

    2013-07-01

    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.

  20. Substrate promiscuity of RdCCD1, a carotenoid cleavage oxygenase from Rosa damascena.

    Science.gov (United States)

    Huang, Fong-Chin; Horváth, Györgyi; Molnár, Péter; Turcsi, Erika; Deli, József; Schrader, Jens; Sandmann, Gerhard; Schmidt, Holger; Schwab, Wilfried

    2009-03-01

    Several of the key flavor compounds in rose essential oil are C(13)-norisoprenoids, such as beta-damascenone, beta-damascone, and beta-ionone which are derived from carotenoid degradation. To search for genes putatively responsible for the cleavage of carotenoids, cloning of carotenoid cleavage (di-)oxygenase (CCD) genes from Rosa damascena was carried out by a degenerate primer approach and yielded a full-length cDNA (RdCCD1). The RdCCD1 gene was expressed in Escherichia coli and recombinant protein was assayed for its cleavage activity with a multitude of carotenoid substrates. The RdCCD1 protein was able to cleave a variety of carotenoids at the 9-10 and 9'-10' positions to produce a C(14) dialdehyde and two C(13) products, which vary depending on the carotenoid substrates. RdCCD1 could also cleave lycopene at the 5-6 and 5'-6' positions to produce 6-methyl-5-hepten-2-one. Expression of RdCCD1 was studied by real-time PCR in different tissues of rose. The RdCCD1 transcript was present predominantly in rose flower, where high levels of volatile C(13)-norisoprenoids are produced. Thus, the accumulation of C(13)-norisoprenoids in rose flower is correlated to the expression of RdCCD1.

  1. Ternary complexes of cobalt cysteinylglycine with histidylserine and histidylphenylalanine-stabilities and DNA cleavage properties

    Indian Academy of Sciences (India)

    Pulimamidi R Reddy; Pallerla Manjula

    2007-11-01

    Interaction of cobalt cysteinylglycine with histidylserine and histidylphenylalanine was investigated in a 1 : 1 : 1 ratio at 35°C and 0.10 mol dm-3 ionic strength. Their stabilities and geometries were determined. Their DNA binding and cleavage properties were investigated. The intrinsic binding constants () for DNA bound 1 and 2 (3.03 × 103 M-1 for 1 and 3.87 × 103 M-1 for 2) were determined. Even though the negative charge on the complexes reduced their affinity for DNA, there was an enhancement of binding through specificity. The degradation of plasmid DNA was achieved by cobalt dipeptide complexes [CoII(CysGly)(HisSer)] (1) and [CoII(CysGly)(HisPhe)] (2). Cleavage experiments revealed that 1 and 2 cleave supercoiled DNA (form I) to nicked circular (form II) through hydrolytic pathway at physiological H. The DNA hydrolytic cleavage rate constants for complexes 1 and 2 were determined to be 0.62 h-1, for 1 and 0.38 h-1 for 2 respectively.

  2. Structural Basis for Accelerated Cleavage of Bovine Pancreatic Trypsin Inhibitor (BPTI) by Human Mesotrypsin

    Energy Technology Data Exchange (ETDEWEB)

    Salameh,M.; Soares, A.; Hockla, A.; Radisky, E.

    2008-01-01

    Human mesotrypsin is an isoform of trypsin that displays unusual resistance to polypeptide trypsin inhibitors and has been observed to cleave several such inhibitors as substrates. Whereas substitution of arginine for the highly conserved glycine 193 in the trypsin active site has been implicated as a critical factor in the inhibitor resistance of mesotrypsin, how this substitution leads to accelerated inhibitor cleavage is not clear. Bovine pancreatic trypsin inhibitor (BPTI) forms an extremely stable and cleavage-resistant complex with trypsin, and thus provides a rigorous challenge of mesotrypsin catalytic activity toward polypeptide inhibitors. Here, we report kinetic constants for mesotrypsin and the highly homologous (but inhibitor sensitive) human cationic trypsin, describing inhibition by, and cleavage of BPTI, as well as crystal structures of the mesotrypsin-BPTI and human cationic trypsin-BPTI complexes. We find that mesotrypsin cleaves BPTI with a rate constant accelerated 350-fold over that of human cationic trypsin and 150,000-fold over that of bovine trypsin. From the crystal structures, we see that small conformational adjustments limited to several side chains enable mesotrypsin-BPTI complex formation, surmounting the predicted steric clash introduced by Arg-193. Our results show that the mesotrypsin-BPTI interface favors catalysis through (a) electrostatic repulsion between the closely spaced mesotrypsin Arg-193 and BPTI Arg-17, and (b) elimination of two hydrogen bonds between the enzyme and the amine leaving group portion of BPTI. Our model predicts that these deleterious interactions accelerate leaving group dissociation and deacylation.

  3. Rubber oxygenase and latex clearing protein cleave rubber to different products and use different cleavage mechanisms.

    Science.gov (United States)

    Birke, Jakob; Jendrossek, Dieter

    2014-08-01

    Two types of enzyme for oxidative cleavage of poly(cis-1,4-isoprene) are known. One is rubber oxygenase (RoxA) that is secreted by Xanthomonas sp. strain 35Y and a few other Gram-negative rubber-degrading bacteria during growth on polyisoprene. RoxA was studied in the past, and the recently solved structure showed a structural relationship to bacterial cytochrome c peroxidases (J. Seidel et al., Proc. Natl. Acad. Sci. U. S. A. 110:13833-13838, 2013, http://dx.doi.org/10.1073/pnas.1305560110). The other enzyme is latex-clearing protein (Lcp) that is secreted by rubber-degrading actinomycetes, but Lcp has not yet been purified. Here, we expressed Lcp of Streptomyces sp. strain K30 in a ΔroxA background of Xanthomonas sp. strain 35Y and purified native (untagged) Lcp. The specific activities of Lcp and RoxA were 0.70 and 0.48 U/mg, respectively. Lcp differed from RoxA in the absence of heme groups and other characteristics. Notably, Lcp degraded polyisoprene via endo-type cleavage to tetra-C20 and higher oligo-isoprenoids with aldehyde and keto end groups, whereas RoxA used an exo-type cleavage mechanism to give the main end product 12-oxo-4,8-dimethyltrideca-4,8-diene-1-al (ODTD). RoxA was able to cleave isolated Lcp-derived oligo-isoprenoid molecules to ODTD. Inhibitor studies, spectroscopic investigations and metal analysis gave no indication for the presence of iron, other metals, or cofactors in Lcp. Our results suggest that Lcp could be a member of the growing group of cofactor-independent oxygenases and differs in the cleavage mechanism from heme-dependent RoxA. In conclusion, RoxA and Lcp represent two different answers to the same biochemical problem, the cleavage of polyisoprene, a polymer that has carbon-carbon double bonds as the only functional groups for enzymatic attack.

  4. 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.

  5. Cleavage of group 1 coronavirus spike proteins: how furin cleavage is traded off against heparan sulfate binding upon cell culture adaptation

    NARCIS (Netherlands)

    Haan, de C.A.M.; Haijema, B.J.; Schellen, P.; Wichgers Schreur, P.J.; Lintelo, te E.; Vennema, H.; Rottier, P.J.M.

    2008-01-01

    A longstanding enigmatic feature of the group 1 coronaviruses is the uncleaved phenotype of their spike protein, an exceptional property among class I fusion proteins. Here, however, we show that some group 1 coronavirus spike proteins carry a furin enzyme recognition motif and can actually be cleav

  6. Enhanced cleavage of double-stranded DNA by artificial zinc-finger nuclease sandwiched between two zinc-finger proteins.

