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Sample records for cell cleavage

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

    DEFF Research Database (Denmark)

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

    1997-01-01

    relative to the reaction in solution. The time course of uPA-catalyzed cleavage of cell-bound uPAR was studied using U937 cells stimulated with phorbol 12-myristate 13-acetate. Only 30 min was required for 10 nM uPA to cleave 50% of the cell-bound uPAR. This uPA-catalyzed cleavage reaction was inhibited...

  2. Stress-induced cleavage of Myc promotes cancer cell survival

    Science.gov (United States)

    Conacci-Sorrell, Maralice; Ngouenet, Celine; Anderson, Sarah; Brabletz, Thomas; Eisenman, Robert N.

    2014-01-01

    Evasion of apoptosis is critical in Myc-induced tumor progression. Here we report that cancer cells evade death under stress by activating calpain-mediated proteolysis of Myc. This generates Myc-nick, a cytoplasmic, transcriptionally inactive cleavage product of Myc. We found conversion of Myc into Myc-nick in cell lines and tissues derived from multiple cancers. In colon cancer, the production of Myc-nick is enhanced under stress conditions such as hypoxia and nutrient deprivation. Under these conditions, ectopic expression of Myc-nick promotes anchorage-independent growth and cell survival at least in part by promoting autophagy. Myc-nick also delays colon cancer cell death after treatment with chemotherapeutic drugs such as etoposide, cisplatin, and imatinib. Furthermore, colon cancer cells expressing a cleavage-resistant form of Myc undergo extensive apoptosis but are rescued by overexpression of Myc-nick. We also found that ectopic expression of Myc-nick results in the induction of the actin-bundling protein fascin, formation of filopodia, and increased cell motility—all mediators of tumor metastasis. Myc-nick-induced survival, autophagy, and motility require Myc box II (MBII), a region of Myc-nick that recruits acetyltransferases that in turn modify cytoplasmic proteins, including α-tubulin and ATG3. Our results suggest that Myc-nick-induced survival and motility contribute to colon cancer progression and metastasis. PMID:24696454

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

    Science.gov (United States)

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

    2015-01-01

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

  4. Cell cycle stage dependent variations in drug-induced topoisomerase II mediated DNA cleavage and cytotoxicity

    International Nuclear Information System (INIS)

    Estey, E.; Adlakha, R.C.; Hittelman, W.N.; Zwelling, L.A.

    1987-01-01

    The DNA cleavage produced by 4'-(9-acridinylamino)methanesulfon-m-anisidide (m-AMSA) in mammalian cells is putatively mediated by topoisomerase II. The authors found that in synchronized HeLa cells the frequency of such cleavage was 4-15-fold greater in mitosis than in S while the DNA of G 1 and G 2 cells exhibited an intermediate susceptibility to cleavage. The hypersensitivity of mitotic DNA to m-AMSA-induced cleavage was acquired relatively abruptly in late G 2 and was lost similarly abruptly in early G 1 . The susceptibility of mitotic cells to m-AMSA-induced DNA cleavage was not clearly paralleled by an increase in topoisomerase II activity in 350 mM NaCl extracts from mitotic cells compared to similar extracts from cells in G 1 , S, or G 2 . Furthermore, equal amounts of decatenating activity from cells in mitosis and S produced equal amounts of m-AMSA-induced cleavage of simian virus 40 (SV40) DNA; i.e., the interaction between m-AMSA and extractable enzyme was similar in mitosis and S. The DNA of mitotic cells was also hypersensitive to cleavage by 4'-demethylepipodophyllotoxin 4-(4,6-O-ethylidene-β-D-glucopyranoside) (etoposide), a drug that produces topoisomerase II mediated DNA cleavage without binding to DNA. Cell cycle stage dependent fluctuations in m-AMSA-induced DNA cleavage may result from fluctuations in the structure of chromatin per se that occur during the cell cycle. Surprisingly, cell cycle stage dependent differences in m-AMSA-induced DNA cleavage did not correlate with differences in the susceptibility to the cytotoxic effects of the drug. In fact, cells in S were most sensitive to these effects. These results are an exception to the previously observed parallel between the susceptibility of mammalian cells to drug-induced DNA cleavage and the susceptibility of the cells to drug-induced cytotoxicity and indicate the complexity of any relationship between the two phenomena

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

    Science.gov (United States)

    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.

  6. Determination of the cell cleavage plane by the LIN-5 complex

    NARCIS (Netherlands)

    Galli, M.

    2011-01-01

    Cells split in two at the final step of each division cycle. This division normally bisects through the middle of the cell and generates two equal daughters. However, developmental signals can change the plane of cell cleavage to facilitate asymmetric segregation of fate determinants and control the

  7. Extracellular cleavage of E-cadherin promotes epithelial cell extrusion

    NARCIS (Netherlands)

    Grieve, Adam G; Rabouille, Catherine

    2014-01-01

    Epithelial cell extrusion and subsequent apoptosis is a key mechanism to prevent the accumulation of excess cells. By contrast, when driven by oncogene expression, apical cell extrusion is followed by proliferation and represents an initial step of tumorigenesis. E-cadherin (E-cad), the main

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

    DEFF Research Database (Denmark)

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

    2004-01-01

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

  9. 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

  10. Electrochemical protein cleavage in a microfluidic cell with integrated boron doped diamond electrodes

    NARCIS (Netherlands)

    van den Brink, Floris Teunis Gerardus; Zhang, Tao; Ma, Liwei; Bomer, Johan G.; 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

  11. BACTERIAL CELL KILLING MEDIATED BY TOPOISOMERASE I DNA CLEAVAGE ACTIVITY

    Science.gov (United States)

    Cheng, Bokun; Shukla, Shikha; Vasunilashorn, Sarinnapha; Mukhopadhyay, Somshuvra; Tse-Dinh, Yuk-Ching

    2005-01-01

    DNA topoisomerases are important clinical targets for antibacterial and anticancer therapy. At least one type IA DNA topoisomerases can be found in every bacterium, making it a logical target for antibacterial agents that can convert the enzyme into poison by trapping its covalent complex with DNA. However, it has not been possible previously to observe the consequence of having such stabilized covalent complex of bacterial topoisomerase I in vivo. We isolated a mutant of recombinant Yersinia pestis topoisomerase I that forms a stabilized covalent complex with DNA by screening for the ability to induce the SOS response in Escherichia coli. Overexpression of this mutant topoisomerase I resulted in bacterial cell death. From sequence analysis and site-directed mutagenesis, it was determined that a single amino acid substitution in the TOPRIM domain changing a strictly conserved glycine residue to serine in either the Y. pestis or E. coli topoisomerase I can result in a mutant enzyme that has the SOS inducing and cell killing properties. Analysis of the purified mutant enzymes showed that they have no relaxation activity but retain the ability to cleave DNA and form a covalent complex. These results demonstrate that perturbation of the active site region of bacterial topoisomerase I can result in stabilization of the covalent intermediate, with the in vivo consequence of bacterial cell death. Small molecules that induce similar perturbation in the enzyme-DNA complex should be candidates as leads for novel antibacterial agents. PMID:16159875

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

    Directory of Open Access Journals (Sweden)

    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.

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

    Science.gov (United States)

    2015-12-01

    event. The discovery that transformed and rapidly proliferating cells use alternative cleavage and polyadenylation ( APA ) to shorten the 3´UTR of their... APA . However, the mechanism that APA is still unknown. The goal of this project is to identify the mechanism of cyclin D1 APA regulation in cancer...for APA in MCL. In addition, by using RNA Seq. CFIm25 has been identified as an important global regulator of shortening of cyclin D1 mRNA and other

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

    International Nuclear Information System (INIS)

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

    2006-01-01

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

  15. Cleavage site of Newcastle disease virus determines viral fitness in persistent infection cells.

    Science.gov (United States)

    Liu, Haijin; Servan de Almeida, Renata; Gil, Patricia; Albina, Emmanuel

    2018-03-01

    Newcastle disease, caused by infection with virulent strains of Newcastle disease virus (NDV), poses a risk for the poultry industry. The virulence of NDV is mainly determined by the cleavage site of F protein. Lentogenic NDV can become velogenic after passages in SPF chicken brain and air sac based on some strains isolated from water birds, because the proportion of virulent-related strains gradually increases. In contrast, this proportion remains unchanged if NDV is passaged via 10-day-old SPF chicken embryos. This information suggests that environmental conditions rather than mutation affect NDV fitness in quasispecies. However, it is unknown how the environment selects virulent-related strains from a viral population. In this study, velogenic and lentogenic NDV marked by green or red fluorescence were used to establish persistent infection (PI) in BHK-21 cells. Monitoring viruses by different methods, we found that, without competition, persistently infected cells harbored lentogenic and velogenic NDV strains similarly in terms of viral release, viral spread and the period of persistent viral infection. In contrast, under competitive co-infection, velogenic NDV became dominant in quasispecies from the fifth passage of PI cells, which resulted in the progressive disappearance of the lentogenic NDV strain. This domination was concomitant with a short-term reduction in the superinfection exclusion and supernatant interference in PI cells resulting in a velogenic virus rebound. We concluded that virulent-related F protein cleavage site facilitates the spread and replication of NDV in conditions under which cells do not secret trypsin-like proteases and do not inhibit free virus infection, resulting in a gradual increase in virulent strains in quasispecies with the number of passages. Copyright © 2018 Elsevier B.V. All rights reserved.

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

    Directory of Open Access Journals (Sweden)

    Alexander Jonathan S

    2010-12-01

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

  17. Caspase-1 cleavage of transcription factor GATA4 and regulation of cardiac cell fate.

    Science.gov (United States)

    Aries, A; Whitcomb, J; Shao, W; Komati, H; Saleh, M; Nemer, M

    2014-12-11

    Caspase-1 or interleukin-1β (IL-1β) converting enzyme is a pro-inflammatory member of the caspase family. An IL-1β-independent role for caspase-1 in cardiomyocyte cell death and heart failure has emerged but the mechanisms underlying these effects are incompletely understood. Here, we report that transcription factor GATA4, a key regulator of cardiomyocyte survival and adaptive stress response is an in vivo and in vitro substrate for caspase-1. Caspase-1 mediated cleavage of GATA4 generates a truncated protein that retains the ability to bind DNA but lacks transcriptional activation domains and acts as a dominant negative regulator of GATA4. We show that caspase-1 is rapidly activated in cardiomyocyte nuclei treated with the cell death inducing drug Doxorubicin. We also find that inhibition of caspase-1 alone is as effective as complete caspase inhibition at rescuing GATA4 degradation and myocyte cell death. Caspase-1 inhibition of GATA4 transcriptional activity is rescued by HSP70, which binds directly to GATA4 and masks the caspase recognition motif. The data identify a caspase-1 nuclear substrate and suggest a direct role for caspase-1 in transcriptional regulation. This mechanism may underlie the inflammation-independent action of caspase-1 in other organs.

  18. Effects of β-Carotene and Its Cleavage Products in Primary Pneumocyte Type II Cells

    Directory of Open Access Journals (Sweden)

    Cornelia Haider

    2017-05-01

    Full Text Available β-Carotene has been shown to increase the risk of developing lung cancer in smokers and asbestos workers in two large scale trails, the Beta-Carotene and Retinol Efficacy Trial (CARET and the Alpha-Tocopherol Beta-carotene Cancer Prevention Trial (ATBC. Based on this observation, it was proposed that genotoxic oxidative breakdown products may cause this effect. In support of this assumption, increased levels of sister chromatid exchanges, micronuclei, and chromosomal aberrations were found in primary hepatocyte cultures treated with a mixture of cleavage products (CPs and the major product apo-8′carotenal. However, because these findings cannot directly be transferred to the lung due to the exceptional biotransformation capacity of the liver, potential genotoxic and cytotoxic effects of β-carotene under oxidative stress and its CPs were investigated in primary pneumocyte type II cells. The results indicate that increased concentrations of β-carotene in the presence of the redox cycling quinone dimethoxynaphthoquinone (DMNQ exhibit a cytotoxic potential, as evidenced by an increase of apoptotic cells and loss of cell density at concentrations > 10 µM. On the other hand, the analysis of micronucleated cells gave no clear picture due to the cytotoxicity related reduction of mitotic cells. Last, although CPs induced significant levels of DNA strand breaks even at concentrations ≥ 1 µM and 5 µM, respectively, β-carotene in the presence of DMNQ did not cause DNA damage. Instead, β-carotene appeared to act as an antioxidant. These findings are in contrast with what was demonstrated for primary hepatocytes and may reflect different sensitivities to and different metabolism of β-carotene in the two cell types.

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

    Science.gov (United States)

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

    2017-06-30

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

  20. Topoisomerase I function during Escherichia coli response to antibiotics and stress enhances cell killing from stabilization of its cleavage complex

    Science.gov (United States)

    Liu, I-Fen; Sutherland, Jeanette H.; Cheng, Bokun; Tse-Dinh, Yuk-Ching

    2011-01-01

    Objectives To explore the role of topoisomerase I in gene activation and increased RecA levels during the bacterial SOS response, as well as the effect of antibiotic treatment and stress challenge on cell killing initiated by trapped topoisomerase I cleavage complex. Methods A mutant Escherichia coli strain with a ΔtopA mutation was used to investigate the role of topoisomerase I function in the SOS response to trimethoprim and mitomycin C. Induction of the recA and dinD1 promoters was measured using luciferase reporters of these promoters fused to luxCDABE. An increase in the RecA level following trimethoprim treatment was quantified directly by western blotting. The effect of stress challenge from trimethoprim and acidified nitrite treatments on cell killing by topoisomerase I cleavage complex accumulation was measured by the decrease in viability following induction of recombinant mutant topoisomerase I that forms a stabilized cleavage complex. Results Topoisomerase I function was found to be required for efficient transcriptional activation of the recA and dinD1 promoters during the E. coli SOS response to trimethoprim and mitomycin C. The role of topoisomerase I in the SOS response was confirmed with quantitative western blot analysis of RecA following trimethoprim treatment. The bactericidal effect from topoisomerase I cleavage complex accumulation was shown to be enhanced by stress challenge from trimethoprim and acidified nitrite. Conclusions Bacterial topoisomerase I function is actively involved in the SOS response to antibiotics and stress challenge. Cell killing initiated by the topoisomerase I cleavage complex would be enhanced by antibiotics and the host response. These findings provide further support for bacterial topoisomerase I as a therapeutic target. PMID:21486853

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

    Science.gov (United States)

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

    2011-11-07

    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.

  2. 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

    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

  3. 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.

  4. Rhinovirus 3C protease facilitates specific nucleoporin cleavage and mislocalisation of nuclear proteins in infected host cells.

    Directory of Open Access Journals (Sweden)

    Erin J Walker

    Full Text Available Human Rhinovirus (HRV infection results in shut down of essential cellular processes, in part through disruption of nucleocytoplasmic transport by cleavage of the nucleoporin proteins (Nups that make up the host cell nuclear pore. Although the HRV genome encodes two proteases (2A and 3C able to cleave host proteins such as Nup62, little is known regarding the specific contribution of each. Here we use transfected as well as HRV-infected cells to establish for the first time that 3C protease is most likely the mediator of cleavage of Nup153 during HRV infection, while Nup62 and Nup98 are likely to be targets of HRV2A protease. HRV16 3C protease was also able to elicit changes in the appearance and distribution of the nuclear speckle protein SC35 in transfected cells, implicating it as a key mediator of the mislocalisation of SC35 in HRV16-infected cells. In addition, 3C protease activity led to the redistribution of the nucleolin protein out of the nucleolus, but did not affect nuclear localisation of hnRNP proteins, implying that complete disruption of nucleocytoplasmic transport leading to relocalisation of hnRNP proteins from the nucleus to the cytoplasm in HRV-infected cells almost certainly requires 2A in addition to 3C protease. Thus, a specific role for HRV 3C protease in cleavage and mislocalisation of host cell nuclear proteins, in concert with 2A, is implicated for the first time in HRV pathogenesis.

  5. 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.

  6. Effect of trastuzumab interchain disulfide bond cleavage on Fcγ receptor binding and antibody-dependent tumour cell phagocytosis.

    Science.gov (United States)

    Suzuki, Mami; Yamanoi, Ayaka; Machino, Yusuke; Ootsubo, Michiko; Izawa, Ken-ichi; Kohroki, Junya; Masuho, Yasuhiko

    2016-01-01

    The Fc domain of human IgG1 binds to Fcγ receptors (FcγRs) to induce effector functions such as phagocytosis. There are four interchain disulfide bonds between the H and L chains. In this study, the disulfide bonds within the IgG1 trastuzumab (TRA), which is specific for HER2, were cleaved by mild S-sulfonation or by mild reduction followed by S-alkylation with three different reagents. The cleavage did not change the binding activities of TRA to HER2-bearing SK-BR-3 cells. The binding activities of TRA to FcγRIIA and FcγRIIB were greatly enhanced by modification with mild reduction and S-alkylation with ICH2CONH2 or N-(4-aminophenyl) maleimide, while the binding activities of TRA to FcγRI and FcγRIIIA were decreased by any of the four modifications. However, the interchain disulfide bond cleavage by the different modifications did not change the antibody-dependent cell-mediated phagocytosis (ADCP) of SK-BR-3 cells by activated THP-1 cells. The order of FcγR expression levels on the THP-1 cells was FcγRII > FcγRI > FcγRIII and ADCP was inhibited by blocking antibodies against FcγRI and FcγRII. These results imply that the effect of the interchain disulfide bond cleavage on FcγRs binding and ADCP is dependent on modifications of the cysteine residues and the FcγR isotypes. © The Authors 2015. Published by Oxford University Press on behalf of the Japanese Biochemical Society. All rights reserved.

  7. 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. Copyright © 2016 Elsevier B.V. All rights reserved.

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

    Directory of Open Access Journals (Sweden)

    Susannah L. Hewitt

    2017-10-01

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

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

    Directory of Open Access Journals (Sweden)

    Xiangyi Kong

    Full Text Available 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 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.

  10. Procalcitonin NH2-terminal cleavage peptide has no mitogenic effect on normal human osteoblast-like cells

    International Nuclear Information System (INIS)

    Hassager, C.; Bonde, S.K.; Anderson, M.A.; Rink, H.; Spelsberg, T.C.; Riggs, B.L.

    1991-01-01

    The NH2-terminal cleavage peptide of procalcitonin (N-proCT) recently was reported to be a bone cell mitogen. The authors have investigated the effect of N-proCT on the proliferation of normal human cells that have the phenotype of mature osteoblasts (hOB cells). N-proCT treatment for 24, 48, or 96 h in concentrations from 1 nM to 1 microM did not significantly increase [3H]thymidine uptake (means ranged from -19% to 38% of control, no significant differences) in hOB cells (6-10 cell strains per experiment) plated at four different densities. However, the hOB cells responded significantly to treatment with transforming growth factor β (3 ng/ml), bovine insulin (300 micrograms/ml), or 30% fetal calf serum, which were included in all experiments as positive controls. The [3H]thymidine uptake data were confirmed in a direct cell count experiment tested at 96 h. Thus they data do not support the hypothesis that N-proCT is a potent mitogen for normal human osteoblasts

  11. Cisplatin-induced caspase activation mediates PTEN cleavage in ovarian cancer cells: a potential mechanism of chemoresistance

    International Nuclear Information System (INIS)

    Singh, Mohan; Chaudhry, Parvesh; Fabi, Francois; Asselin, Eric

    2013-01-01

    The phosphatase and tensin homolog deleted on chromosome 10 (PTEN) tumor suppressor protein is a central negative regulator of the PI3K/AKT signaling cascade and suppresses cell survival as well as cell proliferation. PTEN is found to be either inactivated or mutated in various human malignancies. In the present study, we have investigated the regulation of PTEN during cisplatin induced apoptosis in A2780, A270-CP (cisplatin resistant), OVCAR-3 and SKOV3 ovarian cancer cell lines. Cells were treated with 10μM of cisplatin for 24h. Transcript and protein levels were analysed by quantitative reverse transcriptase-polymerase chain reaction (qRT-PCR) and western blotting, respectively. Immunofluorescence microscopy was used to assess the intracellular localization of PTEN. Proteasome inhibitor and various caspases inhibitors were used to find the mechanism of PTEN degradation. PTEN protein levels were found to be decreased significantly in A2780 cells; however, there was no change in PTEN protein levels in A2780-CP, OVCAR-3 and SKOV3 cells with cisplatin treatment. The decrease in PTEN protein was accompanied with an increase in the levels of AKT phosphorylation (pAKT) in A2780 cells and a decrease of BCL-2. Cisplatin treatment induced the activation/cleavage of caspase-3, -6, -7, -8, -9 in all cell lines tested in this study except the resistant variant A2780-CP cells. In A2780 cells, restoration of PTEN levels was achieved upon pre-treatment with Z-DEVD-FMK (broad range caspases inhibitor) and not with MG132 (proteasome inhibitor) and by overexpression of BCL-2, suggesting that caspases and BCL-2 are involved in the decrease of PTEN protein levels in A2780 cells. The decrease in pro-apoptotic PTEN protein levels and increase in survival factor pAKT in A2780 ovarian cancer cells suggest that cisplatin treatment could further exacerbate drug resistance in A2780 ovarian cancer cells

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

    International Nuclear Information System (INIS)

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

    2016-01-01

    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 1 h after HN2 treatment, high mDPC was achieved and the level maintained for up to 24 h. 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. - Highlights: • Nitrogen mustard-induced MGMT-DNA cross-linking was detected in a living cell. • Concentration- and time-dependent manners of MGMT-DNA cross-linking were revealed. • Proteolysis played an important role in protein (MGMT)-DNA cross-linking repair. • DVC1 acts as a proteolytic enzyme in cross-linking repair in a p

  13. 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.

  14. Oligodendrocyte precursor cells modulate the neuronal network by activity-dependent ectodomain cleavage of glial NG2.

    Directory of Open Access Journals (Sweden)

    Dominik Sakry

    2014-11-01

    Full Text Available The role of glia in modulating neuronal network activity is an important question. Oligodendrocyte precursor cells (OPC characteristically express the transmembrane proteoglycan nerve-glia antigen 2 (NG2 and are unique glial cells receiving synaptic input from neurons. The development of NG2+ OPC into myelinating oligodendrocytes has been well studied, yet the retention of a large population of synapse-bearing OPC in the adult brain poses the question as to additional functional roles of OPC in the neuronal network. Here we report that activity-dependent processing of NG2 by OPC-expressed secretases functionally regulates the neuronal network. NG2 cleavage by the α-secretase ADAM10 yields an ectodomain present in the extracellular matrix and a C-terminal fragment that is subsequently further processed by the γ-secretase to release an intracellular domain. ADAM10-dependent NG2 ectodomain cleavage and release (shedding in acute brain slices or isolated OPC is increased by distinct activity-increasing stimuli. Lack of NG2 expression in OPC (NG2-knockout mice, or pharmacological inhibition of NG2 ectodomain shedding in wild-type OPC, results in a striking reduction of N-methyl-D-aspartate (NMDA receptor-dependent long-term potentiation (LTP in pyramidal neurons of the somatosensory cortex and alterations in the subunit composition of their α-amino-3-hydroxy-5-methyl-4-isoxazolepr opionicacid (AMPA receptors. In NG2-knockout mice these neurons exhibit diminished AMPA and NMDA receptor-dependent current amplitudes; strikingly AMPA receptor currents can be rescued by application of conserved LNS protein domains of the NG2 ectodomain. Furthermore, NG2-knockout mice exhibit altered behavior in tests measuring sensorimotor function. These results demonstrate for the first time a bidirectional cross-talk between OPC and the surrounding neuronal network and demonstrate a novel physiological role for OPC in regulating information processing at neuronal

  15. Vertebrate Embryonic Cleavage Pattern Determination.

    Science.gov (United States)

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

    2017-01-01

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

  16. 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

  17. Site-Specific Cleavage of Ribosomal RNA in Escherichia coli-Based Cell-Free Protein Synthesis Systems.

    Directory of Open Access Journals (Sweden)

    Jurek Failmezger

    Full Text Available Cell-free protein synthesis, which mimics the biological protein production system, allows rapid expression of proteins without the need to maintain a viable cell. Nevertheless, cell-free protein expression relies on active in vivo translation machinery including ribosomes and translation factors. Here, we examined the integrity of the protein synthesis machinery, namely the functionality of ribosomes, during (i the cell-free extract preparation and (ii the performance of in vitro protein synthesis by analyzing crucial components involved in translation. Monitoring the 16S rRNA, 23S rRNA, elongation factors and ribosomal protein S1, we show that processing of a cell-free extract results in no substantial alteration of the translation machinery. Moreover, we reveal that the 16S rRNA is specifically cleaved at helix 44 during in vitro translation reactions, resulting in the removal of the anti-Shine-Dalgarno sequence. These defective ribosomes accumulate in the cell-free system. We demonstrate that the specific cleavage of the 16S rRNA is triggered by the decreased concentrations of Mg2+. In addition, we provide evidence that helix 44 of the 30S ribosomal subunit serves as a point-of-entry for ribosome degradation in Escherichia coli. Our results suggest that Mg2+ homeostasis is fundamental to preserving functional ribosomes in cell-free protein synthesis systems, which is of major importance for cell-free protein synthesis at preparative scale, in order to create highly efficient technical in vitro systems.

  18. Atorvastatin Protects from Aβ1-40-Induced Cell Damage and Depressive-Like Behavior via ProBDNF Cleavage.

    Science.gov (United States)

    Ludka, Fabiana K; Cunha, Maurício P; Dal-Cim, Tharine; Binder, Luisa Bandeira; Constantino, Leandra C; Massari, Caio M; Martins, Wagner C; Rodrigues, Ana Lúcia S; Tasca, Carla I

    2017-10-01

    Intracerebroventricular (icv) amyloid-beta (Aβ) 1-40 infusion to mice has been demonstrated to cause neurotoxicty and depressive-like behavior and it can be used to evaluate antidepressant and neuroprotective effect of drugs. Atorvastatin is a widely used statin that has demonstrated antidepressant-like effect in predictable animal behavioral models and neuroprotective effect against Aβ 1-40 infusion. The purpose of this study was to determine the effect of in vivo atorvastatin treatment against Aβ 1-40 -induced changes in mood-related behaviors and biochemical parameters in ex vivo hippocampal slices from mice. Atorvastatin treatment (10 mg/kg, p.o., once a day for seven consecutive days) abolished depressive-like and anhedonic-like behaviors induced by Aβ 1-40 (400 pmol/site, icv) infusion. Aβ 1-40 -induced hippocampal cell damage was reversed by atorvastatin treatment. Aβ 1-40 infusion decreased glutamate uptake in hippocampal slices, and atorvastatin did not altered it. Glutamine synthetase activity was not altered by any treatment. Atorvastatin also increased hippocampal mature brain-derived neurotrophic factor (mBDNF)/precursor BDNF (proBDNF) ratio, suggesting an increase of proBDNF to mBDNF cleavage. Accordingly, increased tissue-type plasminogen activator (tPA) and p11 genic expression were observed in hippocampus of atorvastatin-treated mice. Atorvastatin displays antidepressant-like and neuroprotective effects against Aβ 1-40 -induced toxicity, and these effects may involve tPA- and p11-mediated cleavage of proBDNF to mBDNF.

  19. Cigarette Smoke Dampens Anti-viral Signaling in Small Airway Epithelial Cells by Disrupting TLR3 Cleavage.

    Science.gov (United States)

    Duffney, Parker F; McCarthy, Claire E; Nogales, Aitor; Thatcher, Thomas H; Martinez-Sobrido, Luis; Phipps, Richard P; Sime, Patricia J

    2017-12-14

    Cigarette smokers and people exposed to secondhand smoke are at an increased risk for pulmonary viral infections, yet the mechanism responsible for this heightened susceptibility is not understood. To understand the effect of cigarette smoke on susceptibility to viral infection we used an air-liquid interface culture system, and exposed primary human small airway epithelial cells (SAEC) to whole cigarette smoke followed by treatment with the viral mimetic polyinosinic polycytidylic acid (poly I:C) or influenza A virus (IAV). We found that prior smoke exposure strongly inhibited production of pro-inflammatory (interleukin 6 and interleukin 8) and anti-viral (interferon gamma induced protein 10, IP-10 and interferons) mediators in SAECs in response to poly I:C and IAV infection. Impaired antiviral responses corresponded to increased infection with IAV. This was associated with a decrease in phosphorylation of the key antiviral transcription factor interferon response factor (IRF3). Here we found that cigarette smoke exposure inhibited activation of toll-like receptor (TLR) 3 by impairing TLR3 cleavage, which was required for downstream phosphorylation of IRF3 and production of IP-10. These results identify a novel mechanism by which cigarette smoke exposure impairs antiviral responses in lung epithelial cells, which may contribute to increased susceptibility to respiratory infections.

  20. Electrochemical protein cleavage in a microfluidic cell with integrated boron doped diamond electrodes

    NARCIS (Netherlands)

    van den Brink, Floris Teunis Gerardus; Zhang, Tao; Ma, Liwei; Odijk, Mathieu; Olthuis, Wouter; Permentier, Hjalmar P.; Bischoff, Rainer P.H.; van den Berg, Albert

    2015-01-01

    We present a microfluidic electrochemical cell with integrated boron doped diamond (BDD) electrodes which is designed for high electrochemical conversion efficiencies. With our newest developments, we aim to exploit the benefits of BDD as a novel electrode material to conduct tyrosine- and

  1. 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

  2. A 'new lease of life': FnCpf1 possesses DNA cleavage activity for genome editing in human cells.

    Science.gov (United States)

    Tu, Mengjun; Lin, Li; Cheng, Yilu; He, Xiubin; Sun, Huihui; Xie, Haihua; Fu, Junhao; Liu, Changbao; Li, Jin; Chen, Ding; Xi, Haitao; Xue, Dongyu; Liu, Qi; Zhao, Junzhao; Gao, Caixia; Song, Zongming; Qu, Jia; Gu, Feng

    2017-11-02

    Cpf1 nucleases were recently reported to be highly specific and programmable nucleases with efficiencies comparable to those of SpCas9. AsCpf1 and LbCpf1 require a single crRNA and recognize a 5'-TTTN-3' protospacer adjacent motif (PAM) at the 5' end of the protospacer for genome editing. For widespread application in precision site-specific human genome editing, the range of sequences that AsCpf1 and LbCpf1 can recognize is limited due to the size of this PAM. To address this limitation, we sought to identify a novel Cpf1 nuclease with simpler PAM requirements. Specifically, here we sought to test and engineer FnCpf1, one reported Cpf1 nuclease (FnCpf1) only requires 5'-TTN-3' as a PAM but does not exhibit detectable levels of nuclease-induced indels at certain locus in human cells. Surprisingly, we found that FnCpf1 possesses DNA cleavage activity in human cells at multiple loci. We also comprehensively and quantitatively examined various FnCpf1 parameters in human cells, including spacer sequence, direct repeat sequence and the PAM sequence. Our study identifies FnCpf1 as a new member of the Cpf1 family for human genome editing with distinctive characteristics, which shows promise as a genome editing tool with the potential for both research and therapeutic applications. © The Author(s) 2017. Published by Oxford University Press on behalf of Nucleic Acids Research.

  3. Oxidative stress, caspase-3 activation and cleavage of ROCK-1 play an essential role in MeHg-induced cell death in primary astroglial cells.

    Science.gov (United States)

    Dos Santos, Alessandra Antunes; López-Granero, Caridad; Farina, Marcelo; Rocha, João B T; Bowman, Aaron B; Aschner, Michael

    2018-03-01

    Methylmercury is a toxic environmental contaminant that elicits significant toxicity in humans. The central nervous system is the primary target of toxicity, and is particularly vulnerable during development. Rho-associated protein kinase 1 (ROCK-1) is a major downstream effector of the small GTPase RhoA and a direct substrate of caspase-3. The activation of ROCK-1 is necessary for membrane blebbing during apoptosis. In this work, we examined whether MeHg could affect the RhoA/ROCK-1 signaling pathway in primary cultures of mouse astrocytes. Exposure of cells with 10 μM MeHg decreased cellular viability after 24 h of incubation. This reduction in viability was preceded by a significant increase in intracellular and mitochondrial reactive oxygen species levels, as well as a reduced NAD + /NADH ratio. MeHg also induced an increase in mitochondrial-dependent caspase-9 and caspase-3, while the levels of RhoA protein expression were reduced or unchanged. We further found that MeHg induced ROCK-1 cleavage/activation and promoted LIMK1 and MYPT1 phosphorylation, both of which are the best characterized ROCK-1 downstream targets. Inhibiting ROCK-1 and caspases activation attenuated the MeHg-induced cell death. Collectively, these findings are the first to show that astrocytes exposed to MeHg showed increased cleavage/activation of ROCK-1, which was independent of the small GTPase RhoA. Copyright © 2018. Published by Elsevier Ltd.

  4. 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)

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

    DEFF Research Database (Denmark)

    Gullberg, Maria; Polacek, Charlotta; Belsham, Graham

    2014-01-01

    The foot-and-mouth disease virus (FMDV) capsid protein precursor P1-2A is cleaved by the virus-encoded 3C protease to VP0, VP3, VP1 and 2A. It was shown previously that modification of a single amino acid residue (K210E) within the VP1 protein and close to the VP1/2A cleavage site, inhibited...... cleavage of this junction and produced 'self-tagged' virus particles. A second site substitution (E83K) within VP1 was also observed within the rescued virus [Gullberg et al. (2013). J Virol 87: , 11591-11603]. It was shown here that introduction of this E83K change alone into a serotype O virus resulted...... in the rapid accumulation of a second site substitution within the 2A sequence (L2P), which also blocked VP1/2A cleavage. This suggests a linkage between the E83K change in VP1 and cleavage of the VP1/2A junction. Cells infected with viruses containing the VP1 K210E or the 2A L2P substitutions contained...

  6. 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.

  7. SaCas9 Requires 5'-NNGRRT-3' PAM for Sufficient Cleavage and Possesses Higher Cleavage Activity than SpCas9 or FnCpf1 in Human Cells.

    Science.gov (United States)

    Xie, Haihua; Tang, Lianchao; He, Xiubin; Liu, Xiexie; Zhou, Chenchen; Liu, Junjie; Ge, Xianglian; Li, Jin; Liu, Changbao; Zhao, Junzhao; Qu, Jia; Song, Zongming; Gu, Feng

    2018-04-01

    CRISPR/Cas9-mediated gene therapy holds great promise for the treatment of human diseases. The protospacer adjacent motif (PAM), the sequence adjacent to the target sequence, is an essential targeting component for the design of CRISPR/Cas9-mediated gene editing. However, currently, very few studies have attempted to directly study the PAM sequence in human cells. To address this issue, the authors develop a dual fluorescence reporter system that could be harnessed for identifying functional PAMs for genome editing endonuclease, including Cas9. With this system, the authors investigate the effects of different PAM sequences for SaCas9, which is small and has the advantage of allowing in vivo genome editing, and found only 5'-NNGRRT-3' PAM could induced sufficient target cleavage with multi-sites. The authors also found SaCas9 possesses higher activity than SpCas9 or FnCpf1 via plasmids (episomal) and chromosomes with integrated eGFP-based comparison. Taken together, the authors show that a dual fluorescence reporter system is a means to identifying a functional PAM and quantitatively comparing the efficiency of different genome editing endonucleases with the similar or identical target sequence in human cells. © 2017 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  8. Individual expression of poliovirus 2Apro and 3Cpro induces activation of caspase-3 and PARP cleavage in HeLa cells.

    Science.gov (United States)

    Calandria, Carlos; Irurzun, Alicia; Barco, Angel; Carrasco, Luis

    2004-08-01

    The expression of individual viral genes enables the study of their effects on cellular functions. Our group previously generated stable HeLa cell lines that efficiently express poliovirus proteases 2A (clone 2A7d) and 3C (clone 3C7) under the control of tetracycline [Virology 266 (2000a) 352; J. Virol. 74 (2000b) 2383]. Upon induction of these proteases, the cells undergo drastic morphological alterations and eventually die. The present paper characterizes, in detail, the cellular and molecular events that lead to cell death in these lines. Several signs of apoptosis were observed in both 2A7d- and 3C7-induced cells, such as nuclear fragmentation, DNA breakdown (as determined by TUNEL), and phosphatidylserine translocation. Protease 2A induces the cleavage of poly-ADP-ribose-polymerase (PARP). This is blocked by the caspase-3 inhibitor DEVD in both 2A7d-On and 3C7-On cells suggesting that this enzyme might account for PARP cleavage in both cell lines. The results indicate that both poliovirus proteases induce apoptosis by mechanisms involving caspase activation, although the kinetics of apoptosis differs.

  9. Fibrinogen cleavage products and Toll-like receptor 4 promote the generation of programmed cell death 1 ligand 2-positive dendritic cells in allergic asthma.

    Science.gov (United States)

    Cho, Minkyoung; Lee, Jeong-Eun; Lim, Hoyong; Shin, Hyun-Woo; Khalmuratova, Roza; Choi, Garam; Kim, Hyuk Soon; Choi, Wahn Soo; Park, Young-Jun; Shim, Inbo; Kim, Byung-Seok; Kang, Chang-Yuil; Kim, Jae-Ouk; Tanaka, Shinya; Kubo, Masato; Chung, Yeonseok

    2017-10-14

    Inhaled protease allergens preferentially trigger T H 2-mediated inflammation in allergic asthma. The role of dendritic cells (DCs) on induction of T H 2 cell responses in allergic asthma has been well documented; however, the mechanism by which protease allergens induce T H 2-favorable DCs in the airway remains unclear. We sought to determine a subset of DCs responsible for T H 2 cell responses in allergic asthma and the mechanism by which protease allergens induce the DC subset in the airway. Mice were challenged intranasally with protease allergens or fibrinogen cleavage products (FCPs) to induce allergic airway inflammation. DCs isolated from mediastinal lymph nodes were analyzed for surface phenotype and T-cell stimulatory function. Anti-Thy1.2 and Mas-TRECK mice were used to deplete innate lymphoid cells and mast cells, respectively. Adoptive cell transfer, bone marrow DC culture, anti-IL-13, and Toll-like receptor (TLR) 4-deficient mice were used for further mechanistic studies. Protease allergens induced a remarkable accumulation of T H 2-favorable programmed cell death 1 ligand 2 (PD-L2) + DCs in mediastinal lymph nodes, which was significantly abolished in mice depleted of mast cells and, to a lesser extent, innate lymphoid cells. Mechanistically, FCPs generated by protease allergens triggered IL-13 production from wild-type mast cells but not from TLR4-deficient mast cells, which resulted in an increase in the number of PD-L2 + DCs. Intranasal administration of FCPs induced an increase in numbers of PD-L2 + DCs in the airway, which was significantly abolished in TLR4- and mast cell-deficient mice. Injection of IL-13 restored the PD-L2 + DC population in mice lacking mast cells. Our findings unveil the "protease-FCP-TLR4-mast cell-IL-13" axis as a molecular mechanism for generation of T H 2-favorable PD-L2 + DCs in allergic asthma and suggest that targeting the PD-L2 + DC pathway might be effective in suppressing allergic T-cell responses in the airway

  10. p75 Neurotrophin Receptor Cleavage by α- and γ-Secretases Is Required for Neurotrophin-mediated Proliferation of Brain Tumor-initiating Cells*

    Science.gov (United States)

    Forsyth, Peter A.; Krishna, Niveditha; Lawn, Samuel; Valadez, J. Gerardo; Qu, Xiaotao; Fenstermacher, David A.; Fournier, Michelle; Potthast, Lisa; Chinnaiyan, Prakash; Gibney, Geoffrey T.; Zeinieh, Michele; Barker, Philip A.; Carter, Bruce D.; Cooper, Michael K.; Kenchappa, Rajappa S.

    2014-01-01

    Malignant gliomas are highly invasive, proliferative, and resistant to treatment. Previously, we have shown that p75 neurotrophin receptor (p75NTR) is a novel mediator of invasion of human glioma cells. However, the role of p75NTR in glioma proliferation is unknown. Here we used brain tumor-initiating cells (BTICs) and show that BTICs express neurotrophin receptors (p75NTR, TrkA, TrkB, and TrkC) and their ligands (NGF, brain-derived neurotrophic factor, and neurotrophin 3) and secrete NGF. Down-regulation of p75NTR significantly decreased proliferation of BTICs. Conversely, exogenouous NGF stimulated BTIC proliferation through α- and γ-secretase-mediated p75NTR cleavage and release of its intracellular domain (ICD). In contrast, overexpression of the p75NTR ICD induced proliferation. Interestingly, inhibition of Trk signaling blocked NGF-stimulated BTIC proliferation and p75NTR cleavage, indicating a role of Trk in p75NTR signaling. Further, blocking p75NTR cleavage attenuated Akt activation in BTICs, suggesting role of Akt in p75NTR-mediated proliferation. We also found that p75NTR, α-secretases, and the four subunits of the γ-secretase enzyme were elevated in glioblastoma multiformes patients. Importantly, the ICD of p75NTR was commonly found in malignant glioma patient specimens, suggesting that the receptor is activated and cleaved in patient tumors. These results suggest that p75NTR proteolysis is required for BTIC proliferation and is a novel potential clinical target. PMID:24519935

  11. 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

  12. 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

    2016-07-01

    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, is 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 erythroblastosis oncogene B 2 (human epidermal growth factor receptor 2) receptor, 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 that 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 epidermal growth factor-treated SKBR3 cells and growth conditions can affect this colocalization. Moreover, Grb7 can affect Caspase3 cleavage of Hax1 isoform 1 in vitro, and Grb7 expression may slow 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. Copyright © 2016 John Wiley & Sons, Ltd. Copyright © 2016 John Wiley & Sons, Ltd.

  13. ADAM17-mediated shedding of FcγRIIIA on human NK cells: identification of the cleavage site and relationship with activation.

    Science.gov (United States)

    Lajoie, Laurie; Congy-Jolivet, Nicolas; Bolzec, Armelle; Gouilleux-Gruart, Valérie; Sicard, Elodie; Sung, Hsueh Cheng; Peiretti, Frank; Moreau, Thierry; Vié, Henri; Clémenceau, Béatrice; Thibault, Gilles

    2014-01-15

    FcγRIIIA/CD16A, the low-affinity receptor for the IgG Fc portion expressed on human CD56(dim) NK cells and involved in Ab-dependent cell cytotoxicity, is shed upon NK cell activation. We found that recombinant a disintegrin and metalloprotease (ADAM) 17 cleaved the ectodomain of FcγRIIIA/CD16A and a peptide for which the sequence encompasses aa 191-201 of the FcγRIIIA/CD16A stalk region but not ADAM10. MALDI-TOF analysis revealed that the peptide was cleaved between Ala(195) and Val(196) (i.e., 1 aa upstream of the expected position). This location of the cleavage site was confirmed by the finding that ADAM17 failed to cleave a peptide in which Ala and Val were reversed. ADAM17 was found to be expressed on NK cells, and stimulation with PMA or N-ethyl-maleimide resulted in the shedding of FcγRIIIA/CD16A and CD62L, a specific substrate of ADAM17. Selective inhibition of ADAM17 prevented the shedding of both molecules. Moreover, the shedding of FcγRIIIA/CD16A was strongly correlated with degranulation when a wide range of CD56(dim) NK cell activating receptors were stimulated, whereas both ADAM17-dependent shedding and internalization were involved in FcγRIIIA/CD16A downmodulation when the latter was engaged. Finally, the shedding of FcγRIIIA/CD16A was restricted to activated cells, suggesting that ADAM17 acts mainly, if not exclusively, in cis. Taken together, our results demonstrated for the first time, to our knowledge, at the molecular level that ADAM17 cleaves the stalk region of FcγRIIIA/CD16A and identified its cleavage site. The shedding of FcγRIIIA/CD16A was at least partially ADAM17 dependent, and it may be considered as a marker of FcγRIIIA/CD16A-independent NK cell activation highly correlated with degranulation.

  14. FADD cleavage by NK cell granzyme M enhances its self-association to facilitate procaspase-8 recruitment for auto-processing leading to caspase cascade.

    Science.gov (United States)

    Wang, S; Xia, P; Shi, L; Fan, Z

    2012-04-01

    Granzyme M (GzmM), an orphan Gzm, is constitutively and abundantly expressed in innate effector natural killer cells. We previously demonstrated that GzmM induces caspase (casp)-dependent apoptosis and cytochrome c release from mitochondria. We also resolved the crystal structure for GzmM and generated its specific inhibitor. However, how GzmM causes casp activation has not been defined. Here we found that casp-8 is an initiator caspase in GzmM-induced casp cascade, which causes other casp activation and Bid cleavage. GzmM does not directly cleave procaspase-3 and Bid, whose processing is casp dependent. Casp-8 knockdown or deficient cells attenuate or abolish GzmM-induced proteolysis of procaspase-3 and Bid. Extrinsic death receptor pathway adaptor Fas-associated protein with death domain (FADD) contributes to GzmM-induced casp-8 activation. GzmM specifically cleaves FADD after Met 196 to generate truncated FADD (tFADD) that enhances its self-association for oligomerization. The oligomerized tFADD facilitates procaspase-8 recruitment to promote its auto-processing leading to casp activation cascade. FADD-deficient cells abrogate GzmM-induced activation of casp-8 and apoptosis as well as significantly inhibit lymphokine-activated killer cell-mediated cytotoxicity. FADD processing by GzmM can potentiate killing efficacy against tumor cells and intracellular pathogens.

  15. A flow cytometry-based method for a high-throughput analysis of drug-stabilized topoisomerase II cleavage complexes in human cells.

    Science.gov (United States)

    de Campos-Nebel, Marcelo; Palmitelli, Micaela; González-Cid, Marcela

    2016-09-01

    Topoisomerase II (Top2) is an important target for anticancer therapy. A variety of drugs that poison Top2, including several epipodophyllotoxins, anthracyclines, and anthracenediones, are widely used in the clinic for both hematologic and solid tumors. The poisoning of Top2 involves the formation of a reaction intermediate Top2-DNA, termed Top2 cleavage complex (Top2cc), which is persistent in the presence of the drug and involves a 5' end of DNA covalently bound to a tyrosine from the enzyme through a phosphodiester group. Drug-induced Top2cc leads to Top2 linked-DNA breaks which are the major responsible for their cytotoxicity. While biochemical detection is very laborious, quantification of drug-induced Top2cc by immunofluorescence-based microscopy techniques is time consuming and requires extensive image segmentation for the analysis of a small population of cells. Here, we developed a flow cytometry-based method for the analysis of drug-induced Top2cc. This method allows a rapid analysis of a high number of cells in their cell cycle phase context. Moreover, it can be applied to almost any human cell type, including clinical samples. The methodology is useful for a high-throughput analysis of drugs that poison Top2, allowing not just the discrimination of the Top2 isoform that is targeted but also to track its removal. © 2016 International Society for Advancement of Cytometry. © 2016 International Society for Advancement of Cytometry.

