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Sample records for proprotein convertase encoded

  1. Deletion of the Gene Encoding Proprotein Convertase 5/6 Causes Early Embryonic Lethality in the Mouse

    PubMed Central

    Essalmani, Rachid; Hamelin, Josée; Marcinkiewicz, Jadwiga; Chamberland, Ann; Mbikay, Majambu; Chrétien, Michel; Seidah, Nabil G.; Prat, Annik

    2006-01-01

    PC5 belongs to the proprotein convertase family and activates precursor proteins by cleavage at basic sites during their transit through the secretory pathway and/or at the cell surface. These precursors include prohormones, proreceptors, growth factors, adhesion molecules, and viral glycoproteins. The Pcsk5 gene encodes two alternatively spliced isoforms, the soluble PC5A and transmembrane PC5B. We have carefully analyzed the expression of PC5 in the mouse during development and in adulthood by in situ hybridization, as well as in mouse tissues and various cell lines by quantitative reverse transcription-PCR. The data show that adrenal cortex and intestine are the richest sources of PC5A and PC5B, respectively. To better define the specific physiological roles of PC5, we have generated a mouse Pcsk5Δ4-deficient allele missing exon 4 that encodes the catalytic Asp173. While Δ4/+ heterozygotes were healthy and fertile, genotyping of progeny obtained from Δ4/+ interbreeding indicated that Δ4/Δ4 embryos died between embryonic days 4.5 and 7.5. These data demonstrate that Pcsk5 is an essential gene. PMID:16354705

  2. Proprotein Convertases in Human Breast Cancer

    DTIC Science & Technology

    2001-03-01

    are a family of serine gests an important role for proprotein convertases in proteinases of the subtilisin /kexin type. To date, human breast...Sambrook J 1989 Extraction, quences. Alternatively, the potential role of propro- purification and analysis of messenger RNA from eucaryotic tein...Chretien M Genetics 12 223-225. & Marcinkiewicz M 1999 Mammalian subtilisin /kexin Steiner DF, Smeekens SP, Ohagi S & Chan SJ 1992 The new isozyme SKI

  3. Proprotein convertase activation of aggrecanases in cartilage in situ.

    PubMed

    Malfait, Anne-Marie; Arner, Elizabeth C; Song, Ruo-Hua; Alston, James T; Markosyan, Stella; Staten, Nicholas; Yang, Zhiyong; Griggs, David W; Tortorella, Micky D

    2008-10-01

    Proteolytic degradation of the major cartilage macromolecules, aggrecan and type II collagen, is a key pathological event in osteoarthritis (OA). ADAMTS-4 and ADAMTS-5, the primary aggrecanases capable of cartilage aggrecan cleavage, are synthesized as latent enzymes and require prodomain removal for activity. The N-termini of the mature proteases suggest that activation involves a proprotein convertase, but the specific family member responsible for aggrecanase activation in cartilage in situ has not been identified. Here we describe purification of a proprotein convertase activity from human OA cartilage. Through biochemical characterization and the use of siRNA, PACE4 was identified as a proprotein convertase responsible for activation of aggrecanases in osteoarthritic and cytokine-stimulated cartilage. Posttranslational activation of ADAMTS-4 and ADAMTS-5 was observed in the extracellular milieu of cartilage, resulting in aggrecan degradation. These findings suggest that PACE4 represents a novel target for the development of OA therapeutics.

  4. Proprotein Convertases in Human Breast Cancer

    DTIC Science & Technology

    1999-10-01

    mammary glands of transgenic mice in affecting Brcal (the mouse homolog) processing and cellular transformation. We will target PC I or firin to the...produce discernable abnormality in the transgenic mouse mammary glands, we will produce "double" transgenic mice between MMVTV-convertase transgenic mice ...with MMTV-neu transgenic mice . The "double" transgenic mice will be used to test the hypothesis that convertases facilitate the oncogenic potentials

  5. Analysis of epigenetic alterations to proprotein convertase genes in disease.

    PubMed

    Fu, YangXin; Nachtigal, Mark W

    2011-01-01

    Epigenetic alterations produce heritable changes in phenotype or gene expression without changing DNA sequence. Modified levels of gene expression contribute to a variety of human diseases encompassing genetic disorders, pediatric syndromes, autoimmune disease, aging, and cancer. Alterations in proprotein convertase gene expression are associated with numerous disease states; however, the underlying mechanism for changes in PC gene expression remains understudied. Epigenetic changes in gene expression profiles can be accomplished through modification of chromatin, specifically via chemical modification of DNA bases (methylation of cytosine) or associated histone proteins (acetylation or methylation). In general, active chromatin is associated with low DNA methylation status and histone acetylation, whereas silenced gene are typically in inactive regions of chromatin exhibiting DNA hypermethylation and histone deacetylation. This chapter will provide in-depth protocols to analyze epigenetic alterations in proprotein convertase gene expression using the PCSK6 gene in the context of human ovarian cancer as a model system.

  6. Myeloid cell expressed proprotein convertase FURIN attenuates inflammation.

    PubMed

    Cordova, Zuzet Martinez; Grönholm, Anna; Kytölä, Ville; Taverniti, Valentina; Hämäläinen, Sanna; Aittomäki, Saara; Niininen, Wilhelmiina; Junttila, Ilkka; Ylipää, Antti; Nykter, Matti; Pesu, Marko

    2016-08-23

    The proprotein convertase enzyme FURIN processes immature pro-proteins into functional end- products. FURIN is upregulated in activated immune cells and it regulates T-cell dependent peripheral tolerance and the Th1/Th2 balance. FURIN also promotes the infectivity of pathogens by activating bacterial toxins and by processing viral proteins. Here, we evaluated the role of FURIN in LysM+ myeloid cells in vivo. Mice with a conditional deletion of FURIN in their myeloid cells (LysMCre-fur(fl/fl)) were healthy and showed unchanged proportions of neutrophils and macrophages. Instead, LysMCre-fur(fl/fl) mice had elevated serum IL-1β levels and reduced numbers of splenocytes. An LPS injection resulted in accelerated mortality, elevated serum pro-inflammatory cytokines and upregulated numbers of pro-inflammatory macrophages. A genome-wide gene expression analysis revealed the overexpression of several pro-inflammatory genes in resting FURIN-deficient macrophages. Moreover, FURIN inhibited Nos2 and promoted the expression of Arg1, which implies that FURIN regulates the M1/M2-type macrophage balance. FURIN was required for the normal production of the bioactive TGF-β1 cytokine, but it inhibited the maturation of the inflammation-provoking TACE and Caspase-1 enzymes. In conclusion, FURIN has an anti-inflammatory function in LysM+ myeloid cells in vivo.

  7. Myeloid cell expressed proprotein convertase FURIN attenuates inflammation

    PubMed Central

    Cordova, Zuzet Martinez; Grönholm, Anna; Kytölä, Ville; Taverniti, Valentina; Hämäläinen, Sanna; Aittomäki, Saara; Niininen, Wilhelmiina; Junttila, Ilkka; Ylipää, Antti; Nykter, Matti; Pesu, Marko

    2016-01-01

    The proprotein convertase enzyme FURIN processes immature pro-proteins into functional end- products. FURIN is upregulated in activated immune cells and it regulates T-cell dependent peripheral tolerance and the Th1/Th2 balance. FURIN also promotes the infectivity of pathogens by activating bacterial toxins and by processing viral proteins. Here, we evaluated the role of FURIN in LysM+ myeloid cells in vivo. Mice with a conditional deletion of FURIN in their myeloid cells (LysMCre-fur(fl/fl)) were healthy and showed unchanged proportions of neutrophils and macrophages. Instead, LysMCre-fur(fl/fl) mice had elevated serum IL-1β levels and reduced numbers of splenocytes. An LPS injection resulted in accelerated mortality, elevated serum pro-inflammatory cytokines and upregulated numbers of pro-inflammatory macrophages. A genome-wide gene expression analysis revealed the overexpression of several pro-inflammatory genes in resting FURIN-deficient macrophages. Moreover, FURIN inhibited Nos2 and promoted the expression of Arg1, which implies that FURIN regulates the M1/M2-type macrophage balance. FURIN was required for the normal production of the bioactive TGF-β1 cytokine, but it inhibited the maturation of the inflammation-provoking TACE and Caspase-1 enzymes. In conclusion, FURIN has an anti-inflammatory function in LysM+ myeloid cells in vivo. PMID:27527873

  8. The proprotein convertase furin is required for trophoblast syncytialization

    PubMed Central

    Zhou, Z; Zhang, Q; Lu, X; Wang, R; Wang, H; Wang, Y-L; Zhu, C; Lin, H-Y; Wang, H

    2013-01-01

    The multinucleated syncytial trophoblast, which forms the outermost layer of the placenta and serves multiple functions, is differentiated from and maintained by cytotrophoblast cell fusion. Deficiencies in syncytial trophoblast differentiation or maintenance likely contribute to intrauterine growth restriction and pre-eclampsia, two common gestational diseases. The cellular and molecular mechanisms governing trophoblast syncytialization are poorly understood. We report here that the proprotein convertase furin is highly expressed in syncytial trophoblast in the first trimester human placentas, and expression of furin in the syncytiotrophoblast is significantly lower in the placentas from pre-eclamptic patients as compared with their gestational age-matched control placentas. Using multiple experimental models including induced fusion of choriocarcinoma BeWo cells and spontaneous fusion of primary cultured cytotrophoblast cells or placental explants, we demonstrate that cytotrophoblast cell fusion and syncytialization are accompanied by furin expression. Furin-specific siRNAs or inhibitors inhibit cell fusion in BeWo cells, as well as trophoblast syncytialization in human placental explants. Furthermore, type 1 IGF receptor (IGF1R) is indicated in this study as a substrate of furin, and processing of IGF1R by furin is an essential mechanism for syncytialization. Finally, using lentivirus-mediated RNAi targeting to mouse trophectoderm, we demonstrate that furin function is required for the development of syncytiotrophoblast structure in the labyrinth layer, as well as for normal embryonic development. PMID:23598405

  9. An Atypical Proprotein Convertase in Giardia lamblia Differentiation

    PubMed Central

    Davids, B. J.; Gilbert, M. A.; Liu, Q.; Reiner, D. S.; Smith, A. J.; Lauwaet, T.; Lee, C.; McArthur, A. G.; Gillin, F. D.

    2010-01-01

    Proteolytic activity is important in the lifecycles of parasites and their interactions with hosts. Cysteine proteases have been best studied in Giardia, but other protease classes have been implicated in growth and/or differentiation. In this study, we employed bioinformatics to reveal the complete set of putative proteases in the Giardia genome. We identified 73 peptidase homologues distributed over 5 catalytic classes in the genome. Serial analysis of gene expression of the G. lamblia lifecycle found thirteen protease genes with significant transcriptional variation over the lifecycle, with only one serine protease transcript upregulated late in encystation. The translated gene sequence of this encystation-specific transcript was most similar to eukaryotic subtilisin-like proprotein convertases (SPC), although the typical catalytic triad was not identified. Epitope-tagged gSPC protein expressed in Giardia under its own promoter was upregulated during encystation with highest expression in cysts and it localized to encystation-specific secretory vesicles (ESV). Total gSPC from encysting cells produced proteolysis in gelatin gels that co-migrated with the epitope-tagged protease in immunoblots. Immuno-purified gSPC also had gelatinase activity. To test whether endogenous gSPC activity is involved in differentiation, trophozoites and cysts were exposed to the specific serine proteinase inhibitor 4-(2-Aminoethyl)-benzenesulfonyl fluoride hydrochloride (AEBSF). After 21 h encystation, a significant decrease in ESV was observed with 1 mM AEBSF and by 42 h the number of cysts was significantly reduced, but trophozoite growth was not inhibited. Concurrently, levels of cyst wall proteins 1 and 2, and AU1-tagged gSPC protein itself were decreased. Excystation of G. muris cysts was also significantly reduced in the presence of AEBSF. These results support the idea that serine protease activity is essential for Giardia encystation and excystation. PMID:21075147

  10. Role of proprotein convertases in prostate cancer progression.

    PubMed

    Couture, Frédéric; D'Anjou, François; Desjardins, Roxane; Boudreau, François; Day, Robert

    2012-11-01

    Better understanding of the distinct and redundant functions of the proprotein convertase (PC) enzyme family within pathophysiological states has a great importance for potential therapeutic strategies. In this study, we investigated the functional redundancy of PCs in prostate cancer in the commonly used androgen-sensitive LNCaP and the androgen-independent DU145 human cell lines. Using a lentiviral-based shRNA delivery system, we examined in vitro and in vivo cell proliferation characteristics of knockdown cell lines for the endogenous PCs furin, PACE4, and PC7 in both cell lines. Of the three PCs, only PACE4 was essential to maintain a high-proliferative status, as determined in vitro using XTT proliferation assays and in vivo using tumor xenografts in nude mice. Furin knockdowns in both cell lines had no effects on cell proliferation or tumor xenograft growth. Paradoxically, PC7 knockdowns reduced in vitro cellular proliferation but had no effect in vivo. Because PCs act within secretion pathways, we showed that conditioned media derived from PACE4 knockdown cells had very poor cell growth-stimulating effects in vitro. Immunohistochemistry of PACE4 knockdown tumors revealed reduced Ki67 and higher p27(KIP) levels (proliferation and cell cycle arrest markers, respectively). Interestingly, we determined that the epidermal growth factor receptor signaling pathway was activated in PC7 knockdown tumors only, providing some explanations of the paradoxical effects of PC7 silencing in prostate cancer cell lines. We conclude that PACE4 has a distinct role in maintaining proliferation and tumor progression in prostate cancer and this positions PACE4 as a relevant therapeutic target for this disease.

  11. Proprotein convertases in high-density lipoprotein metabolism.

    PubMed

    Choi, Seungbum; Korstanje, Ron

    2013-09-18

    The proprotein convertase subtilisin/kexins (PCSKs) are a serine endopeptidase family. PCSK members cleave amino acid residues and modulate the activity of precursor proteins. Evidence from patients and animal models carrying genetic alterations in PCSK members show that PCSK members are involved in various metabolic processes. These studies further revealed the molecular mechanism by which genetic alteration of some PCSK members impairs normal molecular and physiological functions, which in turn lead to cardiovascular disease. High-density lipoprotein (HDL) is anti-atherogenic as it removes excessive amount of cholesterol from blood and peripheral tissues. Several PCSK members are involved in HDL metabolism. PCSK3, PCSK5, and PCSK6 process two triglyceride lipase family members, endothelial lipase and lipoprotein lipase, which are important for HDL remodeling. Recent studies in our lab found evidence that PCSK1 and PCSK9 are also involved in HDL metabolism. A mouse model carrying an amino acid substitution in PCSK1 showed an increase in serum apolipoprotein A1 (APOA1) level. Another mouse model lacking PCSK9 showed a decrease in APOE-containing HDL. In this review, we summarize the role of the five PCSK members in lipid, glucose, and bile acid (BA) metabolism, each of which can influence HDL metabolism. We propose an integrative model in which PCSK members regulate HDL metabolism through various molecular mechanisms and metabolic processes and genetic variation in some PCSK members may affect the efficiency of reverse cholesterol transport. PCSK members are considered as attractive therapeutic targets. A greater understanding of the molecular and physiological functions of PCSK members will improve therapeutic strategies and drug efficacy for cardiovascular disease where PCSK members play critical role, with fewer adverse effects.

  12. Knock-out mouse models of proprotein convertases: unique functions or redundancy?

    PubMed

    Creemers, John W M; Khatib, Abdel-Majid

    2008-05-01

    The members of the proprotein convertase family play a central role in the processing and/or activation of various protein precursors involved in many physiological processes and various pathologies. The proteolysis of these precursors that occur at basic residues within the general motif (K/R)-(X)-(K/R) is mediated by the proprotein convertases PC1/3, PC2, Furin, PACE4, PC4, PC5 and PC7, whereas the proteolysis of precursors within hydrophobic residues performed by the convertase S1P/SKI-1 and the convertase NARC-1/PCSK9 seems to prefer cleavages at the motif LVFAQSIP. Here we provide a comprehensive overview of their remarkable complex roles as revealed by disruption of their genes individually using generalized or conditional approaches.

  13. Proprotein Convertase Subtilisin/Kexin Type 9 Inhibitors: A Brief Overview.

    PubMed

    Noel, Zachary R; Beavers, Craig J

    2017-02-01

    Proprotein convertase subtilisin/kexin type 9 inhibitors serve as a valuable addition to the armamentarium of lipid-lowering agents and have promising potential. By inhibiting the proprotein convertase subtilisin/kexin type 9 enzyme, this novel molecule leads to increased low-density lipoprotein receptor density and decreased circulation of low-density lipoprotein. The fact the agent is a monoclonal antibody has led to limited drug interactions and minimized adverse drug events. It is critical for all providers to have a basic understanding of these novel therapies with their introduction and use for treatment.

  14. Furin Is the Major Proprotein Convertase Required for KISS1-to-Kisspeptin Processing

    PubMed Central

    Harihar, Sitaram; Pounds, Keke M.; Iwakuma, Tomoo; Seidah, Nabil G.; Welch, Danny R.

    2014-01-01

    KISS1 is a broadly functional secreted proprotein that is then processed into small peptides, termed kisspeptins (KP). Since sequence analysis showed cleavage at KR or RR dibasic sites of the nascent protein, it was hypothesized that enzyme(s) belonging to the proprotein convertase family of proteases process KISS1 to generate KP. To this end, cell lines over-expressing KISS1 were treated with the proprotein convertase inhibitors, Dec-RVKR-CMK and α1-PDX, and KISS1 processing was completely inhibited. To identify the specific enzyme(s) responsible for KISS1 processing, mRNA expression was systematically analyzed for six proprotein convertases found in secretory pathways. Consistent expression of the three proteases – furin, PCSK5 and PCSK7 – were potentially implicated in KISS1 processing. However, shRNA-mediated knockdown of furin – but not PCSK5 or PCSK7 – blocked KISS1 processing. Thus, furin appears to be the essential enzyme for the generation of kisspeptins. PMID:24454770

  15. Furin is the major proprotein convertase required for KISS1-to-Kisspeptin processing.

    PubMed

    Harihar, Sitaram; Pounds, Keke M; Iwakuma, Tomoo; Seidah, Nabil G; Welch, Danny R

    2014-01-01

    KISS1 is a broadly functional secreted proprotein that is then processed into small peptides, termed kisspeptins (KP). Since sequence analysis showed cleavage at KR or RR dibasic sites of the nascent protein, it was hypothesized that enzyme(s) belonging to the proprotein convertase family of proteases process KISS1 to generate KP. To this end, cell lines over-expressing KISS1 were treated with the proprotein convertase inhibitors, Dec-RVKR-CMK and α1-PDX, and KISS1 processing was completely inhibited. To identify the specific enzyme(s) responsible for KISS1 processing, mRNA expression was systematically analyzed for six proprotein convertases found in secretory pathways. Consistent expression of the three proteases - furin, PCSK5 and PCSK7 - were potentially implicated in KISS1 processing. However, shRNA-mediated knockdown of furin - but not PCSK5 or PCSK7 - blocked KISS1 processing. Thus, furin appears to be the essential enzyme for the generation of kisspeptins.

  16. Knockdown strategies for the study of proprotein convertases and proliferation in prostate cancer cells.

    PubMed

    D'Anjou, François; Couture, Frédéric; Desjardins, Roxane; Day, Robert

    2014-01-01

    Gene silencing strategies targeting mRNA are suitable methods to validate the functions of specific genes. In this chapter, we sought to compare two knockdown strategies for the study of proprotein convertases and proliferation in prostate cancer cells. We used both SOFA-HDV ribozyme and lentiviral-mediated shRNA delivery system to reduce PACE4 mRNA levels and validate its implication in the proliferation of DU145 prostate cancer cells. The cellular effects of PACE4 knockdown were assessed (1) in vitro using two tetrazolium salts (MTT and XTT assays) and (2) in vivo using a tumor xenograft approach in immunodeficient mice (Nu/Nu). Our results confirm the unique role of the proprotein convertase PACE4 in prostate cancer cell proliferation while demonstrating advantages and disadvantages of each approach. Achieving target validation in an effective manner is critical, as further development using a drug development approach is highly laborious and requires enormous resources.

  17. The isoforms of proprotein convertase PC5 are sorted to different subcellular compartments

    PubMed Central

    1996-01-01

    The proprotein convertase PC5 is encoded by multiple mRNAs, two of which give rise to the COOH-terminal variant isoforms PC5-A (915 amino acids [aa]) and PC5-B (1877 aa). To investigate the differences in biosynthesis and sorting between these two proteins, we generated stably transfected AtT-20 cell lines expressing each enzyme individually and examined their respective processing pattern and subcellular localization. Biosynthetic analyses coupled to immunofluorescence studies demonstrated that the shorter and soluble PC5-A is sorted to regulated secretory granules. In contrast, the COOH- terminally extended and membrane-bound PC5-B is located in the Golgi. The presence of a sorting signal in the COOH-terminal 38 amino acids unique to PC5-A was demonstrated by the inefficient entry into the regulated secretory pathway of a mutant lacking this segment. EM of pancreatic cells established the presence of immunoreactive PC5 in glucagon-containing granules, demonstrating the sorting of this protein to dense core secretory granules in endocrine cells. Thus, a single PC5 gene generates COOH-terminally modified isoforms with different sorting signals directing these proteins to distinct subcellular localization, thereby allowing them to process their appropriate substrates. PMID:8947550

  18. Genetics of the First Seven Proprotein Convertase Enzymes in Health and Disease

    PubMed Central

    Turpeinen, Hannu; Ortutay, Zsuzsanna; Pesu, Marko

    2013-01-01

    Members of the substilisin/kexin like proprotein convertase (PCSK) protease family cleave and convert immature pro-proteins into their biologically active forms. By cleaving for example prohormones, cytokines and cell membrane proteins, PCSKs participate in maintaining the homeostasis in a healthy human body. Conversely, erratic enzymatic function is thought to contribute to the pathogenesis of a wide variety of diseases, including obesity and hypercholestrolemia. The first characterized seven PCSK enzymes (PCSK1-2, FURIN, PCSK4-7) process their substrates at a motif made up of paired basic amino acid residues. This feature results in a variable degree of biochemical redundancy in vitro, and consequently, shared substrate molecules between the different PCSK enzymes. This redundancy has confounded our understanding of the specific biological functions of PCSKs. The physiological roles of these enzymes have been best illustrated by the phenotypes of genetically engineered mice and patients that carry mutations in the PCSK genes. Recent developments in genome-wide methodology have generated a large amount of novel information on the genetics of the first seven proprotein convertases. In this review we summarize the reported genetic alterations and their associated phenotypes. PMID:24396277

  19. Angiopoietin-like protein 3 inhibits lipoprotein lipase activity through enhancing its cleavage by proprotein convertases.

    PubMed

    Liu, Jun; Afroza, Huq; Rader, Daniel J; Jin, Weijun

    2010-09-03

    Lipoprotein lipase (LPL)-mediated lipolysis of triglycerides is the first and rate-limiting step in chylomicron/very low density lipoprotein clearance at the luminal surface of the capillaries. Angiopoietin-like protein 3 (ANGPTL3) is shown to inhibit LPL activity and plays important roles in modulating lipoprotein metabolism in vivo. However, the mechanism by which it inhibits LPL activity remains poorly understood. Using cell-based analysis of the interaction between ANGPTL3, furin, proprotein convertase subtilisin/kexin type 5 (PCSK5), paired amino acid converting enzyme-4 (PACE4), and LPL, we demonstrated that the cleavage of LPL by proprotein convertases is an inactivation process, similar to that seen for endothelial lipase cleavage. At physiological concentrations and in the presence of cells, ANGPTL3 is a potent inhibitor of LPL. This action is due to the fact that ANGPTL3 can enhance LPL cleavage by endogenous furin and PACE4 but not by PCSK5. This effect is specific to LPL but not endothelial lipase. Both N- and C-terminal domains of LPL are required for ANGPTL3-enhanced cleavage, and the N-terminal domain of ANGPTL3 is sufficient to exert its effect on LPL cleavage. Moreover, ANGPTL3 enhances LPL cleavage in the presence of either heparan sulfate proteoglycans or glycosylphosphatidylinositol-anchored high density lipoprotein-binding protein 1 (GPIHBP1). By enhancing LPL cleavage, ANGPTL3 dissociates LPL from the cell surface, inhibiting both the catalytic and noncatalytic functions of LPL. Taken together, our data provide a molecular connection between ANGPTL3, LPL, and proprotein convertases, which may represent a rapid signal communication among different metabolically active tissues to maintain energy homeostasis. These novel findings provide a new paradigm of specific protease-substrate interaction and further improve our knowledge of LPL biology.

  20. The Multifaceted Proprotein Convertases: Their Unique, Redundant, Complementary, and Opposite Functions*

    PubMed Central

    Seidah, Nabil G.; Sadr, Mohamad S.; Chrétien, Michel; Mbikay, Majambu

    2013-01-01

    The secretory proprotein convertase (PC) family comprises nine members: PC1/3, PC2, furin, PC4, PC5/6, PACE4, PC7, SKI-1/S1P, and PCSK9. The first seven PCs cleave their substrates at single or paired basic residues, and SKI-1/S1P cleaves its substrates at non-basic residues in the Golgi. PCSK9 cleaves itself once, and the secreted inactive protease escorts specific receptors for lysosomal degradation. It regulates the levels of circulating LDL cholesterol and is considered a major therapeutic target in phase III clinical trials. In vivo, PCs exhibit unique and often essential functions during development and/or in adulthood, but certain convertases also exhibit complementary, redundant, or opposite functions. PMID:23775089

  1. Processing of alpha4 integrin by the proprotein convertases: histidine at position P6 regulates cleavage.

    PubMed Central

    Bergeron, Eric; Basak, Ajoy; Decroly, Etienne; Seidah, Nabil G

    2003-01-01

    The proprotein convertases (PCs) participate in the limited proteolysis of integrin alpha4 subunit at the H(592)VISKR(597) downward arrow ST site (where underlined residues indicate positively charged amino acids important for PC-mediated cleavage and downward arrow indicates the cleavage site), since this cleavage is inhibited by the serpin alpha1-PDX (alpha1-antitrypsin Portland). Co-expression of alpha4 with each convertase in LoVo (furin-deficient human colon carcinoma) cells revealed that furin and proprotein convertase 5A (PC5A) are the best pro-alpha4 convertases. In agreement, processing of endogenous pro-alpha4 in human lymphoblastoid CEM-T4 cells was enhanced greatly in stable transfectants overexpressing either enzyme. In many leucocyte cell lines, the expression of furin closely correlated with the endogenous processing efficacy, suggesting that furin is a candidate pro-alpha4 convertase. Mutational analysis showed that replacement of P1 Arg(597) with alanine (R597A) abrogated cleavage, whereas the P6 mutant H592R is even better processed by the endogenous convertases of Chinese-hamster ovary CHO-K1 cells. In vitro kinetic studies using synthetic peptides confirmed the importance of a positively charged residue at P6 and showed that wild-type alpha4 processing is performed best by furin and PC5A at acidic and neutral pHs, respectively. Biosynthetic analysis of pro-alpha4 and its H592R and H592K mutants in the presence or absence of the weak base, NH(4)Cl, revealed that the P6 histidine residue renders its processing by furin sensitive to cellular pH. This suggests that pro-alpha4 cleavage occurs preferentially in acidic compartments. In conclusion, although the accepted furin processing motif is Arg-Xaa-(Lys/Arg)-Arg downward arrow, our data further extend it to include a regulatory histidine residue at P6 in precursors that lack a basic residue at P4. PMID:12691605

  2. Processing of alpha4 integrin by the proprotein convertases: histidine at position P6 regulates cleavage.

    PubMed

    Bergeron, Eric; Basak, Ajoy; Decroly, Etienne; Seidah, Nabil G

    2003-07-15

    The proprotein convertases (PCs) participate in the limited proteolysis of integrin alpha4 subunit at the H(592)VISKR(597) downward arrow ST site (where underlined residues indicate positively charged amino acids important for PC-mediated cleavage and downward arrow indicates the cleavage site), since this cleavage is inhibited by the serpin alpha1-PDX (alpha1-antitrypsin Portland). Co-expression of alpha4 with each convertase in LoVo (furin-deficient human colon carcinoma) cells revealed that furin and proprotein convertase 5A (PC5A) are the best pro-alpha4 convertases. In agreement, processing of endogenous pro-alpha4 in human lymphoblastoid CEM-T4 cells was enhanced greatly in stable transfectants overexpressing either enzyme. In many leucocyte cell lines, the expression of furin closely correlated with the endogenous processing efficacy, suggesting that furin is a candidate pro-alpha4 convertase. Mutational analysis showed that replacement of P1 Arg(597) with alanine (R597A) abrogated cleavage, whereas the P6 mutant H592R is even better processed by the endogenous convertases of Chinese-hamster ovary CHO-K1 cells. In vitro kinetic studies using synthetic peptides confirmed the importance of a positively charged residue at P6 and showed that wild-type alpha4 processing is performed best by furin and PC5A at acidic and neutral pHs, respectively. Biosynthetic analysis of pro-alpha4 and its H592R and H592K mutants in the presence or absence of the weak base, NH(4)Cl, revealed that the P6 histidine residue renders its processing by furin sensitive to cellular pH. This suggests that pro-alpha4 cleavage occurs preferentially in acidic compartments. In conclusion, although the accepted furin processing motif is Arg-Xaa-(Lys/Arg)-Arg downward arrow, our data further extend it to include a regulatory histidine residue at P6 in precursors that lack a basic residue at P4.

  3. Proprotein convertase subtilisin/kexin type 9 (PCSK9) in lipid metabolism, atherosclerosis and ischemic stroke.

    PubMed

    Zhang, Lingling; Song, Kangping; Zhu, Mengting; Shi, Jinling; Zhang, Huijuan; Xu, Liang; Chen, Yingzhu

    2016-08-01

    Proprotein convertase subtilisin/kexin 9 (PCSK9) is the ninth member of the proprotein convertase family. It is an important regulator of cholesterol metabolism. PCSK9 can bind to low-density lipoprotein receptors (LDLRs) and induce the degradation of these receptors through the endosome/lysosome pathway, thus decreasing the LDLR levels on the cell surface of hepatocytes, resulting in increased serum low-density lipoprotein cholesterol (LDL-C) concentrations. Recent studies have found that gene polymorphisms of PCSK9 are associated with hypercholesterolemia, risk of atherosclerosis, and ischemic stroke. Furthermore, monoclonal antibodies, peptide mimetics, small molecule inhibitors and gene silencing agents that are associated with PCSK9 are some of the newer pharmaceutical therapeutic strategies and approaches for lowering serum LDL-C levels. In this review, we will discuss recent advances in PCSK9 research, which show that PCSK9 is correlated with lipid metabolism, atherosclerosis, and, in particular, ischemic stroke. We will also discuss the current state of PCSK9 therapeutics and their potential in modulating these diseases.

  4. Hysteretic Behavior of Proprotein Convertase 1/3 (PC1/3)

    PubMed Central

    Icimoto, Marcelo Y.; Barros, Nilana M.; Ferreira, Juliana C.; Marcondes, Marcelo F.; Andrade, Douglas; Machado, Mauricio F.; Juliano, Maria A.; Júdice, Wagner A.; Juliano, Luiz; Oliveira, Vitor

    2011-01-01

    The proprotein convertases (PCs) are calcium-dependent proteases responsible for processing precursor proteins into their active forms in eukariotes. The PC1/3 is a pivotal enzyme of this family that participates in the proteolytic maturation of prohormones and neuropeptides inside the regulated secretory pathway. In this paper we demonstrate that mouse proprotein convertase 1/3 (mPC1/3) has a lag phase of activation by substrates that can be interpreted as a hysteretic behavior of the enzyme for their hydrolysis. This is an unprecedented observation in peptidases, but is frequent in regulatory enzymes with physiological relevance. The lag phase of mPC1/3 is dependent on substrate, calcium concentration and pH. This hysteretic behavior may have implications in the physiological processes in which PC1/3 participates and could be considered an additional control step in the peptide hormone maturation processes as for instance in the transformation of proinsulin to insulin. PMID:21935423

  5. Pro-protein convertases in intermediary metabolism: islet hormones, brain/gut hormones and integrated physiology.

    PubMed

    Bataille, Dominique

    2007-07-01

    Many peptide hormones implicated in the regulation of intermediary metabolism arise from larger precursors called prohormones. These precursors are cut into pieces by proprotein convertases, more precisely those called prohormone convertases (PCs) that cleave at the C terminus of basic doublets. The remaining basic amino acids are eliminated by a specialized carboxypeptidase, leading to the active hormone. This processing may provide, from a single precursor, several peptides with different biological activities depending on the site(s) of cleavage on the precursor. When the processing is tissue-specific, this mechanism allows to produce, from a single protein, different sets of hormones depending on the tissue considered, leading to novel regulatory processes. The archetype of such a pluripotent prohormone in the field of intermediary metabolism is pro-glucagon that, when cut by PC1 in intestinal L cells, produces four different peptides with different specificities [glicentin, oxyntomodulin (OXM), glucagon-like peptide-1, and glucagon-like peptide-2], whereas, when cut by PC2 in the alpha cells of the endocrine pancreas, glucagon is produced and, through the supplementary action of NRD convertase, a fragment of glucagon (miniglucagon) with original properties.

  6. Drugs for hypercholesterolaemia - from statins to pro-protein convertase subtilisin kexin 9 (PCSK9) inhibition.

    PubMed

    Wierzbicki, Anthony S; Grant, Paul

    2016-08-01

    Cardiovascular disease (CVD) remains one of the commonest sources of morbidity and mortality in the world. Lipids and especially low density lipoprotein cholesterol (LDL-C) contribute to the risk of CVD events. Statins are the primary therapy for hypercholesterolaemia and recent evidence supports the use of ezetimibe as a second-line agent. Pro-protein convertase subtilisin kexin 9 (PCSK9) is a regulator of LDL receptor expression. Activating mutations in PCSK9 give rise to a form of familial hypercholesterolaemia, while inactivating mutations lead to lower LDL-C levels and fewer CVD events. Therapies to inhibit PCSK9 are in development and two antibody-based therapies - alirocumab and evolocumab - have recently been licensed. This article reviews the actions of PCSK9, the novel therapeutics targeted on this molecule and how they are likely to be used in clinical practice until large scale CVD outcome studies with PCSK9 inhibitors are published. © 2016 Royal College of Physicians.

  7. The prohormone theory and the proprotein convertases: it is all about serendipity.

    PubMed

    Chrétien, Michel

    2011-01-01

    When I became a physician and an endocrinologist in the early 1960s, peptide hormone sequencing was still in its infancy; it was also far removed from my immediate interests. Through chance encounters with prominent teachers and mentors, I later became increasingly convinced that elucidation of the primary sequence of peptide hormones is key to understanding their production as well as their functions in human health and disease. My interest for pituitary hormones led me to discover that the sequence of β-melanocyte-stimulating hormone was contained within that γ and β-lipotropins and could be released from the latter by limited endoproteolysis. This prohormone theory became the leitmotiv of my career as a clinician/scientist. Through serendipity and the efforts of many laboratories including mine, this theory has now been widely confirmed, extended to various precursor proteins and implicated in many diseases. It has led to our discovery of the proprotein convertases.

  8. Lipoprotein(a) and inhibitors of proprotein convertase subtilisin/kexin type 9

    PubMed Central

    Kotani, Kazuhiko

    2017-01-01

    Lipoprotein(a) [Lp(a)] has been identified as a risk factor for cardiovascular disease. Lp(a) levels are also high under certain clinical conditions, including familial hypercholesterolemia and high blood low-density lipoprotein (LDL) cholesterol levels. Few effective generic therapies for modulating Lp(a) have been developed. However, new therapies involving inhibitors of proprotein convertase subtilisin/kexin type 9 (PCSK9) using monoclonal antibodies have markedly reduced the blood LDL levels—and the Lp(a) levels as well. Much attention has therefore been focused on this therapy and its utility. The mechanism by which PCSK9 inhibitors reduce the Lp(a) levels remains unclear. We here describe the effects of PCSK9 inhibitors on Lp(a) and discuss potential mechanisms and perspectives of this topic. PMID:28203441

  9. The cellular trafficking of the secretory proprotein convertase PCSK9 and its dependence on the LDLR.

    PubMed

    Nassoury, Nasha; Blasiole, Daniel A; Tebon Oler, Angie; Benjannet, Suzanne; Hamelin, Josée; Poupon, Vivianne; McPherson, Peter S; Attie, Alan D; Prat, Annik; Seidah, Nabil G

    2007-06-01

    Mutations in the proprotein convertase PCSK9 gene are associated with autosomal dominant familial hyper- or hypocholesterolemia. These phenotypes are caused by a gain or loss of function of proprotein convertase subtilisin kexin 9 (PCSK9) to elicit the degradation of the low-density lipoprotein receptor (LDLR) protein. Herein, we asked whether the subcellular localization of wild-type PCSK9 or mutants of PCSK9 and the LDLR would provide insight into the mechanism of PCSK9-dependent LDLR degradation. We show that the LDLR is the dominant partner in regulating the cellular trafficking of PCSK9. In cells lacking the LDLR, PCSK9 localized in the endoplasmic reticulum (ER). In cells expressing the LDLR, PCSK9 sorted to post-ER compartments (i.e. endosomes in cell lines and Golgi apparatus in primary hepatocytes), where it colocalized with the LDLR. In cell lines, PCSK9 also colocalized with the LDLR at the cell surface, requiring the presence of the C-terminal Cys/His-rich domain of PCSK9. We provide evidence that PCSK9 promotes the degradation of the LDLR by an endocytic mechanism, as small interfering RNA-mediated knockdown of the clathrin heavy chain reduced the functional activity of PCSK9. We also compared the subcellular localization of natural mutants of PCSK9 with that of the wild-type enzyme in human hepatic (HuH7) cells. Whereas the mutants associated with hypercholesterolemia (S127R, F216L and R218S) localized to endosomes/lysosomes, those associated with hypocholesterolemia did not reach this compartment. We conclude that the sorting of PCSK9 to the cell surface and endosomes is required for PCSK9 to fully promote LDLR degradation and that retention in the ER prevents this activity. Mutations that affect this transport can lead to hyper- or hypocholesterolemia.

  10. Membrane type-1 matrix metalloprotease-independent activation of pro-matrix metalloprotease-2 by proprotein convertases.

    PubMed

    Koo, Bon-Hun; Kim, Hee-Hyun; Park, Michael Y; Jeon, Ok-Hee; Kim, Doo-Sik

    2009-11-01

    Matrix metalloprotease-2 is implicated in many biological processes and degrades extracellular and non-extracellular matrix molecules. Matrix metalloprotease-2 maintains a latent state through a cysteine-zinc ion pairing which, when disrupted, results in full enzyme activation. This pairing can be disrupted by a conformational change or cleavage within the propeptide. The best known activation mechanism for pro-matrix metalloprotease-2 occurs via cleavage of the propeptide by membrane type-1 matrix metalloprotease. However, significant residual activation of pro-matrix metalloprotease-2 is seen in membrane type-1 matrix metalloprotease knockout mice and in fibroblasts treated with metalloprotease inhibitors. These findings indicate the presence of a membrane type-1 matrix metalloprotease-independent activation mechanism for pro-matrix metalloprotease-2 in vivo, which prompted us to explore an alternative activation mechanism for pro-matrix metalloprotese-2. In this study, we demonstrate membrane type-1 matrix metalloprotease-independent propeptide processing of matrix metalloprotease-2 in HEK293F and various tumor cell lines, and show that proprotein convertases can mediate the processing intracellularly as well as extracellularly. Furthermore, processed matrix metalloprotease-2 exhibits enzymatic activity that is enhanced by intermolecular autolytic cleavage. Thus, our experimental data, taken together with the broad expression of proprotein convertases, suggest that the proprotein convertase-mediated processing may be a general activation mechanism for pro-matrix metalloprotease-2 in vivo.

  11. A novel familial mutation in the PCSK1 gene that alters the oxyanion hole residue of proprotein convertase 1/3 and impairs its enzymatic activity.

    PubMed

    Wilschanski, Michael; Abbasi, Montaser; Blanco, Elias; Lindberg, Iris; Yourshaw, Michael; Zangen, David; Berger, Itai; Shteyer, Eyal; Pappo, Orit; Bar-Oz, Benjamin; Martín, Martin G; Elpeleg, Orly

    2014-01-01

    Four siblings presented with congenital diarrhea and various endocrinopathies. Exome sequencing and homozygosity mapping identified five regions, comprising 337 protein-coding genes that were shared by three affected siblings. Exome sequencing identified a novel homozygous N309K mutation in the proprotein convertase subtilisin/kexin type 1 (PCSK1) gene, encoding the neuroendocrine convertase 1 precursor (PC1/3) which was recently reported as a cause of Congenital Diarrhea Disorder (CDD). The PCSK1 mutation affected the oxyanion hole transition state-stabilizing amino acid within the active site, which is critical for appropriate proprotein maturation and enzyme activity. Unexpectedly, the N309K mutant protein exhibited normal, though slowed, prodomain removal and was secreted from both HEK293 and Neuro2A cells. However, the secreted enzyme showed no catalytic activity, and was not processed into the 66 kDa form. We conclude that the N309K enzyme is able to cleave its own propeptide but is catalytically inert against in trans substrates, and that this variant accounts for the enteric and systemic endocrinopathies seen in this large consanguineous kindred.

  12. A Novel Familial Mutation in the PCSK1 Gene That Alters the Oxyanion Hole Residue of Proprotein Convertase 1/3 and Impairs Its Enzymatic Activity

    PubMed Central

    Wilschanski, Michael; Abbasi, Montaser; Blanco, Elias; Lindberg, Iris; Yourshaw, Michael; Zangen, David; Berger, Itai; Shteyer, Eyal; Pappo, Orit; Bar-Oz, Benjamin; Martín, Martin G.; Elpeleg, Orly

    2014-01-01

    Four siblings presented with congenital diarrhea and various endocrinopathies. Exome sequencing and homozygosity mapping identified five regions, comprising 337 protein-coding genes that were shared by three affected siblings. Exome sequencing identified a novel homozygous N309K mutation in the proprotein convertase subtilisin/kexin type 1 (PCSK1) gene, encoding the neuroendocrine convertase 1 precursor (PC1/3) which was recently reported as a cause of Congenital Diarrhea Disorder (CDD). The PCSK1 mutation affected the oxyanion hole transition state-stabilizing amino acid within the active site, which is critical for appropriate proprotein maturation and enzyme activity. Unexpectedly, the N309K mutant protein exhibited normal, though slowed, prodomain removal and was secreted from both HEK293 and Neuro2A cells. However, the secreted enzyme showed no catalytic activity, and was not processed into the 66 kDa form. We conclude that the N309K enzyme is able to cleave its own propeptide but is catalytically inert against in trans substrates, and that this variant accounts for the enteric and systemic endocrinopathies seen in this large consanguineous kindred. PMID:25272002

  13. Identification of proprotein convertase substrates using genome-wide expression correlation analysis

    PubMed Central

    2011-01-01

    Background Subtilisin/kexin-like proprotein convertase (PCSK) enzymes have important regulatory function in a wide variety of biological processes. PCSKs proteolytically process at a target sequence that contains basic amino acids arginine and lysine, which results in functional maturation of the target protein. In vitro assays have showed significant biochemical redundancy between the seven family members, but the phenotypes of PCSK deficient mice and patients carrying an inactive PCSK allele argue for a specific biological function. Modeling the structures of individual PCSK enzymes has offered little insights into the specificity determinants. However, previous studies have shown that there can be a coordinated expression between a PCSK and its target molecule. Here, we have surveyed the putative PCSK target proteins using genome-wide expression correlation analysis and cleavage site prediction algorithms. Results We first performed a gene expression correlation analysis over the whole genome for all PCSK enzymes. PCSKs were found to cluster differently based on the strength of correlations. The screen for putative PCSK target proteins showed a significant enrichment (p-values from 1.2e-4 to < 1.0e-10) of putative targets among the most positively correlating genes for most PCSKs. Interestingly, there was no enrichment in putative targets among the genes that correlated positively with the biologically redundant PCSK7, whereas PCSK5 showed an inverse correlation. PCSKs also showed a highly variable degree of shared target genes that were identified by expression correlation and cleavage site prediction. Multiple alignments were used to evaluate the putative targets to pinpoint the important residues for the substrate recognition. Finally, we validated our approach and identified biochemically PAPPA1 and ADAMTS6 as novel targets for FURIN proteolytic activity. Conclusions Most PCSK enzymes display strong positive expression correlation with predicted target

  14. The proprotein convertase amontillado (amon) is required during Drosophila pupal development.

    PubMed

    Rayburn, Lowell Y M; Rhea, Jeanne; Jocoy, Steven R; Bender, Michael

    2009-09-01

    Peptide hormones governing many developmental processes are generated via endoproteolysis of inactive precursor molecules by a family of subtilisin-like proprotein convertases (SPCs). We previously identified mutations in the Drosophila amontillado (amon) gene, a homolog of the vertebrate neuroendocrine-specific Prohormone Convertase 2 (PC2) gene, and showed that amon is required during embryogenesis, early larval development, and larval molting. Here, we define amon requirements during later developmental stages using a conditional rescue system and find that amon is required during pupal development for head eversion, leg and wing disc extension, and abdominal differentiation. Immuno-localization experiments show that amon protein is expressed in a subset of central nervous system cells but does not co-localize with peptide hormones known to elicit molting behavior, suggesting the involvement of novel regulatory peptides in this process. The amon protein is expressed in neuronal cells that innervate the corpus allatum and corpora cardiaca of the ring gland, an endocrine organ which is the release site for many key hormonal signals. Expression of amon in a subset of these cell types using the GAL4/UAS system in an amon mutant background partially rescues larval molting and growth. Our results show that amon is required for pupal development and identify a subset of neuronal cell types in which amon function is sufficient to rescue developmental progression and growth defects shown by amon mutants. The results are consistent with a model that the amon protein acts to proteolytically process a diverse suite of peptide hormones that coordinate larval and pupal growth and development.