    Science.gov (United States)

    Mineta, Yusuke; Okamoto, Tomoyuki; Takenaka, Kosuke; Doi, Norio; Aoyama, Yasuhiro; Sera, Takashi

    2008-11-25

    To enhance DNA cleavage by zinc-finger nucleases (ZFNs), we sandwiched a DNA cleavage enzyme with two artificial zinc-finger proteins (AZPs). Because the DNA between the two AZP-binding sites is cleaved, the AZP-sandwiched nuclease is expected to bind preferentially to a DNA substrate rather than to cleavage products and thereby cleave it with multiple turnovers. To demonstrate the concept, we sandwiched a staphylococcal nuclease (SNase), which cleaves DNA as a monomer, between two three-finger AZPs. The AZP-sandwiched SNase cleaved large amounts of dsDNA site-specifically. Such multiple-turnover cleavage was not observed with nucleases that possess a single AZP. Thus, AZP-sandwiched nucleases will further refine ZFN technology.

  7. 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.

  8. The action of the bacterial toxin microcin B17. Insight into the cleavage-religation reaction of DNA gyrase.

    Science.gov (United States)

    Pierrat, Olivier A; Maxwell, Anthony

    2003-09-12

    We have examined the effects of the bacterial toxin microcin B17 (MccB17) on the reactions of Escherichia coli DNA gyrase. MccB17 slows down but does not completely inhibit the DNA supercoiling and relaxation reactions of gyrase. A kinetic analysis of the cleavage-religation equilibrium of gyrase was performed to determine the effect of the toxin on the forward (cleavage) and reverse (religation) reactions. A simple mechanism of two consecutive reversible reactions with a nicked DNA intermediate was used to simulate the kinetics of cleavage and religation. The action of MccB17 on the kinetics of cleavage and religation was compared with that of the quinolones ciprofloxacin and oxolinic acid. With relaxed DNA as substrate, only a small amount of gyrase cleavage complex is observed with MccB17 in the absence of ATP, whereas the presence of the nucleotide significantly enhances the effect of the toxin on both the cleavage and religation reactions. In contrast, ciprofloxacin, oxolinic acid, and Ca2+ show lesser dependence on ATP to stabilize the cleavage complex. MccB17 enhances the overall rate of DNA cleavage by increasing the forward rate constant (k2) of the second equilibrium. In contrast, ciprofloxacin increases the amount of cleaved DNA by a combined effect on the forward and reverse rate constants of both equilibria. Based on these results and on the observations that MccB17 only slowly inhibits the supercoiling and relaxation reactions, we suggest a model of the interaction of MccB17 with gyrase.

  9. Synthesis, structural characterization, fluorescence, antimicrobial, antioxidant and DNA cleavage studies of Cu(II) complexes of formyl chromone Schiff bases.

    Science.gov (United States)

    Kavitha, P; Saritha, M; Laxma Reddy, K

    2013-02-01

    Cu(II) complexes have been synthesized from different Schiff bases, such as 3-((2-hydroxy phenylimino)methyl)-4H-chromen-4-one (HL(1)), 2-((4-oxo-4H-chromen-3-yl)methylneamino) benzoicacid (HL(2)), 3-((3-hydroxypyridin-2-ylimino)methyl)-4H-chromen-4-one (HL(3)) and 3-((2-mercaptophenylimino)methyl)-4H-chromen-4-one (HL(4)). The complexes were characterized by analytical, molar conductance, IR, electronic, magnetic, ESR, thermal, powder XRD and SEM studies. The analytical data reveal that metal to ligand molar ratio is 1:2 in all the complexes. Molar conductivity data indicates that all the Cu(II) complexes are neutral. On the basis of magnetic and electronic spectral data, distorted octahedral geometry is proposed for all the Cu(II) complexes. Thermal behaviour of the synthesized complexes illustrates the presence of lattice water molecules in the complexes. X-ray diffraction studies reveal that all the ligands and their Cu(II) complexes have triclinic system with different unit cell parameters. Antimicrobial, antioxidant and DNA cleavage activities indicate that metal complexes exhibited greater activity as compared with ligands.

  10. TamiR159 directed wheat TaGAMYB cleavage and its involvement in anther development and heat response.

    Directory of Open Access Journals (Sweden)

    Yu Wang

    Full Text Available In Arabidopsis and rice, miR159-regulated GAMYB-like family transcription factors function in flower development and gibberellin (GA signaling in cereal aleurone cells. In this study, the involvement of miR159 in the regulation of its putative target TaGAMYB and its relationship to wheat development were investigated. First, we demonstrated that cleavage of TaGAMYB1 and TaGAMYB2 was directed by miR159 using 5'-RACE and a transient expression system. Second, we overexpressed TamiR159, TaGAMYB1 and mTaGAMYB1 (impaired in the miR159 binding site in transgenic rice, revealing that the accumulation in rice of mature miR159 derived from the precursor of wheat resulted in delayed heading time and male sterility. In addition, the number of tillers and primary branches in rice overexpressing mTaGAMYB1 increased relative to the wild type. Our previous study reported that TamiR159 was downregulated after two hours of heat stress treatment in wheat (Triticum aestivum L.. Most notably, the TamiR159 overexpression rice lines were more sensitive to heat stress relative to the wild type, indicating that the downregulation of TamiR159 in wheat after heat stress might participate in a heat stress-related signaling pathway, in turn contributing to heat stress tolerance.

  11. An investigation of the structural requirements for ATP hydrolysis and DNA cleavage by the EcoKI Type I DNA restriction and modification enzyme.

    Science.gov (United States)

    Roberts, Gareth A; Cooper, Laurie P; White, John H; Su, Tsueu-Ju; Zipprich, Jakob T; Geary, Paul; Kennedy, Cowan; Dryden, David T F

    2011-09-01

    Type I DNA restriction/modification systems are oligomeric enzymes capable of switching between a methyltransferase function on hemimethylated host DNA and an endonuclease function on unmethylated foreign DNA. They have long been believed to not turnover as endonucleases with the enzyme becoming inactive after cleavage. Cleavage is preceded and followed by extensive ATP hydrolysis and DNA translocation. A role for dissociation of subunits to allow their reuse has been proposed for the EcoR124I enzyme. The EcoKI enzyme is a stable assembly in the absence of DNA, so recycling was thought impossible. Here, we demonstrate that EcoKI becomes unstable on long unmethylated DNA; reuse of the methyltransferase subunits is possible so that restriction proceeds until the restriction subunits have been depleted. We observed that RecBCD exonuclease halts restriction and does not assist recycling. We examined the DNA structure required to initiate ATP hydrolysis by EcoKI and find that a 21-bp duplex with single-stranded extensions of 12 bases on either side of the target sequence is sufficient to support hydrolysis. Lastly, we discuss whether turnover is an evolutionary requirement for restriction, show that the ATP hydrolysis is not deleterious to the host cell and discuss how foreign DNA occasionally becomes fully methylated by these systems.

  12. Enhanced Protective Efficacy of H5 Subtype Influenza Vaccine with Modification of the Multibasic Cleavage Site of Hemagglutinin in Retroviral Pseudotypes

    Institute of Scientific and Technical Information of China (English)

    Ling Tao; JianJun Chen; Jin Meng; Yao Chen; Hongxia Li; Yan Liu; Zhenhua Zheng

    2013-01-01

    Traditionally,the multibasic cleavage site (MBCS) of surface protein H5-hemagglutinin (HA) is converted to a monobasic one so as to weaken the virulence of recombinant H5N1 influenza viruses and to produce inactivated and live attenuated vaccines.Whether such modification benefits new candidate vaccines has not been adequately investigated.We previously used retroviral vectors to generate wtH5N1 pseudotypes containing the wild-type HA (wtH5) from A/swine/Anhui/ca/2004 (H5N1) virus.Here,we generated mtH5N1 pseudotypes,which contained a mutant-type HA (mtH5) with a modified monobasic cleavage site.Groups of mice were subcutaneously injected with the two types of influenza pseudotypes.Compared to the group immunized with wtH5N1 pseudotypes,the inoculation of mtH5N1 pseudotypes induced significantly higher levels of HA specific IgG and IFN-γ in immunized mice,and enhanced protection against the challenge of mouse-adapted avian influenza virus A/Chicken/Henan/12/2004 (H5N1).This study suggests modification of the H5-hemagglutinin MBCS in retroviral pseudotypes enhances protection efficacy in mice and this information may be helpful for development of vaccines from mammalian cells to fight against H5N1 influenza viruses.