  16. PS1/γ-Secretase-Mediated Cadherin Cleavage Induces β-Catenin Nuclear Translocation and Osteogenic Differentiation of Human Bone Marrow Stromal Cells

    Science.gov (United States)

    Dias, Rhayra B.; Fortuna-Costa, Anneliese; Chicaybam, Leonardo; Lopes, Daiana V.; Dutra, Hélio S.; Borojevic, Radovan; Bonamino, Martin; Mermelstein, Claudia

    2016-01-01

    Bone marrow stromal cells (BMSCs) are considered a promising tool for bone bioengineering. However, the mechanisms controlling osteoblastic commitment are still unclear. Osteogenic differentiation of BMSCs requires the activation of β-catenin signaling, classically known to be regulated by the canonical Wnt pathway. However, BMSCs treatment with canonical Wnts in vitro does not always result in osteogenic differentiation and evidence indicates that a more complex signaling pathway, involving cadherins, would be required to induce β-catenin signaling in these cells. Here we showed that Wnt3a alone did not induce TCF activation in BMSCs, maintaining the cells at a proliferative state. On the other hand, we verified that, upon BMSCs osteoinduction with dexamethasone, cadherins were cleaved by the PS1/γ-secretase complex at the plasma membrane, and this event was associated with an enhanced β-catenin translocation to the nucleus and signaling. When PS1/γ-secretase activity was inhibited, the osteogenic process was impaired. Altogether, we provide evidence that PS1/γ-secretase-mediated cadherin cleavage has as an important role in controlling β-catenin signaling during the onset of BMSCs osteogenic differentiation, as part of a complex signaling pathway responsible for cell fate decision. A comprehensive map of these pathways might contribute to the development of strategies to improve bone repair. PMID:28053606

  17. PS1/γ-Secretase-Mediated Cadherin Cleavage Induces β-Catenin Nuclear Translocation and Osteogenic Differentiation of Human Bone Marrow Stromal Cells

    Directory of Open Access Journals (Sweden)

    Danielle C. Bonfim

    2016-01-01

    Full Text Available Bone marrow stromal cells (BMSCs are considered a promising tool for bone bioengineering. However, the mechanisms controlling osteoblastic commitment are still unclear. Osteogenic differentiation of BMSCs requires the activation of β-catenin signaling, classically known to be regulated by the canonical Wnt pathway. However, BMSCs treatment with canonical Wnts in vitro does not always result in osteogenic differentiation and evidence indicates that a more complex signaling pathway, involving cadherins, would be required to induce β-catenin signaling in these cells. Here we showed that Wnt3a alone did not induce TCF activation in BMSCs, maintaining the cells at a proliferative state. On the other hand, we verified that, upon BMSCs osteoinduction with dexamethasone, cadherins were cleaved by the PS1/γ-secretase complex at the plasma membrane, and this event was associated with an enhanced β-catenin translocation to the nucleus and signaling. When PS1/γ-secretase activity was inhibited, the osteogenic process was impaired. Altogether, we provide evidence that PS1/γ-secretase-mediated cadherin cleavage has as an important role in controlling β-catenin signaling during the onset of BMSCs osteogenic differentiation, as part of a complex signaling pathway responsible for cell fate decision. A comprehensive map of these pathways might contribute to the development of strategies to improve bone repair.

  18. T-cell receptor downregulation by ceramide-induced caspase activation and cleavage of the zeta chain

    DEFF Research Database (Denmark)

    Menné, C; Lauritsen, Jens Peter Holst; Dietrich, J

    2001-01-01

    Regulation of T-cell receptor (TCR) cell surface expression levels is probably an important mechanism by which T-cell responsiveness is controlled. Previously, two distinct pathways for TCR downregulation have been described. One is dependent on protein kinase C (PKC) and the leucine-based recept...

  19. A ‘new lease of life’: FnCpf1 possesses DNA cleavage activity for genome editing in human cells

    Science.gov (United States)

    Tu, Mengjun; Lin, Li; Cheng, Yilu; He, Xiubin; Sun, Huihui; Xie, Haihua; Fu, Junhao; Liu, Changbao; Li, Jin; Chen, Ding; Xi, Haitao; Xue, Dongyu; Liu, Qi; Zhao, Junzhao; Gao, Caixia; Song, Zongming; Qu, Jia

    2017-01-01

    Abstract Cpf1 nucleases were recently reported to be highly specific and programmable nucleases with efficiencies comparable to those of SpCas9. AsCpf1 and LbCpf1 require a single crRNA and recognize a 5′-TTTN-3′ protospacer adjacent motif (PAM) at the 5′ end of the protospacer for genome editing. For widespread application in precision site-specific human genome editing, the range of sequences that AsCpf1 and LbCpf1 can recognize is limited due to the size of this PAM. To address this limitation, we sought to identify a novel Cpf1 nuclease with simpler PAM requirements. Specifically, here we sought to test and engineer FnCpf1, one reported Cpf1 nuclease (FnCpf1) only requires 5′-TTN-3′ as a PAM but does not exhibit detectable levels of nuclease-induced indels at certain locus in human cells. Surprisingly, we found that FnCpf1 possesses DNA cleavage activity in human cells at multiple loci. We also comprehensively and quantitatively examined various FnCpf1 parameters in human cells, including spacer sequence, direct repeat sequence and the PAM sequence. Our study identifies FnCpf1 as a new member of the Cpf1 family for human genome editing with distinctive characteristics, which shows promise as a genome editing tool with the potential for both research and therapeutic applications. PMID:28977650

  20. Endogenous proteolytic cleavage of disease-associated prion protein to produce C2 fragments is strongly cell- and tissue-dependent.

    Science.gov (United States)

    Dron, Michel; Moudjou, Mohammed; Chapuis, Jérôme; Salamat, Muhammad Khalid Farooq; Bernard, Julie; Cronier, Sabrina; Langevin, Christelle; Laude, Hubert

    2010-04-02

    The abnormally folded form of the prion protein (PrP(Sc)) accumulating in nervous and lymphoid tissues of prion-infected individuals can be naturally cleaved to generate a N-terminal-truncated fragment called C2. Information about the identity of the cellular proteases involved in this process and its possible role in prion biology has remained limited and controversial. We investigated PrP(Sc) N-terminal trimming in different cell lines and primary cultured nerve cells, and in the brain and spleen tissue from transgenic mice infected by ovine and mouse prions. We found the following: (i) the full-length to C2 ratio varies considerably depending on the infected cell or tissue. Thus, in primary neurons and brain tissue, PrP(Sc) accumulated predominantly as untrimmed species, whereas efficient trimming occurred in Rov and MovS cells, and in spleen tissue. (ii) Although C2 is generally considered to be the counterpart of the PrP(Sc) proteinase K-resistant core, the N termini of the fragments cleaved in vivo and in vitro can actually differ, as evidenced by a different reactivity toward the Pc248 anti-octarepeat antibody. (iii) In lysosome-impaired cells, the ratio of full-length versus C2 species dramatically increased, yet efficient prion propagation could occur. Moreover, cathepsin but not calpain inhibitors markedly inhibited C2 formation, and in vitro cleavage by cathepsins B and L produced PrP(Sc) fragments lacking the Pc248 epitope, strongly arguing for the primary involvement of acidic hydrolases of the endolysosomal compartment. These findings have implications on the molecular analysis of PrP(Sc) and cell pathogenesis of prion infection.

  1. Endogenous Proteolytic Cleavage of Disease-associated Prion Protein to Produce C2 Fragments Is Strongly Cell- and Tissue-dependent*

    Science.gov (United States)

    Dron, Michel; Moudjou, Mohammed; Chapuis, Jérôme; Salamat, Muhammad Khalid Farooq; Bernard, Julie; Cronier, Sabrina; Langevin, Christelle; Laude, Hubert

    2010-01-01

    The abnormally folded form of the prion protein (PrPSc) accumulating in nervous and lymphoid tissues of prion-infected individuals can be naturally cleaved to generate a N-terminal-truncated fragment called C2. Information about the identity of the cellular proteases involved in this process and its possible role in prion biology has remained limited and controversial. We investigated PrPSc N-terminal trimming in different cell lines and primary cultured nerve cells, and in the brain and spleen tissue from transgenic mice infected by ovine and mouse prions. We found the following: (i) the full-length to C2 ratio varies considerably depending on the infected cell or tissue. Thus, in primary neurons and brain tissue, PrPSc accumulated predominantly as untrimmed species, whereas efficient trimming occurred in Rov and MovS cells, and in spleen tissue. (ii) Although C2 is generally considered to be the counterpart of the PrPSc proteinase K-resistant core, the N termini of the fragments cleaved in vivo and in vitro can actually differ, as evidenced by a different reactivity toward the Pc248 anti-octarepeat antibody. (iii) In lysosome-impaired cells, the ratio of full-length versus C2 species dramatically increased, yet efficient prion propagation could occur. Moreover, cathepsin but not calpain inhibitors markedly inhibited C2 formation, and in vitro cleavage by cathepsins B and L produced PrPSc fragments lacking the Pc248 epitope, strongly arguing for the primary involvement of acidic hydrolases of the endolysosomal compartment. These findings have implications on the molecular analysis of PrPSc and cell pathogenesis of prion infection. PMID:20154089

  2. 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

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

    International Nuclear Information System (INIS)

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

    2010-01-01

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

  4. Proteolytic cleavage of vascular adhesion protein-1 induced by vascular endothelial growth factor in retinal capillary endothelial cells.

    Science.gov (United States)

    Yoshida, Shiho; Murata, Miyuki; Noda, Kousuke; Matsuda, Takashi; Saito, Michiyuki; Saito, Wataru; Kanda, Atsuhiro; Ishida, Susumu

    2018-02-01

    To investigate the mechanism of soluble vascular adhesion protein-1 (sVAP-1) accumulation induced by vascular endothelial growth factor (VEGF) in the vitreous of patients with diabetic retinopathy (DR). Experimental. Protein levels of sVAP-1 and N epsilon-(hexanoyl)lysine (HEL), an oxidative stress marker, in the vitreous samples from patients with proliferative diabetic retinopathy (PDR) with or without intravitreal bevacizumab (IVB) injection were determined by ELISA. The effect of VEGF on both mRNA expression of Vap-1 and secretion of sVAP-1 in rat retinal capillary endothelial cells (TR-iBRB2) was analyzed by real-time PCR and western blotting, respectively. In addition, the impact of VEGF on production and activation ratios of matrix metalloproteinase (MMP)-2 and MMP-9 was examined by gelatin zymography. Hydrogen peroxide production and reactive oxygen species (ROS) levels were assessed in the supernatants of TR-iBRB2 cells treated with VEGF. IVB injection decreased vitreous levels of sVAP-1 and HEL in patients with PDR. VEGF stimulation released sVAP-1 protein from TR-iBRB2 cells as a consequence of membrane-anchored VAP-1 shedding by MMP-2 and MMP-9. In addition, VEGF increased hydrogen peroxide generation and ROS augmentation through spermine oxidation by sVAP-1 as semicarbazide-sensitive amine oxidase (SSAO) in the supernatant of cultured endothelial cells. The current data demonstrate that proangiogenic factor VEGF induces sVAP-1 release from retinal capillary endothelial cells and facilitates hydrogen peroxide generation via enzymatic property of sVAP-1, followed by the increase of oxidative stress, one of the crucial factors in the pathogenesis of DR.

  5. 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

    2017-01-01

    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...... of alternative amino acid substitutions were made at this site and the properties of the mutant viruses determined. Only the presence of a positively charged residue at position P2 in the cleavage site permitted efficient cleavage of the VP1/2A junction, consistent with analyses of diverse FMDV genome sequences...... have implications for the testing of potential antiviral agents targeting the FMDV 3C protease....

  6. 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.

  7. The Polyadenylation Factor Subunit CLEAVAGE AND POLYADENYLATION SPECIFICITY FACTOR30: A Key Factor of Programmed Cell Death and a Regulator of Immunity in Arabidopsis1[W

    Science.gov (United States)

    Bruggeman, Quentin; Garmier, Marie; de Bont, Linda; Soubigou-Taconnat, Ludivine; Mazubert, Christelle; Benhamed, Moussa; Raynaud, Cécile; Bergounioux, Catherine; Delarue, Marianne

    2014-01-01

    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. PMID:24706550

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

    African Journals Online (AJOL)

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

  9. Direct Visualization of RNA-DNA Primer Removal from Okazaki Fragments Provides Support for Flap Cleavage and Exonucleolytic Pathways in Eukaryotic Cells.

    Science.gov (United States)

    Liu, Bochao; Hu, Jiazhi; Wang, Jingna; Kong, Daochun

    2017-03-24

    During DNA replication in eukaryotic cells, short single-stranded DNA segments known as Okazaki fragments are first synthesized on the lagging strand. The Okazaki fragments originate from ∼35-nucleotide-long RNA-DNA primers. After Okazaki fragment synthesis, these primers must be removed to allow fragment joining into a continuous lagging strand. To date, the models of enzymatic machinery that removes the RNA-DNA primers have come almost exclusively from biochemical reconstitution studies and some genetic interaction assays, and there is little direct evidence to confirm these models. One obstacle to elucidating Okazaki fragment processing has been the lack of methods that can directly examine primer removal in vivo In this study, we developed an electron microscopy assay that can visualize nucleotide flap structures on DNA replication forks in fission yeast ( Schizosaccharomyces pombe ). With this assay, we first demonstrated the generation of flap structures during Okazaki fragment processing in vivo The mean and median lengths of the flaps in wild-type cells were ∼51 and ∼41 nucleotides, respectively. We also used yeast mutants to investigate the impact of deleting key DNA replication nucleases on these flap structures. Our results provided direct in vivo evidence for a previously proposed flap cleavage pathway and the critical function of Dna2 and Fen1 in cleaving these flaps. In addition, we found evidence for another previously proposed exonucleolytic pathway involving RNA-DNA primer digestion by exonucleases RNase H2 and Exo1. Taken together, our observations suggest a dual mechanism for Okazaki fragment maturation in lagging strand synthesis and establish a new strategy for interrogation of this fascinating process. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

  10. Newcastle Disease Virus Establishes Persistent Infection in Tumor CellsIn Vitro: Contribution of the Cleavage Site of Fusion Protein and Second Sialic Acid Binding Site of Hemagglutinin-Neuraminidase.

    Science.gov (United States)

    Rangaswamy, Udaya S; Wang, Weijia; Cheng, Xing; McTamney, Patrick; Carroll, Danielle; Jin, Hong

    2017-08-15

    Newcastle disease virus (NDV) is an oncolytic virus being developed for the treatment of cancer. Following infection of a human ovarian cancer cell line (OVCAR3) with a recombinant low-pathogenic NDV, persistent infection was established in a subset of tumor cells. Persistently infected (PI) cells exhibited resistance to superinfection with NDV and established an antiviral state, as demonstrated by upregulation of interferon and interferon-induced genes such as myxoma resistance gene 1 (Mx1) and retinoic acid-inducing gene-I (RIG-I). Viruses released from PI cells induced higher cell-to-cell fusion than the parental virus following infection in two tumor cell lines tested, HT1080 and HeLa, and remained attenuated in chickens. Two mutations, one in the fusion (F) protein cleavage site, F117S (F 117S ), and another in hemagglutinin-neuraminidase (HN), G169R (HN 169R ), located in the second sialic acid binding region, were responsible for the hyperfusogenic phenotype. F 117S improves F protein cleavage efficiency, facilitating cell-to-cell fusion, while HN 169R possesses a multifaceted role in contributing to higher fusion, reduced receptor binding, and lower neuraminidase activity, which together result in increased fusion and reduced viral replication. Thus, establishment of persistent infection in vitro involves viral genetic changes that facilitate efficient viral spread from cell to cell as a potential mechanism to escape host antiviral responses. The results of our study also demonstrate a critical role in the viral life cycle for the second receptor binding region of the HN protein, which is conserved in several paramyxoviruses. IMPORTANCE Oncolytic Newcastle disease virus (NDV) could establish persistent infection in a tumor cell line, resulting in a steady antiviral state reflected by constitutively expressed interferon. Viruses isolated from persistently infected cells are highly fusogenic, and this phenotype has been mapped to two mutations, one each in

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

    International Nuclear Information System (INIS)

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

    1975-01-01

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

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

    DEFF Research Database (Denmark)

    Gullberg, Maria; Polacek, Charlotta; Belsham, Graham

    The foot-and-mouth disease virus (FMDV) capsid protein precursor P1-2A is cleaved by the 3C protease to produce VP0, VP3, VP1 and 2A. It was shown previously that modification of a single amino acid residue (K210) within the VP1 protein, close to the VP1/2A cleavage site, inhibited cleavage...... of this junction and resulted in the production of “self-tagged” virus particles containing the 2A peptide. A second site substitution (E83K) within VP1 was also observed within the rescued virus (Gullberg et al., 2013). It is now shown that introduction of this E83K change alone into a serotype O virus resulted...... in the rapid accumulation of a second site substitution within the 2A sequence (L2P) that also blocked VP1/2A cleavage suggesting a linkage between the E83K change in VP1 and cleavage of the VP1/2A junction. In a serotype A background, the K210E substitution in VP1 rapidly reverted to wild type. However...

  13. 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.

    Directory of Open Access Journals (Sweden)

    Takao Sasado

    Full Text Available 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.

  14. NuMA and nuclear lamins are cleaved during viral infection--inhibition of caspase activity prevents cleavage and rescues HeLa cells from measles virus-induced but not from rhinovirus 1B-induced cell death.

    Science.gov (United States)

    Taimen, Pekka; Berghäll, Heidi; Vainionpää, Raija; Kallajoki, Markku

    2004-03-01

    Nuclear matrix is a structural framework of important nuclear processes. We studied the effect of two different types of viral infections on nuclear matrix. HeLa cells were infected with human rhinovirus 1B (HRV 1B) or measles virus (MV), and Nuclear Mitotic Apparatus protein (NuMA) and lamins A/C and B were used as markers for internal nuclear matrix and peripheral nuclear lamina, respectively. We show that NuMA, lamins, and poly(ADP-ribose) polymerase-1 are cleaved during viral infection in a virus family-specific manner suggesting that these viruses activate different sets of proteases. Morphologically, NuMA was excluded from the condensed chromatin, lamins showed a folded distribution, and both proteins finally remained around the nuclear fragments. A general caspase inhibitor benzyloxycarbonyl-Val-Ala-Asp-fluoromethylketone (z-VAD-FMK) prevented the nuclear disintegration and the cleavage of the proteins studied. Interestingly, z-VAD-FMK rescued MV-infected but not HRV 1B-infected cells from cell death. These results show for the first time that NuMA and lamins are specific target proteins during virus-induced programmed cell death.

  15. NuMA and nuclear lamins are cleaved during viral infection - inhibition of caspase activity prevents cleavage and rescues HeLa cells from measles virus-induced but not from rhinovirus 1B-induced cell death

    International Nuclear Information System (INIS)

    Taimen, Pekka; Berghaell, Heidi; Vainionpaeae, Raija; Kallajoki, Markku

    2004-01-01

    Nuclear matrix is a structural framework of important nuclear processes. We studied the effect of two different types of viral infections on nuclear matrix. HeLa cells were infected with human rhinovirus 1B (HRV 1B) or measles virus (MV), and Nuclear Mitotic Apparatus protein (NuMA) and lamins A/C and B were used as markers for internal nuclear matrix and peripheral nuclear lamina, respectively. We show that NuMA, lamins, and poly(ADP-ribose) polymerase-1 are cleaved during viral infection in a virus family-specific manner suggesting that these viruses activate different sets of proteases. Morphologically, NuMA was excluded from the condensed chromatin, lamins showed a folded distribution, and both proteins finally remained around the nuclear fragments. A general caspase inhibitor benzyloxycarbonyl-Val-Ala-Asp-fluoromethylketone (z-VAD-FMK) prevented the nuclear disintegration and the cleavage of the proteins studied. Interestingly, z-VAD-FMK rescued MV-infected but not HRV 1B-infected cells from cell death. These results show for the first time that NuMA and lamins are specific target proteins during virus-induced programmed cell death

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

    Science.gov (United States)

    Meluzzi, Dario; Arya, Gaurav

    2016-01-08

    Hi-C experiments produce large numbers of DNA sequence read pairs that are typically analyzed to deduce genomewide interactions between arbitrary loci. A key step in these experiments is the cleavage of cross-linked chromatin with a restriction endonuclease. Although this cleavage should happen specifically at the enzyme's recognition sequence, an unknown proportion of cleavage events may involve other sequences, owing to the enzyme's star activity or to random DNA breakage. A quantitative estimation of these non-specific cleavages may enable simulating realistic Hi-C read pairs for validation of downstream analyses, monitoring the reproducibility of experimental conditions and investigating biophysical properties that correlate with DNA cleavage patterns. Here we describe a computational method for analyzing Hi-C read pairs to estimate the fractions of cleavages at different possible targets. The method relies on expressing an observed local target distribution downstream of aligned reads as a linear combination of known conditional local target distributions. We validated this method using Hi-C read pairs obtained by computer simulation. Application of the method to experimental Hi-C datasets from murine cells revealed interesting similarities and differences in patterns of cleavage across the various experiments considered. © The Author(s) 2015. Published by Oxford University Press on behalf of Nucleic Acids Research.

  17. 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.

  18. 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

  19. Mechanisms for ribotoxin-induced ribosomal RNA cleavage.

    Science.gov (United States)

    He, Kaiyu; Zhou, Hui-Ren; Pestka, James J

    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 (≥25ng/ml), the macrocylic trichothecene satratoxin G (SG) (≥10ng/ml) and ribosome-inactivating protein ricin (≥300ng/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. Copyright © 2012 Elsevier Inc. All rights reserved.

  20. 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.

  1. 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.

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

    International Nuclear Information System (INIS)

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

    2013-01-01

    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-Fe 3 O 4 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

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

    Directory of Open Access Journals (Sweden)

    Christian B Lessard

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

  4. Isololiolide, a carotenoid metabolite isolated from the brown alga Cystoseira tamariscifolia, is cytotoxic and able to induce apoptosis in hepatocarcinoma cells through caspase-3 activation, decreased Bcl-2 levels, increased p53 expression and PARP cleavage.

    Science.gov (United States)

    Vizetto-Duarte, Catarina; Custódio, Luísa; Gangadhar, Katkam N; Lago, João Henrique G; Dias, Catarina; Matos, Ana Marta; Neng, Nuno; Nogueira, José Manuel Florêncio; Barreira, Luísa; Albericio, Fernando; Rauter, Amelia P; Varela, João

    2016-05-15

    Brown macroalgae have attracted attention because they display a wide range of biological activities, including antitumoral properties. Inthis study we isolated isololiolide from Cystoseira tamariscifolia for the first time. To examine the therapeutical potential of isololiolide against tumor cell lines. The structure of the compound was established and confirmed by 1D and 2D NMR as well as HRMS spectral analysis. The in vitro cytotoxicity was analyzed by colorimetric 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay in tumoral as well as in non-tumoral cell lines. Cell cycle arrest and induction of apoptosis were assessed by flow cytometry. Alteration of expression levels in proteins important in the apoptotic cascade was analyzed by western blotting. Isololiolidewas isolated for the first time from the brown macroalga C.tamariscifolia. Isololiolide exhibited significant cytotoxic activity against three human tumoral cell lines, namely hepatocarcinoma HepG2 cells, whereas no cytotoxicity was found in non-malignant MRC-5 and HFF-1 human fibroblasts. Isololiolide completely disrupted the HepG2 normal cell cycle and induced significant apoptosis. Moreover, western blot analysis showed that isololiolide altered the expression of proteins that are important in the apoptotic cascade, increasing PARP cleavage and p53 expression while decreasing procaspase-3 and Bcl-2 levels. Isololiolide isolated from C. tamariscifolia is able to exert a selective cytotoxic activity on hepatocarcinoma HepG2 cells as well as induce apoptosis through the modulation of apoptosis-related proteins. Copyright © 2016 Elsevier GmbH. All rights reserved.

  5. A Secreted Protein Promotes Cleavage Furrow Maturation during Cytokinesis

    OpenAIRE

    Xu, Xuehong; Vogel, Bruce E.

    2011-01-01

    Developmental modifications in cell shape depend on dynamic interactions between the extracellular matrix and cytoskeleton. In contrast, existing models of cytokinesis describe substantial cell surface remodeling that involves many intracellular regulatory and structural proteins but includes no contribution from the extracellular matrix [1–3]. Here, we show that extracellular hemicentins assemble at the cleavage furrow of dividing cells in the C. elegans germline and in preimplantation mouse...

  6. SOS Induction by Stabilized Topoisomerase IA Cleavage Complex Occurs via the RecBCD Pathway▿ †

    OpenAIRE

    Sutherland, Jeanette H.; Cheng, Bokun; Liu, I-Fen; Tse-Dinh, Yuk-Ching

    2008-01-01

    Accumulation of mutant topoisomerase I cleavage complex can lead to SOS induction and cell death in Escherichia coli. The single-stranded break associated with mutant topoisomerase I cleavage complex is converted to double-stranded break, which then is processed by the RecBCD pathway, followed by association of RecA with the single-stranded DNA.

  7. VE-cadherin cleavage by LasB protease from Pseudomonas aeruginosa facilitates type III secretion system toxicity in endothelial cells.

    Directory of Open Access Journals (Sweden)

    Guillaume Golovkine

    2014-03-01

    Full Text Available Infection of the vascular system by Pseudomonas aeruginosa (Pa occurs during bacterial dissemination in the body or in blood-borne infections. Type 3 secretion system (T3SS toxins from Pa induce a massive retraction when injected into endothelial cells. Here, we addressed the role of type 2 secretion system (T2SS effectors in this process. Mutants with an inactive T2SS were much less effective than wild-type strains at inducing cell retraction. Furthermore, secretomes from wild-types were sufficient to trigger cell-cell junction opening when applied to cells, while T2SS-inactivated mutants had minimal activity. Intoxication was associated with decreased levels of vascular endothelial (VE-cadherin, a homophilic adhesive protein located at endothelial cell-cell junctions. During the process, the protein was cleaved in the middle of its extracellular domain (positions 335 and 349. VE-cadherin attrition was T3SS-independent but T2SS-dependent. Interestingly, the epithelial (E-cadherin was unaffected by T2SS effectors, indicating that this mechanism is specific to endothelial cells. We showed that one of the T2SS effectors, the protease LasB, directly affected VE-cadherin proteolysis, hence promoting cell-cell junction disruption. Furthermore, mouse infection with Pa to induce acute pneumonia lead to significant decreases in lung VE-cadherin levels, whereas the decrease was minimal with T2SS-inactivated or LasB-deleted mutant strains. We conclude that the T2SS plays a pivotal role during Pa infection of the vascular system by breaching the endothelial barrier, and propose a model in which the T2SS and the T3SS cooperate to intoxicate endothelial cells.

  8. Bortezomib-induced sensitization of malignant human glioma cells to vorinostat-induced apoptosis depends on reactive oxygen species production, mitochondrial dysfunction, Noxa upregulation, Mcl-1 cleavage, and DNA damage.

    Science.gov (United States)

    Premkumar, Daniel R; Jane, Esther P; Agostino, Naomi R; DiDomenico, Joseph D; Pollack, Ian F

    2013-02-01

    Glioblastomas are invasive tumors with poor prognosis despite current therapies. Histone deacetylase inhibitors (HDACIs) represent a class of agents that can modulate gene expression to reduce tumor growth, and we and others have noted some antiglioma activity from HDACIs, such as vorinostat, although insufficient to warrant use as monotherapy. We have recently demonstrated that proteasome inhibitors, such as bortezomib, dramatically sensitized highly resistant glioma cells to apoptosis induction, suggesting that proteasomal inhibition may be a promising combination strategy for glioma therapeutics. In this study, we examined whether bortezomib could enhance response to HDAC inhibition in glioma cells. Although primary cells from glioblastoma multiforme (GBM) patients and established glioma cell lines did not show significant induction of apoptosis with vorinostat treatment alone, the combination of vorinostat plus bortezomib significantly enhanced apoptosis. The enhanced efficacy was due to proapoptotic mitochondrial injury and increased generation of reactive oxygen species. Our results also revealed that combination of bortezomib with vorinostat enhanced apoptosis by increasing Mcl-1 cleavage, Noxa upregulation, Bak and Bax activation, and cytochrome c release. Further downregulation of Mcl-1 using shRNA enhanced cell killing by the bortezomib/vorinostat combination. Vorinostat induced a rapid and sustained phosphorylation of histone H2AX in primary GBM and T98G cells, and this effect was significantly enhanced by co-administration of bortezomib. Vorinostat/bortezomib combination also induced Rad51 downregulation, which plays an important role in the synergistic enhancement of DNA damage and apoptosis. The significantly enhanced antitumor activity that results from the combination of bortezomib and HDACIs offers promise as a novel treatment for glioma patients. Copyright © 2011 Wiley Periodicals, Inc.

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

    Science.gov (United States)

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

    1995-01-01

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

  10. 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.

  11. Cleavage of ST6Gal I by Radiation-Induced BACE1 Inhibits Golgi-Anchored ST6Gal I-Mediated Sialylation of Integrin β1 and Migration in Colon Cancer Cells

    International Nuclear Information System (INIS)

    Lee, Minyoung; Park, Jung-Jin; Ko, Young-Gyu; Lee, Yun-Sil

    2012-01-01

    Previously, we found that β-galactoside α2,6-sialyltransferase (ST6Gal I), an enzyme that adds sialic acids to N-linked oligosaccharides of glycoproteins and is frequently overexpressed in cancer cells, is up-regulated by ionizing radiation (IR) and cleaved to a form possessing catalytic activity comparable to that of the Golgi-localized enzyme. Moreover, this soluble form is secreted into the culture media. Induction of ST6Gal I significantly increased the migration of colon cancer cells via sialylation of integrin β1. Here, we further investigated the mechanisms underlying ST6Gal I cleavage, solubilization and release from cells, and addressed its functions, focusing primarily on cancer cell migration. We performed immunoblotting and lectin affinity assay to analyze the expression of ST6 Gal I and level of sialylated integrin β1. After ionizing radiation, migration of cells was measured by in vitro migration assay. α2, 6 sialylation level of cell surface was analyzed by flow cytometry. Cell culture media were concentrated and then analyzed for soluble ST6Gal I levels using an α2, 6 sialyltransferase sandwich ELISA. We found that ST6Gal I was cleaved by BACE1 (β-site amyloid precursor protein-cleaving enzyme), which was specifically overexpressed in response to IR. The soluble form of ST6Gal I, which also has sialyltransferase enzymatic activity, was cleaved from the Golgi membrane and then released into the culture media. Both non-cleaved and cleaved forms of ST6Gal I significantly increased colon cancer cell migration in a sialylation-dependent manner. The pro-migratory effect of the non-cleaved form of ST6Gal I was dependent on integrin β1 sialylation, whereas that of the cleaved form of ST6Gal I was not, suggesting that other intracellular sialylated molecules apart from cell surface molecules such as integrin β1 might be involved in mediating the pro-migratory effects of the soluble form of ST6Gal I. Moreover, production of soluble form ST6Gal I by

  12. Neuron-Derived ADAM10 Production Stimulates Peripheral Nerve Injury-Induced Neuropathic Pain by Cleavage of E-Cadherin in Satellite Glial Cells.

    Science.gov (United States)

    Li, Jian; Ouyang, Qing; Chen, Cheng-Wen; Chen, Qian-Bo; Li, Xiang-Nan; Xiang, Zheng-Hua; Yuan, Hong-Bin

    2017-09-01

    Increasing evidence suggests the potential involvement of metalloproteinase family proteins in the pathogenesis of neuropathic pain, although the underlying mechanisms remain elusive. Using the spinal nerve ligation model, we investigated whether ADAM10 proteins participate in pain regulation. By implementing invitro methods, we produced a purified culture of satellite glial cells to study the underlying mechanisms of ADAM10 in regulating neuropathic pain. Results showed that the ADAM10 protein was expressed in calcitonin gene-related peptide (CGRP)-containing neurons of the dorsal root ganglia, and expression was upregulated following spinal nerve ligation surgery invivo. Intrathecal administration of GI254023X, an ADAM10 selective inhibitor, to the rats one to three days after spinal nerve ligation surgery attenuated the spinal nerve ligation-induced mechanical allodynia and thermal hyperalgesia. Intrathecal injection of ADAM10 recombinant protein simulated pain behavior in normal rats to a similar extent as those treated by spinal nerve ligation surgery. These results raised a question about the relative contribution of ADAM10 in pain regulation. Further results showed that ADAM10 might act by cleaving E-cadherin, which is mainly expressed in satellite glial cells. GI254023X reversed spinal nerve ligation-induced downregulation of E-cadherin and activation of cyclooxygenase 2 after spinal nerve ligation. β-catenin, which creates a complex with E-cadherin in the membranes of satellite glial cells, was also downregulated by spinal nerve ligation surgery in satellite glial cells. Finally, knockdown expression of β-catenin by lentiviral infection in purified satellite glial cells increased expression of inducible nitric oxide synthase and cyclooxygenase 2. Our findings indicate that neuron-derived ADAM10 production stimulates peripheral nerve injury-induced neuropathic pain by cleaving E-cadherin in satellite glial cells. © 2017 American Academy of Pain Medicine

  13. 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.

  14. 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.

  15. N-CADHERIN PRODOMAIN CLEAVAGE REGULATES SYNAPSE FORMATION IN VIVO

    Science.gov (United States)

    Latefi, Nazlie S.; Pedraza, Liliana; Schohl, Anne; Li, Ziwei; Ruthazer, Edward S.

    2009-01-01

    Cadherins are initially synthesized bearing a prodomain that is thought to limit adhesion during early stages of biosynthesis. Functional cadherins lack this prodomain, raising the intriguing possibility that cells may utilize prodomain cleavage as a means to temporally or spatially regulate adhesion after delivery of cadherin to the cell surface. In support of this idea, immunostaining for the prodomain of zebrafish N-cadherin revealed enriched labeling at neuronal surfaces at the soma and along axonal processes. To determine whether post-translational cleavage of the prodomain affects synapse formation, we imaged Rohon-Beard cells in zebrafish embryos expressing GFP-tagged wild-type N-cadherin (NCAD-GFP) or a GFP-tagged N-cadherin mutant expressing an uncleavable prodomain (PRON-GFP) rendering it non-adhesive. NCAD-GFP accumulated at synaptic microdomains in a developmentally regulated manner, and its overexpression transiently accelerated synapse formation. PRON-GFP was much more diffusely distributed along the axon and its overexpression delayed synapse formation. Our results support the notion that N-cadherin serves to stabilize pre- to postsynaptic contacts early in synapse development and suggests that regulated cleavage of the N-cadherin prodomain may be a mechanism by which the kinetics of synaptogenesis are regulated. PMID:19365814

  16. Targets and intracellular signaling mechanisms for deoxynivalenol-induced ribosomal RNA cleavage.

    Science.gov (United States)

    He, Kaiyu; Zhou, Hui-Ren; Pestka, James J

    2012-06-01

    The trichothecene mycotoxin deoxynivalenol (DON), a known translational inhibitor, induces ribosomal RNA (rRNA) cleavage. Here, we characterized this process relative to (1) specific 18S and 28S ribosomal RNA cleavage sites and (2) identity of specific upstream signaling elements in this pathway. Capillary electrophoresis indicated that DON at concentrations as low as 200 ng/ml evoked selective rRNA cleavage after 6 h and that 1000 ng/ml caused cleavage within 2 h. Northern blot analysis revealed that DON exposure induced six rRNA cleavage fragments from 28S rRNA and five fragments from 18S rRNA. When selective kinase inhibitors were used to identify potential upstream signals, RNA-activated protein kinase (PKR), hematopoietic cell kinase (Hck), and p38 were found to be required for rRNA cleavage, whereas c-Jun N-terminal kinase and extracellular signal-regulated kinase were not. Furthermore, rRNA fragmentation was suppressed by the p53 inhibitors pifithrin-α and pifithrin-μ as well as the pan caspase inhibitor Z-VAD-FMK. Concurrent apoptosis was confirmed by acridine orange/ethidium bromide staining and flow cytometry. DON activated caspases 3, 8, and 9, thus suggesting the possible coinvolvement of both extrinsic and intrinsic apoptotic pathways in rRNA cleavage. Satratoxin G (SG), anisomycin, and ricin also induced specific rRNA cleavage profiles identical to those of DON, suggesting that ribotoxins might share a conserved rRNA cleavage mechanism. Taken together, DON-induced rRNA cleavage is likely to be closely linked to apoptosis activation and appears to involve the sequential activation of PKR/Hck →p38→p53→caspase 8/9→caspase 3.

  17. 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...... algorithms published so far were trained on data from in vitro digestion experiments with constitutive proteasomes. As a result, they did not take into account the characteristics of the structurally modified proteasomes-often called immunoproteasomes-found in cells stimulated by gamma-interferon under...

  18. Cleavages in Serbia and consolidation of democracy

    Directory of Open Access Journals (Sweden)

    Antonić Slobodan

    2007-01-01

    Full Text Available The article discusses the sociological obstacles for consolidation in Serbia after 2000. The author claim that the reason for slow consolidation lies squarely with the type of political cleavages that continue to dominate Serbian politics. Throughout Eastern Europe, symbolic conflicts relatively quickly gave way to distributional conflicts during the 1990s. Distributional conflicts typically result in compromise, which is why they are regarded as favorable to consolidation of democracy. Other type of dominant cleavages is ideological and symbolical. Ideological cleavages divide the body politics to those who were loyal to the previous regime and to those who support the current reformists, and symbolical cleavages are identity-based. The inability to remove the symbolical issues from the political agenda in seven years is what undermines the weak foundation for democracy in Serbia today. Due to the resistance of symbolical and ideological cleavages (patriots/- Europeans, old regime forces/reformers etc. rather than socio-economic cleavages, author defines the party system of Serbia as deeply polarized with the existence of anti-system parties. Deep polarization and the existence of the anti system parties is what undermines consolidation of democracy. The author shows that the existence of anti-system parties is precisely the reason why Serbia cannot get out of the spirit of electoral authoritarianism and why electoral democracy keeps failing to consolidate.

  19. SARS coronavirus nsp1 protein induces template-dependent endonucleolytic cleavage of mRNAs: viral mRNAs are resistant to nsp1-induced RNA cleavage.

    Directory of Open Access Journals (Sweden)

    Cheng Huang

    2011-12-01

    Full Text Available SARS coronavirus (SCoV nonstructural protein (nsp 1, a potent inhibitor of host gene expression, possesses a unique mode of action: it binds to 40S ribosomes to inactivate their translation functions and induces host mRNA degradation. Our previous study demonstrated that nsp1 induces RNA modification near the 5'-end of a reporter mRNA having a short 5' untranslated region and RNA cleavage in the encephalomyocarditis virus internal ribosome entry site (IRES region of a dicistronic RNA template, but not in those IRES elements from hepatitis C or cricket paralysis viruses. By using primarily cell-free, in vitro translation systems, the present study revealed that the nsp1 induced endonucleolytic RNA cleavage mainly near the 5' untranslated region of capped mRNA templates. Experiments using dicistronic mRNAs carrying different IRESes showed that nsp1 induced endonucleolytic RNA cleavage within the ribosome loading region of type I and type II picornavirus IRES elements, but not that of classical swine fever virus IRES, which is characterized as a hepatitis C virus-like IRES. The nsp1-induced RNA cleavage of template mRNAs exhibited no apparent preference for a specific nucleotide sequence at the RNA cleavage sites. Remarkably, SCoV mRNAs, which have a 5' cap structure and 3' poly A tail like those of typical host mRNAs, were not susceptible to nsp1-mediated RNA cleavage and importantly, the presence of the 5'-end leader sequence protected the SCoV mRNAs from nsp1-induced endonucleolytic RNA cleavage. The escape of viral mRNAs from nsp1-induced RNA cleavage may be an important strategy by which the virus circumvents the action of nsp1 leading to the efficient accumulation of viral mRNAs and viral proteins during infection.

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

    Science.gov (United States)

    Marincola, Gabriella; Wolz, Christiane

    2017-06-02

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

  1. 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.

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

    Czech Academy of Sciences Publication Activity Database

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

    2017-01-01

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

  3. Dataset of cocoa aspartic protease cleavage sites

    Directory of Open Access Journals (Sweden)

    Katharina Janek

    2016-09-01

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

  4. Toward the development of metal-based synthetic nucleases: DNA binding and oxidative DNA cleavage of a mixed copper(II) complex with N-(9H-purin-6-yl)benzenesulfonamide and 1,10-phenantroline. Antitumor activity in human Caco-2 cells and Jurkat T lymphocytes. Evaluation of p53 and Bcl-2 proteins in the apoptotic mechanism.

    Science.gov (United States)

    García-Giménez, José Luis; González-Alvarez, Marta; Liu-González, Malva; Macías, Benigno; Borrás, Joaquín; Alzuet, Gloria

    2009-06-01

    The complex [Cu(N9-ABS)(phen)2].3.6H2O, H2N9-ABS = N-(9H-purin-6-yl)benzenesulfonamide and phen = 1,10-phenanthroline, has been synthesized and then characterized with the aid of X-ray diffraction, analytical, and spectroscopic techniques. The geometry of Cu(II) is distorted square pyramidal with the equatorial positions occupied by three N atoms from two phenantroline molecules and one N atom from the adenine ring of the sulfonamide ligand. The interaction of the complex with DNA was studied by means of viscosity measurements and fluorescence spectroscopy. The results pointed to a classic intercalation of the complex between the DNA base pairs. The complex was found to be a very efficient agent of plasmid DNA cleavage in the presence of ascorbate. Both the kinetics and the mechanism of the cleavage reaction were studied. In addition, the cytotoxic properties of the complex were evaluated in human Jurkat T and Caco-2 cell lines. The cytotoxicity of the compound was higher than that of the reference ([Cu(phen)2]2+). The mechanism and type of cell death induced by the compound was determined by flow cytometry and Hoechst dye staining. The compound demonstrated a significant ability to induce cell death by apoptosis. The apoptosis induced by [Cu(N9-ABS)(phen)2].3.6H2O was associated with an increase in p53 protein levels while those of Bcl-2 were reduced.

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

    NARCIS (Netherlands)

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

    2001-01-01

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

  6. 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

  7. A prospective randomized comparison of early embryo cleavage kinetics between two media culture systems

    Science.gov (United States)

    Zhang, Huan; Zheng, Yi; Wu, Yonggen; Ye, Danna; Huang, Xuefeng

    2016-01-01

    Objective: To investigate whether early embryo cleavage kinetics were affected by type of culture media. Methods: In this prospective sibling-split study, 620 oocytes from 37 patients were randomly allocated into two groups: Cook group and Vitrolife group. Oocytes/embryos in Cook group, would be cultured with Cook sequential culture medium, while oocytes/embryos in Vitrolife group, would be cultured with Vitrolife sequential culture medium. Time-lapse imaging technology was used to calculate exact timing of early embryo cleavage events which included time to 2PN breakdown, cleavage to 2-, 3-, 4-, 5- cell and the time duration in the 2-,3-cell stage. Then these timing of early embryo cleavage events were compared between Cook group and Vitrolife group. Moreover, fertilization rate, cleavage rate, high quality embryo rate, usable blastocyst rate, pregnancy rate and implantation rate of these two groups were also analyzed. Results: The results showed there were no differences in all timing of early embryo cleavage events between the two groups. In addition, the two groups were similar in fertilization rate (Cook 71.0% vs. Vitrolife 71.3%, P>0.05), cleavage rate (Cook 98.1% vs. Vitrolife 98.2%, P>0.05), high quality embryo rate (Cook 52.1% vs. Vitrolife 52.7%, P>0.05), usable blastocyst rate (Cook 29.7% vs. Vitrolife 28.0%, P>0.05), pregnancy rate (Cook 46.7% VS. Vitrolife 50.0%, P>0.05) and implantation rate (Cook 30.3% VS. Vitrolife 29.0%, P>0.05). Conclusions: Morphokinetics used for embryo selection are not affected by the two different culture media. PMID:28083029

  8. Can laccases catalyze bond cleavage in lignin?

    DEFF Research Database (Denmark)

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

    2015-01-01

    Modification of lignin is recognized as an important aspect of the successful refining of lignocellulosic biomass, and enzyme-assisted processing and upcycling of lignin is receiving significant attention in the literature. Laccases (EC 1.103.2) are taking the centerstage of this attention, since...... these enzymes may help degrading lignin, using oxygen as the oxidant. Laccases can catalyze polymerization of lignin, but the question is whether and how laccases can directly catalyze modification of lignin via catalytic bond cleavage. Via a thorough review of the available literature and detailed...... illustrations of the putative laccase catalyzed reactions, including the possible reactions of the reactive radical intermediates taking place after the initial oxidation of the phenol-hydroxyl groups, we show that i) Laccase activity is able to catalyze bond cleavage in low molecular weight phenolic lignin...

  9. [Laparoscopic cleavage in splenic symptomatic cyst].

    Science.gov (United States)

    Fernández-López, Antonio-José; Candel-Arenas, Marifé; González-Valverde, Francisco-Miguel; Luján-Martínez, Delia; Medina-Manuel, Esther; Albarracín Marín-Blázquez, Antonio

    2017-12-01

    Splenic cysts are rare diseases that are diagnosed incidentally during imaging studies. When cysts are recognized, surgical treatment is recommended adapted to the particular case, depending on the size and location of the cyst and the age of the patient in order to avoid dangerous complications such as spleen rupture or cyst infection with abscess. We report 2patients with symptomatic splenic epidermoid cyst treated by laparoscopic cleavage. Laparoscopic cleavage is a surgical option for splenic cyst, with the goal of reducing postoperative complications while preserving splenic function. Copyright © 2016 Academia Mexicana de Cirugía A.C. Publicado por Masson Doyma México S.A. All rights reserved.