  15. Cellular processing of the nerve growth factor precursor by the mammalian pro-protein convertases.

    PubMed Central

    Seidah, N G; Benjannet, S; Pareek, S; Savaria, D; Hamelin, J; Goulet, B; Laliberte, J; Lazure, C; Chrétien, M; Murphy, R A

    1996-01-01

    In order to define the enzymes responsible for the maturation of the precursor of nerve growth factor (proNGF), its biosynthesis and intracellular processing by the pro-protein convertases furin, PC1, PC2, PACE4, PC5 and the PC5 isoform PC5/6-B were analysed using the vaccinia virus expression system in cells containing a regulated and/or a constitutive secretory pathway. Results demonstrate that in both cell types furin, and to a lesser extent PACE4 and PC5/6-B, are the best candidate proNGF convertases. Furthermore, two processed NGF forms of 16.5 and 13.5 kDa were evident in constitutively secreting cell lines such as LoVo and BSC40 cells, whereas only the 13.5 kDa form was observed in AtT20 cells, which contain secretory granules. Both forms display the same N-terminal sequence as mature NGF, and were also produced following site-directed mutagenesis of the C-terminal Arg-Arg sequence of NGF into Ala-Ala, suggesting that the difference between them is not at the C-terminus. Co-expression of proNGF with furin and either chromogranin B or secretogranin II (but not chromogranin A) in BSC40 cells eliminated the 16.5 kDa form. Data also show that N-glycosylation of the pro-segment of proNGF and trimming of the oligosaccharide chains are necessary for the exit of this precursor from the endoplasmic reticulum and its eventual processing and secretion. Sulphate labelling experiments demonstrated that proNGF is processed into mature NGF following the arrival of the precursor in the trans-Golgi network. This comparative study shows that the three candidate mammalian subtilisin/kexin-like convertases identified process proNGF into NGF and that the nature of the final processed products is dependent on the intracellular environment. PMID:8615794

  16. The proprotein convertase furin is required to maintain viability of alveolar rhabdomyosarcoma cells

    PubMed Central

    Jaaks, Patricia; Meier, Gianmarco; Alijaj, Nagjie; Brack, Eva; Bode, Peter; Koscielniak, Ewa; Wachtel, Marco; Schäfer, Beat W.; Bernasconi, Michele

    2016-01-01

    Rhabdomyosarcoma (RMS) is the most common soft tissue sarcoma in children. Success of current therapies is still limited and outcome is particularly poor for metastatic alveolar rhabdomyosarcoma (aRMS). We previously identified the proprotein convertase furin as potential target for specific drug delivery with RMS-homing peptides. Furin is a protease that converts inactive precursor proteins into bioactive proteins and peptides. In this study, we investigate the biological role of furin in aRMS progression in vitro and in vivo. Furin expression was confirmed in over 86% RMS biopsies in a tissue microarray (n=89). Inducible furin silencing in vitro led to significant impairment of cell viability and proliferation in all investigated aRMS cell lines, but not in MRC5 fibroblasts. Furthermore, the aRMS cell lines Rh3 and Rh4 revealed to be very sensitive to furin silencing, undergoing caspase-dependent cell death. Notably, furin silencing in vivo led to complete remission of established Rh4 tumors and to delayed growth in Rh30 tumors. Taken together, these findings identify furin as an important factor for aRMS progression and survival. Thus, we propose furin as a novel therapeutic target for treatment of aRMS. PMID:27572312

  17. The Proprotein Convertase Furin Contributes to Rhabdomyosarcoma Malignancy by Promoting Vascularization, Migration and Invasion

    PubMed Central

    Jaaks, Patricia; D’Alessandro, Valentina; Grob, Nicole; Büel, Sina; Hajdin, Katarina; Schäfer, Beat W.; Bernasconi, Michele

    2016-01-01

    The proprotein convertase (PC) furin cleaves precursor proteins, an important step in the activation of many cancer-associated proteins. Substrates of furin and furin-like PCs play a role in proliferation, metastasis and invasion. Some of them are involved in the progression of the pediatric soft tissue sarcoma rhabdomyosarcoma (RMS). In this study, we show that PCs, and in particular furin, are expressed in RMS cell lines. To investigate the functional role of furin, we generated RMS cell lines with modulated furin activity. Silencing or stable inhibition of furin delayed tumor growth in Rh30 and RD xenografts in vivo, and was correlated with lower microvessel density. Reduced furin activity also decreased migration and invasion abilities in vitro, and inhibition of furin in RMS cells diminished processing of IGF1R, VEGF-C, PDGF-B and MT1-MMP, leading to lower levels of mature proteins. Furthermore, we found that furin activity is required for proper IGF signaling in RMS cells, as furin silencing resulted in reduced phosphorylation of Akt upon IGF1 stimulation. Taken together, our results suggest that furin plays an important role in the malignant phenotype of RMS cells by activating proteins involved in tumor growth and vascularization, metastasis and invasion. PMID:27548722

  18. Differential P1 arginine and lysine recognition in the prototypical proprotein convertase Kex2

    PubMed Central

    Wheatley, Joshua L.; Holyoak, Todd

    2007-01-01

    The high-resolution crystal structure of kexin (Kex2) in complex with a peptidyl-chloromethylketone inhibitor containing a noncognate lysine at the P1 position provides the structural basis for the differential lysine/arginine selectivity that defines the prohormone (proprotein) convertase (PC) family. By comparison with the previous structures of Kex2 and furin, this structure of the acylated enzyme provides a basis for the observed decrease in the acylation rate with substrates containing a lysine at P1 and the absence of an effect on the deacylation rate without involving mobility of the S1 lid. The structure of the complex shows that a secondary subsite in the S1 pocket is present, and that this site recognizes and binds the P1 lysine in a more shallow fashion than arginine. This results in a displacement of the bound peptide away from the S385 nucleophile relative to substrates containing a P1 arginine. It is concluded that this alternate binding site and resultant displacement of the scissile bond in the active site results in the observed decrease in the acylation rate. Studies of the inactivation kinetics of Kex2 by two peptidyl chloromethylketone inhibitors demonstrates that the selectivity between lysine and arginine at the P1 position arises at the acylation step, consistent with what was observed with peptidyl substrates [Rockwell NC, Fuller RS (2001) J Biol Chem 276:38394–38399]. PMID:17426142

  19. Proprotein Convertase Subtilisin Kexin Type 9 Inhibition for Autosomal Recessive Hypercholesterolemia-Brief Report.

    PubMed

    Thedrez, Aurélie; Sjouke, Barbara; Passard, Maxime; Prampart-Fauvet, Simon; Guédon, Alexis; Croyal, Mikael; Dallinga-Thie, Geesje; Peter, Jorge; Blom, Dirk; Ciccarese, Milco; Cefalù, Angelo B; Pisciotta, Livia; Santos, Raul D; Averna, Maurizio; Raal, Frederick; Pintus, Paolo; Cossu, Maria; Hovingh, Kees; Lambert, Gilles

    2016-08-01

    Proprotein convertase subtilisin kexin type 9 (PCSK9) inhibitors lower low-density lipoprotein (LDL) cholesterol in the vast majority of patients with autosomal dominant familial hypercholesterolemia. Will PCSK9 inhibition with monoclonal antibodies, in particular alirocumab, be of therapeutic value for patients with autosomal recessive hypercholesterolemia (ARH)? Primary lymphocytes were obtained from 28 genetically characterized ARH patients and 11 controls. ARH lymphocytes treated with mevastatin were incubated with increasing doses of recombinant PCSK9 with or without saturating concentrations of alirocumab. Cell surface LDL receptor expression measured by flow cytometry and confocal microscopy was higher in ARH than in control lymphocytes. PCSK9 significantly reduced LDL receptor expression in ARH lymphocytes albeit to a lower extent than in control lymphocytes (25% versus 76%, respectively), an effect reversed by alirocumab. Fluorescent LDL cellular uptake, also measured by flow cytometry, was reduced in ARH lymphocytes compared with control lymphocytes. PCSK9 significantly lowered LDL cellular uptake in ARH lymphocytes, on average by 18%, compared with a 46% reduction observed in control lymphocytes, an effect also reversed by alirocumab. Overall, the effects of recombinant PCSK9, and hence of alirocumab, on LDL receptor expression and function were significantly less pronounced in ARH than in control cells. PCSK9 inhibition with alirocumab on top of statin treatment has the potential to lower LDL cholesterol in some autosomal recessive hypercholesterolemia patients. © 2016 American Heart Association, Inc.

  20. Proprotein convertase cleavage liberates a fibrillogenic fragment of a resident glycoprotein to initiate melanosome biogenesis.

    PubMed

    Berson, Joanne F; Theos, Alexander C; Harper, Dawn C; Tenza, Danielle; Raposo, Graça; Marks, Michael S

    2003-05-12

    Lysosome-related organelles are cell type-specific intracellular compartments with distinct morphologies and functions. The molecular mechanisms governing the formation of their unique structural features are not known. Melanosomes and their precursors are lysosome-related organelles that are characterized morphologically by intralumenal fibrous striations upon which melanins are polymerized. The integral membrane protein Pmel17 is a component of the fibrils and can nucleate their formation in the absence of other pigment cell-specific proteins. Here, we show that formation of intralumenal fibrils requires cleavage of Pmel17 by a furin-like proprotein convertase (PC). As in the generation of amyloid, proper cleavage of Pmel17 liberates a lumenal domain fragment that becomes incorporated into the fibrils; longer Pmel17 fragments generated in the absence of PC activity are unable to form organized fibrils. Our results demonstrate that PC-dependent cleavage regulates melanosome biogenesis by controlling the fibrillogenic activity of a resident protein. Like the pathologic process of amyloidogenesis, the formation of other tissue-specific organelle structures may be similarly dependent on proteolytic activation of physiological fibrillogenic substrates.

  1. Proprotein convertase cleavage liberates a fibrillogenic fragment of a resident glycoprotein to initiate melanosome biogenesis

    PubMed Central

    Berson, Joanne F.; Theos, Alexander C.; Harper, Dawn C.; Tenza, Danielle; Raposo, Graça; Marks, Michael S.

    2003-01-01

    Lysosome-related organelles are cell type–specific intracellular compartments with distinct morphologies and functions. The molecular mechanisms governing the formation of their unique structural features are not known. Melanosomes and their precursors are lysosome-related organelles that are characterized morphologically by intralumenal fibrous striations upon which melanins are polymerized. The integral membrane protein Pmel17 is a component of the fibrils and can nucleate their formation in the absence of other pigment cell–specific proteins. Here, we show that formation of intralumenal fibrils requires cleavage of Pmel17 by a furin-like proprotein convertase (PC). As in the generation of amyloid, proper cleavage of Pmel17 liberates a lumenal domain fragment that becomes incorporated into the fibrils; longer Pmel17 fragments generated in the absence of PC activity are unable to form organized fibrils. Our results demonstrate that PC-dependent cleavage regulates melanosome biogenesis by controlling the fibrillogenic activity of a resident protein. Like the pathologic process of amyloidogenesis, the formation of other tissue-specific organelle structures may be similarly dependent on proteolytic activation of physiological fibrillogenic substrates. PMID:12732614

  2. Subtilisin-like proprotein convertase PACE4 is required for skeletal muscle differentiation.

    PubMed

    Yuasa, Keizo; Masuda, Tetsuya; Yoshikawa, Chihiro; Nagahama, Masami; Matsuda, Yoshiko; Tsuji, Akihiko

    2009-09-01

    Most growth factors stimulate myoblast proliferation and prevent differentiation, whereas insulin-like growth factors (IGFs) promote myoblast differentiation through the phosphatidylinositol 3-kinase (PI3K) pathway. Subtilisin-like proprotein convertases (SPCs) are involved in cell growth and differentiation via activation of pro-growth factors. However, the role of SPCs in myogenesis remains poorly understood. Here we show that PACE4, a member of the SPC family, plays a critical role in myogenic differentiation of C2C12 cells. PACE4 mRNA levels increased markedly during myogenesis, whereas the expression of other member of SPC family, furin and PC6, remained unchanged. The expression pattern of pro-IGF-II, which is processed extracellularly by SPCs, was similar to that of PACE4. The expression of shRNA targeting PACE4, but not furin, suppressed the expression of the muscle-specific myosin light chain (MLC). Interestingly, reduced expression of MLC was restored following treatment with recombinant mature IGF-II. Finally, we demonstrated that the PI3K inhibitor LY294002 blocked the induction of PACE4 mRNA, a result not observed when another myogenic differentiation inhibitor, SB203580 (p38 MAP kinase inhibitor), was employed, indicating the presence of a positive feedback loop regulating PACE4 expression. These results suggest that PACE4 plays an important role in myogenic differentiation through its association with the IGF-II pathway.

  3. Imaging proprotein convertase activities and their regulation in the implanting mouse blastocyst.

    PubMed

    Mesnard, Daniel; Constam, Daniel B

    2010-10-04

    Axis formation and allocation of pluripotent progenitor cells to the germ layers are governed by the TGF-β-related Nodal precursor and its secreted proprotein convertases (PCs) Furin and Pace4. However, when and where Furin and Pace4 first become active have not been determined. To study the distribution of PCs, we developed a novel cell surface-targeted fluorescent biosensor (cell surface-linked indicator of proteolysis [CLIP]). Live imaging of CLIP in wild-type and Furin- and Pace4-deficient embryonic stem cells and embryos revealed that Furin and Pace4 are already active at the blastocyst stage in the inner cell mass and can cleave membrane-bound substrate both cell autonomously and nonautonomously. CLIP was also cleaved in the epiblast of implanted embryos, in part by a novel activity in the uterus that is independent of zygotic Furin and Pace4, suggesting a role for maternal PCs during embryonic development. The unprecedented sensitivity and spatial resolution of CLIP opens exciting new possibilities to elucidate PC functions in vivo.

  4. Proprotein convertase inhibition results in decreased skin cell proliferation, tumorigenesis, and metastasis.

    PubMed

    Bassi, Daniel E; Zhang, Jirong; Cenna, Jonathan; Litwin, Samuel; Cukierman, Edna; Klein-Szanto, Andres J P

    2010-07-01

    PACE4 is a proprotein convertase (PC) responsible for cleaving and activating proteins that contribute to enhance tumor progression. PACE4 overexpression significantly increased the susceptibility to carcinogenesis, leading to enhanced tumor cell proliferation and premature degradation of the basement membrane. In the present study, we sought to evaluate a novel approach to retard skin tumor progression based on the inhibition of PACE4. We used decanoyl-RVKR-chloromethylketone (CMK), a small-molecule PC inhibitor, for in vitro and in vivo experiments. We found that CMK-dependent blockage of PACE4 activity in skin squamous cell carcinoma cell lines resulted in impaired insulin-like growth factor 1 receptor maturation, diminished its intrinsic tyrosine kinase activity, and decreased tumor cell proliferation. Two-stage skin chemical carcinogenesis experiments, together with topical applications of CMK, demonstrated that this PC inhibitor markedly reduced tumor incidence, tumor multiplicity, and metastasis, pointing to a significant delay in tumor progression in wild-type and PACE4 transgenic mice. These results identify PACE4, together with other PCs, as suitable targets to slow down or block tumor progression, suggesting that PC inhibition is a potential approach for therapy for solid tumors.

  5. [Proprotein convertases - family of serine proteases with a broad spectrum of physiological functions].

    PubMed

    Małuch, Izabela; Walewska, Aleksandra; Sikorska, Emilia; Prahl, Adam

    2016-01-01

    A large group of secretory proteins involved in proper functioning of living organisms, is synthesized as inactive precursor molecules. Their biologically active forms are obtained as a result of numerous post-translational modifications. Some of these processes occur irreversibly, permanently changing the initial compound structure. An example of such modifications is catalytic treatment of proteins performed by proteolytic enzymes. Among five separate classes of these enzymes, the most numerous are serine proteases. Mammalian proprotein convertases (PCs), which include: furin, PC1/3, PC2, PACE4, PC4, PC5/6, PC7, PCSK9, SKI-1, represent serine endoproteases family. PCs play a key role in the activation of a number of precursor proteins causing formation of biologically active forms of enzymes, hormones, signaling molecules, transcription and growth factors. This article summarizes current state of knowledge on biosynthesis, structure and substrate specificity of PCs, identifies the relationship between location and intracellular activity of these enzymes, and their physiological functions in mammals.

  6. Regulation of Bone Morphogenetic Protein Activity by Pro Domains and Proprotein Convertases

    PubMed Central

    Constam, Daniel B.; Robertson, Elizabeth J.

    1999-01-01

    Bone morphogenetic proteins (BMPs) are derived from inactive precursor proteins by endoproteolytic cleavage. Here we show that processing of Nodal and Myc-tagged BMP4 is significantly enhanced by SPC1/Furin or SPC4/PACE4, providing direct evidence that regulation of BMP signaling is likely to be controlled by subtilisin-like proprotein convertase (SPC) activities. Nodal processing is dramatically enhanced if two residues adjacent to the precursor cleavage site are substituted with amino acids found at the equivalent positions of Activin, demonstrating that structural constraints at the precursor cleavage site limit the processing efficiency. However, in transfection assays, mature Nodal is undetectable either in culture supernatants or in cell lysates, despite efficient cleavage of the precursor protein, suggesting that mature Nodal is highly unstable. Domain swap experiments support this conclusion since mature BMP4 or Dorsalin are also destabilized when expressed in conjunction with the Nodal pro domain. By contrast, mature Nodal is stabilized by the Dorsalin pro domain, which mediates the formation of stable complexes. Collectively, these data show that the half-life of mature BMPs is greatly influenced by the identity of their pro regions. PMID:9885250

  7. Fasting reduces plasma proprotein convertase, subtilisin/kexin type 9 and cholesterol biosynthesis in humans

    PubMed Central

    Browning, Jeffrey D.; Horton, Jay D.

    2010-01-01

    Proprotein convertase, subtilisin/kexin type 9 (PCSK9), a key regulator of plasma LDL-cholesterol (LDL-c) and cardiovascular risk, is produced in liver and secreted into plasma where it binds hepatic LDL receptors (LDLR), leading to their degradation. PCSK9 is transcriptionally activated by sterol response element-binding protein (SREBP)-2, a transcription factor that also activates all genes for cholesterol synthesis as well as the LDLR. Here we investigated the relationship between plasma PCSK9 levels and the lathosterol-to-cholesterol ratio, a marker of cholesterol biosynthesis, in 18 healthy subjects during a 48 h fast. In all individuals, plasma PCSK9 levels declined steadily during the fasting period, reaching a nadir at 36 h that was ∼58% lower than levels measured in the fed state (P < 0.001). Similarly, the lathosterol-to-cholesterol ratio declined in parallel with plasma PCSK9 concentrations during the fast, reaching a nadir at 36 h that was ∼28% lower than that measured in the fed state (P = 0.024). In summary, fasting has a marked effect on plasma PCSK9 concentrations, which is mirrored by measures of cholesterol synthesis in humans. Inasmuch as cholesterol synthesis and PCSK9 are both regulated by SREBP-2, these results suggest that plasma PCSK9 levels may serve as a surrogate marker of hepatic SREBP-2 activity in humans. PMID:20716520

  8. Plasminogen activator inhibitor 1 is an intracellular inhibitor of furin proprotein convertase.

    PubMed

    Bernot, Denis; Stalin, Jimmy; Stocker, Pierre; Bonardo, Bernadette; Scroyen, Ilse; Alessi, Marie-Christine; Peiretti, Franck

    2011-04-15

    Proprotein convertases (PCs) are a family of serine proteases that are involved in the post-translational processing and activation of a wide range of regulatory proteins. The upstream role of PCs in the control of many physiological and pathological processes generates a growing interest in understanding their regulation. Here, we demonstrate that the serine protease inhibitor plasminogen activator inhibitor 1 (PAI-1) forms an SDS-stable complex with the PC furin, which leads to the inhibition of the intra-Golgi activity of furin. It is known that elevated PAI-1 plasma levels are correlated with the occurrence of the metabolic syndrome and type 2 diabetes, and we show that PAI-1 reduces the furin-dependent maturation and activity of the insulin receptor and ADAM17: two proteins involved in the onset of these metabolic disorders. In addition to demonstrating that PAI-1 is an intracellular inhibitor of furin, this study also provides arguments in favor of an active role for PAI-1 in the development of metabolic disorders.

  9. The Proprotein Convertase Subtilisin/Kexin FurinA Regulates Zebrafish Host Response against Mycobacterium marinum

    PubMed Central

    Ojanen, Markus J. T.; Turpeinen, Hannu; Cordova, Zuzet M.; Hammarén, Milka M.; Harjula, Sanna-Kaisa E.; Parikka, Mataleena; Rämet, Mika

    2015-01-01

    Tuberculosis is a chronic bacterial disease with a complex pathogenesis. An effective immunity against Mycobacterium tuberculosis requires both the innate and adaptive immune responses, including proper T helper (Th) type 1 cell function. FURIN is a proprotein convertase subtilisin/kexin (PCSK) enzyme, which is highly expressed in Th1 type cells. FURIN expression in T cells is essential for maintaining peripheral immune tolerance, but its role in the innate immunity and infections has remained elusive. Here, we utilized Mycobacterium marinum infection models in zebrafish (Danio rerio) to investigate how furin regulates host responses against mycobacteria. In steady-state furinAtd204e/+ fish reduced furinA mRNA levels associated with low granulocyte counts and elevated Th cell transcription factor expressions. Silencing furin genes reduced the survival of M. marinum-infected zebrafish embryos. A mycobacterial infection upregulated furinA in adult zebrafish, and infected furinAtd204e/+ mutants exhibited a proinflammatory phenotype characterized by elevated tumor necrosis factor a (tnfa), lymphotoxin alpha (lta) and interleukin 17a/f3 (il17a/f3) expression levels. The enhanced innate immune response in the furinAtd204e/+ mutants correlated with a significantly decreased bacterial burden in a chronic M. marinum infection model. Our data show that upregulated furinA expression can serve as a marker for mycobacterial disease, since it inhibits early host responses and consequently promotes bacterial growth in a chronic infection. PMID:25624351

  10. The regulated cell surface zymogen activation of the proprotein convertase PC5A directs the processing of its secretory substrates.

    PubMed

    Mayer, Gaétan; Hamelin, Josée; Asselin, Marie-Claude; Pasquato, Antonella; Marcinkiewicz, Edwidge; Tang, Meiyi; Tabibzadeh, Siamak; Seidah, Nabil G

    2008-01-25

    The proprotein convertases are synthesized as zymogens that acquire activity upon autocatalytic removal of their NH(2)-terminal prosegment. Based on the convertase furin, to fold properly and gain activity, the convertases PC5A, PACE4, and PC7 are presumed to undergo two sequential prosegment cleavages in the endoplasmic reticulum and then in the trans-Golgi network. However, biochemical and immunocytochemical experiments revealed that mouse PC5A is complexed to its prosegment at the plasma membrane. This labeling is lost upon treatment with heparin and is increased by overexpressing members of the syndecan family and CD44, suggesting attachment of secreted PC5A-prosegment complex to heparan sulfate proteoglycans. Following stimulation of Y1 cells with adrenocorticotropic hormone or 8-bromo-cyclic AMP, the cell surface labeling of the prosegment of PC5A is greatly diminished, whereas the signal for mature PC5A is increased. Moreover, after stimulation, the protease activity of PC5A is enhanced, as evidenced by the cleavage of the PC5A substrates Lefty, ADAMTS-4, endothelial lipase, and PCSK9. Our data suggest a novel mechanism for PC5A activation and substrate cleavage at the cell surface, through a regulated removal of its prosegment. A similar mechanism may also apply to the convertase PACE4, thereby extending our knowledge of the molecular details of the zymogen activation and functions of these heparan sulfate proteoglycan-bound convertases.

  11. Mechanism of Fine-tuning pH Sensors in Proprotein Convertases: IDENTIFICATION OF A pH-SENSING HISTIDINE PAIR IN THE PROPEPTIDE OF PROPROTEIN CONVERTASE 1/3.

    PubMed

    Williamson, Danielle M; Elferich, Johannes; Shinde, Ujwal

    2015-09-18

    The propeptides of proprotein convertases (PCs) regulate activation of cognate protease domains by sensing pH of their organellar compartments as they transit the secretory pathway. Earlier experimental work identified a conserved histidine-encoded pH sensor within the propeptide of the canonical PC, furin. To date, whether protonation of this conserved histidine is solely responsible for PC activation has remained unclear because of the observation that various PC paralogues are activated at different organellar pH values. To ascertain additional determinants of PC activation, we analyzed PC1/3, a paralogue of furin that is activated at a pH of ∼5.4. Using biophysical, biochemical, and cell-based methods, we mimicked the protonation status of various histidines within the propeptide of PC1/3 and examined how such alterations can modulate pH-dependent protease activation. Our results indicate that whereas the conserved histidine plays a crucial role in pH sensing and activation of this protease an additional histidine acts as a "gatekeeper" that fine-tunes the sensitivity of the PC1/3 propeptide to facilitate the release inhibition at higher proton concentrations when compared with furin. Coupled with earlier analyses that highlighted the enrichment of the amino acid histidine within propeptides of secreted eukaryotic proteases, our work elucidates how secreted proteases have evolved to exploit the pH of the secretory pathway by altering the spatial juxtaposition of titratable groups to regulate their activity in a spatiotemporal fashion.

  12. T-cell-expressed proprotein convertase FURIN inhibits DMBA/TPA-induced skin cancer development

    PubMed Central

    Vähätupa, Maria; Aittomäki, Saara; Martinez Cordova, Zuzet; May, Ulrike; Prince, Stuart; Uusitalo-Järvinen, Hannele; Pesu, Marko

    2016-01-01

    ABSTRACT Proprotein convertases (PCSK) have a critical role in the body homeostasis as enzymes responsible for processing precursor proteins into their mature forms. FURIN, the first characterized member of the mammalian PCSK family, is overexpressed in multiple malignancies and the inhibition of its activity has been considered potential cancer treatment. FURIN has also an important function in the adaptive immunity, since its deficiency in T cells causes an impaired peripheral immune tolerance and accelerates immune responses. We addressed whether deleting FURIN from the immune cells would strengthen anticancer responses by subjecting mouse strains lacking FURIN from either T cells or macrophages and granulocytes to the DMBA/TPA two-stage skin carcinogenesis protocol. Unexpectedly, deficiency of FURIN in T cells resulted in enhanced and accelerated development of tumors, whereas FURIN deletion in macrophages and granulocytes had no effect. The epidermises of T-cell-specific FURIN deficient mice were significantly thicker with more proliferating Ki67+ cells. In contrast, there were no differences in the numbers of the T cells. The flow cytometric analyses of T-cell populations in skin draining lymph nodes showed that FURIN T-cell KO mice have an inherent upregulation of early activation marker CD69 as well as more CD4+CD25+Foxp3+ positive T regulatory cells. In the early phase of tumor promotion, T cells from the T-cell-specific FURIN knockout animals produced more interferon gamma, whereas at later stage the production of Th2- and Th17-type cytokines was more prominent than in wild-type controls. In conclusion, while PCSK inhibitors are promising therapeutics in cancer treatment, our results show that inhibiting FURIN specifically in T cells may promote squamous skin cancer development. PMID:28123881

  13. Proprotein convertases play an important role in regulating PKGI endoproteolytic cleavage and nuclear transport

    PubMed Central

    Kato, Shin; Zhang, Ruiguang

    2013-01-01

    Nitric oxide and cGMP modulate vascular smooth muscle cell (SMC) phenotype by regulating cell differentiation and proliferation. Recent studies suggest that cGMP-dependent protein kinase I (PKGI) cleavage and the nuclear translocation of a constitutively active kinase fragment, PKGIγ, are required for nuclear cGMP signaling in SMC. However, the mechanisms that control PKGI proteolysis are unknown. Inspection of the amino acid sequence of a PKGI cleavage site that yields PKGIγ and a protease database revealed a putative minimum consensus sequence for proprotein convertases (PCs). Therefore we investigated the role of PCs in regulating PKGI proteolysis. We observed that overexpression of PCs, furin and PC5, but not PC7, which are all expressed in SMC, increase PKGI cleavage in a dose-dependent manner in human embryonic kidney (HEK) 293 cells. Moreover, furin-induced proteolysis of mutant PKGI, in which alanines were substituted into the putative PC consensus sequence, was decreased in these cells. In addition, overexpression of furin increased PKGI proteolysis in LoVo cells, which is an adenocarcinoma cell line expressing defective furin without PC activity. Also, expression of α1-PDX, an engineered serpin-like PC inhibitor, reduced PC activity and decreased PKGI proteolysis in HEK293 cells. Last, treatment of low-passage rat aortic SMC with membrane-permeable PC inhibitor peptides decreased cGMP-stimulated nuclear PKGIγ translocation. These data indicate for the first time that PCs have a role in regulating PKGI proteolysis and the nuclear localization of its active cleavage product, which are important for cGMP-mediated SMC phenotype. PMID:23686857

  14. Loss of Plasma Proprotein Convertase Subtilisin/Kexin 9 (PCSK9) After Lipoprotein Apheresis

    PubMed Central

    Tavori, Hagai; Giunzioni, Ilaria; Linton, MacRae F.; Fazio, Sergio

    2014-01-01

    Rationale Lipoprotein apheresis (LA) reduces low-density lipoprotein (LDL) levels in patients with severe familial hypercholesterolemia (FH). We have recently reported that >30% of plasma proprotein convertase subtilisin/kexin 9 (PCSK9) is bound to LDL, thus we predicted that LA would also reduce plasma PCSK9 levels by removing LDL. Objective Pre- and post-apheresis plasma from 6 patients with familial hypercholesterolemia on 3 consecutive treatment cycles was used to determine changes in PCSK9 levels. Methods and Results LA drastically reduced plasma LDL (by 77±4%). Concomitantly, PCSK9 levels fell by 52±5%, strongly correlating with the LDL drop (P=0.0322; r2=0.26), but not with decreases in triglyceride (49±13%) or high-density lipoprotein levels (18±2%). Levels of albumin, creatinine, and CK-MB did not show significant changes after LA. Similar to LDL, PCSK9 levels returned to pretreatment values between cycles (2-week intervals). Fractionation of pre- and post-apheresis plasma showed that 81±11% of LDL-bound PCSK9 and 48±14% of apolipoprotein B–free PCSK9 were removed. Separation of whole plasma, purified LDL, or the apolipoprotein B–free fraction through a scaled-down, experimental dextran sulfate cellulose beads column produced similar results. Conclusions Our results show, for the first time, that modulation of LDL levels by LA directly affects plasma PCSK9 levels, and suggest that PCSK9 reduction is an additional benefit of LA. Because the loss of PCSK9 could contribute to the LDL-lowering effect of LA, then (1) anti-PCSK9 therapies may reduce frequency of LA in patients currently approved for therapy, and (2) LA and anti-PCSK9 therapies may be used synergistically to reduce treatment burden. PMID:24122718

  15. Enhanced UV-induced skin carcinogenesis in transgenic mice overexpressing proprotein convertases.

    PubMed

    Fu, Jian; Bassi, Daniel E; Zhang, Jirong; Li, Tianyu; Cai, Kathy Q; Testa, Courtney Lyons; Nicolas, Emmanuelle; Klein-Szanto, Andres J

    2013-02-01

    The proprotein convertases (PCs) furin and PACE4 process numerous substrates involved in tumor growth, invasion, and metastasis. We have previously shown that PCs increase the susceptibility to chemical skin carcinogenesis. Because of the human relevancy of UV radiation in the etiopathogenesis of human skin cancer, we investigated whether or not transgenic mice overexpressing either furin alone or both furin and PACE4 show increased susceptibility to UV carcinogenesis. After backcrossing our previously described furin and PACE4 transgenic lines, targeted to the epidermis, into a SKH-1 background, we exposed both single and double transgenic mice to UV radiation for 34 weeks. The results showed an increase in squamous cell carcinoma (SCC) multiplicity of approximately 70% in the single furin transgenic mouse line SF47 (P < .002) and a 30% increase in the other single transgenic line SF49 when compared to wild-type (WT) SKH-1 mice. Interestingly, there was also an increase in the percentage of high histologic grade SCCs in the transgenic lines compared to the WT mice, i.e., WT = 9%, SF47 = 15%, and SF49 = 26% (P < .02). Targeting both furin and PACE4 to the epidermis in double transgenic mice did not have an additive effect on tumor incidence/multiplicity but did enhance the tumor histopathologic grade, i.e., a significant increase in higher grade SCCs was seen in the bigenic mouse line SPF47 (P < .02). Thus, we observed an increased susceptibility to UV in single furin transgenic mice that was not substantially enhanced in the double furin/PACE4 transgenic mice.

  16. Salivary gland development: its mediation by a subtilisin-like proprotein convertase, PACE4.

    PubMed

    Akamatsu, Tetsuya; Azlina, Ahmad; Javkhlan, Purevjav; Hasegawa, Takahiro; Yao, Chenjuan; Hosoi, Kazuo

    2009-01-01

    The submandibular gland (SMG) develops under the epithelial-mesenchymal interaction. Its process is regulated by various growth/differentiation factors, which are synthesized as inactive precursors and activated via the limited proteolysis at their multi basic amino acid site(s) such as Arg-X-Lys/Arg-Arg. Although many of these processing steps are elucidated to be catalyzed by subtilisin-like proprotein convertases (SPCs), little is known about the role of SPCs in the SMG development. Here, we focused upon the physiological role of PACE4 (SPC4), a member of SPC family, in the SMG development. In the organ culture system of rat embryonic SMG (E15), Dec-RVKR-CMK, a potent inhibitor for SPCs, inhibited the salivary branching and the expression of an exocrine gland type water channel, AQP5. However, other peptidyl-CMKs and inhibitors for trypsin-like serine proteases including leupeptin did not affect the salivary branching and AQP5 expression. Dec-RVKR-CMK also suppressed the expression of PACE4, but not furin, another member of the family. The specific antibody for the catalytic domain of PACE4 suppressed the salivary branching and AQP5 expression similarly. These inhibitory effects of Dec-RVKR-CMK were partially rescued by the addition of recombinant BMP2 whose precursor is a candidate for the physiological substrates of PACE4. Further, the transcriptional silencing of PACE4 by its specific siRNAs caused the suppression of both the salivary branching and AQP5 expression in the present organ culture system. These observations strongly support the idea that PACE4 mediates the SMG development.

  17. Novel domain interaction regulates secretion of proprotein convertase subtilisin/kexin type 9 (PCSK9) protein.

    PubMed

    Du, Fen; Hui, Yvonne; Zhang, Michelle; Linton, MacRae F; Fazio, Sergio; Fan, Daping

    2011-12-16

    PCSK9 (proprotein convertase subtilisin/kexin type 9) has emerged as a novel therapeutic target for hypercholesterolemia due to its LDL receptor (LDLR)-reducing activity. Although its structure has been solved, the lack of a detailed understanding of the structure-function relation hinders efforts to develop small molecule inhibitors. In this study, we used mutagenesis and transfection approaches to investigate the roles of the prodomain (PD) and the C-terminal domain (CD) and its modules (CM1-3) in the secretion and function of PCSK9. Deletion of PD residues 31-40, 41-50, or 51-60 did not affect the self-cleavage, secretion, or LDLR-degrading activity of PCSK9, whereas deletion of residues 61-70 abolished all of these functions. Deletion of the entire CD protein did not impair PCSK9 self-cleavage or secretion but completely abolished LDLR-degrading activity. Deletion of any one or two of the CD modules did not affect self-cleavage but influenced secretion and LDLR-reducing activity. Furthermore, in cotransfection experiments, a secretion-defective PD deletion mutant (ΔPD) was efficiently secreted in the presence of CD deletion mutants. This was due to the transfer of PD from the cotransfected CD mutants to the ΔPD mutant. Finally, we found that a discrete CD protein fragment competed with full-length PCSK9 for binding to LDLR in vitro and attenuated PCSK9-mediated hypercholesterolemia in mice. These results show a previously unrecognized domain interaction as a critical determinant in PCSK9 secretion and function. This knowledge should fuel efforts to develop novel approaches to PCSK9 inhibition.

  18. The Proprotein Convertase KPC-1/Furin Controls Branching and Self-avoidance of Sensory Dendrites in Caenorhabditis elegans

    PubMed Central

    Bülow, Hannes E.

    2014-01-01

    Animals sample their environment through sensory neurons with often elaborately branched endings named dendritic arbors. In a genetic screen for genes involved in the development of the highly arborized somatosensory PVD neuron in C. elegans, we have identified mutations in kpc-1, which encodes the homolog of the proprotein convertase furin. We show that kpc-1/furin is necessary to promote the formation of higher order dendritic branches in PVD and to ensure self-avoidance of sister branches, but is likely not required during maintenance of dendritic arbors. A reporter for kpc-1/furin is expressed in neurons (including PVD) and kpc-1/furin can function cell-autonomously in PVD neurons to control patterning of dendritic arbors. Moreover, we show that kpc-1/furin also regulates the development of other neurons in all major neuronal classes in C. elegans, including aspects of branching and extension of neurites as well as cell positioning. Our data suggest that these developmental functions require proteolytic activity of KPC-1/furin. Recently, the skin-derived MNR-1/menorin and the neural cell adhesion molecule SAX-7/L1CAM have been shown to act as a tripartite complex with the leucine rich transmembrane receptor DMA-1 on PVD mechanosensory to orchestrate the patterning of dendritic branches. Genetic analyses show that kpc-1/furin functions in a pathway with MNR-1/menorin, SAX-7/L1CAM and DMA-1 to control dendritic branch formation and extension of PVD neurons. We propose that KPC-1/furin acts in concert with the ‘menorin’ pathway to control branching and growth of somatosensory dendrites in PVD. PMID:25232734

  19. The proprotein convertase KPC-1/furin controls branching and self-avoidance of sensory dendrites in Caenorhabditis elegans.

    PubMed

    Salzberg, Yehuda; Ramirez-Suarez, Nelson J; Bülow, Hannes E

    2014-09-01

    Animals sample their environment through sensory neurons with often elaborately branched endings named dendritic arbors. In a genetic screen for genes involved in the development of the highly arborized somatosensory PVD neuron in C. elegans, we have identified mutations in kpc-1, which encodes the homolog of the proprotein convertase furin. We show that kpc-1/furin is necessary to promote the formation of higher order dendritic branches in PVD and to ensure self-avoidance of sister branches, but is likely not required during maintenance of dendritic arbors. A reporter for kpc-1/furin is expressed in neurons (including PVD) and kpc-1/furin can function cell-autonomously in PVD neurons to control patterning of dendritic arbors. Moreover, we show that kpc-1/furin also regulates the development of other neurons in all major neuronal classes in C. elegans, including aspects of branching and extension of neurites as well as cell positioning. Our data suggest that these developmental functions require proteolytic activity of KPC-1/furin. Recently, the skin-derived MNR-1/menorin and the neural cell adhesion molecule SAX-7/L1CAM have been shown to act as a tripartite complex with the leucine rich transmembrane receptor DMA-1 on PVD mechanosensory to orchestrate the patterning of dendritic branches. Genetic analyses show that kpc-1/furin functions in a pathway with MNR-1/menorin, SAX-7/L1CAM and DMA-1 to control dendritic branch formation and extension of PVD neurons. We propose that KPC-1/furin acts in concert with the 'menorin' pathway to control branching and growth of somatosensory dendrites in PVD.

  20. VACTERL/caudal regression/Currarino syndrome-like malformations in mice with mutation in the proprotein convertase Pcsk5

    PubMed Central

    Szumska, Dorota; Pieles, Guido; Essalmani, Rachid; Bilski, Michal; Mesnard, Daniel; Kaur, Kulvinder; Franklyn, Angela; El Omari, Kamel; Jefferis, Joanna; Bentham, Jamie; Taylor, Jennifer M.; Schneider, Jurgen E.; Arnold, Sebastian J.; Johnson, Paul; Tymowska-Lalanne, Zuzanna; Stammers, Dave; Clarke, Kieran; Neubauer, Stefan; Morris, Andrew; Brown, Steve D.; Shaw-Smith, Charles; Cama, Armando; Capra, Valeria; Ragoussis, Jiannis; Constam, Daniel; Seidah, Nabil G.; Prat, Annik; Bhattacharya, Shoumo

    2008-01-01

    We have identified an ethylnitrosourea (ENU)-induced recessive mouse mutation (Vcc) with a pleiotropic phenotype that includes cardiac, tracheoesophageal, anorectal, anteroposterior patterning defects, exomphalos, hindlimb hypoplasia, a presacral mass, renal and palatal agenesis, and pulmonary hypoplasia. It results from a C470R mutation in the proprotein convertase PCSK5 (PC5/6). Compound mutants (Pcsk5Vcc/null) completely recapitulate the Pcsk5Vcc/Vcc phenotype, as does an epiblast-specific conditional deletion of Pcsk5. The C470R mutation ablates a disulfide bond in the P domain, and blocks export from the endoplasmic reticulum and proprotein convertase activity. We show that GDF11 is cleaved and activated by PCSK5A, but not by PCSK5A-C470R, and that Gdf11-deficient embryos, in addition to having anteroposterior patterning defects and renal and palatal agenesis, also have a presacral mass, anorectal malformation, and exomphalos. Pcsk5 mutation results in abnormal expression of several paralogous Hox genes (Hoxa, Hoxc, and Hoxd), and of Mnx1 (Hlxb9). These include known Gdf11 targets, and are necessary for caudal embryo development. We identified nonsynonymous mutations in PCSK5 in patients with VACTERL (vertebral, anorectal, cardiac, tracheoesophageal, renal, limb malformation OMIM 192350) and caudal regression syndrome, the phenotypic features of which resemble the mouse mutation. We propose that Pcsk5, at least in part via GDF11, coordinately regulates caudal Hox paralogs, to control anteroposterior patterning, nephrogenesis, skeletal, and anorectal development. PMID:18519639

  1. The Role of Proprotein Convertase Subtilisin/Kexin Type 9 in Nephrotic Syndrome-Associated Hypercholesterolemia.

    PubMed

    Haas, Mary E; Levenson, Amy E; Sun, Xiaowei; Liao, Wan-Hui; Rutkowski, Joseph M; de Ferranti, Sarah D; Schumacher, Valerie A; Scherer, Philipp E; Salant, David J; Biddinger, Sudha B

    2016-07-05

    In nephrotic syndrome, damage to the podocytes of the kidney produces severe hypercholesterolemia for which novel treatments are urgently needed. PCSK9 (proprotein convertase subtilisin/kexin type 9) has emerged as an important regulator of plasma cholesterol levels and therapeutic target. Here, we tested the role of PCSK9 in mediating the hypercholesterolemia of nephrotic syndrome. PCSK9 and plasma lipids were studied in nephrotic syndrome patients before and after remission of disease, mice with genetic ablation of the podocyte (Podocyte Apoptosis Through Targeted Activation of Caspase-8, Pod-ATTAC mice) and mice treated with nephrotoxic serum (NTS), which triggers immune-mediated podocyte damage. In addition, mice with hepatic deletion of Pcsk9 were treated with NTS to determine the contribution of PCSK9 to the dyslipidemia of nephrotic syndrome. Patients with nephrotic syndrome showed a decrease in plasma cholesterol and plasma PCSK9 on remission of their disease (P<0.05, n=47-50). Conversely, Pod-ATTAC mice and NTS-treated mice showed hypercholesterolemia and a 7- to 24-fold induction in plasma PCSK9. The induction of plasma PCSK9 appeared to be attributable to increased secretion of PCSK9 from the hepatocyte coupled with decreased clearance. Interestingly, knockout of Pcsk9ameliorated the effects of NTS on plasma lipids. Thus, in the presence of NTS, mice lacking hepatic Pcsk9 showed a 40% to 50% decrease in plasma cholesterol and triglycerides. Moreover, the ability of NTS treatment to increase the percentage of low-density lipoprotein-associated cholesterol (from 9% in vehicle-treated Flox mice to 47% after NTS treatment), was lost in mice with hepatic deletion of Pcsk9 (5% in both the presence and absence of NTS). Podocyte damage triggers marked inductions in plasma PCSK9, and knockout of Pcsk9 ameliorates dyslipidemia in a mouse model of nephrotic syndrome. These data suggest that PCSK9 inhibitors may be beneficial in patients with nephrotic syndrome

  2. Lipopolysaccharide mediated regulation of neuroendocrine associated proprotein convertases and neuropeptide precursor processing in the rat spleen.

    PubMed

    Lansac, Guillaume; Dong, Weijia; Dubois, Claire M; Benlarbi, Nadia; Afonso, Carlos; Fournier, Isabelle; Salzet, Michel; Day, Robert

    2006-02-01

    Within the secretory pathway, the family of proprotein convertases cleave inactive precursors at paired basic residues to generate a myriad of biologically active peptides. Within the PC family, PC1/3 and PC2 are well known for their preferential expression within neuroendocrine cells. However, various data now indicate their potential expression in immune cells. The aim of our study was two fold: (1) survey PC expression in immune tissues, with emphasis on PC1/3 and PC2 and (2) examine PC expression under conditions that mimic an infectious state using lipopolysaccharide, known to activate immune cells via toll-like receptors. Spatial and temporal analyses of tissues from control and lipopolysaccharide treated rats were carried out using in situ hybridization histochemistry, Northern blot, mass spectrometry and antibacterial assays. Our tissue survey showed the basal expression of all PCs in the lymph nodes, thymus and spleen including PC1/3 and PC2. Focusing on the spleen, basal expression of PC1/3 was seen in the red pulp/marginal zone areas, suggesting expression within macrophages. Lipopolysaccharide treatment produced significant changes in PC1/3 expression and notably an induction in B lymphocytes within germinal centers. Similarly, PC2, which was undetectable in control spleens, was induced in germinal centers following lipopolysaccharide treatment. The PC1/3 and PC2 substrate proenkephalin was also induced following lipopolysaccharide treatment in the marginal zone, where PC1/3 expression was also found. Mass spectrometry analysis of spleen extracts demonstrated the presence of the antibacterial peptide enkelytin. Our studies confirmed that PC1/3 and PC2 expression was not restricted to neurons and endocrine cells, but was also found under basal conditions in both macrophage and lymphocytes. Additionally, plasticity of PC expression in immune cells was observed under conditions that mimic pathogen-like infections, suggesting a mechanistic link through Toll

  3. A Homolog of Subtilisin-Like Proprotein Convertase 7 Is Essential to Anterior Neural Development in Xenopus

    PubMed Central

    Senturker, Sema; Thomas, John Terrig; Mateshaytis, Jennifer; Moos, Malcolm

    2012-01-01

    Background Subtilisin-like Proprotein Convertase 7 (SPC7) is a member of the subtilisin/kexin family of pro-protein convertases. It cleaves many pro-proteins to release their active proteins, including members of the bone morphogenetic protein (BMP) family of signaling molecules. Other SPCs are known to be required during embryonic development but corresponding data regarding SPC7 have not been reported previously. Methodology/Principal Findings We demonstrated that Xenopus SPC7 (SPC7) was expressed predominantly in the developing brain and eye, throughout the neural plate initially, then more specifically in the lens and retina primordia as development progressed. Since no prior functional information has been reported for SPC7, we used gain- and loss-of-function experiments to investigate the possibility that it may also convey patterning or tissue specification information similarly to Furin, SPC4, and SPC6. Overexpression of SPC7 was without effect. In contrast, injection of SPC7 antisense morpholino oligonucleotides (MO) into a single blastomere at the 2- or 4-cell stage produced marked disruption of head structures; anophthalmia was salient. Bilateral injections suppressed head and eye formation completely. In parallel with suppression of eye and brain development by SPC7 knockdown, expression of early anterior neural markers (Sox2, Otx2, Rx2, and Pax6) and late eye-specific markers (β-Crystallin and Opsin), and of BMP target genes such as Tbx2 and Tbx3, was reduced or eliminated. Taken together, these findings suggest a critical role for SPC7–perhaps, at least in part, due to activation of one or more BMPs–in early patterning of the anterior neural plate and its derivatives. Conclusion/Significance SPC7 is required for normal development of the eye and brain, possibly through processing BMPs, though other potential substrates cannot be excluded. PMID:22761776

  4. Determination of Histidine pKa Values in the Propeptides of Furin and Proprotein Convertase 1/3 Using Histidine Hydrogen-Deuterium Exchange Mass Spectrometry.

    PubMed

    Elferich, Johannes; Williamson, Danielle M; David, Larry L; Shinde, Ujwal

    2015-08-04

    Propeptides of proprotein convertases regulate activation of their protease domains by sensing the organellar pH within the secretory pathway. Earlier experimental work highlighted the importance of a conserved histidine residue within the propeptide of a widely studied member, furin. A subsequent evolutionary analysis found an increase in histidine content within propeptides of secreted eukaryotic proteases compared with their prokaryotic orthologs. However, furin activates in the trans-golgi network at a pH of 6.5 while a paralog, proprotein convertase 1/3, activates in secretory vesicles at a pH of 5.5. It is unclear how a conserved histidine can mediate activation at two different pH values. In this manuscript, we measured the pKa values of histidines within the propeptides of furin and proprotein convertase 1/3 using a histidine hydrogen-deuterium exchange mass spectrometry approach. The high density of histidine residues combined with an abundance of basic residues provided challenges for generation of peptide ions with unique histidine residues, which were overcome by employing ETD fragmentation. During this analysis, we found slow hydrogen-deuterium exchange in residues other than histidine at basic pH. Finally, we demonstrate that the pKa of the conserved histidine in proprotein convertase 1/3 is acid-shifted compared with furin and is consistent with its lower pH of activation.

  5. In vivo functions of the proprotein convertase PC5/6 during mouse development: Gdf11 is a likely substrate

    PubMed Central

    Essalmani, Rachid; Zaid, Ahmed; Marcinkiewicz, Jadwiga; Chamberland, Ann; Pasquato, Antonella; Seidah, Nabil G.; Prat, Annik

    2008-01-01

    The proprotein convertase PC5/6 cleaves protein precursors after basic amino acids and is essential for implantation in CD1/129/Sv/C57BL/6 mixed-background mice. Conditional inactivation of Pcsk5 in the epiblast but not in the extraembryonic tissue bypassed early embryonic lethality but resulted in death at birth. PC5/6-deficient embryos exhibited Gdf11-related phenotypes such as altered anteroposterior patterning with extra vertebrae and lack of tail and kidney agenesis. They also exhibited Gdf11-independent phenotypes, such as a smaller size, multiple hemorrhages, collapsed alveoli, and retarded ossification. In situ hybridization revealed overlapping PC5/6 and Gdf11 mRNA expression patterns. In vitro and ex vivo analyses showed that the selectivity of PC5/6 for Gdf11 essentially resides in the presence of a P1′ Asn in the RSRR↓N cleavage motif. This work identifies Gdf11 as a likely in vivo specific substrate of PC5/6 and opens the way to the identification of other key substrates of this convertase. PMID:18378898

  6. Molecular Consequences of Proprotein Convertase 1/3 (PC1/3) Inhibition in Macrophages for Application to Cancer Immunotherapy: A Proteomic Study*

    PubMed Central

    Duhamel, Marie; Rodet, Franck; Delhem, Nadira; Vanden Abeele, Fabien; Kobeissy, Firas; Nataf, Serge; Pays, Laurent; Desjardins, Roxanne; Gagnon, Hugo; Wisztorski, Maxence; Fournier, Isabelle; Day, Robert; Salzet, Michel

    2015-01-01

    Macrophages provide the first line of host immune defense. Their activation triggers the secretion of pro-inflammatory cytokines and chemokines recruiting other immune cells. In cancer, macrophages present an M2 anti-inflammatory phenotype promoting tumor growth. In this way, strategies need to be develop to reactivate macrophages. Previously thought to be expressed only in cells with a neural/neuroendocrine phenotype, the proprotein convertase 1/3 has been shown to also be expressed in macrophages and regulated as a function of the Toll-like receptor immune response. Here, we investigated the intracellular impact of the down-regulation of the proprotein convertase 1/3 in NR8383 macrophages and confirmed the results on macrophages from PC1/3 deficient mice. A complete proteomic study of secretomes and intracellular proteins was undertaken and revealed that inhibition of proprotein convertase 1/3 orient macrophages toward an M1 activated phenotype. This phenotype is characterized by filopodial extensions, Toll-like receptor 4 MyD88-dependent signaling, calcium entry augmentation and the secretion of pro-inflammatory factors. In response to endotoxin/lipopolysaccharide, these intracellular modifications increased, and the secreted factors attracted naïve T helper lymphocytes to promote the cytotoxic response. Importantly, the application of these factors onto breast and ovarian cancer cells resulted in a decrease viability or resistance. Under inhibitory conditions using interleukin 10, PC1/3-knockdown macrophages continued to secrete inflammatory factors. These data indicate that targeted inhibition of proprotein convertase 1/3 could represent a novel type of immune therapy to reactivate intra-tumoral macrophages. PMID:26330543

  7. Molecular Consequences of Proprotein Convertase 1/3 (PC1/3) Inhibition in Macrophages for Application to Cancer Immunotherapy: A Proteomic Study.