  13. The Combined Repetitive Oligopeptides of Clostridium difficile Toxin A Counteract Premature Cleavage of the Glucosyl-Transferase Domain by Stabilizing Protein Conformation

    Directory of Open Access Journals (Sweden)

    Alexandra Olling

    2014-07-01

    Full Text Available Toxin A (TcdA and B (TcdB from Clostridium difficile enter host cells by receptor-mediated endocytosis. A prerequisite for proper toxin action is the intracellular release of the glucosyltransferase domain by an inherent cysteine protease, which is allosterically activated by inositol hexaphosphate (IP6. We found that in in vitro assays, the C-terminally-truncated TcdA1–1065 was more efficient at IP6-induced cleavage compared with full-length TcdA. We hypothesized that the C-terminally-located combined repetitive oligopeptides (CROPs interact with the N-terminal part of the toxin, thereby preventing autoproteolysis. Glutathione-S-transferase (GST pull-down assays and microscale thermophoresis confirmed binding between the CROPs and the glucosyltransferase (TcdA1–542 or intermediate (TcdA1102–1847 domain of TcdA, respectively. This interaction between the N- and C-terminus was not found for TcdB. Functional assays revealed that TcdB was more susceptible to inactivation by extracellular IP6-induced cleavage. In vitro autoprocessing and inactivation of TcdA, however, significantly increased, either by acidification of the surrounding milieu or following exchange of its CROP domain by the homologous CROP domain of TcdB. Thus, TcdA CROPs contribute to the stabilization and protection of toxin conformation in addition to function as the main receptor binding domain.

  14. EspC, an Autotransporter Protein Secreted by Enteropathogenic Escherichia coli, Causes Apoptosis and Necrosis through Caspase and Calpain Activation, Including Direct Procaspase-3 Cleavage

    Directory of Open Access Journals (Sweden)

    Antonio Serapio-Palacios

    2016-06-01

    Full Text Available Enteropathogenic Escherichia coli (EPEC has the ability to antagonize host apoptosis during infection through promotion and inhibition of effectors injected by the type III secretion system (T3SS, but the total number of these effectors and the overall functional relationships between these effectors during infection are poorly understood. EspC produced by EPEC cleaves fodrin, paxillin, and focal adhesion kinase (FAK, which are also cleaved by caspases and calpains during apoptosis. Here we show the role of EspC in cell death induced by EPEC. EspC is involved in EPEC-mediated cell death and induces both apoptosis and necrosis in epithelial cells. EspC induces apoptosis through the mitochondrial apoptotic pathway by provoking (i a decrease in the expression levels of antiapoptotic protein Bcl-2, (ii translocation of the proapoptotic protein Bax from cytosol to mitochondria, (iii cytochrome c release from mitochondria to the cytoplasm, (iv loss of mitochondrial membrane potential, (v caspase-9 activation, (vi cleavage of procaspase-3 and (vii an increase in caspase-3 activity, (viii PARP proteolysis, and (ix nuclear fragmentation and an increase in the sub-G1 population. Interestingly, EspC-induced apoptosis was triggered through a dual mechanism involving both independent and dependent functions of its EspC serine protease motif, the direct cleavage of procaspase-3 being dependent on this motif. This is the first report showing a shortcut for induction of apoptosis by the catalytic activity of an EPEC protein. Furthermore, this atypical intrinsic apoptosis appeared to induce necrosis through the activation of calpain and through the increase of intracellular calcium induced by EspC. Our data indicate that EspC plays a relevant role in cell death induced by EPEC.

  15. Characterization and Modeling of the Collision Induced Dissociation Patterns of Deprotonated Glycosphingolipids: Cleavage of the Glycosidic Bond

    Science.gov (United States)

    Rožman, Marko

    2016-01-01

    Glycosphingolipid fragmentation behavior was investigated by combining results from analysis of a series of negative ion tandem mass spectra and molecular modeling. Fragmentation patterns extracted from 75 tandem mass spectra of mainly acidic glycosphingolipid species (gangliosides) suggest prominent cleavage of the glycosidic bonds with retention of the glycosidic oxygen atom by the species formed from the reducing end (B and Y ion formation). Dominant product ions arise from dissociation of sialic acids glycosidic bonds whereas product ions resulting from cleavage of other glycosidic bonds are less abundant. Potential energy surfaces and unimolecular reaction rates of several low-energy fragmentation pathways leading to cleavage of glycosidic bonds were estimated in order to explain observed dissociation patterns. Glycosidic bond cleavage in both neutral (unsubstituted glycosyl group) and acidic glycosphingolipids was the outcome of the charge-directed intramolecular nucleophilic substitution (SN2) mechanism. According to the suggested mechanism, the nucleophile in a form of carboxylate or oxyanion attacks the carbon at position one of the sugar ring, simultaneously breaking the glycosidic bond and yielding an epoxide. For gangliosides, unimolecular reaction rates suggest that dominant product ions related to the cleavage of sialic acid glycosidic bonds are formed via direct dissociation channels. On the other hand, low abundant product ions related to the dissociation of other glycosidic bonds are more likely to be the result of sequential dissociation. Although results from this study mainly contribute to the understanding of glycosphingolipid fragmentation chemistry, some mechanistic findings regarding cleavage of the glycosidic bond may be applicable to other glycoconjugates.

  16. Association of a peptoid ligand with the apical loop of pri-miR-21 inhibits cleavage by Drosha.

    Science.gov (United States)

    Diaz, Jason P; Chirayil, Rachel; Chirayil, Sara; Tom, Martin; Head, Katie J; Luebke, Kevin J

    2014-04-01

    We have found a small molecule that specifically inhibits cleavage of a precursor to the oncogenic miRNA, miR-21, by the microprocessor complex of Drosha and DGCR8. We identified novel ligands for the apical loop of this precursor from a screen of 14,024 N-substituted oligoglycines (peptoids) in a microarray format. Eight distinct compounds with specific affinity were obtained, three having affinities for the targeted loop in the low micromolar range and greater than 15-fold discrimination against a closely related hairpin. One of these compounds completely inhibits microprocessor cleavage of a miR-21 primary transcript at concentrations at which cleavage of another miRNA primary transcript, pri-miR-16, is little affected. The apical loop of pri-miR-21, placed in the context of pri-miR-16, is sufficient for inhibition of microprocessor cleavage by the peptoid. This compound also inhibits cleavage of pri-miR-21 containing the pri-miR-16 apical loop, suggesting an additional site of association within pri-miR-21. The reported peptoid is the first example of a small molecule that inhibits microprocessor cleavage by binding to the apical loop of a pri-miRNA.

  17. Novel insights into the fungal oxidation of monoaromatic and biarylic environmental pollutants by characterization of two new ring cleavage enzymes.

    Science.gov (United States)

    Schlüter, Rabea; Lippmann, Ramona; Hammer, Elke; Gesell Salazar, Manuela; Schauer, Frieder

    2013-06-01

    The phenol-degrading yeast Trichosporon mucoides can oxidize and detoxify biarylic environmental pollutants such as dibenzofuran, diphenyl ether and biphenyl by ring cleavage. The degradation pathways are well investigated, but the enzymes involved are not. The high similarity of hydroxylated biphenyl derivatives and phenol raised the question if the enzymes of the phenol degradation are involved in ring cleavage or whether specific enzymes are necessary. Purification of enzymes from T. mucoides with catechol cleavage activity demonstrated the existence of three different enzymes: a classical catechol-1,2-dioxygenase (CDO), not able to cleave the aromatic ring system of 3,4-dihydroxybiphenyl, and two novel enzymes with a high affinity towards 3,4-dihydroxybiphenyl. The comparison of the biochemical characteristics and mass spectrometric sequence data of these three enzymes demonstrated that they have different substrate specificities. CDO catalyzes the ortho-cleavage of dihydroxylated monoaromatic compounds, while the two novel enzymes carry out a similar reaction on biphenyl derivatives. The ring fission of 3,4-dihydroxybiphenyl by the purified enzymes results in the formation of (5-oxo-3-phenyl-2,5-dihydrofuran-2-yl)acetic acid. These results suggest that the ring cleavage enzymes catalyzing phenol degradation are not involved in the ring cleavage of biarylic compounds by this yeast, although some intermediates of the phenol metabolism may function as inducers.