  10. Can laccases catalyze bond cleavage in lignin?

    Science.gov (United States)

    Munk, Line; Sitarz, Anna K; Kalyani, Dayanand C; Mikkelsen, J Dalgaard; Meyer, Anne S

    2015-01-01

    Modification of lignin is recognized as an important aspect of the successful refining of lignocellulosic biomass, and enzyme-assisted processing and upcycling of lignin is receiving significant attention in the literature. Laccases (EC 1.10.3.2) are taking the centerstage of this attention, since these enzymes may help degrading lignin, using oxygen as the oxidant. Laccases can catalyze polymerization of lignin, but the question is whether and how laccases can directly catalyze modification of lignin via catalytic bond cleavage. Via a thorough review of the available literature and detailed illustrations of the putative laccase catalyzed reactions, including the possible reactions of the reactive radical intermediates taking place after the initial oxidation of the phenol-hydroxyl groups, we show that i) Laccase activity is able to catalyze bond cleavage in low molecular weight phenolic lignin model compounds; ii) For laccases to catalyze inter-unit bond cleavage in lignin substrates, the presence of a mediator system is required. Clearly, the higher the redox potential of the laccase enzyme, the broader the range of substrates, including o- and p-diphenols, aminophenols, methoxy-substituted phenols, benzenethiols, polyphenols, and polyamines, which may be oxidized. In addition, the currently available analytical methods that can be used to detect enzyme catalyzed changes in lignin are summarized, and an improved nomenclature for unequivocal interpretation of the action of laccases on lignin is proposed. Copyright © 2015 Elsevier Inc. All rights reserved.

  11. Controllable laser thermal cleavage of sapphire wafers

    Science.gov (United States)

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

    2018-03-01

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

  12. Calpain cleavage prediction using multiple kernel learning.

    Directory of Open Access Journals (Sweden)

    David A DuVerle

    Full Text Available Calpain, an intracellular Ca²⁺-dependent cysteine protease, is known to play a role in a wide range of metabolic pathways through limited proteolysis of its substrates. However, only a limited number of these substrates are currently known, with the exact mechanism of substrate recognition and cleavage by calpain still largely unknown. While previous research has successfully applied standard machine-learning algorithms to accurately predict substrate cleavage by other similar types of proteases, their approach does not extend well to calpain, possibly due to its particular mode of proteolytic action and limited amount of experimental data. Through the use of Multiple Kernel Learning, a recent extension to the classic Support Vector Machine framework, we were able to train complex models based on rich, heterogeneous feature sets, leading to significantly improved prediction quality (6% over highest AUC score produced by state-of-the-art methods. In addition to producing a stronger machine-learning model for the prediction of calpain cleavage, we were able to highlight the importance and role of each feature of substrate sequences in defining specificity: primary sequence, secondary structure and solvent accessibility. Most notably, we showed there existed significant specificity differences across calpain sub-types, despite previous assumption to the contrary. Prediction accuracy was further successfully validated using, as an unbiased test set, mutated sequences of calpastatin (endogenous inhibitor of calpain modified to no longer block calpain's proteolytic action. An online implementation of our prediction tool is available at http://calpain.org.

  13. 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.

  14. 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.

  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. Copyright © 2015. Published by Elsevier Inc.

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

    Science.gov (United States)

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

    2016-01-01

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

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

    Science.gov (United States)

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

    2016-05-06

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

  18. 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

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

    Science.gov (United States)

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

    2003-11-14

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

  20. Regioselectivity in the Reductive Bond Cleavage of Diarylalkylsulfonium Salts

    DEFF Research Database (Denmark)

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

    2009-01-01

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

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

    Directory of Open Access Journals (Sweden)

    Chenyu Zhang

    2009-05-01

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

  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. 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)

  5. 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.

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

    Directory of Open Access Journals (Sweden)

    Olivier Barré

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

  7. The Restriction Endonuclease Cleavage Map of Rat Liver Mitochondrial DNA

    NARCIS (Netherlands)

    Bakker, H.; Holtrop, M.; Terpstra, P.

    1977-01-01

    Mitochondrial DNA from rat liver contains six sites for cleavage by the restriction endonucleases Hind III and EcoRI. A large stretch of DNA, comprising about 40% of the mitochondrial genome is not cleaved by either of the enzymes; eight cleavage sites are located on a DNA stretch of 35% of the

  8. 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.

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

    International Nuclear Information System (INIS)

    Weber, Nicholas D.; Aubert, Martine; Dang, Chung H.; Stone, Daniel; Jerome, Keith R.

    2014-01-01

    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

  10. The DNA sequence specificity of bleomycin cleavage in a systematically altered DNA sequence.

    Science.gov (United States)

    Gautam, Shweta D; Chen, Jon K; Murray, Vincent

    2017-08-01

    Bleomycin is an anti-tumour agent that is clinically used to treat several types of cancers. Bleomycin cleaves DNA at specific DNA sequences and recent genome-wide DNA sequencing specificity data indicated that the sequence 5'-RTGT*AY (where T* is the site of bleomycin cleavage, R is G/A and Y is T/C) is preferentially cleaved by bleomycin in human cells. Based on this DNA sequence, we constructed a plasmid clone to explore this bleomycin cleavage preference. By systematic variation of single nucleotides in the 5'-RTGT*AY sequence, we were able to investigate the effect of nucleotide changes on bleomycin cleavage efficiency. We observed that the preferred consensus DNA sequence for bleomycin cleavage in the plasmid clone was 5'-YYGT*AW (where W is A/T). The most highly cleaved sequence was 5'-TCGT*AT and, in fact, the seven most highly cleaved sequences conformed to the consensus sequence 5'-YYGT*AW. A comparison with genome-wide results was also performed and while the core sequence was similar in both environments, the surrounding nucleotides were different.

  11. 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. © 2016 by The American Society for Biochemistry and Molecular Biology, Inc.

  12. 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

    2016-01-01

    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. PMID:27317400

  13. Measurement of the cleavage energy of graphite.

    Science.gov (United States)

    Wang, Wen; Dai, Shuyang; Li, Xide; Yang, Jiarui; Srolovitz, David J; Zheng, Quanshui

    2015-08-28

    The basal plane cleavage energy (CE) of graphite is a key material parameter for understanding many of the unusual properties of graphite, graphene and carbon nanotubes. Nonetheless, a wide range of values for the CE has been reported and no consensus has yet emerged. Here we report the first direct, accurate experimental measurement of the CE of graphite using a novel method based on the self-retraction phenomenon in graphite. The measured value, 0.37±0.01 J m(-2) for the incommensurate state of bicrystal graphite, is nearly invariant with respect to temperature (22 °C≤T≤198 °C) and bicrystal twist angle, and insensitive to impurities from the atmosphere. The CE for the ideal ABAB graphite stacking, 0.39±0.02 J m(-2), is calculated based on a combination of the measured CE and a theoretical calculation. These experimental measurements are also ideal for use in evaluating the efficacy of competing theoretical approaches.

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

    International Nuclear Information System (INIS)

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

    2007-01-01

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

  15. 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.

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

    International Nuclear Information System (INIS)

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

    2014-01-01

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

  17. A combinatorial approach to identify calpain cleavage sites in the Machado-Joseph disease protein ataxin-3.

    Science.gov (United States)

    Weber, Jonasz J; Golla, Matthias; Guaitoli, Giambattista; Wanichawan, Pimthanya; Hayer, Stefanie N; Hauser, Stefan; Krahl, Ann-Christin; Nagel, Maike; Samer, Sebastian; Aronica, Eleonora; Carlson, Cathrine R; Schöls, Ludger; Riess, Olaf; Gloeckner, Christian J; Nguyen, Huu P; Hübener-Schmid, Jeannette

    2017-05-01

    Ataxin-3, the disease protein in Machado-Joseph disease, is known to be proteolytically modified by various enzymes including two major families of proteases, caspases and calpains. This processing results in the generation of toxic fragments of the polyglutamine-expanded protein. Although various approaches were undertaken to identify cleavage sites within ataxin-3 and to evaluate the impact of fragments on the molecular pathogenesis of Machado-Joseph disease, calpain-mediated cleavage of the disease protein and the localization of cleavage sites remained unclear. Here, we report on the first precise localization of calpain cleavage sites in ataxin-3 and on the characterization of the resulting breakdown products. After confirming the occurrence of calpain-derived fragmentation of ataxin-3 in patient-derived cell lines and post-mortem brain tissue, we combined in silico prediction tools, western blot analysis, mass spectrometry, and peptide overlay assays to identify calpain cleavage sites. We found that ataxin-3 is primarily cleaved at two sites, namely at amino acid positions D208 and S256 and mutating amino acids at both cleavage sites to tryptophan nearly abolished ataxin-3 fragmentation. Furthermore, analysis of calpain cleavage-derived fragments showed distinct aggregation propensities and toxicities of C-terminal polyglutamine-containing breakdown products. Our data elucidate the important role of ataxin-3 proteolysis in the pathogenesis of Machado-Joseph disease and further emphasize the relevance of targeting this disease pathway as a treatment strategy in neurodegenerative disorders. © The Author (2017). Published by Oxford University Press on behalf of the Guarantors of Brain. All rights reserved. For Permissions, please email: journals.permissions@oup.com.

  18. 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

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

    Science.gov (United States)

    Schiffer, Joshua T; Swan, Dave A; Stone, Daniel; Jerome, Keith R

    2013-01-01

    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 will in turn

  20. Inclusion Complex of Zerumbone with Hydroxypropyl-β-Cyclodextrin Induces Apoptosis in Liver Hepatocellular HepG2 Cells via Caspase 8/BID Cleavage Switch and Modulating Bcl2/Bax Ratio

    Directory of Open Access Journals (Sweden)

    Nabilah Muhammad Nadzri

    2013-01-01

    Full Text Available Zerumbone (ZER isolated from Zingiber zerumbet was previously encapsulated with hydroxypropyl-β-cyclodextrin (HPβCD to enhance ZER’s solubility in water, thus making it highly tolerable in the human body. The anticancer effects of this new ZER-HPβCD inclusion complex via apoptosis cell death were assessed in this study for the first time in liver hepatocellular cells, HepG2. Apoptosis was ascertained by morphological study, nuclear stain, and sub-G1 cell population accumulation with G2/M arrest. Further investigations showed the release of cytochrome c and loss of mitochondrial membrane potential, proving mitochondrial dysfunction upon the ZER-HPβCD treatment as well as modulating proapoptotic and anti-apototic Bcl-2 family members. A significant increase in caspase 3/7, caspase 9, and caspase 8 was detected with the depletion of BID cleaved by caspase 8. Collectively, these results prove that a highly soluble inclusion complex of ZER-HPβCD could be a promising anticancer agent for the treatment of hepatocellular carcinoma in humans.

  1. Cytotoxic, DNA binding, DNA cleavage and antibacterial studies of ...

    Indian Academy of Sciences (India)

    fluoroquinolone complexes. Mohan N ... DNA-binding properties of Ru complexes have been studied by means of absorption spectrophotometry and viscosity measurements as well as their HS DNA cleavage properties by means of agarose gel ...

  2. Detection of nucleic acids by multiple sequential invasive cleavages

    Energy Technology Data Exchange (ETDEWEB)

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

    2012-10-16

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

  3. Structural and functional basis for RNA cleavage by Ire1

    Directory of Open Access Journals (Sweden)

    Stroud Robert M

    2011-07-01

    Full Text Available Abstract Background The unfolded protein response (UPR controls the protein folding capacity of the endoplasmic reticulum (ER. Central to this signaling pathway is the ER-resident bifunctional transmembrane kinase/endoribonuclease Ire1. The endoribonuclease (RNase domain of Ire1 initiates a non-conventional mRNA splicing reaction, leading to the production of a transcription factor that controls UPR target genes. The mRNA splicing reaction is an obligatory step of Ire1 signaling, yet its mechanism has remained poorly understood due to the absence of substrate-bound crystal structures of Ire1, the lack of structural similarity between Ire1 and other RNases, and a scarcity of quantitative enzymological data. Here, we experimentally define the active site of Ire1 RNase and quantitatively evaluate the contribution of the key active site residues to catalysis. Results This analysis and two new crystal structures suggest that Ire1 RNase uses histidine H1061 and tyrosine Y1043 as the general acid-general base pair contributing ≥ 7.6 kcal/mol and 1.4 kcal/mol to transition state stabilization, respectively, and asparagine N1057 and arginine R1056 for coordination of the scissile phosphate. Investigation of the stem-loop recognition revealed that additionally to the stem-loops derived from the classic Ire1 substrates HAC1 and Xbp1 mRNA, Ire1 can site-specifically and rapidly cleave anticodon stem-loop (ASL of unmodified tRNAPhe, extending known substrate specificity of Ire1 RNase. Conclusions Our data define the catalytic center of Ire1 RNase and suggest a mechanism of RNA cleavage: each RNase monomer apparently contains a separate catalytic apparatus for RNA cleavage, whereas two RNase subunits contribute to RNA stem-loop docking. Conservation of the key residues among Ire1 homologues suggests that the mechanism elucidated here for yeast Ire1 applies to Ire1 in metazoan cells, and to the only known Ire1 homologue RNase L.

  4. 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.

  5. Four new dinuclear Cu(ii) hydrazone complexes using various organic spacers: syntheses, crystal structures, DNA binding and cleavage studies and selective cell inhibitory effect towards leukemic and normal lymphocytes.

    Science.gov (United States)

    Banerjee, Sambuddha; Mondal, Susmita; Sen, Soma; Das, Saurabh; Hughes, David L; Rizzoli, Corrado; Desplanches, Cédric; Mandal, Chitra; Mitra, Samiran

    2009-09-14

    Syntheses and crystal structures of four new hydrazone-based Cu(ii) complexes, [{Cu(L(1))(H(2)O)}(2)(mu-pyraz)](ClO(4))(2) (), [{Cu(L(1))(OClO(3))}(2)(mu-4,4'-bipy)] (), [{Cu(L(2)H)}(mu-pyraz){Cu(L(2)H)(OClO(3))}].(ClO(4)) () and [{Cu(L(2))}(2)(mu-bpe)] () [L(1)H = condensation product of benzhydrazide and pyridine-2-carbaldehyde and L(2)H(2) = condensation product of benzoyl acetone and benzhydrazide], bridged by various organic spacers [pyrazine (pyraz), 4,4'-bipyridine (4,4'-bipy) and 1,2-di(4-pyridyl)ethane (bpe)] are reported in this paper. The single-crystal X-ray crystallographic studies reveal that all are dinuclear units where and form strong intermolecular H-bonding to form sheets of interconnected ions, whereas forms sheets of dinuclear chains through pi-pi interactions; in , molecules are linked only through van der Waals interactions. The variable-temperature magnetic moment studies reveal that and show antiferromagnetic coupling between the Cu(ii) centers at lower temperatures. The binding ability of with calf thymus DNA [CT-DNA] is reported using various spectroscopic studies (UV-Vis titration, circular dichroism and fluorescence). The binding constants of with CT-DNA, as calculated by different methodologies, are of the order of 10(5) M(-1). The mode of interaction between and CT-DNA has been predicted using circular dichroic (CD) spectroscopy, where it has been shown that most probably interacts with DNA via intercalation between the base pairs leading to a change in B-DNA conformation. is also able to cleave supercoiled (SC) plasmid DNA pUC19 in a time and dose dependent manner as demonstrated by agarose gel electrophoresis, and also demonstrates its potential to cleave the SC plasmid DNA via both oxidative and hydrolytic mechanisms. Approximately 50% of leukemic cells are found to be dead when two representative leukemic cell lines are exposed to ( approximately 80 muM) even for 24 h as determined by different cell cytotoxicity assays

  6. DNA binding, photo-induced DNA cleavage and cytotoxicity studies of lomefloxacin and its transition metal complexes

    Science.gov (United States)

    Ragheb, Mohamed A.; Eldesouki, Mohamed A.; Mohamed, Mervat S.

    2015-03-01

    This work was focused on a study of the DNA binding and cleavage properties of lomefloxacin (LMF) and its ternary transition metal complexes with glycine. The nature of the binding interactions between compounds and calf thymus DNA (CT-DNA) was studied by electronic absorption spectra, fluorescence spectra and thermal denaturation experiments. The obtained results revealed that LMF and its complexes could interact with CT-DNA via partial/moderate intercalative mode. Furthermore, the DNA cleavage activities of the compounds were investigated by gel electrophoresis. Mechanistic studies of DNA cleavage suggest that singlet oxygen (1O2) is likely to be the cleaving agent via an oxidative pathway, except for Cu(II) complex which proceeds via both oxidative and hydrolytic pathways. Antimicrobial and antitumor activities of the compounds were also studied against some kinds of bacteria, fungi and human cell lines.

  7. Synthesis and crystal structure determination of copper(II)-complex: In vitro DNA and HSA binding, pBR322 plasmid cleavage, cell imaging and cytotoxic studies.

    Science.gov (United States)

    Tabassum, Sartaj; Zaki, Mehvash; Ahmad, Musheer; Afzal, Mohd; Srivastav, Saurabh; Srikrishna, Saripella; Arjmand, Farukh

    2014-08-18

    New Cu(II) complex 1 of indole-3-propionic acid and 1,10-phenanthroline was synthesized and characterized by analytical, spectroscopic and single crystal X-ray diffraction. In vitro DNA binding studies of 1 was performed by employing UV-vis and fluorescence spectroscopic techniques. The binding affinity towards human serum albumin (HSA) was also investigated to understand the carrier role in body system, as the time dependent HPLC experiment of 1 revealed that bonded drug with protein releases slowly in presence of DNA. Complex 1 exhibited good anti-tumor activity (GI50 values <10 μg/ml), and to elucidate the mechanism of tumor inhibition, topoisomerase I enzymatic activity was carried out and further validated by cell imaging studies which clearly showed its nuclear localization. Copyright © 2014 Elsevier Masson SAS. All rights reserved.

  8. Hydrogen peroxide-mediated necrosis induction in HUVECs is associated with an atypical pattern of caspase-3 cleavage.

    Science.gov (United States)

    Csordas, Adam; Wick, Georg; Bernhard, David

    2006-06-10

    Oxidative stress, continuously exerted during chronic inflammation, has been implicated as a major causative agent of cellular dysfunction and cell death. In the present study, we investigated the impact of oxidative stress on the mode of cell death in HUVECs using H2O2 as a model reagent. We found that the predominant form of cell death was necrosis. Necrosis induction was accompanied by a distinct mode of caspase-3 cleavage, yielding a 29-kDa fragment. While inhibition of caspases could not prevent the generation of the 29-kDa fragment, general protease inhibitors, such as leupeptin and LLNL, proved to be effective in inhibiting the distinct processing pattern of caspase-3. These results suggest that caspases can act as substrates for non-caspase proteases in cells primed for necrosis induction. Thus, the pattern of caspase-3 cleavage might reflect the proteolytic system engaged in the cell death machinery in HUVECs.

  9. Timing of first embryonic cleavage is a positive indicator of the in vitro developmental potential of porcine embryos derived from in vitro fertilization, somatic cell nuclear transfer and parthenogenesis.

    Science.gov (United States)

    Isom, S Clay; Li, Rong Feng; Whitworth, Kristin M; Prather, Randall S

    2012-03-01

    Evidence in many species has suggested that those embryos that cleave earliest after fertilization are more developmentally competent than those that cleave relatively later after fertilization. Herein we document this phenomenon in porcine in vitro-fertilized (IVF), somatic cell nuclear transfer (SCNT), and parthenogenetic (PA) embryos. In vitro-matured pig oocytes were used to generate IVF, SCNT, and PA embryos. At 24 hr post-activation (or insemination; hpa/hpi), embryos were visually assessed, and cleaved embryos were moved into a new culture well. This process was repeated at 30 and 48 hpa/hpi. All embryos were allowed to develop 7 days in culture. For IVF embryos, 39.9%, 24.6%, and 10.5% of fast-, intermediate-, or slow-cleaving embryos, respectively, developed into blastocysts by day 7. For SCNT embryos, 31.8% of fast-, 5.7% of intermediate-, and 2.9% of late-cleaving embryos achieved the blastocyst stage of development. For PA embryos, the percentages of those cleaved embryos that developed to blastocyst were 59.3%, 36.7%, and 7.5% for early-, intermediate-, and late-cleaving embryos, respectively. Using RNA collected from early-, intermediate-, and late-cleaving embryos, real-time PCR was performed to assess the transcript levels of 14 different genes of widely varied function. The qPCR results suggest that maternal mRNA degradation may not proceed in an appropriate pattern in slow-cleaving embryos. These findings (1) confirm that, as observed in other species, earlier-cleaving porcine embryos are more successful at developing in culture than are slower-cleaving embryos, and (2) implicate mechanisms of maternal transcript destruction as potential determinants of oocyte/embryo quality. Copyright © 2011 Wiley Periodicals, Inc.

  10. Peptidase specificity from the substrate cleavage collection in the MEROPS database and a tool to measure cleavage site conservation.

    Science.gov (United States)

    Rawlings, Neil D

    2016-03-01

    One peptidase can usually be distinguished from another biochemically by its action on proteins, peptides and synthetic substrates. Since 1996, the MEROPS database (http://merops.sanger.ac.uk) has accumulated a collection of cleavages in substrates that now amounts to 66,615 cleavages. The total number of peptidases for which at least one cleavage is known is 1700 out of a total of 2457 different peptidases. This paper describes how the cleavages are obtained from the scientific literature, how they are annotated and how cleavages in peptides and proteins are cross-referenced to entries in the UniProt protein sequence database. The specificity profiles of 556 peptidases are shown for which ten or more substrate cleavages are known. However, it has been proposed that at least 40 cleavages in disparate proteins are required for specificity analysis to be meaningful, and only 163 peptidases (6.6%) fulfil this criterion. Also described are the various displays shown on the website to aid with the understanding of peptidase specificity, which are derived from the substrate cleavage collection. These displays include a logo, distribution matrix, and tables to summarize which amino acids or groups of amino acids are acceptable (or not acceptable) in each substrate binding pocket. For each protein substrate, there is a display to show how it is processed and degraded. Also described are tools on the website to help with the assessment of the physiological relevance of cleavages in a substrate. These tools rely on the hypothesis that a cleavage site that is conserved in orthologues is likely to be physiologically relevant, and alignments of substrate protein sequences are made utilizing the UniRef50 database, in which in each entry sequences are 50% or more identical. Conservation in this case means substitutions are permitted only if the amino acid is known to occupy the same substrate binding pocket from at least one other substrate cleaved by the same peptidase

  11. 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.

  12. 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.

  13. Cleavage of mispaired heteroduplex DNA substrates by numerous restriction enzymes.

    Science.gov (United States)

    Langhans, Mark T; Palladino, Michael J

    2009-01-01

    The utility of restriction endonucleases as a tool in molecular biology is in large part due to the high degree of specificity with which they cleave well-characterized DNA recognition sequences. The specificity of restriction endonucleases is not absolute, yet many commonly used assays of biological phenomena and contemporary molecular biology techniques rely on the premise that restriction enzymes will cleave only perfect cognate recognition sites. In vitro, mispaired heteroduplex DNAs are commonly formed, especially subsequent to polymerase chain reaction amplification. We investigated a panel of restriction endonucleases to determine their ability to cleave mispaired heteroduplex DNA substrates. Two straightforward, non-radioactive assays are used to evaluate mispaired heteroduplex DNA cleavage: a PCR amplification method and an oligonucleotide-based assay. These assays demonstrated that most restriction endonucleases are capable of site-specific double-strand cleavage with heteroduplex mispaired DNA substrates, however, certain mispaired substrates do effectively abrogate cleavage to undetectable levels. These data are consistent with mispaired substrate cleavage previously reported for Eco RI and, importantly, extend our knowledge of mispaired heteroduplex substrate cleavage to 13 additional enzymes.

  14. Ring cleavage of sulfur heterocycles: how does it happen?

    Science.gov (United States)

    Bressler, D C; Norman, J A; Fedorak, P M

    Sulfur heterocycles are common constituents of petroleum and liquids derived from coal, and they are found in some secondary metabolites of microorganisms and plants. They exist primarily as saturated rings and thiophenes. There are two major objectives driving investigations of the microbial metabolism of organosulfur compounds. One is the quest to develop a process for biodesulfurization of fossil fuels, and the other is to understand the fates of organosulfur compounds in petroleum- or creosote-contaminated environments which is important in assessing bioremediation processes. For these processes to be successful, cleavage of different types of sulfur heterocyclic rings is paramount. This paper reviews the evidence for microbial ring cleavage of a variety of organosulfur compounds and discusses the few well-studied cases which have shown that the C-S bond is most susceptible to breakage leading to disruption of the ring. In most cases, the introduction of one or more oxygen atom(s) onto the adjacent C atom and/or onto the S atom weakens the C-S bond, facilitating its cleavage. Although much is known about the thiophene ring cleavage in dibenzothiophene, there is still a great deal to be learned about the cleavage of other sulfur heterocycles.

  15. Ring cleavage of sulfur heterocycles: how does it happen?

    Energy Technology Data Exchange (ETDEWEB)

    Bressler, D.C.; Norman, J.A.; Fedorak, P.M. [University of Alberta, Edmonton, AB (Canada). Dept. of Biological Sciences

    1997-12-31

    Sulfur heterocycles are common constituents of petroleum and liquids derived from coal, and they are found in some secondary metabolites of microorganisms and plants. They exist primarily as saturated rings and thiophenes. There are two major objectives driving investigations of the microbial metabolism of organosulfur compounds. One is the quest to develop a process for biodesulfurization of fossil fuels, and the other is to understand the fates of organosulfur compounds in petroleum-or-creosote-contaminated environments which is important in assessing bioremediation processes. For these processes to be successful, cleavage of different types of sulfur heterocyclic rings is paramount. This paper reviews the evidence for microbial ring cleavage of a variety of organosulfur compounds and discusses the few well-studied cases which have shown that the C-S bond is most susceptible to breakage leading to disruption of the ring. In most cases, the introduction of one or more oxygen atom(s) onto the adjacent C atom and/or onto the S atom weakens the C-S bond, facilitating its cleavage. Although much is known about the thiophene ring cleavage in dibenzothiophene, there is still a great deal to be learned about the cleavage of other sulfur heterocycles.

  16. Ribosomal Protein S12 and Aminoglycoside Antibiotics Modulate A-site mRNA Cleavage and Transfer-Messenger RNA Activity in Escherichia coli*

    Science.gov (United States)

    Holberger, Laura E.; Hayes, Christopher S.

    2009-01-01

    Translational pausing in Escherichia coli can lead to mRNA cleavage within the ribosomal A-site. A-site mRNA cleavage is thought to facilitate transfer-messenger RNA (tmRNA)·SmpB- mediated recycling of stalled ribosome complexes. Here, we demonstrate that the aminoglycosides paromomycin and streptomycin inhibit A-site cleavage of stop codons during inefficient translation termination. Aminoglycosides also induced stop codon read-through, suggesting that these antibiotics alleviate ribosome pausing during termination. Streptomycin did not inhibit A-site cleavage in rpsL mutants, which express streptomycin-resistant variants of ribosomal protein S12. However, rpsL strains exhibited reduced A-site mRNA cleavage compared with rpsL+ cells. Additionally, tmRNA·SmpB-mediated SsrA peptide tagging was significantly reduced in several rpsL strains but could be fully restored in a subset of mutants when treated with streptomycin. The streptomycin-dependent rpsL(P90K) mutant also showed significantly lower levels of A-site cleavage and tmRNA·SmpB activity. Mutations in rpsD (encoding ribosomal protein S4), which suppressed streptomycin dependence, were able to partially restore A-site cleavage to rpsL(P90K) cells but failed to increase tmRNA·SmpB activity. Taken together, these results show that perturbations to A-site structure and function modulate A-site mRNA cleavage and tmRNA·SmpB activity. We propose that tmRNA·SmpB binds to streptomycin-resistant rpsL ribosomes less efficiently, leading to a partial loss of ribosome rescue function in these mutants. PMID:19776006

  17. Ribosomal protein S12 and aminoglycoside antibiotics modulate A-site mRNA cleavage and transfer-messenger RNA activity in Escherichia coli.

    Science.gov (United States)

    Holberger, Laura E; Hayes, Christopher S

    2009-11-13

    Translational pausing in Escherichia coli can lead to mRNA cleavage within the ribosomal A-site. A-site mRNA cleavage is thought to facilitate transfer-messenger RNA (tmRNA).SmpB- mediated recycling of stalled ribosome complexes. Here, we demonstrate that the aminoglycosides paromomycin and streptomycin inhibit A-site cleavage of stop codons during inefficient translation termination. Aminoglycosides also induced stop codon read-through, suggesting that these antibiotics alleviate ribosome pausing during termination. Streptomycin did not inhibit A-site cleavage in rpsL mutants, which express streptomycin-resistant variants of ribosomal protein S12. However, rpsL strains exhibited reduced A-site mRNA cleavage compared with rpsL(+) cells. Additionally, tmRNA.SmpB-mediated SsrA peptide tagging was significantly reduced in several rpsL strains but could be fully restored in a subset of mutants when treated with streptomycin. The streptomycin-dependent rpsL(P90K) mutant also showed significantly lower levels of A-site cleavage and tmRNA.SmpB activity. Mutations in rpsD (encoding ribosomal protein S4), which suppressed streptomycin dependence, were able to partially restore A-site cleavage to rpsL(P90K) cells but failed to increase tmRNA.SmpB activity. Taken together, these results show that perturbations to A-site structure and function modulate A-site mRNA cleavage and tmRNA.SmpB activity. We propose that tmRNA.SmpB binds to streptomycin-resistant rpsL ribosomes less efficiently, leading to a partial loss of ribosome rescue function in these mutants.

  18. 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.

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

    Science.gov (United States)

    Heo, Jinsol; Kim, Se Hyeuk

    2013-01-01

    Carotenoid cleavage dioxygenases (CCDs) are enzymes that catalyze the oxidative cleavage of carotenoids at a specific double bond to generate apocarotenoids. In this study, we investigated the activity and substrate preferences of NSC3, a CCD of Nostoc sp. strain PCC 7120, in vivo and in vitro using natural and nonnatural carotenoid structures. NSC3 cleaved β-apo-8′-carotenal at 3 positions, C-13C-14, C-15C-15′, and C-13′C-14′, revealing a unique cleavage pattern. NSC3 cleaves the natural structure of carotenoids 4,4′-diaponeurosporene, 4,4′-diaponeurosporen-4′-al, 4,4′-diaponeurosporen-4′-oic acid, 4,4′-diapotorulene, and 4,4′-diapotorulen-4′-al to generate novel cleavage products (apo-14′-diaponeurosporenal, apo-13′-diaponeurosporenal, apo-10′-diaponeurosporenal, apo-14′-diapotorulenal, and apo-10′-diapotorulenal, respectively). The study of carotenoids with natural or nonnatural structures produced by using synthetic modules could provide information valuable for understanding the cleavage reactions or substrate preferences of other CCDs in vivo and in vitro. PMID:23524669

  20. Cryopreservation of biopsied cleavage stage human embryos.

    Science.gov (United States)

    Stachecki, James J; Cohen, Jacques; Munné, Santiago

    2005-12-01

    The aim was to develop a method to optimize cryopreservation of biopsied multi-celled human embryos. Human day 3 embryos that were donated to research, along with those found to be chromosomally abnormal after blastomere biopsy and fluorescence in-situ hyridization (FISH), were cryopreserved using a slow-freezing protocol in either standard embryo cryopreservation solution [embryo transfer freezing medium (ETFM), a conventional sodium-based medium] or CJ3 (a choline-based, sodium-free medium). After thawing, the number of intact cells was recorded and the previously biopsied embryos were re-analysed using FISH. Biopsied embryos had a lower proportion of intact blastomeres after cryopreservation as compared with intact embryos. However, a significantly (P < 0.05) higher proportion of blastomeres from intact and biopsied embryos cryopreserved in CJ3 (84.1 and 80.1% respectively) survived after thaw than those in ETFM (73.6 and 50.5% respectively). The proportion of aneuploid and mosaic embryos was not statistically different between the two groups. In addition, the frequency of lost cells by aneuploid and mosaic embryos was similar. This study describes a new method that improves the survival of cryopreserved biopsied embryos, and shows that it may also be beneficial for the storage of intact human multi-celled embryos.

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

    Science.gov (United States)

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

    2014-12-01

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

  2. Sensitive and fast mutation detection by solid phase chemical cleavage

    DEFF Research Database (Denmark)

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

    1996-01-01

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

  3. Uncovering the Protocatechuate 2,3-Cleavage Pathway Genes▿ †

    OpenAIRE

    Kasai, Daisuke; Fujinami, Toshihiro; Abe, Tomokuni; Mase, Kohei; Katayama, Yoshihiro; Fukuda, Masao; Masai, Eiji

    2009-01-01

    Paenibacillus sp. (formerly Bacillus macerans) strain JJ-1b is able to grow on 4-hydroxybenzoate (4HB) as a sole source of carbon and energy and is known to degrade 4HB via the protocatechuate (PCA) 2,3-cleavage pathway. However, none of the genes involved in this pathway have been identified. In this study, we identified and characterized the JJ-1b genes for the 4HB catabolic pathway via the PCA 2,3-cleavage pathway, which consisted of praR and praABEGFDCHI. Based on the enzyme activities of...

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

    Science.gov (United States)

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

    2014-01-15

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

  5. Inhibition of Cellular Adhesion by Immunological Targeting of Osteopontin Neoepitopes Generated through Matrix Metalloproteinase and Thrombin Cleavage.

    Science.gov (United States)

    Jürets, Alexander; Le Bras, Marie; Staffler, Günther; Stein, Gesine; Leitner, Lukas; Neuhofer, Angelika; Tardelli, Matteo; Turkof, Edvin; Zeyda, Maximilian; Stulnig, Thomas M

    2016-01-01

    Osteopontin (OPN), a secreted protein involved in inflammatory processes and cancer, induces cell adhesion, migration, and activation of inflammatory pathways in various cell types. Cells bind OPN via integrins at a canonical RGD region in the full length form as well as to a contiguous cryptic site that some have shown is unmasked upon thrombin or matrix metalloproteinase cleavage. Thus, the adhesive capacity of osteopontin is enhanced by proteolytic cleavage that may occur in inflammatory conditions such as obesity, atherosclerosis, rheumatoid arthritis, tumor growth and metastasis. Our aim was to inhibit cellular adhesion to recombinant truncated proteins that correspond to the N-terminal cleavage products of thrombin- or matrix metalloproteinase-cleaved OPN in vitro. We specifically targeted the cryptic integrin binding site with monoclonal antibodies and antisera induced by peptide immunization of mice. HEK 293 cells adhered markedly stronger to truncated OPN proteins than to full length OPN. Without affecting cell binding to the full length form, the raised monoclonal antibodies specifically impeded cellular adhesion to the OPN fragments. Moreover, we show that the peptides used for immunization were able to induce antisera, which impeded adhesion either to all OPN forms, including the full-length form, or selectively to the corresponding truncated recombinant proteins. In conclusion, we developed immunological tools to selectively target functional properties of protease-cleaved OPN forms, which could find applications in treatment and prevention of various inflammatory diseases and cancers.

  6. DNA cleavage agents from Schisandra propinqua var. sinensis ...

    African Journals Online (AJOL)

    DNA cleavage agents from Schisandra propinqua var. sinensis. Y Chen, L Hai, Y Liu, X Liao. Abstract. No Abstract. Full Text: EMAIL FREE FULL TEXT EMAIL FREE FULL TEXT · DOWNLOAD FULL TEXT DOWNLOAD FULL TEXT · AJOL African Journals Online. HOW TO USE AJOL... for Researchers · for Librarians ...

  7. 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...

  8. DNA binding and cleavage activity of a structurally characterized Ni ...

    Indian Academy of Sciences (India)

    1375–1381. c Indian Academy of Sciences. DOI 10.1007/s12039-015-0900-4. DNA binding and cleavage activity of a structurally characterized Ni(II). Schiff base complex. SARAT CHANDRA KUMARa, ABHIJIT PALa, MERRY MITRAa,. V M MANIKANDAMATHAVANb, CHIA -HER LINc, BALACHANDRAN UNNI NAIRb,∗.

  9. Early post-cleavage stages and abnormalities identified in the ...

    African Journals Online (AJOL)

    Six early, post-cleavage embryonic stages for chokka squid Loligo vulgaris reynaudii eggs that were developed in an aquarium are identified and described, expanding the embryonic stages for this species from 14 to 20. The influence of water temperature on embryonic development is described. At temperatures  ...

  10. Fe-Catalyzed Oxidative Cleavage of Unsaturated Fatty Acids

    NARCIS (Netherlands)

    Spannring, P.|info:eu-repo/dai/nl/325783802

    2013-01-01

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

  11. Detection of meta - and ortho -cleavage dioxygenases in bacterial ...

    African Journals Online (AJOL)

    In contrast, isolates S-5, Sea-8, W-6, W-15 and Pla-1 showed activity with the enzyme catalyzing the second step in the phenol degradation meta-cleavage pathway, catechol-2,3-dioxygenase. On the basis of our previous and present analysis, the investigated isolates are considered to have a good potential for application ...

  12. Photo-induced antimicrobial and DNA cleavage studies of ...

    Indian Academy of Sciences (India)

    J. Chem. Sci. Vol. 125, No. 5, September 2013, pp. 1015–1027. c Indian Academy of Sciences. Photo-induced antimicrobial and DNA cleavage studies ... †Present address: Department of Chemistry, Bannari Amman Institute of Technology, Sathyamangalam, India. #Present address: ..... C overnight and exam- ined for ...

  13. DNA cleavage agents from Schisandra propinqua var. sinensis

    African Journals Online (AJOL)

    STORAGESEVER

    2009-09-15

    Sep 15, 2009 ... DNA strand breakage process is involved in various bio- logical stages such as inflammation, mutagenesis, carci- nogenesis, or aging (Mibu et al., 2003; Chen et al., 2006). As a consequence of the clinical utility of DNA cleavage ..... and its use in folk medicine to treat fracture, chronic gas- tritis and ...

  14. Definition issues of concepts of social cleavages in Africa | Raphael ...

    African Journals Online (AJOL)

    The main problem is whether the definitions of concepts are coherent to reflect the complex social realities. Based on the theoretical framework and on the analysis of the definitions, it becomes clear that the social cleavage concepts are not consistent once applied to African societies. For the Rwandan society in particular, ...

  15. DNA binding and cleavage activity of a structurally characterized Ni ...

    Indian Academy of Sciences (India)

    Home; Journals; Journal of Chemical Sciences; Volume 127; Issue 8. DNA binding and cleavage activity of a structurally characterized Ni(II) Schiff base complex. Sarat Chandra Kumar Abhijit Pal Merry Mitra V M Manikandamathavan Chia -Her Lin Balachandran Unni Nair Rajarshi Ghosh. Regular Articles Volume 127 ...

  16. 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...

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

    Energy Technology Data Exchange (ETDEWEB)

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

    2015-12-25

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

  18. 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

    2014-01-05

    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. Copyright © 2013 Elsevier B.V. All rights reserved.

  19. Crystal structure of a covalent intermediate in DNA cleavage and rejoining by Escherichia coli DNA topoisomerase I

    Science.gov (United States)

    Zhang, Zhongtao; Cheng, Bokun; Tse-Dinh, Yuk-Ching

    2011-01-01

    DNA topoisomerases control DNA topology by breaking and rejoining DNA strands via covalent complexes with cleaved DNA substrate as catalytic intermediates. Here we report the structure of Escherichia coli topoisomerase I catalytic domain (residues 2–695) in covalent complex with a cleaved single-stranded oligonucleotide substrate, refined to 2.3-Å resolution. The enzyme-substrate intermediate formed after strand cleavage was captured due to the presence of the D111N mutation. This structure of the covalent topoisomerase-DNA intermediate, previously elusive for type IA topoisomerases, shows distinct conformational changes from the structure of the enzyme without bound DNA and provides detailed understanding of the covalent catalysis required for strand cleavage to take place. The portion of cleaved DNA 5′ to the site of cleavage is anchored tightly with extensive noncovalent protein–DNA interactions as predicted by the “enzyme-bridged” model. Distortion of the scissile strand at the -4 position 5′ to the cleavage site allows specific selectivity of a cytosine base in the binding pocket. Many antibacterial and anticancer drugs initiate cell killing by trapping the covalent complexes formed by topoisomerases. We have demonstrated in previous mutagenesis studies that accumulation of the covalent complex of bacterial topoisomerase I is bactericidal. This structure of the covalent intermediate provides the basis for the design of novel antibiotics that can trap the enzyme after formation of the covalent complex. PMID:21482796

  20. Apoptotic cleavage of NuMA at the C-terminal end is related to nuclear disruption and death amplification.

    Science.gov (United States)

    Lin, Hsueh-Hsuan; Hsu, Hsin-Ling; Yeh, Ning-Hsing

    2007-09-01

    NuMA is a nuclear matrix protein in interphase and distributes to the spindle poles during mitosis. While the essential function of NuMA for mitotic spindle assembly is well established, a structural role of NuMA in interphase nucleus has also been proposed. Several observations suggest that the apoptotic degradation of NuMA may relate to chromatin condensation and micronucleation. Here we demonstrate that four apoptotic cleavage sites are clustered at a junction between the globular tail and the central coiled-coil domains of NuMA. Cleavage of a caspase-6-sensitive site at D(1705) produced the R-form, a major tail-less product of NuMA during apoptosis. The other two cleavage sites were defined at D(1726) and D(1747) that were catalyzed, respectively, by caspase-3 and an unknown aspartase. A NuMA deletion mutant missing the entire cleavage region of residues 1701-1828 resisted degradation and protected cells from nuclear disruption upon apoptotic attack. Under such conditions, cytochrome c was released from mitochondria, but the subsequent apoptotic events such as caspase-3 activation, poly(ADP-ribose) polymerase degradation, and DNA fragmentation were attenuated. Conversely, the tail-less NuMA alone, a mutant mimicking the R-form, induced chromatin condensation and activated the death machinery. It supports that intact NuMA is a structural element in maintaining nuclear integrity.

  1. Ion beam modifications of defect sub-structure of calcite cleavages

    Indian Academy of Sciences (India)

    , brought about by He+ ion-bombardment of calcite cleavages (100), have been carried out. Optical and scanning ... stimulation. Planar plastic anisotropy has been studied on irradiated calcite cleavages by measurement of microhardness.

  2. DNA Cleavage and Condensation Activities of Mono- and Binuclear Hybrid Complexes and Regulation by Graphene Oxide

    Directory of Open Access Journals (Sweden)

    Shuo Li

    2016-07-01

    Full Text Available Hybrid complexes with N,N′-bis(2-benzimidazolylmethylamine and cyclen moieties are novel enzyme mimics and controlled DNA release materials, which could interact with DNA through three models under different conditions. In this paper, the interactions between plasmid DNA and seven different complexes were investigated, and the methods to change the interaction patterns by graphene oxide (GO or concentrations were also investigated. The cleavage of pUC19 DNA promoted by target complexes were via hydrolytic or oxidative mechanisms at low concentrations ranging from 3.13 × 10−7 to 6.25 × 10−5 mol/L. Dinuclear complexes 2a and 2b can promote the cleavage of plasmid pUC19 DNA to a linear form at pH values below 7.0. Furthermore, binuclear hybrid complexes could condense DNA as nanoparticles above 3.13 × 10−5 mol/L and partly release DNA by graphene oxide with π-π stacking. Meanwhile, the results also reflected that graphene oxide could prevent DNA from breaking down. Cell viability assays showed dinuclear complexes were safe to normal human hepatic cells at relative high concentrations. The present work might help to develop novel strategies for the design and synthesis of DNA controllable releasing agents, which may be applied to gene delivery and also to exploit the new application for GO.

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

    Science.gov (United States)

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

    2012-02-15

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

  4. MerTK Cleavage on Resident Cardiac Macrophages Compromises Repair After Myocardial Ischemia Reperfusion Injury.

    Science.gov (United States)

    DeBerge, Matthew; Yeap, Xin Yi; Dehn, Shirley; Zhang, Shuang; Grigoryeva, Lubov; Misener, Sol; Procissi, Daniel; Zhou, Xin; Lee, Daniel C; Muller, William A; Luo, Xunrong; Rothlin, Carla; Tabas, Ira; Thorp, Edward B

    2017-09-29

    Clinical benefits of reperfusion after myocardial infarction are offset by maladaptive innate immune cell function, and therapeutic interventions are lacking. We sought to test the significance of phagocytic clearance by resident and recruited phagocytes after myocardial ischemia reperfusion. In humans, we discovered that clinical reperfusion after myocardial infarction led to significant elevation of the soluble form of MerTK (myeloid-epithelial-reproductive tyrosine kinase; ie, soluble MER), a critical biomarker of compromised phagocytosis by innate macrophages. In reperfused mice, macrophage Mertk deficiency led to decreased cardiac wound debridement, increased infarct size, and depressed cardiac function, newly implicating MerTK in cardiac repair after myocardial ischemia reperfusion. More notably, Mertk(CR ) mice, which are resistant to cleavage, showed significantly reduced infarct sizes and improved systolic function. In contrast to other cardiac phagocyte subsets, resident cardiac MHCII LO CCR2 - (major histocompatibility complex II/C-C motif chemokine receptor type 2) macrophages expressed higher levels of MerTK and, when exposed to apoptotic cells, secreted proreparative cytokines, including transforming growth factor-β. Mertk deficiency compromised the accumulation of MHCII LO phagocytes, and this was rescued in Mertk(CR ) mice. Interestingly, blockade of CCR2-dependent monocyte infiltration into the heart reduced soluble MER levels post-ischemia reperfusion. Our data implicate monocyte-induced MerTK cleavage on proreparative MHCII LO cardiac macrophages as a novel contributor and therapeutic target of reperfusion injury. © 2017 American Heart Association, Inc.

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

    Science.gov (United States)

    Hui-Bon-Hoa, Gaston; Kaddour, Hussein; Vergne, Jacques; Kruglik, Sergei G; Maurel, Marie-Christine

    2014-03-21

    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. 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. 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.

  6. Sequence specific inhibition of DNA restriction enzyme cleavage by PNA

    DEFF Research Database (Denmark)

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

    1993-01-01

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

  7. Material grain size and crack size influences on cleavage fracturing.

    Science.gov (United States)

    Armstrong, Ronald W

    2015-03-28

    A review is given of the analogous dependence on reciprocal square root of grain size or crack size of fracture strength measurements reported for steel and other potentially brittle materials. The two dependencies have much in common. For onset of cleavage in steel, attention is focused on relationship of the essentially athermal fracture stress compared with a quite different viscoplastic yield stress behaviour. Both grain-size-dependent stresses are accounted for in terms of dislocation pile-up mechanics. Lowering of the cleavage stress occurs in steel because of carbide cracking. For crack size dependence, there is complication of localized crack tip plasticity in fracture mechanics measurements. Crack-size-dependent conventional and indentation fracture mechanics measurements are described also for results obtained on the diverse materials: polymethylmethacrylate, silicon crystals, alumina polycrystals and WC-Co (cermet) composites. © 2015 The Author(s) Published by the Royal Society. All rights reserved.