    PubMed

    Duhamel, Marie; Rodet, Franck; Delhem, Nadira; Vanden Abeele, Fabien; Kobeissy, Firas; Nataf, Serge; Pays, Laurent; Desjardins, Roxanne; Gagnon, Hugo; Wisztorski, Maxence; Fournier, Isabelle; Day, Robert; Salzet, Michel

    2015-11-01

    Macrophages provide the first line of host immune defense. Their activation triggers the secretion of pro-inflammatory cytokines and chemokines recruiting other immune cells. In cancer, macrophages present an M2 anti-inflammatory phenotype promoting tumor growth. In this way, strategies need to be develop to reactivate macrophages. Previously thought to be expressed only in cells with a neural/neuroendocrine phenotype, the proprotein convertase 1/3 has been shown to also be expressed in macrophages and regulated as a function of the Toll-like receptor immune response. Here, we investigated the intracellular impact of the down-regulation of the proprotein convertase 1/3 in NR8383 macrophages and confirmed the results on macrophages from PC1/3 deficient mice. A complete proteomic study of secretomes and intracellular proteins was undertaken and revealed that inhibition of proprotein convertase 1/3 orient macrophages toward an M1 activated phenotype. This phenotype is characterized by filopodial extensions, Toll-like receptor 4 MyD88-dependent signaling, calcium entry augmentation and the secretion of pro-inflammatory factors. In response to endotoxin/lipopolysaccharide, these intracellular modifications increased, and the secreted factors attracted naïve T helper lymphocytes to promote the cytotoxic response. Importantly, the application of these factors onto breast and ovarian cancer cells resulted in a decrease viability or resistance. Under inhibitory conditions using interleukin 10, PC1/3-knockdown macrophages continued to secrete inflammatory factors. These data indicate that targeted inhibition of proprotein convertase 1/3 could represent a novel type of immune therapy to reactivate intra-tumoral macrophages.

  8. XPACE4 is a localized pro-protein convertase required for mesoderm induction and the cleavage of specific TGFbeta proteins in Xenopus development.

    PubMed

    Birsoy, Bilge; Berg, Linnea; Williams, P Huw; Smith, James C; Wylie, Christopher C; Christian, Jan L; Heasman, Janet

    2005-02-01

    XPACE4 is a member of the subtilisin/kexin family of pro-protein convertases. It cleaves many pro-proteins to release their active proteins, including members of the TGFbeta family of signaling molecules. Studies in mouse suggest it may have important roles in regulating embryonic tissue specification. Here, we examine the role of XPACE4 in Xenopus development and make three novel observations: first, XPACE4 is stored as maternal mRNA localized to the mitochondrial cloud and vegetal hemisphere of the oocyte; second, it is required for the endogenous mesoderm inducing activity of vegetal cells before gastrulation; and third, it has substrate-specific activity, cleaving Xnr1, Xnr2, Xnr3 and Vg1, but not Xnr5, Derriere or ActivinB pro-proteins. We conclude that maternal XPACE4 plays an important role in embryonic patterning by regulating the production of a subset of active mature TGFbeta proteins in specific sites.

  9. Inactivation of endothelial proprotein convertase 5/6 decreases collagen deposition in the cardiovascular system: role of fibroblast autophagy.

    PubMed

    Marchesi, Chiara; Essalmani, Rachid; Lemarié, Catherine A; Leibovitz, Eyal; Ebrahimian, Talin; Paradis, Pierre; Seidah, Nabil G; Schiffrin, Ernesto L; Prat, Annik

    2011-11-01

    Proprotein convertase (PC) 5/6 belongs to a family of secretory proteases involved in proprotein proteolysis. Several studies suggest a role for PC5/6 in cardiovascular disease. Because lethality at birth of mice lacking PC5/6 precluded elucidation of its function in the adult, we generated mice in which the gene of PC5/6 (pcsk5) is specifically inactivated in endothelial cells (ecKO), which are viable and do not exhibit overt abnormalities. In order to uncover the function of PC5/6 in the cardiovascular system, the effect of ecKO was studied in aging mice. In 16 to 18-month-old ecKO mice, the left ventricle (LV) mass, media cross-sectional area of aorta and coronary arteries, and media-to-lumen ratio of mesenteric arteries were decreased. The LV presented decreased diastolic function, and mesenteric arteries showed decreased stiffness. Collagen was decreased in the LV myocardial interstitium and perivascularly in coronary arteries and aorta. Cardiovascular hypotrophy likely develops with aging, since no significant changes were observed in 2-month-old ecKO mice. Fibroblasts, as a source of collagen in myocardium and vasculature, may play a role in the decrease in collagen deposition. Fibroblasts co-cultured with ecKO endothelial cells showed decreased collagen production, decreased insulin-like growth factor (IGF)-1/Akt/mTOR signaling, and enhanced autophagic activation. PC5/6 inactivation in endothelial cells results in cardiovascular hypotrophy associated with decreased collagen deposition, decreased LV diastolic function, and vascular stiffness, suggesting a trophic role of endothelial PC5/6 in the cardiovascular system, likely mediated by IGF-1/Akt/mTOR signaling and control of autophagy.

  10. The secretory proprotein convertases furin, PC5, and PC7 activate VEGF-C to induce tumorigenesis

    PubMed Central

    Siegfried, Geraldine; Basak, Ajoy; Cromlish, James A.; Benjannet, Suzanne; Marcinkiewicz, Jadwiga; Chrétien, Michel; Seidah, Nabil G.; Khatib, Abdel-Majid

    2003-01-01

    The secretory factor VEGF-C has been directly implicated in various physiological processes during embryogenesis and human cancers. However, the importance of the conversion of its precursor proVEGF-C to mature VEGF-C in tumorigenesis, and vessel formation and the identity of the protease(s) that regulate these processes is/are not known. The intracellular processing of proVEGF-C that occurs within the dibasic motif HSIIRR227SL suggests the involvement of the proprotein convertases (PCs) in this process. In addition, furin and VEGF-C were found to be coordinately expressed in adult mouse tissues. Cotransfection of the furin-deficient colon carcinoma cell line LoVo with proVEGF-C and different PC members revealed that furin, PC5, and PC7 are candidate VEGF-C convertases. This finding is consistent with the in vitro digestions of an internally quenched synthetic fluorogenic peptide mimicking the cleavage site of proVEGF-C (220Q-VHSIIRR↓SLP230). The processing of proVEGF-C is blocked by the inhibitory prosegments of furin, PC5, and PACE4, as well as by furin-motif variants of α2-macroglobulin and α1-antitrypsin. Subcutaneous injection of CHO cells stably expressing VEGF-C into nude mice enhanced angiogenesis and lymphangiogenesis, but not tumor growth. In contrast, expression of proVEGF-C obtained following mutation of the cleavage site (HSIIRR227SL to HSIISS227SL) inhibits angiogenesis and lymphangiogenesis as well as tumor growth. Our findings demonstrate the processing of proVEGF-C by PCs and highlight the potential use of PC inhibitors as agents for inhibiting malignancies induced by VEGF-C. PMID:12782675

  11. Engineering of alpha1-antitrypsin variants selective for subtilisin-like proprotein convertases PACE4 and PC6: importance of the P2' residue in stable complex formation of the serpin with proprotein convertase.

    PubMed

    Tsuji, Akihiko; Kanie, Hiroki; Makise, Hirotaka; Yuasa, Keizo; Nagahama, Masami; Matsuda, Yoshiko

    2007-04-01

    Furin and PACE4, members of the subtilisin-like proprotein convertase (SPC) family, have been implicated in the metastatic progression of certain tumors in addition to the activation of viral coat proteins and bacterial toxins, indicating that these enzymes are potential targets for therapeutic agents. Alpha1-Antitrypsin Portland is an engineered alpha1-antitrypsin designed as a furin-specific inhibitor and has been used as a tool in the functional analysis of furin. In this work, we engineered rat alpha1-antitrypsin to create a PACE4-specific inhibitor. Substituting Arg-Arg-Arg-Arg for Ala-Val-Pro-Met(352) at P4-P1 and Ala for Leu(354) at P2' created a potent PACE4- and PC6-specific inhibitor. This variant (RRRRSA) formed an SDS- and heat-stable serpin/proteinase complex with PACE4 or PC6 and inhibited both enzyme activities. The RRRRSA variant was efficiently cleaved by furin without formation of the stable complex. This is the first report of a highly selective protein-based inhibitor of PACE4 and PC6. This inhibitor will be useful in delineating the roles of PACE4 and PC6 localized in the extracellular matrix.

  12. Defective neuropeptide processing and ischemic brain injury: a study on proprotein convertase 2 and its substrate neuropeptide in ischemic brains

    PubMed Central

    Zhan, Shuqin; Zhao, Hongbo; White, Aaron J; Minami, Manabu; Pignataro, Giuseppe; Yang, Tao; Zhu, Xiaorong; Lan, Jingquan; Xiong, Zhigang; Steiner, Donald F; Simon, Roger P; Zhou, An

    2010-01-01

    Using a focal cerebral ischemia model in rats, brain ischemia-induced changes in expression levels of mRNA and protein, and activities of proprotein convertase 2 (PC2) in the cortex were examined. In situ hybridization analyses revealed a transient upregulation of the mRNA level for PC2 at an early reperfusion hour, at which the level of PC2 protein was also high as determined by immunocytochemistry and western blotting. When enzymatic activities of PC2 were analyzed using a synthetic substrate, a significant decrease was observed at early reperfusion hours at which levels of PC2 protein were still high. Also decreased at these reperfusion hours were tissue levels of dynorphin-A(1–8) (DYN-A(1–8)), a PC2 substrate, as determined by radioimmunoassay. Further examination of PC2 protein biosynthesis by metabolic labeling in cultured neuronal cells showed that in ischemic cells, the proteolytic processing of PC2 was greatly attenuated. Finally, in mice, an intracerebroventricular administration of synthetic DYN-A(1–8) significantly reduced the extent of ischemic brain injury. In mice those lack an active PC2, exacerbated brain injury was observed after an otherwise non-lethal focal ischemia. We conclude that brain ischemia attenuates PC2 and PC2-mediated neuropeptide processing. This attenuation may play a role in the pathology of ischemic brain injury. PMID:19142196

  13. Disruption of the expression of the proprotein convertase PC7 reduces BDNF production and affects learning and memory in mice.

    PubMed

    Wetsel, William C; Rodriguiz, Ramona M; Guillemot, Johann; Rousselet, Estelle; Essalmani, Rachid; Kim, Il Hwan; Bryant, Jesse C; Marcinkiewicz, Jadwiga; Desjardins, Roxane; Day, Robert; Constam, Daniel B; Prat, Annik; Seidah, Nabil G

    2013-10-22

    PC7 belongs to the proprotein convertase family, whose members are implicated in the cleavage of secretory precursors. The in vivo function of PC7 is unknown. Herein, we find that the precursor proBDNF is processed into mature BDNF in COS-1 cells coexpressing proBDNF with either PC7 or Furin. Conversely, the processing of proBDNF into BDNF is markedly reduced in the absence of either Furin or PC7 in mouse primary hepatocytes. In vivo we observe that BDNF and PC7 mRNAs are colocalized in mouse hippocampus and amygdala and that mature BDNF protein levels are reduced in these brain areas in PC7 KO mice but not in the hippocampus of PC1/3 KO mice. Various behavioral tests reveal that in PC7 KO mice spatial memory is intact and plasticity of responding is mildly abnormal. Episodic and emotional memories are severely impaired, but both are rescued with the tyrosine receptor kinase B agonist 7,8-dihydroxyflavone. Altogether, these results support an in vivo role for PC7 in the regulation of certain types of cognitive performance, in part via proBDNF processing. Because polymorphic variants of human PC7 are being characterized, it will be important in future studies to determine their effects on additional physiological and behavioral processes.

  14. Disruption of the expression of the proprotein convertase PC7 reduces BDNF production and affects learning and memory in mice

    PubMed Central

    Wetsel, William C.; Rodriguiz, Ramona M.; Guillemot, Johann; Rousselet, Estelle; Essalmani, Rachid; Kim, Il Hwan; Bryant, Jesse C.; Marcinkiewicz, Jadwiga; Desjardins, Roxane; Day, Robert; Constam, Daniel B.; Prat, Annik; Seidah, Nabil G.

    2013-01-01

    PC7 belongs to the proprotein convertase family, whose members are implicated in the cleavage of secretory precursors. The in vivo function of PC7 is unknown. Herein, we find that the precursor proBDNF is processed into mature BDNF in COS-1 cells coexpressing proBDNF with either PC7 or Furin. Conversely, the processing of proBDNF into BDNF is markedly reduced in the absence of either Furin or PC7 in mouse primary hepatocytes. In vivo we observe that BDNF and PC7 mRNAs are colocalized in mouse hippocampus and amygdala and that mature BDNF protein levels are reduced in these brain areas in PC7 KO mice but not in the hippocampus of PC1/3 KO mice. Various behavioral tests reveal that in PC7 KO mice spatial memory is intact and plasticity of responding is mildly abnormal. Episodic and emotional memories are severely impaired, but both are rescued with the tyrosine receptor kinase B agonist 7,8-dihydroxyflavone. Altogether, these results support an in vivo role for PC7 in the regulation of certain types of cognitive performance, in part via proBDNF processing. Because polymorphic variants of human PC7 are being characterized, it will be important in future studies to determine their effects on additional physiological and behavioral processes. PMID:24101515

  15. Development of a high-throughput assay for human proprotein convertase 5/6 for detecting uterine receptivity.

    PubMed

    Heng, Sophea; Dynon, Kemperly; Li, Ying; Edgell, Tracey; Walton, Kelly; Rombauts, Luk J; Vollenhoven, Beverley; Nie, Guiying

    2015-04-15

    Embryo implantation requires a healthy embryo and a receptive uterus. In women, the inner lining of the uterus, the endometrium, remains in a hostile state and becomes receptive for embryo implantation for only a short period during each menstrual cycle. Determining endometrial receptivity is vital in in vitro fertilization (IVF) treatment because the timing of embryo transfer needs to be synchronized with endometrial receptivity. We have previously demonstrated that proprotein convertase 5/6A (PC6) is highly expressed in the receptive endometrium and that PC6 is critical for receptivity establishment in women. Furthermore, endometrial PC6 is secreted into the uterine fluid, and levels correlate with receptivity status. Detection of PC6 in uterine fluids, therefore, would provide a nonsurgical assessment of endometrial receptivity. However, to date no assays are available for human PC6. In this study, we produced three PC6 monoclonal antibodies (mAbs) and developed a sandwich enzyme-linked immunosorbent assay (ELISA) for PC6 detection in human uterine fluids. The PC6 mAbs were confirmed to be highly specific to PC6, and the ELISA detected PC6 in human uterine fluids with a significantly higher level during the receptive phase. This newly established PC6 ELISA provides an important tool in the development of noninvasive strategies to detect endometrial receptivity in women.

  16. Temporospatially regulated expression of subtilisin-like proprotein convertase PACE4 (SPC4) during development of the rat submandibular gland.

    PubMed

    Akamatsu, Tetsuya; Purwanti, Nunuk; Karabasil, Mileva Ratko; Li, Xuefei; Yao, Chenjuan; Kanamori, Norio; Hosoi, Kazuo

    2007-01-01

    The temporospatial expression of PACE4, a member of the mammalian subtilisin-like proprotein convertase family involved in the activation of growth/differentiation factors, was investigated by in situ hybridization during the development of the rat submandibular gland (SMG). At the initiation stage (day 15.5 of gestation; E15), PACE4 was intensely expressed in the submandibular epithelium, but weakly expressed in the mesenchymal cells. At E16 when the branching morphogenesis becomes obvious, the expression of PACE4 in the mesenchyme was further decreased, although its level in the submandibular epithelium had not changed remarkably from that at E15. During the next stage of embryonic development (E17-E20), PACE4 was expressed in the cells derived from the submandibular epithelium, which include the proacinar, terminal tubular, and presumptive ductal cells. In the perinatal SMG, PACE4 was still expressed intensely in the terminal portion of the SMG containing the proacinar and terminal tubular cells, whereas its expression in the ductal cells was obviously decreased at the second postnatal day (P2) and at P6. Acinar cells expressing no PACE4 appeared, and their numbers increased following their development (P9-P20). At P30 when the PACE4 expression in the acinar cells was completely suppressed, its expression in the ductal cells became intense again. This temporospatially regulated expression of PACE4 suggests its apparent association with the proliferation, differentiation, and establishment of functional acinar and ductal cells of the SMG.

  17. Enhanced aggressiveness of benzopyrene-induced squamous carcinomas in transgenic mice overexpressing the proprotein convertase PACE4 (PCSK6)

    PubMed Central

    Bassi, Daniel E.; Cenna, Jonathan; Zhang, Jirong; Cukierman, Edna; Klein-Szanto, Andres J.

    2014-01-01

    PACE4 (PCSK6) is a pro-protein convertase (PC) capable of processing numerous substrates involved in tumor growth, invasion, and metastasis. Because of the human relevancy of the tobacco-associated carcinogen benzo[a]pyrene (B(a)P) we investigated whether transgenic mice in which this PC is targeted to the epidermis (K5-PACE4) may be more susceptible to B(a)P complete carcinogenesis than wild type (WT) mice. In an in vitro experiment, using cell lines derived from skin tumors obtained after B(a)P treatment, we observed that PACE4 overexpression and activity accounts for an increased proliferation rate, exaggerated sensitivity to the PC inhibitor CMK, and interference with IGF-1R autophosphorylation. Squamous cell carcinomas, obtained from K5-PACE4 mice subjected to complete chemical carcinogenesis, were characterized by a 50 % increase in cell proliferation, when compared with similar tumors from WT mice. In addition, tumors from K5-PACE4 mice showed deeper invasion into the underlying dermis. Thus, mice overexpressing PACE4 exhibited tumors of increased growth rate and invasive potential when exposed to the human carcinogen B(a)P, further supporting the significance of PCs in tumor growth and progression. PMID:24845697

  18. Pro-protein convertases (PCs) other than PC6 are not tightly regulated for implantation in the human endometrium.

    PubMed

    Freyer, C; Kilpatrick, L M; Salamonsen, L A; Nie, G

    2007-06-01

    Pro-protein convertases (PCs) are a family of serine proteases (furin, PC1/3, PC2, PACE4, PC4, PC5/6, PC7/8) responsible for post-translational processing and activation of inactive precursors of many regulatory proteins. Endometrial PC6 is critical for implantation in mice and for decidualization of human endometrial stromal cells (ESCs). This study investigated the endometrial expression of other PCs during the menstrual cycle and early pregnancy to elucidate potential redundancies. Furin, PC4, PACE4, and PC7 along with PC6 transcripts were detected in total endometrial RNA, whereas PC1 and PC2 transcription levels were negligible. Quantitative RT-PCR demonstrated highest levels of furin mRNA during menstruation and lowest levels during the proliferative phase. Furin protein was immunolocalized in endometrial luminal and glandular epithelia, stromal fibroblasts, endothelia, and leukocytes. PACE4 and PC7 proteins were also immunodetected in endometrial stroma and glands. Total furin, PC7, and PACE4 proteins were constitutive in both stromal and glandular compartments throughout the cycle and during first trimester pregnancy. Furthermore, Furin and PC7 transcription was unaltered during decidualization of ESCs in vitro in contrast to PC6 which is significantly up-regulated during decidualization. Thus, whereas PC6 is tightly regulated during endometrial preparation for implantation, furin, PACE4, and PC7 are constitutively expressed in human endometrium, but must be considered if PC6 is to be targeted for manipulation of fertility.

  19. Chromosomal assignment of the genes for proprotein convertases PC4, PC5, and PACE 4 in mouse and human.

    PubMed

    Mbikay, M; Seidah, N G; Chrétien, M; Simpson, E M

    1995-03-01

    The genes for three subtilisin/kexin-like proprotein convertases, PC4, PC5, and PACE4, were mapped in the mouse by RFLP analysis of a DNA panel from a (C57BL/6JEi x SPRET/Ei)F1 x SPRET/Ei backcross. The chromosomal locations of the human homologs were determined by Southern blot analysis of a DNA panel from human-rodent somatic cell hybrids, most of which contained a single human chromosome each. The gene for PC4 (Pcsk4 locus) mapped to mouse chromosome 10, close to the Adn (adipsin, a serine protease) locus and near the Amh (anti-müllerian hormone) locus; in human, the gene was localized to chromosome 19. The gene for PC5 (Pcsk5 locus) mapped to mouse chromosome 19 close to the Lpc1 (lipocortin-1) locus and, in human, was localized to chromosome 9. The gene for PACE4 (Pcsk6 locus) mapped to mouse chromosome 7, at a distance of 13 cM from the Pcsk3 locus, which specifies furin, another member of this family of enzymes previously mapped to this chromosome. This is in concordance with the known close proximity of these two loci in the homologous region on human chromosome 15q25-qter. Pcsk3 and Pcsk6 mapped to a region of mouse chromosome 7 that has been associated cytogenetically with postnatal lethality in maternal disomy, suggesting that these genes might be candidates for imprinting.

  20. Enhanced aggressiveness of benzopyrene-induced squamous carcinomas in transgenic mice overexpressing the proprotein convertase PACE4 (PCSK6).

    PubMed

    Bassi, Daniel E; Cenna, Jonathan; Zhang, Jirong; Cukierman, Edna; Klein-Szanto, Andres J

    2015-10-01

    PACE4 (PCSK6) is a proprotein convertase (PC) capable of processing numerous substrates involved in tumor growth, invasion, and metastasis. Because of the human relevancy of the tobacco-associated carcinogen benzo[a]pyrene (B(a)P) we investigated whether transgenic mice in which this PC is targeted to the epidermis (K5-PACE4) may be more susceptible to B(a)P complete carcinogenesis than wild type (WT) mice. In an in vitro experiment, using cell lines derived from skin tumors obtained after B(a)P treatment, we observed that PACE4 overexpression and activity accounts for an increased proliferation rate, exaggerated sensitivity to the PC inhibitor CMK, and interference with IGF-1R autophosphorylation. Squamous cell carcinomas, obtained from K5-PACE4 mice subjected to complete chemical carcinogenesis, were characterized by a 50% increase in cell proliferation, when compared with similar tumors from WT mice. In addition, tumors from K5-PACE4 mice showed deeper invasion into the underlying dermis. Thus, mice overexpressing PACE4 exhibited tumors of increased growth rate and invasive potential when exposed to the human carcinogen B(a)P, further supporting the significance of PCs in tumor growth and progression.

  1. Engineering of α1-antitrypsin variants with improved specificity for the proprotein convertase furin using site-directed random mutagenesis.

    PubMed

    Hada, Koichiro; Isshiki, Kinuka; Matsuda, Shinya; Yuasa, Keizo; Tsuji, Akihiko

    2013-02-01

    Furin, PACE4, PC5/6 and PC7 are members of the subtilisin-like proprotein convertase (SPC) family. Although these enzymes are known to play critical roles in various physiological and pathological events including cell differentiation, tumor growth, virus replication and the activation of bacterial toxins, their distinct functions are yet to be fully delineated. α1-PDX is an engineered α1-antitrypsin variant carrying the RXXR consensus motif for furin within its reactive site loop. However, α1-PDX inhibits other SPCs in addition to furin. In this work, we prepared various rat α1-antitrypsin variants containing Arg at the P1 site within the reactive site loop, and examined their respective selectivity. The novel α1-antitrypsin variant AVNR (AVPM(352)/AVNR) was identified as a highly selective inhibitor of furin. This variant formed a sodium dodecyl sulfate- and heat-stable furin/α1-antitrypsin complex and inhibited furin activity ex vivo and in vitro. Other SPC members including PACE4, PC5/6 and PC7 were not inhibited by the AVNR variant. Furin-mediated maturation of bone morphogenetic protein-4 was completely inhibited by ectopic expression of the AVNR variant. The AVNR variant should prove to be a useful inhibitor in identifying the specific role of furin.

  2. Proprotein convertase subtilisin/kexin type 9 (PCSK9) and metabolic syndrome: insights on insulin resistance, inflammation, and atherogenic dyslipidemia.

    PubMed

    Ferri, Nicola; Ruscica, Massimiliano

    2016-12-01

    Low-density lipoprotein (LDL) cholesterol plays a pivotal role in the pathogenesis of atherosclerotic cardiovascular disease (CVD). The discovery that proprotein convertase subtilisin/kexin type 9 (PCSK9) represents a key regulator pathway for hepatic LDL receptor (LDLR) degradation sheds light on new uncovered issues regarding LDL-C homeostasis. Indeed, as confirmed by phase II and III clinical trials with monoclonal antibodies, targeting PCSK9 represents the newest and most promising pharmacological tool for the treatment of hypercholesterolemia and related CVD. However, clinical, genetic, and experimental evidence indicates that PCSK9 may be either a cause or an effect in the context of metabolic syndrome (MetS), a condition comprising a cluster of risk factors including insulin resistance, obesity, hypertension, and atherogenic dyslipidemia. The latter is characterized by a triad of hypertriglyceridemia, low plasma concentrations of high-density lipoproteins, and qualitative changes in LDLs. PCSK9 levels seem to correlate with many of these lipid parameters as well as with the insulin sensitivity indices, although the molecular mechanisms behind this association are still unknown or not completely elucidated. Nevertheless, this area of research represents an important starting point for a better understanding of the physiological role of PCSK9, also considering the recent approval of new therapies involving anti-PCSK9. Thus, in the present review, we will discuss the current knowledge on the role of PCSK9 in the context of MetS, alteration of lipids, glucose homeostasis, and inflammation.

  3. Lignans from the fruits of Schisandra chinensis (Turcz.) Baill inhibit proprotein convertase subtilisin/kexin type 9 expression.

    PubMed

    Pel, Pisey; Chae, Hee-Sung; Nhoek, Piseth; Yeo, Woojin; Kim, Young-Mi; Chin, Young-Won

    2017-04-01

    Bioactivity-guided fractionation of the fruits of Schisandra chinensis, using the proprotein convertase subtilisin-kexin type 9 (PCSK9) mRNA expression screening assay, led to isolation of two previously unknown lignans, 14-tigloylschinlignan D and rel-(7R, 8R, 7'R, 8'R)-manglisin E, along with 28 known compounds. All structures were established by NMR spectroscopic data as well as CD and MS analysis. All isolates were tested for their inhibitory activities on the mRNA expression of PCSK9. Of the tested compounds, four of the compounds rel-(7R, 8R, 7'R, 8'R)-manglisin E, (-)-schisandrin C, schinlignan D, and (+)-schisandrol B potently inhibited PCSK9 mRNA expression with IC50 values of 3.15, 3.85, 0.36, and 1.10 μM, respectively. Furthermore, schinlignan D and (+)-schisandrol B were found to suppress PCSK9 protein expressions and schinlignan D deemed to increase low density lipoprotein receptor expression.

  4. Proprotein convertase subtilisin/kexin type 9: from the discovery to the development of new therapies for cardiovascular diseases.

    PubMed

    Ferri, Nicola

    2012-01-01

    The identification of the HMG-CoA reductase inhibitors, statins, has represented a dramatic innovation of the pharmacological modulation of hypercholesterolemia and associated cardiovascular diseases. However, not all patients receiving statins achieve guideline-recommended low density lipoprotein (LDL) cholesterol goals, particularly those at high risk. There remains, therefore, an unmet medical need to develop additional well-tolerated and effective agents to lower LDL cholesterol levels. The discovery of proprotein convertase subtilisin/kexin type 9 (PCSK9), a secretory protein that posttranscriptionally regulates levels of low density lipoprotein receptor (LDLR) by inducing its degradation, has opened a new era of pharmacological modulation of cholesterol homeostasis. This paper summarizes the current knowledge of the basic molecular mechanism underlying the regulatory effect of LDLR expression by PCSK9 obtained from in vitro cell-cultured studies and the analysis of the crystal structure of PCSK9. It also describes the epidemiological and experimental evidences of the regulatory effect of PCSK9 on LDL cholesterol levels and cardiovascular diseases and summarizes the different pharmacological approaches under development for inhibiting PCSK9 expression, processing, and the interaction with LDLR.

  5. Arenavirus envelope glycoproteins mimic autoprocessing sites of the cellular proprotein convertase subtilisin kexin isozyme-1/site-1 protease.

    PubMed

    Pasquato, Antonella; Burri, Dominique J; Traba, Esther Gomez-Ibarlucea; Hanna-El-Daher, Layane; Seidah, Nabil G; Kunz, Stefan

    2011-08-15

    A crucial step in the arenavirus life cycle is the proteolytic processing of the viral envelope glycoprotein precursor (GPC) by the cellular proprotein convertase (PC) subtilisin kexin isozyme-1 (SKI-1)/site-1 protease (S1P). Here we conducted a systematic and quantitative analysis of SKI-1/S1P processing of peptides derived from the recognition sites of GPCs of different Old World and New World arenaviruses. We found that SKI-1/S1P showed a strong preference for arenaviral sequences resembling its autoprocessing sites, which are recurrent motifs in arenaviral GPCs. The African arenaviruses Lassa, Mobala, and Mopeia resemble the SKI-1/S1P autoprocessing C-site, whereas sequences derived from Clade B New World viruses Junin and Tacaribe have similarities to the autoprocessing B-site. In contrast, analogous peptides derived from cellular SKI-1/S1P substrates were remarkably poor substrates. The data suggest that arenavirus GPCs evolved to mimic SKI-1/S1P autoprocessing sites, likely ensuring efficient cleavage and perhaps avoiding competition with SKI-1/S1P's cellular substrates.

  6. Effects of niacin, statin, and fenofibrate on circulating proprotein convertase subtilisin/kexin type 9 levels in patients with dyslipidemia.

    PubMed

    Khera, Amit V; Qamar, Arman; Reilly, Muredach P; Dunbar, Richard L; Rader, Daniel J

    2015-01-15

    Recent trials demonstrated substantial improvement in lipid parameters with inhibition of proprotein convertase subtilisin-like/kexin type 9 (PCSK9). Although statins and fibrates have been reported to increase plasma PCSK9 levels, the effect of niacin on PCSK9 is unknown. We investigated the impact of niacin, atorvastatin, and fenofibrate on PCSK9 levels in 3 distinct studies. A statin-only study randomized 74 hypercholesterolemic patients to placebo, atorvastatin 10 mg/day, or atorvastatin 80 mg/day for 16 weeks. A dose-related increase in PCSK9 was noted such that atorvastatin 80 mg increased PCSK9 by a mean +27% (95% confidence interval [CI] +12 to +42), confirming the effect of statin therapy on raising PCSK9. A second study randomized 70 patients with carotid atherosclerosis to simvastatin 20 mg/day, simvastatin 80 mg/day, or simvastatin 20 mg/extended-release (ER) niacin 2 g/day. PCSK9 levels were increased with statin therapy, but decreased with the simvastatin 20 mg/ER niacin combination (mean -13%, CI -3 to -23). A final study involved 19 dyslipidemic participants on atorvastatin 10 mg with serial addition of fenofibric acid 135 mg followed by ER niacin 2 g/day. Fenofibric acid led to a +23% (CI +10 to +36, p = 0.001) increase in PCSK9; the addition of niacin resulted in a subsequent -17% decrease (CI -19 to -5, p = 0.004). A positive association was noted between change in PCSK9 and low-density lipoprotein cholesterol levels (r = 0.62, p = 0.006) with the addition of niacin. In conclusion, niacin therapy offsets the increase in PCSK9 levels noted with statin and fibrate therapy. A portion of the low-density lipoprotein cholesterol reduction seen with niacin therapy may be due to reduction in PCSK9.

  7. Neuroinflammation-Induced Interactions between Protease-Activated Receptor 1 and Proprotein Convertases in HIV-Associated Neurocognitive Disorder

    PubMed Central

    Kim, WooJin; Zekas, Erin; Lodge, Robert; Susan-Resiga, Delia; Marcinkiewicz, Edwidge; Essalmani, Rachid; Mihara, Koichiro; Ramachandran, Rithwik; Asahchop, Eugene; Gelman, Benjamin; Cohen, Éric A.; Power, Christopher; Hollenberg, Morley D.

    2015-01-01

    The proprotein convertases (PCs) furin, PC5, PACE4, and PC7 cleave secretory proteins after basic residues, including the HIV envelope glycoprotein (gp160) and Vpr. We evaluated the abundance of PC mRNAs in postmortem brains of individuals exhibiting HIV-associated neurocognitive disorder (HAND), likely driven by neuroinflammation and neurotoxic HIV proteins (e.g., envelope and Vpr). Concomitant with increased inflammation-related gene expression (interleukin-1β [IL-1β]), the mRNA levels of the above PCs are significantly increased, together with those of the proteinase-activated receptor 1 (PAR1), an inflammation-associated receptor that is cleaved by thrombin at ProArg41↓ (where the down arrow indicates the cleavage location), and potentially by PCs at Arg41XXXXArg46↓. The latter motif in PAR1, but not its R46A mutant, drives its interactions with PCs. Indeed, PAR1 upregulation leads to the inhibition of membrane-bound furin, PC5B, and PC7 and inhibits gp160 processing and HIV infectivity. Additionally, a proximity ligation assay revealed that furin and PC7 interact with PAR1. Reciprocally, increased furin expression reduces the plasma membrane abundance of PAR1 by trapping it in the trans-Golgi network. Furthermore, soluble PC5A/PACE4 can target/disarm cell surface PAR1 through cleavage at Arg46↓. PACE4/PC5A decreased calcium mobilization induced by thrombin stimulation. Our data reveal a new PC-PAR1-interaction pathway, which offsets the effects of HIV-induced neuroinflammation, viral infection, and potentially the development of HAND. PMID:26283733

  8. Policosanol Attenuates Statin-Induced Increases in Serum Proprotein Convertase Subtilisin/Kexin Type 9 When Combined with Atorvastatin

    PubMed Central

    Guo, Yuan-Lin; Xu, Rui-Xia; Zhu, Cheng-Gang; Wu, Na-Qiong; Cui, Zhi-Ping; Li, Jian-Jun

    2014-01-01

    Objective. Statin treatment alone has been demonstrated to significantly increase plasma proprotein convertase subtilisin/kexin type 9 (PCSK9) levels. The effect of policosanol combined with statin on PCSK9 is unknown. Methods. Protocol I: 26 patients with atherosclerosis were randomly assigned to receive either atorvastatin 20 mg/d or policosanol 20 mg/d + atorvastatin 20 mg/d for 8 weeks. Protocol II: 15 healthy volunteers were randomly assigned to either policosanol 20 mg/d or a control group for 12 weeks. Serum levels of PCSK9 were determined at day 0 and the end of each protocol. Results. Protocol I: atorvastatin 20 mg/d significantly increased serum PCSK9 level by 39.4% (256 ± 84 ng/mL versus 357 ± 101 ng/mL, P = 0.002). However, policosanol 20 mg/d + atorvastatin 20 mg/d increased serum PCSK9 level by only 17.4% without statistical significance (264 ± 60 ng/mL versus 310 ± 86 ng/mL, P = 0.184). Protocol II: there was a trend toward decreasing serum PCSK9 levels in the policosanol group (289 ± 71 ng/mL versus 235 ± 46 ng/mL, P = 0.069). Conclusion. Policosanol combined with statin attenuated the statin-induced increase in serum PCSK9 levels. This finding indicates that policosanol might have a modest effect of lowering serum PCSK9 levels. PMID:25478000

  9. Alterations in gene expression of proprotein convertases in human lung cancer have a limited number of scenarios.

    PubMed

    Demidyuk, Ilya V; Shubin, Andrey V; Gasanov, Eugene V; Kurinov, Alexander M; Demkin, Vladimir V; Vinogradova, Tatyana V; Zinovyeva, Marina V; Sass, Alexander V; Zborovskaya, Irina B; Kostrov, Sergey V

    2013-01-01

    Proprotein convertases (PCs) is a protein family which includes nine highly specific subtilisin-like serine endopeptidases in mammals. The system of PCs is involved in carcinogenesis and levels of PC mRNAs alter in cancer, which suggests expression status of PCs as a possible marker for cancer typing and prognosis. The goal of this work was to assess the information value of expression profiling of PC genes. Quantitative polymerase chain reaction was used for the first time to analyze mRNA levels of all PC genes as well as matrix metalloproteinase genes MMP2 and MMP14, which are substrates of PCs, in 30 matched pairs of samples of human lung cancer tumor and adjacent tissues without pathology. Significant changes in the expression of PCs have been revealed in tumor tissues: increased FURIN mRNA level (p<0.00005) and decreased mRNA levels of PCSK2 (p<0.007), PCSK5 (p<0.0002), PCSK7 (p<0.002), PCSK9 (p<0.00008), and MBTPS1 (p<0.00004) as well as a tendency to increase in the level of PCSK1 mRNA. Four distinct groups of samples have been identified by cluster analysis of the expression patterns of PC genes in tumor vs. normal tissue. Three of these groups covering 80% of samples feature a strong elevation in the expression of a single gene in cancer: FURIN, PCSK1, or PCSK6. Thus, the changes in the expression of PC genes have a limited number of scenarios, which may reflect different pathways of tumor development and cryptic features of tumors. This finding allows to consider the mRNAs of PC genes as potentially important tumor markers.

  10. Neuroinflammation-Induced Interactions between Protease-Activated Receptor 1 and Proprotein Convertases in HIV-Associated Neurocognitive Disorder.

    PubMed

    Kim, WooJin; Zekas, Erin; Lodge, Robert; Susan-Resiga, Delia; Marcinkiewicz, Edwidge; Essalmani, Rachid; Mihara, Koichiro; Ramachandran, Rithwik; Asahchop, Eugene; Gelman, Benjamin; Cohen, Éric A; Power, Christopher; Hollenberg, Morley D; Seidah, Nabil G

    2015-11-01

    The proprotein convertases (PCs) furin, PC5, PACE4, and PC7 cleave secretory proteins after basic residues, including the HIV envelope glycoprotein (gp160) and Vpr. We evaluated the abundance of PC mRNAs in postmortem brains of individuals exhibiting HIV-associated neurocognitive disorder (HAND), likely driven by neuroinflammation and neurotoxic HIV proteins (e.g., envelope and Vpr). Concomitant with increased inflammation-related gene expression (interleukin-1β [IL-1β]), the mRNA levels of the above PCs are significantly increased, together with those of the proteinase-activated receptor 1 (PAR1), an inflammation-associated receptor that is cleaved by thrombin at ProArg41↓ (where the down arrow indicates the cleavage location), and potentially by PCs at Arg41XXXXArg46↓. The latter motif in PAR1, but not its R46A mutant, drives its interactions with PCs. Indeed, PAR1 upregulation leads to the inhibition of membrane-bound furin, PC5B, and PC7 and inhibits gp160 processing and HIV infectivity. Additionally, a proximity ligation assay revealed that furin and PC7 interact with PAR1. Reciprocally, increased furin expression reduces the plasma membrane abundance of PAR1 by trapping it in the trans-Golgi network. Furthermore, soluble PC5A/PACE4 can target/disarm cell surface PAR1 through cleavage at Arg46↓. PACE4/PC5A decreased calcium mobilization induced by thrombin stimulation. Our data reveal a new PC-PAR1-interaction pathway, which offsets the effects of HIV-induced neuroinflammation, viral infection, and potentially the development of HAND.

  11. Relation between proprotein convertase subtilisin/kexin type 9 and directly measured low-density lipoprotein cholesterol

    PubMed Central

    Tecson, Kristen M.; Panettiere-Kennedy, Katherine S.; Won, Jane I.; Garg, Puja; Olugbode, Oluseun

    2017-01-01

    Proprotein convertase subtilisin/kexin type 9 (PCSK9) is a regulator of low-density lipoprotein cholesterol (LDL-C) receptor (LDL-R) recycling and, thus, is a determinant of plasma LDL-C concentration. We sought to determine the relation between serum concentrations of PCSK9 and LDL-C while considering a variety of influential variables, including treatment for dyslipidemia. Using a prospective lipid clinic registry, we evaluated clinical variables, the results of advanced lipid testing, and PCSK9 concentrations determined by immunoassay. We evaluated the relationship between directly measured LDL-C and PCSK9 in serum by performing a simple linear regression. Correlation analyses were performed to examine the relationships of PCSK9 to other clinical and laboratory values and to test for differences in median PCSK9 across patient groups. Factors identified as potential predictors were considered jointly in a multivariate model. For the 26 patients in the analyses, a relationship was not detected between LDL-C and PCSK9 (r = 0.009, P = 0.97); however, PCSK9 was correlated with C-peptide (r = 0.48; P = 0.01) and heart rate (r = 0.52; P = 0.006). Median PCSK9 values differed between statin users (284.0 ng/mL [quartile 1 = 241.0, quartile 3 = 468.0]) and nonusers (219.0 ng/mL [quartile 1 = 151.0, quartile 3 = 228.0]; P = 0.02). More investigation is needed to evaluate the relationship between LDL and PCSK9, as well as the determinants of PCSK9, a major factor regulating cholesterol concentrations. PMID:28127122

  12. Treatment with Proprotein Convertase Subtilisin/Kexin type 9 (PCSK9) Inhibitors to reduce cardiovascular inflammation and outcomes.

    PubMed

    Bonaventura, Aldo; Carbone, Federico; Vecchié, Alessandra; Dallegri, Franco; Camici, Giovanni G; Montecucco, Fabrizio; Liberale, Luca

    2017-03-03

    Proprotein Convertase Subtilisin/Kexin type 9 (PCSK9) is a serine protease involved in cholesterol homeostasis. After binding to the complex low-density lipoprotein (LDL)-receptor, PCSK9 induces its intracellular degradation, thus reducing serum LDL clearance. PCSK9 is mainly secreted by the liver, but it is also expressed to a lesser extent in other organs. Apart from the well-known activity concerning hepatic LDL receptor-mediated pathway, PCSK9 has been supposed to potentially interfere with vascular inflammation in atherogenesis. Vascular smooth muscle cells have been demonstrated to produce higher amounts of PCSK9 as compared to endothelial cells especially in an inflammatory microenvironment. Low shear stress regions increase PCSK9 expression within SMCs, while higher shear stress gradually reduced PCSK9 expression. Moreover, a crosstalk between PCSK9 and reactive oxygen species has been also described. Oxidized LDL were shown to up regulate the expression of PCKS9 by influencing dose-dependently the secretion of interleukin (IL)-1α, IL-6, and tumor necrosis factor-α. After the identification of gene loss-of-function mutations and no detectable circulating protein levels, PCSK9 has attracted a great interest as an effective target for cholesterol-lowering therapies. Different strategies have been implemented to block the effects of both intracellular and circulating PCSK9. In particular, monoclonal antibodies represent the most promising approach and two of these, alirocumab and evolocumab, have been approved for clinical use in patients affected by familial hypercholesterolemia with encouraging results. In the next future, the improvement of the knowledge of the "pleiotropic" effects of PCSK9 inhibitors might unveil therapeutic potential on cardiovascular outcome independently on the cholesterol lowering activity.

  13. Proprotein convertase subtilisin/kexin type 9 inhibition: a new therapeutic mechanism for reducing cardiovascular disease risk.

    PubMed

    Bergeron, Nathalie; Phan, Binh An P; Ding, Yunchen; Fong, Aleyna; Krauss, Ronald M

    2015-10-27

    Proprotein convertase subtilisin/kexin type 9 (PCSK9) plays an important role in the regulation of cholesterol homeostasis. By binding to hepatic low-density lipoprotein (LDL) receptors and promoting their lysosomal degradation, PCSK9 reduces LDL uptake, leading to an increase in LDL cholesterol concentrations. Gain-of-function mutations in PCSK9 associated with high LDL cholesterol and premature cardiovascular disease have been causally implicated in the pathophysiology of autosomal-dominant familial hypercholesterolemia. In contrast, the more commonly expressed loss-of-function mutations in PCSK9 are associated with reduced LDL cholesterol and cardiovascular disease risk. The development of therapeutic approaches that inhibit PCSK9 function has therefore attracted considerable attention from clinicians and the pharmaceutical industry for the management of hypercholesterolemia and its associated cardiovascular disease risk. This review summarizes the effects of PCSK9 on hepatic and intestinal lipid metabolism and the more recently explored functions of PCSK9 in extrahepatic tissues. Therapeutic approaches that prevent interaction of PCSK9 with hepatic LDL receptors (monoclonal antibodies, mimetic peptides), inhibit PCSK9 synthesis in the endoplasmic reticulum (antisense oligonucleotides, siRNAs), and interfere with PCSK9 function (small molecules) are also described. Finally, clinical trials testing the safety and efficacy of monoclonal antibodies to PCSK9 are reviewed. These have shown dose-dependent decreases in LDL cholesterol (44%-65%), apolipoprotein B (48%-59%), and lipoprotein(a) (27%-50%) without major adverse effects in various high-risk patient categories, including those with statin intolerance. Initial reports from 2 of these trials have indicated the expected reduction in cardiovascular events. Hence, inhibition of PCSK9 holds considerable promise as a therapeutic option for decreasing cardiovascular disease risk.

  14. Structure of the unliganded form of the proprotein convertase furin suggests activation by a substrate-induced mechanism

    PubMed Central

    Dahms, Sven O.; Arciniega, Marcelino; Steinmetzer, Torsten; Huber, Robert; Than, Manuel E.

    2016-01-01

    Proprotein convertases (PCs) are highly specific proteases required for the proteolytic modification of many secreted proteins. An unbalanced activity of these enzymes is connected to pathologies like cancer, atherosclerosis, hypercholesterolaemia, and infectious diseases. Novel protein crystallographic structures of the prototypical PC family member furin in different functional states were determined to 1.8–2.0 Å. These, together with biochemical data and modeling by molecular dynamics calculations, suggest essential elements underlying its unusually high substrate specificity. Furin shows a complex activation mechanism and exists in at least four defined states: (i) the “off state,” incompatible with substrate binding as seen in the unliganded enzyme; (ii) the active “on state” seen in inhibitor-bound furin; and the respective (iii) calcium-free and (iv) calcium-bound forms. The transition from the off to the on state is triggered by ligand binding at subsites S1 to S4 and appears to underlie the preferential recognition of the four-residue sequence motif of furin. The molecular dynamics simulations of the four structural states reflect the experimental observations in general and provide approximations of the respective stabilities. Ligation by calcium at the PC-specific binding site II influences the active-site geometry and determines the rotamer state of the oxyanion hole-forming Asn295, and thus adds a second level of the activity modulation of furin. The described crystal forms and the observations of different defined functional states may foster the development of new tools and strategies for pharmacological intervention targeting furin. PMID:27647913

  15. Subtilisin-like Proprotein Convertase Expression, Localization, and Activity in the Human Retina and Optic Nerve Head

    PubMed Central

    Fuller, John A.; Brun-Zinkernagel, Anne-Marie; Clark, Abbot F.; Wordinger, Robert J.

    2014-01-01

    Purpose Subtilisin-like proprotein convertases (SPCs) are a family of calcium-dependent cleavage enzymes that act on dibasic sites of various peptide/protein substrates. The purpose of this study was to investigate the expression, localization, and activity of SPCs in the human retina and optic nerve head. Methods mRNA expression of the SPC family in the human retina and optic nerve head tissues was evaluated by quantitative reverse transcription polymerase chain reaction (QRT-PCR). Double immunofluorescence staining was performed on paraffin-embedded human posterior sections to localize SPC family members. Western blot analysis was used to identify PACE4 isoform expression within the optic nerve head and retina. In addition, a fluorogenic SPC substrate-based assay was used to elucidate SPC enzyme activity within human retina and optic nerve head (ONH) tissues. Results QPCR results indicated that PC1 and PC2 were expressed 4.1- and 5.7-fold higher in retina compared to optic nerve head, whereas PACE4 was expressed 4.1-fold higher in the ONH. PC1 and PC2 were localized primarily in neuronal cells, whereas PACE4 and PC5 were limited to the glia of the retina and optic nerve head. SPC activity in ONH lysate was significantly higher than that of retinal lysate; however, when an SPC inhibitor was added, activity in ONH decreased more than that in retina. Conclusions These results indicate that the SPCs are expressed in distinct patterns throughout the human retina and ONH. PC1 and PC2 were primarily expressed in neurons, whereas PACE4 appeared to be largely restricted to glia. Thus, elevated PACE4 may modulate the bioactivity of proteins secreted in the ONH and retina. PMID:19339735

  16. Characterization and expression of proprotein convertases in CHO cells: Efficient proteolytic maturation of human bone morphogenetic protein-7.

    PubMed

    Sathyamurthy, Madhavi; Kim, Che Lin; Bang, You Lim; Kim, Young Sik; Jang, Ju Woong; Lee, Gyun Min

    2015-03-01

    Bone morphogenetic protein-7 (BMP-7) is synthesized as a precursor that requires proteolytic cleavage of the propeptide by proprotein convertases (PCs) for its functional activity. A high-level expression of BMP-7 in CHO cells (CHO-BMP-7) resulted in secretion of a mixture of inactive precursor and active BMP-7. In an effort to achieve efficient processing of BMP-7 in CHO cells, PCs responsible for cleavage of the precursors in CHO cells were characterized. Analysis of the mRNA expression levels of four PCs (furin, PACE4, PC5/6, and PC7) revealed that only furin and PC7 genes are expressed in CHO-BMP-7 cells. Specific inhibition of the PCs by hexa-D-arginine (D6R) or decanoyl-RVKR-chloromethyl ketone (RVKR-CMK) further revealed that furin is mainly responsible for the proteolytic processing of BMP-7. To identify a more efficient PC for BMP-7 processing, the four PC genes were transiently expressed in CHO-BMP-7 cells, respectively. Among these, PC5/6 was found to be the most efficient in BMP-7 processing. Stable overexpression of PC5/6ΔC, a secreted form of PC5/6, significantly improved mature BMP-7 production in CHO-BMP-7 cells. When the maximum BMP-7 concentration was obtained in the culture of CHO-BMP-7 cells, approximately 88% of BMP-7 was unprocessed. In contrast, no precursor was found in the culture of PC5/6ΔC-overexpressing cells (clone #97). Furthermore, the in vitro biological activity of the mature BMP-7 from PC5/6ΔC-overexpressing cells was comparable to that from CHO-BMP-7 cells. Taken together, the present results indicate that overexpression of PC5/6ΔC in CHO-BMP-7 cells is an efficient means of increasing the yield of BMP-7.