  18. 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.

  19. 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.

  20. Site-specific O-glycosylation by Polypeptide GalNAc-transferase T2 Co-regulates Beta1-adrenergic Receptor N-terminal Cleavage.

    Science.gov (United States)

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

    2017-02-06

    The β1-adrenergic receptor (β1AR) is a G protein-coupled receptor (GPCR) and the predominant adrenergic receptor subtype in the heart, where it mediates cardiac contractility and the force of contraction. Although it is the most important target for β-adrenergic antagonists, such as beta-blockers, relatively little is still known about its regulation. We have previously shown that β1AR undergoes constitutive and regulated N-terminal cleavage participating in receptor down-regulation, and moreover that the receptor is modified by O-glycosylation. Here we demonstrate that the polypeptide GalNAc-transferase 2 (GalNAc-T2) specifically O-glycosylates β1AR at five residues in the extracellular N-terminus, including the Ser49 residue at a location of the common Ser49Gly single-nucleotide polymorphism. Using in vitro O-glycosylation and proteolytic cleavage assays, a cell line deficient in O-glycosylation, GalNAc-T edited cell line model systems, and a GalNAc-T2 knockout rat model, we show that GalNAc-T2 co-regulates the metalloproteinase-mediated limited proteolysis of β1AR. Furthermore, we demonstrate that impaired O-glycosylation and enhanced proteolysis leads to attenuated receptor signaling, as the maximal response elicited by the βAR agonist isoproterenol and it potency in a cAMP accumulation assay was decreased in HEK293 cells lacking GalNAc-T2. Our findings reveal, for the first time, a GPCR as a target for co-regulatory functions of site-specific O-glycosylation mediated by a unique GalNAc-T isoform. The results provide a new level of β1AR regulation that may open up possibilities for new therapeutic strategies for cardiovascular diseases.

  1. RHEED study of the (1 1 0) cleavage surface of CdTe:Cr single crystals

    Science.gov (United States)

    Sagan, P.; Kuzma, M.

    2007-03-01

    The structure of (1 1 0) plane of Cr-doped CdTe single crystals has been studied by reflection high energy electron diffraction and scanning electron microscopy. Diffraction patterns consist of diffraction spots and Kikuchi lines. However, for very small incident angle, the Debye rings are observed. The constant lattice attributed to these rings is 0.8% less then for pure CdTe. These anomalous properties of the near surface layer are likely to occur due to the concentration of Cr atoms creating compressive surface strains or the effect of crystal cleavage.

  2. Stereoelectronic Control of Cleavage of Dioxolane Five-membered Ring on Carbohydrates

    Institute of Scientific and Technical Information of China (English)

    PAN Xiao-liang; ZHOU Yi-xuan; LIU Wei; LIU Jing-yao; DONG Hai

    2013-01-01

    A mechanism about the origin of the selectivities for the cleavage of dioxolane five-membered rings on pyranoside rings was suggested.Quantum chemical studies were performed to testify the rationality of the mechanism.It is thus suggested that the selectivities should be dependent on the differences of the free energy at the transition states when the five-membered ring cleaves.Natural bond orbital(NBO) analysis was further made to assess the influence of stereoelectronic effects on the selectivities.

  3. Adsorption and enzymatic cleavage of osteopontin at interfaces with different surface chemistries

    DEFF Research Database (Denmark)

    Malmström, Jenny; Shipovskov, Stepan; Christensen, Brian;

    2009-01-01

    was able to bind and cleave the surface bound osteopontin at the hydrophobic surface. The altered levels of osteopontin binding, hydration of the layer, and susceptibility to thrombin cleavage suggest that osteopontin adopts different conformations and/or orientations at the different material surfaces....... with respect to post-translational modifications. Osteopontin adsorbed at all the surfaces formed thin (approximately 2-5 nm) hydrated layers with the highest amount of protein and the highest density layers observed at the hydrophobic surface. Less protein and a higher level of hydration was observed...

  4. Autocatalytic cyclization of an excised intervening sequence RNA is a cleavage-ligation reaction.

    Science.gov (United States)

    Zaug, A J; Grabowski, P J; Cech, T R

    The intervening sequence (IVS) of the Tetrahymena ribosomal RNA precursor is excised as a linear RNA molecule which subsequently cyclizes itself in a protein-independent reaction. Cyclization involves cleavage of the linear IVS RNA 15 nucleotides from its 5' end and formation of a phosphodiester bond between the new 5' phosphate and the original 3'-hydroxyl terminus of the IVS. This recombination mechanism is analogous to that by which splicing of the precursor RNA is achieved. The circular molecules appear to have no direct function in RNA splicing, and we propose the cyclization serves to prevent unwanted RNA from driving the splicing reactions backwards.

  5. Proteolytic cleavage of the Chlamydia pneumoniae major outer membrane protein in the absence of Pmp10

    DEFF Research Database (Denmark)

    Juul, Nicolai Stefan; Timmerman, E; Gevaert, K;

    2007-01-01

    that Pmp10 is differentially expressed in the C. pneumoniae CWL029 isolate. To evaluate whether the absence of Pmp10 in the outer membrane causes further changes to the C. pneumoniae protein profile, we subcloned the CWL029 isolate and selected a clone with minimal Pmp10 expression. Subsequently, we...... compared the proteome of the CWL029 isolate with the proteome of the subcloned strain and identified a specific cleavage of the C-terminal part of the major outer membrane protein (MOMP), which occurred only in the absence of Pmp10. In contrast, when Pmp10 was expressed we predominantly observed full...

  6. Multicistronic lentiviral vectors containing the FMDV 2A cleavage factor demonstrate robust expression of encoded genes at limiting MOI

    Directory of Open Access Journals (Sweden)

    Margison Geoffrey P

    2006-03-01

    Full Text Available Abstract Background A number of gene therapy applications would benefit from vectors capable of expressing multiple genes. In this study we explored the feasibility and efficiency of expressing two or three transgenes in HIV-1 based lentiviral vector. Bicistronic and tricistronic self-inactivating lentiviral vectors were constructed employing the internal ribosomal entry site (IRES sequence of encephalomyocarditis virus (EMCV and/or foot-and-mouth disease virus (FMDV cleavage factor 2A. We employed enhanced green fluorescent protein (eGFP, O6-methylguanine-DNA-methyltransferase (MGMT, and homeobox transcription factor HOXB4 as model genes and their expression was detected by appropriate methods including fluorescence microscopy, flow cytometry, immunocytochemistry, biochemical assay, and western blotting. Results All the multigene vectors produced high titer virus and were able to simultaneously express two or three transgenes in transduced cells. However, the level of expression of individual transgenes varied depending on: the transgene itself; its position within the construct; the total number of transgenes expressed; the strategy used for multigene expression and the average copy number of pro-viral insertions. Notably, at limiting MOI, the expression of eGFP in a bicistronic vector based on 2A was ~4 times greater than that of an IRES based vector. Conclusion The small and efficient 2A sequence can be used alone or in combination with an IRES for the construction of multicistronic lentiviral vectors which can express encoded transgenes at functionally relevant levels in cells containing an average of one pro-viral insert.

  7. Neurodegeneration in Autoimmune Optic Neuritis Is Associated with Altered APP Cleavage in Neurons and Up-Regulation of p53.