  8. 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.

  9. Fracture behaviour and cleavage initiation in hypoeutectoid pearlitic steel

    Czech Academy of Sciences Publication Activity Database

    Holzmann, Miloslav; Jurášek, L.; Dlouhý, Ivo

    2007-01-01

    Roč. 148, č. 1 (2007), s. 13-28 ISSN 0376-9429 R&D Projects: GA AV ČR IAA200410502; GA AV ČR 1QS200410502 Institutional research plan: CEZ:AV0Z20410507 Keywords : cleavage fracture * fracture initiation * pearlitic steel Subject RIV: JL - Materials Fatigue, Friction Mechanics Impact factor: 1.003, year: 2007 http://www.springerlink.com/content/2h8167l167436165/fulltext.pdf

  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. Domainal cleavage as an Anisotropic Reaction-diffusion Process

    Science.gov (United States)

    Mulchrone, Kieran; Meere, Patrick

    2017-04-01

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

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

    International Nuclear Information System (INIS)

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

    2012-01-01

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

  13. DNAzyme-Controlled Cleavage of Dimer and Trimer Origami Tiles.

    Science.gov (United States)

    Wu, Na; Willner, Itamar

    2016-04-13

    Dimers of origami tiles are bridged by the Pb(2+)-dependent DNAzyme sequence and its substrate or by the histidine-dependent DNAzyme sequence and its substrate to yield the dimers T1-T2 and T3-T4, respectively. The dimers are cleaved to monomer tiles in the presence of Pb(2+)-ions or histidine as triggers. Similarly, trimers of origami tiles are constructed by bridging the tiles with the Pb(2+)-ion-dependent DNAzyme sequence and the histidine-dependent DNAzyme sequence and their substrates yielding the trimer T1-T5-T4. In the presence of Pb(2+)-ions and/or histidine as triggers, the programmed cleavage of trimer proceeds. Using Pb(2+) or histidine as trigger cleaves the trimer to yield T5-T4 and T1 or the dimer T1-T5 and T4, respectively. In the presence of Pb(2+)-ions and histidine as triggers, the cleavage products are the monomer tiles T1, T5, and T4. The different cleavage products are identified by labeling the tiles with 0, 1, or 2 streptavidin labels and AFM imaging.

  14. Numerical modeling of ductile tearing effects on cleavage fracture toughness

    International Nuclear Information System (INIS)

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

    1994-05-01

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

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

    Science.gov (United States)

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

    2005-01-01

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

  16. 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

  17. 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.

  18. Serum Collagen Type II Cleavage Epitope and Serum Hyaluronic Acid as Biomarkers for Treatment Monitoring of Dogs with Hip Osteoarthritis.

    Directory of Open Access Journals (Sweden)

    José M Vilar

    Full Text Available The aim of this study was to evaluate the use of serum type II collagen cleavage epitope and serum hyaluronic acid as biomarkers for treatment monitoring in osteoarthritic dogs. For this purpose, a treatment model based on mesenchymal stem cells derived from adipose tissue combined with plasma rich in growth factors was used. This clinical study included 10 dogs with hip osteoarthritis. Both analytes were measured in serum at baseline, just before applying the treatment, and 1, 3, and 6 months after treatment. These results were compared with those obtained from force plate analysis using the same animals during the same study period. Levels of type II collagen cleavage epitope decreased and those of hyaluronic acid increased with clinical improvement objectively verified via force plate analysis, suggesting these two biomarkers could be effective as indicators of clinical development of joint disease in dogs.

  19. 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.

  20. Inhibition of Human Cytomegalovirus pUL89 Terminase Subunit Blocks Virus Replication and Genome Cleavage.

    Science.gov (United States)

    Wang, Yan; Mao, Lili; Kankanala, Jayakanth; Wang, Zhengqiang; Geraghty, Robert J

    2017-02-01

    The human cytomegalovirus terminase complex cleaves concatemeric genomic DNA into unit lengths during genome packaging and particle assembly. This process is an attractive drug target because cleavage of concatemeric DNA is not required in mammalian cell DNA replication, indicating that drugs targeting the terminase complex could be safe and selective. One component of the human cytomegalovirus terminase complex, pUL89, provides the endonucleolytic activity for genome cleavage, and the domain responsible is reported to have an RNase H-like fold. We hypothesize that the pUL89 endonuclease activity is inhibited by known RNase H inhibitors. Using a novel enzyme-linked immunosorbent assay (ELISA) format as a screening assay, we found that a hydroxypyridonecarboxylic acid compound, previously reported to be an inhibitor of human immunodeficiency virus RNase H, inhibited pUL89 endonuclease activity at low-micromolar concentrations. Further characterization revealed that this pUL89 endonuclease inhibitor blocked human cytomegalovirus replication at a relatively late time point, similarly to other reported terminase complex inhibitors. Importantly, this inhibitor also prevented the cleavage of viral genomic DNA in infected cells. Taken together, these results substantiate our pharmacophore hypothesis and validate our ligand-based approach toward identifying novel inhibitors of pUL89 endonuclease. Human cytomegalovirus infection in individuals lacking a fully functioning immune system, such as newborns and transplant patients, can have severe and debilitating consequences. The U.S. Food and Drug Administration-approved anti-human cytomegalovirus drugs mainly target the viral polymerase, and resistance to these drugs has appeared. Therefore, anti-human cytomegalovirus drugs from novel targets are needed for use instead of, or in combination with, current polymerase inhibitors. pUL89 is a viral ATPase and endonuclease and is an attractive target for anti-human cytomegalovirus

  1. Severe teratozoospermia and its influence on pronuclear morphology, embryonic cleavage and compaction

    Directory of Open Access Journals (Sweden)

    Russell Helena

    2011-03-01

    Full Text Available Abstract Background Fertilization, cell division and embryo development depend on genomic contributions from male and female gametes. We hypothesize that teratozoospermic sperm influences early embryo development and embryo compaction. Methods We conducted a retrospective analysis of embryos derived from intracytoplasmic sperm injection (ICSI cycles. Two hundred thirty-five consecutive ICSI cycles were included in the study; all treatment was provided at the Cleveland Clinic Fertility Center. Patient cycles were divided by sperm morphology based on Kruger's strict criteria: Group A, embryos where teratozoospermic sperm (0-2% normal were used for ICSI and Group B, embryos where dysmorphic sperm (5-13% normal were used for ICSI. All cycles analyzed were of patients doing day 3 embryo transfers. Outcome measures assessed included pronuclear (PN pattern, syngamy, early cleavage, cell number, rate of compaction and blastulation of embryos left in culture and not transferred on day 3. Results A total of 1762 embryos were analyzed. PN patterns were similar in Group A and Group B embryos. No differences were noted in syngamy, cleavage, cell number or blastulation rate. Studying the development of embryos in culture after day 3 transfer revealed a difference in the timeline for compaction. By day 4, 25% of Group A embryos had compacted compared to 36% in Group B (P = 0.0007. There was no difference found between Group A and Group B embryos in regards to blastulation. Conclusions We did not find an association between sperm morphology and clinical outcomes. The impact of teratozoospermia may be masked in ICSI cycles where fertilization, implantation rate and clinical pregnancy rate are the primary outcome measures. However, by examining the timeline of development, we were better able to discern a potential paternal effect at critical transition points from fertilization through activation.

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

    Science.gov (United States)

    Ningrum, Andriati; Schreiner, Matthias

    2014-11-01

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

  3. Drosha regulates gene expression independently of RNA cleavage function

    DEFF Research Database (Denmark)

    Gromak, Natalia; Dienstbier, Martin; Macias, Sara

    2013-01-01

    Drosha is the main RNase III-like enzyme involved in the process of microRNA (miRNA) biogenesis in the nucleus. Using whole-genome ChIP-on-chip analysis, we demonstrate that, in addition to miRNA sequences, Drosha specifically binds promoter-proximal regions of many human genes in a transcription......-terminal protein-interaction domain, which associates with the RNA-binding protein CBP80 and RNA Polymerase II. Consequently, we uncover a previously unsuspected RNA cleavage-independent function of Drosha in the regulation of human gene expression....

  4. Cleavage of the JunB Transcription Factor by Caspases Generates a Carboxyl-terminal Fragment That Inhibits Activator Protein-1 Transcriptional Activity*

    Science.gov (United States)

    Lee, Jason K. H.; Pearson, Joel D.; Maser, Brandon E.; Ingham, Robert J.

    2013-01-01

    The activator protein-1 (AP-1) family transcription factor, JunB, is an important regulator of proliferation, apoptosis, differentiation, and the immune response. In this report, we show that JunB is cleaved in a caspase-dependent manner in apoptotic anaplastic lymphoma kinase-positive, anaplastic large cell lymphoma cell lines and that ectopically expressed JunB is cleaved in murine RAW 264.7 macrophage cells treated with the NALP1b inflammasome activator, anthrax lethal toxin. In both cases, we identify aspartic acid 137 as the caspase cleavage site and demonstrate that JunB can be directly cleaved in vitro by multiple caspases at this site. Cleavage of JunB at aspartic acid 137 separates the N-terminal transactivation domain from the C-terminal DNA binding and dimerization domains, and we show that the C-terminal cleavage fragment retains both DNA binding activity and the ability to interact with AP-1 family transcription factors. Furthermore, this fragment interferes with the binding of full-length JunB to AP-1 sites and inhibits AP-1-dependent transcription. In summary, we have identified and characterized a novel mechanism of JunB post-translational modification and demonstrate that the C-terminal JunB caspase cleavage product functions as a potent inhibitor of AP-1-dependent transcription. PMID:23749999

  5. Cleavage of the JunB transcription factor by caspases generates a carboxyl-terminal fragment that inhibits activator protein-1 transcriptional activity.

    Science.gov (United States)

    Lee, Jason K H; Pearson, Joel D; Maser, Brandon E; Ingham, Robert J

    2013-07-26

    The activator protein-1 (AP-1) family transcription factor, JunB, is an important regulator of proliferation, apoptosis, differentiation, and the immune response. In this report, we show that JunB is cleaved in a caspase-dependent manner in apoptotic anaplastic lymphoma kinase-positive, anaplastic large cell lymphoma cell lines and that ectopically expressed JunB is cleaved in murine RAW 264.7 macrophage cells treated with the NALP1b inflammasome activator, anthrax lethal toxin. In both cases, we identify aspartic acid 137 as the caspase cleavage site and demonstrate that JunB can be directly cleaved in vitro by multiple caspases at this site. Cleavage of JunB at aspartic acid 137 separates the N-terminal transactivation domain from the C-terminal DNA binding and dimerization domains, and we show that the C-terminal cleavage fragment retains both DNA binding activity and the ability to interact with AP-1 family transcription factors. Furthermore, this fragment interferes with the binding of full-length JunB to AP-1 sites and inhibits AP-1-dependent transcription. In summary, we have identified and characterized a novel mechanism of JunB post-translational modification and demonstrate that the C-terminal JunB caspase cleavage product functions as a potent inhibitor of AP-1-dependent transcription.

  6. Proteolytic processing of the cilium adhesin MHJ_0194 (P123J ) in Mycoplasma hyopneumoniae generates a functionally diverse array of cleavage fragments that bind multiple host molecules.

    Science.gov (United States)

    Raymond, Benjamin B A; Jenkins, Cheryl; Seymour, Lisa M; Tacchi, Jessica L; Widjaja, Michael; Jarocki, Veronica M; Deutscher, Ania T; Turnbull, Lynne; Whitchurch, Cynthia B; Padula, Matthew P; Djordjevic, Steven P

    2015-03-01

    Mycoplasma hyopneumoniae, the aetiological agent of porcine enzootic pneumonia, regulates the presentation of proteins on its cell surface via endoproteolysis, including those of the cilial adhesin P123 (MHJ_0194). These proteolytic cleavage events create functional adhesins that bind to proteoglycans and glycoproteins on the surface of ciliated and non-ciliated epithelial cells and to the circulatory host molecule plasminogen. Two dominant cleavage events of the P123 preprotein have been previously characterized; however, immunoblotting studies suggest that more complex processing events occur. These extensive processing events are characterized here. The functional significance of the P97 cleavage fragments is also poorly understood. Affinity chromatography using heparin, fibronectin and plasminogen as bait and peptide arrays were used to expand our knowledge of the adhesive capabilities of P123 cleavage fragments and characterize a novel binding motif in the C-terminus of P123. Further, we use immunohistochemistry to examine in vivo, the biological significance of interactions between M. hyopneumoniae and fibronectin and show that M. hyopneumoniae induces fibronectin deposition at the site of infection on the ciliated epithelium. Our data supports the hypothesis that M. hyopneumoniae possesses the molecular machinery to influence key molecular communication pathways in host cells. © 2014 John Wiley & Sons Ltd.

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

    International Nuclear Information System (INIS)

    Craft, Willie Warren; Dutch, Rebecca Ellis

    2005-01-01

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

  8. Synthesis, antimicrobial, DNA cleavage and antioxidant activities of tricyclic sultams derived from saccharin.

    Science.gov (United States)

    Elghamry, Ibrahim; Youssef, Magdy M; Al-Omair, Mohammed A; Elsawy, Hany

    2017-10-20

    Two series of fused tricyclic sultams (carboxylates, 3a, b and 5a, f, g and anilides 5b-e) were synthesized from saccharin and their chemical structures were confirmed by spectroscopic tools. Then, their antibacterial activities and MIC were evaluated against two strains of gram positive and gram-negative bacteria. The MIC values of the tested compounds are in the of range 8-33 μg/ml. In addition, their DNA cleavage ability, binding affinity and their anticancer activities against hepatic cancer cell were tested. And their antioxidant activities were also measured. Four carboxylate derivatives (3a, 5a, 5f and 5g) and one anilide (5d) of the tested compounds proved to be the highest activity all over the study. Copyright © 2017 Elsevier Masson SAS. All rights reserved.

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

    Directory of Open Access Journals (Sweden)

    Ramesh Tati

    2017-02-01

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

  10. [Copper-catalyzed cleavage of DNA by arenes].

    Science.gov (United States)

    Koval', O A; Boguslavskiĭ, E G; Oleĭnikova, S B; Chernolovskaia, E L; Litvak, V V; Nadolinnyĭ, V A; Blasov, V V

    2003-01-01

    DNA was found to be cleaved in neutral solutions containing arenes and copper (II) salts. The reaction is comparable in efficiency with the DNA cleavage by such systems as Cu(II)-phenanthroline and Cu(II)-ascorbic acid, but, in contrast to the latter, the system Cu(2+)-arene does not require the presence of an exogenous reducing agent or hydrogen peroxide. The system Cu(2+)-arene does not cleave DNA under anaerobic conditions. Catalase, sodium azide, and bathocuproine, which is a specific chelator of Cu(I), completely inhibit the reaction. The data obtained allow one to suppose that Cu(I) ions, superoxide radical, and singlet oxygen participate in the reaction. It has been shown by the EPR method using spin traps that the reaction proceeds with formation of alkoxyl radicals, which can insert breaks in the DNA molecule. For effective cleavage of DNA in the Cu(II)-o-bromobenzoic acid system, the radicals have to be generated by a specific copper-DNA-o-bromobenzoic acid complex, in which copper ions are most probably coordinated with oxygen atoms of the DNA phosphate groups. The English version of the paper: Russian Journal of Bioorganic Chemistry, 2003, vol. 29, no. 6; see also http://www.maik.ru.

  11. Carotenoids biosynthesis and cleavage related genes from bacteria to plants.

    Science.gov (United States)

    Liang, Ming-Hua; Zhu, Jianhua; Jiang, Jian-Guo

    2017-06-13

    Carotenoids are essential for photosynthesis and photoprotection in photosynthetic organisms and beneficial for human health. Apocarotenoids derived from carotenoid degradation can serve critical functions including hormones, volatiles, and signals. They have been used commercially as food colorants, animal feed supplements, and nutraceuticals for cosmetic and pharmaceutical purposes. This review focuses on the molecular evolution of carotenogenic enzymes and carotenoid cleavage oxygenases (CCOs) from bacteria, fungi, cyanobacteria, algae, and plants. The diversity of carotenoids and apocarotenoids as well as their complicated biosynthetic pathway in different species can shed light on the history of early molecular evolution. Some carotenogenic genes (such as phytoene synthases) have high protein sequence similarity from bacteria to land plants, but some (such as phytoene desaturases, lycopene cyclases, carotenoid hydroxylases, and CCOs) have low similarity. The broad diversity of apocarotenoid volatile compounds can be attributed to large numbers of carotenoid precursors and the various cleavage sites catalyzed by CCOs enzymes. A variety of carotenogenic enzymes and CCOs indicate the functional diversification of carotenoids and apocrotenoids in different species. New carotenoids, new apocarotenoids, new carotenogenic enzymes, new CCOs, and new pathways still need to be explored.

  12. Cleavage stage versus blastocyst stage embryo transfer in assisted reproductive technology.

    Science.gov (United States)

    Glujovsky, Demián; Farquhar, Cindy; Quinteiro Retamar, Andrea Marta; Alvarez Sedo, Cristian Roberto; Blake, Deborah

    2016-06-30

    Advances in cell culture media have led to a shift in in vitro fertilisation (IVF) practice from cleavage stage embryo transfer to blastocyst stage transfer. The rationale for blastocyst transfer is to improve both uterine and embryonic synchronicity and enable self selection of viable embryos, thus resulting in better live birth rates. To determine whether blastocyst stage (day 5 to 6) embryo transfers improve the live birth rate, and other associated outcomes, compared with cleavage stage (day 2 to 3) embryo transfers. We searched the Cochrane Gynaecology and Fertility Group Specialised Register of controlled trials, Cochrane Central Register of Controlled Trials (CENTRAL; the Cochrane Library; 2016, Issue 4), MEDLINE, EMBASE, PsycINFO, CINAHL, and Bio extracts from inception to 4th April 2016. We also searched registers of ongoing trials and the reference lists of studies retrieved. We included randomised controlled trials (RCTs) which compared the effectiveness of blastocyst versus cleavage stage transfers. We used standard methodological procedures recommended by Cochrane. Our primary outcomes were live birth and cumulative clinical pregnancy rates. Secondary outcomes were clinical pregnancy, multiple pregnancy, high order pregnancy, miscarriage, failure to transfer embryos, and embryo freezing. We assessed the overall quality of the evidence for the main comparisons using GRADE methods. We included 27 RCTs (4031 couples or women).The live birth rate following fresh transfer was higher in the blastocyst transfer group (odds ratio (OR) 1.48, 95% confidence interval (CI) 1.20 to 1.82; 13 RCTs, 1630 women, I(2) = 45%, low quality evidence) following fresh transfer. This suggests that if 29% of women achieve live birth after fresh cleavage stage transfer, between 32% and 42% would do so after fresh blastocyst stage transfer.There was no evidence of a difference between the groups in rates per couple of cumulative pregnancy following fresh and frozen

  13. Identification of E-cadherin signature motifs functioning as cleavage sites for Helicobacter pylori HtrA

    Science.gov (United States)

    Schmidt, Thomas P.; Perna, Anna M.; Fugmann, Tim; Böhm, Manja; Jan Hiss; Haller, Sarah; Götz, Camilla; Tegtmeyer, Nicole; Hoy, Benjamin; Rau, Tilman T.; Neri, Dario; Backert, Steffen; Schneider, Gisbert; Wessler, Silja

    2016-03-01

    The cell adhesion protein and tumour suppressor E-cadherin exhibits important functions in the prevention of gastric cancer. As a class-I carcinogen, Helicobacter pylori (H. pylori) has developed a unique strategy to interfere with E-cadherin functions. In previous studies, we have demonstrated that H. pylori secretes the protease high temperature requirement A (HtrA) which cleaves off the E-cadherin ectodomain (NTF) on epithelial cells. This opens cell-to-cell junctions, allowing bacterial transmigration across the polarised epithelium. Here, we investigated the molecular mechanism of the HtrA-E-cadherin interaction and identified E-cadherin cleavage sites for HtrA. Mass-spectrometry-based proteomics and Edman degradation revealed three signature motifs containing the [VITA]-[VITA]-x-x-D-[DN] sequence pattern, which were preferentially cleaved by HtrA. Based on these sites, we developed a substrate-derived peptide inhibitor that selectively bound and inhibited HtrA, thereby blocking transmigration of H. pylori. The discovery of HtrA-targeted signature sites might further explain why we detected a stable 90 kDa NTF fragment during H. pylori infection, but also additional E-cadherin fragments ranging from 105 kDa to 48 kDa in in vitro cleavage experiments. In conclusion, HtrA targets E-cadherin signature sites that are accessible in in vitro reactions, but might be partially masked on epithelial cells through functional homophilic E-cadherin interactions.

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

    Directory of Open Access Journals (Sweden)

    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.

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

    International Nuclear Information System (INIS)

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

    2005-01-01

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

  16. Cleavage leads to expansion of bacteriophage P4 procapsids in vitro

    International Nuclear Information System (INIS)

    Wang Sifang; Chandramouli, Preethi; Butcher, Sarah; Dokland, Terje

    2003-01-01

    Proteolytic cleavage of the structural proteins is an important part of the maturation process for most bacteriophages and other viruses. In the double-stranded DNA bacteriophages this cleavage is associated with DNA packaging, capsid expansion, and scaffold removal. To understand the role of protein cleavage in the expansion of bacteriophages P2 and P4, we have experimentally cleaved P4 procapsids produced by overexpression of the capsid and scaffolding proteins. The cleavage leads to particle expansion and scaffold removal in vitro. The resulting expanded capsid has a thin-shelled structure similar, but not identical, to that of mature virions

  17. 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

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

    Science.gov (United States)

    Kegel, Kimberly B; Sapp, Ellen; Alexander, Jonathan; Reeves, Patrick; Bleckmann, Dorothee; Sobin, Linsday; Masso, Nicholas; Valencia, Antonio; Jeong, Hyunkyung; Krainc, Dimitri; Palacino, James; Curtis, Daniel; Kuhn, Rainer; Betschart, Claudia; Sena-Esteves, Miguel; Aronin, Neil; Paganetti, Paolo; Difiglia, Marian

    2010-12-14

    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. 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. 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-D like properties in immortalized neurons and gamma

  19. Roles of metalloproteinase-3 and aggrecanase 1 and 2 in aggrecan cleavage during human meniscus degeneration

    Directory of Open Access Journals (Sweden)

    Naoki Ishiguro

    2009-10-01

    Full Text Available The meniscus plays important roles in proper knee function. It was recently reported that meniscus degeneration was associated with progression of osteoarthritis (OA. However, little is known about the effects of degradative enzymes on the meniscus matrix, primarily collagen type I and aggrecan, during OA. This study examined the effects of metalloproteinase (MMP and aggrecanase on the destruction of aggrecan in the human meniscus. Eighteen trimmed meniscus portions were collected during partial menisectomy. Specimens were categorized into 3 groups according to the modified Copenhaver classification based on the degrees of damage to collagen bundles. Histological and immunohistochemical studies were conducted. Sections were stained with antibodies against MMP-3, aggrecanase 1 and 2, and their specific cleavage sites of aggrecan. Their localizations and staining ratios in the inner superficial and outer deep zones of the meniscus were determined separately. The population of chondrocyte-like cells increased with degeneration of the meniscus. MMP-3 and aggrecanase 1 and 2 are primarily expressed and activated in chondrocyte-like cells. MMP-3 expression and activation increased with degeneration and the population of chondrocyte-like cells. Changes in aggrecanase 1 expression with the degeneration were not clearly detected, whereas the expression of aggrecanase 2 was associated with progression of degeneration. MMP-3 and aggrecanases, particularly aggrecanase 2, expressed in chondrocyte-like cells could play important roles in aggrecan degradation in the human meniscus.

  20. Enantiopure copper(II) complex of natural product rosin derivative: DNA binding, DNA cleavage and cytotoxicity.

    Science.gov (United States)

    Fei, Bao-Li; Yin, Bin; Li, Dong-Dong; Xu, Wu-Shuang; Lu, Yang

    2016-12-01

    To develop chiral anticancer drug candidates for molecular target DNA, the synthesis and characterization of a novel enantiomerically pure copper(II) complex [Cu 1 Cl 2 ] (2) of an optically pure ligand N-(pyridin-2-ylmethylene) dehydroabietylamine (1) was carried out. The coordination geometry of the copper center is a distorted square-planar arrangement. The interactions of 1 and 2 with salmon sperm DNA were investigated by viscosity measurements, UV, fluorescence and circular dichroism (CD) spectroscopic techniques. All the results reveal that 1 and 2 interacted with DNA through intercalation and 2 exhibited a higher DNA binding ability. Further, 1 and 2 could cleave supercoiled pBR322 DNA by single strand and 2 displayed stronger cleavage ability in the presence of ascorbic acid. In vitro cytotoxicity of 1 and 2 against HeLa, SiHa, HepG-2 and A431 cancer cell lines was studied using CCK-8 assay. The results indicate that 2 had a superior cytotoxicity than 1 and the widely used drug cisplatin under identical conditions. Flow cytometry analysis demonstrates 2 produced death of HeLa cancer cells through an apoptotic pathway. Cell cycle analysis shows that 2 mainly arrested HeLa cells at the S phase. A novel enantiomerically pure copper(II) complex [Cu 1 Cl 2 ] (2) of an optically pure ligand N-(pyridin-2-ylmethylene) dehydroabietylamine (1), based on natural product rosin has been synthesized. 2 has the potential to act as effective anticancer drug.

  1. 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.

  2. Cloning and functional characterization of carotenoid cleavage dioxygenase 4 genes.

    Science.gov (United States)

    Huang, Fong-Chin; Molnár, Péter; Schwab, Wilfried

    2009-01-01

    Although a number of plant carotenoid cleavage dioxygenase (CCD) genes have been functionally characterized in different plant species, little is known about the biochemical role and enzymatic activities of members of the subclass 4 (CCD4). To gain insight into their biological function, CCD4 genes were isolated from apple (Malus x domestica, MdCCD4), chrysanthemum (Chrysanthemum x morifolium, CmCCD4a), rose (Rosa x damascena, RdCCD4), and osmanthus (Osmanthus fragrans, OfCCD4), and were expressed, together with AtCCD4, in Escherichia coli. In vivo assays showed that CmCCD4a and MdCCD4 cleaved beta-carotene well to yield beta-ionone, while OfCCD4, RdCCD4, and AtCCD4 were almost inactive towards this substrate. No cleavage products were found for any of the five CCD4 genes when they were co-expressed in E. coli strains that accumulated cis-zeta-carotene and lycopene. In vitro assays, however, demonstrated the breakdown of 8'-apo-beta-caroten-8'-al by AtCCD4 and RdCCD4 to beta-ionone, while this apocarotenal was almost not degraded by OfCCD4, CmCCD4a, and MdCCD4. Sequence analysis of genomic clones of CCD4 genes revealed that RdCCD4, like AtCCD4, contains no intron, while MdCCD, OfCCD4, and CmCCD4a contain introns. These results indicate that plants produce at least two different forms of CCD4 proteins. Although CCD4 enzymes cleave their substrates at the same position (9,10 and 9',10'), they might have different biochemical functions as they accept different (apo)-carotenoid substrates, show various expression patterns, and are genomically differently organized.

  3. 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.

  4. Memorizing Shape to Orient Cell Division.

    Science.gov (United States)

    Michel, Marcus; Dahmann, Christian

    2016-03-21

    A century ago, Oscar Hertwig discovered that cells orient their cleavage plane orthogonal to their long axis. Reporting recently in Nature, Bosveld et al. (2016) shed light on how, showing that NuMA/Mud localization at tricellular junctions provides mitotic cells with the memory of interphase shape used to orient cleavage plane. Copyright © 2016 Elsevier Inc. All rights reserved.

  5. Ion beam modifications of defect sub-structure of calcite cleavages

    Indian Academy of Sciences (India)

    WINTEC

    (a) Optical micrograph of calcite cleavage chemically etched with propionic acid with water (1 : 100) (rhombic etch pits; time 30 s). Scanning electron micrographs of calcite cleavages chemically etched after ion bombardment with different ener- gies: (b) 100 KeV, (c) 120 KeV and (d) 140 KeV. connecting Keithley (Model ...

  6. DNA binding and cleavage activity by a mononuclear iron(II)Schiff ...

    Indian Academy of Sciences (India)

    Spectroscopic and hydrodynamic investigations revealed intercalative mode of binding of 1 with DNA. 1 is also found to induce oxidative cleavage of the supercoiled pUC 18 DNA to its nicked circular form in a concentration dependent manner. Keywords. Iron(II); Schiff base; X-ray structure; DNA binding; DNA cleavage. 1.

  7. DNA binding and cleavage activity by a mononuclear iron (II) Schiff ...

    Indian Academy of Sciences (India)

    DNA binding and cleavage activity by a mononuclear iron(II)Schiff base complex: Synthesis and structural characterization. Abhijit Pal Bhaskar ... Iron(II); Schiff base; X-ray structure; DNA binding; DNA cleavage. ... Spectroscopic and hydrodynamic investigations revealed intercalative mode of binding of 1 with DNA. 1 is also ...

  8. Low-temperature side-chain cleavage and decarboxylation of polythiophene esters by acid catalysis

    DEFF Research Database (Denmark)

    Søndergaard, Roar; Norrman, Kion; Krebs, Frederik C

    2012-01-01

    substituents have been examined by TGA‐MS using different sulphonic acids. A substantial lowering of the cleavage temperature is observed, and the ester cleavage can even be performed in situ on roll‐to‐roll‐coated films on polyethylene terephthalate (PET). © 2011 Wiley Periodicals, Inc. J Polym Sci Part A...

  9. The dynactin complex is required for cleavage plane specification in early Caenorhabditis elegans embryos

    Science.gov (United States)

    Skop, Ahna R.; White, John G.

    2013-01-01

    Background During metazoan development, cell diversity arises primarily from asymmetric cell divisions which are executed in two phases: segregation of cytoplasmic factors and positioning of the mitotic spindle – and hence the cleavage plane – relative to the axis of segregation. When polarized cells divide, spindle alignment probably occurs through the capture and subsequent shortening of astral microtubules by a site in the cortex. Results Here, we report that dynactin, the dynein-activator complex, is localized at cortical microtubule attachment sites and is necessary for mitotic spindle alignment in early Caenorhabditis elegans embryos. Using RNA interference techniques, we eliminated expression in early embryos of dnc-1 (the ortholog of the vertebrate gene for p150Glued) and dnc-2 (the ortholog of the vertebrate gene for p50/Dynamitin). In both cases, misalignment of mitotic spindles occurred, demonstrating that two components of the dynactin complex, DNC-1 and DNC-2, are necessary to align the spindle. Conclusions Dynactin complexes may serve as a tether for dynein at the cortex and allow dynein to produce forces on the astral microtubules required for mitotic spindle alignment. PMID:9778526

  10. 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.

  11. Prediction of in-vitro developmental competence of early cleavage-stage mouse embryos with compact time-lapse equipment.

    Science.gov (United States)

    Pribenszky, Csaba; Losonczi, Eszter; Molnár, Miklós; Lang, Zsolt; Mátyás, Szabolcs; Rajczy, Klára; Molnár, Katalin; Kovács, Péter; Nagy, Péter; Conceicao, Jason; Vajta, Gábor

    2010-03-01

    Single blastocyst transfer is regarded as an efficient way to achieve high pregnancy rates and to avoid multiple pregnancies. Risk of cancellation of transfer due to a lack of available embryos may be reduced by early prediction of blastocyst development. Time-lapse investigation of mouse embryos shows that the time of the first and second cleavage (to the 2- and 3-cell stages, respectively) has a strong predictive value for further development in vitro, while cleavage from the 3-cell to the 4-cell stage has no predictive value. In humans, embryo fragmentation during preimplantation development has been associated with lower pregnancy rates and a higher incidence of developmental abnormalities. Analysis of time-lapse records shows that most fragmentation is reversible in the mouse and is resorbed in an average of 9 h. Daily or bi-daily microscopic checks of embryo development, applied routinely in human IVF laboratories, would fail to detect 36 or 72% of these fragmentations, respectively. Fragmentation occurring in a defined time frame has a strong predictive value for in-vitro embryo development. The practical compact system used in the present trial, based on the 'one camera per patient' principle, has eliminated the usual disadvantages of time-lapse investigations and is applicable for the routine follow-up of in-vitro embryo development. Copyright 2009 Reproductive Healthcare Ltd. Published by Elsevier Ltd. All rights reserved.

  12. Proteolytic receptor cleavage in the pathogenesis of blood rheology and co-morbidities in metabolic syndrome. Early forms of autodigestion.

    Science.gov (United States)

    Mazor, Rafi; Schmid-Schönbein, Geert W

    2015-01-01

    Abnormal blood rheological properties seldom occur in isolation and instead are accompanied by other complications, often designated as co-morbidities. In the metabolic syndrome with complications like hypertension, diabetes and lack of normal microvascular blood flow, the underlying molecular mechanisms that simultaneously lead to elevated blood pressure and diabetes as well as abnormal microvascular rheology and other cell dysfunctions have remained largely unknown. In this review, we propose a new hypothesis for the origin of abnormal cell functions as well as multiple co-morbidities. Utilizing experimental models for the metabolic disease with diverse co-morbidities we summarize evidence for the presence of an uncontrolled extracellular proteolytic activity that causes ectodomain receptor cleavage and loss of their associated cell function. We summarize evidence for unchecked degrading proteinase activity, e.g. due to matrix metalloproteases, in patients with hypertension, Type II diabetes and obesity, in addition to evidence for receptor cleavage in the form of receptor fragments and decreased extracellular membrane expression levels. The evidence suggest that a shift in blood rheological properties and other co-morbidities may in fact be derived from a common mechanism that is due to uncontrolled proteolytic activity, i.e. an early form of autodigestion. Identification of the particular proteases involved and the mechanisms of their activation may open the door to treatment that simultaneously targets multiple co-morbidities in the metabolic syndrome.

  13. 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.

  14. p38 Activation Is Required Upstream of Potassium Current Enhancement and Caspase Cleavage in Thiol Oxidant-Induced Neuronal Apoptosis

    Science.gov (United States)

    McLaughlin, BethAnn; Pal, Sumon; Tran, Minhnga P.; Parsons, Andrew A.; Barone, Frank C.; Erhardt, Joseph A.; Aizenman, Elias

    2013-01-01

    Oxidant-induced neuronal apoptosis has been shown to involve potassium and zinc dysregulation, energetic dysfunction, activation of stress-related kinases, and caspase cleavage. The temporal ordering and interdependence of these events was investigated in primary neuronal cultures exposed to the sulfhydryl oxidizing agent 2,2′-dithiodipyridine (DTDP), a compound that induces the intracellular release of zinc. We previously observed that tetraethylammonium (TEA), high extracellular potassium, or cysteine protease inhibitors block apoptosis induced by DTDP. We now report that both p38 and extracellular signal-regulated kinase phosphorylation are evident in neuronal cultures within 2 hr of a brief exposure to 100 μm DTDP. However, only p38 inhibition is capable of blocking oxidant-induced toxicity. Cyclohexamide or actinomycin D does not attenuate DTDP-induced cell death, suggesting that posttranslational modification of existing targets, rather than transcriptional activation, is responsible for the deleterious effects of p38. Indeed, an early robust increase in TEA-sensitive potassium channel currents induced by DTDP is attenuated by p38 inhibition but not by caspase inhibition. Moreover, we found that activation of p38 is required for caspase 3 and 9 cleavage, suggesting that potassium currents enhancement is required for caspase activation. Finally, we observed that DTDP toxicity could be blocked with niacinamide or benzamide, inhibitors of poly (ADP-ribose) synthetase. Based on these findings, we conclude that oxidation of sulfhydryl groups on intracellular targets results in intracellular zinc release, p38 phosphorylation, enhancement of potassium currents, caspase cleavage, energetic dysfunction, and translationally independent apoptotic cell death. PMID:11331359

  15. Influence of the antimitotic phenanthrenequinone on the cleavage of the eggs of the mollusc,Mytilus edulis L.

    Science.gov (United States)

    Abd-El-Wahab, A

    1958-07-01

    1. Phenanthrenequinone, in concentrations above 1∶2 M, produces cytolysis. 2. It causes blockage in the single cell stage in concentrations down to 1∶200 M. 3. The main area of antimitotic activity lies in between 1∶2 M to 1∶20 M. 4. Most of the abnormal forms are like those produced by naphthoquinone. 5. A new and so far unobserved "linear type of cleavage" is found at the three- and four-cell stages. 6. Phenanthrenequinone is relatively more toxic than naphthoquinone. 7. Phenanthrenequinone can also affect the nuclear apparatus, producing pyenosis. 8. It can also inhibit the cell cortex in the same manner as naphthoquinone: there is no evidence that in Mytilus one quinone stimulates cortical expansion and the other cortical contraction.

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

    Science.gov (United States)

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

    2016-01-01

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

  17. 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.

  18. Genetic stability of attenuated mengovirus vectors with duplicate primary cleavage sequences

    International Nuclear Information System (INIS)

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

    2003-01-01

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

  19. Dengue and Zika viruses subvert reticulophagy by NS2B3-mediated cleavage of FAM134B.

    Science.gov (United States)

    Lennemann, Nicholas J; Coyne, Carolyn B

    2017-02-01

    The endoplasmic reticulum (ER) is exploited by several diverse viruses during their infectious life cycles. Flaviviruses, including dengue virus (DENV) and Zika virus (ZIKV), utilize the ER as a source of membranes to establish their replication organelles and to facilitate their assembly and eventual maturation along the secretory pathway. To maintain normal homeostasis, host cells have evolved highly efficient processes to dynamically regulate the ER, such as through reticulophagy, a selective form of autophagy that leads to ER degradation. Here, we identify the ER-localized reticulophagy receptor FAM134B as a host cell restriction factor for both DENV and ZIKV. We show that RNAi-mediated depletion of FAM134B significantly enhances both DENV and ZIKV replication at an early stage of the viral life cycle. Consistent with its role as an antiviral host factor, we found that several flaviviruses including DENV, ZIKV, and West Nile virus (WNV), utilize their NS3 virally-encoded proteases to directly cleave FAM134B at a single site within its reticulon homology domain (RHD). Mechanistically, we show that NS3-mediated cleavage of FAM134B blocks the formation of ER and viral protein-enriched autophagosomes, suggesting that the cleavage of FAM134B serves to specifically suppress the reticulophagy pathway. These findings thus point to an important role for FAM134B and reticulophagy in the regulation of flavivirus infection and suggest that these viruses specifically target these pathways to promote viral replication.

  20. 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.

  1. Characterization of clade 2.3.4.4 H5N8 highly pathogenic avian influenza viruses from wild birds possessing atypical hemagglutinin polybasic cleavage sites.

    Science.gov (United States)

    Usui, Tatsufumi; Soda, Kosuke; Tomioka, Yukiko; Ito, Hiroshi; Yabuta, Toshiyo; Takakuwa, Hiroki; Otsuki, Koichi; Ito, Toshihiro; Yamaguchi, Tsuyoshi

    2017-02-01

    Since 2014, clade 2.3.4.4 H5 subtype highly pathogenic avian influenza viruses (HPAIVs) have been distributed worldwide. These viruses, which were reported to be highly virulent in chickens by intravenous inoculation, have a consensus HPAI motif PLRERRRKR at the HA cleavage site. However, two-clade 2.3.4.4 H5N8 viruses which we isolated from wild migratory birds in late 2014 in Japan possessed atypical HA cleavage sequences. A swan isolate, Tottori/C6, had a novel polybasic cleavage sequence, PLGERRRKR, and another isolate from a dead mandarin duck, Gifu/01, had a heterogeneous mixture of consensus PLRERRRKR and variant PLRERRRRKR sequences. The polybasic HA cleavage site is the prime virulence determinant of AIVs. Therefore, in the present study, we examined the pathogenicity of these H5N8 isolates in chickens by intravenous inoculation. When 10 6 EID 50 of these viruses were intravenously inoculated into chickens, the mean death time associated with Tottori/C6 was substantially longer (>6.1 days) than that associated with Gifu/01 (2.5 days). These viruses had comparable abilities to replicate in tissue culture cells in the presence and absence of exogenous trypsin, but the growth of Tottori/C6 was hampered. These results indicate that the novel cleavage motif of Tottori/C6 did not directly affect the infectivity of the virus, but Tottori/C6 caused attenuated pathogenicity in chickens because of hampered replication efficiency. It is important to test for the emergence of diversified HPAIVs, because introduction of HPAIVs with a lower virulence like Tottori/C6 might hinder early detection of affected birds in poultry farms.

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

    International Nuclear Information System (INIS)

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

    2011-01-01

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

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

    Directory of Open Access Journals (Sweden)

    Jennifer L Fuentes

    2010-12-01

    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.

  4. 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.

  5. Copper(II) complex of methionine conjugated bis-pyrazole based ligand promotes dual pathway for DNA cleavage.

    Science.gov (United States)

    Bhattacharyya, Sudipta; Sarkar, Amrita; Dey, Suman Kr; Jose, Gregor P; Mukherjee, Arindam; Sengupta, Tapas K

    2013-08-28

    Three Cu(II) complexes of bis-pyrazole based ligands have been synthesized and structurally characterized by X-ray crystallography. One of the ligand (L2) contains a methionine ester conjugated to a bis-pyrazole carboxylate through an amide linkage. The binding constant for complexes 1-3 with CT DNA are of the order of 10(4) M(-1). The crystal structure suggests that the axial Cu-O bonds (ca. 2.31(4) Å) are relatively labile and hence during the redox cycle with ascorbic acid and oxygen one or both the axial Cu-O bonds might open to promote copper oxygen reaction and generate ROS. The chemical nuclease activity of complexes 1-3 in dark, show complete relaxation of supercoiled DNA at 100 μM concentration in presence of ascorbic acid (H2A). The mechanistic investigation suggests that the complexes 1 and 2 show involvement of peroxo species whereas 3 shows involvement of both singlet oxygen and peroxo species in DNA cleavage. The singlet oxygen formation in dark is otherwise unfavourable but the presence of methionine as pendant arm in L2 might activate the generation of singlet oxygen from the metal generated peroxo species. The results of DNA cleavage studies suggest that methionine based copper(II) complexes can promote dual pathway for DNA cleavage. Probing the cytotoxic activity of these complexes on MCF-7, human breast cancer cell line shows that 3 is the most effective one with an IC50 of 70(2) μM.

  6. Reactivity of hydropersulfides toward the hydroxyl radical unraveled: disulfide bond cleavage, hydrogen atom transfer, and proton-coupled electron transfer.

    Science.gov (United States)

    Anglada, Josep M; Crehuet, Ramon; Adhikari, Sarju; Francisco, Joseph S; Xia, Yu

    2018-02-14

    Hydropersulfides (RSSH) are highly reactive as nucleophiles and hydrogen atom transfer reagents. These chemical properties are believed to be key for them to act as antioxidants in cells. The reaction involving the radical species and the disulfide bond (S-S) in RSSH, a known redox-active group, however, has been scarcely studied, resulting in an incomplete understanding of the chemical nature of RSSH. We have performed a high-level theoretical investigation on the reactions of the hydroxyl radical (˙OH) toward a set of RSSH (R = -H, -CH 3 , -NH 2 , -C(O)OH, -CN, and -NO 2 ). The results show that S-S cleavage and H-atom abstraction are the two competing channels. The electron inductive effect of R induces selective ˙OH substitution at one sulfur atom upon S-S cleavage, forming RSOH and ˙SH for the electron donating groups (EDGs), whereas producing HSOH and ˙SR for the electron withdrawing groups (EWGs). The H-Atom abstraction by ˙OH follows a classical hydrogen atom transfer (hat) mechanism, producing RSS˙ and H 2 O. Surprisingly, a proton-coupled electron transfer (pcet) process also occurs for R being an EDG. Although for RSSH having EWGs hat is the leading channel, S-S cleavage can be competitive or even dominant for the EDGs. The overall reactivity of RSSH toward ˙OH attack is greatly enhanced with the presence of an EDG, with CH 3 SSH being the most reactive species found in this study (overall rate constant: 4.55 × 10 12 M -1 s -1 ). Our results highlight the complexity in RSSH reaction chemistry, the extent of which is closely modulated by the inductive effect of the substituents in the case of the oxidation by hydroxyl radicals.

  7. Amine Oxidative N-Dealkylation via Cupric Hydroperoxide Cu–OOH Homolytic Cleavage Followed by Site-Specific Fenton Chemistry

    Science.gov (United States)

    Kim, Sunghee; Ginsbach, Jake W.; Lee, Jung Yoon; Peterson, Ryan L.; Liu, Jeffrey J.; Siegler, Maxime A.; Sarjeant, Amy A.; Solomon, Edward I.; Karlin, Kenneth D.

    2015-01-01

    Copper(II)-hydroperoxide species are significant intermediates in processes such as fuel cells and (bio)chemical oxidations, all involving stepwise reduction of molecular oxygen. We previously reported a CuII-OOH species that performs oxidative N-dealkylation on a dibenzylamino group that is appended to the 6-position of a pyridyl donor of a tripodal tetradentate ligand. To obtain insights into the mechanism of this process, reaction kinetics and products were determined employing ligand substrates with various para- substituent dibenzyl pairs (-H,-H; -H,-Cl; -H,-OMe and -Cl,-OMe), or with partially or fully deuterated dibenzyl N-(CH2Ph)2 moieties. A series of ligand-copper(II) bis-perchlorate complexes were synthesized, characterized, and the X-ray structures of the -H, -OMe analog was were determined. The corresponding metastable CuII-OOH species were generated by addition of H2O2/base in acetone at –90 °C. These convert (t1/2 ~ 53 s) to oxidatively N-dealkylated products, producing para-substituted benzaldehydes. Based on the experimental observations and supporting DFT calculations, a reaction mechanism involving dibenzylamine H-atom abstraction or electron-transfer oxidation by the CuII-OOH entity could be ruled out. It is concluded that the chemistry proceeds by rate limiting Cu–O homolytic cleavage of the CuII–(OOH) species, followed by site-specific copper Fenton chemistry. As a process of broad interest in copper as well as iron oxidative (bio)chemistries, a detailed computational analysis was performed, indicating that a CuIOOH species undergoes O–O homolytic cleavage to yield a hydroxyl radical and CuIIOH rather than heterolytic cleavage to yield water and a CuII-O•−. PMID:25706825

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

    Directory of Open Access Journals (Sweden)

    Anna Konopka

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

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

    Science.gov (United States)

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

    2016-01-01

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

  10. Dynamic propagation and cleavage crack arrest in bainitic steel

    International Nuclear Information System (INIS)

    Hajjaj, M.