  17. Furin and proprotein convertase 7 (PC7)/lymphoma PC endogenously expressed in rat liver can be resolved into distinct post-Golgi compartments.

    PubMed

    Wouters, S; Leruth, M; Decroly, E; Vandenbranden, M; Creemers, J W; van de Loo, J W; Ruysschaert, J M; Courtoy, P J

    1998-12-01

    The intracellular compartmentalization in rat liver of the membrane-associated convertases furin and proprotein convertase 7 (PC7)/lymphoma PC (LPC) was investigated by analytical subcellular fractionation. In control animals, both enzymes were found to localize in fractions depleted of endoplasmic reticulum, cis-Golgi and lysosomal markers, but to co-distribute with the Golgi marker galactosyltransferase and the trans-Golgi network (TGN) marker TGN38. After overloading Golgi-derived vesicles with very-low-density lipoproteins (VLDL) by feeding rats with ethanol, the distribution of PC7/LPC was shifted markedly towards lower densities, in contrast with those of furin and the TGN marker. This provides support for the TGN localization of endogenously expressed furin and indicates that, at steady state, a considerable proportion of PC7/LPC may be associated with vesicles derived from the TGN.

  18. Furin and proprotein convertase 7 (PC7)/lymphoma PC endogenously expressed in rat liver can be resolved into distinct post-Golgi compartments.

    PubMed Central

    Wouters, S; Leruth, M; Decroly, E; Vandenbranden, M; Creemers, J W; van de Loo, J W; Ruysschaert, J M; Courtoy, P J

    1998-01-01

    The intracellular compartmentalization in rat liver of the membrane-associated convertases furin and proprotein convertase 7 (PC7)/lymphoma PC (LPC) was investigated by analytical subcellular fractionation. In control animals, both enzymes were found to localize in fractions depleted of endoplasmic reticulum, cis-Golgi and lysosomal markers, but to co-distribute with the Golgi marker galactosyltransferase and the trans-Golgi network (TGN) marker TGN38. After overloading Golgi-derived vesicles with very-low-density lipoproteins (VLDL) by feeding rats with ethanol, the distribution of PC7/LPC was shifted markedly towards lower densities, in contrast with those of furin and the TGN marker. This provides support for the TGN localization of endogenously expressed furin and indicates that, at steady state, a considerable proportion of PC7/LPC may be associated with vesicles derived from the TGN. PMID:9820806

  19. Long-Term Follow-up of a Case with Proprotein Convertase 1/3 Deficiency: Transient Diabetes Mellitus with Intervening Diabetic Ketoacidosis During Growth Hormone Therapy.

    PubMed

    Tuli, Gerdi; Tessaris, Daniele; Einaudi, Silvia; De Sanctis, Luisa; Matarazzo, Patrizia

    2017-09-01

    Proprotein convertase 1/3 (PC1/3) deficiency is a very rare disease characterized by severe intractable diarrhea in the first years of life, followed by obesity and several hormonal deficiencies later. Diabetes mellitus requiring insulin treatment and diabetic ketoacidosis have not been reported in this disorder. We herein present a girl with PC1/3 deficiency who has been followed from birth to 17 years of age. She developed deficiencies of all pituitary hormones over time as well as diabetes mellitus while receiving growth hormone (GH) therapy. She was complicated with diabetic ketoacidosis during dietary management of diabetes mellitus, thus insulin treatment was initiated. Insulin requirement to regulate hyperglycemia was short-lived. Repeat oral glucose tolerance test five years later was normal. The findings of this patient show that diabetes mellitus can develop at any time during follow-up of cases with proportein convertase 1/3 deficiency especially under GH therapy.

  20. The prosegments of furin and PC7 as potent inhibitors of proprotein convertases. In vitro and ex vivo assessment of their efficacy and selectivity.

    PubMed

    Zhong, M; Munzer, J S; Basak, A; Benjannet, S; Mowla, S J; Decroly, E; Chrétien, M; Seidah, N G

    1999-11-26

    All proprotein convertases (PCs) of the subtilisin/kexin family contain an N-terminal prosegment that is presumed to act both as an intramolecular chaperone and an inhibitor of its parent enzyme. In this work, we examined inhibition by purified, recombinant bacterial prosegments of furin and PC7 on the in vitro processing of either the fluorogenic peptide pERTKR-MCA or the human immunodeficiency virus envelope glycoprotein gp160. These propeptides are potent inhibitors that display measurable selectivity toward specific proprotein convertases. Small, synthetic decapeptides derived from the C termini of the prosegments are also potent inhibitors, albeit less so than the full-length proteins, and the C-terminal P1 arginine is essential for inhibition. The bacterial, recombinant prosegments were also used to generate specific antisera, allowing us to study the intracellular metabolic fate of the prosegments of furin and PC7 expressed via vaccinia virus constructs. These vaccinia virus recombinants, along with transient transfectants of the preprosegments of furin and PC7, efficiently inhibited the ex vivo processing of the neurotrophins nerve growth factor and brain-derived neurotrophic factor. Thus, we have demonstrated for the first time that PC prosegments, expressed ex vivo as independent domains, can act in trans to inhibit precursor maturation by intracellular PCs.

  1. Mature proprotein convertase subtilisin/kexin type 9, coronary atheroma burden, and vessel remodeling in heterozygous familial hypercholesterolemia.

    PubMed

    Kataoka, Yu; Harada-Shiba, Mariko; Nakao, Kazuhiro; Nakashima, Takahiro; Kawakami, Shoji; Fujino, Masashi; Kanaya, Tomoaki; Nagai, Toshiyuki; Tahara, Yoshio; Asaumi, Yasuhide; Hori, Mika; Ogura, Masatsune; Goto, Yoichi; Noguchi, Teruo; Yasuda, Satoshi

    Proprotein convertase subtilisin/kexin type 9 (PCSK9), an important contributor to low-density lipoprotein metabolism in heterozygous familial hypercholesterolemia (HeFH), exhibits direct proatherogenic effects. PCSK9 circulates as mature and furin-cleaved forms, which differ in its biological activity. However, it remains to be elucidated whether each PCSK9 subtype has different atherogenic properties. To investigate the association of each PCSK9 subtype with coronary atherosclerosis in HeFH. About 204 nonculprit segments in 138 HeFH subjects with coronary artery disease were evaluated by using intravascular ultrasound. Mature, furin-cleaved PCSK9 and total concentration of PCSK9 subtypes were measured by using enzyme-linked immunosorbent assay (BML Inc., Tokyo, Japan). The relationship of these PCSK9 values with intravascular ultrasound measures was investigated. Mature PCSK9 level was positively associated with percent atheroma volume (PAV: r = 0.78, P = .003). Despite extensive atheroma under a higher mature PCSK9 level, vessel volume did not change across any mature PCSK9 levels (r = 0.05, P = .78). These responses resulted in smaller lumen volume, which was negatively correlated to mature PCSK9 level (r = 0.65, P = .009). By contrast, there were no significant relationships of PAV with furin-cleaved (r = 0.12, P = .45) and total PCSK9 (r = 0.37, P = .25) levels. On multivariate analysis, mature PCSK9 level independently contributed to PAV (odds ratio: 1.45, 95% confidence interval: 1.11-1.67, P = .01). Even in subjects with low-density lipoprotein cholesterol level <2.6 mmol/L, greater PAV was still observed in association with an elevated mature PCSK9 level (P = .003). Mature PCSK9 associated with atheroma volume and impaired vessel remodeling in HeFH patients with coronary artery disease. These findings suggest the potential role of mature PCSK9 in propagation of coronary atherosclerosis in HeFH. Copyright © 2017 National Lipid

  2. In vitro elucidation of substrate specificity and bioassay of proprotein convertase 4 using intramolecularly quenched fluorogenic peptides.

    PubMed Central

    Basak, Sarmistha; Chrétien, Michel; Mbikay, Majambu; Basak, Ajoy

    2004-01-01

    The fourth member of Ca2+-dependent mammalian secretory subtilase, PC4 (proprotein convertase 4), is primarily expressed in testicular germ cell and ovarian macrophage. Its role in sperm fertilization and in early embryonic development has been demonstrated earlier through several studies, including those with PC4 null mice. A number of physiological substrates found in reproductive tissues have been postulated or identified for PC4 by various biochemical studies. These include growth factors IGF-1 (insulin-like growth factor-1) and IGF-2, hormonal polypeptide proPACAP (where PACAP stands for pituitary adenylate cyclase-activating polypeptide) and a number of surface proteins of ADAM (ADisintegrin And Metalloproteinase-like) family such as ADAM-1 (fertilin a), ADAM-2 (fertilin b), ADAM-3 (procyritestin) and ADAM-5. To provide further evidence in support of this notion and also to study the substrate specificity and bioassay of PC4, a series of intramolecularly quenched fluorogenic peptides containing the cleavage sites and several mutants were prepared. A comparative kinetic analysis and measurement of Vmax (app)/Km (app) ratio of these fluorogenic substrates against PC4 and PC7 revealed that the mutant variants of h (human) proPACAP and m (mouse) ADAM-5 derived peptides Q-PACAP141-151-mutant [Abz-141RVKNKGRRI150P151SY(NO2)-A-CONH2] (150A151Y replaced by PS) and Q-ADAM-5380-388-mutant [Abz-380E381PKPARRP388RY(NO2)A-CONH2] (381R replaced by P) are most efficiently and selectively cleaved by PC4. Using these two and Q-IGF-263-71 peptides, we showed that the sperm extract of normal adult mice is much higher when compared with that of PC4-null mice. This suggests that these fluorogenic peptides are useful for specific bioassay of PC4 activity. In addition, kinetic studies with various peptidyl-MCA indicate that the hexapeptide Ac-KTKQLR-MCA (where MCA stands for 4-methyl coumaryl-7-amide) is most efficiently and selectively cleaved by PC4 at RMCA, making it another

  3. Embryo implantation is closely associated with dynamic expression of proprotein convertase 5/6 in the rabbit uterus

    PubMed Central

    2011-01-01

    Background Proprotein convertase 5/6 (PC5/6) is critical for embryo implantation in women, regulating both uterine epithelial receptivity and stromal cell decidualization. PC5/6 is likewise essential for implantation in mice, but involved only in decidualization. An alternative animal model is required to address the function of PC5/6 in the uterine epithelium. This study aimed to establish whether PC5/6 is associated with embryo implantation in rabbits. Methods Virgin New-Zealand white rabbits aged 3-4 moths were mated with males of the same strain, or pseudo-pregnancy induced. After mating, uterine tissues were collected over a 10 day (d) period (n = 3 per time point) for RNA, protein and histological analyses to determine the temporal and spatial uterine expression pattern of PC5/6 during the initial stages of pregnancy or induced pseudo-pregnancy. Results PC5/6 mRNA was up-regulated just prior to embryo attachment on d6, and the elevated expression was maintained throughout implantation on d6.5-10. Western analysis revealed a preferential up-regulation of PC5/6 in the implantation sites. Immunohistochemical analysis identified that both the amount and cellular localization of PC5/6 changed with increasing pregnancy stages. Before embryo attachment, PC5/6 was low and localised in the luminal and glandular epithelium. It increased on d6.5 in the basal glands and mucosal folds, and then strongly intensified on d7-10 in the multinucleated luminal symplasma and decidual cells at the site of embryo implantation. In contrast, the pseudo-pregnant uterus displayed relatively low and static PC6 mRNA expression throughout the 10 days, with no obvious changes in either PC5/6 level or cellular localization. Conclusions These findings demonstrate that embryo implantation in the rabbit is closely associated with dynamic expression of uterine PC5/6, and that the rabbit may be an appropriate model to investigate the function of PC5/6 in the uterine epithelium during embryo

  4. The impact of proprotein convertase subtilisin-kexin type 9 serine protease inhibitors on lipid levels and outcomes in patients with primary hypercholesterolaemia: a network meta-analysis.

    PubMed

    Lipinski, Michael J; Benedetto, Umberto; Escarcega, Ricardo O; Biondi-Zoccai, Giuseppe; Lhermusier, Thibault; Baker, Nevin C; Torguson, Rebecca; Brewer, H Bryan; Waksman, Ron

    2016-02-07

    We performed a network meta-analysis of randomized controlled trials (RCTs) in patients with primary hypercholesterolaemia to compare the impact of proprotein convertase subtilisin-kexin type 9 serine protease (PCSK9) inhibitors with placebo and ezetimibe on lipid levels and outcomes. MEDLINE/PubMed, Cochrane CENTRAL, and ClinicalTrials.gov were searched for RCTs assessing PCSK9 inhibitors vs. other therapies in patients with primary hypercholesterolaemia. Network meta-analysis with both a frequentist approach and a Bayesian framework was performed to directly and indirectly compare PCSK9 inhibition on lipid levels with ezetimibe and placebo. Odds ratios with 95% confidence intervals (OR [95% CIs]) were generated with random-effects models to compare outcomes. Our meta-analysis included 17 RCTs with 13 083 patients that were randomized to PCSK9 inhibitors (n = 8250), placebo (n = 3957), ezetimibe (n = 846), or PCSK9 inhibitors and ezetimibe (n = 30). The mean age was 59 ± 10, 52% were male, 34% had coronary artery disease, 51% had hypertension, 19% had diabetes mellitus, baseline LDL of 122 ± 36 mg/dL, total cholesterol of 199 ± 39 mg/dL, and HDL of 51 ± 14 mg/dL. inhibitors significantly reduced LDL cholesterol by 57% relative to placebo (P < 0.001) and 36.1% relative to ezetimibe (P < 0.001). Proprotein convertase subtilisin-kexin type 9 serine protease inhibitors reduced the incidence of all-cause mortality [OR 0.43 (95% CI 0.22-0.82), P = 0.01] but was associated with an increased incidence of neurocognitive adverse events [OR 2.34 (95% CI 1.11-4.93), I(2) = 4%, P = 0.02] when compared with placebo. Proprotein convertase subtilisin-kexin type 9 serine protease inhibition significantly improved lipid profiles and reduced the incidence of all-cause mortality compared with placebo but had a higher rate of neurocognitive adverse events. Thus, PCSK9 inhibitor therapy may serve as an alternative for patients with statin intolerance and for those who do not

  5. Two proprotein convertase subtilisin/kexin type 9 (PCSK9) paralogs from the tropical sea cucumber (Stichopus monotuberculatus): Molecular characterization and inducible expression with immune challenge.

    PubMed

    Ren, Chunhua; Chen, Ting; Jiang, Xiao; Sun, Hongyan; Qian, Jing; Hu, Chaoqun; Wang, Yanhong

    2016-09-01

    Proprotein convertase subtilisin/kexin type 9 (PCSK9) is a multifunctional protein that widely exists in eukaryotic species. In this study, two PCSK9 paralogs, named StmPCSK9-1 and StmPCSK9-2, were identified from the tropical sea cucumber (Stichopus monotuberculatus). The cDNAs of StmPCSK9-1 and StmPCSK9-2 are 1330 kb and 1508 kb in size, respectively. The open reading frames (ORF) for StmPCSK9-1 and StmPCSK9-2 cDNAs are 1128 and 1167 bp in length, encoding the proteins of 375 and 388 amino acids with the deduced molecular weights of 38.76 and 41.07 kDa, respectively. In accord with other members in PCSK9 family, the two StmPCSK9 paralogs possessed the inhibitor_I9 and peptidase_S8 functional domains, seven active sites, a catalytic triad and two calcium binding sites. For the gene structure, the splicing of the two StmPCSK9 paralogs was relatively conserved. In addition, the mRNA expression of StmPCSK9-1 and StmPCSK9-2 was only detected in the sea cucumber intestine and coelomocytes, and the expression levels of both the two StmPCSK9 paralogs were higher in intestine. Moreover, StmPCSK9-2 was found to be a cytoplasm protein without signal peptide, and show no response to the immune challenge. On the contrary, StmPCSK9-1 was a secreted protein and the transcriptional expression of StmPCSK9-1 was significantly up-regulated by lipopolysaccharides (LPS) treatment and slightly down-regulated by polyriboinosinic polyribocytidylic acid [Poly (I:C)] challenge in in vitro experiments performed in the cultural primary coelomocytes, suggesting that the StmPCSK9-1 may play critical roles in the innate immune defense of sea cucumber, S. monotuberculatus, against bacterial and/or viral infections.

  6. Proprotein convertase 5/6 cleaves platelet-derived growth factor A in the human endometrium in preparation for embryo implantation.

    PubMed

    Paule, Sarah; Nebl, Thomas; Webb, Andrew I; Vollenhoven, Beverley; Rombauts, Luk J F; Nie, Guiying

    2015-03-01

    Establishment of endometrial receptivity is vital for successful embryo implantation. Proprotein convertase 5/6 (referred to as PC6) is up-regulated in the human endometrium specifically at the time of epithelial receptivity. PC6, a serine protease of the proprotein convertase family, plays an important role in converting precursor proteins into their active forms through specific proteolysis. The proform of platelet-derived growth factor A (pro-PDGFA) requires PC cleavage to convert to the active-PDGFA. We investigated the PC6-mediated activation of PDGFA in the human endometrium during the establishment of receptivity. Proteomic analysis identified that the pro-PDGFA was increased in the conditioned medium of HEC1A cells in which PC6 was stably knocked down by small interfering RNA (PC6-siRNA). Western blot analysis demonstrated an accumulation of the pro-PDGFA but a reduction in the active-PDGFA in PC6-siRNA cell lysates and medium compared with control. PC6 cleavage of pro-PDGFA was further confirmed in vitro by incubation of recombinant pro-PDGFA with PC6. Immunohistochemistry revealed cycle-stage-specific localization of the active-PDGFA in the human endometrium. During the non-receptive phase, the active-PDGFA was barely detectable. In contrast, it was localized specifically to the apical surface of the luminal and glandular epithelium in the receptive phase. Furthermore, the active-PDGFA was detected in uterine lavage with levels being significantly higher in the receptive than the non-receptive phase. We thus identified that the secreted PDGFA may serve as a biomarker for endometrial receptivity. This is also the first study demonstrating that the active-PDGFA localizes to the apical surface of the endometrium during receptivity.

  7. Proprotein convertase 5/6 is critical for embryo implantation in women: regulating receptivity by cleaving EBP50, modulating ezrin binding, and membrane-cytoskeletal interactions.

    PubMed

    Heng, Sophea; Cervero, Ana; Simon, Carlos; Stephens, Andrew N; Li, Ying; Zhang, Jin; Paule, Sarah; Rainczuk, Adam; Singh, Harmeet; Quinonero, Alicia; Tapia, Alejandro; Velasquez, Luis; Salamonsen, Lois; Rombauts, Luk J F; Nie, Guiying

    2011-12-01

    Establishment of endometrial receptivity is vital for successful embryo implantation; its failure causes infertility. Epithelial receptivity acquisition involves dramatic structural changes in the plasma membrane and cytoskeleton. Proprotein convertase 5/6 (PC6), a serine protease of the proprotein convertase (PC) family, is up-regulated in the human endometrium specifically at the time of epithelial receptivity and stromal cell decidualization. PC6 is the only PC member tightly regulated in this manner. The current study addressed the importance and mechanisms of PC6 action in regulating receptivity in women. PC6 was dysregulated in the endometrial epithelium during the window of implantation in infertile women of three demographically different cohorts. Its critical role in receptivity was evidenced by a significant reduction in mouse blastocyst attachment of endometrial epithelial cells after PC6 knockdown by small interfering RNA. Using a proteomic approach, we discovered that PC6 cleaved the key scaffolding protein, ezrin-radixin-moesin binding phosphoprotein 50 (EBP50), thereby profoundly affecting its interaction with binding protein ezrin (a key protein bridging actin filaments and plasma membrane), EBP50/ezrin cellular localization, and cytoskeleton-membrane connections. We further validated this novel PC6 regulation of receptivity in human endometrium in vivo in fertile vs. infertile patients. These results strongly indicate that PC6 plays a key role in regulating fundamental cellular remodeling processes, such as plasma membrane transformation and membrane-cytoskeletal interface reorganization. PC6 cleavage of a crucial scaffolding protein EBP50, thereby profoundly regulating membrane-cytoskeletal reorganization, greatly extends the current knowledge of PC biology and provides substantial new mechanistic insight into the fields of reproduction, basic cellular biology, and PC biochemistry.

  8. Furin-cleaved proprotein convertase subtilisin/kexin type 9 (PCSK9) is active and modulates low density lipoprotein receptor and serum cholesterol levels.

    PubMed

    Lipari, Michael T; Li, Wei; Moran, Paul; Kong-Beltran, Monica; Sai, Tao; Lai, Joyce; Lin, S Jack; Kolumam, Ganesh; Zavala-Solorio, Jose; Izrael-Tomasevic, Anita; Arnott, David; Wang, Jianyong; Peterson, Andrew S; Kirchhofer, Daniel

    2012-12-21

    Proprotein convertase subtilisin/kexin 9 (PCSK9) regulates plasma LDL cholesterol levels by regulating the degradation of LDL receptors. Another proprotein convertase, furin, cleaves PCSK9 at Arg(218)-Gln(219) in the surface-exposed "218 loop." This cleaved form circulates in blood along with the intact form, albeit at lower concentrations. To gain a better understanding of how cleavage affects PCSK9 function, we produced recombinant furin-cleaved PCSK9 using antibody Ab-3D5, which binds the intact but not the cleaved 218 loop. Using Ab-3D5, we also produced highly purified hepsin-cleaved PCSK9. Hepsin cleaves PCSK9 at Arg(218)-Gln(219) more efficiently than furin but also cleaves at Arg(215)-Phe(216). Further analysis by size exclusion chromatography and mass spectrometry indicated that furin and hepsin produced an internal cleavage in the 218 loop without the loss of the N-terminal segment (Ser(153)-Arg(218)), which remained attached to the catalytic domain. Both furin- and hepsin-cleaved PCSK9 bound to LDL receptor with only 2-fold reduced affinity compared with intact PCSK9. Moreover, they reduced LDL receptor levels in HepG2 cells and in mouse liver with only moderately lower activity than intact PCSK9, consistent with the binding data. Single injection into mice of furin-cleaved PCSK9 resulted in significantly increased serum cholesterol levels, approaching the increase by intact PCSK9. These findings indicate that circulating furin-cleaved PCSK9 is able to regulate LDL receptor and serum cholesterol levels, although somewhat less efficiently than intact PCSK9. Therapeutic anti-PCSK9 approaches that neutralize both forms should be the most effective in preserving LDL receptors and in lowering plasma LDL cholesterol.

  9. Propeptides are sufficient to regulate organelle-specific pH-dependent activation of furin and proprotein convertase 1/3.

    PubMed

    Dillon, Stephanie L; Williamson, Danielle M; Elferich, Johannes; Radler, David; Joshi, Rajendra; Thomas, Gary; Shinde, Ujwal

    2012-10-12

    The proprotein convertases (PCs) furin and proprotein convertase 1/3 (PC1) cleave substrates at dibasic residues along the eukaryotic secretory/endocytic pathway. PCs are evolutionarily related to bacterial subtilisin and are synthesized as zymogens. They contain N-terminal propeptides (PRO) that function as dedicated catalysts that facilitate folding and regulate activation of cognate proteases through multiple-ordered cleavages. Previous studies identified a histidine residue (His69) that functions as a pH sensor in the propeptide of furin (PRO(FUR)), which regulates furin activation at pH~6.5 within the trans-Golgi network. Although this residue is conserved in the PC1 propeptide (PRO(PC1)), PC1 nonetheless activates at pH~5.5 within the dense core secretory granules. Here, we analyze the mechanism by which PRO(FUR) regulates furin activation and examine why PRO(FUR) and PRO(PC1) differ in their pH-dependent activation. Sequence analyses establish that while both PRO(FUR) and PRO(PC1) are enriched in histidines when compared with cognate catalytic domains and prokaryotic orthologs, histidine content in PRO(FUR) is ~2-fold greater than that in PRO(PC1), which may augment its pH sensitivity. Spectroscopy and molecular dynamics establish that histidine protonation significantly unfolds PRO(FUR) when compared to PRO(PC1) to enhance autoproteolysis. We further demonstrate that PRO(FUR) and PRO(PC1) are sufficient to confer organelle sensing on folding and activation of their cognate proteases. Swapping propeptides between furin and PC1 transfers pH-dependent protease activation in a propeptide-dictated manner in vitro and in cells. Since prokaryotes lack organelles and eukaryotic PCs evolved from propeptide-dependent, not propeptide-independent prokaryotic subtilases, our results suggest that histidine enrichment may have enabled propeptides to evolve to exploit pH gradients to activate within specific organelles.

  10. [Blood Levels of Proprotein Convertase Subtilisin/Kexin Type 9 (PCSK9) in Men from Different Population Groups and Its Relation to Unfavorable Long-Term Prognosis].

    PubMed

    Ragino, Yu I; Astrakova, K S; Shakhtshneider, E V; Stakhneva, E M; Gafarov, V V; Bogatyrev, S N; Voevoda, M I

    2017-04-01

    of the study was to investigate blood levels of proprotein convertase subtilisin/kexin type 9 (PCSK9) in men from different population subgroups, their associations with cardiovascular risk factors and with unfavorable 7-years long-term prognosis. The study included three subgroups of men from a population sample of residents of Novosibirsk, 44-73 years old, not receiving lipid-lowering drugs: subgroup of population proper (183 men), subgroup with hypercholesterolemia (46 men), and subgroup with hypocholesterolemia (18 men). Blood level of PCSK9 was determined by ELISA using the test-systems "Human Proprotein Convertase 9/PCSK9 Immunoassay". Study endpoints (myocardial infarction, cardiovascular death) were registered during 7 years after baseline examination of subgroups using the data of the Registers of myocardial infarction and cardiovascular mortality. Distribution of PCSK9 protein in subgroups with hyper- and hypocholesterolemia was normal. In the subgroup of population proper it was abnormal with leftward shift. PCSK9 protein concentration in the subgroup with hypercholesterolemia was 1.2 times higher than in the population subgroup. PCSK9 protein level correlated significantly with blood levels of total cholesterol (CH), low density lipoprotein (LDL) CH, and glucose. Only 15% of PCSK9 variability was due to the influence of other factors (R Square=0.155, p<0.001). Factors with significant influence on blood level of PCSK9 protein were levels of high density lipoprotein CH (=0.238, p=0.023), triglycerides (=0.253, p=0.049) and LDL CH (=0.751, p=0.009). Multivariate regression analysis revealed significant independent association of PCSK9 protein levels with cardiovascular death during period of registration (7-years) (p=0.048, OR=1.01). This result indicates that in men increase of blood level of PCSK9 protein by 1ng/ml independently of other parameters increases relative risk of cardiovascular death during following 7 years by 1%.

  11. Proprotein convertase subtilisin/kexin type 7 (PCSK7) is essential for the zebrafish development and bioavailability of transforming growth factor β1a (TGFβ1a).

    PubMed

    Turpeinen, Hannu; Oksanen, Anna; Kivinen, Virpi; Kukkurainen, Sampo; Uusimäki, Annemari; Rämet, Mika; Parikka, Mataleena; Hytönen, Vesa P; Nykter, Matti; Pesu, Marko

    2013-12-20

    Proprotein convertase subtilisin/kexin (PCSK) enzymes convert proproteins into bioactive end products. Although other PCSK enzymes are known to be essential for biological processes ranging from cholesterol metabolism to host defense, the in vivo importance of the evolutionarily ancient PCSK7 has remained enigmatic. Here, we quantified the expressions of all pcsk genes during the 1st week of fish development and in several tissues. pcsk7 expression was ubiquitous and evident already during the early development. To compare mammalian and zebrafish PCSK7, we prepared homology models, which demonstrated remarkable structural conservation. When the PCSK7 function in developing larvae was inhibited, we found that PCSK7-deficient fish have defects in various organs, including the brain, eye, and otic vesicle, and these result in mortality within 7 days postfertilization. A genome-wide analysis of PCSK7-dependent gene expression showed that, in addition to developmental processes, several immune system-related pathways are also regulated by PCSK7. Specifically, the PCSK7 contributed to the mRNA expression and proteolytic cleavage of the cytokine TGFβ1a. Consequently, tgfβ1a morphant fish displayed phenotypical similarities with pcsk7 morphants, underscoring the importance of this cytokine in the zebrafish development. Targeting PCSK activity has emerged as a strategy for treating human diseases. Our results suggest that inhibiting PCSK7 might interfere with normal vertebrate development.

  12. Proprotein convertase PACE4 is down-regulated by the basic helix-loop-helix transcription factor hASH-1 and MASH-1.

    PubMed

    Yoshida, I; Koide, S; Hasegawa, S I; Nakagawara, A; Tsuji, A; Matsuda, Y

    2001-12-15

    PACE4 is a mammalian subtilisin-like proprotein convertase that activates transforming growth factor (TGF)-beta-related proteins such as bone morphogenetic protein 2 (BMP2), BMP4 and Nodal and exhibits a dynamic expression pattern during embryogenesis. We recently determined that the 1 kb 5'-upstream region of the PACE4 gene contains 12 E-box (E1-E12) elements and that an E-box cluster (E4-E9) acts as a negative regulator [Tsuji, Yoshida, Hasegawa, Bando, Yoshida, Koide, Mori and Matsuda (1999) J. Biochem. (Tokyo) 126, 494-502]. It is known that the mammalian achaete-scute homologue 1 (MASH-1) binds specifically to an E-box (CACCTG) sequence in collaboration with E47, a ubiquitously expressed basic helix-loop-helix (bHLH) factor. To identify the roles of the bHLH factor and E-box elements in regulating PACE4 gene expression in neural development, we analysed the effects of human achaete-scute homologue 1 (hASH-1) on PACE4 gene expression with various neuroblastoma cell lines. The expressions of PACE4 and hASH-1 are correlated inversely in these cell lines. The overexpression of hASH-1 or MASH-1 causes a marked decrease in endogenous PACE4 gene expression but has no effect on the expression of other subtilisin-like proprotein convertases such as furin, PC5/6 and PC7/8. In contrast, other neural bHLH factors (MATH-1, MATH-2, neurogenin 1, neurogenin 2, neurogenin 3 and E47) did not affect PACE4 gene expression. Furthermore, an E-box cluster was a negative regulatory element for the promoter activity in NBL-S cells expressing hASH-1 at high level as determined by a luciferase assay. Binding of hASH-1 to the E-box cluster was confirmed by gel mobility-shift assay. In the present study we identified the PACE4 gene as one of the targets of hASH-1, which is a key factor in the initiation of neural differentiation. These results suggest that the alteration of PACE4 gene expression by hASH-1 causes rapid changes in the biological activities of TGF-beta-related proteins via

  13. A proteomic approach reveals transient association of reticulocalbin-3, a novel member of the CREC family, with the precursor of subtilisin-like proprotein convertase, PACE4.

    PubMed

    Tsuji, Akihiko; Kikuchi, Yayoi; Sato, Yukimi; Koide, Shizuyo; Yuasa, Keizo; Nagahama, Masami; Matsuda, Yoshiko

    2006-05-15

    SPCs (subtilisin-like proprotein convertases) are a family of seven structurally related serine endoproteases that are involved in the proteolytic activation of proproteins. In an effort to examine the substrate protein for PACE4 (paired basic amino-acid-cleaving enzyme-4), an SPC, a potent protein inhibitor of PACE4, an alpha1-antitrypsin RVRR (Arg-Val-Arg-Arg) variant, was expressed in GH4C1 cells. Ectopic expression of the RVRR variant caused accumulation of the 48 kDa protein in cells. Sequence analysis indicates that the 48 kDa protein is a putative Ca2+-binding protein, RCN-3 (reticulocalbin-3), which had previously been predicted by bioinformatic analysis of cDNA from the human hypothalamus. RCN-3 is a member of the CREC (Cab45/reticulocalbin/ERC45/calumenin) family of multiple EF-hand Ca2+-binding proteins localized to the secretory pathway. The most interesting feature of the RCN-3 sequence is the presence of five Arg-Xaa-Xaa-Arg motifs, which represents the target sequence of SPCs. Biosynthetic studies showed that RCN-3 is transiently associated with proPACE4, but not with mature PACE4. Inhibition of PACE4 maturation by a Ca2+ ionophore resulted in accumulation of the proPACE4-RCN-3 complex in cells. Furthermore, autoactivation and secretion of PACE4 was increased upon co-expression with RCN-3. Our findings suggest that selective and transient association of RCN-3 with the precursor of PACE4 plays an important role in the biosynthesis of PACE4.

  14. Estrogen stimuli promote osteoblastic differentiation via the subtilisin-like proprotein convertase PACE4 in MC3T3-E1 cells.

    PubMed

    Kim, Hyejin; Tabata, Atsushi; Tomoyasu, Toshifumi; Ueno, Tomomi; Uchiyama, Shigeto; Yuasa, Keizo; Tsuji, Akihiko; Nagamune, Hideaki

    2015-01-01

    Estrogenic compounds include endogenous estrogens such as estradiol as well as soybean isoflavones, such as daidzein and its metabolite equol, which are known phytoestrogens that prevent osteoporosis in postmenopausal women. Indeed, mineralization of MC3T3-E1 cells, a murine osteoblastic cell line, was significantly decreased in medium containing fetal bovine serum treated with charcoal-dextran to deplete endogenous estrogens, but estradiol and these soybean isoflavones dose-dependently restored the differentiation of MC3T3-E1 cells; equol was tenfold more effective than daidzein. These differentiation-promoting effects were inhibited by the addition of fulvestrant, which is a selective downregulator of estrogen receptors. Analysis of the expression pattern of bone-related genes by reverse transcription PCR (RT-PCR)/quantitative real-time PCR (qRT-PCR), which focused on responsiveness to the estrogen stimuli, revealed that the transcription of PACE4, a subtilisin-like proprotein convertase, was tightly linked with the differentiation of MC3T3-E1 cells induced by estrogen stimuli. Moreover, treatment with RNAi of PACE4 in MC3T3-E1 cells resulted in a drastic decrease of mineralization in the presence of estrogen stimuli. These results strongly suggest that PACE4 participates in bone formation at least in osteoblast differentiation, and estrogen receptor-mediated stimuli induce osteoblast differentiation through the upregulation of PACE4 expression.

  15. Evaluation of proprotein convertase subtilisin/kexin type 9: focus on potential clinical and therapeutic implications for low-density lipoprotein cholesterol lowering.

    PubMed

    Lose, Jennifer M; Dorsch, Michael P; Bleske, Barry E

    2013-04-01

    Reduction in low-density lipoprotein cholesterol (LDL-C) is associated with a decrease in coronary heart disease (CHD). Statins are currently the most effective medications for LDL-C lowering; however, there continues to be a residual risk for cardiovascular events. Proprotein convertase subtilisin/kexin type 9 (PCSK9) is a protease that promotes LDL receptor degradation, leading to an increase in LDL-C blood levels. Patients with PCSK9 gain-of-function mutations can have up to a 20-fold increase in associated CHD compared with patients without these mutations. Conversely, patients with PCSK9 loss-of-function mutations can have up to an 88% reduction in CHD without any deficits in neurologic or physiologic functions. PCSK9 can be modulated by current antihyperlipidemic therapies. In particular, statins lead to an increase in PCSK9, which may attenuate their full lipid-lowering effects. These attributes have made PCSK9 inhibition a desirable target for future drug therapies. Current investigational modalities inhibiting PCSK9 will also be discussed. © 2013 Pharmacotherapy Publications, Inc.

  16. Development of proprotein convertase subtilisin/kexin type 9 inhibitors and the clinical potential of monoclonal antibodies in the management of lipid disorders

    PubMed Central

    Gupta, Sanjiv

    2016-01-01

    The aim of this manuscript is to review available data to evaluate the present status of proprotein convertase subtilisin/kexin type 9 (PCSK9) inhibitors in the treatment of hypercholesterolemia. Relevant literature since 2003 is reviewed. The effectiveness of PCSK9 inhibitors in lowering low-density lipoprotein cholesterol and other atherogenic lipid fractions was studied in various Phase 2 and Phase 3 trials of Alirocumab, Evolocumab, and Bococizumab. The results of published long-term ODYSSEY and OSLER studies are summarized. There have been three excellent meta-analysis studies on PCSK9 inhibitors which are outlined. The complex problem of cost-effectiveness was carefully evaluated by the Institute for Clinical and Economic Review (ICER). The draft report (ICER-2015) is summarized herewith. The cardiovascular outcome trials with Evolocumab (FOURIER), Alirocumab (ODYSSEY OUTCOME) and Bococizumab (SPIRE-1 and SPIRE-2) are the ongoing clinical trials, and their results are expected in 2017–2018. The search for new cost-effective analogs of PCSK9 inhibitors is ongoing. PMID:27877050

  17. Current and emerging treatments for hypercholesterolemia: A focus on statins and proprotein convertase subtilisin/kexin Type 9 inhibitors for perioperative clinicians

    PubMed Central

    Trentman, Terrence L.; Avey, Steven G.; Ramakrishna, Harish

    2016-01-01

    Statins are a mainstay of hyperlipidemia treatment. These drugs inhibit the enzyme 3-hydroxy-3-methylglutaryl coenzyme A reductase and have beneficial effects on atherosclerosis including plaque stabilization, reduction of platelet activation, and reduction of plaque proliferation and inflammation. Statins also have a benefit beyond atherosclerotic plaque, including anticoagulation, vasodilatation, antioxidant effects, and reduction of mediators of inflammation. In the perioperative period, statins appear to contribute to improved outcomes via these mechanisms. Both vascular and nonvascular surgery patients have been shown in prospective studies to have lower risk of adverse cardiac outcomes when initiated on statins preoperatively. However, not all patients can tolerate statins; the search for novel lipid-lowering therapies led to the discovery of the proprotein convertase subtilisin/kexin Type 9 (PCSK9) inhibitors. These drugs are fully-humanized, injectable monoclonal antibodies. With lower PCSK9 activity, low-density lipoprotein cholesterol (LDL-C) receptors are more likely to be recycled to the hepatocyte surface, where they serve to clear plasma LDL-C. Evidence from several prospective studies shows that these new agents can significantly lower LDL-C levels. While PCSK9 inhibitors offer hope of effective therapy for patients with familial hyperlipidemia or intolerance of statins, several important questions remain, including the results of long term cardiovascular outcome studies. The perioperative effects of new LDL-C-lowering drugs are unknown at present but are likely to be similar to the older agents. PMID:28096572

  18. Relation of resistin to proprotein convertase subtilisin-kexin type 9 levels in coronary artery disease patients with different nutritional status.

    PubMed

    Li, S; Xu, R X; Zhang, Y; Guo, Y L; Zhu, C G; Liu, G; Dong, Q; Li, J J

    2015-12-01

    To investigate the association of resistin with proprotein convertase subtilisin-kexin type 9 (PCSK9) levels, another novel regulator of atherosclerosis, in the condition of coronary artery disease (CAD). We prospectively enrolled a total of 356 consecutive stable CAD patients who were not treated with lipid-lowering drugs in the present study. The baseline clinical characteristics were collected. Plasma PCSK9 and resistin levels were determined by ELISA. The relationship between plasma PCSK9 and resistin levels was investigated. Overall, plasma resistin exhibited a positive nonparametric correlation with PCSK9 levels (r = 0.123, p = 0.02). When the patients were classified into groups based on body mass index (BMI), the resistin correlated significantly to the PCSK9 levels in patients with BMI < 25 kg/m(2) (r = 0.162, p = 0.026) but not in patients with BMI ≥ 25 kg/m(2) (r = 0.087, p = 0.205). Multivariate regression analysis corroborated the relation between the PCSK9 and an elevated resistin level in patients with BMI < 25 kg/m(2) independently of traditional parameters including age, sex, BMI, smoking, family history of CAD, systolic blood pressure, glucose, low density lipoprotein cholesterol, white blood cell, neutrophil to lymphocyte ratio, and high-sensitive C-reactive protein. Plasma resistin was positively related to PCSK9 levels in CAD patients with normal weight, suggesting that the circulating resistin might represent a link with PCSK9 level variations in CAD progression of normal body weight.

  19. The Functional Maturation of A Disintegrin and Metalloproteinase (ADAM) 9, 10, and 17 Requires Processing at a Newly Identified Proprotein Convertase (PC) Cleavage Site*

    PubMed Central

    Wong, Eitan; Maretzky, Thorsten; Peleg, Yoav; Blobel, Carl P.; Sagi, Irit

    2015-01-01

    Proenzyme maturation is a general mechanism to control the activation of enzymes. Catalytically active members of the A Disintegrin And Metalloprotease (ADAM) family of membrane-anchored metalloproteases are synthesized as proenzymes, in which the latency is maintained by their autoinhibitory pro-domains. A proteolytic processing then transforms the proenzyme into a catalytically active form. The removal of the pro-domain of ADAMs is currently thought to depend on processing at a canonical consensus site for the proprotein convertase Furin (RXXR) between the pro- and the catalytic domain. Here, we demonstrate that this previously described canonical site is a secondary cleavage site to a prerequisite cleavage in a newly characterized upstream PC site embedded within the pro-domain sequence. The novel upstream regulatory site is important for the maturation of several ADAM proenzymes. Mutations in the upstream regulatory site of ADAM17, ADAM10, and ADAM9 do not prevent pro-domain processing between the pro- and metalloprotease domain, but nevertheless, cause significantly reduced catalytic activity. Thus, our results have uncovered a novel functionally relevant PC processing site in the N-terminal part of the pro-domain that is important for the activation of these ADAMs. These results suggest that the novel PC site is part of a general mechanism underlying proenzyme maturation of ADAMs that is independent of processing at the previously identified canonical Furin cleavage site. PMID:25795784

  20. Opposing function of the proprotein convertases furin and PACE4 on breast cancer cells' malignant phenotypes: role of tissue inhibitors of metalloproteinase-1.

    PubMed

    Lapierre, Marion; Siegfried, Geraldine; Scamuffa, Nathalie; Bontemps, Yannick; Calvo, Fabien; Seidah, Nabil G; Khatib, Abdel-Majid

    2007-10-01

    Proteolytic cleavage of various cancer-related substrates by the proprotein convertases (PC) was reported to be important in the processes of neoplasia. These enzymes are inhibited by their naturally occurring inhibitors, the prosegments (ppPC), and by the engineered general PC inhibitor, the serpin variant alpha1-PDX. In the present study, we sought to compare the effect of these PC inhibitors on malignant phenotypes of breast cancer cells. Overexpression in a stable manner of alpha1-PDX and the prosegment ppPACE4 in MDA-MB-231 breast cancer cells resulted in increased matrix metalloproteinase (MMP)-9 (but not MMP-2) activity and a reduced secretion of tissue inhibitor of metalloproteinase 1 (TIMP-1). This was associated with significant enhancement in cell motility, migration, and invasion of collagen in vitro. In contrast, ppFurin expression in these cells decreased MMP-9 activity and diminished these biological functions, but had no significant effect on TIMP-1 secretion. Taken together, these data showed the specific and opposing roles of Furin and PACE4 in the regulation of MMP-9/TIMP-1-mediated cell motility and invasion.

  1. Inhibition of pro-protein convertase subtilisin/kexin type 6 has a protective role against synovitis in a rat model of rheumatoid arthritis.

    PubMed

    Jiang, Huiyu; Wang, Lin; Pan, Jihong

    2015-11-01

    The aim of the present study was to assess the effects of pro-protein convertase subtilisin/kexin type 6 (PCSK6), a proteinase implicated in the proteolytic activity of various precursor proteins and involved in the regulation of protein maturation, in fibroblast‑like synoviocytes (FLS) of a rat model of collagen‑induced arthritis (CIA). Cultured FLS from CIA models were subjected to small interfering RNA mediated PCSK6 knockdown, followed by assessment of the proliferation, invasive and migratory capacity, the secretion of inflammation factors and the cell cycle. Expression of genes associated with proliferation, invasion, migration and inflammation was detected by reverse transcription polymerase chain reaction. The results showed that PCSK6 knockdown significantly decreased the cell proliferation, invasion and migration of FLS from rats with CIA. ELISA showed an obvious decrease of tumor necrosis factor α and interleukin 1β secretion, and flow cytometric analysis revealed G0/G1 arrest of FLS following PCSK6 knockdown. Furthermore, a decrease in the mRNA levels of inflammation‑associated chemokine CXCL9, angiogenesis‑associated genes MMP‑2, MMP‑9 and NOSTRIN, hypoxia‑associated gene HIF‑1α, adhesion‑associated gene MPZL2, proliferation‑associated gene IGF‑2 and citrullination‑associated gene PADI4 was detected after PCSK6 knockdown. The results of the present study indicated that inhibition of PCSK6 may have a protective role against synovitis in rheumatoid arthritis.

  2. Subtilisin-like proprotein convertase paired basic amino acid-cleaving enzyme 4 is required for chondrogenic differentiation in ATDC5 cells.

    PubMed

    Yuasa, Keizo; Futamatsu, Go; Kawano, Tsuyoshi; Muroshita, Masaki; Kageyama, Yoko; Taichi, Hiromi; Ishikawa, Hiroshi; Nagahama, Masami; Matsuda, Yoshiko; Tsuji, Akihiko

    2012-11-01

    Bone morphogenetic proteins (BMPs) have been implicated in the regulation of multiple stages of endochondral bone development. BMPs are synthesized as inactive precursors, and activated by removal of the propeptide. The subtilisin-like proprotein convertase (SPC) family comprises seven members [furin/SPC1, PC2/SPC2, PC1/PC3/SPC3, paired basic amino acid-cleaving enzyme 4 (PACE4)/SPC4, PC4/SPC5, PC6/PC5/SPC6, and PC8/PC7/LPC/SPC7], and activates various signaling molecules, including BMPs. In this study, we analyzed the role of this family in chondrogenic differentiation by using the mouse embryonal carcinoma-derived clonal cell line ATDC5. Both SPC-specific inhibitors, decanoyl-Arg-Val-Lys-Arg-chloromethylketone and α1-antitrypsin Portland variant, suppressed chondrogenic differentiation. RT-PCR analysis revealed that PACE4 mRNA levels increased markedly during chondrogenic differentiation, whereas furin expression remained unchanged. Knockdown of PACE4 expression significantly reduced chondrogenic differentiation. Furthermore, proBMP6, which shows an expression pattern similar to that of PACE4, was efficiently processed into its mature form by PACE4, whereas furin could not process proBMP6. These results suggest that PACE4 may regulate the rate of hypertrophic conversion of ATDC5 cells through activation of proBMP6.

  3. The proprotein convertase subtilisin/kexin type 9 (PCSK9) active site and cleavage sequence differentially regulate protein secretion from proteolysis.

    PubMed

    Chorba, John S; Shokat, Kevan M

    2014-10-17

    Biologic-based strategies to inhibit proprotein convertase subtilisin/kexin type 9 (PCSK9) show promise as anti-hypercholesterolemic and, therefore, anti-atherosclerotic therapies. Despite substantial effort, no small molecule strategy to inhibit PCSK9 has demonstrated feasibility. In this study we interrogated the chemistry of the PCSK9 active site and its adjacent residues to identify a foothold with which to drug the PCSK9 processing pathway and ultimately disrupt the interaction with the LDL receptor. Here, we develop a system in which we amplify the readout of PCSK9 proteolysis with a highly specific substrate in cells, showing that the PCSK9 catalytic domain is capable of proteolysis in trans. We use this system to show that the substrate specificity for PCSK9 proteolysis is distinct from the specificity for PCSK9 secretion, demonstrating that PCSK9 processing occurs in two separate sequential steps: that of proteolysis followed by secretion. We show that specific residues in the protease recognition sequence can differentially modulate the effects on proteolysis and secretion. Additionally, we demonstrate that the clinically described, dominant negative Q152H mutation restricts proteolysis and secretion independently. Our results suggest that the PCSK9 active site and its adjacent residues serve as an allosteric modulator of protein secretion independent of its role in proteolysis, revealing a new strategy for intracellular PCSK9 inhibition. © 2014 by The American Society for Biochemistry and Molecular Biology, Inc.

  4. The Role of Proprotein Convertase Subtilisin/Kexin Type 9 (PCSK9) in Cardiovascular Homeostasis: A Non-Systematic Literature Review.