    Directory of Open Access Journals (Sweden)

    Sabine Herold

    Full Text Available Multiple Sclerosis (MS is a chronic autoimmune inflammatory disease of the central nervous system (CNS. Histopathological and radiological analysis revealed that neurodegeneration occurs early in the disease course. However, the pathological mechanisms involved in neurodegeneration are poorly understood. Myelin oligodendrocyte glycoprotein (MOG-induced experimental autoimmune encephalomyelitis (EAE in Brown Norway rats (BN-rats is a well-established animal model, especially of the neurodegenerative aspects of MS. Previous studies in this animal model indicated that loss of retinal ganglion cells (RGCs, the neurons that form the axons of the optic nerve, occurs in the preclinical phase of the disease and is in part independent of overt histopathological changes of the optic nerve. Therefore, the aim of this study was to identify genes which are involved in neuronal cell loss at different disease stages of EAE. Furthermore, genes that are highly specific for autoimmune-driven neurodegeneration were compared to those regulated in RGCs after optic nerve axotomy at corresponding time points. Using laser capture micro dissection we isolated RNA from unfixed RGCs and performed global transcriptome analysis of retinal neurons. In total, we detected 582 genes sequentially expressed in the preclinical phase and 1150 genes in the clinical manifest EAE (P 1.5. Furthermore, using ingenuity pathway analysis (IPA, we identified amyloid precursor protein (APP as a potential upstream regulator of changes in gene expression in the preclinical EAE but neither in clinical EAE, nor at any time point after optic nerve transection. Therefore, the gene pathway analysis lead to the hypothesis that altered cleavage of APP in neurons in the preclinical phase of EAE leads to the enhanced production of APP intracellular domain (AICD, which in turn acts as a transcriptional regulator and thereby initiates an apoptotic signaling cascade via up-regulation of the target gene p

  8. CTAG-containing cleavage site profiling to delineate Salmonella into natural clusters.

    Directory of Open Access Journals (Sweden)

    Le Tang

    Full Text Available The bacterial genus Salmonella contains thousands of serotypes that infect humans or other hosts, causing mild gastroenteritis to potentially fatal systemic infections in humans. Pathogenically distinct Salmonella serotypes have been classified as individual species or as serological variants of merely one or two species, causing considerable confusion in both research and clinical settings. This situation reflects a long unanswered question regarding whether the Salmonella serotypes exist as discrete genetic clusters (natural species of organisms or as phenotypic (e.g. pathogenic variants of a single (or two natural species with a continuous spectrum of genetic divergence among them. Our recent work, based on genomic sequence divergence analysis, has demonstrated that genetic boundaries exist among Salmonella serotypes, circumscribing them into clear-cut genetic clusters of bacteria.To further test the genetic boundary concept for delineating Salmonella into clearly defined natural lineages (e.g., species, we sampled a small subset of conserved genomic DNA sequences, i.e., the endonuclease cleavage sites that contain the highly conserved CTAG sequence such as TCTAGA for XbaI. We found that the CTAG-containing cleavage sequence profiles could be used to resolve the genetic boundaries as reliably and efficiently as whole genome sequence comparisons but with enormously reduced requirements for time and resources.Profiling of CTAG sequence subsets reflects genetic boundaries among Salmonella lineages and can delineate these bacteria into discrete natural clusters.

  9. 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.

  10. 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”.

  11. The Prediction of Calpain Cleavage Sites with the mRMR and IFS Approaches

    Directory of Open Access Journals (Sweden)

    Wenyi Zhang

    2013-01-01

    Full Text Available Calpains are an important family of the Ca2+-dependent cysteine proteases which catalyze the limited proteolysis of many specific substrates. Calpains play crucial roles in basic physiological and pathological processes, and identification of the calpain cleavage sites may facilitate the understanding of the molecular mechanisms and biological function. But traditional experiment approaches to predict the sites are accurate, and are always labor-intensive and time-consuming. Thus, it is common to see that computational methods receive increasing attention due to their convenience and fast speed in recent years. In this study, we develop a new predictor based on the support vector machine (SVM with the maximum relevance minimum redundancy (mRMR method followed by incremental feature selection (IFS. And we concern the feature of physicochemical/biochemical properties, sequence conservation, residual disorder, secondary structure, and solvent accessibility to represent the calpain cleavage sites. Experimental results show that the performance of our predictor is better than several other state-of- the-art predictors, whose average prediction accuracy is 79.49%, sensitivity is 62.31%, and specificity is 88.12%. Since user-friendly and publicly accessible web servers represent the future direction for developing practically more useful predictors, here we have provided a web-server for the method presented in this paper.

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

    Energy Technology Data Exchange (ETDEWEB)

    Selmer, B.L. (State Univ. of New York, Stony Brook); 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.

  13. NMR-spectroscopic characterization of phosphodiester bond cleavage catalyzed by the minimal hammerhead ribozyme.

    Science.gov (United States)

    Fürtig, Boris; Richter, Christian; Schell, Peter; Wenter, Philipp; Pitsch, Stefan; Schwalbe, Harald

    2008-01-01

    In order to relate the conformational dynamics of the hammerhead ribozyme to its biological function the cleavage reaction catalyzed by the hammerhead ribozyme was monitored by time-resolved nuclear magnetic resonance (NMR) spectroscopy. For this purpose, the two nucleosides around the scissile phosphodiester bond were selectively (13)C labelled in multi-step organic syntheses starting from uniformly (13)C-labelled glucose. The phosphoamidites were incorporated using phosphoamidite chemistry in the hammerhead substrate strand. In addition, the 2'-OH group on the 5'-side of the hammerhead substrate strand was labelled with a photolabile protecting group. This labelling strategy enabled a detailed characterisation of the nucleotides around the scissile phosphodiester bond in the ground state conformation of the hammerhead ribozyme in the absence and presence of Mg(2+) ions as well as of the product state. Photochemical induction of the reaction in situ was further characterized by time-resolved NMR spectroscopy. The detailed structural and dynamic investigations revealed that the conformation of the hammerhead ribozyme is significantly affected by addition of Mg(2+) leading to an ensemble of conformations where dynamic transitions between energetically similar conformations occur on the ms-timescale in the presence of Mg(2+). The dynamic transitions are localized around the catalytic core. Cleavage from this ensemble cannot be described by mono-exponential kinetics but follows bi-exponential kinetics. A model is described to take into account these experimental data.

  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. Translocation-coupled DNA cleavage by the Type ISP restriction-modification enzymes.

    Science.gov (United States)

    Chand, Mahesh K; Nirwan, Neha; Diffin, Fiona M; van Aelst, Kara; Kulkarni, Manasi; Pernstich, Christian; Szczelkun, Mark D; Saikrishnan, Kayarat

    2015-11-01

    Production of endonucleolytic double-strand DNA breaks requires separate strand cleavage events. Although catalytic mechanisms for simple, dimeric endonucleases are known, there are many complex nuclease machines that are poorly understood. Here we studied the single polypeptide Type ISP restriction-modification (RM) enzymes, which cleave random DNA between distant target sites when two enzymes collide after convergent ATP-driven translocation. We report the 2.7-Å resolution X-ray crystal structure of a Type ISP enzyme-DNA complex, revealing that both the helicase-like ATPase and nuclease are located upstream of the direction of translocation, an observation inconsistent with simple nuclease-domain dimerization. Using single-molecule and biochemical techniques, we demonstrate that each ATPase remodels its DNA-protein complex and translocates along DNA without looping it, leading to a collision complex in which the nuclease domains are distal. Sequencing of the products of single cleavage events suggests a previously undescribed endonuclease model, where multiple, stochastic strand-nicking events combine to produce DNA scission.

  16. Synthesis, structure, DNA binding and cleavage activity of a new copper(Ⅱ) complex of bispyridylpyrrolide

    Institute of Scientific and Technical Information of China (English)

    MIN Rui; HU Xiao-hui; YI Xiao-yi; ZHANG Shou-chun

    2015-01-01

    A copper-bispyridylpyrrolide complex [Cu(PDPH)Cl] (PDPH = 2,5-bis(2′-pyridyl)pyrrole) was synthesized and characterized. The complex crystallizes in the orthorhombic system with space groupPccn,a = 0.9016(3) nm,b = 1.0931(4) nm,c = 2.5319(8) nm, andV = 2.4951(15) nm3. The copper center is situated in a square planar geometry. The interaction of the copper(Ⅱ) complexwith calf thymus DNA (CT-DNA) was investigated by electronic absorption, circular dichroism (CD) and fluorescence spectra. It is proposed that the complex binds to CT-DNA through groove binding mode. Nuclease activity of the complex was also studied by gel electrophoresis method. The complex can efficiently cleave supercoiled pBR322 DNA in the presence of ascorbate (H2A) via oxidative pathway. The preliminary mechanism of DNA cleavage by the complex with different inhibiting reagents indicates that the hydroxyl radicals were involved as the active species in the DNA cleavage process.