    2006-06-01

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

  11. 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

  12. Parvovirus Capsid Structures Required for Infection: Mutations Controlling Receptor Recognition and Protease Cleavages.

    Science.gov (United States)

    Callaway, Heather M; Feng, Kurtis H; Lee, Donald W; Allison, Andrew B; Pinard, Melissa; McKenna, Robert; Agbandje-McKenna, Mavis; Hafenstein, Susan; Parrish, Colin R

    2017-01-15

    Parvovirus capsids are small but complex molecular machines responsible for undertaking many of the steps of cell infection, genome packing, and cell-to-cell as well as host-to-host transfer. The details of parvovirus infection of cells are still not fully understood, but the processes must involve small changes in the capsid structure that allow the endocytosed virus to escape from the endosome, pass through the cell cytoplasm, and deliver the single-stranded DNA (ssDNA) genome to the nucleus, where viral replication occurs. Here, we examine capsid substitutions that eliminate canine parvovirus (CPV) infectivity and identify how those mutations changed the capsid structure or altered interactions with the infectious pathway. Amino acid substitutions on the exterior surface of the capsid (Gly299Lys/Ala300Lys) altered the binding of the capsid to transferrin receptor type 1 (TfR), particularly during virus dissociation from the receptor, but still allowed efficient entry into both feline and canine cells without successful infection. These substitutions likely control specific capsid structural changes resulting from TfR binding required for infection. A second set of changes on the interior surface of the capsid reduced viral infectivity by >100-fold and included two cysteine residues and neighboring residues. One of these substitutions, Cys270Ser, modulates a VP2 cleavage event found in ∼10% of the capsid proteins that also was shown to alter capsid stability. A neighboring substitution, Pro272Lys, significantly reduced capsid assembly, while a Cys273Ser change appeared to alter capsid transport from the nucleus. These mutants reveal additional structural details that explain cell infection processes of parvovirus capsids. Parvoviruses are commonly found in both vertebrate and invertebrate animals and cause widespread disease. They are also being developed as oncolytic therapeutics and as gene therapy vectors. Most functions involved in infection or transduction

  13. RNA synthesis during cleavage of the Lymnaea egg

    NARCIS (Netherlands)

    Biggelaar, J.A.M. van den

    In eggs of Lymnaea RNA synthesis can be detected autoradiographically from the 8- to the 16-cell stage. From the 16- to the 24-cell stage distinct nucleoli reappear which are immediately engaged in RNA synthesis.

  14. Dinitrogen cleavage and functionalization by carbon monoxide promoted by a hafnium complex.

    Science.gov (United States)

    Knobloch, Donald J; Lobkovsky, Emil; Chirik, Paul J

    2010-01-01

    Molecular nitrogen (N(2)) and carbon monoxide (CO) have the two strongest bonds in chemistry and present significant challenges in developing new transformations that exploit these two abundant feedstocks. At the core of this objective is the discovery of transition-metal compounds that promote the six-electron reductive cleavage of N(2) at ambient temperature and pressure and also promote new nitrogen-element bond formation. Here we show that an organometallic hafnium compound induces N(2) cleavage on the addition of CO, with a simultaneous assembly of new nitrogen-carbon and carbon-carbon bonds. Subsequent addition of a weak acid liberates oxamide, which demonstrates that an important agrochemical can be synthesized directly from N(2) and CO. These studies introduce an alternative paradigm for N(2) cleavage and functionalization in which the six-electron reductive cleavage is promoted by both the transition metal and the incoming ligand, CO, used for the new bond formations.

  15. Effect of chilling on the development of in vitro produced bovine embryos at various cleavage stages

    OpenAIRE

    Balasubramanian, S.; Rho, Gyu-Jin

    2006-01-01

    Purpose: Bovine embryos and zygotes are known to be sensitive to “temperature shock” when cooled to temperatures near 0°C. The effect of chilling on in vitro derived embryos at various cleavage stages was investigated.

  16. 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.

  17. How to find simple and accurate rules for viral protease cleavage specificities

    Directory of Open Access Journals (Sweden)

    Garwicz Daniel

    2009-05-01

    Full Text Available Abstract Background Proteases of human pathogens are becoming increasingly important drug targets, hence it is necessary to understand their substrate specificity and to interpret this knowledge in practically useful ways. New methods are being developed that produce large amounts of cleavage information for individual proteases and some have been applied to extract cleavage rules from data. However, the hitherto proposed methods for extracting rules have been neither easy to understand nor very accurate. To be practically useful, cleavage rules should be accurate, compact, and expressed in an easily understandable way. Results A new method is presented for producing cleavage rules for viral proteases with seemingly complex cleavage profiles. The method is based on orthogonal search-based rule extraction (OSRE combined with spectral clustering. It is demonstrated on substrate data sets for human immunodeficiency virus type 1 (HIV-1 protease and hepatitis C (HCV NS3/4A protease, showing excellent prediction performance for both HIV-1 cleavage and HCV NS3/4A cleavage, agreeing with observed HCV genotype differences. New cleavage rules (consensus sequences are suggested for HIV-1 and HCV NS3/4A cleavages. The practical usability of the method is also demonstrated by using it to predict the location of an internal cleavage site in the HCV NS3 protease and to correct the location of a previously reported internal cleavage site in the HCV NS3 protease. The method is fast to converge and yields accurate rules, on par with previous results for HIV-1 protease and better than previous state-of-the-art for HCV NS3/4A protease. Moreover, the rules are fewer and simpler than previously obtained with rule extraction methods. Conclusion A rule extraction methodology by searching for multivariate low-order predicates yields results that significantly outperform existing rule bases on out-of-sample data, but are more transparent to expert users. The approach

  18. The spt5 C-terminal region recruits yeast 3' RNA cleavage factor I.

    Science.gov (United States)

    Mayer, Andreas; Schreieck, Amelie; Lidschreiber, Michael; Leike, Kristin; Martin, Dietmar E; Cramer, Patrick

    2012-04-01

    During transcription elongation, RNA polymerase II (Pol II) binds the general elongation factor Spt5. Spt5 contains a repetitive C-terminal region (CTR) that is required for cotranscriptional recruitment of the Paf1 complex (D. L. Lindstrom et al., Mol. Cell. Biol. 23:1368-1378, 2003; Z. Zhang, J. Fu, and D. S. Gilmour, Genes Dev. 19:1572-1580, 2005). Here we report a new role of the Spt5 CTR in the recruitment of 3' RNA-processing factors. Chromatin immunoprecipitation (ChIP) revealed that the Spt5 CTR is required for normal recruitment of pre-mRNA cleavage factor I (CFI) to the 3' ends of Saccharomyces cerevisiae genes. RNA contributes to CFI recruitment, as RNase treatment prior to ChIP further decreases CFI ChIP signals. Genome-wide ChIP profiling detected occupancy peaks of CFI subunits around 100 nucleotides downstream of the polyadenylation (pA) sites of genes. CFI recruitment to this defined region may result from simultaneous binding to the Spt5 CTR, to nascent RNA containing the pA sequence, and to the elongating Pol II isoform that is phosphorylated at serine 2 (S2) residues in its C-terminal domain (CTD). Consistent with this model, the CTR interacts with CFI in vitro but is not required for pA site recognition and transcription termination in vivo.

  19. CRISPR/Cas9 nuclease cleavage combined with Gibson assembly for seamless cloning.

    Science.gov (United States)

    Wang, Jia-Wang; Wang, Amy; Li, Kunyu; Wang, Bangmei; Jin, Shunqian; Reiser, Michelle; Lockey, Richard F

    2015-04-01

    Restriction enzymes have two major limitations for cloning: they cannot cleave at any desired location in a DNA sequence and may not cleave uniquely within a DNA sequence. In contrast, the clustered regularly interspaced short palindromic repeat (CRISPR)-associated enzyme 9 (Cas9), when coupled with single guide RNAs (sgRNA), has been used in vivo to cleave the genomes of many species at a single site, enabling generation of mutated cell lines and animals. The Cas9/sgRNA complex recognizes a 17-20 base target site, which can be of any sequence as long as it is located 5' of the protospacer adjacent motif (PAM; sequence 5'-NRG, where R = G or A). Thus, it can be programmed to cleave almost anywhere with a stringency higher than that of one cleavage in a sequence of human genome size. Here, the Cas9 enzyme and a specific sgRNA were used to linearize a 22 kb plasmid in vitro. A DNA fragment was then inserted into the linearized vector seamlessly through Gibson assembly. Our technique can be used to directly, and seamlessly, clone fragments into vectors of any size as well as to modify existing constructs where no other methods are available.

  20. Understanding in-line probing experiments by modeling cleavage of nonreactive RNA nucleotides.

    Science.gov (United States)

    Mlýnský, Vojtěch; Bussi, Giovanni

    2017-05-01

    Ribonucleic acid (RNA) is involved in many regulatory and catalytic processes in the cell. The function of any RNA molecule is intimately related with its structure. In-line probing experiments provide valuable structural data sets for a variety of RNAs and are used to characterize conformational changes in riboswitches. However, the structural determinants that lead to differential reactivities in unpaired nucleotides have not been investigated yet. In this work, we used a combination of theoretical approaches, i.e., classical molecular dynamics simulations, multiscale quantum mechanical/molecular mechanical calculations, and enhanced sampling techniques in order to compute and interpret the differential reactivity of individual residues in several RNA motifs, including members of the most important GNRA and UNCG tetraloop families. Simulations on the multinanosecond timescale are required to converge the related free-energy landscapes. The results for uGAAAg and cUUCGg tetraloops and double helices are compared with available data from in-line probing experiments and show that the introduced technique is able to distinguish between nucleotides of the uGAAAg tetraloop based on their structural predispositions toward phosphodiester backbone cleavage. For the cUUCGg tetraloop, more advanced ab initio calculations would be required. This study is the first attempt to computationally classify chemical probing experiments and paves the way for an identification of tertiary structures based on the measured reactivity of nonreactive nucleotides. © 2017 Mlýnský and Bussi; Published by Cold Spring Harbor Laboratory Press for the RNA Society.

  1. Selective formation of discrete versus polymeric copper organophosphates: DNA cleavage and cytotoxic activity.

    Science.gov (United States)

    Bhat, Gulzar A; Maqbool, Raihana; Dar, Aijaz A; Ul Hussain, Mahboob; Murugavel, Ramaswamy

    2017-10-10

    Copper phosphate metalloligands [Cu(X-dipp)(Pyterpy)] 2 [X = H (1), Br (2)], exemplifying expanded 4,4'-bipyridine type molecules, have been synthesized by reacting 4'-(4-pyridyl)-2,2':6',2''-terpyridine (Pyterpy) and para substituted 2,6-diisopropylphenyl phosphate (X-dippH 2 ) with copper acetate. The pendant N,N-ends of dimeric copper phosphates 1 and 2 have been forced to engage in further coordination by limiting the concentration of Pyterpy in the reaction mixture to yield rare Pyterpy bridged corner-shared polymeric copper phosphates [Cu 2 (X-dippH)(X-dipp)(Pyterpy)(H 2 O)] n [X = Cl (3), Br (4), I (5)]. The formation of 1-5 is supported by spectroscopic and analytical data. The solid state structures of these compounds have further been confirmed by single-crystal X-ray diffraction studies. Soluble dimeric complexes 1 and 2 have been assessed for their in vitro anti-tumour properties against human breast and colorectal cancer cell lines. The DNA cleavage, protein cleaving and cytotoxicity assays revealed that these compounds are effective in cleaving DNA, while the activity of 1 as an anti-tumor agent is better than 2.

  2. 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.

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

    Science.gov (United States)

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

    2015-10-01

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

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

    OpenAIRE

    Hui-Bon-Hoa, Gaston; Kaddour, Hussein; Vergne, Jacques; Kruglik, Sergei G; Maurel, Marie-Christine

    2014-01-01

    International audience; 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 ...

  5. Structural insight into the quinolone-DNA cleavage complex of type IIA topoisomerases.

    Science.gov (United States)

    Laponogov, Ivan; Sohi, Maninder K; Veselkov, Dennis A; Pan, Xiao-Su; Sawhney, Ritica; Thompson, Andrew W; McAuley, Katherine E; Fisher, L Mark; Sanderson, Mark R

    2009-06-01

    Type II topoisomerases alter DNA topology by forming a covalent DNA-cleavage complex that allows DNA transport through a double-stranded DNA break. We present the structures of cleavage complexes formed by the Streptococcus pneumoniae ParC breakage-reunion and ParE TOPRIM domains of topoisomerase IV stabilized by moxifloxacin and clinafloxacin, two antipneumococcal fluoroquinolones. These structures reveal two drug molecules intercalated at the highly bent DNA gate and help explain antibacterial quinolone action and resistance.

  6. 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....

  7. Analysis of Thisbe and Pyramus functional domains reveals evidence for cleavage of Drosophila FGFs

    Directory of Open Access Journals (Sweden)

    Stathopoulos Angelike

    2010-08-01

    Full Text Available Abstract Background As important regulators of developmental and adult processes in metazoans, Fibroblast Growth Factor (FGF proteins are potent signaling molecules whose activities must be tightly regulated. FGFs are known to play diverse roles in many processes, including mesoderm induction, branching morphogenesis, organ formation, wound healing and malignant transformation; yet much more remains to be learned about the mechanisms of regulation used to control FGF activity. Results In this work, we conducted an analysis of the functional domains of two Drosophila proteins, Thisbe (Ths and Pyramus (Pyr, which share homology with the FGF8 subfamily of ligands in vertebrates. Ths and Pyr proteins are secreted from Drosophila Schneider cells (S2 as smaller N-terminal fragments presumably as a result of intracellular proteolytic cleavage. Cleaved forms of Ths and Pyr can be detected in embryonic extracts as well. The FGF-domain is contained within the secreted ligand portion, and this domain alone is capable of functioning in the embryo when ectopically expressed. Through targeted ectopic expression experiments in which we assay the ability of full-length, truncated, and chimeric proteins to support cell differentiation, we find evidence that (1 the C-terminal domain of Pyr is retained inside the cell and does not seem to be required for receptor activation and (2 the C-terminal domain of Ths is secreted and, while also not required for receptor activation, this domain does plays a role in limiting the activity of Ths when present. Conclusions We propose that differential protein processing may account for the previously observed inequalities in signaling capabilities between Ths and Pyr. While the regulatory mechanisms are likely complex, studies such as ours conducted in a tractable model system may be able to provide insights into how ligand processing regulates growth factor activity.

  8. Synthesis, DNA binding, photo-induced DNA cleavage, cytotoxicity studies of a family of heavy rare earth complexes.

    Science.gov (United States)

    Chen, Gong-Jun; Wang, Zhi-Gang; Qiao, Xin; Xu, Jing-Yuan; Tian, Jin-Lei; Yan, Shi-Ping

    2013-10-01

    As a continuing investigation of our previous studies about the influence of the different rare earth metal ions on the bioactivity, a family of heavy rare earth metal complexes, [RE(acac)3(dpq)] (RE=Tb (1), Dy (2), Ho (3), Er (4), Tm (5), Yb (6), Lu (7)) and [RE(acac)3(dppz)]·CH3OH (RE=Tb (8), Dy (9), Ho (10), Er (11), Tm (12), Yb (13), Lu (14) viz. acetylacetonate (acac), dipyrido[3,2-d:20,30-f]quinoxaline (dpq), dipyrido[3,2-a:20,30-c] phenazine (dppz)), has been synthesized and their biological activities were also investigated. On the irradiation with UV-A light of 365nm or ambient light, all complexes exhibit efficient DNA cleavage activity via the mechanistic pathway involving the formation of singlet oxygen and hydroxyl radical as the reactive species. In addition, the in vitro cytotoxicity of these complexes on HeLa cells has been examined by MTT assay, which indicate that these compounds have the potential to act as effective anticancer drugs. The results of the above biological experiments also reveal that the choice of different rare earth metal ions has little influence on the DNA binding, DNA cleavage and cytotoxicity. Copyright © 2013 Elsevier Inc. All rights reserved.

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

    Directory of Open Access Journals (Sweden)

    Nguyen Albert T

    2011-12-01

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

  10. 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

    2017-07-01

    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.

  11. Alanine substitutions in the GXXXG motif alter C99 cleavage by γ-secretase but not its dimerization.

    Science.gov (United States)

    Higashide, Hidekazu; Ishihara, Seiko; Nobuhara, Mika; Ihara, Yasuo; Funamoto, Satoru

    2017-03-01

    The amyloid β (Aβ) protein is a major component of senile plaques, one of the neuropathological hallmarks of Alzheimer's disease. Amyloidogenic processing of amyloid precursor protein (APP) by β- and γ-secretases leads to production of Aβ. APP contains tandem triple repeats of the GXXXG motif in its extracellular juxtamembrane and transmembrane regions. It is reported that the GXXXG motif is related to protein-protein interactions, but it remains controversial whether the GXXXG motif in APP is involved in substrate dimerization and whether dimerization affects γ-secretase-dependent cleavage. Therefore, the relationship between the GXXXG motifs, substrate dimerization, and γ-secretase-dependent cleavage sites remains unclear. Here, we applied blue native poly acrylamide gel electrophoresis to examine the effect of alanine substitutions within the GXXXG motifs of APP carboxyl terminal fragment (C99) on its dimerization and Aβ production. Surprisingly, alanine substitutions in the motif failed to alter C99 dimerization in detergent soluble state. Cell-based and solubilized γ-secretase assays demonstrated that increasing alanine substitutions in the motif tended to decrease long Aβ species such as Aβ42 and Aβ43 and to increase in short Aβ species concomitantly. Our data suggest that the GXXXG motif is crucial for Aβ production, but not for C99 dimerization. © 2016 International Society for Neurochemistry.

  12. Pathological phenotypes and in vivo DNA cleavage by unrestrained activity of a phosphorothioate-based restriction system in Salmonella

    Science.gov (United States)

    Cao, Bo; Cheng, Qiuxiang; Gu, Chen; Yao, Fen; DeMott, Michael S.; Zheng, Xiaoqing; Deng, Zixin; Dedon, Peter C.; You, Delin

    2015-01-01

    Summary Prokaryotes protect their genomes from foreign DNA with a diversity of defense mechanisms, including a widespread restriction-modification (R-M) system involving phosphorothioate (PT) modification of the DNA backbone. Unlike classical R-M systems, highly partial PT-modification of consensus motifs in bacterial genomes suggests an unusual mechanism of PT-dependent restriction. In Salmonella enterica, PT modification is mediated by four genes dptB-E, while restriction involves additional three genes dptF-H. Here, we performed a series of studies to characterize the PT-dependent restriction, and found that it presented several features distinct with traditional R-M systems. The presence of restriction genes in a PT-deficient mutant was not lethal, but instead resulted in several pathological phenotypes. Subsequent transcriptional profiling revealed the expression of >600 genes was affected by restriction enzymes in cells lacking PT, including induction of bacteriophage, SOS response and DNA repair-related genes. These transcriptional responses are consistent with the observation that restriction enzymes caused extensive DNA cleavage in the absence of PT modifications in vivo. However, over-expression of restriction genes was lethal to the host in spite of the presence PT modifications. These results point to an unusual mechanism of PT-dependent DNA cleavage by restriction enzymes in the face of partial PT modification. PMID:25040300

  13. Cleavage of phosphorothioated DNA and methylated DNA by the type IV restriction endonuclease ScoMcrA.

    Directory of Open Access Journals (Sweden)

    Guang Liu

    2010-12-01

    Full Text Available Many taxonomically diverse prokaryotes enzymatically modify their DNA by replacing a non-bridging oxygen with a sulfur atom at specific sequences. The biological implications of this DNA S-modification (phosphorothioation were unknown. We observed that simultaneous expression of the dndA-E gene cluster from Streptomyces lividans 66, which is responsible for the DNA S-modification, and the putative Streptomyces coelicolor A(32 Type IV methyl-dependent restriction endonuclease ScoA3McrA (Sco4631 leads to cell death in the same host. A His-tagged derivative of ScoA3McrA cleaved S-modified DNA and also Dcm-methylated DNA in vitro near the respective modification sites. Double-strand cleavage occurred 16-28 nucleotides away from the phosphorothioate links. DNase I footprinting demonstrated binding of ScoA3McrA to the Dcm methylation site, but no clear binding could be detected at the S-modified site under cleavage conditions. This is the first report of in vitro endonuclease activity of a McrA homologue and also the first demonstration of an enzyme that specifically cleaves S-modified DNA.

  14. 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.

  15. High-level expression of an antimicrobial peptide histonin as a natural form by multimerization and furin-mediated cleavage.

    Science.gov (United States)

    Kim, Jung Min; Jang, Su A; Yu, Byung Jo; Sung, Bong Hyun; Cho, Ju Hyun; Kim, Sun Chang

    2008-02-01

    Direct expression of an antimicrobial peptide (AMP) in Escherichia coli causes several problems such as the toxicity of AMP to the host cell, its susceptibility to proteolytic degradation, and decreased antimicrobial activity due to the additional residue(s) introduced after cleavage of AMPs from fusion partners. To overcome these problems and produce a large quantity of a potent AMP histonin (RAGLQFPVGKLLKKLLKRLKR) in E. coli, an efficient expression system was developed, in which the toxicity of histonin was neutralized by a fusion partner F4 (a truncated fragment of PurF protein) and the productivity was increased by a multimeric expression of a histonin gene. The expression level of the fusion proteins reached a maximum with a 12-mer of a histonin gene. In addition, because of the RLKR residues present at the C terminus of histonin, furin cleavage of the multimeric histonin expressed produces an intact, natural histonin. The AMP activity of the histonin produced in E. coli was identical to that of a synthetic histonin. With our expression system, 167 mg of histonin was obtained from 1 l of E. coli culture. These results may lead to a cost-effective solution for the mass production of AMPs that are toxic to a host.

  16. Pathological phenotypes and in vivo DNA cleavage by unrestrained activity of a phosphorothioate-based restriction system in Salmonella.

    Science.gov (United States)

    Cao, Bo; Cheng, Qiuxiang; Gu, Chen; Yao, Fen; DeMott, Michael S; Zheng, Xiaoqing; Deng, Zixin; Dedon, Peter C; You, Delin

    2014-08-01

    Prokaryotes protect their genomes from foreign DNA with a diversity of defence mechanisms, including a widespread restriction-modification (R-M) system involving phosphorothioate (PT) modification of the DNA backbone. Unlike classical R-M systems, highly partial PT modification of consensus motifs in bacterial genomes suggests an unusual mechanism of PT-dependent restriction. In Salmonella enterica, PT modification is mediated by four genes dptB-E, while restriction involves additional three genes dptF-H. Here, we performed a series of studies to characterize the PT-dependent restriction, and found that it presented several features distinct with traditional R-M systems. The presence of restriction genes in a PT-deficient mutant was not lethal, but instead resulted in several pathological phenotypes. Subsequent transcriptional profiling revealed the expression of > 600 genes was affected by restriction enzymes in cells lacking PT, including induction of bacteriophage, SOS response and DNA repair-related genes. These transcriptional responses are consistent with the observation that restriction enzymes caused extensive DNA cleavage in the absence of PT modifications in vivo. However, overexpression of restriction genes was lethal to the host in spite of the presence PT modifications. These results point to an unusual mechanism of PT-dependent DNA cleavage by restriction enzymes in the face of partial PT modification. © 2014 John Wiley & Sons Ltd.

  17. 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.

  18. 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.

  19. 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

  20. 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,

  1. Increasing on-target cleavage efficiency for CRISPR/Cas9-induced large fragment deletion in Myxococcus xanthus.

    Science.gov (United States)

    Yang, Ying-Jie; Wang, Ye; Li, Zhi-Feng; Gong, Ya; Zhang, Peng; Hu, Wen-Chao; Sheng, Duo-Hong; Li, Yue-Zhong

    2017-08-16

    The CRISPR/Cas9 system is a powerful tool for genome editing, in which the sgRNA binds and guides the Cas9 protein for the sequence-specific cleavage. The protocol is employable in different organisms, but is often limited by cell damage due to the endonuclease activity of the introduced Cas9 and the potential off-target DNA cleavage from incorrect guide by the 20 nt spacer. In this study, after resolving some critical limits, we have established an efficient CRISPR/Cas9 system for the deletion of large genome fragments related to the biosynthesis of secondary metabolites in Myxococcus xanthus cells. We revealed that the high expression of a codon-optimized cas9 gene in M. xanthus was cytotoxic, and developed a temporally high expression strategy to reduce the cell damage from high expressions of Cas9. We optimized the deletion protocol by using the tRNA-sgRNA-tRNA chimeric structure to ensure correct sgRNA sequence. We found that, in addition to the position-dependent nucleotide preference, the free energy of a 20 nt spacer was a key factor for the deletion efficiency. By using the developed protocol, we achieved the CRISPR/Cas9-induced deletion of large biosynthetic gene clusters for secondary metabolites in M. xanthus DK1622 and its epothilone-producing mutant. The findings and the proposals described in this paper were suggested to be workable in other organisms, for example, other Gram negative bacteria with high GC content.

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

    Science.gov (United States)

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

    2017-07-06

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

  3. Photosensitized cleavage of dynein heavy chains. Cleavage at the V1 site by irradiation at 365 nm in the presence of ATP and vanadate

    International Nuclear Information System (INIS)

    Gibbons, I.R.; Lee-Eiford, A.; Mocz, G.; Phillipson, C.A.; Tang, W.J.; Gibbons, B.H.

    1987-01-01

    Irradiation of soluble dynein 1 from sea urchin sperm flagella at 365 nm in the presence of MgATP and 0.05-50 microM vanadate (Vi) cleaves the alpha and beta heavy chains (Mr 428,000) at their V1 sites to give peptides of Mr 228,000 and 200,000, without the nonspecific side effects produced by irradiation at 254 nm as described earlier. The decrease in intact heavy chain material is biphasic; in 10 microM Vi, approximately 80% occurs with a half-time of 7 min and the remainder with a half-time of about 90 min, and the yield of cleavage peptides is better than 90%. Loss of dynein ATPase activity appears to be a direct result of the cleavage process and is not significantly affected by the presence of up to 0.1 M cysteamine (CA, 60-23-1) or 2-aminoethyl carbamimidothioic acid dihydrobromide (CA, 56-10-0) as free radical trapping agents. The concentration of Vi required for 50% maximal initial cleavage rate is 4.5 microM, while that for 50% ATPase inhibition is 0.8 microM, both in a 0.6 M NaCl medium. In the presence of 20 microM Vi, CTP and UTP support cleavage at about half the rate of ATP, whereas GTP and ITP support cleavage only if the Vi concentration is raised to about 200 microM. Substitution of any of the transition metal cations Cr2+, Mn2+, Fe2+, or Co2+ for the usual Mg2+ suppresses the photocleavage, presumably by quenching the excited chromophore prior to scission of the heavy chain. The photocleaved dynein 1 binds to dynein-depleted flagella similarly to intact dynein 1, but upon reactivation of the flagella with 1 mM ATP their motility is partially inhibited, rather than being augmented as with intact dynein

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

    Science.gov (United States)

    Defendi, Federica; Charignon, Delphine; Ghannam, Arije; Habib, Mohammed; Drouet, Christian

    2016-01-01

    Background Angioedema without wheals (AE) is a symptom characterised by localised episodes of oedema presumably caused by kinin release from kininogen cleavage. It can result from a hereditary deficiency in C1 Inhibitor (C1Inh), but it can present with normal level of C1Inh. These forms are typically difficult to diagnose although enhanced kinin production is suspected or demonstrated in some cases. Objectives We wanted to investigate bradykinin overproduction in all AE condition with normal C1Inh, excluding cases with enhanced kinin catabolism, and to propose this parameter as a disease biomarker. Methods We retrospectively investigated high molecular weight kininogen (HK) cleavage pattern, using gel electrophoresis and immunorevelation. Plasma samples were drawn using the same standardised procedure from blood donors or AE patients with normal C1Inh conditions, normal kinin catabolism, and without prophylaxis. Results Circulating native HK plasma concentrations were similar in the healthy men (interquartile range: 98–175μg/mL, n = 51) and in healthy women (90–176μg/mL, n = 74), while HK cleavage was lower (p14.4% HK cleavage for men; 33.0% HK cleavage for women, with >98% specificity achieved for all parameters. In plasma from patients undergoing recovery two months after oestrogen/progestin combination withdrawal (n = 13) or two weeks after AE attack (n = 2), HK cleavage was not fully restored, suggesting its use as a post-attack assay. Conclusion As a diagnostic tool, HK cleavage can offer physicians supportive arguments for kinin production in suspected AE cases and improve patient follow-up in clinical trials or prophylactic management. PMID:27685806

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

    Directory of Open Access Journals (Sweden)

    Rémi Baroso

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

  6. Evolution of the R2 retrotransposon ribozyme and its self-cleavage site.

    Directory of Open Access Journals (Sweden)

    Danna G Eickbush

    Full Text Available R2 is a non-long terminal repeat retrotransposon that inserts site-specifically in the tandem 28S rRNA genes of many animals. Previously, R2 RNA from various species of Drosophila was shown to self-cleave from the 28S rRNA/R2 co-transcript by a hepatitis D virus (HDV-like ribozyme encoded at its 5' end. RNA cleavage was at the precise 5' junction of the element with the 28S gene. Here we report that RNAs encompassing the 5' ends of R2 elements from throughout its species range fold into HDV-like ribozymes. In vitro assays of RNA self-cleavage conducted in many R2 lineages confirmed activity. For many R2s, RNA self-cleavage was not at the 5' end of the element but at 28S rRNA sequences up to 36 nucleotides upstream of the junction. The location of cleavage correlated well with the types of endogenous R2 5' junctions from different species. R2 5' junctions were uniform for most R2s in which RNA cleavage was upstream in the rRNA sequences. The 28S sequences remaining on the first DNA strand synthesized during retrotransposition are postulated to anneal to the target site and uniformly prime second strand DNA synthesis. In species where RNA cleavage occurred at the R2 5' end, the 5' junctions were variable. This junction variation is postulated to result from the priming of second strand DNA synthesis by chance microhomologies between the target site and the first DNA strand. Finally, features of R2 ribozyme evolution, especially changes in cleavage site and convergence on the same active site sequences, are discussed.

  7. Characterization of cleavage events in the multifunctional cilium adhesin Mhp684 (P146) reveals a mechanism by which Mycoplasma hyopneumoniae regulates surface topography.

    Science.gov (United States)

    Bogema, Daniel R; Deutscher, Ania T; Woolley, Lauren K; Seymour, Lisa M; Raymond, Benjamin B A; Tacchi, Jessica L; Padula, Matthew P; Dixon, Nicholas E; Minion, F Chris; Jenkins, Cheryl; Walker, Mark J; Djordjevic, Steven P

    2012-01-01

    Mycoplasma hyopneumoniae causes enormous economic losses to swine production worldwide by colonizing the ciliated epithelium in the porcine respiratory tract, resulting in widespread damage to the mucociliary escalator, prolonged inflammation, reduced weight gain, and secondary infections. Protein Mhp684 (P146) comprises 1,317 amino acids, and while the N-terminal 400 residues display significant sequence identity to the archetype cilium adhesin P97, the remainder of the molecule is novel and displays unusual motifs. Proteome analysis shows that P146 preprotein is endogenously cleaved into three major fragments identified here as P50(P146), P40(P146), and P85(P146) that reside on the cell surface. Liquid chromatography with tandem mass spectrometry (LC-MS/MS) identified a semitryptic peptide that delineated a major cleavage site in Mhp684. Cleavage occurred at the phenylalanine residue within sequence (672)ATEF↓QQ(677), consistent with a cleavage motif resembling S/T-X-F↓X-D/E recently identified in Mhp683 and other P97/P102 family members. Biotinylated surface proteins recovered by avidin chromatography and separated by two-dimensional gel electrophoresis (2-D GE) showed that more-extensive endoproteolytic cleavage of P146 occurs. Recombinant fragments F1(P146)-F3(P146) that mimic P50(P146), P40(P146), and P85(P146) were constructed and shown to bind porcine epithelial cilia and biotinylated heparin with physiologically relevant affinity. Recombinant versions of F3(P146) generated from M. hyopneumoniae strain J and 232 sequences strongly bind porcine plasminogen, and the removal of their respective C-terminal lysine and arginine residues significantly reduces this interaction. These data reveal that P146 is an extensively processed, multifunctional adhesin of M. hyopneumoniae. Extensive cleavage coupled with variable cleavage efficiency provides a mechanism by which M. hyopneumoniae regulates protein topography. Vaccines used to control Mycoplasma

  8. Intracellular trapping of cycloSal-pronucleotides by enzymatic cleavage.

    Science.gov (United States)

    Jessen, H J; Tonn, V; Meier, C

    2007-01-01

    A new synthesis for cycloSal-pronucleotides bearing enzymatically cleavable triggers is presented. This trigger is introduced to trap the pronucleotide inside cells. The general concept and hydrolysis data in different media are discussed.

  9. Water-soluble Manganese and Iron Mesotetrakis(carboxyl)porphyrin: DNA Binding, Oxidative Cleavage, and Cytotoxic Activities.

    Science.gov (United States)

    Shi, Lei; Jiang, Yi-Yu; Jiang, Tao; Yin, Wei; Yang, Jian-Ping; Cao, Man-Li; Fang, Yu-Qi; Liu, Hai-Yang

    2017-06-29

    Two new water-soluble metal carboxyl porphyrins, manganese (III) meso -tetrakis (carboxyl) porphyrin and iron (III) meso -tetrakis (carboxyl) porphyrin, were synthesized and characterized. Their interactions with ct-DNA were investigated by UV-Vis titration, fluorescence spectra, viscosity measurement and CD spectra. The results showed they can strongly bind to ct-DNA via outside binding mode. Electrophoresis experiments revealed that both complexes can cleave pBR322 DNA efficiently in the presence of hydrogen peroxide, albeit 2-Mn exhibited a little higher efficiency. The inhibitor tests suggest the oxidative DNA cleavage by these two complexes may involve hydroxyl radical active intermediates. Notably, 2-Mn exhibited considerable photocytotoxicity against Hep G2 cell via triggering a significant generation of ROS and causing disruption of MMP after irradiation.

  10. Cytoprotective effects of Glycyrrhizae radix extract and its active component liquiritigenin against cadmium-induced toxicity (effects on bad translocation and cytochrome c-mediated PARP cleavage)

    International Nuclear Information System (INIS)

    Kim, Sang Chan; Byun, Sung Hui; Yang, Chae Ha; Kim, Chul Young; Kim, Jin Woong; Kim, Sang Geon

    2004-01-01

    Glycyrrhizae radix has been popularly used as one of the oldest and most frequently employed botanicals in herbal medicine in Asian countries, and currently occupies an important place in food products. Cadmium (Cd) induces both apoptotic and non-apoptotic cell death, in which alterations in cellular sulfhydryls participate. In the present study, we determined the effects of G. radix extract (GRE) and its representative active components on cell death induced by Cd and explored the mechanistic basis of cytoprotective effects of G. radix. Incubation of H4IIE cells with GRE inhibited cell death induced by 10 μM Cd. Also, GRE effectively blocked Cd (1 μM)-induced cell death potentiated by buthionine sulfoximine (BSO) without restoration of cellular GSH. GRE prevented both apoptotic and non-apoptotic cell injury induced by Cd (10 μM) or Cd (0.3-1 μM) + BSO. Inhibition of Cd-induced cell injury by pretreatment of cells with GRE suggested that the cytoprotective effect result from alterations in the levels of the protein(s) responsible for cell viability. GRE inhibited mitochondrial Bad translocation by Cd or Cd+BSO, and caused restoration of mitochondrial Bcl xL and cytochrome c levels. Cd-induced poly(ADP-ribose)polymerase cleavage in control cells or in cells deprived of sulfhydryls was prevented by GRE treatment. Among the major components present in GRE, liquiritigenin, but not liquiritin, isoliquiritigenin or glycyrrhizin, exerted cytoprotective effect. These results demonstrated that GRE blocked Cd-induced cell death by inhibiting the apoptotic processes involving translocation of Bad into mitochondria, decreases in mitochondrial Bcl xL and cytochrome c, and poly(ADP-ribose)polymerase cleavage

  11. Theoretical study on isomerization and peptide bond cleavage at aspartic residue.

    Science.gov (United States)

    Sang-aroon, Wichien; Ruangpornvisuti, Vithaya

    2013-09-01

    Isomerization and peptide bond cleavage at aspartic residue (Asp) in peptide models have been reported. In this study, the mechanisms and energies concerning the isomerization and peptide bond cleavage at Asp residue were investigated by the density functional theory (DFT) at B3LYP/6-311++G(d,p). The integral equation formalism-polarizable continuum model (IEF-PCM) was utilized to calculate solvation effect by single-point calculation of the gas-phase B3LYP/6-311++G(d,p)-optimized structure. Mechanisms and energies of the dehydration in isomerization reaction of Asp residue were comparatively analyzed with the deamidation reaction of Asn residue. The results show that the succinimide intermediate was formed preferentially through the step-wise reaction via the tetrahedral intermediate. The cleavage at C-terminus is more preferential than those at N-terminus. In comparison to isomerization, peptide bond cleavage is ≈ 20 kcal mol(-1) and lower in activation barrier than the isomerization. So, in this case, the isomerization of Asp is inhibited by the peptide bond cleavage.

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

    Directory of Open Access Journals (Sweden)

    Naganori Nao

    2017-02-01

    Full Text Available 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.

  13. CRISPR/Cas9 cleavages in budding yeast reveal templated insertions and strand-specific insertion/deletion profiles.

    Science.gov (United States)

    Lemos, Brenda R; Kaplan, Adam C; Bae, Ji Eun; Ferrazzoli, Alexander E; Kuo, James; Anand, Ranjith P; Waterman, David P; Haber, James E

    2018-02-13

    Harnessing CRISPR-Cas9 technology provides an unprecedented ability to modify genomic loci via DNA double-strand break (DSB) induction and repair. We analyzed nonhomologous end-joining (NHEJ) repair induced by Cas9 in budding yeast and found that the orientation of binding of Cas9 and its guide RNA (gRNA) profoundly influences the pattern of insertion/deletions (indels) at the site of cleavage. A common indel created by Cas9 is a 1-bp (+1) insertion that appears to result from Cas9 creating a 1-nt 5' overhang that is filled in by a DNA polymerase and ligated. The origin of +1 insertions was investigated by using two gRNAs with PAM sequences located on opposite DNA strands but designed to cleave the same sequence. These templated +1 insertions are dependent on the X-family DNA polymerase, Pol4. Deleting Pol4 also eliminated +2 and +3 insertions, which are biased toward homonucleotide insertions. Using inverted PAM sequences, we also found significant differences in overall NHEJ efficiency and repair profiles, suggesting that the binding of the Cas9:gRNA complex influences subsequent NHEJ processing. As with events induced by the site-specific HO endonuclease, CRISPR-Cas9-mediated NHEJ repair depends on the Ku heterodimer and DNA ligase 4. Cas9 events are highly dependent on the Mre11-Rad50-Xrs2 complex, independent of Mre11's nuclease activity. Inspection of the outcomes of a large number of Cas9 cleavage events in mammalian cells reveals a similar templated origin of +1 insertions in human cells, but also a significant frequency of similarly templated +2 insertions.

  14. 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.

  15. A Molecular Sensor To Characterize Arenavirus Envelope Glycoprotein Cleavage by Subtilisin Kexin Isozyme 1/Site 1 Protease.

    Science.gov (United States)

    Oppliger, Joel; da Palma, Joel Ramos; Burri, Dominique J; Bergeron, Eric; Khatib, Abdel-Majid; Spiropoulou, Christina F; Pasquato, Antonella; Kunz, Stefan

    2016-01-15

    Arenaviruses are emerging viruses including several causative agents of severe hemorrhagic fevers in humans. The advent of next-generation sequencing technology has greatly accelerated the discovery of novel arenavirus species. However, for many of these viruses, only genetic information is available, and their zoonotic disease potential remains unknown. During the arenavirus life cycle, processing of the viral envelope glycoprotein precursor (GPC) by the cellular subtilisin kexin isozyme 1 (SKI-1)/site 1 protease (S1P) is crucial for productive infection. The ability of newly emerging arenaviruses to hijack human SKI-1/S1P appears, therefore, to be a requirement for efficient zoonotic transmission and human disease potential. Here we implement a newly developed cell-based molecular sensor for SKI-1/S1P to characterize the processing of arenavirus GPC-derived target sequences by human SKI-1/S1P in a quantitative manner. We show that only nine amino acids flanking the putative cleavage site are necessary and sufficient to accurately recapitulate the efficiency and subcellular location of arenavirus GPC processing. In a proof of concept, our sensor correctly predicts efficient processing of the GPC of the newly emergent pathogenic Lujo virus by human SKI-1/S1P and defines the exact cleavage site. Lastly, we employed our sensor to show efficient GPC processing of a panel of pathogenic and nonpathogenic New World arenaviruses, suggesting that GPC cleavage represents no barrier for zoonotic transmission of these pathogens. Our SKI-1/S1P sensor thus represents a rapid and robust test system for assessment of the processing of putative cleavage sites derived from the GPCs of newly discovered arenavirus by the SKI-1/S1P of humans or any other species, based solely on sequence information. Arenaviruses are important emerging human pathogens that can cause severe hemorrhagic fevers with high mortality in humans. A crucial step in productive arenavirus infection of human

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

    Energy Technology Data Exchange (ETDEWEB)

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

    2016-01-01

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

  17. A physical map of human Alu repeats cleavage by restriction endonucleases

    Directory of Open Access Journals (Sweden)

    Chernukhin Valery A

    2008-06-01

    Full Text Available Abstract Background Alu repetitive elements are the abundant sequences in human genome. Diversity of DNA sequences of these elements makes difficult the construction of theoretical patterns of Alu repeats cleavage by restriction endonucleases. We have proposed a method of restriction analysis of Alu repeats sequences in silico. Results Simple software to analyze Alu repeats database has been suggested and Alu repeats digestion patterns for several restriction enzymes' recognition sites have been constructed. Restriction maps of Alu repeats cleavage for corresponding restriction enzymes have been calculated and plotted. Theoretical data have been compared with experimental results on DNA hydrolysis with restriction enzymes, which we obtained earlier. Conclusion Alu repeats digestions provide the main contribution to the patterns of human chromosomal DNA cleavage. This corresponds to the experimental data on total human DNA hydrolysis with restriction enzymes.

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

    Science.gov (United States)

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

    2013-08-16

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

  19. Sulfur mustard causes caspase-mediated cleavage of cytoskeletal keratins

    NARCIS (Netherlands)

    Mol, M.A.E.; Berg, R.M. van den; Dijk, C. van; Jong, A.L. de

    2004-01-01

    In order to study the toxic mechanism of action of sulfur mustard (HD), we examined protein expression in control and HD-treated cultured human epidermal keratinocytes (HEK) at 24 h after exposure to 100 μM HD. Protein patterns of cell lysates were prepared using two-dimensional gel electrophoresis

  20. 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.

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

    International Nuclear Information System (INIS)

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

    1983-01-01

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

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

    OpenAIRE

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

    1989-01-01

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

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

    Science.gov (United States)

    Crouzier, Lucile; Dubois, Camille; Wengel, Jesper; Veedu, Rakesh N

    2012-07-15

    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. Copyright © 2012 Elsevier Ltd. All rights reserved.

  4. Inhibition of hepatitis B virus replication via HBV DNA cleavage by Cas9 from Staphylococcus aureus.

    Science.gov (United States)

    Liu, Yu; Zhao, Miaoxian; Gong, Mingxing; Xu, Ying; Xie, Cantao; Deng, Haohui; Li, Xueying; Wu, Hongkai; Wang, Zhanhui

    2018-04-01

    Chronic hepatitis B virus (HBV) infection is difficult to cure due to the presence of covalently closed circular DNA (cccDNA). Accumulating evidence indicates that the CRISPR/Cas9 system effectively disrupts HBV genome, including cccDNA, in vitro and in vivo. However, efficient delivery of CRISPR/Cas9 system to the liver or hepatocytes using an adeno-associated virus (AAV) vector remains challenging due to the large size of Cas9 from Streptococcus pyogenes (Sp). The recently identified Cas9 protein from Staphylococcus aureus (Sa) is smaller than SpCas9 and thus is able to be packaged into the AAV vector. To examine the efficacy of SaCas9 system on HBV genome destruction, we designed 5 guide RNAs (gRNAs) that targeted different HBV genotypes, 3 of which were shown to be effective. The SaCas9 system significantly reduced HBV antigen expression, as well as pgRNA and cccDNA levels, in Huh7, HepG2.2.15 and HepAD38 cells. The dual expression of gRNAs/SaCas9 in these cell lines resulted in more efficient HBV genome cleavage. In the mouse model, hydrodynamic injection of gRNA/SaCas9 plasmids resulted in significantly lower levels of HBV protein expression. We also delivered the SaCas9 system into mice with persistent HBV replication using an AAV vector. Both the AAV vector and the mRNA of Cas9 could be detected in the C3H mouse liver cells. Decreased hepatitis B surface antigen (HBsAg), HBV DNA and pgRNA levels were observed when a higher titer of AAV was injected, although this decrease was not significantly different from the control. In summary, the SaCas9 system accurately and efficiently targeted the HBV genome and inhibited HBV replication both in vitro and in vivo. The system was delivered by an AAV vector and maybe used as a novel therapeutic strategy against chronic HBV infection. Copyright © 2018 Elsevier B.V. All rights reserved.

  5. The highly pathogenic H7N3 avian influenza strain from July 2012 in Mexico acquired an extended cleavage site through recombination with host 28S rRNA.