    PubMed

    Hachem, Ahmad; Hariri, Essa; Saoud, Perla; Lteif, Christelle; Lteif, Louis; Welty, Francine

    2017-08-04

    Proprotein convertase subtilisin/kexin type 9 (PCSK9) has been gaining major attention recently after the emergence of data showing the promising role of these proteins in lipid homeostasis and atherosclerosis process, glucose and blood pressure regulation. PubMed, EMBASE, Scholar and Scopus databases were searched to identify randomized controlled trials, observational studies, in-vitro trials and reviews about the role of PCSK9 in cardiovascular homeostasis. PCSK9 was found to have major impact on lipid homeostasis and inflammatory process through regulation of low-density lipoprotein receptors. Furthermore, inflammation was found to stimulate the expression of PCSK9 in various cells. As for glomerular proteinuria, a positive correlation was determined between PCSK9 levels and the degree of proteinuria. Hypertension, a major cardiovascular risk factor, is likely affected by PCSK9 levels through their role on epithelial sodium channel (ENaC) surface expression. Likewise, some studies show that PCSK9 is associated with higher fasting blood glucose and plasma insulin, demonstrating a potential role of PCSK9 in glucose homeostasis. The role of PCSK9 in cardiovascular homeostasis is one that is still not completely unraveled. Studies have clearly shown the implication of PCSK9 in the cardiovascular risk factors: the higher the PCSK9 levels, the higher the risk of atherosclerosis, fasting plasma glucose and insulin resistance. Inhibiting PCSK9 may therefore theoretically prove to present great benefits in diabetic patients with high cardiovascular risk. Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.org.

  5. Safety and efficacy of LY3015014, a monoclonal antibody to proprotein convertase subtilisin/kexin type 9 (PCSK9): a randomized, placebo-controlled Phase 2 study

    PubMed Central

    Kastelein, John J.P.; Nissen, Steven E.; Rader, Daniel J.; Hovingh, G. Kees; Wang, Ming-Dauh; Shen, Tong; Krueger, Kathryn A.

    2016-01-01

    Aims The objective of this study was to evaluate the efficacy, safety, and tolerability of LY3015014 (LY), a neutralizing antibody of proprotein convertase subtilisin/kexin type 9 (PCSK9), administered every 4 or 8 weeks in patients with primary hypercholesterolaemia, when added to a background of standard-of-care lipid-lowering therapy, including statins. Methods and results Double-blind, placebo-controlled trial randomized 527 patients with primary hypercholesterolaemia from June 2013 to January 2014 at 61 community and academic centres in North America, Europe, and Japan. Patients were randomized to subcutaneous injections of LY 20, 120, or 300 mg every 4 weeks (Q4W); 100 or 300 mg every 8 weeks (Q8W) alternating with placebo Q4W; or placebo Q4W. The primary endpoint was percentage change from baseline in low-density lipoprotein cholesterol (LDL-C) by beta quantification at Week 16. The mean baseline LDL-C by beta quantification was 136.3 (SD, 45.0)mg/dL. LY3015014 dose-dependently decreased LDL-C, with a maximal reduction of 50.5% with 300 mg LY Q4W and 37.1% with 300 mg LY Q8W compared with a 7.6% increase with placebo maintained at the end of the dosing interval. There were no treatment-related serious adverse events (AEs). The most common AE terms (>10% of any treatment group) reported more frequently with LY compared with placebo were injection site (IS) pain and IS erythema. No liver or muscle safety issues emerged. Conclusions LY3015014 dosed every 4 or 8 weeks, resulted in robust and durable reductions in LDL-C. No clinically relevant safety issues emerged with the administration of LY. The long-term effects on cardiovascular outcomes require further investigation. PMID:26757788

  6. Implications of Proprotein Convertases in Ovarian Cancer Cell Proliferation and Tumor Progression: Insights for PACE4 as a Therapeutic Target1

    PubMed Central

    Longuespée, Rémi; Couture, Frédéric; Levesque, Christine; Kwiatkowska, Anna; Desjardins, Roxane; Gagnon, Sandra; Vergara, Daniele; Maffia, Michelle; Fournier, Isabelle; Salzet, Michel; Day, Robert

    2014-01-01

    Proprotein convertases are a family of kexin-like serine proteases that process proteins at single and multiple basic residues. Among the predicted and identified PC substrates, an increasing number of proteins having functions in cancer progression indicate that PCs may be potential targets for antineoplastic drugs. In support of this notion, we identified PACE4 as a vital PC involved in prostate cancer proliferation and progression, contrasting with the other co-expressed PCs. The aim of the present study was to test the importance of PCs in ovarian cancer cell proliferation and tumor progression. Based on tissue-expression profiles, furin, PACE4, PC5/6 and PC7 all displayed increased expression in primary tumor, ascites cells and metastases. These PCs were also expressed in variable levels in three model ovarian cell lines tested, namely SKOV3, CAOV3 and OVCAR3 cells. Since SKOV3 cells closely represented the PC expression profile of ovarian cancer cells, we chose them to test the effects of PC silencing using stable gene-silencing shRNA strategy to generate knockdown SKOV3 cells for each expressed PC. In vitro and in vivo assays confirmed the role of PACE4 in the sustainment of SKOV3 cell proliferation, which was not observed with the other three PCs. We also tested PACE4 peptide inhibitors on all three cell lines and observed consequent reduced cell proliferation which was correlated with PACE4 expression. Overall, these data support a role of PACE4 in promoting cell proliferation in ovarian cancer and provides further evidence for PACE4 as a potential therapeutic target. PMID:24818756

  7. cDNA structure of the mouse and rat subtilisin/kexin-like PC5: a candidate proprotein convertase expressed in endocrine and nonendocrine cells.

    PubMed

    Lusson, J; Vieau, D; Hamelin, J; Day, R; Chrétien, M; Seidah, N G

    1993-07-15

    By using reverse transcriptase/PCR and oligonucleotide sequences derived from conserved segments (including the conserved RRGDL sequence) of the known proprotein convertases (PCs) PC1, PC2, furin, and PC4, we identified a subtilisin/kexin-like PC called PC5 in both mouse and rat tissues. The composite structure (2.85 kb) was deduced from the analysis of the reverse transcription/PCR products combined with the sequence from a clone isolated from a cDNA library made from corticotropin-activated mouse adrenocortical Y1 cells. The deduced cDNA structures of mouse PC5 and rat PC5 showed that the closest homologue is PACE4. Furthermore, like furin, Drosophila melanogaster (d) dfurin2, and PACE4, PC5 shows the presence of a C-terminal Cys-rich domain containing either 5 (PC5 and PACE4) or 10 (dfurin2) repeats of the consensus motif Cys-Xaa2-Cys-Xaa3-Cys-Xaa(5-7)-Cys-Xaa2-Cys-Xaa (8-15)-Cys-Xaa3-Cys-Xaa(9-16). The richest sources of rat PC5 mRNA (3.8 kb) are the adrenal and gut, but it can also be detected in many endocrine and nonendocrine tissues. Corticotropin-stimulated adrenocortical Y1 cells showed an increased expression of PC5 mRNA, suggesting an upregulation by cAMP. In situ hybridization of rat brain sections demonstrated a unique distribution of PC5 compared to PC1, PC2, and furin.

  8. cDNA structure of the mouse and rat subtilisin/kexin-like PC5: a candidate proprotein convertase expressed in endocrine and nonendocrine cells.

    PubMed Central

    Lusson, J; Vieau, D; Hamelin, J; Day, R; Chrétien, M; Seidah, N G

    1993-01-01

    By using reverse transcriptase/PCR and oligonucleotide sequences derived from conserved segments (including the conserved RRGDL sequence) of the known proprotein convertases (PCs) PC1, PC2, furin, and PC4, we identified a subtilisin/kexin-like PC called PC5 in both mouse and rat tissues. The composite structure (2.85 kb) was deduced from the analysis of the reverse transcription/PCR products combined with the sequence from a clone isolated from a cDNA library made from corticotropin-activated mouse adrenocortical Y1 cells. The deduced cDNA structures of mouse PC5 and rat PC5 showed that the closest homologue is PACE4. Furthermore, like furin, Drosophila melanogaster (d) dfurin2, and PACE4, PC5 shows the presence of a C-terminal Cys-rich domain containing either 5 (PC5 and PACE4) or 10 (dfurin2) repeats of the consensus motif Cys-Xaa2-Cys-Xaa3-Cys-Xaa(5-7)-Cys-Xaa2-Cys-Xaa (8-15)-Cys-Xaa3-Cys-Xaa(9-16). The richest sources of rat PC5 mRNA (3.8 kb) are the adrenal and gut, but it can also be detected in many endocrine and nonendocrine tissues. Corticotropin-stimulated adrenocortical Y1 cells showed an increased expression of PC5 mRNA, suggesting an upregulation by cAMP. In situ hybridization of rat brain sections demonstrated a unique distribution of PC5 compared to PC1, PC2, and furin. Images Fig. 3 Fig. 4 PMID:8341687

  9. Proprotein Convertase Subtilisin/Kexin Type 9 (PCSK9) Single Domain Antibodies Are Potent Inhibitors of Low Density Lipoprotein Receptor Degradation.

    PubMed

    Weider, Elodie; Susan-Resiga, Delia; Essalmani, Rachid; Hamelin, Josée; Asselin, Marie-Claude; Nimesh, Surendra; Ashraf, Yahya; Wycoff, Keith L; Zhang, Jianbing; Prat, Annik; Seidah, Nabil G

    2016-08-05

    Single domain antibodies (sdAbs) correspond to the antigen-binding domains of camelid antibodies. They have the same antigen-binding properties and specificity as monoclonal antibodies (mAbs) but are easier and cheaper to produce. We report here the development of sdAbs targeting human PCSK9 (proprotein convertase subtilisin/kexin type 9) as an alternative to anti-PCSK9 mAbs. After immunizing a llama with human PCSK9, we selected four sdAbs that bind PCSK9 with a high affinity and produced them as fusion proteins with a mouse Fc. All four sdAb-Fcs recognize the C-terminal Cys-His-rich domain of PCSK9. We performed multiple cellular assays and demonstrated that the selected sdAbs efficiently blocked PCSK9-mediated low density lipoprotein receptor (LDLR) degradation in cell lines, in human hepatocytes, and in mouse primary hepatocytes. We further showed that the sdAb-Fcs do not affect binding of PCSK9 to the LDLR but rather block its induced cellular LDLR degradation. Pcsk9 knock-out mice expressing a human bacterial artificial chromosome (BAC) transgene were generated, resulting in plasma levels of ∼300 ng/ml human PCSK9. Mice were singly or doubly injected with the best sdAb-Fc and analyzed at day 4 or 11, respectively. After 4 days, mice exhibited a 32 and 44% decrease in the levels of total cholesterol and apolipoprotein B and ∼1.8-fold higher liver LDLR protein levels. At 11 days, the equivalent values were 24 and 46% and ∼2.3-fold higher LDLR proteins. These data constitute a proof-of-principle for the future usage of sdAbs as PCSK9-targeting drugs that can efficiently reduce LDL-cholesterol, and as tools to study the Cys-His-rich domain-dependent sorting the PCSK9-LDLR complex to lysosomes.

  10. Proprotein convertase subtilisin/kexin type 9 (PCSK9) can mediate degradation of the low density lipoprotein receptor-related protein 1 (LRP-1).

    PubMed

    Canuel, Maryssa; Sun, Xiaowei; Asselin, Marie-Claude; Paramithiotis, Eustache; Prat, Annik; Seidah, Nabil G

    2013-01-01

    Elevated LDL-cholesterol (LDLc) levels are a major risk factor for cardiovascular disease and atherosclerosis. LDLc is cleared from circulation by the LDL receptor (LDLR). Proprotein convertase subtilisin/kexin 9 (PCSK9) enhances the degradation of the LDLR in endosomes/lysosomes, resulting in increased circulating LDLc. PCSK9 can also mediate the degradation of LDLR lacking its cytosolic tail, suggesting the presence of as yet undefined lysosomal-targeting factor(s). Herein, we confirm this, and also eliminate a role for the transmembrane-domain of the LDLR in mediating its PCSK9-induced internalization and degradation. Recent findings from our laboratory also suggest a role for PCSK9 in enhancing tumor metastasis. We show herein that while the LDLR is insensitive to PCSK9 in murine B16F1 melanoma cells, PCSK9 is able to induce degradation of the low density lipoprotein receptor-related protein 1 (LRP-1), suggesting distinct targeting mechanisms for these receptors. Furthermore, PCSK9 is still capable of acting upon the LDLR in CHO 13-5-1 cells lacking LRP-1. Conversely, PCSK9 also acts on LRP-1 in the absence of the LDLR in CHO-A7 cells, where re-introduction of the LDLR leads to reduced PCSK9-mediated degradation of LRP-1. Thus, while PCSK9 is capable of inducing degradation of LRP-1, the latter is not an essential factor for LDLR regulation, but the LDLR effectively competes with LRP-1 for PCSK9 activity. Identification of PCSK9 targets should allow a better understanding of the consequences of PCSK9 inhibition for lowering LDLc and tumor metastasis.

  11. Activation of Adiponectin Receptor Regulates Proprotein Convertase Subtilisin/Kexin Type 9 Expression and Inhibits Lesions in ApoE-Deficient Mice.

    PubMed

    Sun, Lei; Yang, Xiaoxiao; Li, Qi; Zeng, Peng; Liu, Ying; Liu, Lipei; Chen, Yuanli; Yu, Miao; Ma, Chuanrui; Li, Xiaoju; Li, Yan; Zhang, Rongxin; Zhu, Yan; Miao, Qing Robert; Han, Jihong; Duan, Yajun

    2017-07-01

    The reduced adiponectin levels are associated with atherosclerosis. Adiponectin exerts its functions by activating adiponectin receptor (AdipoR). Proprotein convertase subtilisin kexin type 9 (PCSK9) degrades LDLR protein (low-density lipoprotein receptor) to increase serum LDL-cholesterol levels. PCSK9 expression can be regulated by PPARγ (peroxisome proliferator-activated receptor γ) or SREBP2 (sterol regulatory element-binding protein 2). The effects of AdipoR agonists on PCSK9 and LDLR expression, serum lipid profiles, and atherosclerosis remain unknown. At cellular levels, AdipoR agonists (ADP355 and AdipoRon) induced PCSK9 transcription/expression that solely depended on activation of PPAR-responsive element in the PCSK9 promoter. AdipoR agonists induced PPARγ expression; thus, the AdipoR agonist-activated PCSK9 expression/production was impaired in PPARγ deficient hepatocytes. Meanwhile, AdipoR agonists transcriptionally activated LDLR expression by activating SRE in the LDLR promoter. Moreover, AMP-activated protein kinase α (AMPKα) was involved in AdipoR agonist-activated PCSK9 expression. In wild-type mice, ADP355 increased PCSK9 and LDLR expression and serum PCSK9 levels, which was associated with activation of PPARγ, AMPKα and SREBP2 and reduction of LDL-cholesterol levels. In contrast, ADP355 reduced PCSK9 expression/secretion in apoE-deficient (apoE(-/-)) mice, but it still activated hepatic LDLR, PPARγ, AMPKα, and SREBP2. More importantly, ADP355 inhibited lesions in en face aortas and sinus lesions in aortic root in apoE(-/-) mice with amelioration of lipid profiles. Our study demonstrates that AdipoR activation by agonists regulated PCSK9 expression differently in wild-type and apoE(-/-) mice. However, ADP355 activated hepatic LDLR expression and ameliorated lipid metabolism in both types of mice and inhibited atherosclerosis in apoE(-/-) mice. © 2017 American Heart Association, Inc.

  12. PEGylation of a proprotein convertase peptide inhibitor for vaginal route of drug delivery: in vitro bioactivity, stability and in vivo pharmacokinetics.

    PubMed

    Ho, Huiting; Nero, Tracy L; Singh, Harmeet; Parker, Michael W; Nie, Guiying

    2012-12-01

    Uterine proprotein convertase (PC) 6 is critical for embryo implantation in mice and women. It is also one of the PC family members that play a vital role in HIV infectivity. We hypothesized that inhibiting PC6 in the female reproductive tract (vagina, cervix and uterus), may protect women from both pregnancy and HIV infection. One key requirement to prove this concept in an animal model is a vaginally deliverable PC6 inhibitor. Nona-D-arginine (Poly R) is a potent peptide PC inhibitor and is able to inhibit HIV in cell culture. We modified Poly R by PEGylation with different strategies and determined their biochemical properties in vitro and in vivo. PEGylation at the C-terminus, regardless of the PEG size (30 kDa or 1239 Da) did not compromise the inhibitory potency of Poly R. In contrast, PEGylation at both termini (1239 Da) dramatically reduced its inhibitory activity. Poly R and C-PEGylated Poly Rs also showed equal potency in inhibiting a PC6-dependent cellular process critical for embryo implantation. Poly R and the equipotent C-PEGylated Poly Rs were further tested for their serum stability in vitro and pharmacokinetics in vivo following vaginal administration in mice. All Poly Rs were equally stable in mouse serum in vitro for 24h; C-PEGylated Poly Rs showed enhanced vaginal absorption and penetration across the vaginal mucosa/epithelium. This is the first report that C-terminal PEGylation significantly enhances the therapeutic properties of Poly R for vaginal drug delivery. Our findings also provide important insights into future design of Poly R derivatives.

  13. Proprotein Convertase Subtilisin/Kexin type 9, C-Reactive Protein, Coronary Severity, and Outcomes in Patients With Stable Coronary Artery Disease

    PubMed Central

    Li, Jian-Jun; Li, Sha; Zhang, Yan; Xu, Rui-Xia; Guo, Yuan-Lin; Zhu, Cheng-Gang; Wu, Na-Qiong; Qing, Ping; Gao, Ying; Sun, Jing; Liu, Geng; Dong, Qian

    2015-01-01

    Abstract Proprotein convertase subtilisin/kexin type 9 (PCSK9) is suggested as a novel factor associated with coronary artery disease (CAD). However, few studies have comprehensively evaluated plasma PCSK9 with cardiovascular risk till now. Hence, we aimed to prospectively investigate the association between baseline PCSK9 and cardiovascular risk graded with number of risk factors (RFs), coronary severity, and outcomes in patients with stable CAD. Baseline characteristics and biomarkers were measured in 616 consecutive, nontreated patients with stable CAD. Coronary severity was measured using SYNTAX, Gensini, and Jeopardy scoring systems. Patients were then received treatment and followed for a median of 17 months. The primary endpoints were cardiac death, stroke, myocardial infarction (MI), post-discharge revascularization, or unstable angina (UA). Overall, follow-up data were obtained from 603 patients. A total of 72 (11.9%) patients presented with at least 1 major adverse cardiovascular event (MACE) (4 cardiac deaths, 4 strokes, 6 MIs, 28 revascularizations, and 30 UAs). At baseline, PCSK9 was increased with an increasing number of RFs and positively associated with coronary severity scores (P < 0.05, all). After follow-up, those with MACE had a higher baseline PCSK9, hs-CRP, and coronary scores than those without (P < 0.05, all). Multivariate analysis showed that PCSK9, hs-CRP, and coronary scores were independently predictive for MACEs (P < 0.05, all). Interestingly, more significant predictive values of PCSK9 in medical-alone-treated population but no such associations in revascularization-treated patients were found. Together, plasma PCSK9, as well as hs-CRP and coronary scores, could independently predict MACEs in patients with stable CAD. PMID:26717403

  14. Peroxisome Proliferator-activated Receptor γ Activation by Ligands and Dephosphorylation Induces Proprotein Convertase Subtilisin Kexin Type 9 and Low Density Lipoprotein Receptor Expression*

    PubMed Central

    Duan, Yajun; Chen, Yuanli; Hu, Wenquan; Li, Xiaoju; Yang, Xiaoxiao; Zhou, Xin; Yin, Zhinan; Kong, Deling; Yao, Zhi; Hajjar, David P.; Liu, Lin; Liu, Qiang; Han, Jihong

    2012-01-01

    Proprotein convertase subtilisin kexin type 9 (PCSK9) plays an important role in cholesterol homeostasis by enhancing the degradation of LDL receptor (LDLR) protein. Peroxisome proliferator-activated receptor γ (PPARγ) has been shown to be atheroprotective. PPARγ can be activated by ligands and/or dephosphorylation with ERK1/2 inhibitors. The effect of PPARγ on PCSK9 and LDLR expression remains unknown. In this study, we investigated the effects of PPARγ on PCSK9 and LDLR expression. At the cellular levels, PPARγ ligands induced PCSK9 mRNA and protein expression in HepG2 cells. PCSK9 expression was induced by inhibition of ERK1/2 activity but inhibited by ERK1/2 activation. The mutagenic study and promoter activity assay suggested that the induction of PCSK9 expression by ERK1/2 inhibitors was tightly linked to PPARγ dephosphorylation. However, PPARγ activation by ligands or ERK1/2 inhibitors induced hepatic LDLR expression. The promoter assay indicated that the induction of LDLR expression by PPARγ was sterol regulatory element-dependent because PPARγ enhanced sterol regulatory element-binding protein 2 (SREBP2) processing. In vivo, administration of pioglitazone or U0126 alone increased PCSK9 expression in mouse liver but had little effect on PCSK9 secretion. However, the co-treatment of pioglitazone and U0126 enhanced both PCSK9 expression and secretion. Similar to in vitro, the increased PCSK9 expression by pioglitazone and/or U0126 did not result in decreased LDLR expression and function. In contrast, pioglitazone and/or U0126 increased LDLR protein expression and membrane translocation, SREBP2 processing, and CYP7A1 expression in the liver, which led to decreased total and LDL cholesterol levels in serum. Our results indicate that although PPARγ activation increased PCSK9 expression, PPARγ activation induced LDLR and CYP7A1 expression that enhanced LDL cholesterol metabolism. PMID:22593575

  15. 60 YEARS OF POMC: From the prohormone theory to pro-opiomelanocortin and to proprotein convertases (PCSK1 to PCSK9).

    PubMed

    Chrétien, Michel; Mbikay, Majambu

    2016-05-01

    Pro-opiomelanocortin (POMC), is a polyprotein expressed in the pituitary and the brain where it is proteolytically processed into peptide hormones and neuropeptides with distinct biological activities. It is the prototype of multipotent prohormones. The prohormone theory was first suggested in 1967 when Chrétien and Li discovered γ-lipotropin and observed that (i) it was part of β-lipotropin (β-LPH), a larger polypeptide characterized 2 years earlier and (ii) its C-terminus was β-melanocyte-stimulating hormone (β-MSH). This discovery led them to propose that the lipotropins might be related biosynthetically to the biologically active β-MSH in a precursor to end product relationship. The theory was widely confirmed in subsequent years. As we celebrate the 50th anniversary of the sequencing of β-LPH, we reflect over the lessons learned from the sequencing of those proteins; we explain their extension to the larger POMC precursor; we examine how the theory of precursor endoproteolysis they inspired became relevant for vast fields in biology; and how it led, after a long and arduous search, to the novel proteolytic enzymes called proprotein convertases. This family of nine enzymes plays multifaceted functions in growth, development, metabolism, endocrine, and brain functions. Their genetics has provided many insights into health and disease. Their therapeutic targeting is foreseeable in the near future. Thus, what started five decades ago as a theory based on POMC fragments, has opened up novel and productive avenues of biological and medical research, including, for our own current interest, a highly intriguing hypocholesterolemic Gln152His PCSK9 mutation in French-Canadian families.

  16. Endosome to trans-Golgi network transport of Proprotein Convertase 7 is mediated by a cluster of basic amino acids and palmitoylated cysteines.

    PubMed

    Declercq, Jeroen; Ramos-Molina, Bruno; Sannerud, Ragna; Brouwers, Bas; Pruniau, Vincent P E G; Meulemans, Sandra; Plets, Evelyn; Annaert, Wim; Creemers, John W M

    2017-08-01

    Proprotein Convertase 7 (PC7) is a Furin-like endoprotease that cleaves precursor proteins at basic amino acids. PC7 is concentrated in the trans-Golgi network (TGN) but it shuttles between the plasma membrane and the TGN depending on sequences in the cytoplasmic tail. A short region containing a three amino acids motif, P(724)-L(725)-C(726), is essential and sufficient for internalization of PC7 but not for TGN localization, which requires the additional presence of the juxtamembrane region. In this study we have investigated the contribution of a cluster of basic amino acids and two reversibly palmitoylated cysteine residues to endocytic trafficking. Stable cell lines overexpressing chimeric proteins (CD25 and CD46) containing the cytoplasmic domain of PC7 in which the basic cluster alone or together with both palmitoylated cysteines are mutated showed enhanced surface expression as demonstrated by immunofluorescence experiments and surface biotinylation. The mutant proteins no longer recycled to the TGN in antibody uptake experiments and accumulated in an endosomal compartment. Recycling of wild type PC7 to the TGN is blocked by nocodazole, suggesting that PC7 shuttles to the TGN via late endosomes, similar to Furin. Unlike furin, however, PC7 was found to recycle to a region within the TGN, which is deficient in sialyltransferase, as shown by resialylation experiments. In conclusion, a novel motif, composed of a basic amino acid cluster and two palmitoylated cysteines are essential for TGN localization and endocytic trafficking. Copyright © 2017 Elsevier GmbH. All rights reserved.

  17. Recent advances in the understanding and care of familial hypercholesterolaemia: significance of the biology and therapeutic regulation of proprotein convertase subtilisin/kexin type 9.

    PubMed

    Page, Michael M; Stefanutti, Claudia; Sniderman, Allan; Watts, Gerald F

    2015-07-01

    Familial hypercholesterolaemia (FH) is an autosomal co-dominant disorder that markedly raises plasma low-density lipoprotein-cholesterol (LDL-C) concentration, causing premature atherosclerotic coronary artery disease (CAD). FH has recently come under intense focus and, although there is general consensus in recent international guidelines regarding diagnosis and treatment, there is debate about the value of genetic studies. Genetic testing can be cost-effective as part of cascade screening in dedicated centres, but the full mutation spectrum responsible for FH has not been established in many populations, and its use in primary care is not at present logistically feasible. Whether using genetic testing or not, cholesterol screening of family members of index patients with an abnormally raised LDL-C must be used to determine the need for early treatment to prevent the development of CAD. The metabolic defects in FH extend beyond LDL, and may affect triacylglycerol-rich and high-density lipoproteins, lipoprotein(a) and oxidative stress. Achievement of the recommended targets for LDL-C with current treatments is difficult, but this may be resolved by new drug therapies. Lipoprotein apheresis remains an effective treatment for severe FH and, although expensive, it costs less than the two recently introduced orphan drugs (lomitapide and mipomersen) for homozygous FH. Recent advances in understanding of the biology of proprotein convertase subtilisin/kexin type 9 (PCSK9) have further elucidated the regulation of lipoprotein metabolism and led to new drugs for effectively treating hypercholesterolaemia in FH and related conditions, as well as for treating many patients with statin intolerance. The mechanisms of action of PCSK9 inhibitors on lipoprotein metabolism and atherosclerosis, as well as their impact on cardiovascular outcomes and cost-effectiveness, remain to be established.

  18. Efficiency and Safety of Proprotein Convertase Subtilisin/Kexin 9 Monoclonal Antibody on Hypercholesterolemia: A Meta-Analysis of 20 Randomized Controlled Trials

    PubMed Central

    Li, Chuanwei; Lin, Ling; Zhang, Wen; Zhou, Liang; Wang, Hongyong; Luo, Xiaoli; Luo, Hao; Cai, Yue; Zeng, Chunyu

    2015-01-01

    Background Proprotein convertase subtilisin/kexin9 (PCSK9) monoclonal antibody significantly reduces low-density lipoprotein cholesterol level in patients with hypercholesterolemia. The goal of this study was to review recently reported randomized controlled trials to investigate the therapeutic effects and safety of PCSK9 inhibitors. Methods and Results The clinical randomized controlled trials published from inception to March 19, 2015 were identified from The Cochrane Library databases, PUBMED, and EBASE. Randomized controlled trials of at least 8 weeks duration using PCSK9 inhibitors in treating patients with hypercholesterolemia were included. Mean difference (MD) with a 95% CI was used to calculate the continuous data, the standardized mean difference with a 95% CI was used when the unit was not unified, and risk ratio with a 95% CI was used for dichotomous data. After screening, 20 trials fulfilled the inclusion criteria. PCSK9 inhibitors significantly decreased the levels of low-density lipoprotein cholesterol (MD=−65.29 mg/dL, 95% CI: −72.08 to −58.49), total cholesterol (MD=−60.04 mg/dL, 95% CI: −69.95 to −50.13), triglycerides (MD=−12.21 mg/dL, 95% CI: −16.21 to −8.22) and apolipoprotein-B (MD=−41.01 mg/dL, 95% CI: −46.07 to −35.94), lipoprotein(a) (standardized mean difference=−0.94, 95% CI: −1.12 to −0.77) and increased the levels of high-density lipoprotein cholesterol (MD=3.40 mg/dL, 95% CI: 3.12 to 3.68) and apolipoprotein-A1 (MD=6.75 mg/dL, 95% CI: 4.64 to 8.86). There was no significant difference in the incidence of treatment-emergent adverse events (risk ratio=1.01, 95% CI: 0.98 to 1.04), serious treatment-emergent adverse events (risk ratio=1.01, 95% CI: 0.88 to 1.17), and the discontinuation of treatment between the 2 groups (risk ratio=1.07, 95% CI: 0.86 to 1.34). Conclusions The meta-analysis indicated that PCSK9 inhibitors had a strong effect in lowering low-density lipoprotein cholesterol and other

  19. Ectopically Expressed Pro-group X Secretory Phospholipase A2 Is Proteolytically Activated in Mouse Adrenal Cells by Furin-like Proprotein Convertases

    PubMed Central

    Layne, Joseph D.; Shridas, Preetha; Webb, Nancy R.

    2015-01-01

    Group X secretory phospholipase A2 (GX sPLA2) hydrolyzes mammalian cell membranes, liberating free fatty acids and lysophospholipids. GX sPLA2 is produced as a pro-enzyme (pro-GX sPLA2) that contains an N-terminal 11-amino acid propeptide ending in a dibasic motif, suggesting cleavage by a furin-like proprotein convertase (PC). Although propeptide cleavage is clearly required for enzymatic activity, the protease(s) responsible for pro-GX sPLA2 activation have not been identified. We previously reported that GX sPLA2 negatively regulates adrenal glucocorticoid production, likely by suppressing liver X receptor-mediated activation of steroidogenic acute regulatory protein expression. In this study, using a FLAG epitope-tagged pro-GX sPLA2 expression construct (FLAG-pro-GX sPLA2), we determined that adrenocorticotropic hormone (ACTH) enhanced FLAG-pro-GX sPLA2 processing and phospholipase activity secreted by Y1 adrenal cells. ACTH increased the expression of furin and PCSK6, but not other members of the PC family, in Y1 cells. Overexpression of furin and PCSK6 in HEK 293 cells significantly enhanced FLAG-pro-GX sPLA2 processing, whereas siRNA-mediated knockdown of both PCs almost completely abolished FLAG-pro-GX sPLA2 processing in Y1 cells. Expression of either furin or PCSK6 enhanced the ability of GX sPLA2 to suppress liver X receptor reporter activity. The PC inhibitor decanoyl-Arg-Val-Lys-Arg-chloromethyl ketone significantly suppressed FLAG-pro-GX sPLA2 processing and sPLA2 activity in Y1 cells, and it significantly attenuated GX sPLA2-dependent inhibition of steroidogenic acute regulatory protein expression and progesterone production. These findings provide strong evidence that pro-GX sPLA2 is a substrate for furin and PCSK6 proteolytic processing and define a novel mechanism for regulating corticosteroid production in adrenal cells. PMID:25623068

  20. Suppressor of Cytokine Signaling-3 (SOCS-3) Induces Proprotein Convertase Subtilisin Kexin Type 9 (PCSK9) Expression in Hepatic HepG2 Cell Line.

    PubMed

    Ruscica, Massimiliano; Ricci, Chiara; Macchi, Chiara; Magni, Paolo; Cristofani, Riccardo; Liu, Jingwen; Corsini, Alberto; Ferri, Nicola

    2016-02-12

    The suppressor of cytokine signaling (SOCS) proteins are negative regulators of the JAK/STAT pathway activated by proinflammatory cytokines, including the tumor necrosis factor-α (TNF-α). SOCS3 is also implicated in hypertriglyceridemia associated to insulin resistance. Proprotein convertase subtilisin kexin type 9 (PCSK9) levels are frequently found to be positively correlated to insulin resistance and plasma very low density lipoprotein (VLDL) triglycerides concentrations. The present study aimed to investigate the possible role of TNF-α and JAK/STAT pathway on de novo lipogenesis and PCSK9 expression in HepG2 cells. TNF-α induced both SOCS3 and PCSK9 in a concentration-dependent manner. This effect was inhibited by transfection with siRNA anti-STAT3, suggesting the involvement of the JAK/STAT pathway. Retroviral overexpression of SOCS3 in HepG2 cells (HepG2(SOCS3)) strongly inhibited STAT3 phosphorylation and induced PCSK9 mRNA and protein, with no effect on its promoter activity and mRNA stability. Consistently, siRNA anti-SOCS3 reduced PCSK9 mRNA levels, whereas an opposite effect was observed with siRNA anti-STAT3. In addition, HepG2(SOCS3) express higher mRNA levels of key enzymes involved in the de novo lipogenesis, such as fatty-acid synthase, stearoyl-CoA desaturase (SCD)-1, and apoB. These responses were associated with a significant increase of SCD-1 protein, activation of sterol regulatory element-binding protein-1c (SREBP-1), accumulation of cellular triglycerides, and secretion of apoB. HepG2(SOCS3) show lower phosphorylation levels of insulin receptor substrate 1 (IRS-1) Tyr(896) and Akt Ser(473) in response to insulin. Finally, insulin stimulation produced an additive effect with SOCS3 overexpression, further inducing PCSK9, SREBP-1, fatty acid synthase, and apoB mRNA. In conclusion, our data candidate PCSK9 as a gene involved in lipid metabolism regulated by proinflammatory cytokine TNF-α in a SOCS3-dependent manner.

  1. Suppressor of Cytokine Signaling-3 (SOCS-3) Induces Proprotein Convertase Subtilisin Kexin Type 9 (PCSK9) Expression in Hepatic HepG2 Cell Line*

    PubMed Central

    Ruscica, Massimiliano; Ricci, Chiara; Macchi, Chiara; Magni, Paolo; Cristofani, Riccardo; Liu, Jingwen; Corsini, Alberto; Ferri, Nicola

    2016-01-01

    The suppressor of cytokine signaling (SOCS) proteins are negative regulators of the JAK/STAT pathway activated by proinflammatory cytokines, including the tumor necrosis factor-α (TNF-α). SOCS3 is also implicated in hypertriglyceridemia associated to insulin resistance. Proprotein convertase subtilisin kexin type 9 (PCSK9) levels are frequently found to be positively correlated to insulin resistance and plasma very low density lipoprotein (VLDL) triglycerides concentrations. The present study aimed to investigate the possible role of TNF-α and JAK/STAT pathway on de novo lipogenesis and PCSK9 expression in HepG2 cells. TNF-α induced both SOCS3 and PCSK9 in a concentration-dependent manner. This effect was inhibited by transfection with siRNA anti-STAT3, suggesting the involvement of the JAK/STAT pathway. Retroviral overexpression of SOCS3 in HepG2 cells (HepG2SOCS3) strongly inhibited STAT3 phosphorylation and induced PCSK9 mRNA and protein, with no effect on its promoter activity and mRNA stability. Consistently, siRNA anti-SOCS3 reduced PCSK9 mRNA levels, whereas an opposite effect was observed with siRNA anti-STAT3. In addition, HepG2SOCS3 express higher mRNA levels of key enzymes involved in the de novo lipogenesis, such as fattyacid synthase, stearoyl-CoA desaturase (SCD)-1, and apoB. These responses were associated with a significant increase of SCD-1 protein, activation of sterol regulatory element-binding protein-1c (SREBP-1), accumulation of cellular triglycerides, and secretion of apoB. HepG2SOCS3 show lower phosphorylation levels of insulin receptor substrate 1 (IRS-1) Tyr896 and Akt Ser473 in response to insulin. Finally, insulin stimulation produced an additive effect with SOCS3 overexpression, further inducing PCSK9, SREBP-1, fatty acid synthase, and apoB mRNA. In conclusion, our data candidate PCSK9 as a gene involved in lipid metabolism regulated by proinflammatory cytokine TNF-α in a SOCS3-dependent manner. PMID:26668321

  2. Efficacy and safety of proprotein convertase subtilisin-kexin type 9 (PCSK9) inhibitors, alirocumab and evolocumab, a post-commercialization study.

    PubMed

    Choi, Joshua; Khan, Amir M; Jarmin, Michael; Goldenberg, Naila; Glueck, Charles J; Wang, Ping

    2017-07-24

    Efficacy-safety of proprotein convertase subtilisin-kexin type 9 (PCSK9) inhibitors, alirocumab (ALI) and evolocumab (EVO), have previously been evaluated through controlled clinical trials with selective patient groups. Post-commercially, in 69 patients with heterozygous familial hypercholesterolemia (HeFH) and/or cardiovascular disease (CVD) with suboptimal LDL cholesterol (LDLC) lowering on maximal tolerated LDLC therapy, we assessed efficacy and safety of ALI and EVO. Post-commercially, we started 29 patients on ALI 75 mg, 18 on ALI 150 mg, and 22 on EVO 140 mg every 2 weeks added to a maximally tolerated LDLC-lowering regimen. Since LDLC lowering did not differ between ALI 150 and EVO 140 mg, ALI 150-EVO 140 data were pooled (ALI-EVO). Changes in LDLC and AHA and NIH calculated 10-year CVD risks were assessed. Of the 69 patients, 25 had HeFH, 25 CVD, and 19 had both. At entry, 23 (33%) took statins and 46 (67%) were statin-intolerant. Mean ± SD and median follow-up were 49 ± 13 and 49 weeks on ALI 75 mg, and 37 ± 12 and 33 weeks on ALI-EVO. In the ALI-EVO group (n = 40), median LDLC fell from 165 mg/dl at entry to 70 mg/dl (median - 59%, p < .0001). AHA 10-year calculated CVD risk fell from 10.2 to 5.5% (median - 28%, p < .0001), and by the NIH calculator from 14.2 to 3.6% (median - 78%, p < .0001). In the ALI 75 mg group (n = 29), entry LDLC fell from 115 to 68 mg/dl (median - 39%, p < .0001). AHA 10-year calculated CVD risk fell from 11.5 to 7.3% (median - 20%, p = .004), and NIH 10-year risk from 12.9 to 5.1% (median 67%, p < .0001). Absolute and percent change in LDLC was independent of statin use. There were flu-like symptoms in 14% of patients. Adverse events did not differ (p > 0.05) between ALI 75 mg and ALI-EVO. In patients with HeFH and/or CVD, LDLC decreased from 115 to 68 mg/dl (39%) on ALI 75 mg with mean follow-up of 49 weeks, and from 165 to 70 mg/dl (59%) on ALI-EVO over 37 weeks, p

  3. The proprotein convertase subtilisin/kexin type 9 gene E670G polymorphism and serum lipid levels in the Guangxi Bai Ku Yao and Han populations

    PubMed Central

    2011-01-01

    Background Proprotein convertase subtilisin-like kexin type 9 (PCSK9) plays a key role in regulating plasma low-density lipoprotein cholesterol (LDL-C) levels. However, the association of E670G (rs505151) polymorphism in the PCSK9 gene and serum lipid levels is inconsistent in several previous studies. The present study was undertaken to detect the association of PCSK9 E670G polymorphism and several environmental factors with serum lipid levels in the Guangxi Bai Ku Yao and Han populations. Methods A total of 649 subjects of Bai Ku Yao and 646 participants of Han were randomly selected from our previous samples. Genotypes of the PCSK9 E670G polymorphism were determined via polymerase chain reaction and restriction fragment length polymorphism combined with gel electrophoresis, and then confirmed by direct sequencing. Results Serum levels of total cholesterol, high-density lipoprotein cholesterol (HDL-C), LDL-C, and apolipoprotein (Apo) AI were lower in Bai Ku Yao than in Han (P < 0.01 for all). The frequency of G allele was 2.00% in Bai Ku Yao and 4.80% in Han (P < 0.01). There was significant difference in the genotypic and allelic frequencies between Bai Ku Yao and Han (P < 0.01); between normal LDL-C (≤ 3.20 mmol/L) and high LDL-C subgroups (> 3.20 mmol/L, P < 0.01) in Bai Ku Yao; and between normal HDL-C (≥ 0.91 mmol/L) and low HDL-C (< 0.91 mmol/L, P < 0.05), between normal ApoAI (≥ 1.00 g/L) and low ApoAI (< 1.00 g/L, P < 0.05), or between normal ApoAI/ApoB ratio (≥ 1.00) and low ApoAI/ApoB ratio (< 1.00, P < 0.01) subgroups in Han. The G allele carriers in Han had higher serum HDL-C levels and the ratio of ApoAI to ApoB than the G allele noncarriers. The G allele carriers in Han had higher serum HDL-C and ApoAI levels than the G allele noncarriers in males (P < 0.05 for each), whereas the G allele carriers had lower serum ApoB levels and higher the ratio of ApoAI to ApoB than the G allele noncarriers in females (P < 0.05 for all). Serum HDL-C and Apo

  4. The proteolytic processing of pro-platelet-derived growth factor-A at RRKR(86) by members of the proprotein convertase family is functionally correlated to platelet-derived growth factor-A-induced functions and tumorigenicity.

    PubMed

    Siegfried, Géraldine; Khatib, Abdel-Majid; Benjannet, Suzanne; Chrétien, Michel; Seidah, Nabil G

    2003-04-01

    Although altered expression of platelet-derived growth factor (PDGF)-A is a hallmark of many cancers, the importance of pro-PDGF-A conversion to PDGF-A in tumorigenesis and the cognate protease(s) is unknown. Pro-PDGF-A processing occurs at pairs of basic residues, likely involving the proprotein convertases (PCs). In the colon carcinoma cell line LoVo, we found that Furin is the most potent PDGF-A convertase. Mutation of the PC-site RRKR(86) to ARKA(86) inhibited pro-PDGF-A processing, its receptor tyrosine phosphorylation, and cell proliferation. This processing is also blocked by the PC preprosegments (pps) ppFurin, ppPC5, and ppPACE4, and by the Furin-variants of alpha2-macroglobulin and alpha1-antitrypsin. Chinese hamster ovary cells overexpressing pro-PDGF-A (ARKA(86)) failed to induce tumors in nude mice. Thus, PC-directed inhibitors might represent new agents for therapy in neoplasia induced by PDGF-A.

  5. Implication of the proprotein convertases furin, PC5 and PC7 in the cleavage of surface glycoproteins of Hong Kong, Ebola and respiratory syncytial viruses: a comparative analysis with fluorogenic peptides.

    PubMed Central

    Basak, A; Zhong, M; Munzer, J S; Chrétien, M; Seidah, N G

    2001-01-01

    Fluorogenic peptides encompassing the processing sites of envelope glycoproteins of the infectious influenza A Hong Kong virus (HKV), Ebola virus (EBOV) and respiratory syncytial virus (RSV) were tested for cleavage by soluble recombinants of the proprotein convertases furin, PC5 and PC7. Kinetic studies with these intramolecularly quenched fluorogenic peptides revealed selective cleavages at the physiological dibasic sites. The HKV peptide is cleaved by both furin and PC5 with similar efficacy; in comparison, PC7 cleaves this substrate poorly. In contrast with the basic tetrapeptide insertion within the haemagglutinin sequence of HKV, two other dipeptide insertions revealed a poorer cleavage with a similar rank order of potency. These results demonstrate that the N-terminal RERR insertion to the wild-type avian RKKR downward arrow sequence is functionally significant, and suggest that the approx. 5-fold increase in cleavage efficacy contributes to the high infectivity of the H5N1 virus subtype. With regard to RSV peptide processing, PC7 is twice as effective as PC5 and furin. The EBOV peptide was processed with similar efficiency by the three enzymes. Our observations that all of these cleavages can be effectively inhibited by a plant andrographolide derivative at 250 microM or less might aid in the design of potent convertase inhibitors as alternative antiviral therapies. PMID:11171050

  6. A Mechanism-Based Pharmacokinetic/Pharmacodynamic Model for Bococizumab, a Humanized Monoclonal Antibody Against Proprotein Convertase Subtilisin/Kexin Type 9, and Its Application in Early Clinical Development.

    PubMed

    Udata, Chandrasekhar; Garzone, Pamela D; Gumbiner, Barry; Joh, Tenshang; Liang, Hong; Liao, Kai-Hsin; Williams, Jason H; Meng, Xu

    2017-07-01

    Bococizumab (RN316/PF-04950615), a humanized monoclonal antibody, binds to secreted proprotein convertase subtilisin/kexin type 9 (PCSK9) and prevents its downregulation of low-density lipoprotein receptor, leading to improved clearance and reduction of low-density lipoprotein cholesterol (LDL-C) in plasma. A mechanism-based drug-target binding model was developed, accounting for bococizumab, PCSK9, and LDL-C concentrations and the effects of concomitant administration of statins. This model was utilized to better understand the pharmacokinetic/pharmacodynamic (PK/PD) data obtained from 3 phase 1 and 2 phase 2a clinical studies. First, simulations performed with this model demonstrated that the conventional method of the area-under-the-curve ratio for bioavailability determination underestimated the subcutaneous bioavailability of bococizumab due to its target-mediated disposition. Second, a covariate model component for statin effects on bococizumab PK/PD was characterized, including a description of the decreased baseline LDL-C, increased baseline PCSK9, and increased LDL-C lowering with concomitant use of statins. Last, the impact of the dosing regimens with and without a dose holiday on bococizumab's LDL-C-lowering effectiveness was shown to be predictable due to the well-characterized PK-PD relationship. © 2017, The American College of Clinical Pharmacology.

  7. The cysteine-rich domain of the secreted proprotein convertases PC5A and PACE4 functions as a cell surface anchor and interacts with tissue inhibitors of metalloproteinases.

    PubMed

    Nour, Nadia; Mayer, Gaétan; Mort, John S; Salvas, Alexandre; Mbikay, Majambu; Morrison, Charlotte J; Overall, Christopher M; Seidah, Nabil G

    2005-11-01

    The proprotein convertases PC5, PACE4 and furin contain a C-terminal cysteine-rich domain (CRD) of unknown function. We demonstrate that the CRD confers to PC5A and PACE4 properties to bind tissue inhibitors of metalloproteinases (TIMPs) and the cell surface. Confocal microscopy and biochemical analyses revealed that the CRD is essential for cell surface tethering of PC5A and PACE4 and that it colocalizes and coimmunoprecipitates with the full-length and C-terminal domain of TIMP-2. Surface-bound PC5A in TIMP-2 null fibroblasts was only observed upon coexpression with TIMP-2. In COS-1 cells, plasma membrane-associated PC5A can be displaced by heparin, suramin, or heparinases I and III and by competition with excess exogenous TIMP-2. Furthermore, PC5A and TIMP-2 are shown to be colocalized over the surface of enterocytes in the mouse duodenum and jejunum, as well as in liver sinusoids. In conclusion, the CRD of PC5A and PACE4 functions as a cell surface anchor favoring the processing of their cognate surface-anchored substrates, including endothelial lipase.

  8. Development of selectivity of alpha1-antitrypsin variant by mutagenesis in its reactive site loop against proprotein convertase. A crucial role of the P4 arginine in PACE4 inhibition.

    PubMed

    Tsuji, Akihiko; Ikoma, Takayuki; Hashimoto, Emi; Matsuda, Yoshiko

    2002-02-01

    PACE4, furin and PC6 are Ca2+-dependent serine endoproteases that belong to the subtilisin-like proprotein convertase (SPC) family. Recent reports have supported the involvement of these enzymes in processing of growth/differentiation factors, viral replication, activation of bacterial toxins and tumorigenesis, indicating that these enzymes are a fascinating target for therapeutic agents. In this work, we evaluated the sensitivity and selectivity of three rat alpha1-antitrypsin variants which contained RVPR352, AVRR352 and RVRR352, respectively, within their reactive site loop using both inhibition of enzyme activity toward a fluorogenic substrate in vitro and formation of a SDS-stable protease/inhibitor complex ex vivo. The RVPR variant showed relatively broad selectivity, whereas the AVRR and RVRR variants were more selective than the RVPR variant. The AVRR variant inhibited furin and PC6 but not PACE4. This selectivity was further confirmed by complex formation and inhibition of pro-complement C3 processing. On the other hand, although the RVRR variant inhibited both PACE4 and furin effectively, it needed a 600-fold higher concentration than the RVPR variant to inhibit PC6 in vitro. These inhibitors will be useful tools in helping us to understand the roles of PACE4, furin and PC6.

  9. Transcriptional regulation of subtilisin-like proprotein convertase PACE4 by E2F: possible role of E2F-mediated upregulation of PACE4 in tumor progression.

    PubMed

    Yuasa, Keizo; Suzue, Kaori; Nagahama, Masami; Matsuda, Yoshiko; Tsuji, Akihiko

    2007-11-01

    PACE4, a member of the subtilisin-like proprotein convertase (SPC) family, is expressed at high levels in certain tumor cells and plays a role in metastatic progression through activation of matrix metalloproteinases. The mechanism leading to overexpression of PACE4 in tumor cells remains unclear. In this study, we show that the E2F1 transcription factor, which is implicated in carcinoma invasiveness, upregulates the expression of PACE4. HT1080 (highly tumorigenic and invasive) cells expressed much higher levels of PACE4 and E2F family (E2F1 and E2F2) transcripts than IMR90 (normal fibroblast) cells. Expression levels of other SPCs (furin and PC6) remained unchanged in these cells. Promoter analysis indicated that two E2F consensus binding sites (-117/-110 and -86/-79) in the 5'-flanking region of the human PACE4 gene function as positive regulatory elements. Mutation of these sites abolished PACE4 promoter response to E2F1 as well as binding of E2F1 in electrophoretic mobility-shift assays. Other E2F members, E2F2 and E2F3, also activated PACE4 expression, as in the case of E2F1. These results indicate a novel mechanism for E2F family-mediated promotion of carcinoma invasiveness through PACE4.