  17. 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.

  18. Ion beam modifications of defect sub-structure of calcite cleavages

    Indian Academy of Sciences (India)

    E Venkateshwar Rao; M Ramakrishna Murthy

    2008-04-01

    Experimental investigations on the defect sub-structure and surface modifications, brought about by He+ ion-bombardment of calcite cleavages (100), have been carried out. Optical and scanning electron microscopic investigations revealed drastic modifications on the surface morphology, local symmetry and defect concentration. Additional structural defects on ion-bombardment of calcite surfaces also have been observed. Changes in shape and form of chemical etch pits are found to be a function of ion-beam energy, as studied by optical microscopy. Radiation damage in calcite has been attributed mainly due to desorption of CO$^{-2}_{3}$ ions from the calcite surfaces, on irradiation. Measurements of surface conductivity on irradiated calcite surfaces have been made employing a four-probe technique. Enhancement of surface conductivity has been considered to be due to an increase in concentration of CO$^{-2}_{3}$ ions formed, on ion irradiation and subsequent thermal stimulation. Planar plastic anisotropy has been studied on irradiated calcite cleavages by measurement of microhardness.

  19. DNA cleavage system of nanosized graphene oxide sheets and copper ions.

    Science.gov (United States)

    Ren, Hongliu; Wang, Chong; Zhang, Jiali; Zhou, Xuejiao; Xu, Dafeng; Zheng, Jing; Guo, Shouwu; Zhang, Jingyan

    2010-12-28

    The exploration of efficient DNA intercalative agents (intercalators) is essential for understanding DNA scission, repair, and signal transduction. In this work, we explored systematically the graphene oxide (GO) interaction with DNA molecules using fluorescence spectroscopic (FL) and circular dichroism (CD) studies, gel electrophoresis, and DNA thermal denaturation. We demonstrated that the GO nanosheets could intercalate efficiently into DNA molecules. Significantly, we illustrated that the scission of DNA by GO sheets combining with copper ions could take place pronouncedly. The scission of DNA by the GO/Cu(2+) system is critically dependent on the concentrations of GO and Cu(2+) and their ratio. DNA cleavage ability exhibited by the GO with several other metal ions and the fact that GO/Cu(2+)-cleaved DNA fragments can be partially relegated suggest that the mechanism of DNA cleavage by the GO/metal ion system is oxidative and hydrolytic. The result reveals that the GO/Cu(2+) could be used as a DNA cleaving system that should find many practical applications in biotechnology and as therapeutic agents.

  20. 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.

  1. Method of Evaluating Delayed Fracture Susceptibility of Tempered Martensitic Steel Showing Quasi-Cleavage Fracture

    Science.gov (United States)

    Matsumoto, Yu; Takai, Kenichi

    2017-02-01

    The difference in the hydrogen charging methods, immersion in a NH4SCN aqueous solution, and cathodic electrolysis in a NaOH aqueous solution, did not affect the hydrogen state present in the steel, but it did affect the surface state of the specimens through corrosion, causing fracture strength to fluctuate in tensile testes. As for stress application method, the fracture strength at lower crosshead speeds in tensile tests was consistent with that found for hydrogen precharging prior to stress application in CLTs as long as hydrogen charging was conducted by cathodic electrolysis. However, the fracture strength obtained with concurrent hydrogen charging without precharging prior to stress application in CLTs was higher than that with hydrogen precharging prior to stress application in CLTs regardless of the same hydrogen content. In other words, delayed fracture susceptibility was affected by the order of hydrogen charging and stress application for quasi-cleavage fracture associated with local plastic deformation, i.e., dislocation motion. Therefore, by taking into account the cathodic electrolysis in the NaOH solution, the low crosshead speed and the order of hydrogen charging and stress application, the fracture strength in CLTs, and tensile tests coincided with respect to quasi-cleavage fracture even though the stress application methods were different.

  2. Caspase cleavage of cytochrome c1 disrupts mitochondrial function and enhances cytochrome c release

    Institute of Scientific and Technical Information of China (English)

    Yushan Zhu; Min Li; Xiaohui Wang; Haijing Jin; Shusen Liu; Jianxin Xu; Quan Chen

    2012-01-01

    Mitochondrial catastrophe can be the cause or consequence of apoptosis and is associated with a number of pathophysiological conditions.The exact relationship between mitochondrial catastrophe and caspase activation is not completely understood.Here we addressed the underlying mechanism,explaining how activated caspase could feedback to attack mitochondria to amplify further cytochrome e (cyto.c) release.We discovered that cytochrome c1 (cyto.c1) in the bc1 complex of the mitochondrial respiration chain was a novel substrate of caspase 3 (casp.3).We found that cyto.c1 was cleaved at the site of D106,which is critical for binding with cyto.c,following apoptotic stresses or targeted expression of casp.3 into tbe mitochondrial intermembrane space.We demonstrated that this cleavage was closely linked with further cyto.c release and mitochondrial catastrophe.These mitochondrial events could be effectively blocked by expressing non-cleavable cyto.c1 (D106A) or by caspase inhibitor z-VAD-fmk.Our results demonstrate that the cleavage of cyto.c1 represents a critical step for the feedback amplification of cyto.c release by caspases and subsequent mitochondrial catastrophe.

  3. Study of interface formation on the cleavage surfaces of A3{B}6 layered semiconductors

    Science.gov (United States)

    Galiy, P. V.; Nenchuk, T. M.; Stakhira, J. M.

    2001-01-01

    The adsorption activity of In4Se3, In4Se3(Ag), InSe, GaSe and TlGaSe2 semiconductor crystal interlayer cleavage surfaces relatively to N2, O2, CO gases and water vapour has been studied by Auger electron spectroscopy and mass spectrometry. It has been determined that atomically clean layered crystal surfaces do not adsorb N2 and water vapour but reveal a low activity with respect to O2. The kinetics of CO adsorption on surfaces obtained by cleavage in an UHV have been investigated. Indium and gallium selenides adsorb CO with the tendency increasing in the sequence GaSe→TlGaSe2→ InSe→In4Se3 crystals; In4Se3 is essentially more active than the others. The adsorption model with dissociations of the CO molecule and carbon adsorption resulting from the layered structure and peculiarities in the electron-energy spectra of the crystals and their surfaces is discussed with the In4Se3 crystal serving as example.

  4. Fluorescence quenching of graphene oxide combined with the site-specific cleavage of restriction endonuclease for deoxyribonucleic acid demethylase activity assay

    Energy Technology Data Exchange (ETDEWEB)

    Ji, Lijuan; Qian, Yingdan; Wu, Ping; Zhang, Hui; Cai, Chenxin, E-mail: cxcai@njnu.edu.cn

    2015-04-15

    Highlights: • An approach for sensitive and selective DNA demethylase activity assay is reported. • This assay is based on the fluorescence quenching of GO and site-specific cleavage of endonuclease. • It can determine as low as 0.05 ng mL{sup −1} of MBD2 with a linear range of 0.2–300 ng mL{sup −1}. • It has an ability to recognize MBD2 from other possibly coexisting proteins and cancer cell extracts. • It can avoid false signals, requiring no bisulfite conversion, PCR amplification, radioisotope-labeling. - Abstract: We report on the development of a sensitive and selective deoxyribonucleic acid (DNA) demethylase (using MBD2 as an example) activity assay by coupling the fluorescence quenching of graphene oxide (GO) with the site-specific cleavage of HpaII endonuclease to improve the selectivity. This approach was developed by designing a single-stranded probe (P1) that carries a binding region to facilitate the interaction with GO, which induces fluorescence quenching of the labeled fluorophore (FAM, 6-carboxyfluorescein), and a sensing region, which contains a hemi-methylated site of 5′-CmCGG-3′, to specifically recognize the target (T1, a 32-mer DNA from the promoter region of p53 gene) and hybridize with it to form a P1/T1 duplex. After demethylation with MBD2, the duplex can be specifically cleaved using HpaII, which releases the labeled FAM from the GO surface and results in the recovery of fluorescence. However, this cleavage is blocked by the hemi-methylation of this site. Thus, the magnitude of the recovered fluorescence signal is related to the MBD2 activity, which establishes the basis of the DNA demethylase activity assay. This assay can determine as low as ∼(0.05 ± 0.01) ng mL{sup −1} (at a signal/noise of 3) of MBD2 with a linear range of 0.2–300 ng mL{sup −1} and recognize MBD2 from other possibly coexisting proteins and cancer cell extracts. The advantage of this assay is its ability to avoid false signals and no

  5. Mixed ligand copper(II) dicarboxylate complexes: the role of co-ligand hydrophobicity in DNA binding, double-strand DNA cleavage, protein binding and cytotoxicity.