    Science.gov (United States)

    Maurer-Stroh, Sebastian; Lee, Raphael T C; Gunalan, Vithiagaran; Eisenhaber, Frank

    2013-05-01

    A characteristic difference between highly and non-highly pathogenic avian influenza strains is the presence of an extended, often multibasic, cleavage motif insertion in the hemagglutinin protein. Such motif is found in H7N3 strains from chicken farm outbreaks in 2012 in Mexico. Through phylogenetic, sequence and structural analysis, we try to shed light on the role, prevalence, likelihood of appearance and origin of the inserted cleavage motifs in these H7N3 avian influenza strains. The H7N3 avian influenza strain which caused outbreaks in chicken farms in June/July 2012 in Mexico has a new extended cleavage site which is the likely reason for its high pathogenicity in these birds. This cleavage site appears to have been naturally acquired and was not present in the closest low pathogenic precursors. Structural modeling shows that insertion of a productive cleavage site is quite flexible to accept insertions of different length and with sequences from different possible origins. Different from recent cleavage site insertions, the origin of the insert here is not from the viral genome but from host 28S ribosomal RNA (rRNA) instead. This is a novelty for a natural acquisition as a similar insertion has so far only been observed in a laboratory strain before. Given the abundance of viral and host RNA in infected cells, the acquisition of a pathogenicity-enhancing extended cleavage site through a similar route by other low-pathogenic avian strains in future does not seem unlikely. Important for surveillance of these H7N3 strains, the structural sites known to enhance mammalian airborne transmission are dominated by the characteristic avian residues and the risk of human to human transmission should currently be low but should be monitored for future changes accordingly. This highly pathogenic H7N3 avian influenza strain acquired a novel extended cleavage site which likely originated from recombination with 28S rRNA from the avian host. Notably, this new virus can

  6. The Role of Trop2 Cleavage Products in Prostate Tumorigenesis

    Science.gov (United States)

    2014-10-01

    prostatic tubules when combined with urogenital sinus mesen- chyme (UGSM) and implanted under the kidney capsule of severe combined immunodeficiency (SCID...progenitor self-renewal and prostate tubule formation. We have also determined that Trop2 is activated by regulated intramembrane proteolysis (RIP...combined with UGSM. The grafts composed of prostate cells and UGSM were engrafted under the kidney capsule of SCID mice, allowing regeneration of

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

    Directory of Open Access Journals (Sweden)

    Ting Wei

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

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

    African Journals Online (AJOL)

    NJD

    2004-04-22

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

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

    Czech Academy of Sciences Publication Activity Database

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

    2013-01-01

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

  10. Cleavage strongly influences whether soluble HIV-1 envelope glycoprotein trimers adopt a native-like conformation

    NARCIS (Netherlands)

    Ringe, Rajesh P.; Sanders, Rogier W.; Yasmeen, Anila; Kim, Helen J.; Lee, Jeong Hyun; Cupo, Albert; Korzun, Jacob; Derking, Ronald; van Montfort, Thijs; Julien, Jean-Philippe; Wilson, Ian A.; Klasse, Per Johan; Ward, Andrew B.; Moore, John P.

    2013-01-01

    We compare the antigenicity and conformation of soluble, cleaved vs. uncleaved envelope glycoprotein (Env gp) 140 trimers from the subtype A HIV type 1 (HIV-1) strain BG505. The impact of gp120-gp41 cleavage on trimer structure, in the presence or absence of trimer-stabilizing modifications (i.e., a

  11. 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.

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

    International Nuclear Information System (INIS)

    Cleaver, J.E.

    1983-01-01

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

  13. Anaerobic DNA cleavage in red light by dicopper(II) complexes on ...

    Indian Academy of Sciences (India)

    Administrator

    *For correspondence. Anaerobic DNA cleavage in red light by dicopper(II) complexes on disulphide bond activation. DEBOJYOTI LAHIRI a. , RITANKAR MAJUMDAR b. , ASHIS K PATRA a and. AKHIL R CHAKRAVARTY a,. *. aDepartment of Inorganic and Physical Chemistry, Indian Institute of Science, Bangalore 560 012.

  14. Horizontal Cleavage Meniscus Tear Treated With All-inside Circumferential Compression Stitches

    NARCIS (Netherlands)

    Woodmass, Jarret M.; Johnson, Joshua D.; Wu, Isabella T.; Saris, Daniel B.F.; Stuart, Michael J.; Krych, Aaron J.

    2017-01-01

    Horizontal cleavage meniscus tears are a common orthopedic injury often treated with partial or total meniscectomy versus repair. This Technical Note presents a technique for all-inside repair with uniform compression of the superior and inferior leaflets by placement of multiple circumferential

  15. DNA binding and cleavage activity of a structurally characterized Ni(II)

    Indian Academy of Sciences (India)

    1375–1381. c Indian Academy of Sciences. DOI 10.1007/s12039-015-0900-4. DNA binding and cleavage activity of a structurally characterized Ni(II). Schiff base complex. SARAT CHANDRA KUMARa, ABHIJIT PALa, MERRY MITRAa,. V M MANIKANDAMATHAVANb, CHIA -HER LINc, BALACHANDRAN UNNI NAIRb,∗.

  16. Targeting cleavage and polyadenylation specific factor 1 via shRNA ...

    Indian Academy of Sciences (India)

    Cleavage and polyadenylation specificity factor 1 (CPSF1), a member of CPSF complex, has been reported to play a keyrole in pre-mRNA 30-end formation, but its possible role in ovarian cancer remains unclear. In the present study, we foundthe mRNA level of CPSF1 was overexpressed in ovarian cancer tissues using ...

  17. Guest–host interactions in the cleavage of phenylphenyl acetates by ...

    Indian Academy of Sciences (India)

    WINTEC

    in the inhibition on the hydrolysis rate. This is attri- buted to the inclusion of the alkyl part of the ester in the CD cavity. In phenylphenyl acetate (PPA) both side of the ester group is flanked by aryl groups. Therefore it was thought of interest to investigate the cleavage of PPA catalysed by β-cyclodextrin. (CD). 2. Experimental.

  18. Metal-organic framework catalysts for selective cleavage of aryl-ether bonds

    Science.gov (United States)

    Allendorf, Mark D.; Stavila, Vitalie

    2017-08-01

    The present invention relates to methods of employing a metal-organic framework (MOF) as a catalyst for cleaving chemical bonds. In particular instances, the MOF results in selective bond cleavage that results in hydrogenolyzis. Furthermore, the MOF catalyst can be reused in multiple cycles. Such MOF-based catalysts can be useful, e.g., to convert biomass components.

  19. Rhodium-catalyzed C-C Bond Cleavage Reactions - An Update

    Czech Academy of Sciences Publication Activity Database

    Korotvička, A.; Nečas, D.; Kotora, Martin

    2012-01-01

    Roč. 16, č. 10 (2012), s. 1170-1214 ISSN 1385-2728 Grant - others:GA MŠk(CZ) LC06070 Program:LC Institutional research plan: CEZ:AV0Z40550506 Keywords : rhodium * C-C bond cleavage * catalysis * synthesis Subject RIV: CC - Organic Chemistry Impact factor: 3.039, year: 2012

  20. Nickel-Catalyzed Synthesis of Primary Aryl and Heteroaryl Amines via C–O Bond Cleavage

    KAUST Repository

    Yue, Huifeng

    2017-03-13

    A nickel-catalyzed protocol for the conversion of aryl and heteroaryl alcohol derivatives to primary and secondary aromatic amines via C(sp2)-O bond cleavage is described. The new amination protocol can be applied to a range of substrates bearing diverse functional groups and uses readily available benzophenone imines as an effective nitrogen source.

  1. Rendering one autolysis site in Bacillus subtilis neutral protease resistant to cleavage reveals a new fission

    NARCIS (Netherlands)

    Van den Burg, B; Eijsink, VGH; Vriend, G; Veltman, OR; Venema, G

    Autolytic degradation of the thermolysin-like proteinase of Bacillus subtilis (TLP-sub) is responsible for the irreversible inactivation of the enzyme at elevated temperatures. Previously we have reported five cleavage sites in Tip-sub [Van den Burg et al, (1990) Biochem. J. 272, 93-97]. In an

  2. Use of divalent metal ions in the DNA cleavage reaction of topoisomerase IV

    Science.gov (United States)

    Pitts, Steven L.; Liou, Grace F.; Mitchenall, Lesley A.; Burgin, Alex B.; Maxwell, Anthony; Neuman, Keir C.; Osheroff, Neil

    2011-01-01

    It has long been known that type II topoisomerases require divalent metal ions in order to cleave DNA. Kinetic, mutagenesis and structural studies indicate that the eukaryotic enzymes utilize a novel variant of the canonical two-metal-ion mechanism to promote DNA scission. However, the role of metal ions in the cleavage reaction mediated by bacterial type II enzymes has been controversial. Therefore, to resolve this critical issue, this study characterized the DNA cleavage reaction of Escherichia coli topoisomerase IV. We utilized a series of divalent metal ions with varying thiophilicities in conjunction with oligonucleotides that replaced bridging and non-bridging oxygen atoms at (and near) the scissile bond with sulfur atoms. DNA scission was enhanced when thiophilic metal ions were used with substrates that contained bridging sulfur atoms. In addition, the metal-ion dependence of DNA cleavage was sigmoidal in nature, and rates and levels of DNA cleavage increased when metal ion mixtures were used in reactions. Based on these findings, we propose that topoisomerase IV cleaves DNA using a two-metal-ion mechanism in which one of the metal ions makes a critical interaction with the 3′-bridging atom of the scissile phosphate and facilitates DNA scission by the bacterial type II enzyme. PMID:21300644

  3. Human topoisomerase IIα uses a two-metal-ion mechanism for DNA cleavage

    Science.gov (United States)

    Deweese, Joseph E.; Burgin, Alex B.; Osheroff, Neil

    2008-01-01

    The DNA cleavage reaction of human topoisomerase IIα is critical to all of the physiological and pharmacological functions of the protein. While it has long been known that the type II enzyme requires a divalent metal ion in order to cleave DNA, the role of the cation in this process is not known. To resolve this fundamental issue, the present study utilized a series of divalent metal ions with varying thiophilicities in conjunction with DNA cleavage substrates that replaced the 3′-bridging oxygen of the scissile bond with a sulfur atom (i.e. 3′-bridging phosphorothiolates). Rates and levels of DNA scission were greatly enhanced when thiophilic metal ions were included in reactions that utilized sulfur-containing substrates. Based on these results and those of reactions that employed divalent cation mixtures, we propose that topoisomerase IIα mediates DNA cleavage via a two-metal-ion mechanism. In this model, one of the metal ions makes a critical interaction with the 3′-bridging atom of the scissile phosphate. This interaction greatly accelerates rates of enzyme-mediated DNA cleavage, and most likely is needed to stabilize the leaving 3′-oxygen. PMID:18653531

  4. DNA binding and cleavage studies of copper(II) complexes with 2'-deoxyadenosine modified histidine moiety.

    Science.gov (United States)

    Borowska, Justyna; Sierant, Malgorzata; Sochacka, Elzbieta; Sanna, Daniele; Lodyga-Chruscinska, Elzbieta

    2015-09-01

    This work is focused on the study of DNA binding and cleavage properties of 2'-deoxyadenosines modified with ester/amide of histidine (his(6)dA ester, his(6)dA amide) and their copper(II) complexes. To determine the coordination mode of the complex species potentiometric and spectroscopic (UV-visible, CD, EPR) studies have been performed. The analysis of electronic absorption and fluorescence spectra has been used to find the nature of the interactions between the compounds and calf thymus DNA (CT-DNA). There is significant influence of the -NH2 and -OCH3 groups on binding of the ligands or the complexes to DNA. Only amide derivative and its complex reveal intercalative ability. In the case of his(6)dA ester and Cu(II)-his(6)dA ester the main interactions can be groove binding. DNA cleavage activities of the compounds have been examined by gel electrophoresis. The copper complexes have promoted the cleavage of plasmid DNA, but none of the ligands exhibited any chemical nuclease activity. The application of different scavengers of reactive oxygen species provided a conclusion that DNA cleavage caused by copper complexes might occur via hydrolytic pathway.

  5. HAI-2 stabilizes, inhibits and regulates SEA-cleavage-dependent secretory transport of matriptase

    DEFF Research Database (Denmark)

    Nonboe, Annika Weile; Krigslund, Oliver; Søndergård, Christoffer

    2017-01-01

    of matriptase, and as a result the SEA domain cleavage of matriptase. Two of the HAI-2 Kunitz domain 1 mutants investigated (C47F, R48L and C47F/R48L) also displayed a reduced ability to proteolytically silence matriptase. Hence, HAI-2 separately stabilizes matriptase, regulates the secretory transport...

  6. 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

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

    Czech Academy of Sciences Publication Activity Database

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

    2016-01-01

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

  8. Tolloid cleavage activates latent GDF8 by priming the pro-complex for dissociation.

    Science.gov (United States)

    Le, Viet Q; Iacob, Roxana E; Tian, Yuan; McConaughy, William; Jackson, Justin; Su, Yang; Zhao, Bo; Engen, John R; Pirruccello-Straub, Michelle; Springer, Timothy A

    2018-02-01

    Growth differentiation factor 8 (GDF8)/myostatin is a latent TGF-β family member that potently inhibits skeletal muscle growth. Here, we compared the conformation and dynamics of precursor, latent, and Tolloid-cleaved GDF8 pro-complexes to understand structural mechanisms underlying latency and activation of GDF8. Negative stain electron microscopy (EM) of precursor and latent pro-complexes reveals a V-shaped conformation that is unaltered by furin cleavage and sharply contrasts with the ring-like, cross-armed conformation of latent TGF-β1. Surprisingly, Tolloid-cleaved GDF8 does not immediately dissociate, but in EM exhibits structural heterogeneity consistent with partial dissociation. Hydrogen-deuterium exchange was not affected by furin cleavage. In contrast, Tolloid cleavage, in the absence of prodomain-growth factor dissociation, increased exchange in regions that correspond in pro-TGF-β1 to the α1-helix, latency lasso, and β1-strand in the prodomain and to the β6'- and β7'-strands in the growth factor. Thus, these regions are important in maintaining GDF8 latency. Our results show that Tolloid cleavage activates latent GDF8 by destabilizing specific prodomain-growth factor interfaces and primes the growth factor for release from the prodomain. © 2018 The Authors.

  9. Endonuclease active site plasticity allows DNA cleavage with diverse alkaline Earth and transition metal ions.

    Science.gov (United States)

    Vasu, Kommireddy; Saravanan, Matheshwaran; Nagaraja, Valakunja

    2011-09-16

    A majority of enzymes show a high degree of specificity toward a particular metal ion in their catalytic reaction. However, Type II restriction endonuclease (REase) R.KpnI, which is the first member of the HNH superfamily of REases, exhibits extraordinary diversity in metal ion dependent DNA cleavage. Several alkaline earth and transition group metal ions induce high fidelity and promiscuous cleavage or inhibition depending upon their concentration. The metal ions having different ionic radii and co-ordination geometries readily replace each other from the enzyme's active site, revealing its plasticity. Ability of R.KpnI to cleave DNA with both alkaline earth and transition group metal ions having varied ionic radii could imply utilization of different catalytic site(s). However, mutation of the invariant His residue of the HNH motif caused abolition of the enzyme activity with all of the cofactors, indicating that the enzyme follows a single metal ion catalytic mechanism for DNA cleavage. Indispensability of His in nucleophile activation together with broad cofactor tolerance of the enzyme indicates electrostatic stabilization function of metal ions during catalysis. Nevertheless, a second metal ion is recruited at higher concentrations to either induce promiscuity or inhibit the DNA cleavage. Regulation of the endonuclease activity and fidelity by a second metal ion binding is a unique feature of R.KpnI among REases and HNH nucleases. The active site plasticity of R.KpnI opens up avenues for redesigning cofactor specificities and generation of mutants specific to a particular metal ion.

  10. 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.

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

    DEFF Research Database (Denmark)

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

    2010-01-01

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

  12. 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...

  13. Multisubunit RNA Polymerase Cleavage Factors Modulate the Kinetics and Energetics of Nucleotide Incorporation: An RNA Polymerase I Case Study.

    Science.gov (United States)

    Appling, Francis D; Schneider, David A; Lucius, Aaron L

    2017-10-24

    All cellular RNA polymerases are influenced by protein factors that stimulate RNA polymerase-catalyzed cleavage of the nascent RNA. Despite divergence in amino acid sequence, these so-called "cleavage factors" appear to share a common mechanism of action. Cleavage factors associate with the polymerase through a conserved structural element of the polymerase known as the secondary channel or pore. This mode of association enables the cleavage factor to reach through the secondary channel into the polymerase active site to reorient the active site divalent metal ions. This reorientation converts the polymerase active site into a nuclease active site. Interestingly, eukaryotic RNA polymerases I and III (Pols I and III, respectively) have incorporated their cleavage factors as bona fide subunits known as A12.2 and C11, respectively. Although it is clear that A12.2 and C11 dramatically stimulate the polymerase's cleavage activity, it is not known if or how these subunits affect the polymerization mechanism. In this work we have used transient-state kinetic techniques to characterize a Pol I isoform lacking A12.2. Our data clearly demonstrate that the A12.2 subunit profoundly affects the kinetics and energetics of the elementary steps of Pol I-catalyzed nucleotide incorporation. Given the high degree of conservation between polymerase-cleavage factor interactions, these data indicate that cleavage factor-modulated nucleotide incorporation mechanisms may be common to all cellular RNA polymerases.

  14. Allyl deprotection of galacturonic acid derivatives: mechanistic aspects of mercuric-catalyzed prop-1-enyl acetal cleavage.

    Science.gov (United States)

    Barbier, Maximilien; Grand, Eric; Kovensky, José

    2007-12-10

    Different deallylation methods were assayed for selective deprotection of allyl galactopyranosiduronic acid derivatives. A two-step procedure using DABCO and (Ph(3)P)(3)RhCl followed by mercuric-assisted cleavage gave quantitative yields. Reaction in the presence of [(18)O]water allowed us to obtain evidence about the mechanism of prop-1-enyl cleavage.

  15. 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.

  16. Low-Density Lipoprotein Receptor-Related Protein-1 (LRP1) C4408R Mutant Promotes Amyloid Precursor Protein (APP) α-Cleavage in Vitro.

    Science.gov (United States)

    Hou, Huayan; Habib, Ahsan; Zi, Dan; Tian, Kathy; Tian, Jun; Giunta, Brian; Sawmiller, Darrell; Tan, Jun

    2017-09-01

    Previous studies have demonstrated that the low-density lipoprotein receptor-related protein-1 (LRP1) plays conflicting roles in Alzheimer's disease (AD) pathogenesis, clearing β-amyloid (Aβ) from the brain while also enhancing APP endocytosis and resultant amyloidogenic processing. We have recently discovered that co-expression of mutant LRP1 C-terminal domain (LRP1-CT C4408R) with Swedish mutant amyloid precursor protein (APPswe) in Chinese hamster ovary (CHO) cells decreases Aβ production, while also increasing sAPPα and APP α-C-terminal fragment (α-CTF), compared with CHO cells expressing APPswe alone. Surprisingly, the location of this mutation on LRP1 corresponded with the α-secretase cleavage site of APP. Further experimentation confirmed that in CHO cells expressing APPswe or wild-type APP (APPwt), co-expression of LRP1-CT C4408R decreases Aβ and increases sAPPα and α-CTF compared with co-expression of wild-type LRP1-CT. In addition, LRP1-CT C4408R enhanced the unglycosylated form of LRP1-CT and reduced APP endocytosis as determined by flow cytometry. This finding identifies a point mutation in LRP1 which slows LRP1-CT-mediated APP endocytosis and amyloidogenic processing, while enhancing APP α-secretase cleavage, thus demonstrating a potential novel target for slowing AD pathogenesis.

  17. DNA and protein binding, double-strand DNA cleavage and cytotoxicity of mixed ligand copper(II) complexes of the antibacterial drug nalidixic acid.

    Science.gov (United States)

    Loganathan, Rangasamy; Ganeshpandian, Mani; Bhuvanesh, Nattamai S P; Palaniandavar, Mallayan; Muruganantham, Amsaveni; Ghosh, Swapan K; Riyasdeen, Anvarbatcha; Akbarsha, Mohammad Abdulkader

    2017-09-01

    The water soluble mixed ligand complexes [Cu(nal)(diimine)(H 2 O)](ClO 4 ) 1-4, where H(nal) is nalidixic acid and diimine is 2,2'-bipyridine (1), 1,10-phenanthroline (2), 5,6-dimethyl-1,10-phenanthroline (3), and 3,4,7,8-tetramethyl-1,10-phenanthroline (4), have been isolated. The coordination geometry around Cu(II) in 1 and that in the Density Functional Theory optimized structures of 1-4 has been assessed as square pyramidal. The trend in DNA binding constants (K b ) determined using absorption spectral titration (K b : 1, 0.79±0.1base pair. In contrast, 3 and 4 are involved in intimate hydrophobic interaction with DNA through the methyl substituents on phen ring, which is supported by viscosity and protein binding studies. DNA docking studies imply that 4 is involved preferentially in DNA major groove binding while 1-3 in minor groove binding and that all the complexes, upon removing the axially coordinated water molecule, bind in the major groove. Interestingly, 3 and 4 display prominent double-strand DNA cleavage while 1 and 2 effect only single-strand DNA cleavage in the absence of an activator. The complexes 3 and 4 show cytotoxicity higher than 1 and 2 against human breast cancer cell lines (MCF-7). The complex 4 induces apoptotic mode of cell death in cancer cells. Copyright © 2017 Elsevier Inc. All rights reserved.

  18. Efficient cleavage of single and clustered AP site lesions within mono-nucleosome templates by CHO-K1 nuclear extract contrasts with retardation of incision by purified APE1

    Science.gov (United States)

    Eccles, Laura J.; Menoni, Hervé; Angelov, Dimitar; Lomax, Martine E.; O’Neill, Peter

    2015-01-01

    Clustered DNA damage is a unique characteristic of radiation-induced DNA damage and the formation of these sites poses a serious challenge to the cell’s repair machinery. Within a cell DNA is compacted, with nucleosomes being the first order of higher level structure. However, few data are reported on the efficiency of clustered-lesion processing within nucleosomal DNA templates. Here, we show retardation of cleavage of a single AP site by purified APE1 when contained in nucleosomal DNA, compared to cleavage of an AP site in non-nucleosomal DNA. This retardation seen in nucleosomal DNA was alleviated by incubation with CHO-K1 nuclear extract. When clustered DNA damage sites containing bistranded AP sites were present in nucleosomal DNA, efficient cleavage of the AP sites was observed after treatment with nuclear extract. The resultant DSB formation led to DNA dissociating from the histone core and nucleosomal dispersion. Clustered damaged sites containing bistranded AP site/8-oxoG residues showed no retardation of cleavage of the AP site but retardation of 8-oxoG excision, compared to isolated lesions, thus DSB formation was not seen. An increased understanding of processing of clustered DNA damage in a nucleosomal environment may lead to new strategies to enhance the cytotoxic effects of radiotherapeutics. PMID:26439176

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

    Science.gov (United States)

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

    2013-07-16

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

  20. Longitudinal cleavage of the penis in chronic spinal cord injury: two case reports.

    Science.gov (United States)

    Mansoor, Sahibzada Nasir; Ayaz, Saeed Bin; Rathore, Farooq Azam; New, Peter

    2016-05-01

    Penile cleavage is a rare complication of spinal cord injury (SCI) in patients with a chronic indwelling catheter. We report two cases of chronic SCI who developed penile urethral cleavage after prolonged use of an indwelling catheter for bladder management. A 25-year-old wheelchair mobile male with T7 American Spinal Injury Association (ASIA) Impairment Scale (AIS) grade A paraplegia developed a 4 × 1.5 cm ventral urethral cleavage after using an indwelling catheter for four months with inadequate care. He had an associated urinary tract infection and undiagnosed diabetes mellitus. A suprapubic catheter was inserted and surgical repair recommended after resolution of UTI and adequate control of his diabetes mellitus. After initial treatment he was lost to follow-up. The second patient was a 15-year-old male with AIS grade B tetraplegia who presented with a 2.5 cm cleavage on the ventral aspect of penis for the preceding three months. He had been using an indwelling catheter for bladder management for the previous 18 months. He had modified Ashworth scale grade III spasticity in lower limbs resistant to conservative management. There was no history of trauma, infection or diabetes mellitus. The patient was advised penile urethral repair surgery but was lost to follow-up. Penile cleavage is a rare complication of neurogenic bladder in SCI patients. Patients and care givers should be trained in proper bladder management techniques during the hospital stay, counseled regarding the need for regular follow up, and be taught identification and prevention of common complications.

  1. Sequence-specific cleavage of dsRNA by Mini-III RNase.

    Science.gov (United States)

    Głów, Dawid; Pianka, Dariusz; Sulej, Agata A; Kozłowski, Łukasz P; Czarnecka, Justyna; Chojnowski, Grzegorz; Skowronek, Krzysztof J; Bujnicki, Janusz M

    2015-03-11

    Ribonucleases (RNases) play a critical role in RNA processing and degradation by hydrolyzing phosphodiester bonds (exo- or endonucleolytically). Many RNases that cut RNA internally exhibit substrate specificity, but their target sites are usually limited to one or a few specific nucleotides in single-stranded RNA and often in a context of a particular three-dimensional structure of the substrate. Thus far, no RNase counterparts of restriction enzymes have been identified which could cleave double-stranded RNA (dsRNA) in a sequence-specific manner. Here, we present evidence for a sequence-dependent cleavage of long dsRNA by RNase Mini-III from Bacillus subtilis (BsMiniIII). Analysis of the sites cleaved by this enzyme in limited digest of bacteriophage Φ6 dsRNA led to the identification of a consensus target sequence. We defined nucleotide residues within the preferred cleavage site that affected the efficiency of the cleavage and were essential for the discrimination of cleavable versus non-cleavable dsRNA sequences. We have also determined that the loop α5b-α6, a distinctive structural element in Mini-III RNases, is crucial for the specific cleavage, but not for dsRNA binding. Our results suggest that BsMiniIII may serve as a prototype of a sequence-specific dsRNase that could possibly be used for targeted cleavage of dsRNA. © The Author(s) 2015. Published by Oxford University Press on behalf of Nucleic Acids Research.

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

    Science.gov (United States)

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

    2017-09-01

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

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

    Directory of Open Access Journals (Sweden)

    Jiangning Song

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

  4. 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.

  5. Collision Induced Dissociation Products of Disulfide-Bonded Peptides: Ions Result from the Cleavage of More Than One Bond

    Science.gov (United States)

    Clark, Daniel F.; Go, Eden P.; Toumi, Melinda L.; Desaire, Heather

    2011-03-01

    Disulfide bonds are a post-translational modification (PTM) that can be scrambled or shuffled to non-native bonds during recombinant expression, sample handling, or sample purification. Currently, mapping of disulfide bonds is not easy because of various sample requirements and data analysis difficulties. One step towards facilitating this difficult work is developing a better understanding of how disulfide-bonded peptides fragment during collision induced dissociation (CID). Most automated analysis algorithms function based on the assumption that the preponderance of product ions observed during the dissociation of disulfide-bonded peptides result from the cleavage of just one peptide bond, and in this report we tested that assumption by extensively analyzing the product ions generated when several disulfide-bonded peptides are subjected to CID on a quadrupole time of flight (QTOF) instrument. We found that one of the most common types of product ions generated resulted from two peptide bond cleavages, or a double cleavage. We found that for several of the disulfide-bonded peptides analyzed, the number of double cleavage product ions outnumbered those of single cleavages. The influence of charge state and precursor ion size was investigated, to determine if those parameters dictated the amount of double cleavage product ions formed. It was found in this sample set that no strong correlation existed between the charge state or peptide size and the portion of product ions assigned as double cleavages. These data show that these ions could account for many of the product ions detected in CID data of disulfide bonded peptides. We also showed the utility of double cleavage product ions on a peptide with multiple cysteines present. Double cleavage products were able to fully characterize the bonding pattern of each cysteine where typical single b/ y cleavage products could not.

  6. Copper complexes based on chiral Schiff-base ligands: DNA/BSA binding ability, DNA cleavage activity, cytotoxicity and mechanism of apoptosis.

    Science.gov (United States)

    Zhou, Xue-Quan; Li, Yang; Zhang, Dong-Yan; Nie, Yan; Li, Zong-Jin; Gu, Wen; Liu, Xin; Tian, Jin-Lei; Yan, Shi-Ping

    2016-05-23

    Four copper(II) complexes with chiral Schiff-base ligands, [Cu(R-L(1))2]·EtOAc (1) and [Cu(S-L(1))2]·EtOAc (2), [Cu(R-L(2))2]·EtOAc (3) and [Cu(S-L(2))2]·EtOAc (4), (R/S-HL(1) = (R/S)-(1-naththyl)-salicylaldimine, R/S-HL(2) = (R/S)-(1-naththyl)-3-methoxysalicylaldimine, EtOAc = ethyl acetate) were synthesized to serve as artificial nucleases and anticancer drugs. All complexes and R/S-HL(1) ligands were structurally characterized by X-ray crystallography. The interaction of these complexes with CT-DNA was researched via several spectroscopy methods, which indicates that complexes bind to CT-DNA by moderate intercalation binding mode. Moreover, DNA cleavage experiments revealed that the complexes exhibited remarkable DNA cleavage activities in the presence of H2O2via the generation of hydroxyl radical. Particularly, complex 4 also could nick DNA with the production of (1)O2. And all complexes exhibited excellent cytotoxicity to MDA-MB-231, A549 and Hela human cancer cells in micromole magnitude. Furthermore, complex 4 exhibited comparable cytotoxic effect to cisplatin against the proliferation of MDA-MB-231 and A549 cancer cells, as well as showed better anticancer ability to the three cancer cells than the other complexes. The results of cell cycle analysis indicated that complexes 3-4 could induce G2/M phase cell cycle arrest. Furthermore, MDA-MB-231 cells treated with 3 and 4 were subjected to apoptosis and death by generation of ROS and the activation of caspase-3. Interestingly, the chiral complexes 3 and 4 may induce cell apoptosis through extrinsic and mitochondrial intrinsic pathway, respectively. Copyright © 2016 Elsevier Masson SAS. All rights reserved.

  7. Microbial side-chain cleavage of phytosterols by mycobacteria in vegetable oil/aqueous two-phase system.

    Science.gov (United States)

    Xu, Yang-Guang; Guan, Yi-Xin; Wang, Hai-Qing; Yao, Shan-Jing

    2014-09-01

    Microbial side-chain cleavage of natural sterols to 4-androstene-3,17-dione (AD) and 1,4-androstadiene-3,17-dione (ADD) by Mycobacteria has received much attention in pharmaceutical industry, while low yield of the reaction owing to the strong hydrophobicity of sterols is a tough problem to be solved urgently. Eight kinds of vegetable oils, i.e., sunflower, peanut, corn, olive, linseed, walnut, grape seed, and rice oil, were used to construct oil/aqueous biphasic systems in the biotransformation of phytosterols by Mycobacterium sp. MB 3683 cells. The results indicated that vegetable oils are suitable for phytosterol biotransformation. Specially, the yield of AD carried out in sunflower biphasic system (phase ratio of 1:9, oil to aqueous) was greatly increased to 84.8 % with 10 g/L feeding concentration after 120-h transformation at 30 °C and 200 rpm. Distribution coefficients of AD in different oil/aqueous systems were also determined. Because vegetable oils are of low cost and because of their eco-friendly characters, there is a great potential for the application of oil/aqueous two-phase systems in bacteria whole cell biocatalysis.

  8. 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.

  9. Reactive oxygen species-dependent HSP90 protein cleavage participates in arsenical As+3- and MMA+3-induced apoptosis through inhibition of telomerase activity via JNK activation

    International Nuclear Information System (INIS)

    Shen, S.-C.; Yang, L.-Y.; Lin, H.-Y.; Wu, C.-Y.; Su, T.-H.; Chen, Y.-C.

    2008-01-01

    The effects of six arsenic compounds including As +3 , MMA +3 , DMA +3 , As +5 , MMA +5 , and DMA +5 on the viability of NIH3T3 cells were examined. As +3 and MMA +3 , but not the others, exhibited significant cytotoxic effects in NIH3T3 cells through apoptosis induction. The apoptotic events such as DNA fragmentation and chromosome condensation induced by As +3 and MMA +3 were prevented by the addition of NAC and CAT, and induction of HO-1 gene expression in accordance with cleavage of the HSP90 protein, and suppression of telomerase activity were observed in NIH3T3 cells under As +3 and MMA +3 treatments. An increase in the intracellular peroxide level was examined in As +3 - and MMA +3 -treated NIH3T3 cells, and As +3 - and MMA +3 -induced apoptotic events were blocked by NAC, CAT, and DPI addition. HSP90 inhibitors, GA and RD, significantly attenuated the telomerase activity in NIH3T3 cells with an enhancement of As +3 - and MMA +3 -induced cytotoxicity. Suppression of JNKs significantly inhibited As +3 - and MMA +3 -induced apoptosis by blocking HSP90 protein cleavage and telomerase reduction in NIH3T3 cells. Furthermore, Hb, SnPP, and dexferosamine showed no effect against As +3 - and MMA +3 -induced apoptosis, and overexpression of HO-1 protein or inhibition of HO-1 protein expression did not affect the apoptosis induced by As +3 or MMA +3 . These data provide the first evidence to indicate that apoptosis induced by As +3 and MMA +3 is mediated by an ROS-dependent degradation of HSP90 protein and reduction of telomerase via JNK activation, and HO-1 induction might not be involved

  10. Reactive oxygen species-dependent HSP90 protein cleavage participates in arsenical As(+3)- and MMA(+3)-induced apoptosis through inhibition of telomerase activity via JNK activation.

    Science.gov (United States)

    Shen, Shing-Chuan; Yang, Liang-Yo; Lin, Hui-Yi; Wu, Chin-Yen; Su, Tsung-Hsien; Chen, Yen-Chou

    2008-06-01

    The effects of six arsenic compounds including As(+3), MMA(+3), DMA(+3), As(+5), MMA(+5), and DMA(+5) on the viability of NIH3T3 cells were examined. As(+3) and MMA(+3), but not the others, exhibited significant cytotoxic effects in NIH3T3 cells through apoptosis induction. The apoptotic events such as DNA fragmentation and chromosome condensation induced by As(+3) and MMA(+3) were prevented by the addition of NAC and CAT, and induction of HO-1 gene expression in accordance with cleavage of the HSP90 protein, and suppression of telomerase activity were observed in NIH3T3 cells under As(+3) and MMA(+3) treatments. An increase in the intracellular peroxide level was examined in As(+3)- and MMA(+3)-treated NIH3T3 cells, and As(+3)- and MMA(+3)-induced apoptotic events were blocked by NAC, CAT, and DPI addition. HSP90 inhibitors, GA and RD, significantly attenuated the telomerase activity in NIH3T3 cells with an enhancement of As(+3)- and MMA(+3)-induced cytotoxicity. Suppression of JNKs significantly inhibited As(+3)- and MMA(+3)-induced apoptosis by blocking HSP90 protein cleavage and telomerase reduction in NIH3T3 cells. Furthermore, Hb, SnPP, and dexferosamine showed no effect against As(+3)- and MMA(+3)-induced apoptosis, and overexpression of HO-1 protein or inhibition of HO-1 protein expression did not affect the apoptosis induced by As(+3) or MMA(+3). These data provide the first evidence to indicate that apoptosis induced by As(+3) and MMA(+3) is mediated by an ROS-dependent degradation of HSP90 protein and reduction of telomerase via JNK activation, and HO-1 induction might not be involved.

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

    Science.gov (United States)

    Hilbert, Brendan J.; Hayes, Janelle A.; Stone, Nicholas P.; Xu, Rui-Gang

    2017-01-01

    Abstract Many viruses use a powerful terminase motor to pump their genome inside an empty procapsid shell during virus maturation. The large terminase (TerL) protein contains both enzymatic activities necessary for packaging in such viruses: the adenosine triphosphatase (ATPase) that powers DNA translocation and an endonuclease that cleaves the concatemeric genome at both initiation and completion of genome packaging. However, how TerL binds DNA during translocation and cleavage remains mysterious. Here we investigate DNA binding and cleavage using TerL from the thermophilic phage P74-26. We report the structure of the P74-26 TerL nuclease domain, which allows us to model DNA binding in the nuclease active site. We screened a large panel of TerL variants for defects in binding and DNA cleavage, revealing that the ATPase domain is the primary site for DNA binding, and is required for nuclease activity. The nuclease domain is dispensable for DNA binding but residues lining the active site guide DNA for cleavage. Kinetic analysis of DNA cleavage suggests flexible tethering of the nuclease domains during DNA cleavage. We propose that interactions with the procapsid during DNA translocation conformationally restrict the nuclease domain, inhibiting cleavage; TerL release from the capsid upon completion of packaging unlocks the nuclease domains to cleave DNA. PMID:28082398

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

    NARCIS (Netherlands)

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

    2003-01-01

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

  13. Culture of Impure Human Islet Fractions in the Presence of Alpha-1-Antitrypsin Prevents Insulin Cleavage and Improves Islet Recovery

    Science.gov (United States)

    Loganathan, G.; Dawra, R.K.; Pugazhenthi, S.; Wiseman, A.C.; Sanders, M.A.; Saluja, A.K.; Sutherland, D.E.R.; Hering, B.J.; Balamurugan, A.N.

    2010-01-01

    Background Exocrine tissue is commonly cotransplanted with islets in autografting and allotransplantation of impure preparations. Proteases and insulin are released by acinar cells and islets, respectively, during pretransplantation culture and also systemically after transplantation. We hypothesized that released proteases could cleave insulin molecules and that addition of alpha 1 antitrypsin (A1AT) to impure islet cultures would block this cleavage, improving islet recovery and function. Methods Trypsin, chymotrypsin, and elastase (TCE) activity and insulin levels were measured in culture supernates of pure (n = 5) and impure (n = 5) islet fractions, which were isolated from deceased donors. SDS-PAGE was used to detect insulin after incubation with proteases. We assessed the effects of A1AT supplementation (0.5 mg/mL; n = 4] on TCE activity, insulin levels, culture recovery, and islet quality. The ultrastructure of islets exposed to TCE versus control medium was examined using electron microscopy (EM). Results Protease (TCE) activity in culture supernates was directly proportional to the percentage purity of islets: pure, impure, or highly impure. Increasingly lower levels of insulin were detected in culture supernates with higher protease activity levels. Insulin levels measured in supernates of 2000 IE aliquots of impure and highly impure islet preparations were 61 ± 23.7% and 34 ± 33% of that in pure preparations, respectively. Incubation with commercially available proteases (TCE) or exocrine acinar cell supernates cleaved insulin molecules as assessed using SDS-PAGE. Addition of A1AT to impure islet preparations reduced protease activity and restored normal insulin levels as detected using enzyme-linked immunosorbent assay (ELISA) and SDS-PAGE of culture supernates. A1AT improved insulin levels to 98% ± 1.3% in impure and 78% ± 34.2% in highly impure fractions compared with pure islet fractions. A1AT supplementation improved postculture recovery of

  14. 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.

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

    Science.gov (United States)

    Ahrazem, Oussama; Gómez-Gómez, Lourdes; Rodrigo, María J; Avalos, Javier; Limón, María Carmen

    2016-10-26

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

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

    International Nuclear Information System (INIS)

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

    1975-01-01

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

  17. Cleavage of DNA containing 5-fluorocytosine or 5-fluorouracil by type II restriction endonucleases.

    Science.gov (United States)

    Olszewska, Agata; Dadová, Jitka; Mačková, Michaela; Hocek, Michal

    2015-11-01

    A systematic study of the cleavage of DNA sequences containing 5-fluorocytosine or 5-fluorouracil by type II restriction endonucleases (REs) was performed and the results compared with the same sequences containing natural pyrimidine bases, uracil or 5-methylcytosine. The results show that some REs recognize fluorine as a hydrogen on cytosine and cleave the corresponding sequences where the presence of m5dC leads to blocking of the cleavage. However, on uracil, the same REs recognize the F as a methyl surrogate and cleave the sequences which are not cleaved if uracil is incorporated instead of thymine. These results are interesting for understanding the recognition of DNA sequences by REs and for manipulation of the specific DNA cutting. Copyright © 2015 Elsevier Ltd. All rights reserved.

  18. 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

    . 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...... 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.......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...

  19. 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.

  20. The relation between surgical cleavage and preoperative neuroradiological findings in intracranial meningiomas

    Energy Technology Data Exchange (ETDEWEB)

    Celikoglu, Erhan [Neurosurgery Clinic, Dr. Lutfi Kirdar Kartal Research and Training Hospital, Petrol Is Mahallesi, Raman Sokak, Kartal, Istanbul (Turkey); Suslu, Hikmet Turan, E-mail: hikmets1972@yahoo.com [Neurosurgery Clinic, Dr. Lutfi Kirdar Kartal Research and Training Hospital, Petrol Is Mahallesi, Raman Sokak, Kartal, Istanbul (Turkey); Hazneci, Julide; Bozbuga, Mustafa [Neurosurgery Clinic, Dr. Lutfi Kirdar Kartal Research and Training Hospital, Petrol Is Mahallesi, Raman Sokak, Kartal, Istanbul (Turkey)

    2011-11-15

    Background: Meningiomas are generally benign masses, and in many cases they do not invade the brain. Therefore their potential to provide cures is high. The most important cause of the development of recurrence in the post-operative period is subtotal resection. Any information that will allow us to perform total mass resection will be beneficial in terms of long-term good clinical procedure. Our aim in this study is to obtain the radiological data from which we can obtain accurate information in terms of the surgical cleavage between the tumor and parenchyma during the surgical planning of the meningiomas. Methods: We evaluated 85 cases with intracranial meningioma that were treated by the same microsurgical technique. All posterior fossa and skull base meningiomas were not included in the study. Results: Tumor size was smaller than 3 cm in 19 cases, between 3 and 6 cm in 46 cases, and bigger than 6 cm in 20 cases. The cleavage line between the tumor capsule and the cortex underneath was extrapial in 32 cases, subpial in 29 cases, and mixed in 24 cases. Dominant arterial supply was dural in 46 cases. Thirty-three cases were predominantly mixed and 6 cases were predominantly corticopial. At magnetic resonance images, 16 of 28 cases which showed clear tumor-cortex interface, had an extrapial cleavage line. Conclusions: When surgical treatment of intracranial meningiomas are considered, it is necessary to examine if there is a surgically safe border between the cortex underneath in the preoperative images. It can be concluded that it is appropriate to operate small meningiomas which are on the sensitive regions of the brain when they are in their earlier stages and still have an extrapial cleavage.

  1. The DNA gyrase-quinolone complex. ATP hydrolysis and the mechanism of DNA cleavage

    DEFF Research Database (Denmark)

    Kampranis, S C; Maxwell, A

    1998-01-01

    , S. C., and Maxwell, A. (1998) J. Biol. Chem. 269, 22606-22614). The kinetics of ATP hydrolysis via this pathway have been studied and found to differ from those of the reaction of the drug-free enzyme. The quinolone-characteristic ATPase rate is DNA-dependent and can be induced in the presence...... binding and drug-induced DNA cleavage are separate processes constituting two sequential steps in the mechanism of action of quinolones on DNA gyrase....

  2. Multinuclear non-heme iron complexes for double-strand DNA cleavage

    NARCIS (Netherlands)

    Megens, Rik P.; van den Berg, Tieme A.; de Bruijn, A. Dowine; Feringa, Ben L.; Roelfes, Gerard

    2009-01-01

    The cytotoxicity of the antitumor drug BLM is believed to be related to the ability of the corresponding iron complex (Fe-BLM) to engage in oxidative double-strand DNA cleavage. The iron complex of the ligand N4Py (Fe-N4Py; N4Py - N,N-bis(2-pyridyl)-N-bis(2-pyridyl)methylamine has proven to be a

  3. DNA binding and cleavage activity by a mononuclear iron(II)Schiff ...

    Indian Academy of Sciences (India)

    C. One experiment was carried out incubating DNA with. 20μM metal complex, 1μM H2O2 and DMSO. A load- ing buffer containing 0.25% bromophenol blue, 40%. (w/v) sucrose and 0.5M EDTA was added and the electrophoresis of the DNA cleavage products were performed on agarose gel containing ethidium bro- mide.

  4. Metal ion dependence of DNA cleavage by SepMI and EhoI restriction endonucleases.

    Science.gov (United States)

    Belkebir, Abdelkarim; Azeddoug, Houssine

    2013-02-22

    Most of type II restriction endonucleases show an absolute requirement for divalent metal ions as cofactors for DNA cleavage. While Mg(2+) is the natural cofactor other metal ions can substitute it and mediate the catalysis, however Ca(2+) (alone) only supports DNA binding. To investigate the role of Mg(2+) in DNA cleavage by restriction endonucleases, we have studied the Mg(2+) and Mn(2+) concentration dependence of DNA cleavage by SepMI and EhoI. Digestion reactions were carried out at different Mg(2+) and Mn(2+) concentrations at constant ionic strength. These enzymes showed different behavior regarding the ions requirement, SepMI reached near maximal level of activity between 10 and 20mM while no activity was detected in the presence of Mn(2+) and in the presence of Ca(2+) cleavage activity was significantly decreased. However, EhoI was more highly active in the presence of Mn(2+) than in the presence of Mg(2+) and can be activated by Ca(2+). Our results propose the two-metal ion mechanism for EhoI and the one-metal ion mechanism for SepMI restriction endonuclease. The analysis of the kinetic parameters under steady state conditions showed that SepMI had a K(m) value for pTrcHisB DNA of 6.15 nM and a V(max) of 1.79×10(-2)nM min(-1), while EhoI had a K(m) for pUC19 plasmid of 8.66 nM and a V(max) of 2×10(-2)nM min(-1). Copyright © 2012 Elsevier GmbH. All rights reserved.

  5. Thiophene antibacterials that allosterically stabilize DNA-cleavage complexes with DNA gyrase

    OpenAIRE

    Chan, Pan F.; Germe, Thomas; Bax, Benjamin D.; Huang, Jianzhong; Thalji, Reema K.; Bacqu��, Eric; Checchia, Anna; Chen, Dongzhao; Cui, Haifeng; Ding, Xiao; Ingraham, Karen; McCloskey, Lynn; Raha, Kaushik; Srikannathasan, Velupillai; Maxwell, Anthony

    2017-01-01

    The spread of multidrug-resistant bacteria constitutes a significant unmet medical need. Fluoroquinolone antibiotics have been compromised by resistance mutations in their targets: DNA gyrase and topoisomerase IV. Using biochemical and genetic techniques, we have identified and characterized a class of antibacterials which transforms DNA gyrase into toxic DNA-cleavage complexes, similar to fluoroquinolones, but with a distinct mechanism of action. X-ray crystallography shows that the inhibito...