  10. A visible light induced photoelectrochemical aptsensor constructed by aligned ZnO@CdTe core shell nanocable arrays/carboxylated g-C3N4 for the detection of Proprotein convertase subtilisin/kexin type 6 gene.

    PubMed

    Pang, Xuehui; Pan, Jihong; Gao, Picheng; Wang, Youying; Wang, Liguo; Du, Bin; Wei, Qin

    2015-12-15

    It was reported that Proprotein convertase subtilisin/kexin type 6 (PCSK6) can promote the progression of rheumatoid arthritis to a higher aggressive status. In this work, a novel visible light induced photoelectrochemical (PEC) platform was designed to detect PCSK6 gene. ZnO@CdTe nanocable arrays/carboxylated g-C3N4 used as the PEC signal generator. Hexagonal ZnO nanorods grew on ITO electrode firstly. CdTe were then electrodeposited on the ZnO nanorods surface to enhance the photogenerated h(+)/e(-) separation efficiency. Carboxylated g-C3N4 was utilized to improve h(+)/e(-) separation efficiency and anchor the capture probes of PCSK6 gene by the covalent bonding effect. The 5' and 3' primers captured PCSK6 ssDNA by the specific recognition. The linear range was 10 pg/mL to 20.0 ng/mL with a detection limit of 2 pg/mL.

  11. Inhibition and transcriptional silencing of a subtilisin-like proprotein convertase, PACE4/SPC4, reduces the branching morphogenesis of and AQP5 expression in rat embryonic submandibular gland.

    PubMed

    Akamatsu, Tetsuya; Azlina, Ahmad; Purwanti, Nunuk; Karabasil, Mileva Ratko; Hasegawa, Takahiro; Yao, Chenjuan; Hosoi, Kazuo

    2009-01-15

    The submandibular gland (SMG) develops through the epithelial-mesenchymal interaction mediated by many growth/differentiation factors including activin and BMPs, which are synthesized as inactive precursors and activated by subtilisin-like proprotein convertases (SPC) following cleavage at their R-X-K/R-R site. Here, we found that Dec-RVKR-CMK, a potent inhibitor of SPC, inhibited the branching morphogenesis of the rat embryonic SMG, and caused low expression of a water channel AQP5, in an organ culture system. Dec-RVKR-CMK also decreased the expression of PACE4, a SPC member, but not furin, another SPC member, suggesting the involvement of PACE4 in the SMG development. Heparin, which is known to translocate PACE4 in the extracellular matrix into the medium, and an antibody specific for the catalytic domain of PACE4, both reduced the branching morphogenesis and AQP5 expression in the SMG. The inhibitory effects of Dec-RVKR-CMK were partially rescued by the addition of recombinant BMP2, whose precursor is one of the candidate substrates for PACE4 in vivo. Further, the suppression of PACE4 expression by siRNAs resulted in decreased expression of AQP5 and inhibition of the branching morphogenesis in the present organ culture system. These observations suggest that PACE4 regulates the SMG development via the activation of some growth/differentiation factors.

  12. Fasting induces hyperlipidemia in mice overexpressing proprotein convertase subtilisin kexin type 9: lack of modulation of very-low-density lipoprotein hepatic output by the low-density lipoprotein receptor.

    PubMed

    Lambert, Gilles; Jarnoux, Anne-Laure; Pineau, Thierry; Pape, Olivier; Chetiveaux, Maud; Laboisse, Christian; Krempf, Michel; Costet, Philippe

    2006-10-01

    Several proprotein convertase subtilisin kexin type 9 (PCSK9) mutations lead to familial hypercholesterolemia by virtue of its role as a negative modulator of the low-density lipoprotein receptor (LDLr). Here, we uncover that upon dietary challenge, the down-regulation of the LDLr is also a key mechanism by which PCSK9 modulates the hepatic production of apolipoprotein-B-containing lipoproteins. Thus, adenoviral-mediated overexpression of PCSK9 in 24-h fasted mice results in massive hyperlipidemia, due to a striking increase in very-low-density lipoprotein (VLDL) triglycerides and apolipoprotein B100 hepatic output. Similar studies in LDLr (-/-) mice demonstrate that PCSK9-mediated alteration of VLDL output in the fasted state requires the LDLr. This increased production of VLDL was associated with a concomitant reduction of intrahepatic lipid stores as well as a lack of down-regulation of peroxisome proliferator-activated receptor-alpha activity and target genes expression. Finally, we show that PCSK9 hepatic expression is inhibited by the hypotriglyceridemic peroxisome proliferator-activated receptor-alpha agonist fenofibrate. In summary, the negative modulation of LDLr expression by PCSK9, which decreases plasma LDL clearance, also promotes an overproduction of nascent VLDL in vivo upon fasting.

  13. CdSe quantum dot-functionalized TiO2 nanohybrids as a visible light induced photoelectrochemical platform for the detection of proprotein convertase subtilisin/kexin type 6.

    PubMed

    Pang, Xuehui; Pan, Jihong; Wang, Lin; Ren, Wei; Gao, Picheng; Wei, Qin; Du, Bin

    2015-09-15

    Proprotein convertase subtilisin/kexin type 6 (PCSK6) plays a major role in promoting the progression of rheumatoid arthritis to a higher aggressive status. A novel highly sensitive photoelectrochemical platform was developed for the detection of PCSK6 by using CdSe quantum dots (QDs)-functionalized TiO2 nanoparticles (NPs) nanohybrids (TiO2@CdSe) as the photo-to-electron conversion medium. TiO2@CdSe showed excellent visible-light absorbency, and much higher photoelectrochemical activity than both CdSe QDs and TiO2 NPs. The 5' and 3' primers of PCSK6 ssDNA acted as capture probes to realize the detection of PCSK6 ssDNA by the specific recognition. The capture probes can be fixed by poly-l-lysine (PLL) through positively strong electrostatic attraction and the carboxyl group of TiO2@CdSe nanohybrids. PLL was electropolymerized on ITO electrode by cyclic voltammetry (CV). Simultaneously, the amino group of PLL can interact with the carboxyl group of TiO2@CdSe nanohybrids to enhance the stability of the photoelectrochemical signal. The fabricated aptsensor exhibited excellent performance towards PCSK6 with a wide linear range (0.5 pg/mL to 80.0 ng/mL) and a detection limit of 0.1 fg/mL. This work opens up a new detection platform for PCSK6 with good sensitivity, reproducibility and stability.

  14. The expression of proprotein convertase PACE4 is highly regulated by Hash-2 in placenta: possible role of placenta-specific basic helix-loop-helix transcription factor, human achaete-scute homologue-2.

    PubMed

    Koide, Shizuyo; Yoshida, Ichiro; Tsuji, Akihiko; Matsuda, Yoshiko

    2003-09-01

    PACE4 is a member of the mammalian subtilisin-like proprotein convertase (SPC) family, which contribute to the activation of transforming growth factor (TGF) beta family proteins. We previously reported that PACE4 is highly expressed in syncytiotrophoblasts of human placenta [Tsuji et al. (2003) BIOCHIM: Biophys. Acta 1645, 95-104]. In this study, the regulatory mechanism for PACE4 expression in placenta was analyzed using a human placental choriocarcinoma cell line, BeWo cells. Promoter analysis indicated that an E-box cluster (E4-E9) in the 5'-flanking region of the PACE4 gene acts as a negative regulatory element. The binding of human achaete-scute homologue 2 (Hash-2) to the E-box cluster was shown by gel mobility-shift assay. The overexpression of Hash-2 caused a marked decrease in PACE4 gene expression. When BeWo cells were grown under low oxygen (2%) conditions, the expression of Hash-2 decreased, while that of PACE4 increased. In both cases, other SPCs, such as furin, PC5/6, and PC7/8, were not affected. Further, PACE4 expression was found to be developmentally regulated in rat placenta. By in situ hybridization, Mash-2 (mammalian achaete-scute homologue 2) mRNA was found to be expressed in the spongiotrophoblast layer where PACE4 was not expressed. In contrast, the PACE4 mRNA was expressed mainly in the labyrinthine layer where Mash-2 was not detected. These results suggest that PACE4 expression is down-regulated by Hash-2/Mash-2 in both human and rat placenta and that many bioactive proteins might be regulated by PACE4 activity.

  15. Dietary high oleic canola oil supplemented with docosahexaenoic acid attenuates plasma proprotein convertase subtilisin kexin type 9 (PCSK9) levels in participants with cardiovascular disease risk: A randomized control trial.

    PubMed

    Pu, Shuaihua; Rodríguez-Pérez, Celia; Ramprasath, Vanu Ramkumar; Segura-Carretero, Antonio; Jones, Peter J H

    2016-12-01

    Proprotein convertase subtilisin/kexin type 9 (PCSK9) is a novel circulating protein which plays an important role in regulation of cholesterol metabolism by promoting hepatic LDL receptor degradation. However, the action of dietary fat composition on PCSK9 levels remains to be fully elucidated. The objective was to investigate the action of different dietary oils on circulating PCSK9 levels in the Canola Oil Multicenter Intervention Trial (COMIT). COMIT employed a double-blinded crossover randomized control design, consisting of five 30-d treatment periods. Diets were provided based on a 3000Kcal/d intake, including a 60g/d treatment of conventional canola oil (Canola), a high oleic canola/DHA oil blend (CanolaDHA), a corn/safflower oil blend (CornSaff), a flax/safflower oil blend (FlaxSaff) or a high oleic canola oil (CanolaOleic). Plasma PCSK9 levels were assessed using ELISA at the end of each phase. Lipid profiles (n=84) showed that CanolaDHA feeding resulted in the highest (P<0.05) serum total cholesterol (TC, 5.06±0.09mmol/L) and LDL-cholesterol levels (3.15±0.08mmol/L) across all five treatments. CanolaDHA feeding also produced the lowest (P<0.05) plasma PCSK9 concentrations (216.42±8.77ng/mL) compared to other dietary oil treatments. Plasma PCSK9 levels positively correlated (P<0.05) with serum TC, LDL-cholesterol, apolipoprotein A, and apolipoprotein B levels but did not correlate to HDL-cholesterol levels. Results indicate that post-treatment response in PCSK9 may be altered with the CanolaDHA diet. In conclusion, the elevated LDL-C levels from a DHA oil treatment may not be relevant for the observed decline in PCSK9 levels.

  16. Plasma Membrane Tetraspanin CD81 Complexes with Proprotein Convertase Subtilisin/Kexin Type 9 (PCSK9) and Low Density Lipoprotein Receptor (LDLR), and Its Levels Are Reduced by PCSK9.

    PubMed

    Le, Quoc-Tuan; Blanchet, Matthieu; Seidah, Nabil G; Labonté, Patrick

    2015-09-18

    Proprotein convertase subtilisin/kexin type 9 (PCSK9) is an important factor in plasma cholesterol regulation through modulation of low density lipoprotein receptor (LDLR) levels. Naturally occurring mutations can lead to hyper- or hypocholesterolemia in human. Recently, we reported that PCSK9 was also able to modulate CD81 in Huh7 cells. In the present study, several gain-of-function and loss-of-function mutants as well as engineered mutants of PCSK9 were compared for their ability to modulate the cell surface expression of LDLR and CD81. Although PCSK9 gain-of-function D374Y enhanced the degradation both receptors, D374H and D129N seemed to only reduce LDLR levels. In contrast, mutations in the C-terminal hinge-cysteine-histidine-rich domain segment primarily affected the PCSK9-induced CD81 degradation. Furthermore, when C-terminally fused to an ACE2 transmembrane anchor, the secretory N-terminal catalytic or hinge-cysteine-histidine-rich domain domains of PCSK9 were able to reduce CD81 and LDLR levels. These data confirm that PCSK9 reduces CD81 levels via an intracellular pathway as reported for LDLR. Using immunocytochemistry, a proximity ligation assay, and co-immunoprecipitation, we found that the cell surface level of PCSK9 was enhanced upon overexpression of CD81 and that both PCSK9 and LDLR interact with this tetraspanin protein. Interestingly, using CHO-A7 cells lacking LDLR expression, we revealed that LDLR was not required for the degradation of CD81 by PCSK9, but its presence strengthened the PCSK9 effect.

  17. New hypotheses about the structure-function of proprotein convertase subtilisin/kexin type 9: analysis of the epidermal growth factor-like repeat A docking site using WaterMap.

    PubMed

    Pearlstein, Robert A; Hu, Qi-Ying; Zhou, Jing; Yowe, David; Levell, Julian; Dale, Bethany; Kaushik, Virendar K; Daniels, Doug; Hanrahan, Susan; Sherman, Woody; Abel, Robert

    2010-09-01

    LDL cholesterol (LDL-C) is cleared from plasma via cellular uptake and internalization processes that are largely mediated by the low-density lipoprotein cholesterol receptor (LDL-R). LDL-R is targeted for lysosomal degradation by association with proprotein convertase subtilisin-kexin type 9 (PCSK9). Gain of function mutations in PCSK9 can result in excessive loss of receptors and dyslipidemia. On the other hand, receptor-sparing phenomena, including loss-of-function mutations or inhibition of PCSK9, can lead to enhanced clearance of plasma lipids. We hypothesize that desolvation and resolvation processes, in many cases, constitute rate-determining steps for protein-ligand association and dissociation, respectively. To test this hypothesis, we analyzed and compared the predicted desolvation properties of wild-type versus gain-of-function mutant Asp374Tyr PCSK9 using WaterMap, a new in silico method for predicting the preferred locations and thermodynamic properties of water solvating proteins ("hydration sites"). We compared these results with binding kinetics data for PCSK9, full-length LDL-R ectodomain, and isolated EGF-A repeat. We propose that the fast k(on) and entropically driven thermodynamics observed for PCSK9-EGF-A binding stem from the functional replacement of water occupying stable PCSK9 hydration sites (i.e., exchange of PCSK9 H-bonds from water to polar EGF-A groups). We further propose that the relatively fast k(off) observed for EGF-A unbinding stems from the limited displacement of solvent occupying unstable hydration sites. Conversely, the slower k(off) observed for EGF-A and LDL-R unbinding from Asp374Tyr PCSK9 stems from the destabilizing effects of this mutation on PCSK9 hydration sites, with a concomitant increase in the persistence of the bound complex.

  18. Plasma Membrane Tetraspanin CD81 Complexes with Proprotein Convertase Subtilisin/Kexin Type 9 (PCSK9) and Low Density Lipoprotein Receptor (LDLR), and Its Levels Are Reduced by PCSK9*

    PubMed Central

    Le, Quoc-Tuan; Blanchet, Matthieu; Seidah, Nabil G.; Labonté, Patrick

    2015-01-01

    Proprotein convertase subtilisin/kexin type 9 (PCSK9) is an important factor in plasma cholesterol regulation through modulation of low density lipoprotein receptor (LDLR) levels. Naturally occurring mutations can lead to hyper- or hypocholesterolemia in human. Recently, we reported that PCSK9 was also able to modulate CD81 in Huh7 cells. In the present study, several gain-of-function and loss-of-function mutants as well as engineered mutants of PCSK9 were compared for their ability to modulate the cell surface expression of LDLR and CD81. Although PCSK9 gain-of-function D374Y enhanced the degradation both receptors, D374H and D129N seemed to only reduce LDLR levels. In contrast, mutations in the C-terminal hinge-cysteine-histidine-rich domain segment primarily affected the PCSK9-induced CD81 degradation. Furthermore, when C-terminally fused to an ACE2 transmembrane anchor, the secretory N-terminal catalytic or hinge-cysteine-histidine-rich domain domains of PCSK9 were able to reduce CD81 and LDLR levels. These data confirm that PCSK9 reduces CD81 levels via an intracellular pathway as reported for LDLR. Using immunocytochemistry, a proximity ligation assay, and co-immunoprecipitation, we found that the cell surface level of PCSK9 was enhanced upon overexpression of CD81 and that both PCSK9 and LDLR interact with this tetraspanin protein. Interestingly, using CHO-A7 cells lacking LDLR expression, we revealed that LDLR was not required for the degradation of CD81 by PCSK9, but its presence strengthened the PCSK9 effect. PMID:26195630

  19. Effect of alirocumab, a monoclonal proprotein convertase subtilisin/kexin 9 antibody, on lipoprotein(a) concentrations (a pooled analysis of 150 mg every two weeks dosing from phase 2 trials).

    PubMed

    Gaudet, Daniel; Kereiakes, Dean J; McKenney, James M; Roth, Eli M; Hanotin, Corinne; Gipe, Daniel; Du, Yunling; Ferrand, Anne-Catherine; Ginsberg, Henry N; Stein, Evan A

    2014-09-01

    Lipoprotein(a) [Lp(a)] is an independent risk factor for cardiovascular disease, with limited treatment options. This analysis evaluated the effect of a monoclonal antibody to proprotein convertase subtilisin/kexin 9, alirocumab 150 mg every 2 weeks (Q2W), on Lp(a) levels in pooled data from 3 double-blind, randomized, placebo-controlled, phase 2 studies of 8 or 12 weeks' duration conducted in patients with hypercholesterolemia on background lipid-lowering therapy (NCT01266876, NCT01288469, and NCT01288443). Data were available for 102 of 108 patients who received alirocumab 150 mg Q2W and 74 of 77 patients who received placebo. Alirocumab resulted in a significant reduction in Lp(a) from baseline compared with placebo (-30.3% vs -0.3%, p <0.0001). Median percentage Lp(a) reductions in the alirocumab group were of a similar magnitude across a range of baseline Lp(a) levels, resulting in greater absolute reductions in Lp(a) in patients with higher baseline levels. Regression analysis indicated that <5% of the variance in the reduction of Lp(a) was explained by the effect of alirocumab on low-density lipoprotein cholesterol. In conclusion, pooled data from 3 phase 2 trials demonstrate substantive reduction in Lp(a) with alirocumab 150 mg Q2W, including patients with baseline Lp(a) >50 mg/dl. Reductions in Lp(a) only weakly correlated with the magnitude of low-density lipoprotein cholesterol lowering.

  20. Results of bococizumab, a monoclonal antibody against proprotein convertase subtilisin/kexin type 9, from a randomized, placebo-controlled, dose-ranging study in statin-treated subjects with hypercholesterolemia.

    PubMed

    Ballantyne, Christie M; Neutel, Joel; Cropp, Anne; Duggan, William; Wang, Ellen Q; Plowchalk, David; Sweeney, Kevin; Kaila, Nitin; Vincent, John; Bays, Harold

    2015-05-01

    Bococizumab is a humanized monoclonal antibody binding proprotein convertase subtilisin/kexin type 9, which may be a potential therapeutic option for reducing low-density lipoprotein cholesterol (LDL-C) levels in patients with hypercholesterolemia. In this 24-week, multicenter, double-blind, placebo-controlled, dose-ranging study (NCT01592240), subjects with LDL-C levels≥80 mg/dl on stable statin therapy were randomized to Q14 days subcutaneous placebo or bococizumab 50, 100, or 150 mg or Q28 days subcutaneous placebo or bococizumab 200 or 300 mg. Doses of bococizumab were reduced if LDL-C levels persistently decreased to ≤25 mg/dl. The primary end point was the absolute change in LDL-C levels from baseline to week 12 after placebo or bococizumab administration. Continuation of bococizumab administration through to week 24 enabled the collection of safety data over an extended period. Of the 354 subjects randomized, 351 received treatment (placebo [n=100] or bococizumab [n=251]). The most efficacious bococizumab doses were 150 mg Q14 days and 300 mg Q28 days. Compared with placebo, bococizumab 150 mg Q14 days reduced LDL-C at week 12 by 53.4 mg/dl and bococizumab 300 mg Q28 days reduced LDL-C by 44.9 mg/dl; this was despite dose reductions in 32.5% and 34.2% of subjects at week 10 or 8, respectively. Pharmacokinetic/pharmacodynamic model-based simulation assuming no dose reductions predicted that bococizumab would lower LDL-C levels by 72.2 and 55.4 mg/dl, respectively. Adverse events were similar across placebo and bococizumab groups. Few subjects (n=7; 2%) discontinued treatment because of treatment-related adverse events. In conclusion, bococizumab significantly reduced LDL-C across all doses despite dose reductions in many subjects. Model-based simulations predicted greater LDL-C reduction in the absence of bococizumab dose reduction. The Q14 days regimen is being evaluated in phase 3 clinical trials.

  1. Efficacy, safety, and tolerability of a monoclonal antibody to proprotein convertase subtilisin/kexin type 9 in combination with a statin in patients with hypercholesterolaemia (LAPLACE-TIMI 57): a randomised, placebo-controlled, dose-ranging, phase 2 study

    PubMed Central

    Giugliano, Robert P; Desai, Nihar R; Kohli, Payal; Rogers, William J; Somaratne, Ransi; Huang, Fannie; Liu, Thomas; Mohanavelu, Satishkumar; Hoffman, Elaine B; McDonald, Shannon T; Abrahamsen, Timothy E; Wasserman, Scott M; Scott, Robert; Sabatine, Marc S

    2013-01-01

    Summary Background LDL cholesterol (LDL-C) is a well established risk factor for cardiovascular disease. Proprotein convertase subtilisin/kexin type 9 (PCSK9) binds LDL receptors, targeting them for degradation. We therefore assessed the efficacy, safety, and tolerability of AMG 145, a human monoclonal IgG2 antibody against PCSK9, in stable patients with hypercholesterolemia on a statin. Methods In a phase 2, dose-ranging study done in 78 centres in the USA, Canada, Denmark, Hungary, and Czech Republic, patients (aged 18–80 years) with LDL-C greater than 2.2 mmol/L on a stable dose of statin (with or without ezetimibe), were randomly assigned equally, through an interactive voice response system, to subcutaneous injections of AMG 145 70 mg, 105 mg, or 140 mg, or matching placebo every 2 weeks; or subcutaneous injections of AMG 145 280 mg, 350 mg, or 420 mg, or matching placebo every 4 weeks. Everyone was masked to treatment assignment within the every 2 weeks and every 4 weeks schedules. The primary endpoint was the percentage change in LDL-C concentration from baseline after 12 weeks. Analysis was by modified intention to treat. This study is registered with ClinicalTrials.gov, number NCT01380730. Findings 631 patients with hypercholesterolaemia were randomly assigned to AMG 145 70 mg (n=79), 105 mg (n=79), or 140 mg (n=78), or matching placebo (n=78) every 2 weeks; or AMG 145 280 mg (n=79), 350 mg (n=79), and 420 mg (n=80), and matching placebo (n=79) every 4 weeks. At the end of the dosing interval at week 12, the mean LDL-C concentrations were reduced generally dose dependently by AMG 145 every 2 weeks (ranging from 41.8% to 66.1%; p<0.0001 for each dose vs placebo) and AMG 145 every 4 weeks (ranging from 41.8% to 50.3%; p<0.0001). No treatment-related serious adverse events occurred. The frequencies of treatment-related adverse events were similar in the AMG 145 and placebo groups (39 [8%] of 474 vs 11 [7%] of 155); none of these events were severe or life

  2. Ectopically expressed pro-group X secretory phospholipase A2 is proteolytically activated in mouse adrenal cells by furin-like proprotein convertases: implications for the regulation of adrenal steroidogenesis.

    PubMed

    Layne, Joseph D; Shridas, Preetha; Webb, Nancy R

    2015-03-20

    Group X secretory phospholipase A2 (GX sPLA2) hydrolyzes mammalian cell membranes, liberating free fatty acids and lysophospholipids. GX sPLA2 is produced as a pro-enzyme (pro-GX sPLA2) that contains an N-terminal 11-amino acid propeptide ending in a dibasic motif, suggesting cleavage by a furin-like proprotein convertase (PC). Although propeptide cleavage is clearly required for enzymatic activity, the protease(s) responsible for pro-GX sPLA2 activation have not been identified. We previously reported that GX sPLA2 negatively regulates adrenal glucocorticoid production, likely by suppressing liver X receptor-mediated activation of steroidogenic acute regulatory protein expression. In this study, using a FLAG epitope-tagged pro-GX sPLA2 expression construct (FLAG-pro-GX sPLA2), we determined that adrenocorticotropic hormone (ACTH) enhanced FLAG-pro-GX sPLA2 processing and phospholipase activity secreted by Y1 adrenal cells. ACTH increased the expression of furin and PCSK6, but not other members of the PC family, in Y1 cells. Overexpression of furin and PCSK6 in HEK 293 cells significantly enhanced FLAG-pro-GX sPLA2 processing, whereas siRNA-mediated knockdown of both PCs almost completely abolished FLAG-pro-GX sPLA2 processing in Y1 cells. Expression of either furin or PCSK6 enhanced the ability of GX sPLA2 to suppress liver X receptor reporter activity. The PC inhibitor decanoyl-Arg-Val-Lys-Arg-chloromethyl ketone significantly suppressed FLAG-pro-GX sPLA2 processing and sPLA2 activity in Y1 cells, and it significantly attenuated GX sPLA2-dependent inhibition of steroidogenic acute regulatory protein expression and progesterone production. These findings provide strong evidence that pro-GX sPLA2 is a substrate for furin and PCSK6 proteolytic processing and define a novel mechanism for regulating corticosteroid production in adrenal cells.

  3. Effect of an RNA interference drug on the synthesis of proprotein convertase subtilisin/kexin type 9 (PCSK9) and the concentration of serum LDL cholesterol in healthy volunteers: a randomised, single-blind, placebo-controlled, phase 1 trial.

    PubMed

    Fitzgerald, Kevin; Frank-Kamenetsky, Maria; Shulga-Morskaya, Svetlana; Liebow, Abigail; Bettencourt, Brian R; Sutherland, Jessica E; Hutabarat, Renta M; Clausen, Valerie A; Karsten, Verena; Cehelsky, Jeffrey; Nochur, Saraswathy V; Kotelianski, Victor; Horton, Jay; Mant, Timothy; Chiesa, Joseph; Ritter, James; Munisamy, Malathy; Vaishnaw, Akshay K; Gollob, Jared A; Simon, Amy

    2014-01-04

    Proprotein convertase subtilisin/kexin type 9 (PCSK9) binds to LDL receptors, leading to their degradation. Genetics studies have shown that loss-of-function mutations in PCSK9 result in reduced plasma LDL cholesterol and decreased risk of coronary heart disease. We aimed to investigate the safety and efficacy of ALN-PCS, a small interfering RNA that inhibits PCSK9 synthesis, in healthy volunteers with raised cholesterol who were not on lipid-lowering treatment. We did a randomised, single-blind, placebo-controlled, phase 1 dose-escalation study in healthy adult volunteers with serum LDL cholesterol of 3·00 mmol/L or higher. Participants were randomly assigned in a 3:1 ratio by computer algorithm to receive one dose of intravenous ALN-PCS (with doses ranging from 0·015 to 0·400 mg/kg) or placebo. The primary endpoint was safety and tolerability of ALN-PCS. Secondary endpoints were the pharmacokinetic characteristics of ALN-PCS and its pharmacodynamic effects on PCSK9 and LDL cholesterol. Study participants were masked to treatment assignment. Analysis was per protocol and we used ANCOVA to analyse pharmacodynamic endpoint data. This trial is registered with ClinicalTrials.gov, number NCT01437059. Of 32 participants, 24 were randomly allocated to receive a single dose of ALN-PCS (0·015 mg/kg [n=3], 0·045 mg/kg [n=3], 0·090 mg/kg [n=3], 0·150 mg/kg [n=3], 0·250 mg/kg [n=6], or 0·400 mg/kg [n=6]) and eight to placebo. The proportions of patients affected by treatment-emergent adverse events were similar in the ALN-PCS and placebo groups (19 [79%] vs seven [88%]). ALN-PCS was rapidly distributed, with peak concentration and area under the curve (0 to last measurement) increasing in a roughly dose-proportional way across the dose range tested. In the group given 0·400 mg/kg of ALN-PCS, treatment resulted in a mean 70% reduction in circulating PCSK9 plasma protein (p<0·0001) and a mean 40% reduction in LDL cholesterol from baseline relative to placebo (p<0

  4. Treatment-Induced Viral Cure of Hepatitis C Virus-Infected Patients Involves a Dynamic Interplay among three Important Molecular Players in Lipid Homeostasis: Circulating microRNA (miR)-24, miR-223, and Proprotein Convertase Subtilisin/Kexin Type 9.

    PubMed

    Hyrina, Anastasia; Olmstead, Andrea D; Steven, Paul; Krajden, Mel; Tam, Edward; Jean, François

    2017-08-24

    In patients with chronic hepatitis C virus (HCV) infection, viral hijacking of the host-cell biosynthetic pathways is associated with altered lipid metabolism, which contributes to disease progression and may influence antiviral response. We investigated the molecular interplay among four key regulators of lipid homeostasis [microRNA (miR)-122, miR-24, miR-223, and proprotein convertase subtilisin/kexin type 9 (PCSK9)] in HCV-infected patients (n=72) who achieved a treatment-based viral cure after interferon-based therapy with first-generation direct-acting antivirals. Real-time PCR was used to quantify microRNA plasma levels, and ELISA assays were used to determine plasma concentrations of PCSK9. We report that levels of miR-24 and miR-223 significantly increased in patients achieving sustained virologic response (SVR), whereas the levels of miR-122, a liver-specific cofactor for HCV infection, decreased in these patients. PCSK9 concentrations were significantly increased in SVRs, suggesting that PCSK9 may help impede viral infection. The modulatory effect of PCSK9 on HCV infection was also demonstrated in the context of HCV-infected Huh-7.5.1 cells employing recombinant human PCSK9 mutants. Together, these results provide insights into a novel coordinated interplay among three important molecular players in lipid homeostasis - circulating miR-24, miR-223 and PCSK9 - whose regulation is affected by HCV infection and treatment-based viral cure. Copyright © 2017. Published by Elsevier B.V.

  5. Conservation of the prohormone convertase gene family in metazoa: analysis of cDNAs encoding a PC3-like protein from hydra.

    PubMed Central

    Chan, S J; Oliva, A A; LaMendola, J; Grens, A; Bode, H; Steiner, D F

    1992-01-01

    A subclass of proteolytic enzymes that correctly cleave precursor proteins at paired basic residues and are structurally related to the bacterial subtilisins has recently been identified. In yeast, a single membrane-bound proteolytic processing enzyme encoded by the kex2 gene has been found, whereas in higher vertebrates cDNAs encoding four distinct enzymes (PC2, PC3, furin, and PACE 4) have been identified. Like kex2, furin (also known as PACE) contains a hydrophobic transmembrane domain, but PC2, PC3, and PACE 4 lack this feature. All five enzymes exhibit striking similarities in their catalytic domains, and this suggests that they have arisen from a common ancestral subtilisin-like gene. We report here the identification of cDNAs encoding a protein that is similar in structure to PC3 from a simple metazoan, Hydra vulgaris (formerly Hydra attenuata). cDNAs encoding two isoforms of this PC3-like enzyme were obtained that differ only in their carboxyl-terminal sequences, probably due to alternative splicing of a common pre-mRNA. Neither form contains a transmembrane domain. Predicted amino acid sequence comparisons revealed that the hydra PC3-like enzyme is 55.4% and 56.7% identical in the catalytic domain to mouse PC3 and human furin, respectively. RNA blot analyses revealed that the PC3-like RNA is expressed predominantly in the hydra body column and not in the head region, although the hydra head contains a high density of nerve cells, which synthesize a variety of neuropeptides. For this reason, we suspect that another proprotein cleavage enzyme isoform may be expressed in head nerve cells. The isolation of a PC3-like cDNA from hydra is consistent with the presence of neuroendocrine cells and indicates that the PC/furin gene family has been well conserved in all metazoa. A simplified nomenclature for the group of mammalian processing proteases is proposed. Images PMID:1495957

  6. Identification of a cDNA encoding a second putative prohormone convertase related to PC2 in AtT20 cells and islets of Langerhans.

    PubMed Central

    Smeekens, S P; Avruch, A S; LaMendola, J; Chan, S J; Steiner, D F

    1991-01-01

    PC2 and furin are two recently identified members of a class of mammalian proteins homologous to the yeast precursor processing protease kex2 and the bacterial subtillisins. We have used the polymerase chain reaction to identify and clone a cDNA (PC3) from the mouse AtT20 anterior pituitary cell line that represents an additional member of this growing family of mammalian proteases. PC3 encodes a 753-residue protein that begins with a signal peptide and contains a 292-residue domain closely related to the catalytic modules of PC2, furin, and kex2. Within this region 58%, 65%, and 50% of the amino acids of PC3 are identical to those of the aligned PC2, furin, and kex2 sequences, respectively, and the catalytically important Asp, His, and Ser residues are all conserved. On Northern blots, PC3 hybridizes to two transcripts of 3 and 5 kilobases. Tissue distribution studies indicate that both PC2 and PC3 are expressed in a variety of neuroendocrine tissues, including pancreatic islets and brain, but are not expressed in liver, kidney, skeletal muscle, and spleen. The high degree of similarity of PC3, PC2, and furin suggests that they are all members of a superfamily of mammalian proteases that are involved in the processing of prohormones and/or other protein precursors. In contrast to furin, PC3, like PC2, lacks a hydrophobic transmembrane anchor, but it has a potential C-terminal amphipathic helical segment similar to the putative membrane anchor of carboxypeptidase H. These and other differences suggest that these proteins carry out compartmentalized proteolysis within cells, such as processing within regulated versus constitutive secretory pathways. Images PMID:1988934

  7. Convertase Inhibitory Properties of Staphylococcal Extracellular Complement-binding Protein*

    PubMed Central

    Jongerius, Ilse; Garcia, Brandon L.; Geisbrecht, Brian V.; van Strijp, Jos A. G.; Rooijakkers, Suzan H. M.

    2010-01-01

    The human pathogen Staphylococcus aureus secretes several complement evasion molecules to combat the human immune response. Extracellular complement-binding protein (Ecb) binds to the C3d domain of C3 and thereby blocks C3 convertases of the alternative pathway and C5 convertases via all complement pathways. Inhibition of C5 convertases results in complete inhibition of C5a generation and subsequent neutrophil migration. Here, we show that binding of Ecb to the C3d domain of C3b is crucial for inhibition of C5 convertases. Ecb does not interfere with substrate binding to convertases but prevents formation of an active convertase enzyme. PMID:20304920

  8. Propeptides of eukaryotic proteases encode histidines to exploit organelle pH for regulation.

    PubMed

    Elferich, Johannes; Williamson, Danielle M; Krishnamoorthy, Bala; Shinde, Ujwal

    2013-08-01

    Eukaryotic cells maintain strict control over protein secretion, in part by using the pH gradient maintained within their secretory pathway. How eukaryotic proteins evolved from prokaryotic orthologs to exploit the pH gradient for biological functions remains a fundamental question in cell biology. Our laboratory previously demonstrated that protein domains located within precursor proteins, propeptides, encode histidine-driven pH sensors to regulate organelle-specific activation of the eukaryotic proteases furin and proprotein convertase-1/3. Similar findings have been reported in other unrelated protease families. By analyzing >10,000 unique proteases within evolutionarily unrelated families, we show that eukaryotic propeptides are enriched in histidines compared with prokaryotic orthologs. On this basis, we hypothesize that eukaryotic proteins evolved to enrich histidines within their propeptides to exploit the tightly controlled pH gradient of the secretory pathway, thereby regulating activation within specific organelles. Enrichment of histidines in propeptides may therefore be used to predict the presence of pH sensors in other proteases or even protease substrates.

  9. Mammalian subtilisin/kexin isozyme SKI-1: A widely expressed proprotein convertase with a unique cleavage specificity and cellular localization

    PubMed Central

    Seidah, Nabil G.; Mowla, Seyed J.; Hamelin, Josée; Mamarbachi, Aida M.; Benjannet, Suzanne; Touré, Barry B.; Basak, Ajoy; Munzer, Jon Scott; Marcinkiewicz, Jadwiga; Zhong, Mei; Barale, Jean-Christophe; Lazure, Claude; Murphy, Richard A.; Chrétien, Michel; Marcinkiewicz, Mieczyslaw

    1999-01-01

    Using reverse transcriptase–PCR and degenerate oligonucleotides derived from the active-site residues of subtilisin/kexin-like serine proteinases, we have identified a highly conserved and phylogenetically ancestral human, rat, and mouse type I membrane-bound proteinase called subtilisin/kexin-isozyme-1 (SKI-1). Computer databank searches reveal that human SKI-1 was cloned previously but with no identified function. In situ hybridization demonstrates that SKI-1 mRNA is present in most tissues and cells. Cleavage specificity studies show that SKI-1 generates a 28-kDa product from the 32-kDa brain-derived neurotrophic factor precursor, cleaving at an RGLT↓SL bond. In the endoplasmic reticulum of either LoVo or HK293 cells, proSKI-1 is processed into two membrane-bound forms of SKI-1 (120 and 106 kDa) differing by the nature of their N-glycosylation. Late along the secretory pathway some of the membrane-bound enzyme is shed into the medium as a 98-kDa form. Immunocytochemical analysis of stably transfected HK293 cells shows that SKI-1 is present in the Golgi apparatus and within small punctate structures reminiscent of endosomes. In vitro studies suggest that SKI-1 is a Ca2+-dependent serine proteinase exhibiting a wide pH optimum for cleavage of pro-brain-derived neurotrophic factor. PMID:9990022

  10. Family of prohormone convertases in Lymnaea: characterization of two alternatively spliced furin-like transcripts and cell-specific regulation of their expression.

    PubMed

    Spijker, S; Smit, A B; Sharp-Baker, H E; Van Elk, R; Van Kesteren, E R; Van Minnen, J; Kurosky, A; Geraerts, W P

    1999-11-15

    The majority of neuropeptides in Lymnaea stagnalis are proteolytically processed from larger precursors at sites composed of single or multiple basic amino acid residues. Previous studies have identified several putative prohormone convertases in the brain of Lymnaea. To characterize the complete family, we undertook three independent approaches: reverse-transcribed polymerase chain reaction screening, and low-stringency cDNA and genomic library screenings. The central nervous system cDNA library screening yielded two cDNAs encoding Lfurin1 and its variant form, Lfurin1-X. Both proteins show the characteristic organization of (human) furin with a putative catalytic domain, a P domain, a Cys-rich domain, a transmembrane domain, and a cytoplasmic tail. Lfurin1 and Lfurin1-X are identical, apart from a putative alternatively spliced noncatalytic luminal protein domain, which is present exclusively in Lfurin1-X. In situ hybridization revealed that the Lfur1 gene is expressed throughout the Lymnaea brain, but that the level varies considerably from one neuron to another. Quantitative analysis of the expression level of the two alternatively spliced transcripts revealed that it is neuron type-specifically regulated. This probably indicates the functional importance of noncatalytic luminal protein domains in these enzymes. In addition, our findings suggest that apart from the identified convertases LPC2, Lfurin1/Lfurin1-X, and Lfurin2, additional prohormone convertase diversity is either not present or present only at low levels in the Lymnaea brain. Alternatively, additional prohormone convertases could exist with a lower degree of sequence conservation than the other Lymnaea prohormone convertase members. From our findings, it appears that the majority of prohormone processing in Lymnaea is carried out by the three thus far identified types of Kex2-related prohormone convertases despite the large number of neuropeptide precursors and diverse multiple basic cleavage

  11. The Streptomyces coelicolor Developmental Transcription Factor σBldN Is Synthesized as a Proprotein

    PubMed Central

    Bibb, Maureen J.; Buttner, Mark J.

    2003-01-01

    bldN is one of a set of genes required for the formation of specialized, spore-bearing aerial hyphae during differentiation in the mycelial bacterium Streptomyces coelicolor. Previous analysis (M. J. Bibb et al., J. Bacteriol. 182:4606-4616, 2000) showed that bldN encodes a member of the extracytoplasmic function subfamily of RNA polymerase σ factors and that translation from the most strongly predicted start codon (GTG1) would give rise to a σ factor having an unusual N-terminal extension of ca. 86 residues. Here, by using a combination of site-directed mutagenesis and immunoblot analysis, we provide evidence that all bldN translation arises from initiation at GTG1 and that the primary translation product is a proprotein (pro-σBldN) that is proteolytically processed to a mature species (σBldN) by removal of most of the unusual N-terminal extension. A time course taken during differentiation of the wild type on solid medium showed early production of pro-σBldN and the subsequent appearance of mature σBldN, which was concomitant with aerial mycelium formation and the disappearance of pro-σBldN. Two genes encoding members of a family of metalloproteases that are involved in the regulated proteolytic processing of transcription factors in other organisms were identified in the S. coelicolor genome, but their disruption did not affect differentiation or pro-σBldN processing. PMID:12644505

  12. Occurrence of an HIV-1 gp160 endoproteolytic activity in low-density vesicles and evidence for a distinct density distribution from endogenously expressed furin and PC7/LPC convertases.

    PubMed

    Wouters, S; Decroly, E; Vandenbranden, M; Shober, D; Fuchs, R; Morel, V; Leruth, M; Seidah, N G; Courtoy, P J; Ruysschaert, J M

    1999-07-30

    Human immunodeficiency virus (HIV) glycoprotein (gp) 160 processing by host cell proteinases is an essential step for viral fusion and infectivity. We have identified a rat liver subcellular fraction which specifically processes gp160 into gp120 and gp41. Using equilibration of microsomes in sucrose gradients, the gp160 cleavage activity was associated with particles equilibrating at low densities, well-separated from the endoplasmic reticulum, cis-Golgi network, Golgi stacks, lysosomes and plasma membrane. Its density distribution was compatible with light secretory vesicles derived from the trans-Golgi network (TGN) or to endosomes, but association with endosomes was not supported by free flow electrophoresis. Although furin and pro-protein convertase (PC) 7/LPC have been proposed as the major gp160 processing convertases, the rat liver microsomal gp160 processing activity was essentially resolved from furin and only partially overlapped PC7/LPC. These data suggest that proteinase(s) other than furin and PC7/LPC, presumably located in TGN-derived vesicles, may participate in the gp160 processing into gp120 and gp41.

  13. Prohormone convertase PC5 is a candidate processing enzyme for prorenin in the human adrenal cortex.

    PubMed

    Mercure, C; Jutras, I; Day, R; Seidah, N G; Reudelhuber, T L

    1996-11-01

    We isolated a cDNA clone encoding the human prohormone convertase PC5 from human adrenal gland mRNA. The deduced protein sequence would encode a 915 amino acid preproPC5 that shares a very high degree of homology with previously cloned rat and mouse homologues. PC5 mRNA was detected in multiple human tissues, including the brain, adrenal and thyroid glands, heart, placenta, lung, and testes. PC5 mRNA was undetectable in the liver and was present at lower levels in skeletal muscle, kidney, pancreas, small intestine, and stomach. Co-transfection of human PC5 and human prorenin expression vectors in cultured GH4C1 cells led to secretion of active renin. The activation of human prorenin by PC5 depended on a pair of basic amino acids at positions 42 and 43 of the prorenin prosegment and occurred only in cells containing dense core secretory granules. Human PC5 was colocalized with renin by immunohistochemistry in the zona glomerulosa of the adrenal gland, suggesting that it could participate in the activation of a local renin-angiotensin system in the human adrenal cortex.

  14. [Severe hypercholesterolaemia--when to use the proprotein convertase subtilisin-kexin type 9 protease inhibitors (PCSK9 inhibitors)? Polish Society of Cardiology experts' group statement].

    PubMed

    Cybulska, Barbara; Gaciong, Zbigniew; Hoffman, Piotr; Jankowski, Piotr; Kłosiewicz-Latoszek, Longina; Kaźmierczak, Jarosław; Mitręga, Katarzyna; Opolski, Grzegorz; Pająk, Andrzej; Ponikowski, Piotr; Rynkiewicz, Andrzej; Stępińska, Janina; Średniawa, Beata; Kalarus, Zbigniew

    2016-01-01

    The severe hypercholesterolaemia can be recognised when low density lipoprotein cholesterol (LDL-C) serum levels are equal to or above 5 mmol/L (≥ 190 mg/dL). The prevalence of LDL-C ≥ 5 mmol/L is 3.8% in Polish population aged 18-79 years. Among these adults there are patients with familial hypercholesterolaemia (FH). According to meta-analysis of 6 Polish population surveys prevalence of heterozygous FH (HeFH) diagnosed using Dutch Lipid Clinic criteria is 0.4% (95% Cl 0.28-0.53%) in men and women aged 20-74 years, i.e. one in every 250 people. As HeFH is a wellknown cause of premature coronary heart disease the rigorous treatment targets for LDL-C have been established in clinical guidelines. Their achievements, even with a high dose of high efficacy statin therapy is difficult or even impossible. New strong hypolipidaemic drugs i.e. PCSK9 inhibitors have been initiated against this chalange. Both drugs, evolocumab and alirocumab, have been extensively studied in numerous phase 2 and phase 3 trials. Fewer studies with bococizumab are available until now. The PCSK9 inhibitors, as monotherapy as well in combination with statins were associated with mean LDL-C reduction about 60%. It means that the majority of patients (70-90%) with severe hypercholesterolaemia (including HeFH), treated with statins, after addition of PCSK9 inhibitors were able to achieve an LDL-C < 2.5 mmol/L (< 100 mg/dL) or < 1.8 mmol/L (< 70 mg/dL) level. Another group of patients who may benefit from PCSK9 inhibitors include those who need lipid lowering therapy, but who are statin intolerant, especially because of statin-associated muscle symptoms (SAMS). In our statement we have accepted the diagnosis of SAMS proposed recently by European Atherosclerosis Society. Today the longest clinical trial with evolocumab (11 months) was the open OSLER study, and with alirocumab ODYSSEY LONG TERM (78 weeks). In the first one the reduction of cardiovascular events by 53% (95% Cl 22-72%) was observed, and in the second one by 48% (10-69%). Neurocognitive events were reported more frequently with both drugs than with placebo. This adverse effect will be the subject of observation in ongoing studies. We still await the results of 4 ongoing large placebo controlled phase 3 trials investigating whether PCSK9 inhibitors on background of statin therapy reduce cardiovascular events. Meanwhile evolocumab, as well as alirocumab have been accepted to use in clinical practice by European Medicine Agency. In this situation the experts of Polish Society of Cardiology have prepared the statement on the use PCSK9 inhibitors with indication in the first place for HeFH patients, statin intolerant and those at high risk who are not able to reach LDL-C target level with a high potent high dose statin.

  15. Two independent targeting signals in the cytoplasmic domain determine trans-Golgi network localization and endosomal trafficking of the proprotein convertase furin.

    PubMed Central

    Schäfer, W; Stroh, A; Berghöfer, S; Seiler, J; Vey, M; Kruse, M L; Kern, H F; Klenk, H D; Garten, W

    1995-01-01

    Furin, a subtilisin-like eukaryotic endoprotease, is responsible for proteolytic cleavage of cellular and viral proteins transported via the constitutive secretory pathway. Cleavage occurs at the C-terminus of basic amino acid sequences, such as R-X-K/R-R and R-X-X-R. Furin was found predominantly in the trans-Golgi network (TGN), but also in clathrin-coated vesicles dispatched from the TGN, on the plasma membrane as an integral membrane protein and in the medium as an anchorless enzyme. When furin was vectorially expressed in normal rat kidney (NRK) cells it accumulated in the TGN similarly to the endogenous glycoprotein TGN38, often used as a TGN marker protein. The signals determining TGN targeting of furin were investigated by mutational analysis of the cytoplasmic tail of furin and by using the hemagglutinin (HA) of fowl plague virus, a protein with cell surface destination, as a reporter molecule, in which membrane anchor and cytoplasmic tail were replaced by the respective domains of furin. The membrane-spanning domain of furin grafted to HA does not localize the chimeric molecule to the TGN, whereas the cytoplasmic domain does. Results obtained on furin mutants with substitutions and deletions of amino acids in the cytoplasmic tail indicate that wild-type furin is concentrated in the TGN by a mechanism involving two independent targeting signals, which consist of the acidic peptide CPSDSEEDEG783 and the tetrapeptide YKGL765. The acidic signal in the cytoplasmic domain of a HA-furin chimera is necessary and sufficient to localize the reporter molecule to the TGN, whereas YKGL is a determinant for targeting to the endosomes. The data support the concept that the acidic signal, which is the dominant one, retains furin in the TGN, whereas the YKGL motif acts as a retrieval signal for furin that has escaped to the cell surface. Images PMID:7781597

  16. Deficiency in prohormone convertase PC1 impairs prohormone processing in Prader-Willi syndrome

    PubMed Central

    Burnett, Lisa C.; LeDuc, Charles A.; Sulsona, Carlos R.; Paull, Daniel; Rausch, Richard; Eddiry, Sanaa; Carli, Jayne F. Martin; Morabito, Michael V.; Skowronski, Alicja A.; Hubner, Gabriela; Zimmer, Matthew; Wang, Liheng; Day, Robert; Levy, Brynn; Dubern, Beatrice; Poitou, Christine; Clement, Karine; Rosenbaum, Michael; Salles, Jean Pierre; Tauber, Maithe; Egli, Dieter

    2016-01-01

    Prader-Willi syndrome (PWS) is caused by a loss of paternally expressed genes in an imprinted region of chromosome 15q. Among the canonical PWS phenotypes are hyperphagic obesity, central hypogonadism, and low growth hormone (GH). Rare microdeletions in PWS patients define a 91-kb minimum critical deletion region encompassing 3 genes, including the noncoding RNA gene SNORD116. Here, we found that protein and transcript levels of nescient helix loop helix 2 (NHLH2) and the prohormone convertase PC1 (encoded by PCSK1) were reduced in PWS patient induced pluripotent stem cell–derived (iPSC-derived) neurons. Moreover, Nhlh2 and Pcsk1 expression were reduced in hypothalami of fasted Snord116 paternal knockout (Snord116p–/m+) mice. Hypothalamic Agrp and Npy remained elevated following refeeding in association with relative hyperphagia in Snord116p–/m+ mice. Nhlh2-deficient mice display growth deficiencies as adolescents and hypogonadism, hyperphagia, and obesity as adults. Nhlh2 has also been shown to promote Pcsk1 expression. Humans and mice deficient in PC1 display hyperphagic obesity, hypogonadism, decreased GH, and hypoinsulinemic diabetes due to impaired prohormone processing. Here, we found that Snord116p–/m+ mice displayed in vivo functional defects in prohormone processing of proinsulin, pro-GH–releasing hormone, and proghrelin in association with reductions in islet, hypothalamic, and stomach PC1 content. Our findings suggest that the major neuroendocrine features of PWS are due to PC1 deficiency. PMID:27941249

  17. The developmental expression in the rat CNS and peripheral tissues of proteases PC5 and PACE4 mRNAs: comparison with other proprotein processing enzymes.