    Science.gov (United States)

    Loganathan, Rangasamy; Ramakrishnan, Sethu; Ganeshpandian, Mani; Bhuvanesh, Nattamai S P; Palaniandavar, Mallayan; Riyasdeen, Anvarbatcha; Akbarsha, Mohamad Abdulkadhar

    2015-06-14

    A few water soluble mixed ligand copper(ii) complexes of the type [Cu(bimda)(diimine)] , where bimda is N-benzyliminodiacetic acid and diimine is 2,2'-bipyridine (bpy, ) or 1,10-phenanthroline (phen, ) or 5,6-dimethyl-1,10-phenanthroline (5,6-dmp, ) or 3,4,7,8-tetramethyl-1,10-phenanthroline (3,4,7,8-tmp, ) and dipyrido[3,2-d: 2',3'-f]quinoxaline (dpq, ), have been successfully isolated and characterized by elemental analysis and other spectral techniques. The coordination geometry around copper(ii) in is described as distorted square based pyramidal while that in is described as square pyramidal. Absorption spectral titrations and competitive DNA binding studies reveal that the intrinsic DNA binding affinity of the complexes depends upon the diimine co-ligand, dpq () > 3,4,7,8-tmp () > 5,6-dmp () > phen () > bpy (). The phen and dpq co-ligands are involved in the π-stacking interaction with DNA base pairs while the 3,4,7,8-tmp/5,6-dmp and bpy co-ligands are involved in respectively hydrophobic and surface mode of binding with DNA. The small enhancement in the relative viscosity of DNA upon binding to supports the DNA binding modes proposed. Interestingly, and are selective in exhibiting a positive induced CD band (ICD) upon binding to DNA suggesting that they induce B to A conformational change. In contrast, and show CD responses which reveal their involvement in strong DNA binding. The complexes are unique in displaying prominent double-strand DNA cleavage while effects only single-strand DNA cleavage, and their ability to cleave DNA in the absence of an activator varies as > > > > . Also, all the complexes exhibit oxidative double-strand DNA cleavage activity in the presence of ascorbic acid, which varies as > > > > . The ability of the complexes to bind and cleave the protein BSA varies in the order > > > > . Interestingly, and cleave the protein non-specifically in the presence of H2O2 as an activator suggesting that they can act also as chemical proteases

  6. In vivo proof-of-concept of removal of the huntingtin caspase cleavage motif-encoding exon 12 approach in the YAC128 mouse model of Huntington's disease.

    Science.gov (United States)

    Casaca-Carreira, João; Toonen, Lodewijk J A; Evers, Melvin M; Jahanshahi, Ali; van-Roon-Mom, Willeke M C; Temel, Yasin

    2016-12-01

    Huntington's disease (HD) is a progressive autosomal dominant disease, caused by a CAG repeat expansion in the HTT gene, resulting in an expanded polyglutamine stretch at the N-terminal of the huntingtin protein. An important event in HD pathogenesis appears to be the proteolysis of the mutant protein, which forms N-terminal huntingtin fragments. These fragments form insoluble aggregates and are found in nuclei and cytoplasm of affected neurons where they interfere with normal cell functioning. Important cleavage sites are encoded by exon 12 of HTT. A novel approach is Htt protein modification through exon skipping, which has recently been proven effective both in vitro and in vivo. Here we report proof-of-concept of AON 12.1 in vivo using the YAC128 mouse model of HD. Our results support and encourage future longitudinal studies exploring the therapeutic effects of sustained infusions in the YAC128 mouse model.

  7. The vhs1 mutant form of herpes simplex virus virion host shutoff protein retains significant internal ribosome entry site-directed RNA cleavage activity.

    Science.gov (United States)

    Lu, P; Saffran, H A; Smiley, J R

    2001-01-01

    The virion host shutoff (vhs) protein of herpes simplex virus (HSV) triggers global shutoff of host protein synthesis and accelerated turnover of host and viral mRNAs during HSV infection. As well, it induces endoribonucleolytic cleavage of RNA substrates when produced in a rabbit reticulocyte lysate (RRL) in vitro translation system. The vhs1 point mutation (Thr 214-->Ile) eliminates vhs function during virus infection and in transiently transfected mammalian cells and was therefore previously considered to abolish vhs activity. Here we demonstrate that the vhs1 mutant protein induces readily detectable endoribonuclease activity on RNA substrates bearing the internal ribosome entry site of encephalomyocarditis virus in the RRL assay system. These data document that the vhs1 mutation does not eliminate catalytic activity and raise the possibility that the vhs-dependent endoribonuclease employs more than one mode of substrate recognition.

  8. Heterolytic OO bond cleavage: Functional role of Glu113 during bis-Fe(IV) formation in MauG.

    Science.gov (United States)

    Geng, Jiafeng; Huo, Lu; Liu, Aimin

    2017-02-01

    The diheme enzyme MauG utilizes H2O2 to perform oxidative posttranslational modification on a protein substrate. A bis-Fe(IV) species of MauG was previously identified as a key intermediate in this reaction. Heterolytic cleavage of the OO bond of H2O2 drives the formation of the bis-Fe(IV) intermediate. In this work, we tested a hypothesis that a glutamate residue, Glu113 in the distal pocket of the pentacoordinate heme of MauG, facilitates heterolytic OO bond cleavage, thereby leading to bis-Fe(IV) formation. This hypothesis was proposed based on sequence alignment and structural comparison with other H2O2-utilizing hemoenzymes, especially those from the diheme enzyme superfamily that MauG belongs to. Electron paramagnetic resonance (EPR) characterization of the reaction between MauG and H2O2 revealed that mutation of Glu113 inhibited heterolytic OO bond cleavage, in agreement with our hypothesis. This result was further confirmed by the HPLC study in which an analog of H2O2, cumene hydroperoxide, was used to probe the pattern of OO bond cleavage. Together, our data suggest that Glu113 functions as an acid-base catalyst to assist heterolytic OO bond cleavage during the early stage of the catalytic reaction. This work advances our mechanistic understanding of the H2O2-activation process during bis-Fe(IV) formation in MauG.

  9. Triple helix-forming oligonucleotides conjugated to indolocarbazole poisons direct topoisomerase I-mediated DNA cleavage to a specific site.

    Science.gov (United States)

    Arimondo, P B; Bailly, C; Boutorine, A S; Moreau, P; Prudhomme, M; Sun, J S; Garestier, T; Hélène, C

    2001-01-01

    Topoisomerase I is an ubiquitous DNA-cleaving enzyme and an important therapeutic target in cancer chemotherapy for camptothecins as well as for indolocarbazole antibiotics such as rebeccamycin. To achieve a sequence-specific cleavage of DNA by topoisomerase I, a triple helix-forming oligonucleotide was covalently linked to indolocarbazole-type topoisomerase I poisons. The three indolocarbazole-oligonucleotide conjugates investigated were able to direct topoisomerase I cleavage at a specific site based upon sequence recognition by triplex formation. The efficacy of topoisomerase I-mediated DNA cleavage depends markedly on the intrinsic potency of the drug. We show that DNA cleavage depends also upon the length of the linker arm between the triplex-forming oligonucleotide and the drug. Based on a known structure of the DNA-topoisomerase I complex, a molecular model of the oligonucleotide conjugates bound to the DNA-topoisomerase I complex was elaborated to facilitate the design of a potent topoisomerase I inhibitor-oligonucleotide conjugate with an optimized linker between the two moieties. The resulting oligonucleotide-indolocarbazole conjugate at 10 nM induced cleavage at the triple helix site 2-fold more efficiently than 5 microM of free indolocarbazole, while the other drug-sensitive sites were not cleaved. The rational design of drug-oligonucleotide conjugates carrying a DNA topoisomerase poison may be exploited to improve the efficacy and selectivity of chemotherapeutic cancer treatments by targeting specific genes and reducing drug toxicity.