  6. An MLP-based feature subset selection for HIV-1 protease cleavage site analysis.

    Science.gov (United States)

    Kim, Gilhan; Kim, Yeonjoo; Lim, Heuiseok; Kim, Hyeoncheol

    2010-01-01

    In recent years, several machine learning approaches have been applied to modeling the specificity of the human immunodeficiency virus type 1 (HIV-1) protease cleavage domain. However, the high dimensional domain dataset contains a small number of samples, which could misguide classification modeling and its interpretation. Appropriate feature selection can alleviate the problem by eliminating irrelevant and redundant features, and thus improve prediction performance. We introduce a new feature subset selection method, FS-MLP, that selects relevant features using multi-layered perceptron (MLP) learning. The method includes MLP learning with a training dataset and then feature subset selection using decompositional approach to analyze the trained MLP. Our method is able to select a subset of relevant features in high dimensional, multi-variate and non-linear domains. Using five artificial datasets that represent four data types, we verified the FS-MLP performance with seven other feature selection methods. Experimental results showed that the FS-MLP is superior at high dimensional, multi-variate and non-linear domains. In experiments with HIV-1 protease cleavage dataset, the FS-MLP selected a set of 14 highly relevant features among 160 original features. On a validation set of 131 test instances, classifiers that used the 14 features showed about 95% accuracy which outperformed other seven methods in terms of accuracy and the number of features. Our experimental results indicate that the FS-MLP is effective in analyzing multi-variate, non-linear and high dimensional datasets such as HIV-1 protease cleavage dataset. The 14 relevant features which were selected by the FS-MLP provide us with useful insights into the HIV-1 cleavage site domain as well. The FS-MLP is a useful method for computational sequence analysis in general. 2009 Elsevier B.V. All rights reserved.

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

    Science.gov (United States)

    Vann, Kendra R; Sedgeman, Carl A; Gopas, Jacob; Golan-Goldhirsh, Avi; Osheroff, Neil

    2015-07-28

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

  8. Cleavage of Functionalized DNA Containing 5-Modified Pyrimidines by Type II Restriction Endonucleases

    Czech Academy of Sciences Publication Activity Database

    Macíčková-Cahová, Hana; Pohl, Radek; Hocek, Michal

    2011-01-01

    Roč. 12, č. 3 (2011), s. 431-438 ISSN 1439-4227 R&D Projects: GA MŠk LC512; GA ČR GA203/09/0317 Institutional research plan: CEZ:AV0Z40550506 Keywords : base-modified DNA * DNA cleavage * DNA polymerases * nucleosides * restriction endonucleases Subject RIV: CC - Organic Chemistry Impact factor: 3.944, year: 2011

  9. Cleavage of a RNA analog containing uridine by a bifunctional dinuclear Zn(II) catalyst.

    Science.gov (United States)

    Rossiter, Clifford S; Mathews, Ryan A; del Mundo, Imee Marie A; Morrow, Janet R

    2009-01-01

    The macrocyclic ligand, 1,4-bis((1-oxa-4,7,10-triazacyclododecan-7-yl)methyl)benzene (L1) is prepared. L1 binds two Zn(II) ions at neutral pH to form Zn(2)(L1) as studied by using pH-potentiometric titrations. Zn(2)(L1) binds two uridines at pH 7.0, I=0.100M (NaCl) and the mononuclear analog Zn(L2) (L2=1-oxa-4,7,10-triazacyclododecane) binds a single uridine; dissociation constants for both complexes are in the millimolar range. Both complexes promote the cleavage of a simple RNA analog lacking a nucleobase (HpPNP=2-hydroxypropyl-4-nitrophenylphosphate), and a uridine containing RNA analog UpPNP (uridine-3'-4-nitrophenylphosphate). Plots of the first-order rate constant for cleavage of HpPNP as a function of Zn(L2) concentration from 0.5mM to 20.0mM are linear, consistent with weak complexation to substrate K(d)>20mM. In contrast, first-order rate constants for cleavage of UpPNP by Zn(L2) or Zn(2)(L1) over similar concentration ranges exhibit a downward curvature, consistent with the formation of a complex between catalyst and UpPNP. Comparison of second-order rate constants (k(2)=k(cat)/K(d)) shows that the dinuclear complex Zn(2)(L1) is a better catalyst than Zn(L2) for both HpPNP and UpPNP cleavage.

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

    Science.gov (United States)

    2016-01-01

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

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

    Science.gov (United States)

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

    2017-07-14

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

  12. 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.

  13. D-β-aspartyl residue exhibiting uncommon high resistance to spontaneous peptide bond cleavage.

    Science.gov (United States)

    Aki, Kenzo; Okamura, Emiko

    2016-02-15

    Although L-amino acids were selected as main constituents of peptides and proteins during chemical evolution, D-aspartyl (Asp) residue is found in a variety of living tissues. In particular, D-β-Asp is thought to be stable than any other Asp isomers, and this could be a reason for gradual accumulation in abnormal proteins and peptides to modify their structures and functions. It is predicted that D-β-Asp shows high resistance to biomolecular reactions. For instance, less reactivity of D-β-Asp is expected to bond cleavage, although such information has not been provided yet. In this work, the spontaneous peptide bond cleavage was compared between Asp isomers, by applying real-time solution-state NMR to eye lens αΑ-crystallin 51-60 fragment, S(51)LFRTVLD(58)SG(60) and αΒ-crystallin 61-67 analog, F(61)D(62)TGLSG(67) consisting of L-α- and D-β-Asp 58 and 62, respectively. Kinetic analysis showed how tough the uncommon D-β-Asp residue was against the peptide bond cleavage as compared to natural L-α-Asp. Differences in pKa and conformation between L-α- and D-β-Asp side chains were plausible factors to determine reactivity of Asp isomers. The present study, for the first time, provides a rationale to explain less reactivity of D-β-Asp to allow abnormal accumulation.

  14. Experimental study and local approach of cleavage crack arrest in a bainitic steel

    International Nuclear Information System (INIS)

    Dahl, A.

    2012-01-01

    EDF wants to complete the assessment of reactor pressure vessels, usually based on crack initiation concept, by crack arrest concept. The work aims at improving the knowledge of cleavage crack arrest in a reactor pressure vessel steel. For that purpose, isothermal crack arrest experiments were performed for temperatures ranging from - 150 C up to - 50 C on compact tensile specimens and on pre-cracked rings submitted to compressive loading. Fractographic observations revealed that the whole crack propagation and arrest occurs by cleavage even if ductile tearing occurs before initiation of the unstable crack propagation. A local cleavage crack arrest criterion is applied in finite element computations carried out in elasto-visco-plasticity and in full dynamics: the crack propagates since the largest principal stress reaches a critical stress. The application of this criterion on the experiments leads to a good prediction of the crack speed and of the crack length and shows that the critical stress increases with the temperature in relation with dissipation features observed on the fracture surfaces. Dependence to the geometry is observed; it can be due to the assumption used for the 2D computations. The study of the structural dynamic shows that the crack arrest phenomenon is very linked to the global dynamics of the structure: crack arrest and crack closure occur approximately at the same time. (author)

  15. Simulating multiplexed SNP discovery rates using base-specific cleavage and mass spectrometry.

    Science.gov (United States)

    Böcker, Sebastian

    2007-01-15

    Single Nucleotide Polymorphisms (SNPs) are believed to contribute strongly to the genetic variability in living beings, and SNP and mutation discovery are of great interest in today's Life Sciences. A comparatively new method to discover such polymorphisms is based on base-specific cleavage, where resulting cleavage products are analyzed by mass spectrometry (MS). One particular advantage of this method is the possibility of multiplexing the biochemical reactions, i.e. examining multiple genomic regions in parallel. Simulations can help estimating the performance of a method for polymorphism discovery, and allow us to evaluate the influence of method parameters on the discovery rate, and also to investigate whether the method is well suited for a certain genomic region. We show how to efficiently conduct such simulations for polymorphism discovery using base-specific cleavage and MS. Simulating multiplexed polymorphism discovery leads us to the problem of uniformly drawing a multiplex. Given a multiset of natural numbers we want to uniformly draw a subset of fixed cardinality so that the elements sum up to some fixed total length. We show how to enumerate multiplex layouts using dynamic programming, which allows us to uniformly draw a multiplex.

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

    Science.gov (United States)

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

    2017-02-01

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

  17. Catalysis and co-catalysis of bond cleavages in coal and coal analogs

    Energy Technology Data Exchange (ETDEWEB)

    Miller, B.

    1990-01-01

    Progress is reported on the cleavage of bonds in coal and coal analogs. This quarter, 9-Phenylthioanthracene (9-PTA) was chosen as an appropriate substrate to test the hypothesis that reductive cleavage of bonds to aromatic rings caused by heating in hydrogen donor solvents, such as tetralin, may be catalyzed by electron donating agents in the absence of hydrogen bonding or acidic reagents. Ca. 0.02 M solutions of 9-PTA in tetralin (purified by chromatography on neutral alumina) were heated in glass ampoules. It was observed that decomposition of 9-PTA occurs on heating at temperatures as low as 250{degree}C, even in the absence of additives or catalysts. Reductive cleavage (measured by yields of anthracene), however, was invariably a minor process. Two major products, obtained at retention times of ca. 6.5 and 9.0 min., respectively, were observed in all runs. However, these products were obtained in rather variable yields, with an increase in the yield of one product often being accompanied by a decrease in the yield of the other. The nature of the products is unknown. GC-MS analysis is awaited. Addition of 2,6-xylenol, 1,3,5-trimethoxybenzene, or 1,4-dimethoxynaphthalene to the thermolysis mixtures appeared to offer, at best, only slight increases in reaction rates, and little change in product compositions.

  18. BspRI restriction endonuclease: cloning, expression in Escherichia coli and sequential cleavage mechanism.

    Science.gov (United States)

    Raskó, Tamás; Dér, András; Klement, Eva; Slaska-Kiss, Krystyna; Pósfai, Eszter; Medzihradszky, Katalin F; Marshak, Daniel R; Roberts, Richard J; Kiss, Antal

    2010-11-01

    The GGCC-specific restriction endonuclease BspRI is one of the few Type IIP restriction endonucleases, which were suggested to be a monomer. Amino acid sequence information obtained by Edman sequencing and mass spectrometry analysis was used to clone the gene encoding BspRI. The bspRIR gene is located adjacently to the gene of the cognate modification methyltransferase and encodes a 304 aa protein. Expression of the bspRIR gene in Escherichia coli was dependent on the replacement of the native TTG initiation codon with an ATG codon, explaining previous failures in cloning the gene using functional selection. A plasmid containing a single BspRI recognition site was used to analyze kinetically nicking and second-strand cleavage under steady-state conditions. Cleavage of the supercoiled plasmid went through a relaxed intermediate indicating sequential hydrolysis of the two strands. Results of the kinetic analysis of the first- and second-strand cleavage are consistent with cutting the double-stranded substrate site in two independent binding events. A database search identified eight putative restriction-modification systems in which the predicted endonucleases as well as the methyltransferases share high sequence similarity with the corresponding protein of the BspRI system. BspRI and the related putative restriction endonucleases belong to the PD-(D/E)XK nuclease superfamily.

  19. Modulation of mutant huntingtin N-terminal cleavage and its effect on aggregation and cell death

    NARCIS (Netherlands)

    Juenemann, Katrin; Weisse, Christina; Reichmann, Denise; Kaether, Christoph; Calkhoven, Cornelis F.; Schilling, Gabriele

    2011-01-01

    Huntington's disease (HD) is a neurodegenerative disorder caused by a polyglutamine expansion near the N-terminus of huntingtin. A neuropathological hallmark of Huntington's disease is the presence of intracellular aggregates composed of mutant huntingtin N-terminal fragments in human postmortem

  20. Rubber Oxygenase and Latex Clearing Protein Cleave Rubber to Different Products and Use Different Cleavage Mechanisms

    Science.gov (United States)

    Birke, Jakob

    2014-01-01

    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. PMID:24907333

  1. 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.

  2. Toward the design of a catalytic metallodrug: selective cleavage of G-quadruplex telomeric DNA by an anticancer copper-acridine-ATCUN complex.

    Science.gov (United States)

    Yu, Zhen; Han, Menglu; Cowan, James A

    2015-02-02

    Telomeric DNA represents a novel target for the development of anticancer drugs. By application of a catalytic metallodrug strategy, a copper-acridine-ATCUN complex (CuGGHK-Acr) has been designed that targets G-quadruplex telomeric DNA. Both fluorescence solution assays and gel sequencing demonstrate the CuGGHK-Acr catalyst to selectively bind and cleave the G-quadruplex telomere sequence. The cleavage pathway has been mapped by matrix assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS) experiments. CuGGHK-Acr promotes significant inhibition of cancer cell proliferation and shortening of telomere length. Both senescence and apoptosis are induced in the breast cancer cell line MCF7. © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  3. Blocking Internalization of Phosphatidylethanolamine at Cleavage Furrow of Mitosis as a Novel Mechanism of Anti-Breast Cancer Strategy

    National Research Council Canada - National Science Library

    Cui, Zhen

    2002-01-01

    During the formation of cleavage furrow of mitosis, phosphatidylethanolamine (PE) flips from inner leaflet of the plasma membrane to the outer leaflet specifically in the furrow region near the contractile ring...

  4. Blocking Internalization of Phosphatidylethanolamine at Cleavage Furrow of Mitosis as a Novel Mechanism of Anti-Breast-Cancer Strategy

    National Research Council Canada - National Science Library

    Cui, Zheng

    2003-01-01

    During the formation of cleavage furrow of mitosis, phosphatidylethanolamine (PE) flips from inner leaflet of the plasma membrane to the outer leaflet specifically in the furrow region near the contractile ring...

  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

  6. Seneca Valley Virus Suppresses Host Type I Interferon Production by Targeting Adaptor Proteins MAVS, TRIF, and TANK for Cleavage.

    Science.gov (United States)

    Qian, Suhong; Fan, Wenchun; Liu, Tingting; Wu, Mengge; Zhang, Huawei; Cui, Xiaofang; Zhou, Yun; Hu, Junjie; Wei, Shaozhong; Chen, Huanchun; Li, Xiangmin; Qian, Ping

    2017-08-15

    Seneca Valley virus (SVV) is an oncolytic RNA virus belonging to the Picornaviridae family. Its nucleotide sequence is highly similar to those of members of the Cardiovirus genus. SVV is also a neuroendocrine cancer-selective oncolytic picornavirus that can be used for anticancer therapy. However, the interaction between SVV and its host is yet to be fully characterized. In this study, SVV inhibited antiviral type I interferon (IFN) responses by targeting different host adaptors, including mitochondrial antiviral signaling (MAVS), Toll/interleukin 1 (IL-1) receptor domain-containing adaptor inducing IFN-β (TRIF), and TRAF family member-associated NF-κB activator (TANK), via viral 3C protease (3C pro ). SVV 3C pro mediated the cleavage of MAVS, TRIF, and TANK at specific sites, which required its protease activity. The cleaved MAVS, TRIF, and TANK lost the ability to regulate pattern recognition receptor (PRR)-mediated IFN production. The cleavage of TANK also facilitated TRAF6-induced NF-κB activation. SVV was also found to be sensitive to IFN-β. Therefore, SVV suppressed antiviral IFN production to escape host antiviral innate immune responses by cleaving host adaptor molecules. IMPORTANCE Host cells have developed various defenses against microbial pathogen infection. The production of IFN is the first line of defense against microbial infection. However, viruses have evolved many strategies to disrupt this host defense. SVV, a member of the Picornavirus genus, is an oncolytic virus that shows potential functions in anticancer therapy. It has been demonstrated that IFN can be used in anticancer therapy for certain tumors. However, the relationship between oncolytic virus and innate immune response in anticancer therapy is still not well known. In this study, we showed that SVV has evolved as an effective mechanism to inhibit host type I IFN production by using its 3C pro to cleave the molecules MAVS, TRIF, and TANK directly. These molecules are crucial for

  7. Pro-domain removal in ASP-2 and the cleavage of the amyloid precursor are influenced by pH

    Directory of Open Access Journals (Sweden)

    Austen Brian

    2002-08-01

    Full Text Available Abstract Background One of the signatures of Alzheimer's disease is the accumulation of aggregated amyloid protein, Aβ, in the brain. Aβ arises from cleavage of the Amyloid Precursor protein by β and γ secretases, which present attractive candidates for therapeutic targeting. Two β-secretase candidates, ASP-1 and ASP-2, were identified as aspartic proteases, both of which cleave the amyloid precursor at the β-site. These are produced as immature transmembrane proteins containing a pro-segment. Results ASP-2 expressed in HEK293-cells cleaved the Swedish mutant amyloid precursor at different β-sites at different pHs in vitro. Recent reports show that furin cleaves the pro-peptide of ASP-2, whereas ASP-1 undergoes auto-catalysis. We show that purified recombinant ASP-2 cleaves its own pro-peptide at ph 5 but not pH 8.5 as seen by mass spectrometry, electrophoresis and N-terminal sequencing. Conclusion We suggest that ASP-2 processing as well as activity are influenced by pH, and hence the cellular localisation of the protein may have profound effects on the production of Aβ. These factors should be taken into consideration in the design of potential inhibitors for these enzymes.

  8. Cleavage of Type I Collagen by Fibroblast Activation Protein-α Enhances Class A Scavenger Receptor Mediated Macrophage Adhesion.

    Directory of Open Access Journals (Sweden)

    Anna Mazur

    Full Text Available Pathophysiological conditions such as fibrosis, inflammation, and tumor progression are associated with modification of the extracellular matrix (ECM. These modifications create ligands that differentially interact with cells to promote responses that drive pathological processes. Within the tumor stroma, fibroblasts are activated and increase the expression of type I collagen. In addition, activated fibroblasts specifically express fibroblast activation protein-α (FAP, a post-prolyl peptidase. Although FAP reportedly cleaves type I collagen and contributes to tumor progression, the specific pathophysiologic role of FAP is not clear. In this study, the possibility that FAP-mediated cleavage of type I collagen modulates macrophage interaction with collagen was examined using macrophage adhesion assays. Our results demonstrate that FAP selectively cleaves type I collagen resulting in increased macrophage adhesion. Increased macrophage adhesion to FAP-cleaved collagen was not affected by inhibiting integrin-mediated interactions, but was abolished in macrophages lacking the class A scavenger receptor (SR-A/CD204. Further, SR-A expressing macrophages localize with activated fibroblasts in breast tumors of MMTV-PyMT mice. Together, these results demonstrate that FAP-cleaved collagen is a substrate for SR-A-dependent macrophage adhesion, and suggest that by modifying the ECM, FAP plays a novel role in mediating communication between activated fibroblasts and macrophages.

  9. Life and death of proteins after protease cleavage: protein degradation by the N-end rule pathway.

    Science.gov (United States)

    Dissmeyer, Nico; Rivas, Susana; Graciet, Emmanuelle

    2018-05-01

    Contents Summary 929 I. conservation and diversity of N-end rule pathways 929 II. Defensive functions of the N-end rule pathway in plants 930 III. Proteases and degradation by the N-end rule pathway 930 IV. New proteomics approaches for the identification of N-end rule substrates 932 V. Concluding remarks 932 Acknowledgements 934 References 934 SUMMARY: The N-end rule relates the stability of a protein to the identity of its N-terminal residue and some of its modifications. Since its discovery in the 1980s, the repertoire of N-terminal degradation signals has expanded, leading to a diversity of N-end rule pathways. Although some of these newly discovered N-end rule pathways remain largely unexplored in plants, recent discoveries have highlighted roles of N-end rule-mediated protein degradation in plant defense against pathogens and in cell proliferation during organ growth. Despite this progress, a bottleneck remains the proteome-wide identification of N-end rule substrates due to the prerequisite for endoproteolytic cleavage and technical limitations. Here, we discuss the recent diversification of N-end rule pathways and their newly discovered functions in plant defenses, stressing the role of proteases. We expect that novel proteomics techniques (N-terminomics) will be essential for substrate identification. We review these methods, their limitations and future developments. © 2017 The Authors. New Phytologist © 2017 New Phytologist Trust.

  10. 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.

  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.

    2011-01-01

    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. PMID:21685455

  12. 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.

  13. Amplification of bleomycin-induced DNA cleavage at cytosine residues 3' to GGG sequences by pyrrole triamide.

    Science.gov (United States)

    Hiraku, Y; Oikawa, S; Kuroki, K; Sugiyama, H; Saito, I; Kawanishi, S

    2001-02-01

    We investigated the amplification of bleomycin-induced DNA cleavage by synthetic triamides containing N-methylpyrrole (Py) and/or N-methylimidazole (Im), PyPyPy, PyPyIm, PyImPy, and PyImIm, using 32P-labeled DNA fragments obtained from the human c-Ha-ras-1 and p53 genes. Peplomycin, a bleomycin analog, plus Fe(II) caused DNA cleavage at the 5'-GC-3' and 5'-GT-3' sequences (damaged bases are underlined). The addition of PyPyPy dramatically enhanced the cleavage, particularly at cytosine residues 3' to consecutive guanines. Alteration in the site specificity was not observed with other triamides (PyPyIm, PyImPy, and PyImIm). DNase I footprinting revealed that PyPyPy bound to the sites adjacent to the sites where DNA cleavage was enhanced by PyPyPy, and that PyPyPy enhanced DNase I-induced cleavage in GC-rich regions. These findings suggest that binding of PyPyPy to the DNA minor groove changes the DNA conformation to allow peplomycin to cleave DNA more efficiently at GC-rich sequences, resulting in intensive site-specific DNA cleavage particularly at cytosines at the 3'-side of polyguanines. The present study on amplifiers of antitumor drugs would appear to offer a novel approach to the establishment of more effective chemotherapy.

  14. Chemoproteomics Reveals Unexpected Lysine/Arginine-Specific Cleavage of Peptide Chains as a Potential Protein Degradation Machinery.

    Science.gov (United States)

    Tian, Caiping; Liu, Keke; Sun, Rui; Fu, Ling; Yang, Jing

    2018-01-02

    Proteins can undergo oxidative cleavage by in vitro metal-catalyzed oxidation (MCO) in either the α-amidation or the diamide pathway. However, whether oxidative cleavage of polypeptide-chain occurs in biological systems remains unexplored. We describe a chemoproteomic approach to globally and site-specifically profile electrophilic protein degradants formed from peptide backbone cleavages in human proteomes, including the known N-terminal α-ketoacyl products and >1000 unexpected N-terminal formyl products. Strikingly, such cleavages predominantly occur at the carboxyl side of lysine (K) and arginine (R) residues across native proteomes in situ, while MCO-induced oxidative cleavages randomly distribute on peptide/protein sequences in vitro. Furthermore, ionizing radiation-induced reactive oxygen species (ROS) also generate random oxidative cleavages in situ. These findings suggest that the endogenous formation of N-formyl and N-α-ketoacyl degradants in biological systems is more likely regulated by a previously unknown mechanism with a trypsin-like specificity, rather than the random oxidative damage as previously thought. More generally, our study highlights the utility of quantitative chemoproteomics in combination with unrestricted search tools as a viable strategy to discover unexpected chemical modifications of proteins labeled with active-based probes.

  15. Enzymatic cleavage of type II restriction endonucleases on the 2'-O-methyl nucleotide and phosphorothioate substituted DNA.

    Directory of Open Access Journals (Sweden)

    Guojie Zhao

    Full Text Available The effects of nucleotide analogue substitution on the cleavage efficiencies of type II restriction endonucleases have been investigated. Six restriction endonucleases (EcoRV, SpeI, XbaI, XhoI, PstI and SphI were investigated respectively regarding their cleavage when substrates were substituted by 2'-O-methyl nucleotide (2'-OMeN and phosphorothioate (PS. Substitutions were made in the recognition sequence and the two nucleotides flanking the recognition sequence for each endonuclease. The endonuclease cleavage efficiencies were determined using FRET-based assay. Results demonstrated a position-dependent inhibitory effect of substitution on the cleavage efficiency for all the six endonucleases. In general, the 2'-OMeN substitutions had greater impact than the PS substitutions on the enzymatic activities. Nucleotides of optimal substitutions for protection against RE cleavage were identified. Experimental results and conclusions in this study facilitate our insight into the DNA-protein interactions and the enzymatic cleavage mechanism, particularly for those whose detailed structure information is not available. In addition, the information could benefit the development of bioengineering and synthetic biology.

  16. Enzymatic cleavage of type II restriction endonucleases on the 2'-O-methyl nucleotide and phosphorothioate substituted DNA.

    Science.gov (United States)

    Zhao, Guojie; Li, Jun; Tong, Zhaoxue; Zhao, Bin; Mu, Runqing; Guan, Yifu

    2013-01-01

    The effects of nucleotide analogue substitution on the cleavage efficiencies of type II restriction endonucleases have been investigated. Six restriction endonucleases (EcoRV, SpeI, XbaI, XhoI, PstI and SphI) were investigated respectively regarding their cleavage when substrates were substituted by 2'-O-methyl nucleotide (2'-OMeN) and phosphorothioate (PS). Substitutions were made in the recognition sequence and the two nucleotides flanking the recognition sequence for each endonuclease. The endonuclease cleavage efficiencies were determined using FRET-based assay. Results demonstrated a position-dependent inhibitory effect of substitution on the cleavage efficiency for all the six endonucleases. In general, the 2'-OMeN substitutions had greater impact than the PS substitutions on the enzymatic activities. Nucleotides of optimal substitutions for protection against RE cleavage were identified. Experimental results and conclusions in this study facilitate our insight into the DNA-protein interactions and the enzymatic cleavage mechanism, particularly for those whose detailed structure information is not available. In addition, the information could benefit the development of bioengineering and synthetic biology.

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

    Science.gov (United States)

    Lewis, John G; Elder, Peter A

    2017-07-01

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

  18. Site-specificO-Glycosylation by PolypeptideN-Acetylgalactosaminyltransferase 2 (GalNAc-transferase T2) Co-regulates β1-Adrenergic Receptor N-terminal Cleavage.

    Science.gov (United States)

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

    2017-03-17

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

  19. Granzyme B mediates both direct and indirect cleavage of extracellular matrix in skin after chronic low-dose ultraviolet light irradiation.

    Science.gov (United States)

    Parkinson, Leigh G; Toro, Ana; Zhao, Hongyan; Brown, Keddie; Tebbutt, Scott J; Granville, David J

    2015-02-01

    Extracellular matrix (ECM) degradation is a hallmark of many chronic inflammatory diseases that can lead to a loss of function, aging, and disease progression. Ultraviolet light (UV) irradiation from the sun is widely considered as the major cause of visible human skin aging, causing increased inflammation and enhanced ECM degradation. Granzyme B (GzmB), a serine protease that is expressed by a variety of cells, accumulates in the extracellular milieu during chronic inflammation and cleaves a number of ECM proteins. We hypothesized that GzmB contributes to ECM degradation in the skin after UV irradiation through both direct cleavage of ECM proteins and indirectly through the induction of other proteinases. Wild-type and GzmB-knockout mice were repeatedly exposed to minimal erythemal doses of solar-simulated UV irradiation for 20 weeks. GzmB expression was significantly increased in wild-type treated skin compared to nonirradiated controls, colocalizing to keratinocytes and to an increased mast cell population. GzmB deficiency significantly protected against the formation of wrinkles and the loss of dermal collagen density, which was related to the cleavage of decorin, an abundant proteoglycan involved in collagen fibrillogenesis and integrity. GzmB also cleaved fibronectin, and GzmB-mediated fibronectin fragments increased the expression of collagen-degrading matrix metalloproteinase-1 (MMP-1) in fibroblasts. Collectively, these findings indicate a significant role for GzmB in ECM degradation that may have implications in many age-related chronic inflammatory diseases. © 2014 The Authors. Aging Cell published by the Anatomical Society and John Wiley & Sons Ltd.

  20. Cleavage-based hybridization chain reaction for electrochemical detection of thrombin.

    Science.gov (United States)

    Chang, Yuanyuan; Chai, Yaqin; Xie, Shunbi; Yuan, Yali; Zhang, Juan; Yuan, Ruo

    2014-09-07

    In the present work, we constructed a new label-free "inter-sandwich" electrochemical aptasensor for thrombin (TB) detection by employing a cleavage-based hybridization chain reaction (HCR). The designed single-stranded DNA (defined as binding DNA), which contained the thrombin aptamer binding sequence, a DNAzyme cleavage site and a signal reporter sequence, was first immobilized on the electrode. In the absence of a target TB, the designed DNAzymes could combine with the thrombin aptamer binding sequence via complementary base pairing, and then Cu(2+) could cleave the binding DNA. In the presence of a target TB, TB could combine with the thrombin aptamer binding sequence to predominantly form an aptamer-protein complex, which blocked the DNAzyme cleavage site and prevented the binding DNA from being cleaved by Cu(2+)-dependent DNAzyme. As a result, the signal reporter sequence could leave the electrode surface to trigger HCR with the help of two auxiliary DNA single-strands, A1 and A2. Then, the electron mediator hexaammineruthenium (III) chloride ([Ru(NH3)6](3+)) was embedded into the double-stranded DNA (dsDNA) to produce a strong electrochemical signal for the quantitative measurement of TB. For further amplification of the electrochemical signal, graphene reduced by dopamine (PDA-rGO) was introduced as a platform in this work. With this strategy, the aptasensor displayed a wide linearity in the range of 0.0001 nM to 50 nM with a low detection limit of 0.05 pM. Moreover, the resulting aptasensor exhibited good specificity and acceptable reproducibility and stability. Because of these factors, the fabrication protocol proposed in this work may be extended to clinical application.

  1. 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.

  2. 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.

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

    Science.gov (United States)

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

    2017-11-01

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

  4. Salicylate, a catalytic inhibitor of topoisomerase II, inhibits DNA cleavage and is selective for the α isoform.

    Science.gov (United States)

    Bau, Jason T; Kang, Zhili; Austin, Caroline A; Kurz, Ebba U

    2014-02-01

    Topoisomerase II (topo II) is a ubiquitous enzyme that is essential for cell survival through its role in regulating DNA topology and chromatid separation. Topo II can be poisoned by common chemotherapeutics (such as doxorubicin and etoposide), leading to the accumulation of cytotoxic enzyme-linked DNA double-stranded breaks. In contrast, nonbreak-inducing topo II catalytic inhibitors have also been described and have more limited use in clinical chemotherapy. These agents, however, may alter the efficacy of regimens incorporating topo II poisons. We previously identified salicylate, the primary metabolite of aspirin, as a novel catalytic inhibitor of topo II. We have now determined the mechanism by which salicylate inhibits topo II. As catalytic inhibitors can act at a number of steps in the topo II catalytic cycle, we used multiple independent, biochemical approaches to interrogate the catalytic cycle. Furthermore, as mammalian cells express two isoforms of topo II (α and β), we examined whether salicylate was isoform selective. Our results demonstrate that salicylate is unable to intercalate DNA, and does not prevent enzyme-DNA interaction, nor does it promote stabilization of topo IIα in closed clamps on DNA. Although salicylate decreased topo IIα ATPase activity in a dose-dependent noncompetitive manner, this was secondary to salicylate-mediated inhibition of DNA cleavage. Surprisingly, comparison of salicylate's effects using purified human topo IIα and topo IIβ revealed that salicylate selectively inhibits the α isoform. These findings provide a definitive mechanism for salicylate-mediated inhibition of topo IIα and provide support for further studies determining the basis for its isoform selectivity.

  5. 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.

  6. 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

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

    Science.gov (United States)

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

    2014-10-20

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

  8. Cleavage of C-O bonds in lignin model compounds catalyzed by methyldioxorhenium in homogeneous phase.

    Science.gov (United States)

    Harms, Reentje G; Markovits, Iulius I E; Drees, Markus; Herrmann, H C Mult Wolfgang A; Cokoja, Mirza; Kühn, Fritz E

    2014-02-01

    Methyldioxorhenium (MDO)-catalyzed C-O bond cleavage of a variety of lignin β-O-4-model compounds yields phenolic and aldehydic compounds in homogeneous phase under mild reaction conditions. MDO is in situ generated by reduction of methyltrioxorhenium (MTO) and is remarkably stable under the applied reaction conditions allowing its reuse for least five times without significant activity loss. Based on the observed and isolated intermediates, 17 O- and 2 H-isotope labeling experiments, DFT calculations, and several spectroscopic studies, a reaction mechanism is proposed. Copyright © 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

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

    International Nuclear Information System (INIS)

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

    2007-01-01

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

  10. Mechanism of dihydrogen cleavage by high-valent metal oxo compounds: experimental and computational studies.

    Science.gov (United States)

    Collman, J P; Slaughter, L M; Eberspacher, T A; Strassner, T; Brauman, J I

    2001-11-19

    The oxidation of dihydrogen by metal tetraoxo compounds was investigated. Kinetic measurements of the oxidations of H(2) by MnO(4)(-) and RuO(4), performed by UV-vis spectroscopy, showed these reactions to be quite rapid at 25 degrees C (k(1) approximately (3-6) x 10(-2) M(-1) s(-1)). Rates measured for H(2) oxidation by MnO(4)(-) in aqueous solution (using KMnO(4)) and in chlorobenzene (using (n)Bu(4)NMnO(4)) revealed only a minor solvent effect on the reaction rate. Substantial kinetic isotope effects [(k(H)2/k(D)2 = 3.8(2) (MnO(4)(-), aq), 4.5(5) (MnO(4)(-), C(6)H(5)Cl soln), and 1.8(6) (RuO(4), CCl(4) soln)] indicated that H-H bond cleavage is rate determining and that the mechanism of dihydrogen cleavage is likely similar in aqueous and organic solutions. Third-row transition-metal oxo compounds, such as OsO(4), ReO(4)(-), and MeReO(3), were found to be completely unreactive toward H(2). Experiments were performed to probe for a catalytic hydrogen/deuterium exchange between D(2) and H(2)O as possible evidence of dihydrogen sigma-complex intermediates, but no H/D exchange was observed in the presence of various metal oxo compounds at various pH values. In addition, no inhibition of RuO(4)-catalyzed hydrocarbon oxidation by H(2) was observed. On the basis of the available evidence, a concerted mechanism for the cleavage of H(2) by metal tetraoxo compounds is proposed. Theoretical models were developed for pertinent MnO(4)(-) + H(2) transition states using density functional theory in order to differentiate between concerted [2 + 2] and [3 + 2] scissions of H(2). The density functional theory calculations strongly favor the [3 + 2] mechanism and show that the H(2) cleavage shares some mechanistic features with related hydrocarbon oxidation reactions. The calculated activation energy for the [3 + 2] pathway (DeltaH(++) = 15.4 kcal mol(-1)) is within 2 kcal mol(-1) of the experimental value.

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

    Science.gov (United States)

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

    2015-01-15

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

  12. Site accessibility tailors DNA cleavage by restriction enzymes in DNA confined monolayers.

    Science.gov (United States)

    Rotella, Chiara; Doni, Giovanni; Bosco, Alessandro; Castronovo, Matteo; De Vita, Alessandro; Casalis, Loredana; Pavan, Giovanni M; Parisse, Pietro

    2017-05-18

    Density-tunable nanografted monolayers (NAMs) of short oligonucleotide sequences on gold surfaces show novel properties that make them suitable for advanced biosensing applications, and in particular to study the effects of crowding and confinement on biomolecular interactions. Here, combining atomic force microscopy nanolithography, topography measurements and coarse-grained molecular dynamics simulations, we investigated restriction enzyme reaction mechanisms within confined DNA brushes highlighting the role played by the DNA sequence conformation and restriction site position along the chain, respectively, in determining the accessibility of the enzyme, and its consequent cleavage efficiency.

  13. Efficient hydrolytic cleavage of plasmid DNA by chloro-cobalt(II) complexes based on sterically hindered pyridyl tripod tetraamine ligands: synthesis, crystal structure and DNA cleavage.

    Science.gov (United States)

    Massoud, Salah S; Perkins, Richard S; Louka, Febee R; Xu, Wu; Le Roux, Anne; Dutercq, Quentin; Fischer, Roland C; Mautner, Franz A; Handa, Makoto; Hiraoka, Yuya; Kreft, Gabriel L; Bortolotto, Tiago; Terenzi, Hernán

    2014-07-14

    Four new cobalt(ii) complexes [Co(6-MeTPA)Cl]ClO4/PF6 (2/2a), [Co(6-Me2TPA)Cl]ClO4/PF6 (3/3a), [Co(BPQA)Cl]ClO4/PF6 (4/4a) and [Co(BQPA)Cl]ClO4/PF6 (5/5a) as well as [Co(TPA)Cl]ClO4 (1) where TPA = tris(2-pyridylmethyl)amine, 6-MeTPA = ((6-methyl-2-pyridyl)methyl)bis(2-pyridylmethyl)amine, 6-Me2TPA = bis(6-methyl-2-pyridyl)methyl)-(2-pyridylmethyl)amine, BPQA = bis(2-pyridylmethyl)-(2-quinolylmethyl)-amine and BQPA = bis(2-quinolylmethyl)-(2-pyridylmethyl)amine were synthesized and structurally characterized. Single crystal X-ray crystallography confirmed the distorted trigonal bipyramidal geometries of complexes 2a-5a. Spectrophotometric titrations and conductivity measurements of the complexes in the CH3CN-H2O mixture showed that the chloro complexes exist in equilibrium with the corresponding hydrolyzed aqua species, [Co(L)(H2O)](2+). The pKa values of the coordinated H2O in aqua complexes vary from 8.4 to 8.7 (37 °C). The interactions of the complexes (1-5) with DNA have been investigated at pH = 7.0 and 9.0 (10 mM Tris-HCl buffer) and 37 °C where very high catalytic cleavage was observed. Under pseudo Michaelis-Menten kinetic conditions, the catalytic rate constants, kcat, decrease in the order 4>2>5>1>3. At pH 7.0 (10 mM Tris-HCl buffer) and 37 °C, the kcat value for complex 4 (6.02 h(-1)), where [Co(BPQA)(H2O)](2+) is the major species, corresponds to 170 million rate enhancement over the non-catalyzed DNA. Electrophoretic experiments conducted in the presence and absence of radical scavengers (DMSO, KI, NaN3) ruled out the oxidative mechanistic pathway of the reaction and suggested that the hydrolytic mechanism is the preferred one. This finding was in agreement with the observed increase in the kcat values at pH 9.0 compared to the corresponding values at pH 7.0 as a result of the increased concentration of the reactive hydroxo species, [Co(L)(OH)](+). The reactivity of the synthesized complexes in catalyzing the DNA cleavage is discussed in relation to

  14. 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

  15. Tyrosine phosphorylation and proteolytic cleavage of Notch are required for non-canonical Notch/Abl signaling inDrosophilaaxon guidance.

    Science.gov (United States)

    Kannan, Ramakrishnan; Cox, Eric; Wang, Lei; Kuzina, Irina; Gu, Qun; Giniger, Edward

    2018-01-17

    Notch signaling is required for the development and physiology of nearly every tissue in metazoans. Much of Notch signaling is mediated by transcriptional regulation of downstream target genes, but Notch controls axon patterning in Drosophila by local modulation of Abl tyrosine kinase signaling, via direct interactions with the Abl co-factors Disabled and Trio. Here, we show that Notch-Abl axonal signaling requires both of the proteolytic cleavage events that initiate canonical Notch signaling. We further show that some Notch protein is tyrosine phosphorylated in Drosophila , that this form of the protein is selectively associated with Disabled and Trio, and that relevant tyrosines are essential for Notch-dependent axon patterning but not for canonical Notch-dependent regulation of cell fate. Based on these data, we propose a model for the molecular mechanism by which Notch controls Abl signaling in Drosophila axons. © 2018. Published by The Company of Biologists Ltd.

  16. DNA binding, DNA cleavage and cytotoxicity studies of a new water soluble copper(II) complex: the effect of ligand shape on the mode of binding.

    Science.gov (United States)

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

    2012-02-01

    The interaction of native calf thymus DNA (CT-DNA) with [Cu(ph(2)phen)(phen-dione)Cl]Cl was studied at physiological pH by spectrophotometric, spectrofluorometric, circular dichroism, and viscometric techniques. Considerable hypochromicity and red shift are observed in the UV absorption band of the Cu complex. Binding constants (K(b)) of DNA with the complex were calculated at different temperatures. Thermodynamic parameters, enthalpy and entropy changes were calculated according to Van't Hoff equation, which indicated that reaction is predominantly enthalpically driven. All these results indicate that Cu(II) complex interacts with CT-DNA via intercalative mode. Also, this new complex induced cleavage in pUC18 plasmid DNA as indicated in gel electrophoresis and showed excellent antitumor activity against K562 (human chronic myeloid leukemia) and human T lymphocyte carcinoma-Jurkat cell lines. Copyright © 2011 Elsevier B.V. All rights reserved.

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

    Science.gov (United States)

    Vodonik, S A; Gray, G R

    1988-04-01

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

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

    Science.gov (United States)

    Yang, Mengyi; Peng, Sijia; Sun, Ruirui; Lin, Jingdi; Wang, Nan; Chen, Chunlai

    2018-01-09

    Off-target binding and cleavage by Cas9 pose major challenges in its application. How the conformational dynamics of Cas9 govern its nuclease activity under on- and off-target conditions remains largely unknown. Here, using intra-molecular single-molecule fluorescence resonance energy transfer measurements, we revealed that Cas9 in apo, sgRNA-bound, and dsDNA/sgRNA-bound forms spontaneously transits among three major conformational states, mainly reflecting significant conformational mobility of the catalytic HNH domain. We also uncovered surprising long-range allosteric communication between the HNH domain and the RNA/DNA heteroduplex at the PAM-distal end to ensure correct positioning of the catalytic site, which demonstrated that a unique proofreading mechanism served as the last checkpoint before DNA cleavage. Several Cas9 residues were likely to mediate the allosteric communication and proofreading step. Modulating interactions between Cas9 and heteroduplex at the PAM-distal end by introducing mutations on these sites provides an alternative route to improve and optimize the CRISPR/Cas9 toolbox. Copyright © 2017 The Author(s). Published by Elsevier Inc. All rights reserved.

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

    Science.gov (United States)

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

    2015-01-01

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

  20. N-H bond cleavage of ammonia on graphene-like B36 borophene: DFT studies.

    Science.gov (United States)

    Rostami, Zahra; Soleymanabadi, Hamed

    2016-04-01

    Ammonia N-H bond cleavage at metal-free substrates has attracted great attention because of its industrial importance. Here, we investigate the dissociative adsorption of ammonia onto the surface of a B36 borophene sheet by means of density functional theory calculations. We show that the N-H bond may be broken at the edges of B36 even at room temperature, regarding the small energy barrier of 14.1-19.3 kcal mol(-1) at different levels of theory, and more negative Gibbs free energy change. Unlike basis set size, the kind of exchange correlation functional significantly affects the electronic properties of the studied systems. Also, by increasing the percentage of Hartree Fock (HF) exchange of density functionals, the activation and adsorption energies are lowered. A linear relationship between the highest occupied molecular orbital or lowest unoccupied molecular orbital of B36 borophene and the %HF exchange of functionals is predicted. Our work reveals that pure whole boron nanosheets may be promising metal-free materials in N-H bond cleavage, which would raise the potential application of these sheets.

  1. Peripheral regions of natural hammerhead ribozymes greatly increase their self-cleavage activity

    Science.gov (United States)

    De la Peña, Marcos; Gago, Selma; Flores, Ricardo

    2003-01-01

    Natural hammerhead ribozymes are mostly found in some viroid and viroid-like RNAs and catalyze their cis cleavage during replication. Hammerheads have been manipulated to act in trans and assumed to have a similar catalytic behavior in this artificial context. However, we show here that two natural cis-acting hammerheads self-cleave much faster than trans-acting derivatives and other reported artificial hammerheads. Moreover, modifications of the peripheral loops 1 and 2 of one of these natural hammerheads induced a >100-fold reduction of the self-cleavage constant, whereas engineering a trans-acting artificial hammerhead into a cis derivative by introducing a loop 1 had no effect. These data show that regions external to the central conserved core of natural hammerheads play a role in catalysis, and suggest the existence of tertiary interactions between these peripheral regions. The interactions, determined by the sequence and size of loops 1 and 2 and most likely of helices I and II, must result from natural selection and should be studied in order to better understand the hammerhead requirements in vivo. PMID:14532128

  2. Receptor cleavage and P-selectin-dependent reduction of leukocyte adhesion in the spontaneously hypertensive rat

    Science.gov (United States)

    Chen, Angela Y.; Ha, Jessica N.; DeLano, Frank A.; Schmid-Schönbein, Geert W.

    2012-01-01

    The SHR, a genetic model for hypertension and the metabolic syndrome, has attenuated leukocyte adhesion to the postcapillary endothelium by an unknown mechanism. Based on recent evidence of elevated levels of MMPs in plasma and on microvascular endothelium of the SHR with cleavage of several receptor types, we hypothesize that the reduced leukocyte-endothelial interaction is a result of enhanced proteolytic cleavage of P-selectin on the postcapillary endothelium and PSGL-1 on leukocytes. The attenuated rolling interactions of SHR leukocytes with the endothelium were restored by chronic treatment with a broad-spectrum MMP inhibitor (CGS) for 24 weeks. The SHR MMP levels, in plasma and mesentery, as well as the systolic blood pressure, decreased significantly with treatment. In the SHR mesentery, labeling of P-selectin in the postcapillary venules by immunohistochemistry demonstrated, on average, a 31% lower extracellular P-selectin density compared with the normotensive WKY. A significantly lower extracellular PSGL-1 density on the membranes of SHR neutrophils compared with the WKY also supported our hypothesis. In vivo stimulation of the mesenteric postcapillary venules with histamine demonstrated that the SHR had an attenuated response, as measured by leukocyte rolling velocity on the endothelium. The reduced P-selectin and PSGL-1 density, on SHR postcapillary endothelium and on SHR leukocytes, respectively, was restored significantly by chronic MMP inhibition. The impaired ability of SHR leukocytes to reduce rolling velocity upon inflammatory stimulation led to fewer firmly adhered leukocytes to the endothelium as a contributor to immune suppression. PMID:22566571

  3. Cathepsin-Mediated Cleavage of Peptides from Peptide Amphiphiles Leads to Enhanced Intracellular Peptide Accumulation

    Energy Technology Data Exchange (ETDEWEB)

    Acar, Handan [Institute; Department; Samaeekia, Ravand [Institute; Department; Schnorenberg, Mathew R. [Institute; Department; Medical; Sasmal, Dibyendu K. [Institute; Huang, Jun [Institute; Tirrell, Matthew V. [Institute; Institute; LaBelle, James L. [Department

    2017-08-24

    Peptides synthesized in the likeness of their native interaction domain(s) are natural choices to target protein protein interactions (PPIs) due to their fidelity of orthostatic contact points between binding partners. Despite therapeutic promise, intracellular delivery of biofunctional peptides at concentrations necessary for efficacy remains a formidable challenge. Peptide amphiphiles (PAs) provide a facile method of intracellular delivery and stabilization of bioactive peptides. PAs consisting of biofunctional peptide headgroups linked to hydrophobic alkyl lipid-like tails prevent peptide hydrolysis and proteolysis in circulation, and PA monomers are internalized via endocytosis. However, endocytotic sequestration and steric hindrance from the lipid tail are two major mechanisms that limit PA efficacy to target intracellular PPIs. To address these problems, we have constructed a PA platform consisting of cathepsin-B cleavable PAs in which a selective p53-based inhibitory peptide is cleaved from its lipid tail within endosomes, allowing for intracellular peptide accumulation and extracellular recycling of the lipid moiety. We monitor for cleavage and follow individual PA components in real time using a resonance energy transfer (FRET)-based tracking system. Using this platform, components in real time using a Forster we provide a better understanding and quantification of cellular internalization, trafficking, and endosomal cleavage of PAs and of the ultimate fates of each component.