    PubMed

    Zheng, M; Seidah, N G; Pintar, J E

    1997-01-15

    Many peptides modulating cellular growth and differentiation in development are first synthesized as precursors that require proteolytic processing by the "prohormone convertase" (PC) family of endoproteases. Using in situ hybridization, we have here determined that two recently identified PC members, PC5 and PACE4, are expressed prenatally in spatial and temporal patterns that are each unique and distinct from those of previously characterized PCs. PC5 mRNA is first detected at e9 in highly restricted regions of the neural tube, in caudal myotomes, and at the materno-embryonic junction of the uterus. At e10, restricted PC5 mRNA expression is detected in the optic and otic vesicles, the roof of midbrain, and trunk myotomes. By midgestation (e13-e16), PC5 mRNA expression in the developing nervous system has expanded to multiple regions including hippocampus, thalamus, hypothalamus, brain stem, and spinal cord. By midgestational stages, PACE4 mRNA is expressed in multiple regions of the developing nervous system, generally distinct from PC5, and including a uniquely high level of expression in the ventricular zone of the hippocampus. In several peripheral organ systems, including lung and gut, we observed remarkably complementary patterns of PC5 and PACE4 expression. In addition, PACE4 transcripts are expressed in the heart and liver, whereas PC5 is expressed in the adrenal and kidney primordia. These results suggest that both PC5 and PACE4 may be involved in neuropeptide precursor processing in the developing nervous system and peripheral tissues with the general nonoverlapping expression patterns suggesting that PC5 and PACE4 may process distinct sets of proprotein substrates.

  18. Molecular insights into the surface-specific arrangement of complement C5 convertase enzymes.

    PubMed

    Berends, Evelien T M; Gorham, Ronald D; Ruyken, Maartje; Soppe, Jasper A; Orhan, Hatice; Aerts, Piet C; de Haas, Carla J C; Gros, Piet; Rooijakkers, Suzan H M

    2015-11-09

    Complement is a large protein network in plasma that is crucial for human immune defenses and a major cause of aberrant inflammatory reactions. The C5 convertase is a multi-molecular protease complex that catalyses the cleavage of native C5 into its biologically important products. So far, it has been difficult to study the exact molecular arrangement of C5 convertases, because their non-catalytic subunits (C3b) are covalently linked to biological surfaces through a reactive thioester. Through development of a highly purified model system for C5 convertases, we here aim to provide insights into the surface-specific nature of these important protease complexes. Alternative pathway (AP) C5 convertases were generated on small streptavidin beads that were coated with purified C3b molecules. Site-specific biotinylation of C3b via the thioester allowed binding of C3b in the natural orientation on the surface. In the presence of factor B and factor D, these C3b beads could effectively convert C5. Conversion rates of surface-bound C3b were more than 100-fold higher than fluid-phase C3b, confirming the requirement of a surface. We determine that high surface densities of C3b, and its attachment via the thioester, are essential for C5 convertase formation. Combining our results with molecular modeling explains how high C3b densities may facilitate intermolecular interactions that only occur on target surfaces. Finally, we define two interfaces on C5 important for its recognition by surface-bound C5 convertases. We establish a highly purified model that mimics the natural arrangement of C5 convertases on a surface. The developed model and molecular insights are essential to understand the molecular basis of deregulated complement activity in human disease and will facilitate future design of therapeutic interventions against these critical enzymes in inflammation.

  19. RNAi-mediated silencing of prohormone convertase (PC) 5/6 expression leads to impairment in processing of cocaine- and amphetamine-regulated transcript (CART) precursor

    PubMed Central

    Stein, Jeffrey; Shah, Rohan; Steiner, Donald F.; Dey, Arunangsu

    2006-01-01

    Understanding the functions of the widely expressed PCs (prohormone/proprotein convertases), including PC5/6, furin and PACE4 (paired basic amino acid cleaving enzyme 4), in animal models is difficult since individual knockouts of these PCs in mice exhibit early embryonic lethality. To investigate the roles of PC5/6 in processing pro-CART (pro-cocaine- and amphetamine-regulated transcript), an important anorexigenic peptide precursor, we have generated GH3 cells silenced for PC5/6 expression by RNAi (RNA interference). We show, following transient knockdown of PC5/6 in these neuroendocrine cells, that generation of the two bioactive forms, CART I (amino acids 42–89/55–102) and CART II (amino acids 49–89/62–102), from pro-CART is impaired due to a lack particularly of the A isoform of PC5/6. The results indicate that PC5/6A shares specificities primarily with PC2 (PC5/6A

  20. Specificity of prohormone convertase endoproteolysis of progastrin in AtT-20 cells.

    PubMed Central

    Dickinson, C J; Sawada, M; Guo, Y J; Finniss, S; Yamada, T

    1995-01-01

    Biologically active peptide hormones are synthesized from larger precursor proteins by a variety of posttranslational processing reactions. Endoproteolytic cleavage at the Lys74-Lys75 dibasic processing site of progastrin is the major determinant for the relative distribution of gastrin heptadecapeptide and tetratriacontapeptide in tissues. Thus, we explored the ability of two prohormone convertases, PC1/PC3 and PC2, to cleave this important site within progastrin. We expressed wild-type human gastrin cDNA and mutant cDNAs in which the Lys74Lys75 site was changed to Lys74Arg75, Arg74Arg75, and Arg74Lys75 residues in AtT-20 cells. Because AtT-20 cells express Pc1/PC3 but not PC2, we also coexpressed a cDNA encoding PC2 in both wild-type and mutant gastrin-producing AtT-20 cells. Wild-type Lys74Lys75 and mutant Arg74Arg75 progastrin processing sites were efficiently cleaved in AtT-20 cells only after coexpression of PC2. Mutant Lys74Arg75 progastrin was readily processed in cells in the presence or absence of PC2 coexpression, but, in contrast, mutant Arg74Lys75 progastrin was inefficiently cleaved regardless of PC2 coexpression. Northern analysis revealed the presence of PC2 but not PC1/ PC3 in canine antral gastrin-producing G cells. These data suggest that PC2 but not PC1/PC3 is responsible for the cleavage of the Lys74Lys75 site in wild-type progastrin. Images PMID:7657815

  1. Molecular cloning and characterization of prohormone convertase 1 gene in abalone (Haliotis diversicolor supertexta).

    PubMed

    Zhou, Jin; Cai, Zhong-hua

    2010-03-01

    Prohormone convertases (PCs) are calcium-dependent serine endoproteases of the subtilisin family that play a key role in the posttranslational processing of precursors for bioactive peptides. In this study, the cDNA of PC1 from abalone (Haliotis diversicolor supertexta) was cloned and sequenced. The PC1 cDNA consisted of 2216 bp with an open reading frame of 2010 bp encoding a 670 amino acid peptide. Comparative structural analysis revealed that abalone PC1 shared high similarity and identity with most PC counterparts. The profile of deduced peptide of PC1 was composed of an N-terminal signal peptide, a prosegment domain, a catalytic domain and a P domain, which were common in many species. Sequence analysis indicated that the abalone PC1 was highly conserved in catalytic domain, including three conserved serine catalytic signatures that comprised a catalytic triad active center. Also conserved were the potential cleavage site for release of the mature peptide, a cognate integrin binding site RGD in P domain, and four cysteine residues involved in forming an intrachain disulfide bridge. To further investigate the functions of PC1 in abalone, real-time quantitative PCR was performed to determine the expression level of this gene at three different reproduction stages (i.e. pre-, during- and post-breeding). Results indicated that PC1 was expressed throughout the three stages but the expression levels varied with the timepoints and different tissues in abalone. The expression levels of PC1 in digestive gland were much higher than those of the gonad. In female abalone, the expression of PC1 was higher at pre-breeding and during-breeding stages (P<0.05), and the expression declined at the subsequent stage. Whereas, the level of PC1 in male individual did not exhibit a significant difference in various reproduction stages. Also, the natural enzyme activity of PC1 partially exhibited a similar tendency with the mRNA expression. According to the results, it can be

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

    PubMed Central

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

    2013-01-01

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

  3. A humanized antibody that regulates the alternative pathway convertase: potential for therapy of renal disease associated with nephritic factors.

    PubMed

    Paixão-Cavalcante, Danielle; Torreira, Eva; Lindorfer, Margaret A; Rodriguez de Cordoba, Santiago; Morgan, B Paul; Taylor, Ronald P; Llorca, Oscar; Harris, Claire L

    2014-05-15

    Dysregulation of the complement alternative pathway can cause disease in various organs that may be life-threatening. Severe alternative pathway dysregulation can be triggered by autoantibodies to the C3 convertase, termed nephritic factors, which cause pathological stabilization of the convertase enzyme and confer resistance to innate control mechanisms; unregulated complement consumption followed by deposition of C3 fragments in tissues ensues. The mAb, 3E7, and its humanized derivative, H17, have been shown previously to specifically bind activated C3 and prevent binding of both the activating protein, factor B, and the inhibitor, factor H, which are opposite effects that complicate its potential for therapy. Using ligand binding assays, functional assays, and electron microscopy, we show that these Abs bind C3b via a site that overlaps the binding site on C3 for the Ba domain within factor B, thereby blocking an interaction essential for convertase formation. Both Abs also bind the preformed convertase, C3bBb, and provide powerful inhibition of complement activation by preventing cleavage of C3. Critically, the Abs also bound and inhibited C3 cleavage by the nephritic factor-stabilized convertase. We suggest that by preventing enzyme formation and/or cleavage of C3 to its active downstream fragments, H17 may be an effective therapy for conditions caused by severe dysregulation of the C3 convertase and, in particular, those that involve nephritic factors, such as dense deposit disease.

  4. Insights into complement convertase formation based on the structure of the factor B-cobra venom factor complex.

    PubMed

    Janssen, Bert J C; Gomes, Lucio; Koning, Roman I; Svergun, Dmitri I; Koster, Abraham J; Fritzinger, David C; Vogel, Carl-Wilhelm; Gros, Piet

    2009-08-19

    Immune protection by the complement system critically depends on assembly of C3 convertases on the surface of pathogens and altered host cells. These short-lived protease complexes are formed through pro-convertases, which for the alternative pathway consist of the complement component C3b and the pro-enzyme factor B (FB). Here, we present the crystal structure at 2.2-A resolution, small-angle X-ray scattering and electron microscopy (EM) data of the pro-convertase formed by human FB and cobra venom factor (CVF), a potent homologue of C3b that generates more stable convertases. FB is loaded onto CVF through its pro-peptide Ba segment by specific contacts, which explain the specificity for the homologous C3b over the native C3 and inactive products iC3b and C3c. The protease segment Bb binds the carboxy terminus of CVF through the metal-ion dependent adhesion site of the Von Willebrand factor A-type domain. A possible dynamic equilibrium between a 'loading' and 'activation' state of the pro-convertase may explain the observed difference between the crystal structure of CVFB and the EM structure of C3bB. These insights into formation of convertases provide a basis for further development of complement therapeutics.

  5. Isolation and Characterization of a Thionin Proprotein-processing Enzyme from Barley*

    PubMed Central

    Plattner, Stephan; Gruber, Clemens; Stadlmann, Johannes; Widmann, Stefan; Gruber, Christian W.; Altmann, Friedrich; Bohlmann, Holger

    2015-01-01

    Thionins are plant-specific antimicrobial peptides that have been isolated from the endosperm and leaves of cereals, from the leaves of mistletoes, and from several other plant species. They are generally basic peptides with three or four disulfide bridges and a molecular mass of ∼5 kDa. Thionins are produced as preproproteins consisting of a signal peptide, the thionin domain, and an acidic domain. Previously, only mature thionin peptides have been isolated from plants, and in addition to removal of the signal peptide, at least one cleavage processing step between the thionin and the acidic domain is necessary to release the mature thionin. In this work, we identified a thionin proprotein-processing enzyme (TPPE) from barley. Purification of the enzyme was guided by an assay that used a quenched fluorogenic peptide comprising the amino acid sequence between the thionin and the acidic domain of barley leaf-specific thionin. The barley TPPE was identified as a serine protease (BAJ93208) and expressed in Escherichia coli as a strep tag-labeled protein. The barley BTH6 thionin proprotein was produced in E. coli using the vector pETtrx1a and used as a substrate. We isolated and sequenced the BTH6 thionin from barley to confirm the N and C terminus of the peptide in planta. Using an in vitro enzymatic assay, the recombinant TPPE was able to process the quenched fluorogenic peptide and to cleave the acidic domain at least at six sites releasing the mature thionin from the proprotein. Moreover, it was found that the intrinsic three-dimensional structure of the BTH6 thionin domain prevents cleavage of the mature BTH6 thionin by the TPPE. PMID:26013828

  6. Isolation and Characterization of a Thionin Proprotein-processing Enzyme from Barley.

    PubMed

    Plattner, Stephan; Gruber, Clemens; Stadlmann, Johannes; Widmann, Stefan; Gruber, Christian W; Altmann, Friedrich; Bohlmann, Holger

    2015-07-17

    Thionins are plant-specific antimicrobial peptides that have been isolated from the endosperm and leaves of cereals, from the leaves of mistletoes, and from several other plant species. They are generally basic peptides with three or four disulfide bridges and a molecular mass of ~5 kDa. Thionins are produced as preproproteins consisting of a signal peptide, the thionin domain, and an acidic domain. Previously, only mature thionin peptides have been isolated from plants, and in addition to removal of the signal peptide, at least one cleavage processing step between the thionin and the acidic domain is necessary to release the mature thionin. In this work, we identified a thionin proprotein-processing enzyme (TPPE) from barley. Purification of the enzyme was guided by an assay that used a quenched fluorogenic peptide comprising the amino acid sequence between the thionin and the acidic domain of barley leaf-specific thionin. The barley TPPE was identified as a serine protease (BAJ93208) and expressed in Escherichia coli as a strep tag-labeled protein. The barley BTH6 thionin proprotein was produced in E. coli using the vector pETtrx1a and used as a substrate. We isolated and sequenced the BTH6 thionin from barley to confirm the N and C terminus of the peptide in planta. Using an in vitro enzymatic assay, the recombinant TPPE was able to process the quenched fluorogenic peptide and to cleave the acidic domain at least at six sites releasing the mature thionin from the proprotein. Moreover, it was found that the intrinsic three-dimensional structure of the BTH6 thionin domain prevents cleavage of the mature BTH6 thionin by the TPPE.

  7. Genome-wide census and expression profiling of chicken neuropeptide and prohormone convertase genes.

    PubMed

    Delfino, K R; Southey, B R; Sweedler, J V; Rodriguez-Zas, S L

    2010-02-01

    Neuropeptides regulate cell-cell signaling and influence many biological processes in vertebrates, including development, growth, and reproduction. The complex processing of neuropeptides from prohormone proteins by prohormone convertases, combined with the evolutionary distance between the chicken and mammalian species that have experienced extensive neuropeptide research, has led to the empirical confirmation of only 18 chicken prohormone proteins. To expand our knowledge of the neuropeptide and prohormone convertase gene complement, we performed an exhaustive survey of the chicken genomic, EST, and proteomic databases using a list of 95 neuropeptide and 7 prohormone convertase genes known in other species. Analysis of the EST resources and 22 microarray studies offered a comprehensive portrait of gene expression across multiple conditions. Five neuropeptide genes (apelin, cocaine-and amphetamine-regulated transcript protein, insulin-like 5, neuropeptide S, and neuropeptide B) previously unknown in chicken were identified and 62 genes were confirmed. Although most neuropeptide gene families known in human are present in chicken, there are several gene not present in the chicken. Conversely, several chicken neuropeptide genes are absent from mammalian species, including C-RF amide, c-type natriuretic peptide 1 precursor, and renal natriuretic peptide. The prohormone convertases, with one exception, were found in the chicken genome. Bioinformatic models used to predict prohormone cleavages confirm that the processing of prohormone proteins into neuropeptides is similar between species. Neuropeptide genes are most frequently expressed in the brain and head, followed by the ovary and small intestine. Microarray analyses revealed that the expression of adrenomedullin, chromogranin-A, augurin, neuromedin-U, platelet-derived growth factor A and D, proenkephalin, relaxin-3, prepronociceptin, and insulin-like growth factor I was most susceptible (P-value<0.005) to changes

  8. Genome-wide Census and Expression Profiling of Chicken Neuropeptide and Prohormone Convertase Genes

    PubMed Central

    Delfino, K. R.; Southey, B. R.; Sweedler, J. V.; Rodriguez-Zas, S. L.

    2009-01-01

    Neuropeptides regulate cell-cell signaling and influence many biological processes in vertebrates, including development, growth, and reproduction. The complex processing of neuropeptides from prohormone proteins by prohormone convertases, combined with the evolutionary distance between the chicken and mammalian species that have experienced extensive neuropeptide research, has led to the empirical confirmation of only 18 chicken prohormone proteins. To expand our knowledge of the neuropeptide and prohormone convertase gene complement, we performed an exhaustive survey of the chicken genomic, EST, and proteomic databases using a list of 95 neuropeptide and 7 prohormone convertase genes known in other species. Analysis of the EST resources and 22 microarray studies offered a comprehensive portrait of gene expression across multiple conditions. Five neuropeptide genes (apelin, cocaine-and amphetamine-regulated transcript protein, insulin-like 5, neuropeptide S, and neuropeptide B) previously unknown in chicken were identified and 62 genes were confirmed. Although most neuropeptide gene families known in human are present in chicken, there are several gene not present in the chicken. Conversely, several chicken neuropeptide genes are absent from mammalian species, including C-RF amide, c-type natriuretic peptide 1 precursor, and renal natriuretic peptide. The prohormone convertases, with one exception, were found in the chicken genome. Bioinformatic models used to predict prohormone cleavages confirm that the processing of prohormone proteins into neuropeptides is similar between species. Neuropeptide genes are most frequently expressed in the brain and head, followed by the ovary and small intestine. Microarray analyses revealed that the expression of adrenomedullin, chromogranin-A, augurin, neuromedin-U, platelet-derived growth factor A and D, proenkephalin, relaxin-3, prepronociceptin, and insulin-like growth factor I was most susceptible (P-value < 0.001) to

  9. Role of Endoproteolytic Dibasic Proprotein Processing in Maturation of Secretory Proteins in Trichoderma reesei

    PubMed Central

    Goller, Sabine P.; Schoisswohl, Doris; Baron, Michel; Parriche, Martine; Kubicek, Christian P.

    1998-01-01

    Cell extracts of Trichoderma reesei exhibited dibasic endopeptidase activity toward the carboxylic side of KR, RR, and PR sequences. This activity was stimulated by the presence of Ca2+ ions and localized in vesicles of low bouyant density; it therefore exhibited some similarity to yeast Kex2. Analytical chromatofocusing revealed a single peak of activity. The dibasic endopeptidase activity was strongly and irreversibly inhibited in vitro as well as in vivo by 1 mM p-amidinophenylmethylsulfonyl fluoride (pAPMSF) but not by PMSF at concentrations up to 5 mM. We therefore used pAPMSF to study the role of the dibasic endopeptidase in the secretion of protein by T. reesei. Secretion of xylanase I (proprotein processing sequence -R-R-↓-R-↓-A-) and xylanase II (-K-R-↓-Q-) was strongly inhibited by 1 mM pAPMSF, and a larger, unprocessed enzyme form was detected intracellularly under these conditions. Secretion of cellobiohydrolase II (CBH II; -E-R-↓-Q-) was only slightly inhibited by pAPMSF, and no accumulation of unprocessed precursors was detected. In contrast, secretion of CBH I (-R-A-↓-Q-) was stimulated by pAPMSF addition, and a simultaneous decrease in the concentration of intracellular CBH I was detected. Similar experiments were also carried out with a single heterologous protein, ShBLE, the phleomycin-binding protein from Streptoalloteichus hindustanus, fused to a series of model proprotein-processing sequences downstream of the expression signals of the Aspergillus nidulans gpdA promoter. Consistent with the results obtained with homologous proteins, pAPMSF inhibited the secretion of ShBLE with fusions containing dibasic (RK and KR) target sequences, but it even stimulated secretion in fusions to LR, NHA, and EHA target sequences. Addition of 5 mM PMSF, a nonspecific inhibitor of serine protease, nonspecifically inhibited the secretion of heterologous proteins from fusions bearing the NHA and LR targets. These data point to the existence of different

  10. An aspartyl cathepsin, CTH3, is essential for proprotein processing during secretory granule maturation in Tetrahymena thermophila

    PubMed Central

    Kumar, Santosh; Briguglio, Joseph S.; Turkewitz, Aaron P.

    2014-01-01

    In Tetrahymena thermophila, peptides secreted via dense-core granules, called mucocysts, are generated by proprotein processing. We used expression profiling to identify candidate processing enzymes, which localized as cyan fluorescent protein fusions to mucocysts. Of note, the aspartyl cathepsin Cth3p plays a key role in mucocyst-based secretion, since knockdown of this gene blocked proteolytic maturation of the entire set of mucocyst proproteins and dramatically reduced mucocyst accumulation. The activity of Cth3p was eliminated by mutation of two predicted active-site mutations, and overexpression of the wild-type gene, but not the catalytic-site mutant, partially rescued a Mendelian mutant defective in mucocyst proprotein processing. Our results provide the first direct evidence for the role of proprotein processing in this system. Of interest, both localization and the CTH3 disruption phenotype suggest that the enzyme provides non–mucocyst-related functions. Phylogenetic analysis of the T. thermophila cathepsins, combined with prior work on the role of sortilin receptors in mucocyst biogenesis, suggests that repurposing of lysosomal enzymes was an important step in the evolution of secretory granules in ciliates. PMID:24943840

  11. First survey and functional annotation of prohormone and convertase genes in the pig

    PubMed Central

    2012-01-01

    Background The pig is a biomedical model to study human and livestock traits. Many of these traits are controlled by neuropeptides that result from the cleavage of prohormones by prohormone convertases. Only 45 prohormones have been confirmed in the pig. Sequence homology can be ineffective to annotate prohormone genes in sequenced species like the pig due to the multifactorial nature of the prohormone processing. The goal of this study is to undertake the first complete survey of prohormone and prohormone convertases genes in the pig genome. These genes were functionally annotated based on 35 gene expression microarray experiments. The cleavage sites of prohormone sequences into potentially active neuropeptides were predicted. Results We identified 95 unique prohormone genes, 2 alternative calcitonin-related sequences, 8 prohormone convertases and 1 cleavage facilitator in the pig genome 10.2 assembly and trace archives. Of these, 11 pig prohormone genes have not been reported in the UniProt, UniGene or Gene databases. These genes are intermedin, cortistatin, insulin-like 5, orexigenic neuropeptide QRFP, prokineticin 2, prolactin-releasing peptide, parathyroid hormone 2, urocortin, urocortin 2, urocortin 3, and urotensin 2-related peptide. In addition, a novel neuropeptide S was identified in the pig genome correcting the previously reported pig sequence that is identical to the rabbit sequence. Most differentially expressed prohormone genes were under-expressed in pigs experiencing immune challenge relative to the un-challenged controls, in non-pregnant relative to pregnant sows, in old relative to young embryos, and in non-neural relative to neural tissues. The cleavage prediction based on human sequences had the best performance with a correct classification rate of cleaved and non-cleaved sites of 92% suggesting that the processing of prohormones in pigs is similar to humans. The cleavage prediction models did not find conclusive evidence supporting the

  12. Activation of bean (Phaseolus vulgaris) [alpha]-amylase inhibitor requires proteolytic processing of the proprotein

    SciTech Connect

    Pueyo, J.J.; Hunt, D.C.; Chrispeels, M.J. )

    1993-04-01

    Seeds of the common bean (Phaseolus vulgaris) contain a plant defense protein that inhibits the [alpha]-amylases of mammals and insects. This [alpha]-amylase inhibitor ([alpha]Al) is synthesized as a proprotein on the endoplasmic reticulum and is proteolytically processed after arrival in the protein storage vacuoles to polypeptides of relative molecular weight (M[sub r]) 15,000 to 18,000. The authors report two types of evidence that proteolytic processing is linked to activation of the inhibitory activity. First, by surveying seed extracts of wild accessions of P. vulgaris and other species in the genus Phaseolus, they found that antibodies to [alpha]Al recognize large (M[sub r] 30,000-35,000) polypeptides as well as typical [alpha]Al processing products (M[sub r] 15,000-18,000). [alpha]Al activity was found in all extracts that had the typical [alpha]Al processed polypeptides, but was absent from seed extracts that lacked such polypeptides. Second, they made a mutant [alpha]Al in which asparagine-77 is changed to aspartic acid-77. This mutation slows down the proteolytic processing of pro-[alpha]Al when the gene is expressed in tobacco. When pro-[alpha]Al was separated from mature [alpha]Al by gel filtration, pro-[alpha]Al was found not to have [alpha]-amylase inhibitory activity. The authors interpret these results to mean that formation of the active inhibitor is causally related to proteolytic processing of the proprotein. They suggest that the polypeptide cleavage removes a conformation constraint on the precursor to produce the biochemically active molecule. 43 refs., 5 figs., 1 tab.

  13. Bioactivity of the putative apelin proprotein expands the repertoire of apelin receptor ligands.

    PubMed

    Shin, Kyungsoo; Chapman, Nigel A; Sarker, Muzaddid; Kenward, Calem; Huang, Shuya K; Weatherbee-Martin, Nathan; Pandey, Aditya; Dupré, Denis J; Rainey, Jan K

    2017-08-01

    Apelin is a peptide ligand for a class A G-protein coupled receptor called the apelin receptor (AR or APJ) that regulates angiogenesis, the adipoinsular axis, and cardiovascular functions. Apelin has been shown to be bioactive as 13, 17, and 36 amino acid isoforms, C-terminal fragments of the putatively inactive 55-residue proprotein (proapelin or apelin-55). Although intracellular proprotein processing has been proposed, isolation of apelin-55 from colostrum and milk demonstrates potential for secretion prior to processing and the possibility of proapelin-AR interaction. Apelin isoform activity and potency were compared by an In-Cell Western™ assay for ERK phosphorylation using a stably AR-transfected HEK293A cell line. Conformational comparison of apelin isoforms was carried out by circular dichroism and heteronuclear solution-state nuclear magnetic resonance spectroscopy. Apelin-55 is shown to activate the AR, with similar maximum ERK phophorylation response and potency to the shorter isoforms except for apelin-13, which exhibited a greater potency. Correlating to this shared activity, highly similar conformations are exhibited in all apelin isoforms for the shared C-terminal region responsible for receptor binding and activation. AR activation by all apelin isoforms likely hinges upon shared conformation and dynamics in the C-terminus, with apelin-55 providing an alternative bioactive isoform despite the addition of 19N-terminal residues relative to apelin-36. Beyond providing novel insight into the physiology of this system, re-annotation of proapelin to the bioactive apelin-55 isoform adds to the molecular toolkit for dissection of apelin-AR interactions and expands the repertoire of therapeutic targets for the apelinergic system. Copyright © 2017 Elsevier B.V. All rights reserved.

  14. A proprotein convertase subtilisin-like/kexin type 9 (PCSK9) C-terminal domain antibody antigen-binding fragment inhibits PCSK9 internalization and restores low density lipoprotein uptake.

    PubMed

    Ni, Yan G; Condra, Jon H; Orsatti, Laura; Shen, Xun; Di Marco, Stefania; Pandit, Shilpa; Bottomley, Matthew J; Ruggeri, Lionello; Cummings, Richard T; Cubbon, Rose M; Santoro, Joseph C; Ehrhardt, Anka; Lewis, Dale; Fisher, Timothy S; Ha, Sookhee; Njimoluh, Leila; Wood, Dana D; Hammond, Holly A; Wisniewski, Douglas; Volpari, Cinzia; Noto, Alessia; Lo Surdo, Paola; Hubbard, Brian; Carfí, Andrea; Sitlani, Ayesha

    2010-04-23

    PCSK9 binds to the low density lipoprotein receptor (LDLR) and leads to LDLR degradation and inhibition of plasma LDL cholesterol clearance. Consequently, the role of PCSK9 in modulating circulating LDL makes it a promising therapeutic target for treating hypercholesterolemia and coronary heart disease. Although the C-terminal domain of PCSK9 is not involved in LDLR binding, the location of several naturally occurring mutations within this region suggests that it has an important role for PCSK9 function. Using a phage display library, we identified an anti-PCSK9 Fab (fragment antigen binding), 1G08, with subnanomolar affinity for PCSK9. In an assay measuring LDL uptake in HEK293 and HepG2 cells, 1G08 Fab reduced 50% the PCSK9-dependent inhibitory effects on LDL uptake. Importantly, we found that 1G08 did not affect the PCSK9-LDLR interaction but inhibited the internalization of PCSK9 in these cells. Furthermore, proteolysis and site-directed mutagenesis studies demonstrated that 1G08 Fab binds a region of beta-strands encompassing Arg-549, Arg-580, Arg-582, Glu-607, Lys-609, and Glu-612 in the PCSK9 C-terminal domain. Consistent with these results, 1G08 fails to bind PCSK9DeltaC, a truncated form of PCSK9 lacking the C-terminal domain. Additional studies revealed that lack of the C-terminal domain compromised the ability of PCSK9 to internalize into cells, and to inhibit LDL uptake. Together, the present study demonstrate that the PCSK9 C-terminal domain contribute to its inhibition of LDLR function mainly through its role in the cellular uptake of PCSK9 and LDLR complex. 1G08 Fab represents a useful new tool for delineating the mechanism of PCSK9 uptake and LDLR degradation.

  15. Identification of the paired basic convertases implicated in HIV gp160 processing based on in vitro assays and expression in CD4(+) cell lines.

    PubMed

    Decroly, E; Wouters, S; Di Bello, C; Lazure, C; Ruysschaert, J M; Seidah, N G

    1996-11-29

    The human immunodeficiency virus HIV envelope glycoprotein gp160 is synthesized as an inactive precursor, which is processed into its fusiogenic form gp120/gp41 by host cell proteinases during its intracellular trafficking. Kexin/subtilisin-related endoproteases have been proposed to be enzyme candidates for this maturation process. In the present study, 1) we examined the ability of partially purified precursor convertases and their isoforms to cleave gp160 in vitro. The data demonstrate that all the convertases tested specifically cleave the HIV envelope glycoprotein into gp120 and gp41. 2) We demonstrated that a 19-amino acid model peptide spanning the gp120/gp41 junction is cleaved by all convertases at the same gp160 site as that recognized in HIV-infected cells. 3) In an effort to evaluate specific convertase inhibitors, we showed that the alpha1-antitrypsin variant, alpha1-PDX, inhibits equally well the ability of the tested convertases to cleave gp160 in vitro. 4) Three lymphocyte cell lines were screened by reverse transcription polymerase chain reaction in an effort to identify which are the convertases expressed in the most common HIV target, the CD4(+) lymphocytes. The data demonstrate that furin, PC5/6, and the newly cloned PC7 are the main transcribed convertases, suggesting that these proteinases are the major gp160-converting enzymes in T4 lymphocytes.

  16. Manipulating the mediator: modulation of the alternative complement pathway C3 convertase in health, disease and therapy.

    PubMed

    Ricklin, Daniel

    2012-11-01

    The complement network is increasingly recognized as an important triage system that is able to differentiate between healthy host cells, microbial intruders, cellular debris and immune complexes, and tailor its actions accordingly. At the center of this triage mechanism is the alternative pathway C3 convertase (C3bBb), a potent enzymatic protein complex capable of rapidly converting the inert yet abundant component C3 into powerful effector fragments (C3a and C3b), thereby amplifying the initial response on unprotected surfaces and inducing a variety of effector functions. A fascinating molecular mechanism of convertase assembly and intrinsic regulation, as well as the interplay with a panel of cell surface-bound and soluble inhibitors are essential for directing complement attack to intruders and protecting healthy host cells. While efficiently keeping immune surveillance and homeostasis on track, the reliance on an intricate cascade of interaction and conversion steps also renders the C3 convertase vulnerable to derail. On the one hand, tissue damage, accumulation of debris, or polymorphisms in complement genes may unfavorably shift the balance between activation and regulation, thereby contributing to a variety of clinical conditions. On the other hand, pathogens developed powerful evasion strategies to avoid complement attack by targeting the convertase. Finally, we increasingly challenge our bodies with foreign materials such as biomaterial implants or drug delivery vehicles that may induce adverse effects that are at least partially caused by complement activation and amplification via the alternative pathway. The involvement of the C3 convertase in a range of pathological conditions put this complex into the spotlight of complement-targeted drug discovery efforts. Fortunately, the physiological regulation and microbial evasion approaches provide a rich source of inspiration for the development of powerful treatment options. This review provides insight into

  17. Sensitive radiometric assay for enkephalin convertase and other carboxypeptidase B-like enzymes

    SciTech Connect

    Stack, G.; Fricker, L.D.; Snyder, S.H.

    1984-01-09

    A sensitive radiometric assay for carboxypeptidase B-like enzymes has been developed using enkephalin convertase, an enkephalin synthesizing carboxypeptidase. The assay is based on the differential solubility of /sup 3/H-labeled substrate and product in chloroform. The substrates /sup 3/H-benzoyl-Phe-Ala-Arg or /sup 3/H-benzoyl-Phe-Leu-Arg are poorly soluble in chloroform due to the charged arginine. The products of carboxypeptidase B-like activity on these substrates, /sup 3/H-benzoyl-Phe-Ala or /sup 3/H-benzoyl Phe-Leu partition quantitatively into chloroform, allowing rapid separation of product from substrate. This assay is approximately 100 times more sensitive than a similar fluorometric assay utilizing dansyl-Phe-Ala-Arg as a substrate.

  18. Shared functions in vivo of a glycosyl-phosphatidylinositol-linked aspartyl protease, Mkc7, and the proprotein processing protease Kex2 in yeast.

    PubMed Central

    Komano, H; Fuller, R S

    1995-01-01

    The MKC7 gene was isolated as a multicopy suppressor of the cold-sensitive growth phenotype of a yeast kex2 mutant, which lacks the protease that cleaves pro-alpha-factor and other secretory proproteins at pairs of basic residues in a late Golgi compartment in yeast. MKC7 encodes an aspartyl protease most closely related to product of the YAP3 gene, a previously isolated multicopy suppressor of the pro-alpha-factor processing defect of a kex2 null. Multicopy MKC7 suppressed the alpha-specific mating defect of a kex2 null as well as multicopy YAP3 did, but multicopy YAP3 was a relatively weak suppressor of kex2 cold sensitivity. Overexpression of MKC7 resulted in production of a membrane-associated proteolytic activity that cleaved an internally quenched fluorogenic peptide substrate on the carboxyl side of a Lys-Arg site. Treatment with phosphatidylinositol-specific phospholipase C shifted Mkc7 activity from the detergent to the aqueous phase in a Triton X-114 phase separation, indicating that membrane attachment of Mkc7 is mediated by a glycosyl-phosphatidylinositol anchor. Although disruption of MKC7 or YAP3 alone resulted in no observable phenotype, mkc7 yap3 double disruptants exhibited impaired growth at 37 degrees C. Disruption of MKC7 and YAP3 in a kex2 null mutant resulted in profound temperature sensitivity and more generalized cold sensitivity. The synergism of mkc7, yap3, and kex2 null mutations argues that Mkc7 and Yap3 are authentic processing enzymes whose functions overlap those of Kex2 in vivo. Images Fig. 1 Fig. 3 Fig. 4 PMID:7479877

  19. The C5 convertase is not required for activation of the terminal complement pathway in murine experimental cerebral malaria.

    PubMed

    Ramos, Theresa N; Darley, Meghan M; Weckbach, Sebastian; Stahel, Philip F; Tomlinson, Stephen; Barnum, Scott R

    2012-07-13

    Cerebral malaria (CM) is the most severe manifestation of clinical malaria syndromes and has a high fatality rate especially in the developing world. Recent studies demonstrated that C5(-/-) mice are resistant to experimental CM (ECM) and that protection was due to the inability to form the membrane attack complex. Unexpectedly, we observed that C4(-/-) and factor B(-/-) mice were fully susceptible to disease, indicating that activation of the classical or alternative pathways is not required for ECM. C3(-/-) mice were also susceptible to ECM, indicating that the canonical C5 convertases are not required for ECM development and progression. Abrogation of ECM by treatment with anti-C9 antibody and detection of C5a in serum of C3(-/-) mice confirmed that C5 activation occurs in ECM independent of C5 convertases. Our data indicate that activation of C5 in ECM likely occurs via coagulation enzymes of the extrinsic protease pathway.

  20. Relative resistance of the F-42-stabilized classical pathway C3 convertase to inactivation by C4-binding protein.

    PubMed

    Daha, M R; van Es, L A

    1980-11-01

    The sera of some patients with SLE contain an IgG antibody (F-42) directed against the classical pathway C3 convertase (C-42), which is capable of stabilizing C42 in a dose-dependent manner. The half-life (T 1/2) of C42 is prolonged by F-42. In order to determine whether C4-binding protein was capable of reversing stabilization of C42, stabilized and unstabilized cell-bound C42 were exposed to purified C4-bp and the convertase activity was assessed. C4-bp was capable of accelerating the decay of C42 in a dose-dependent manner; 2 microgram/ml C4-bp reduced the T 1/2 of C42 from 5 to 2.5 min at 30 degrees C. On the other hand, 16 microgram C4-bp could reverse stabilization of C42 by F-42 from T 1/2 = 78 min to a T 1/2 - 40 min; 128 microgram C4-bp reduced the T 1/2 of stabilized C42 to 4 min. Functional inactivation of C42 occurs via enhanced decay-dissociation of C2 from the convertase by C4-bp, as shown by the release of 125I-C2i from the cell-bound convertase. Stabilization of C42 by F-42 is caused by prevention of decay-dissociation of 125I-C2. F-42 was also capable of stabilizing C4oxy2 even further, as shown by prolongation of the T 1/2 of cell-bound C4 oxy2 to a T 1/2 of at least 300 min at 30 degrees C.

  1. Regulation of prohormone convertase 2 protein expression via GPR40/FFA1 in the hypothalamus.

    PubMed

    Nakamoto, Kazuo; Aizawa, Fuka; Nishinaka, Takashi; Tokuyama, Shogo

    2015-09-05

    Previous studies have shown that the administration of docosahexaenoic acid (DHA) or GW9508, a GPR40/FFA1 (free fatty acid receptor) agonist, facilitates β-endorphin release in the arcuate nucleus of the hypothalamus in mice. However, the mechanisms mediating β-endorphin release induced by GPR40/FFA1 agonists remain unknown. In this study, we focused on the changes in expression of hypothalamic prohormone convertase (PC) 2, which is a calcium-dependent subtilisin-related proteolytic enzyme. The intracerebroventricular injection of DHA or GW9508 significantly increased PC2 protein expression in the hypothalamus. This increase in PC2 expression was inhibited by pretreatment with GW1100, a GPR40/FFA1 antagonist. Furthermore, PC2 protein expression gradually increased over time after complete Freund's adjuvant. These increase in PC2 expression were inhibited by pretreatment with GW1100. However, GW1100 by itself had no effect on PC2 levels. Taken together, our findings suggest that activation of the hypothalamic GPR40/FFA1 signaling pathway may regulate β-endorphin release via PC2, and regulate the endogenous pain control system.

  2. Enkephalin convertase: Characterization and localization with ( sup 3 H)-guanidinoethylmercap-tosuccinic acid

    SciTech Connect

    Lynch, D.R.

    1988-01-01

    Enkephalin convertase (EC) has been characterized by the binding of its selective inhibitor ({sup 3}H)-guanidinoethylmercaptosuccinic acid (GEMSA). The pharmacology and affinity of ({sup 3}H)-GEMSA binding match the pharmacology of EC activity and the inhibition of EC activity by GEMSA. EC activity and ({sup 3}H)-GEMSA binding activity copurify to homogeneity demonstrating that ({sup 3}H)-GEMSA binds selectively to EC. The selective association of ({sup 3}H)-GEMSA for EC allows localization of membrane bound EC by in vitro autoradiography. EC is heterogeneously distributed in the rat brain with the highest levels in the outer zone of the median eminence and in the hypothalamic magnocellular nuclei. In the pituitary gland, autoradiography localizes EC to all three lobes with the highest levels in the intermediate lobe. In the adrenal EC is found exclusively in the medulla. In the gastrointestinal tract, EC is localized to epithelial surfaces where its function cannot be that of propeptide processing. In the heart EC is localized to atrium where it likely processes precursors of atrial natriuretic factor.

  3. The structure of C2b, a fragment of complement component C2 produced during C3 convertase formation

    SciTech Connect

    Krishnan, Vengadesan; Xu, Yuanyuan; Macon, Kevin; Volanakis, John E.; Narayana, Sthanam V. L.

    2009-03-01

    The crystal structure of C2b has been determined at 1.8 Å resolution, which reveals the arrangement of its three complement control protein (CCP) modules. A model for complement component C2 is presented and its conformational changes during the C3-convertase formation are also discussed. The second component of complement (C2) is a multi-domain serine protease that provides catalytic activity for the C3 and C5 convertases of the classical and lectin pathways of human complement. The formation of these convertases requires the Mg{sup 2+}-dependent binding of C2 to C4b and the subsequent cleavage of C2 by C1s or MASP2, respectively. The crystal structure of full-length C2 is not yet available, although the structure of its C-terminal catalytic segment C2a has been determined. The crystal structure of the N-terminal segment C2b of C2 determined to 1.8 Å resolution presented here reveals the arrangement of its three CCP domains. The domains are arranged differently compared with most other CCP-domain assemblies, but their arrangement is similar to that found in the Ba part of the full-length factor B structure. The crystal structures of C2a, C2b and full-length factor B are used to generate a model for C2 and a discussion of the domain association and possible interactions with C4b during formation of the C4b–C2 complex is presented. The results of this study also suggest that upon cleavage by C1s, C2a domains undergo conformational rotation while bound to C4b and the released C2b domains may remain folded together similar to as observed in the intact protein.

  4. Factor C acts as a lipopolysaccharide-responsive C3 convertase in horseshoe crab complement activation.

    PubMed

    Ariki, Shigeru; Takahara, Shusaku; Shibata, Toshio; Fukuoka, Takaaki; Ozaki, Aya; Endo, Yuichi; Fujita, Teizo; Koshiba, Takumi; Kawabata, Shun-ichiro

    2008-12-01

    The complement system in vertebrates plays an important role in host defense against and clearance of invading microbes, in which complement component C3 plays an essential role in the opsonization of pathogens, whereas the molecular mechanism underlying C3 activation in invertebrates remains unknown. In an effort to understand the molecular activation mechanism of invertebrate C3, we isolated and characterized an ortholog of C3 (designated TtC3) from the horseshoe crab Tachypleus tridentatus. Flow cytometric analysis using an Ab against TtC3 revealed that the horseshoe crab complement system opsonizes both Gram-negative and Gram-positive bacteria. Evaluation of the ability of various pathogen-associated molecular patterns to promote the proteolytic conversion of TtC3 to TtC3b in hemocyanin-depleted plasma indicated that LPS, but not zymosan, peptidoglycan, or laminarin, strongly induces this conversion, highlighting the selective response of the complement system to LPS stimulation. Although originally characterized as an LPS-sensitive initiator of hemolymph coagulation stored within hemocytes, we identified factor C in hemolymph plasma. An anti-factor C Ab inhibited various LPS-induced phenomena, including plasma amidase activity, the proteolytic activation of TtC3, and the deposition of TtC3b on the surface of Gram-negative bacteria. Moreover, activated factor C present on the surface of Gram-negative bacteria directly catalyzed the proteolytic conversion of the purified TtC3, thereby promoting TtC3b deposition. We conclude that factor C acts as an LPS-responsive C3 convertase on the surface of invading Gram-negative bacteria in the initial phase of horseshoe crab complement activation.

  5. Gene expression of the dibasic-pair cleaving enzyme NRD convertase (N-arginine dibasic convertase) is differentially regulated in the GH3 pituitary and Mat-Lu prostate cell lines.

    PubMed

    Winter, A G; Pierotti, A R

    2000-11-01

    NRD convertase (N-arginine dibasic convertase, NRD-C) is a dibasic selective metalloprotease which cleaves on the N-terminal side of an arginine residue in a dibasic pair. Abundant in endocrine tissues, the highest levels are found in testis. The mechanism whereby NRD-C expression is regulated at the transcriptional level has been examined by reporter-gene assay and electrophoretic-mobility-shift assays. Analysis of the rat and human promoters show that they are highly conserved, containing a number of motifs which may correspond to transcription-factor binding sites. The rat promoter has been cloned into a luciferase reporter vector and analysed in a number of cell lines. Full functionality of the promoter is observed with 5' deletions to 411 bp upstream of the transcriptional start site in spermatid, prostate and pituitary cell lines. Further deletion to 101 bp causes a complete loss of activity in spermatid and prostate lines. By contrast, GH3 pituitary cells display no reduction in promoter activity with deletion to 101 bp of upstream sequence. A number of transcription-factor binding sites have been identified by electrophoretic-mobility-shift assays in the region 411-101; however, no differences in binding between the cell lines were observed.

  6. The C5 Convertase Is Not Required for Activation of the Terminal Complement Pathway in Murine Experimental Cerebral Malaria*

    PubMed Central

    Ramos, Theresa N.; Darley, Meghan M.; Weckbach, Sebastian; Stahel, Philip F.; Tomlinson, Stephen; Barnum, Scott R.

    2012-01-01

    Cerebral malaria (CM) is the most severe manifestation of clinical malaria syndromes and has a high fatality rate especially in the developing world. Recent studies demonstrated that C5−/− mice are resistant to experimental CM (ECM) and that protection was due to the inability to form the membrane attack complex. Unexpectedly, we observed that C4−/− and factor B−/− mice were fully susceptible to disease, indicating that activation of the classical or alternative pathways is not required for ECM. C3−/− mice were also susceptible to ECM, indicating that the canonical C5 convertases are not required for ECM development and progression. Abrogation of ECM by treatment with anti-C9 antibody and detection of C5a in serum of C3−/− mice confirmed that C5 activation occurs in ECM independent of C5 convertases. Our data indicate that activation of C5 in ECM likely occurs via coagulation enzymes of the extrinsic protease pathway. PMID:22689574

  7. Expression and localization of prohormone convertase PC1 in the calcitonin-producing cells of the bullfrog ultimobranchial gland.

    PubMed

    Yaoi, Yuichi; Suzuki, Masakazu; Tomura, Hideaki; Kurabuchi, Shingo; Sasayama, Yuichi; Tanaka, Shigeyasu

    2003-11-01

    We examined the expression and localization of the prohormone convertases, PC1 and PC2, in the ultimobranchial gland of the adult bullfrog using immunohistochemical (IHC) and in situ hybridization (ISH) techniques. In the ultimobranchial gland, PC1-immunoreactive cells were columnar, and were present in the follicular epithelium. When serial sections were immunostained with anti-calcitonin, anti-CGRP, anti-PC1, and anti-PC2 sera, PC1 was found only in the calcitonin/CGRP-producing cells. No PC2-immunopositive cells were detected. In the ISH, PC1 mRNA-positive cells were detected in the follicle cells in the ultimobranchial gland. No PC2 mRNA-positive cells were detected. RT-PCR revealed expression of the mRNAs of PC1 and the PC2 in the ultimobranchial gland. However, very little of the PC2 mRNA is probably translated because no PC2 protein was detected either by IHC staining or by Western blotting analysis. We conclude that the main prohormone convertase that is involved in the proteolytic cleavage of procalcitonin in the bullfrog is PC1.