  10. Synthesis, characterization, DNA interactions, DNA cleavage, radical scavenging activity, antibacterial, anti-proliferative and docking studies of new transition metal complexes.

    Science.gov (United States)

    Chennam, Kishan Prasad; Ravi, Mudavath; Ushaiah, B; Srinu, V; Eslavath, Ravi Kumar; Devi, Ch Sarala

    2016-01-01

    The compound N-(2-hydroxybenzylidene)-1-ethyl-1, 4-dihydro-7-methyl-4-oxo-1, 8 naphthyridine-3-carbohydrazide (LH) and its Cu (II), Co (II) and Zn (II) complexes were synthesized and characterized. The absorption spectral titrations and competitive DNA binding studies depicted those complexes of title compound bind to CT-DNA through intercalation. Interestingly [Cu (II)-(L2)] showed relatively high binding constant value (6.61 x 10(5) M(-1)) compared to [Co (II)-(L2)] (4.378× 10(5) M(-1)) and [Zn (II)-(L2)] (3.1x10(5) M(-1)). Ligand and its complexes were also examined for DNA nuclease activity against pBR-322 plasmid DNA, which showed that [Cu (II)-(L2)] had the best hydrolytic cleavage property displaying prominent double-strand DNA cleavage. In addition, antioxidant activities of the ligand and its metal complexes investigated through scavenging effects for DPPH radical in- vitro, indicated their potentiality as good antioxidants. The in vitro anti-bacterial study inferred the better anti-bacterial activity of [Cu (II)-(L2)] and this was also correlated theoretically by employing docking studies wherein [Cu (II)-(L2)] displayed good Gold score and Chem score. Finally the in vitro anti- proliferative activity of studied compounds was tested against HeLa and MCF-7 cell lines. Interestingly [Cu (II)-(L2)] displayed lower IC50 value and lower percentage of viability in both HeLa and MCF-7 cell lines.

  11. Secreted aspartic protease cleavage of Candida albicans Msb2 activates Cek1 MAPK signaling affecting biofilm formation and oropharyngeal candidiasis.

    Directory of Open Access Journals (Sweden)

    Sumant Puri

    Full Text Available Perception of external stimuli and generation of an appropriate response are crucial for host colonization by pathogens. In pathogenic fungi, mitogen activated protein kinase (MAPK pathways regulate dimorphism, biofilm/mat formation, and virulence. Signaling mucins, characterized by a heavily glycosylated extracellular domain, a transmembrane domain, and a small cytoplasmic domain, are known to regulate various signaling pathways. In Candida albicans, the mucin Msb2 regulates the Cek1 MAPK pathway. We show here that Msb2 is localized to the yeast cell wall and is further enriched on hyphal surfaces. A msb2Δ/Δ strain formed normal hyphae but had biofilm defects. Cek1 (but not Mkc1 phosphorylation was absent in the msb2Δ/Δ mutant. The extracellular domain of Msb2 was shed in cells exposed to elevated temperature and carbon source limitation, concomitant with germination and Cek1 phosphorylation. Msb2 shedding occurred differentially in cells grown planktonically or on solid surfaces in the presence of cell wall and osmotic stressors. We further show that Msb2 shedding and Cek1 phosphorylation were inhibited by addition of Pepstatin A (PA, a selective inhibitor of aspartic proteases (Saps. Analysis of combinations of Sap protease mutants identified a sap8Δ/Δ mutant with reduced MAPK signaling along with defects in biofilm formation, thereby suggesting that Sap8 potentially serves as a major regulator of Msb2 processing. We further show that loss of either Msb2 (msb2Δ/Δ or Sap8 (sap8Δ/Δ resulted in higher C. albicans surface β-glucan exposure and msb2Δ/Δ showed attenuated virulence in a murine model of oral candidiasis. Thus, Sap-mediated proteolytic cleavage of Msb2 is required for activation of the Cek1 MAPK pathway in response to environmental cues including those that induce germination. Inhibition of Msb2 processing at the level of Saps may provide a means of attenuating MAPK signaling and reducing C. albicans virulence.

  12. Sequence/structure selective thermal and photochemical cleavage of yeast-tRNA(Phe) by UO(2)2+

    DEFF Research Database (Denmark)

    Nielsen, Peter E.; Møllegaard, N E

    1997-01-01

    The uranyl(VI) ion, UO(2)2+, cleaves yeast tRNA(Phe) both thermally and photochemically. Photochemical cleavage takes place at all positions but exhibits maxima at G10, G18, G30, A38, C49 and A62. Furthermore, in the presence of stoichiometric concentrations of citrate, the cleavage is generally...... suppressed except that strong cleavage at positions G10 and C48-U50 persists, indicating the presence of a high-affinity metal-ion binding site. It is proposed that these photocleavage sites reflect the tertiary structure of the yeast tRNA(Phe) molecule in terms of D-loop/T-loop interaction and anticodon...

  13. Regulation of aromatics biodegradation by rhl quorum sensing system through induction of catechol meta-cleavage pathway.

    Science.gov (United States)

    Yong, Yang-Chun; Zhong, Jian-Jiang

    2013-05-01

    The mechanism for quorum sensing (QS) regulation on aromatics degradation was investigated. Deletion of rhl QS system resulted in a significant decrease in aromatics biodegradation as well as the activity of catechol 2,3-dioxygenase (C23O, key enzyme for catechol meta-cleavage pathway) in Pseudomonas aeruginosa CGMCC1.860. Interestingly, this repression could be relieved by N-butyryl homoserine lactone (the signaling molecule of rhl QS system) addition. In accordance, the transcription level of nahH (the gene encoding C23O) and nahR (transcriptional activator) also responded to rhl perturbation in a similar way. The results indicated that rhl QS system positively controlled the catechol meta-cleavage pathway, and hence improved aromatics biodegradation. It suggested manipulation of QS system could be a promising strategy to tune the catechol cleavage pathway and to control aromatics biodegradation.

  14. The bacterial toxin RelE induces specific mRNA cleavage in the A site of the eukaryote ribosome

    Science.gov (United States)

    Andreev, Dmitri; Hauryliuk, Vasili; Terenin, Ilya; Dmitriev, Sergey; Ehrenberg, Måns; Shatsky, Ivan

    2008-01-01

    RelE/RelB is a well-characterized toxin–anti-toxin pair involved in nutritional stress responses in Bacteria and Archae. RelE lacks any eukaryote homolog, but we demonstrate here that it efficiently and specifically cleaves mRNA in the A site of the eukaryote ribosome. The cleavage mechanism is similar to that in bacteria, showing the feasibility of A-site cleavage of mRNA for regulatory purposes also in eukaryotes. RelE cleavage in the A-site codon of a stalled eukaryote ribosome is precise and easily monitored, making “RelE printing” a useful complement to toeprinting to determine the exact mRNA location on the eukaryote ribosome and to probe the occupancy of its A site. PMID:18083838

  15. 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.

  16. Efficient Production of a Bioactive Bevacizumab Monoclonal Antibody Using the 2A Self-cleavage Peptide in Transgenic Rice Callus

    Science.gov (United States)

    Chen, Lei; Yang, Xiaoyu; Luo, Da; Yu, Weichang

    2016-01-01

    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 Bevacizumab heavy chain (BHC) genes were designed, synthesized as a polyprotein with a 2A self-cleavage linker peptide from the Foot-and-mouth disease virus, 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 BL