  4. 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.

  5. 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.

  6. Mechanisms of catalytic cleavage of benzyl phenyl ether in aqueous and apolar phases

    Energy Technology Data Exchange (ETDEWEB)

    He, Jiayue; Lu, Lu; Zhao, Chen; Mei, Donghai; Lercher, Johannes A.

    2014-03-01

    Catalytic pathways for the cleavage of ether bonds in benzyl phenyl ether (BPE) in liquid phase using Ni- and zeolite-based catalysts are explored. In the absence of catalysts, the C-O bond is selectively cleaved in water by hydrolysis, forming phenol and benzyl alcohol as intermediates, followed by alkylation. The hydronium ions catalyzing the reactions are provided by the dissociation of water at 523 K. Upon addition of HZSM-5, rates of hydrolysis and alkylation are markedly increased in relation to proton concentrations. In the presence of Ni/SiO2, the selective hydrogenolysis dominates for cleaving the Caliphatic-O bond. Catalyzed by the dual-functional Ni/HZSM-5, hydrogenolysis occurs as the major route rather than hydrolysis (minor route). In apolar undecane, the non-catalytic thermal pyrolysis route dominates. Hydrogenolysis of BPE appears to be the major reaction pathway in undecane in the presence of Ni/SiO2 or Ni/HZSM-5, almost completely suppressing radical reactions. Density functional theory (DFT) calculations strongly support the proposed C-O bond cleavage mechanisms on BPE in aqueous and apolar phases. These calculations show that BPE is initially protonated and subsequently hydrolyzed in the aqueous phase. Finally, DFT calculations suggest that the radical reactions in non-polar solvents lead to primary benzyl and phenoxy radicals in undecane, which leads to heavier condensation products as long as metals are absent for providing dissociated hydrogen.

  7. 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”.

  8. 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.

  9. Urokinase receptor cleavage correlates with tumor volume in a transgenic mouse model of breast cancer

    DEFF Research Database (Denmark)

    Thurison, Tine; Almholt, Kasper; Gårdsvoll, Henrik

    2015-01-01

    PAR(I)]. The level of muPAR(I) is significantly increased in mammary tumor-bearing mice compared to controls and, notably, there is a strong correlation to tumor volume. In contrast, the tumor volume is only weakly correlated to the level of intact muPAR(I-III), indicating that cleavage of muPAR is a more specific...... marker for cancer than increased expression of muPAR per se. The levels of the muPAR forms are dramatically affected by in vivo challenge with a urokinase -blocking antibody, demonstrating a functional role of uPA in uPAR cleavage. The levels of the muPAR forms are, however, unaffected by u......PA-deficiency, suggesting that redundant proteases maintains the task of cleaving uPAR(I-III) when uPA is absent. Our findings emphasize the significance of the cleaved uPAR forms as cancer biomarkers. The strong correlation between muPAR(I) and the tumor volume in our experimental setup may motivate investigations...

  10. Arthroscopic repair of horizontal meniscal cleavage tears with marrow-stimulating technique.

    Science.gov (United States)

    Ahn, Ji-Hyun; Kwon, Oh-Jin; Nam, Tae-Seok

    2015-01-01

    The purpose of this study was to evaluate patients after arthroscopic repair of meniscal horizontal tears with a marrow-stimulating technique through clinical signs and second-look arthroscopy. We retrospectively reviewed a consecutive series of 32 meniscal repairs with horizontal cleavage tears and evaluated them through clinical assessment and second-look arthroscopic examinations. Arthroscopic meniscal repair and a marrow-stimulating technique were performed. Functional outcomes were evaluated using the visual analog scale (VAS) pain score, Lysholm knee scoring scale, and Tegner activity scale. Assessment of meniscal healing was evaluated clinically by the presence of meniscal signs; second-look arthroscopy was performed in 11 patients. Correlation between chronicity of a meniscal lesion (time from initial symptom [TFIS]) and meniscal healing was evaluated. The mean follow-up period was 45.6 ± 13.9 months. Improvements in mean VAS scores from 6.7 to 1.9 (P meniscal healing was clinically significant (P = .001) but arthroscopically insignificant (P = .085) on second-look arthroscopy. The meniscal repair procedure for horizontal cleavage tears in the present study suggests an alternative treatment option to approach the treatment of meniscal tears extending into the avascular zone and degenerative tissue. The marrow-stimulating technique using a cannulated reamer can be considered as an alternative method for the augmentation of meniscal healing. Level IV, therapeutic case series. Copyright © 2015 Arthroscopy Association of North America. Published by Elsevier Inc. All rights reserved.

  11. Isolation and Functional Characterization of Carotenoid Cleavage Dioxygenase-1 from Laurus nobilis L. (Bay Laurel) Fruits.

    Science.gov (United States)

    Yahyaa, Mosaab; Berim, Anna; Isaacson, Tal; Marzouk, Sally; Bar, Einat; Davidovich-Rikanati, Rachel; Lewinsohn, Efraim; Ibdah, Mwafaq

    2015-09-23

    Bay laurel (Laurus nobilis L.) is an agriculturally important tree used in food, drugs, and the cosmetics industry. Many of the health beneficial properties of bay laurel are due to volatile terpene metabolites that they contain, including various norisoprenoids. Despite their importance, little is known about the norisoprenoid biosynthesis in Laurus nobilis fruits. We found that the volatile norisoprenoids 6-methyl-5-hepten-2-one, pseudoionone, and β-ionone accumulated in Laurus nobilis fruits in a pattern reflecting their carotenoid content. A full-length cDNA encoding a potential carotenoid cleavage dioxygenase (LnCCD1) was isolated. The LnCCD1 gene was overexpressed in Escherichia coli, and recombinant protein was assayed for its cleavage activity with an array of carotenoid substrates. The LnCCD1 protein was able to cleave a variety of carotenoids at the 9,10 (9',10') and 5,6 (5',6') positions to produce 6-methyl-5-hepten-2-one, pseudoionone, β-ionone, and α-ionone. Our results suggest a role for LnCCD1 in Laurus nobilis fruit flavor biosynthesis.

  12. Identification and expression pattern of a new carotenoid cleavage dioxygenase gene member from Bixa orellana

    Science.gov (United States)

    Rodríguez-Ávila, N. L.; Narváez-Zapata, J. A.; Ramírez-Benítez, J. E.; Aguilar-Espinosa, M. L.; Rivera-Madrid, R.

    2011-01-01

    Carotenoid cleavage dioxygenases (CCDs) are a class of enzymes involved in the biosynthesis of a broad diversity of secondary metabolites known as apocarotenoids. In plants, CCDs are part of a genetic family with members which cleave specific double bonds of carotenoid molecules. CCDs are involved in the production of diverse and important metabolites such as vitamin A and abscisic acid (ABA). Bixa orellana L. is the main source of the natural pigment annatto or bixin, an apocarotenoid accumulated in large quantities in its seeds. Bixin biosynthesis has been studied and the involvement of a CCD has been confirmed in vitro. However, the CCD genes involved in the biosynthesis of the wide variety of apocarotenoids found in this plant have not been well documented. In this study, a new CCD1 gene member (BoCCD1) was identified and its expression was charaterized in different plant tissues of B. orellana plantlets and adult plants. The BoCCD1 sequence showed high homology with plant CCD1s involved mainly in the cleavage of carotenoids in several sites to generate multiple apocarotenoid products. Here, the expression profiles of the BoCCD1 gene were analysed and discussed in relation to total carotenoids and other important apocarotenoids such as bixin. PMID:21813796

  13. 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...... in the presence of H(2)O(2) were characterized by EPR spectroscopy. The detected signals were assigned to Cr(V)-catechol, Cr(V)-peroxo, and mixed Cr(V)-catechol-peroxo complexes. Oxygen consumption during the reactions of Cr(VI) with 1, 2, 4, and 5 was studied, and H(2)O(2) production was quantified. Reactions...... of Cr(VI) with 1 and 2, but not 4 and 5, consume considerable amounts of dissolved O(2), and give extensive H(2)O(2) production. Extents of oxygen consumption and H(2)O(2) production during the reaction of Cr(VI) with enzymatically generated 1 and N-acetyl-DOPA (from the reaction of Tyr and N...

  14. 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.

  15. Cleavage-independent HIV-1 Env trimers engineered as soluble native spike mimetics for vaccine design.

    Science.gov (United States)

    Sharma, Shailendra Kumar; de Val, Natalia; Bale, Shridhar; Guenaga, Javier; Tran, Karen; Feng, Yu; Dubrovskaya, Viktoriya; Ward, Andrew B; Wyatt, Richard T

    2015-04-28

    Viral glycoproteins mediate entry by pH-activated or receptor-engaged activation and exist in metastable pre-fusogenic states that may be stabilized by directed rational design. As recently reported, the conformationally fixed HIV-1 envelope glycoprotein (Env) trimers in the pre-fusion state (SOSIP) display molecular homogeneity and structural integrity at relatively high levels of resolution. However, the SOSIPs necessitate full Env precursor cleavage, which requires endogenous furin overexpression. Here, we developed an alternative strategy using flexible peptide covalent linkage of Env subdomains to produce soluble, homogeneous, and cleavage-independent Env mimics, called native flexibly linked (NFL) trimers, as vaccine candidates. This simplified design avoids the need for furin co-expression and, in one case, antibody affinity purification to accelerate trimer scale-up for preclinical and clinical applications. We have successfully translated the NFL design to multiple HIV-1 subtypes, establishing the potential to become a general method of producing native-like, well-ordered Env trimers for HIV-1 or other viruses. Copyright © 2015 The Authors. Published by Elsevier Inc. All rights reserved.

  16. 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.

  17. Thiophene antibacterials that allosterically stabilize DNA-cleavage complexes with DNA gyrase.

    Science.gov (United States)

    Chan, Pan F; Germe, Thomas; Bax, Benjamin D; Huang, Jianzhong; Thalji, Reema K; Bacqué, Eric; Checchia, Anna; Chen, Dongzhao; Cui, Haifeng; Ding, Xiao; Ingraham, Karen; McCloskey, Lynn; Raha, Kaushik; Srikannathasan, Velupillai; Maxwell, Anthony; Stavenger, Robert A

    2017-05-30

    A paucity of novel acting antibacterials is in development to treat the rising threat of antimicrobial resistance, particularly in Gram-negative hospital pathogens, which has led to renewed efforts in antibiotic drug discovery. Fluoroquinolones are broad-spectrum antibacterials that target DNA gyrase by stabilizing DNA-cleavage complexes, but their clinical utility has been compromised by resistance. We have identified a class of antibacterial thiophenes that target DNA gyrase with a unique mechanism of action and have activity against a range of bacterial pathogens, including strains resistant to fluoroquinolones. Although fluoroquinolones stabilize double-stranded DNA breaks, the antibacterial thiophenes stabilize gyrase-mediated DNA-cleavage complexes in either one DNA strand or both DNA strands. X-ray crystallography of DNA gyrase-DNA complexes shows the compounds binding to a protein pocket between the winged helix domain and topoisomerase-primase domain, remote from the DNA. Mutations of conserved residues around this pocket affect activity of the thiophene inhibitors, consistent with allosteric inhibition of DNA gyrase. This druggable pocket provides potentially complementary opportunities for targeting bacterial topoisomerases for antibiotic development.

  18. Probing cleavage promiscuity of heparinase III towards chemoenzymatically synthetic heparan sulfate oligosaccharides.

    Science.gov (United States)

    Hu, Guixin; Shao, Meng; Gao, Xin; Wang, Fengshan; Liu, Chunhui

    2017-10-01

    An insightful investigation into specificity of bacterial heparinase III has been intriguingly difficult due to heterogeneity of polymeric substrates. Herein, we chemoenzymatically synthesized a tailored library of HS oligosaccharides as substrates. A ∼15-fold reactivity difference to heparinase III was found between trisaccharides bearing different primary cleavage sites. Variable glucosamine modification decreased reactivity of trisaccharides by >20-fold compared with their counterpart primary substrates, while iduronate-containing secondary linkage showed slightly less sensitivity. The 2-sulfated iduronate residue extremely reduced reactivity to its adjacent primary site at reducing end of oligosaccharides, but showed marginal influence on the non-reducing site. Moreover, oligosaccharide susceptibility to digestion was size-dependent and had an obvious preference for the internal linkages over those near to non-reducing/reducing ends. Surface plasmon resonance revealed cleavage promiscuity attributed to different affinities or incorrect binding of substrates to the enzyme. The attractive information on heparinase III will be valuable in characterizing heparin and HS. Copyright © 2017 Elsevier Ltd. All rights reserved.

  19. Two combinatorial optimization problems for SNP discovery using base-specific cleavage and mass spectrometry.

    Science.gov (United States)

    Chen, Xin; Wu, Qiong; Sun, Ruimin; Zhang, Louxin

    2012-01-01

    The discovery of single-nucleotide polymorphisms (SNPs) has important implications in a variety of genetic studies on human diseases and biological functions. One valuable approach proposed for SNP discovery is based on base-specific cleavage and mass spectrometry. However, it is still very challenging to achieve the full potential of this SNP discovery approach. In this study, we formulate two new combinatorial optimization problems. While both problems are aimed at reconstructing the sample sequence that would attain the minimum number of SNPs, they search over different candidate sequence spaces. The first problem, denoted as SNP - MSP, limits its search to sequences whose in silico predicted mass spectra have all their signals contained in the measured mass spectra. In contrast, the second problem, denoted as SNP - MSQ, limits its search to sequences whose in silico predicted mass spectra instead contain all the signals of the measured mass spectra. We present an exact dynamic programming algorithm for solving the SNP - MSP problem and also show that the SNP - MSQ problem is NP-hard by a reduction from a restricted variation of the 3-partition problem. We believe that an efficient solution to either problem above could offer a seamless integration of information in four complementary base-specific cleavage reactions, thereby improving the capability of the underlying biotechnology for sensitive and accurate SNP discovery.

  20. Optimization schemes for selective molecular cleavage with tailored ultrashort laser pulses

    International Nuclear Information System (INIS)

    Krieger, Kevin; Castro, Alberto; Gross, E.K.U.

    2011-01-01

    Graphical abstract: Isosurface plot of the electronic density, and positions of the nuclei, during the irradiation of a formaldiminium ion with an optimized laser pulse, leading to the selective cleavage of a proton. Display Omitted Highlights: ► We use QOCT in combination with mixed quantum-classical molecular models. ► TDDFT models the electrons coupled to the nuclei through Ehrenfest equations. ► Both gradient-free and gradient-based algorithms are explored for the optimization. ► We explore different options for the definition of the target functional. - Abstract: We present some approaches to the computation of ultra-fast laser pulses capable of selectively breaking molecular bonds. The calculations are based on a mixed quantum-classical description: The electrons are treated quantum mechanically (making use of time-dependent density-functional theory), whereas the nuclei are treated classically. The temporal shape of the pulses is tailored to maximize a control target functional which is designed to produce the desired molecular cleavage. The precise definition of this functional is a crucial ingredient: we explore expressions based on the forces, on the momenta and on the velocities of the nuclei. The algorithm used to find the optimum pulse is also relevant; we test both direct gradient-free algorithms, as well as schemes based on formal optimal control theory. The tests are performed both on one dimensional models of atomic chains, and on first-principles descriptions of molecules.

  1. 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.

  2. Residues of E. coli topoisomerase I conserved for interaction with a specific cytosine base to facilitate DNA cleavage

    Science.gov (United States)

    Narula, Gagandeep; Tse-Dinh, Yuk-Ching

    2012-01-01

    Bacterial and archaeal topoisomerase I display selectivity for a cytosine base 4 nt upstream from the DNA cleavage site. Recently, the solved crystal structure of Escherichia coli topoisomerase I covalently linked to a single-stranded oligonucleotide revealed that R169 and R173 interact with the cytosine base at the −4 position via hydrogen bonds while the phenol ring of Y177 wedges between the bases at the −4 and the −5 position. Substituting R169 to alanine changed the selectivity of the enzyme for the base at the −4 position from a cytosine to an adenine. The R173A mutant displayed similar sequence selectivity as the wild-type enzyme, but weaker cleavage and relaxation activity. Mutation of Y177 to serine or alanine rendered the enzyme inactive. Although mutation of each of these residues led to different outcomes, R169, R173 and Y177 work together to interact with a cytosine base at the −4 position to facilitate DNA cleavage. These strictly conserved residues might act after initial substrate binding as a Molecular Ruler to form a protein–DNA complex with the scissile phosphate positioned at the active site for optimal DNA cleavage by the tyrosine hydroxyl nucleophile to facilitate DNA cleavage in the reaction pathway. PMID:22833607

  3. Core-shell silica nanoparticles synthesized for quantitative study of DNA cleavage by laser-induced fluorescence microscopy.

    Science.gov (United States)

    Ko, Jungaa; Lim, H B

    2011-02-01

    Dye-doped silica nanoparticles (C dots) were synthesized in reverse microemulsions and used to quantitatively examine DNA cleavage in the presence of transition metal ions. The cores were synthesized as fluorescein isothiocyanate (FITC)-doped silica nanoparticles and the shells' surfaces were modified with single-stranded DNA oligomers tagged with Cy5 fluorophores. DNA cleavage induced by heavy metal ions was estimated by comparing the fluorescence of Cy5 before and after reaction with metal ions. For this, a lab-built laser-induced fluorescence microscope equipped with a charge coupled device (CCD) camera, for imaging, and photomultiplier tube, for photon counting, was used. FITC fluorescence from the core was measured as an internal standard to compensate for possible loss of the beads during the treatment. The cleavage of DNA in air in the presence of Pb(2+), Cd(2+), and Hg(2+) at 1 ng/mL was found to be 14%, 6%, and 20%, respectively, and was significantly reduced to below 9% under N(2) gas, indicating that the main cleavage source was oxygen in air. The most significant DNA cleavage was observed with the addition of hydrogen peroxide. This analytical method using dye-doped C dots provided convenient handling and quantification of the estimation of metal-DNA interaction with a detection limit of 34.9 pmol/mL.

  4. Secreted Aspartic Protease Cleavage of Candida albicans Msb2 Activates Cek1 MAPK Signaling Affecting Biofilm Formation and Oropharyngeal Candidiasis

    Science.gov (United States)

    Chadha, Sonia; Tati, Swetha; Conti, Heather R.; Hube, Bernhard; Cullen, Paul J.; Edgerton, Mira

    2012-01-01

    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. PMID:23139737

  5. Ribosomal Protein S12 and Aminoglycoside Antibiotics Modulate A-site mRNA Cleavage and Transfer-Messenger RNA Activity in Escherichia coli*

    OpenAIRE

    Holberger, Laura E.; Hayes, Christopher S.

    2009-01-01

    Translational pausing in Escherichia coli can lead to mRNA cleavage within the ribosomal A-site. A-site mRNA cleavage is thought to facilitate transfer-messenger RNA (tmRNA)·SmpB- mediated recycling of stalled ribosome complexes. Here, we demonstrate that the aminoglycosides paromomycin and streptomycin inhibit A-site cleavage of stop codons during inefficient translation termination. Aminoglycosides also induced stop codon read-through, suggesting that these antibiotics alleviate ribosome pa...

  6. 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.

  7. Transcription elongation rate has a tissue-specific impact on alternative cleavage and polyadenylation in Drosophila melanogaster.

    Science.gov (United States)

    Liu, Xiaochuan; Freitas, Jaime; Zheng, Dinghai; Oliveira, Marta S; Hoque, Mainul; Martins, Torcato; Henriques, Telmo; Tian, Bin; Moreira, Alexandra

    2017-12-01

    Alternative polyadenylation (APA) is a mechanism that generates multiple mRNA isoforms with different 3'UTRs and/or coding sequences from a single gene. Here, using 3' region extraction and deep sequencing (3'READS), we have systematically mapped cleavage and polyadenylation sites (PASs) in Drosophila melanogaster , expanding the total repertoire of PASs previously identified for the species, especially those located in A-rich genomic sequences. Cis -element analysis revealed distinct sequence motifs around fly PASs when compared to mammalian ones, including the greater enrichment of upstream UAUA elements and the less prominent presence of downstream UGUG elements. We found that over 75% of mRNA genes in Drosophila melanogaster undergo APA. The head tissue tends to use distal PASs when compared to the body, leading to preferential expression of APA isoforms with long 3'UTRs as well as with distal terminal exons. The distance between the APA sites and intron location of PAS are important parameters for APA difference between body and head, suggesting distinct PAS selection contexts. APA analysis of the RpII215 C4 mutant strain, which harbors a mutant RNA polymerase II (RNAPII) with a slower elongation rate, revealed that a 50% decrease in transcriptional elongation rate leads to a mild trend of more usage of proximal, weaker PASs, both in 3'UTRs and in introns, consistent with the "first come, first served" model of APA regulation. However, this trend was not observed in the head, suggesting a different regulatory context in neuronal cells. Together, our data expand the PAS collection for Drosophila melanogaster and reveal a tissue-specific effect of APA regulation by RNAPII elongation rate. © 2017 Liu et al.; Published by Cold Spring Harbor Laboratory Press for the RNA Society.

  8. Exploring both sequence detection and restriction endonuclease cleavage kinetics by recognition site via single-molecule microfluidic trapping.

    Science.gov (United States)

    Xu, Weilin; Muller, Susan J

    2011-02-07

    We demonstrate the feasibility of a single-molecule microfluidic approach to both sequence detection and obtaining kinetic information for restriction endonucleases on dsDNA. In this method, a microfluidic stagnation point flow is designed to trap, hold, and linearize double-stranded (ds) genomic DNA to which a restriction endonuclease has been pre-bound sequence-specifically. By introducing the cofactor magnesium, we determine the binding location of the enzyme by the cleavage process of dsDNA as in optical restriction mapping, however here the DNA need not be immobilized on a surface. We note that no special labeling of the enzyme is required, which makes it simpler than our previous scheme using stagnation point flows for sequence detection. Our accuracy in determining the location of the recognition site is comparable to or better than other single molecule techniques due to the fidelity with which we can control the linearization of the DNA molecules. In addition, since the cleavage process can be followed in real time, information about the cleavage kinetics, and subtle differences in binding and cleavage frequencies among the recognition sites, may also be obtained. Data for the five recognition sites for the type II restriction endonuclease EcoRI on λ-DNA are presented as a model system. While the roles of the varying fluid velocity and tension along the chain backbone on the measured kinetics remain to be determined, we believe this new method holds promise for a broad range of studies of DNA-protein interactions, including the kinetics of other DNA cleavage processes, the dissociation of a restriction enzyme from the cleaved substrate, and other macromolecular cleavage processes.

  9. Determination of the key microstructural parameter for the cleavage fracture toughness of reactor pressure vessel steels in the transition region

    International Nuclear Information System (INIS)

    Kim, M.C.; Lee, B.S.; Hong, J.H.; Yang, W.J.

    2005-01-01

    The effects of the microstructural parameters, such as the prior austenite grain size and carbide size, on the cleavage fracture toughness were investigated in the transition region of Mn-Mo-Ni bainitic low alloy steels. Cleavage fracture toughness was evaluated by the ASTM standard E 1921 Master curve method. In order to clarify the effects of each microstructure, the grain size and carbide size of the test materials were independently controlled by modifying the heat treatment process. Firstly, the grain sizes were changed from 25 to 110 without any significant changes in the carbide size and shape. Secondly, the average carbide sizes were changed from 0.20 to 0.29 but maintaining the initial grain sizes. As a result, the fracture toughness in the transition region did not show any significant dependency on the austenite grain size, while the carbide size showed a close relation to the fracture toughness. Fracture toughness was decreased with an increase of the average carbide size. From the microscopic observation of the fractured surface, the cleavage initiation distance (CID) from the original crack tip showed no direct relationship to the prior austenite grain sizes but a strong relationship to the carbide sizes. However, the measured cleavage fracture toughness was strongly related to the distance from the crack tip to the cleavage initiation site. From the viewpoint of the weakest link theory, the particle size and their distribution in front of the crack tip is probably more important than the grain size in the transition temperature range where the fracture was controlled by the cleavage crack initiation. (orig.)

  10. Tailoring Bond Cleavage in Gas-Phase Biomolecules by Low Energy Electrons

    Science.gov (United States)

    Ptasinska, Sylwia

    2014-10-01

    The high energy quanta of impinging radiation can generate a large number (about 5x104) of secondary electrons per 1 MeV of energy deposited. When ejected in condensed phase water, the kinetic energy distribution of these free or quasi-free electrons is peaked below 10 eV. Low energy electrons also dominate in the secondary emission from biomolecular targets exposed to different energies of primary radiation. Due to the complexity of the radiation-induced processes in the condensed-phase environment, mechanisms of secondary electrons induced damage in biomolecules (BM) still need to be investigated. However, based on results from theory and different experiments accumulated within the last decade, it is now possible to determine the fundamental mechanisms that are involved in many chemical reactions induced in isolated gas-phase biomolecules by low energy electrons. The central finding of earlier research was the discovery of the bond- and site- selectivity in the dissociative electron attachment (DEA) process to biomolecules. It has been demonstrated that by tuning the energy of the incoming electron we can gain control over the location of the bond cleavage. These studies showed the selectivity in single bond cleavage reactions leading to the formation of the dehydrogenated closed shell anion (BM-H)- or the complementary reaction leading to H-. The loss of a hydrogen atom or an anion is fast compared with ring cleavage and the excision of heavier fragments and, hence, this reaction can compete efficiently with autodetachment. Moreover, site selectivity has been also observed in the metastable anion formation via the DEA process. Such delayed fragmentation was studied recently for the dehydrogenated closed-shell anion conversion into NCO- upon DEA proceeded a few μ sec after electron attachment, indicating a rather slow unimolecular decomposition. Interestingly, site selectivity was observed in the prompt as well as the metastable NCO- formation in DEA. The

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

    Science.gov (United States)

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

    2015-09-01

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

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

    Directory of Open Access Journals (Sweden)

    M. Sunita

    2017-05-01

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

  13. Structural basis for specific cleavage of Lys6-linked polyubiquitin chains by USP30.

    Science.gov (United States)

    Sato, Yusuke; Okatsu, Kei; Saeki, Yasushi; Yamano, Koji; Matsuda, Noriyuki; Kaiho, Ai; Yamagata, Atsushi; Goto-Ito, Sakurako; Ishikawa, Minoru; Hashimoto, Yuichi; Tanaka, Keiji; Fukai, Shuya

    2017-11-01

    Parkin ubiquitin (Ub) ligase (also known as PARK2) ubiquitinates damaged mitochondria for their clearance and quality control. USP30 deubiquitinase opposes parkin-mediated Ub-chain formation on mitochondria by preferentially cleaving Lys6-linked Ub chains. Here, we report the crystal structure of zebrafish USP30 in complex with a Lys6-linked diubiquitin (diUb or Ub 2 ) at 1.87-Å resolution. The distal Ub-recognition mechanism of USP30 is similar to those of other USP family members, whereas Phe4 and Thr12 of the proximal Ub are recognized by a USP30-specific surface. Structure-based mutagenesis showed that the interface with the proximal Ub is critical for the specific cleavage of Lys6-linked Ub chains, together with the noncanonical catalytic triad composed of Cys-His-Ser. The structural findings presented here reveal a mechanism for Lys6-linkage-specific deubiquitination.

  14. Effect of women's age on embryo morphology, cleavage rate and competence-A multicenter cohort study

    DEFF Research Database (Denmark)

    Grøndahl, Marie Louise; Christiansen, Sofie Lindgren; Kesmodel, Ulrik Schiøler

    2017-01-01

    .0001) with increasing age. Maternal age had no effect on cleavage parameters or on the morphology of the embryo day 2 post insemination. Interestingly, initial hCG value after single embryo transfer followed by ongoing pregnancy was increased with age in both IVF (p = 0.007) and ICSI (p = 0.001) cycles. For the first...... time, we show that a woman's age does impose a significant footprint on early embryo morphological development (3PN). In addition, the developmentally competent embryos were associated with increased initial hCG values as the age of the women increased. Further studies are needed to elucidate......, if this increase in initial hCG value with advancing maternal age is connected to the embryo or the uterus....

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

    Directory of Open Access Journals (Sweden)

    G. H. PHILIP

    2010-01-01

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

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

    Directory of Open Access Journals (Sweden)

    Tatsuki Miyamoto

    2016-06-01

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

  17. Stress wave radiation from the cleavage crack extension in 3D bcc iron crystals

    Czech Academy of Sciences Publication Activity Database

    Uhnáková, Alena; Machová, Anna; Hora, Petr; Červ, Jan; Kroupa, Tomáš

    2010-01-01

    Roč. 50, č. 2 (2010), s. 678-685 ISSN 0927-0256 R&D Projects: GA ČR(CZ) GA101/09/1630; GA AV ČR(CZ) IAA200760611 Institutional research plan: CEZ:AV0Z20760514 Keywords : bcc iron * cleavage * acoustic emission sources * FEM * MD Subject RIV: BI - Acoustics Impact factor: 1.458, year: 2010 http://www.sciencedirect.com/science?_ob=MImg&_imagekey=B6TWM-5196KMK-3-16&_cdi=5566&_user=640952&_pii=S0927025610005379&_origin=search&_zone=rslt_list_item&_coverDate=12%2F31%2F2010&_sk=999499997&wchp=dGLbVtb-zSkWA&md5=d89705973ea4893248499205ae317b76&ie=/sdarticle.pdf

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

    Science.gov (United States)

    Bullock, R Morris; Chambers, Geoffrey M

    2017-08-28

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

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

    Directory of Open Access Journals (Sweden)

    Reshef Tal

    2012-01-01

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

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

    International Nuclear Information System (INIS)

    Kimura, Hiroshi

    1975-01-01

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

  1. Streptococcal Cysteine Protease-Mediated Cleavage of Desmogleins Is Involved in the Pathogenesis of Cutaneous Infection

    Directory of Open Access Journals (Sweden)

    Tomoko Sumitomo

    2018-01-01

    Full Text Available Streptococcus pyogenes is responsible for a wide variety of cutaneous infections ranging from superficial impetigo to fulminant invasive necrotizing fasciitis. Dysfunction of desmosomes is associated with the pathogenesis of cutaneous diseases. We identified streptococcal pyrogenic exotoxin B (SpeB as a proteolytic factor that cleaves the extracellular domains of desmoglein 1 and 3. In an epicutaneous infection model, lesional skin infected with an speB deletion mutant were significantly smaller as compared to those caused by the wild-type strain. Furthermore, immunohistological analysis indicated cleavage of desmogleins that developed around the invasion site of the wild-type strain. In contrast, the speB mutant was preferentially found on the epidermis surface layer. Taken together, our findings provide evidence that SpeB-mediated degradation of desmosomes has a pathogenic role in development of S. pyogenes cutaneous infection.

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

    Energy Technology Data Exchange (ETDEWEB)

    Bullock, R. Morris; Chambers, Geoffrey M.

    2017-07-24

    This Perspective examines the field of Frustrated Lewis Pairs (FLPs) in the context of transition metal mediated heterolytic cleavage of H2, with a particular emphasis on molecular complexes bearing an intramolecular Lewis base. FLPs have traditionally been associated with group compounds, yet many transition metal reactions support a broader classification of FLPs to include certain types of transition metal complexes with reactivity resembling main group based FLPs. This article surveys transition metal complexes that heterolytically cleave H2, which vary in the degree that the Lewis pairs within these systems interact. Particular attention is focused on complexes bearing a pendant amine function as the base. Consideration of transition metal compounds in the context of FLPs can inspire new innovations and improvements in transition metal catalysis.

  3. A colorimetric and ratiometric fluorescent probe for sulfite based on an intramolecular cleavage mechanism.

    Science.gov (United States)

    Hou, Peng; Chen, Song; Voitchovsky, Kislon; Song, Xiangzhi

    2014-11-01

    A colorimetric and ratiometric fluorescent sulfite probe, the levulinate of 4-hydroxynaphthalimide, was successfully synthesized from 4-hydroxy-naphthalimide and levulinic acid. Through sulfite-mediated intramolecular cleavage, the probe was converted into 4-hydroxynaphthalimide, which when excited at 450 nm, displayed a large Stokes shift due to the intramolecular charge transfer process. The probe exhibited high selectivity and sensitivity towards sulfite over other typical anionic species (F(-), Cl(-), Br(-), I(-), HPO(4)(2-), SO(4)(2-), NO(3)(-), AcO(-), ClO(4)(-), HCO(3)(-)) in HEPES-buffered solution (25 mm, pH 7.4, 50% acetonitrile, v/v). Copyright © 2013 John Wiley & Sons, Ltd.

  4. On the Mechanisms of Hydrogen Implantation Induced Silicon Surface Layer Cleavage

    Energy Technology Data Exchange (ETDEWEB)

    Hochbauer, Tobias [Univ. of Marburg (Germany)

    2001-11-01

    The “Ion-Cut”, a layer splitting process by hydrogen ion implantation and subsequent annealing is a versatile and efficient technique of transferring thin silicon surface layers from bulk substrates onto other substrates, thus enabling the production of silicon-oninsulator (SOI) materials. Cleavage is induced by the coalescence of the highly pressurized sub-surface H2-gas bubbles, which form upon thermal annealing. A fundamental understanding of the basic mechanisms on how the cutting process occurs is still unclear, inhibiting further optimization of the Ion-Cut process. This work elucidates the physical mechanisms behind the Ion-Cut process in hydrogen-implanted silicon. The investigation of the cleavage process reveals the cut to be largely controlled by the lattice damage, generated by the hydrogen ion irradiation process, and its effects on the local stress field and the fracture toughness within the implantation zone rather than by the depth of maximum H-concentration. Furthermore, this work elucidates the different kinetics in the H-complex formations in silicon crystals with different conductivity types, and examines the mechanically induced damage accumulation caused by the crack propagation through the silicon sample in the splitting step of the Ion-Cut process. Additionally, the influence of boron pre-implantation on the Ion-Cut in hydrogen implanted silicon is investigated. These studies reveal, that both, the atomic interaction between the boron implant and the hydrogen implant and the shift of the Fermi level due to the electrical activation of the implanted boron have a tremendous enhancing effect on the Ion-Cut process.

  5. On the Mechanisms of Hydrogen Implantation Induced Silicon Surface Layer Cleavage

    Energy Technology Data Exchange (ETDEWEB)

    Hochbauer, Tobias Franz [Univ. of Marburg (Germany)

    2002-08-01

    The “Ion-Cut”, a layer splitting process by hydrogen ion implantation and subsequent annealing is a versatile and efficient technique of transferring thin silicon surface layers from bulk substrates onto other substrates, thus enabling the production of silicon-oninsulator (SOI) materials. Cleavage is induced by the coalescence of the highly pressurized sub-surface H2-gas bubbles, which form upon thermal annealing. A fundamental understanding of the basic mechanisms on how the cutting process occurs is still unclear, inhibiting further optimization of the Ion-Cut process. This work elucidates the physical mechanisms behind the Ion-Cut process in hydrogen-implanted silicon. The investigation of the cleavage process reveals the cut to be largely controlled by the lattice damage, generated by the hydrogen ion irradiation process, and its effects on the local stress field and the fracture toughness within the implantation zone rather than by the depth of maximum H-concentration. Furthermore, this work elucidates the different kinetics in the H-complex formations in silicon crystals with different conductivity types, and examines the mechanically induced damage accumulation caused by the crack propagation through the silicon sample in the splitting step of the Ion-Cut process. Additionally, the influence of boron pre-implantation on the Ion-Cut in hydrogen implanted silicon is investigated. These studies reveal, that both, the atomic interaction between the boron implant and the hydrogen implant and the shift of the Fermi level due to the electrical activation of the implanted boron have a tremendous enhancing effect on the Ion-Cut process.

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

    Science.gov (United States)

    Stella, Stefano; Alcón, Pablo; Montoya, Guillermo

    2017-06-22

    Cpf1 is an RNA-guided endonuclease that is emerging as a powerful genome-editing tool. Here we provide insight into its DNA-targeting mechanism by determining the structure of Francisella novicida Cpf1 with the triple-stranded R-loop generated after DNA cleavage. The structure reveals the machinery involved in DNA unwinding to form a CRISPR RNA (crRNA)-DNA hybrid and a displaced DNA strand. The protospacer adjacent motif (PAM) is recognized by the PAM-interacting domain. The loop-lysine helix-loop motif in this domain contains three conserved lysine residues that are inserted in a dentate manner into the double-stranded DNA. Unzipping of the double-stranded DNA occurs in a cleft arranged by acidic and hydrophobic residues facilitating the crRNA-DNA hybrid formation. The PAM single-stranded DNA is funnelled towards the nuclease site through a mixed hydrophobic and basic cavity. In this catalytic conformation, the PAM-interacting domain and the helix-loop-helix motif in the REC1 domain adopt a 'rail' shape and 'flap-on' conformations, respectively, channelling the PAM strand into the cavity. A steric barrier between the RuvC-II and REC1 domains forms the 'septum', separating the displaced PAM strand and the crRNA-DNA hybrid, avoiding DNA re-annealing. Mutations in key residues reveal a mechanism linking the PAM and DNA nuclease sites. Analysis of the Cpf1 structures proposes a singular working model of RNA-guided DNA cleavage, suggesting new avenues for redesign of Cpf1.

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

    International Nuclear Information System (INIS)

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

    2011-01-01

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

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

    Directory of Open Access Journals (Sweden)

    Mengyi Yang

    2018-01-01

    Full Text Available Summary: Off-target binding and cleavage by Cas9 pose major challenges in its application. How the conformational dynamics of Cas9 govern its nuclease activity under on- and off-target conditions remains largely unknown. Here, using intra-molecular single-molecule fluorescence resonance energy transfer measurements, we revealed that Cas9 in apo, sgRNA-bound, and dsDNA/sgRNA-bound forms spontaneously transits among three major conformational states, mainly reflecting significant conformational mobility of the catalytic HNH domain. We also uncovered surprising long-range allosteric communication between the HNH domain and the RNA/DNA heteroduplex at the PAM-distal end to ensure correct positioning of the catalytic site, which demonstrated that a unique proofreading mechanism served as the last checkpoint before DNA cleavage. Several Cas9 residues were likely to mediate the allosteric communication and proofreading step. Modulating interactions between Cas9 and heteroduplex at the PAM-distal end by introducing mutations on these sites provides an alternative route to improve and optimize the CRISPR/Cas9 toolbox. : Yang et al. revealed significant conformational dynamics of Cas9 at global and local scales using single-molecule FRET. They uncovered surprising long-range allosteric communication between the HNH nuclease domain and the RNA/DNA heteroduplex at the PAM-distal end that serves as a proofreading checkpoint to govern the nuclease activity and specificity of Cas9. Keywords: CRISPR, Cas9, single-molecule, FRET, conformational dynamics, proofreading, off-target, allosteric communication, genome editing

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

    Science.gov (United States)

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

    2017-07-20

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

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

    Energy Technology Data Exchange (ETDEWEB)

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

    2011-04-30

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

  11. Mitochondrial DNA cleavage patterns distinguish independent origin of Chinese domestic geese and Western domestic geese.

    Science.gov (United States)

    Shi, X-W; Wang, J-W; Zeng, F-T; Qiu, X-P

    2006-06-01

    It has generally been assumed, based on morphology, that Chinese domestic goose breeds were derived from the swan goose (Anser cygnoides) and that European and American breeds were derived from the graylag goose (Anser anser). To test the validity of this assumption, we investigated the mtDNA cleavage patterns of 16 Chinese breeds and 2 European breeds as well as hybrids produced between a Chinese breed and a European breed. After 224 mtDNAs, isolated from the Chinese and European breeds, were digested by 19 restriction endonucleases, variations of the cleavage patterns were observed for four enzymes (EcoRV, HaeII, HincII, and KpnI). All Chinese breeds and their maternal hybrids except the Yili breed showed an identical haplotype, named haplotype I or the Chinese haplotype; the European breeds and the Yili breed showed another haplotype, named haplotype II or the western haplotype. None of the haplotype found in the Chinese type was detectable in the western type and vice versa. The two haplotypes were found to differ from each other at 8.0% of the sites surveyed and with a 0.72% sequence divergence. Using 2% substitution per million years calibrated from the genera Anser and Branta, the two domestic geese haplotypes were estimated to have diverged approximately 360,000 years ago, well outside the 3000-6000 years in domestic history. Our findings provide the first molecular genetic evidence to support the dual origin assumption of domestic geese in the world. Meanwhile, the four mtDNA restriction fragment length polymorphisms can be used as maternal genetic markers to distinguish the two types of domestic geese.

  12. Systematic Profiling of Poly(A)+ Transcripts Modulated by Core 3’ End Processing and Splicing Factors Reveals Regulatory Rules of Alternative Cleavage and Polyadenylation

    Science.gov (United States)

    Li, Wencheng; You, Bei; Hoque, Mainul; Zheng, Dinghai; Luo, Wenting; Ji, Zhe; Park, Ji Yeon; Gunderson, Samuel I.; Kalsotra, Auinash; Manley, James L.; Tian, Bin

    2015-01-01

    Alternative cleavage and polyadenylation (APA) results in mRNA isoforms containing different 3’ untranslated regions (3’UTRs) and/or coding sequences. How core cleavage/polyadenylation (C/P) factors regulate APA is not well understood. Using siRNA knockdown coupled with deep sequencing, we found that several C/P factors can play significant roles in 3’UTR-APA. Whereas Pcf11 and Fip1 enhance usage of proximal poly(A) sites (pAs), CFI-25/68, PABPN1 and PABPC1 promote usage of distal pAs. Strong cis element biases were found for pAs regulated by CFI-25/68 or Fip1, and the distance between pAs plays an important role in APA regulation. In addition, intronic pAs are substantially regulated by splicing factors, with U1 mostly inhibiting C/P events in introns near the 5’ end of gene and U2 suppressing those in introns with features for efficient splicing. Furthermore, PABPN1 inhibits expression of transcripts with pAs near the transcription start site (TSS), a property possibly related to its role in RNA degradation. Finally, we found that groups of APA events regulated by C/P factors are also modulated in cell differentiation and development with distinct trends. Together, our results support an APA code where an APA event in a given cellular context is regulated by a number of parameters, including relative location to the TSS, splicing context, distance between competing pAs, surrounding cis elements and concentrations of core C/P factors. PMID:25906188

  13. Systematic profiling of poly(A+ transcripts modulated by core 3' end processing and splicing factors reveals regulatory rules of alternative cleavage and polyadenylation.

    Directory of Open Access Journals (Sweden)

    Wencheng Li

    2015-04-01

    Full Text Available Alternative cleavage and polyadenylation (APA results in mRNA isoforms containing different 3' untranslated regions (3'UTRs and/or coding sequences. How core cleavage/polyadenylation (C/P factors regulate APA is not well understood. Using siRNA knockdown coupled with deep sequencing, we found that several C/P factors can play significant roles in 3'UTR-APA. Whereas Pcf11 and Fip1 enhance usage of proximal poly(A sites (pAs, CFI-25/68, PABPN1 and PABPC1 promote usage of distal pAs. Strong cis element biases were found for pAs regulated by CFI-25/68 or Fip1, and the distance between pAs plays an important role in APA regulation. In addition, intronic pAs are substantially regulated by splicing factors, with U1 mostly inhibiting C/P events in introns near the 5' end of gene and U2 suppressing those in introns with features for efficient splicing. Furthermore, PABPN1 inhibits expression of transcripts with pAs near the transcription start site (TSS, a property possibly related to its role in RNA degradation. Finally, we found that groups of APA events regulated by C/P factors are also modulated in cell differentiation and development with distinct trends. Together, our results support an APA code where an APA event in a given cellular context is regulated by a number of parameters, including relative location to the TSS, splicing context, distance between competing pAs, surrounding cis elements and concentrations of core C/P factors.

  14. Fluorescence quenching of graphene oxide combined with the site-specific cleavage of restriction endonuclease for deoxyribonucleic acid demethylase activity assay.

    Science.gov (United States)

    Ji, Lijuan; Qian, Yingdan; Wu, Ping; Zhang, Hui; Cai, Chenxin

    2015-04-15

    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(-1) (at a signal/noise of 3) of MBD2 with a linear range of 0.2-300 ng mL(-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 requirement of bisulfite conversion, PCR amplification, radioisotope labeling, or separation. Copyright © 2015 Elsevier B.V. All rights reserved.

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

    DEFF Research Database (Denmark)

    Belsham, Graham; Hua Guan, Su

    2017-01-01

    amino acid residues in the functions of this protein. These selected residues are either highly conserved or are charged and exposed on the protein surface. Using transient expression assays within BHK cells, it was found that residues around the active site (W52, L53 and A149) of Lpro and others...... located elsewhere (K38, K39, R44, H138 and W159) are involved in the induction of eIF4G cleavage but not in the processing of the L/P1 junction. Modified viruses, encoding such amino acid substitutions within Lpro can replicate in BHK cells but did not grow well in primary bovine thyroid cells. This study...... characterizes mutant viruses that are deficient in blocking host cell responses to infection (e.g. interferon induction) and can assist in the rational design of antiviral agents targeting this process and in the production of attenuated viruses....

  16. Avian paramyxovirus serotype 1 strains of low virulence with unusual fusion protein cleavage sites isolated from poultry species

    Science.gov (United States)

    Avian paramyxo-serotype-1 viruses (APMV1) with fusion cleavage sites containing two basic amino acids and a phenylalanine (F) at position 117 have been isolated from poultry species in two states from 2007-2009. The intracerebral pathogenicity indices for these viruses are of low virulence at 0.00 ...

  17. A neural network method for identification of prokaryotic and eukaryotic signal peptides and prediction of their cleavage sites

    DEFF Research Database (Denmark)

    Nielsen, Henrik; Engelbrecht, Jacob; Brunak, Søren

    1997-01-01

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

  18. Overexpression of the rice carotenoid cleavage dioxygenase 1 gene in Golden Rice endosperm suggests apocarotenoids as substrates in planta.

    Science.gov (United States)

    Ilg, Andrea; Yu, Qiuju; Schaub, Patrick; Beyer, Pete