  8. Expression and Localization of Prohormone Convertase PC1 in the Calcitonin-producing Cells of the Bullfrog Ultimobranchial Gland

    PubMed Central

    Yaoi, Yuichi; Suzuki, Masakazu; Tomura, Hideaki; Kurabuchi, Shingo; Sasayama, Yuichi; Tanaka, Shigeyasu

    2003-01-01

    We examined the expression and localization of the prohormone convertases, PC1 and PC2, in the ultimobranchial gland of the adult bullfrog using immunohistochemical (IHC) and in situ hybridization (ISH) techniques. In the ultimobranchial gland, PC1-immunoreactive cells were columnar, and were present in the follicular epithelium. When serial sections were immunostained with anti-calcitonin, anti-CGRP, anti-PC1, and anti-PC2 sera, PC1 was found only in the calcitonin/CGRP-producing cells. No PC2-immunopositive cells were detected. In the ISH, PC1 mRNA-positive cells were detected in the follicle cells in the ultimobranchial gland. No PC2 mRNA-positive cells were detected. RT-PCR revealed expression of the mRNAs of PC1 and the PC2 in the ultimobranchial gland. However, very little of the PC2 mRNA is probably translated because no PC2 protein was detected either by IHC staining or by Western blotting analysis. We conclude that the main prohormone convertase that is involved in the proteolytic cleavage of procalcitonin in the bullfrog is PC1. PMID:14566018

  9. Gene family encoding the major toxins of lethal Amanita mushrooms.

    PubMed

    Hallen, Heather E; Luo, Hong; Scott-Craig, John S; Walton, Jonathan D

    2007-11-27

    Amatoxins, the lethal constituents of poisonous mushrooms in the genus Amanita, are bicyclic octapeptides. Two genes in A. bisporigera, AMA1 and PHA1, directly encode alpha-amanitin, an amatoxin, and the related bicyclic heptapeptide phallacidin, a phallotoxin, indicating that these compounds are synthesized on ribosomes and not by nonribosomal peptide synthetases. alpha-Amanitin and phallacidin are synthesized as proproteins of 35 and 34 amino acids, respectively, from which they are predicted to be cleaved by a prolyl oligopeptidase. AMA1 and PHA1 are present in other toxic species of Amanita section Phalloidae but are absent from nontoxic species in other sections. The genomes of A. bisporigera and A. phalloides contain multiple sequences related to AMA1 and PHA1. The predicted protein products of this family of genes are characterized by a hypervariable "toxin" region capable of encoding a wide variety of peptides of 7-10 amino acids flanked by conserved sequences. Our results suggest that these fungi have a broad capacity to synthesize cyclic peptides on ribosomes.

  10. Differences in Cryptococcus neoformans capsular polysaccharide structure influence assembly of alternative complement pathway C3 convertase on fungal surfaces.

    PubMed

    Washburn, R G; Bryant-Varela, B J; Julian, N C; Bennett, J E

    1991-01-01

    Binding of complement component C3 and Factor B to Cryptococcus neoformans serotypes A through D via the alternative complement pathway was measured in a system containing fresh nonimmune human serum. Serotypes B and C (C. neoformans var. gattii) bound approximately half as many molecules of both complement components as serotypes A and D (C. neoformans var. neoformans). In contrast, removal of xylosyl and glucuronyl side chains from the mannan main chain of capsular polysaccharide by the Smith degradation procedure resulted in binding of similar quantities of C3 to each of the four serotypes. We conclude that the relatively high degree of side chain substitution of capsular polysaccharide from C. neoformans variety gattii contributes to inefficient surface assembly of the alternative pathway C3 convertase. Inefficient binding of alternative pathway complement components to serotypes B and C may contribute to the relative difficulty in successfully treating infections caused by these organisms.

  11. Inhibition of Prohormone Convertases PC1/3 and PC2 by 2,5-Dideoxystreptamine Derivatives

    PubMed Central

    Vivoli, Mirella; Caulfield, Thomas R.; Martínez-Mayorga, Karina; Johnson, Alan T.; Jiao, Guan-Sheng

    2012-01-01

    The prohormone convertases PC1/3 and PC2 are eukaryotic serine proteases involved in the proteolytic maturation of peptide hormone precursors and are implicated in a variety of pathological conditions, including obesity, diabetes, and neurodegenerative diseases. In this work, we screened 45 compounds obtained by derivatization of a 2,5-dideoxystreptamine scaffold with guanidinyl and aryl substitutions for convertase inhibition. We identified four promising PC1/3 competitive inhibitors and three PC2 inhibitors that exhibited various inhibition mechanisms (competitive, noncompetitive, and mixed), with sub- and low micromolar inhibitory potency against a fluorogenic substrate. Low micromolar concentrations of certain compounds blocked the processing of the physiological substrate proglucagon. The best PC2 inhibitor effectively inhibited glucagon synthesis, a known PC2-mediated process, in a pancreatic cell line; no cytotoxicity was observed. We also identified compounds that were able to stimulate both 87 kDa PC1/3 and PC2 activity, behavior related to the presence of aryl groups on the dideoxystreptamine scaffold. By contrast, inhibitory activity was associated with the presence of guanidinyl groups. Molecular modeling revealed interactions of the PC1/3 inhibitors with the active site that suggest structural modifications to further enhance potency. In support of kinetic data suggesting that PC2 inhibition probably occurs via an allosteric mechanism, we identified several possible allosteric binding sites using computational searches. It is noteworthy that one compound was found to both inhibit PC2 and stimulate PC1/3. Because glucagon acts in functional opposition to insulin in blood glucose homeostasis, blocking glucagon formation and enhancing proinsulin cleavage with a single compound could represent an attractive therapeutic approach in diabetes. PMID:22169851

  12. Posttranslocation chaperone PrsA2 regulates the maturation and secretion of Listeria monocytogenes proprotein virulence factors.

    PubMed

    Forster, Brian M; Zemansky, Jason; Portnoy, Daniel A; Marquis, Hélène

    2011-11-01

    PrsA2 is a conserved posttranslocation chaperone and a peptidyl prolyl cis-trans isomerase (PPIase) that contributes to the virulence of the Gram-positive intracellular pathogen Listeria monocytogenes. One of the phenotypes associated with a prsA2 mutant is decreased activity of the broad-range phospholipase C (PC-PLC). PC-PLC is made as a proenzyme whose maturation is mediated by a metalloprotease (Mpl). The proforms of PC-PLC and Mpl accumulate at the membrane-cell wall interface until a decrease in pH triggers their maturation and rapid secretion into the host cell. In this study, we examined the mechanism by which PrsA2 regulates the activity of PC-PLC. We observed that in the absence of PrsA2, the proenzymes are secreted at physiological pH and do not mature upon a decrease in pH. The sensitivity of the prsA2 mutant to cell wall hydrolases was modified. However, no apparent changes in cell wall porosity were detected. Interestingly, synthesis of PC-PLC in the absence of its propeptide lead to the secretion of a fully active enzyme in the cytosol of host cells independent of PrsA2, indicating that neither the propeptide of PC-PLC nor PrsA2 is required for native folding of the catalytic domain, although both influence secretion of the enzyme. Taken together, these results suggest that PrsA2 regulates compartmentalization of Mpl and PC-PLC, possibly by influencing cell wall properties and interacting with the PC-PLC propeptide. Moreover, the ability of these proproteins to respond to a decrease in pH during intracellular growth depends on their localization at the membrane-cell wall interface.

  13. Posttranslocation Chaperone PrsA2 Regulates the Maturation and Secretion of Listeria monocytogenes Proprotein Virulence Factors ▿

    PubMed Central

    Forster, Brian M.; Zemansky, Jason; Portnoy, Daniel A.; Marquis, Hélène

    2011-01-01

    PrsA2 is a conserved posttranslocation chaperone and a peptidyl prolyl cis-trans isomerase (PPIase) that contributes to the virulence of the Gram-positive intracellular pathogen Listeria monocytogenes. One of the phenotypes associated with a prsA2 mutant is decreased activity of the broad-range phospholipase C (PC-PLC). PC-PLC is made as a proenzyme whose maturation is mediated by a metalloprotease (Mpl). The proforms of PC-PLC and Mpl accumulate at the membrane-cell wall interface until a decrease in pH triggers their maturation and rapid secretion into the host cell. In this study, we examined the mechanism by which PrsA2 regulates the activity of PC-PLC. We observed that in the absence of PrsA2, the proenzymes are secreted at physiological pH and do not mature upon a decrease in pH. The sensitivity of the prsA2 mutant to cell wall hydrolases was modified. However, no apparent changes in cell wall porosity were detected. Interestingly, synthesis of PC-PLC in the absence of its propeptide lead to the secretion of a fully active enzyme in the cytosol of host cells independent of PrsA2, indicating that neither the propeptide of PC-PLC nor PrsA2 is required for native folding of the catalytic domain, although both influence secretion of the enzyme. Taken together, these results suggest that PrsA2 regulates compartmentalization of Mpl and PC-PLC, possibly by influencing cell wall properties and interacting with the PC-PLC propeptide. Moreover, the ability of these proproteins to respond to a decrease in pH during intracellular growth depends on their localization at the membrane-cell wall interface. PMID:21908675

  14. Structures of adamalysin II with peptidic inhibitors. Implications for the design of tumor necrosis factor alpha convertase inhibitors.

    PubMed Central

    Gomis-Rüth, F. X.; Meyer, E. F.; Kress, L. F.; Politi, V.

    1998-01-01

    Crotalus adamanteus snake venom adamalysin II is the structural prototype of the adamalysin or ADAM family comprising proteolytic domains of snake venom metalloproteinases, multimodular mammalian reproductive tract proteins, and tumor necrosis factor alpha convertase, TACE, involved in the release of the inflammatory cytokine, TNFalpha. The structure of adamalysin II in noncovalent complex with two small-molecule right-hand side peptidomimetic inhibitors (Pol 647 and Pol 656) has been solved using X-ray diffraction data up to 2.6 and 2.8 A resolution. The inhibitors bind to the S'-side of the proteinase, inserting between two protein segments, establishing a mixed parallel-antiparallel three-stranded beta-sheet and coordinate the central zinc ion in a bidentate manner via their two C-terminal oxygen atoms. The proteinase-inhibitor complexes are described in detail and are compared with other known structures. An adamalysin-based model of the active site of TACE reveals that these small molecules would probably fit into the active site cleft of this latter metalloproteinase, providing a starting model for the rational design of TACE inhibitors. PMID:9521103

  15. ENCODE data at the ENCODE portal

    PubMed Central

    Sloan, Cricket A.; Chan, Esther T.; Davidson, Jean M.; Malladi, Venkat S.; Strattan, J. Seth; Hitz, Benjamin C.; Gabdank, Idan; Narayanan, Aditi K.; Ho, Marcus; Lee, Brian T.; Rowe, Laurence D.; Dreszer, Timothy R.; Roe, Greg; Podduturi, Nikhil R.; Tanaka, Forrest; Hong, Eurie L.; Cherry, J. Michael

    2016-01-01

    The Encyclopedia of DNA Elements (ENCODE) Project is in its third phase of creating a comprehensive catalog of functional elements in the human genome. This phase of the project includes an expansion of assays that measure diverse RNA populations, identify proteins that interact with RNA and DNA, probe regions of DNA hypersensitivity, and measure levels of DNA methylation in a wide range of cell and tissue types to identify putative regulatory elements. To date, results for almost 5000 experiments have been released for use by the scientific community. These data are available for searching, visualization and download at the new ENCODE Portal (www.encodeproject.org). The revamped ENCODE Portal provides new ways to browse and search the ENCODE data based on the metadata that describe the assays as well as summaries of the assays that focus on data provenance. In addition, it is a flexible platform that allows integration of genomic data from multiple projects. The portal experience was designed to improve access to ENCODE data by relying on metadata that allow reusability and reproducibility of the experiments. PMID:26527727

  16. The fsr Quorum-Sensing System and Cognate Gelatinase Orchestrate the Expression and Processing of Proprotein EF_1097 into the Mature Antimicrobial Peptide Enterocin O16.

    PubMed

    Dundar, Halil; Brede, Dag A; La Rosa, Sabina Leanti; El-Gendy, Ahmed Osama; Diep, Dzung B; Nes, Ingolf F

    2015-07-01

    A novel antimicrobial peptide designated enterocin O16 was purified from Enterococcus faecalis. Mass spectrometry showed a monoisotopic mass of 7,231 Da, and N-terminal Edman degradation identified a 29-amino-acid sequence corresponding to residues 90 to 119 of the EF_1097 protein. Bioinformatic analysis showed that enterocin O16 is composed of the 68 most C-terminal residues of the EF_1097 protein. Introduction of an in-frame isogenic deletion in the ef1097 gene abolished the production of enterocin O16. Enterocin O16 has a narrow inhibitory spectrum, as it inhibits mostly lactobacilli. Apparently, E. faecalis is intrinsically resistant to the antimicrobial peptide, as no immunity connected to the production of enterocin O16 could be identified. ef1097 has previously been identified as one of three loci regulated by the fsr quorum-sensing system. The introduction of a nonsense mutation into fsrB consistently impaired enterocin O16 production, but externally added gelatinase biosynthesis-activating pheromone restored the antimicrobial activity. Functional genetic analysis showed that the EF_1097 proprotein is processed extracellularly into enterocin O16 by the metalloprotease GelE. Thus, it is evident that the fsr quorum-sensing system constitutes the regulatory unit that controls the expression of the EF_1097 precursor protein and the protease GelE and that the latter is required for the formation of enterocin O16. On the basis of these results, this study identified antibacterial antagonism as a novel aspect related to the function of fsr and provides a rationale for why ef1097 is part of the fsr regulon. The fsr quorum-sensing system modulates important physiological functions in E. faecalis via the activity of GelE. The present study presents a new facet of fsr signaling. The system controls the expression of three primary target operons (fsrABCD, gelE-sprE, and ef1097-ef1097b). We demonstrate that the concerted expression of these operons constitutes the

  17. Comparative cellular processing of the human immunodeficiency virus (HIV-1) envelope glycoprotein gp160 by the mammalian subtilisin/kexin-like convertases.

    PubMed Central

    Vollenweider, F; Benjannet, S; Decroly, E; Savaria, D; Lazure, C; Thomas, G; Chrétien, M; Seidah, N G

    1996-01-01

    We present here the pulse and pulse-chase analysis of the biosynthesis of the envelope glycoprotein gp160 and its intracellular processing by the subtilisin/kexin-like convertases furin, PACE4, PC1, PC5 and its isoform PC5/6-B. We demonstrate that furin and to a much lesser extent PACE4, PC5/6-B and PC1 are candidate enzymes capable of processing gp160 intracellularly. Furthermore we show that furin can also process gp160/gp120 into gp77/gp53 products by cleavage at the sequence RIQR/GPGR just preceding the conserved GPGR structure found at the tip of the hypervariable V3 loop. The results show that processing into gp120 could occur at or before the trans-Golgi network (TGN) where sulphation of the oligosaccharide moieties of gp160 was detected. In contrast, the formation of gp77/gp53 by furin is a late event occurring after exit from the TGN. Our data also revealed that the alpha glucosidase I inhibitor N-butyldeoxynojirimycin, although affecting the oligosaccharide composition of gp160, does not impair the processing of either gp160 or gp120 by either furin or PACE4. Finally, the co-expression of the [Arg355, Arg358]-alpha-1-antitrypsin Portland variant was shown to potently inhibit the processing of both gp160 and gp120 by these convertases. PMID:8670066

  18. The metabolic sensor Sirt1 and the hypothalamus: Interplay between peptide hormones and pro-hormone convertases.

    PubMed

    Nillni, Eduardo A

    2016-12-15

    The last decade had witnessed a tremendous progress in our understanding of the causes of metabolic diseases including obesity. Among the contributing factors regulating energy balance are nutrient sensors such as sirtuins. Sirtuin1 (Sirt1), a NAD + - dependent deacetylase is affected by diet, environmental stress, and also plays a critical role in metabolic health by deacetylating proteins in many tissues, including liver, muscle, adipose tissue, heart, endothelium, and in the complexity of the hypothalamus. Because of its dependence on NAD+, Sirt1 also functions as a nutrient/redox sensor, and new novel data show a function of this enzyme in the maturation of hypothalamic peptide hormones controlling energy balance either through regulation of specific nuclear transcription factors or by regulating specific pro-hormone convertases (PCs) involved in the post-translational processing of pro-hormones. The post-translational processing mechanism of pro-hormones is critical in the pathogenesis of obesity as recently shown that metabolic and physiological triggers affect the biosynthesis and processing of many peptides hormones. Specific regulation of pro-hormone processing is likely another key step where final amounts of bioactive peptides can be tightly regulated. Different factors stimulate or inhibit pro-hormones biosynthesis in concert with an increase in the PCs involved in the maturation of bioactive hormones. Adding more complexity to the system, the new studies describe here suggest that Sirt1 could also regulate the fate of peptide hormone biosynthesis. The present review summarizes the recent progress in hypothalamic SIRT1 research with a particular emphasis on the tissue-specific control of neuropeptide hormone maturation. The series of studies done in mouse and rat models strongly advocate for the first time that a deacetylating enzyme could be a regulator in the maturation of peptide hormones and their processing enzymes. These discoveries are the

  19. Miniaturised optical encoder

    NASA Astrophysics Data System (ADS)

    Carr, John; Desmulliez, Marc P. Y.; Weston, Nick; McKendrick, David; Cunningham, Graeme; McFarland, Geoff; Meredith, Wyn; McKee, Andrew; Langton, Conrad; Eddie, Iain

    2008-08-01

    Optical encoders are pervasive in many sectors of industry including metrology, motion systems, electronics, medical, scanning/ printing, scientific instruments, space research and specialist machine tools. The precision of automated manufacture and assembly has been revolutionised by the adoption of optical diffractive measurement methods. Today's optical encoders comprise discrete components: light source(s), reference and analyser gratings, and a photodiode array that utilise diffractive optic methods to achieve high resolution. However the critical alignment requirements between the optical gratings and to the photodiode array, the bulky nature of the encoder devices and subsequent packaging mean that optical encoders can be prohibitively expensive for many applications and unsuitable for others. We report here on the design, manufacture and test of a miniaturised optical encoder to be used in precision measurement systems. Microsystems manufacturing techniques facilitate the monolithic integration of the traditional encoder components onto a single compound semiconductor chip, radically reducing the size, cost and set-up time. Fabrication of the gratings at the wafer level, by standard photo-lithography, allows for the simultaneous alignment of many devices in a single process step. This development coupled with a unique photodiode configuration not only provides increased performance but also significantly improves the alignment tolerances in both manufacture and set-up. A National Research and Development Corporation type optical encoder chip has been successfully demonstrated under test conditions on both amplitude and phase scales with pitches of 20 micron, 8 micron and 4 micron, showing significantly relaxed alignment tolerances with signal-to-noise ratios greater than 60:1. Various reference mark schemes have also been investigated. Results are presented here.

  20. Polarization encoded color camera.

    PubMed

    Schonbrun, Ethan; Möller, Guðfríður; Di Caprio, Giuseppe

    2014-03-15

    Digital cameras would be colorblind if they did not have pixelated color filters integrated into their image sensors. Integration of conventional fixed filters, however, comes at the expense of an inability to modify the camera's spectral properties. Instead, we demonstrate a micropolarizer-based camera that can reconfigure its spectral response. Color is encoded into a linear polarization state by a chiral dispersive element and then read out in a single exposure. The polarization encoded color camera is capable of capturing three-color images at wavelengths spanning the visible to the near infrared.

  1. Video time encoding machines.

    PubMed

    Lazar, Aurel A; Pnevmatikakis, Eftychios A

    2011-03-01

    We investigate architectures for time encoding and time decoding of visual stimuli such as natural and synthetic video streams (movies, animation). The architecture for time encoding is akin to models of the early visual system. It consists of a bank of filters in cascade with single-input multi-output neural circuits. Neuron firing is based on either a threshold-and-fire or an integrate-and-fire spiking mechanism with feedback. We show that analog information is represented by the neural circuits as projections on a set of band-limited functions determined by the spike sequence. Under Nyquist-type and frame conditions, the encoded signal can be recovered from these projections with arbitrary precision. For the video time encoding machine architecture, we demonstrate that band-limited video streams of finite energy can be faithfully recovered from the spike trains and provide a stable algorithm for perfect recovery. The key condition for recovery calls for the number of neurons in the population to be above a threshold value.

  2. Plasmids encoding therapeutic agents

    DOEpatents

    Keener, William K.

    2007-08-07

    Plasmids encoding anti-HIV and anti-anthrax therapeutic agents are disclosed. Plasmid pWKK-500 encodes a fusion protein containing DP178 as a targeting moiety, the ricin A chain, an HIV protease cleavable linker, and a truncated ricin B chain. N-terminal extensions of the fusion protein include the maltose binding protein and a Factor Xa protease site. C-terminal extensions include a hydrophobic linker, an L domain motif peptide, a KDEL ER retention signal, another Factor Xa protease site, an out-of-frame buforin II coding sequence, the lacZ.alpha. peptide, and a polyhistidine tag. More than twenty derivatives of plasmid pWKK-500 are described. Plasmids pWKK-700 and pWKK-800 are similar to pWKK-500 wherein the DP178-encoding sequence is substituted by RANTES- and SDF-1-encoding sequences, respectively. Plasmid pWKK-900 is similar to pWKK-500 wherein the HIV protease cleavable linker is substituted by a lethal factor (LF) peptide-cleavable linker.

  3. Genetically-encoded Reporters

    NASA Astrophysics Data System (ADS)

    Isacoff, Ehud

    2002-03-01

    One of the principle goals of neuroscience has been to understand the cellular basis of information processing and the plasticity that underlies learning and memory. Efforts in this area have mainly relied on electrical recording and optical imaging with chemical dyes. Over the last few years we and others have begun to develop genetically-encoded optical reporter "dyes" which should provide several important advantages over the classical methods for monitoring signal transmission in the nervous system. The advantages are that genetically-encoded reporters can be molecularly targeted a) to specific cell types via cell-specific promoters, and b) to specific subcellular compartments by peptides that are recognized by the protein sorting machinery of the cell. This makes it possible, in principle, to exclude signals from non-neuronal cells and to visualize selectively, in a brain region that contains many cell types with numerous kinds of synaptic connections, the activity of specific types of neurons (e.g. GABAergic interneurons) and specific synaptic elements (e.g. nerve terminals or dendrites), something that has hitherto not been possible. An additional advantage is that protein reporters may be rationally and irrationally "tuned" with mutations in functional domains known to control their dynamic range of operation. The general idea behind genetically-encoded reporters of cell signaling is to encode a protein that is either intrinsically fluorescent, or that can be labeled orthogonally with a fluorescent probe, and where the physiological signal changes fluorescence emission. I will describe recent progress employing both kinds of approaches.

  4. Time-Encoded Imagers.

    SciTech Connect

    Marleau, Peter; Brubaker, Erik

    2014-11-01

    This report provides a short overview of the DNN R&D funded project, Time-Encoded Imagers. The project began in FY11 and concluded in FY14. The Project Description below provides the overall motivation and objectives for the project as well as a summary of programmatic direction. It is followed by a short description of each task and the resulting deliverables.

  5. On the origin of C3 nephritic factor (antibody to the alternative pathway C3 convertase): evidence for the Adam and Eve concept of autoantibody production.

    PubMed

    Spitzer, R E; Stitzel, A E; Tsokos, G

    1992-09-01

    The antibody to the alternative pathway C3 convertase, designated C3 nephritic factor or C3NeF, is an autoantibody that is produced in everyone from the time of birth. The elaboration of C3NeF utilizes germline V-region genes which undergo antigen-driven affinity maturation, resulting in an autoantibody that is produced in large amounts with high affinity and narrow specificity. Our data also suggest that under normal conditions, the idiotypic network may play an important part in the control of this autoantibody. Further, a defect in the network with loss of control or inappropriate stimulation may be an underlying mechanism in the unrestricted production of C3NeF in patients with membranoproliferative glomerulonephritis.

  6. Time-Encoded Imagers

    SciTech Connect

    Marleau, Peter; Brubaker, Erik; Brennan, James S.; Nowack, Aaron

    2014-09-01

    We have developed two neutron detector systems based on time-encoded imaging and demonstrated their applicability toward non-proliferation missions. The 1D-TEI system was designed for and evaluated against the ability to detect Special Nuclear Material (SNM) in very low signal to noise environments; in particular, very large stand-off and/or weak sources that may be shielded. We have demonstrated significant detection (>5 sigma) of a 2.8e5 n/s neutron fission source at 100 meters stand-off in 30 min. If scaled to an IAEA significant quantity of Pu, we estimate that this could be reduced to as few as ~5 minutes. In contrast to simple counting detectors, this was accomplished without the need of previous background measurements. The 2D-TEI system was designed for high resolution spatial mapping of distributions of SNM and proved feasibility of twodimensional fast neutron imaging using the time encoded modulation of rates on a single pixel detector. Because of the simplicity of the TEI design, there is much lower systematic uncertainty in the detector response typical coded apertures. Other imaging methods require either multiple interactions (e.g. neutron scatter camera or Compton imagers), leading to intrinsically low efficiencies, or spatial modulation of the signal (e.g., Neutron Coded Aperture Imager (Hausladen, 2012)), which requires a complicated, high channel count, and expensive position sensitive detector. In contrast, a single detector using a time-modulated collimator can encode directional information in the time distribution of detected events. This is the first investigation of time-encoded imaging for nuclear nonproliferation applications.

  7. Spectrally encoded confocal microscopy

    SciTech Connect

    Tearney, G.J.; Webb, R.H.; Bouma, B.E.

    1998-08-01

    An endoscope-compatible, submicrometer-resolution scanning confocal microscopy imaging system is presented. This approach, spectrally encoded confocal microscopy (SECM), uses a quasi-monochromatic light source and a transmission diffraction grating to detect the reflectivity simultaneously at multiple points along a transverse line within the sample. Since this method does not require fast spatial scanning within the probe, the equipment can be miniaturized and incorporated into a catheter or endoscope. Confocal images of an electron microscope grid were acquired with SECM to demonstrate the feasibility of this technique. {copyright} {ital 1998} {ital Optical Society of America}

  8. Plasmodium falciparum subtilisin-like protease 2, a merozoite candidate for the merozoite surface protein 1–42 maturase

    PubMed Central

    Barale, Jean-Christophe; Blisnick, Thierry; Fujioka, Hisashi; Alzari, Pedro M.; Aikawa, Masamishi; Braun-Breton, Catherine; Langsley, Gordon

    1999-01-01

    The process of human erythrocyte invasion by Plasmodium falciparum parasites involves a calcium-dependent serine protease with properties consistent with a subtilisin-like activity. This enzyme achieves the last crucial maturation step of merozoite surface protein 1 (MSP1) necessary for parasite entry into the host erythrocyte. In eukaryotic cells, such processing steps are performed by subtilisin-like maturases, known as proprotein convertases. In an attempt to characterize the MSP1 maturase, we have identified a gene that encodes a P. falciparum subtilisin-like protease (PfSUB2) whose deduced active site sequence resembles more bacterial subtilisins. Therefore, we propose that PfSUB2 belongs to a subclass of eukaryotic subtilisins different from proprotein convertases. Pfsub2 is expressed during merozoite differentiation and encodes an integral membrane protein localized in the merozoite dense granules, a secretory organelle whose contents are believed to participate in a late step of the erythrocyte invasion. PfSUB2’s subcellular localization, together with its predicted enzymatic properties, leads us to propose that PfSUB2 could be responsible for the late MSP1 maturation step and thus is an attractive target for the development of new antimalarial drugs. PMID:10339607

  9. Time Encoded Radiation Imaging

    SciTech Connect

    Marleau, Peter; Brubaker, Erik; Gerling, Mark D.; Schuster, Patricia Frances; Steele, John T.

    2011-09-01

    Passive detection of special nuclear material (SNM) at long range or under heavy shielding can only be achieved by observing the penetrating neutral particles that it emits: gamma rays and neutrons in the MeV energy range. The ultimate SNM standoff detector system would have sensitivity to both gamma and neutron radiation, a large area and high efficiency to capture as many signal particles as possible, and good discrimination against background particles via directional and energy information. Designing such a system is a daunting task. Using timemodulated collimators could be a transformative technique leading to practical gamma-neutron imaging detector systems that are highly efficient with the potential to exhibit simultaneously high angular and energy resolution. A new technique using time encoding to make a compact, high efficiency imaging detector was conceived. Design considerations using Monte Carlo modeling and the construction and demonstration of a prototype imager are described.

  10. Time encoded radiation imaging

    SciTech Connect

    Marleau, Peter; Brubaker, Erik; Kiff, Scott

    2014-10-21

    The various technologies presented herein relate to detecting nuclear material at a large stand-off distance. An imaging system is presented which can detect nuclear material by utilizing time encoded imaging relating to maximum and minimum radiation particle counts rates. The imaging system is integrated with a data acquisition system that can utilize variations in photon pulse shape to discriminate between neutron and gamma-ray interactions. Modulation in the detected neutron count rates as a function of the angular orientation of the detector due to attenuation of neighboring detectors is utilized to reconstruct the neutron source distribution over 360 degrees around the imaging system. Neutrons (e.g., fast neutrons) and/or gamma-rays are incident upon scintillation material in the imager, the photons generated by the scintillation material are converted to electrical energy from which the respective neutrons/gamma rays can be determined and, accordingly, a direction to, and the location of, a radiation source identified.

  11. Rotary encoding device

    NASA Technical Reports Server (NTRS)

    Leviton, Douglas B. (Inventor)

    1993-01-01

    A device for position encoding of a rotating shaft in which a polygonal mirror having a number of facets is mounted to the shaft and a light beam is directed towards the facets is presented. The facets of the polygonal mirror reflect the light beam such that a light spot is created on a linear array detector. An analog-to-digital converter is connected to the linear array detector for reading the position of the spot on the linear array detector. A microprocessor with memory is connected to the analog-to-digital converter to hold and manipulate the data provided by the analog-to-digital converter on the position of the spot and to compute the position of the shaft based upon the data from the analog-to-digital converter.

  12. Linear encoding device

    NASA Technical Reports Server (NTRS)

    Leviton, Douglas B. (Inventor)

    1993-01-01

    A Linear Motion Encoding device for measuring the linear motion of a moving object is disclosed in which a light source is mounted on the moving object and a position sensitive detector such as an array photodetector is mounted on a nearby stationary object. The light source emits a light beam directed towards the array photodetector such that a light spot is created on the array. An analog-to-digital converter, connected to the array photodetector is used for reading the position of the spot on the array photodetector. A microprocessor and memory is connected to the analog-to-digital converter to hold and manipulate data provided by the analog-to-digital converter on the position of the spot and to compute the linear displacement of the moving object based upon the data from the analog-to-digital converter.

  13. Peroxidase activity of selenoprotein GrdB of glycine reductase and stabilisation of its integrity by components of proprotein GrdE from Eubacterium acidaminophilum.

    PubMed

    Gröbe, Tina; Reuter, Michael; Gursinsky, Torsten; Söhling, Brigitte; Andreesen, Jan R

    2007-01-01

    The anaerobe Eubacterium acidaminophilum has been shown to contain an uncharacterized peroxidase, which may serve to protect the sensitive selenoproteins in that organism. We purified this peroxidase and found that it was identical with the substrate-specific "protein B"-complex of glycine reductase. The "protein B"-complex consists of the selenocysteine-containing GrdB subunit and two subunits, which derive from the GrdE proprotein. The specific peroxidase activity was 1.7 U (mg protein)(-1) with DTT and cumene hydroperoxide as substrates. Immunoprecipitation experiments revealed that GrdB was important for DTT- and NADH-dependent peroxidase activities in crude extracts, whereas the selenoperoxiredoxin PrxU could be depleted without affecting these peroxidase activities. GrdB could be heterologously produced in Escherichia coli with coexpression of selB and selC from E. acidaminophilum for selenocysteine insertion. Although GrdB was sensitive to proteolysis, some full-size protein was present which accounted for a peroxidase activity of about 0.5 U (mg protein)(-1) in these extracts. Mutation of the potentially redox-active UxxCxxC motif in GrdB resulted in still significant, but decreased activity. Heterologous GrdB was protected from degradation by full-length GrdE or by GrdE-domains. The GrdB-GrdE interaction was confirmed by copurification of GrdE with Strep-tagged GrdB. The data suggest that GrdE domains serve to stabilise GrdB.

  14. Space vehicle onboard command encoder

    NASA Technical Reports Server (NTRS)

    1975-01-01

    A flexible onboard encoder system was designed for the space shuttle. The following areas were covered: (1) implementation of the encoder design into hardware to demonstrate the various encoding algorithms/code formats, (2) modulation techniques in a single hardware package to maintain comparable reliability and link integrity of the existing link systems and to integrate the various techniques into a single design using current technology. The primary function of the command encoder is to accept input commands, generated either locally onboard the space shuttle or remotely from the ground, format and encode the commands in accordance with the payload input requirements and appropriately modulate a subcarrier for transmission by the baseband RF modulator. The following information was provided: command encoder system design, brassboard hardware design, test set hardware and system packaging, and software.

  15. Effects of labor on pituitary expression of proopiomelanocortin, prohormone convertase (PC)-1, PC-2, and glucocorticoid receptor mRNA in fetal sheep.

    PubMed

    Holloway, A C; Gyomorey, S; Challis, J R

    2000-08-01

    We hypothesized that the concurrent prepartum rise in adrenocorticotropic hormone (ACTH) and cortisol in the plasma of fetal sheep might be attributable to altered expression of pituitary endoproteases, prohormone convertase (PC)-1, and PC-2, or to changes in pituitary expression of glucocorticoid receptor (GR) that would influence negative feedback potential. We obtained pituitary tissue from fetal sheep during late pregnancy (d 100-d 145, term) and at precise times during the process of labor and used in situ hybridization to localize and quantify mRNA levels. Proopiomelanocortin (POMC) mRNA was regionally distributed (pars intermedia > inferior pars distalis > superior pars distalis) and increased within the pars distalis during late pregnancy and with labor. At term, levels of PC-1 and PC-2 mRNA were higher in the pars intermedia than pars distalis; PC-1 but not PC-2 in the pars distalis increased with gestational age, although it did not change further at labor. GR mRNA levels in the pars distalis increased between d 135 and term, then decreased during labor. We suggest that the concomitant rise in plasma ACTH and cortisol of fetal sheep during late gestation may be attributable, in part, to increased expression of PC-1 leading to increased POMC processing. Furthermore, the negative feedback effects of cortisol on pituitary POMC synthesis and/or ACTH release during active parturition may be lessened by downregulation of anterior pituitary GR.

  16. Stabilization of the classical pathway C3 convertase C42, by a factor F-42, isolated from serum of patients with systemic lupus erythematosus.

    PubMed

    Daha, M R; Hazevoet, H M; Vanes, L A; Cats, A

    1980-07-01

    Sera from sixteen patients with SLE were investigated for the presence of a factor which would conserve convertase activity on preformed EAClgp 4hu2hu for 30 min at 30 degrees in EDTA. Although such a factor could not be detected readily in the sera, chromatography on DE-52 cellulose yielded fractions appearing as three peaks in one patient and as two peaks in a second patient. These peaks were capable of conserving C42 activity and were designated as F-42. Purification of F-42 from the second peak eluting between 4 and 7 mS on DE-52 was obtained by SP-C50, S-300 and QAE-A50 chromatography. F-42 exhibited charge heterogeneity upon SP-C50 chromatography. On polyacrylamide gel electrophoresis the final material migrated as one band, which coincided with the position of F-42 activity upon eluation from a parallel gel. F-42 had an apparent molecular weight of 150,000 and reacted with anti-IgG in Ouchterlony analysis. Sepharose-bound anti-IgG was capable of neutralizing F-42 activity. The purified material was shown to prolong the half-life (T 1/2) of performed cell-bound C42 in GVB-EDTA at 30 degrees from 5 to 80 min.

  17. Aurin tricarboxylic acid self-protects by inhibiting aberrant complement activation at the C3 convertase and C9 binding stages.

    PubMed

    Lee, Moonhee; Guo, Jian-Ping; McGeer, Edith G; McGeer, Patrick L

    2013-05-01

    Aberrant complement activation is known to exacerbate the pathology in a spectrum of degenerative diseases of aging. We previously reported that aurin tricarboxylic acid (ATA) is an orally effective agent which prevents formation of the membrane attack complex of complement. It inhibits C9 attachment to tissue bound C5b678 and thus prevents bystander lysis of host cells. In this study, we investigated the effects of ATA on the alternative complement pathway. We found that ATA prevented cleavage of the tissue bound properdin-C3b-Factor B complex into the active C3 convertase enzyme properdin-C3b-Factor Bb. This inhibition was reversed by adding Factor D to the serum. Using enzyme-linked immunosorbent type assays, we established that ATA binds directly to Factor D and C9 but not to properdin or other complement proteins. We conclude that ATA, by inhibiting at two stages of the alternative pathway, might be a particularly effective therapeutic agent in conditions such as macular degeneration, paroxysmal nocturnal hemoglobinemia, and rheumatoid arthritis, in which activation of the alternative complement pathway initiates self damage.

  18. Tranexamic acid suppresses ultraviolet B eye irradiation-induced melanocyte activation by decreasing the levels of prohormone convertase 2 and alpha-melanocyte-stimulating hormone.

    PubMed

    Hiramoto, Keiichi; Yamate, Yurika; Sugiyama, Daijiro; Takahashi, Yumi; Mafune, Eiichi

    2014-12-01

    Tranexamic acid (trans-4-aminomethylcyclohexanecarboxylic acid) is a medicinal amino acid used in skin whitening care. This study examined the effects of tranexamic acid on the melanocyte activation of the skin induced by an ultraviolet (UV) B eye irradiation. The eye or ear was locally exposed to UVB at a dose of 1.0 kJ/m(2) using a 20SE sunlamp after covering the remaining body surface with aluminum foil. UVB eye irradiation induced melanocyte activation of the skin, similar to that observed following UVB ear irradiation, which was suppressed by the administration of tranexamic acid treatment. The plasma α-melanocyte-stimulating hormone (α-MSH) content was increased by UVB irradiation of the eye; however, the increase in α-MSH was suppressed by tranexamic acid treatment. In addition, UVB eye irradiation induced the up-regulation of prohormone convertase (PC) 2 in the pituitary gland. Meanwhile, the increase in PC2 induced by UVB eye irradiation was suppressed by tranexamic acid treatment. These results clearly indicate that tranexamic acid decreases the expression of PC2, which cleavages from proopiomelanocortin to α-MSH in the pituitary gland, thereby suppressing melanocyte activation. © 2014 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

  19. N-Consecutive-Phase Encoder

    NASA Technical Reports Server (NTRS)

    Divsalar, Dariush; Lee, Ho-Kyoung; Weber, Charles

    1995-01-01

    N-consecutive-phase encoder (NCPE) is conceptual encoder for generating alphabet of N consecutive full-response continuous-phase-modulation (CPM) signals. Enables use of binary preencoder of higher rate than used with simple continuous-phase encoder (CPE). NCPE makes possible to achieve power efficiencies and bandwidth efficiencies greater than conventional trellis coders with continuous-phase frequency-shift keying (CPFSK).

  20. Genetically encoding new bioreactivity.

    PubMed

    Wang, Lei

    2017-09-25

    The genetic code can be expanded to include unnatural amino acids (Uaas) by engineering orthogonal components involved in protein translation. To be compatible with live cells, side chains of Uaas have been limited to either chemically inert or bio-orthogonal (i.e., nonreactive toward biomolecules) functionalities. To introduce bioreactivity into live systems, the genetic code has recently been engineered to encode a new class of Uaas, the bioreactive Uaas. These Uaas, after being incorporated into proteins, specifically react with target natural amino acid residues via proximity-enabled bioreactivity, enabling the selective formation of new covalent linkages within and between proteins both in vitro and in live systems. The new covalent bonding ability has been harnessed within proteins to enhance photostability, increase thermostability, staple proteins recombinantly, and build optical nano-switches, and between proteins to pinpoint ligand-receptor interaction, target native receptors irreversibly, and generate covalent macromolecular inhibitors. These diverse bioreactivities, inaccessible to natural proteins, thus open doors to novel protein engineering and provide new avenues for biological studies, biotherapeutics and synthetic biology. Copyright © 2016 Elsevier B.V. All rights reserved.

  1. Missense mutations in the PCSK9 gene are associated with hypocholesterolemia and possibly increased response to statin therapy.

    PubMed

    Berge, Knut Erik; Ose, Leiv; Leren, Trond P

    2006-05-01

    The proprotein convertase subtilisin/kexin type 9 (PCSK9) gene encodes a proprotein convertase that causes degradation of cell surface low-density lipoprotein receptors (LDLRs). Mutations in the PCSK9 gene that disrupt the normal function of PCSK9 could therefore result in increased number of LDLRs and hypocholesterolemia. Also, the cholesterol-lowering effect of statins could be increased in subjects carrying mutations in the PCSK9 gene. We have screened 38 unrelated hypocholesterolemic subjects as well as 25 unrelated familial hypercholesterolemia (FH) heterozygotes who responded particularly well to statin therapy for mutations in the 12 exons of the PCSK9 gene by DNA sequencing. Six of the 38 (15.8%) hypocholesterolemic subjects were heterozygous for 1 of the 3 mutations R46L, G106R, or R237W in the PCSK9 gene. In the group of 25 FH heterozygotes who responded particularly well to statin therapy, 3 (8.8%) were heterozygous for mutations R46L or N157K in the PCSK9 gene. None of 441 hypercholesterolemic subjects without mutations in the LDLR gene or in the apolipoprotein B-100 gene possessed any of the 4 mutations. The 4 missense mutations R46L, G106R, N157K, and R237W are associated with hypocholesterolemia and possibly increased response to statin therapy.

  2. Prosodic Encoding in Silent Reading.

    ERIC Educational Resources Information Center

    Wilkenfeld, Deborah

    In silent reading, short-memory tasks, such as semantic and syntactic processing, require a stage of phonetic encoding between visual representation and the actual extraction of meaning, and this encoding includes prosodic as well as segmental features. To test for this suprasegmental coding, an experiment was conducted in which subjects were…

  3. Prosodic Encoding in Silent Reading.

    ERIC Educational Resources Information Center

    Wilkenfeld, Deborah

    In silent reading, short-memory tasks, such as semantic and syntactic processing, require a stage of phonetic encoding between visual representation and the actual extraction of meaning, and this encoding includes prosodic as well as segmental features. To test for this suprasegmental coding, an experiment was conducted in which subjects were…

  4. Peri-encoding predictors of memory encoding and consolidation.

    PubMed

    Cohen, Noga; Pell, Liat; Edelson, Micah G; Ben-Yakov, Aya; Pine, Alex; Dudai, Yadin

    2015-03-01

    We review reports of brain activations that occur immediately prior to the onset or following the offset of to-be-remembered information and can predict subsequent mnemonic success. Memory-predictive pre-encoding processes, occurring from fractions of a second to minutes prior to event onset, are mainly associated with activations in the medial temporal lobe (MTL), amygdala and midbrain, and with enhanced theta oscillations. These activations may be considered as the neural correlates of one or more cognitive operations, including contextual processing, attention, and the engagement of distinct computational modes associated with prior encoding or retrieval. Post-encoding activations that correlate with subsequent memory performance are mainly observed in the MTL, sensory cortices and frontal regions. These activations may reflect binding of elements of the encoded information and initiation of memory consolidation. In all, the findings reviewed here illustrate the importance of brain states in the immediate peri-encoding time windows in determining encoding success. Understanding these brain states and their specific effects on memory may lead to optimization of the encoding of desired memories and mitigation of undesired ones. Copyright © 2014 Elsevier Ltd. All rights reserved.

  5. Information encoder/decoder using chaotic systems

    DOEpatents

    Miller, S.L.; Miller, W.M.; McWhorter, P.J.

    1997-10-21

    The present invention discloses a chaotic system-based information encoder and decoder that operates according to a relationship defining a chaotic system. Encoder input signals modify the dynamics of the chaotic system comprising the encoder. The modifications result in chaotic, encoder output signals that contain the encoder input signals encoded within them. The encoder output signals are then capable of secure transmissions using conventional transmission techniques. A decoder receives the encoder output signals (i.e., decoder input signals) and inverts the dynamics of the encoding system to directly reconstruct the original encoder input signals. 32 figs.

  6. Information encoder/decoder using chaotic systems

    DOEpatents

    Miller, Samuel Lee; Miller, William Michael; McWhorter, Paul Jackson

    1997-01-01

    The present invention discloses a chaotic system-based information encoder and decoder that operates according to a relationship defining a chaotic system. Encoder input signals modify the dynamics of the chaotic system comprising the encoder. The modifications result in chaotic, encoder output signals that contain the encoder input signals encoded within them. The encoder output signals are then capable of secure transmissions using conventional transmission techniques. A decoder receives the encoder output signals (i.e., decoder input signals) and inverts the dynamics of the encoding system to directly reconstruct the original encoder input signals.

  7. Prohormone convertases 1/3 and 2 together orchestrate the site-specific cleavages of progastrin to release gastrin-34 and gastrin-17.

    PubMed

    Rehfeld, Jens F; Zhu, Xiaorong; Norrbom, Christina; Bundgaard, Jens R; Johnsen, Anders H; Nielsen, John E; Vikesaa, Jonas; Stein, Jeffrey; Dey, Arunangsu; Steiner, Donald F; Friis-Hansen, Lennart

    2008-10-01

    Cellular synthesis of peptide hormones requires PCs (prohormone convertases) for the endoproteolysis of prohormones. Antral G-cells synthesize the most gastrin and express PC1/3, 2 and 5/6 in the rat and human. But the cleavage sites in progastrin for each PC have not been determined. Therefore, in the present study, we measured the concentrations of progastrin, processing intermediates and alpha-amidated gastrins in antral extracts from PC1/3-null mice and compared the results with those in mice lacking PC2 and wild-type controls. The expression of PCs was examined by immunocytochemistry and in situ hybridization of mouse G-cells. Finally, the in vitro effect of recombinant PC5/6 on progastrin and progastrin fragments containing the relevant dibasic cleavage sites was also examined. The results showed that mouse G-cells express PC1/3, 2 and 5/6. The concentration of progastrin in PC1/3-null mice was elevated 3-fold. Chromatography showed that cleavage of the Arg(36)Arg(37) and Arg(73)Arg(74) sites were grossly decreased. Accordingly, the concentrations of progastrin products were markedly reduced, alpha-amidated gastrins (-34 and -17) being 25% of normal. Lack of PC1/3 was without effect on the third dibasic site (Lys(53)Lys(54)), which is the only processing site for PC2. Recombinant PC5/6 did not cleave any of the dibasic processing sites in progastrin and fragments containing the relevant dibasic processing sites. The complementary cleavages of PC1/3 and 2, however, suffice to explain most of the normal endoproteolysis of progastrin. Moreover, the results show that PCs react differently to the same dibasic sequences, suggesting that additional structural factors modulate the substrate specificity.

  8. Islets of Langerhans from prohormone convertase-2 knockout mice show α-cell hyperplasia and tumorigenesis with elevated α-cell neogenesis.

    PubMed

    Jones, Huw B; Reens, Jaimini; Brocklehurst, Simon R; Betts, Catherine J; Bickerton, Sue; Bigley, Alison L; Jenkins, Richard P; Whalley, Nicky M; Morgan, Derrick; Smith, David M

    2014-02-01

    Antagonism of the effects of glucagon as an adjunct therapy with other glucose-lowering drugs in the chronic treatment of diabetes has been suggested to aggressively control blood glucose levels. Antagonism of glucagon effects, by targeting glucagon secretion or disabling the glucagon receptor, is associated with α-cell hyperplasia. We evaluated the influence of total glucagon withdrawal on islets of Langerhans using prohormone convertase-2 knockout mice (PC2-ko), in which α-cell hyperplasia is present from a young age and persists throughout life, in order to understand whether or not sustained glucagon deficit would lead to islet tumorigenesis. PC2-ko and wild-type (WT) mice were maintained drug-free, and cohorts of these groups sampled at 3, 12 and 18 months for plasma biochemical and morphological (histological, immunohistochemical, electron microscopical and image analytical) assessments. WT mice showed no islet tumours up to termination of the study, but PC2-ko animals displayed marked changes in islet morphology from α-cell hypertrophy/hyperplasia/atypical hyperplasia, to adenomas and carcinomas, these latter being first encountered at 6-8 months. Islet hyperplasias and tumours primarily consisted of α-cells associated to varying degrees with other islet endocrine cell types. In addition to substantial increases in islet neoplasia, increased α-cell neogenesis associated primarily with pancreatic duct(ule)s was present. We conclude that absolute blockade of the glucagon signal results in tumorigenesis and that the PC2-ko mouse represents a valuable model for investigation of islet tumours and pancreatic ductal neogenesis.

  9. Immunocytochemical localization of prohormone convertases PC1 and PC2 in the anuran pituitary gland: subcellular localization in corticotrope and melanotrope cells.

    PubMed

    Kurabuchi, S; Tanaka, S

    1997-06-01

    Specific antisera against mammalian prohormone convertases PC1 and PC2 have been used to examine, light-immunocytochemically, the distribution of these enzymes in the pituitary gland of five different species of anuran amphibians (Rana catesbeiana, Bufo japonicus formosus, Xenopus laevis, Rana brevipoda porosa, and Buergeria japonica). A differential pattern of immunoreactivity of PC1 and PC2 was found among these species. Only PC1 was found in the corticotrope cells of the pars distalis in R. catesbeiana, B. japonicus formosus, and X. laevis. Only PC2 was observed in these cells in B. japonica, whereas both PC1 and PC2 were present in these cells in R. brevipoda porosa. PC2 immunoreactivity was always observed in melanotrope cells in the pars intermedia of all of the species, but it coexisted with PC1 immunoreactivity only in R. catesbeiana and X. laevis. The nerve fibers and terminals in the pars nervosa in all of the species were intensely immunopositive with both PC1 and PC2 antibodies. Immunoelectron microscopy on B. japonicus formosus and B. japonica, by means of double-labeling with gold particles of different sizes, revealed that almost all the adrenocorticotropin-positive secretory granules in the corticotrope cells and alpha-melanophore-stimulating-hormone-positive secretory granules in the melanotrope cells were also labeled with either PC1 or PC2 antibodies. This study suggests that PC1 and PC2 are involved in the intracellular proteolytic cleavage of proopiomelanocortin in amphibian pituitary glands, a situation similar to that proposed for mammals.

  10. Processing of synthetic pro-islet amyloid polypeptide (proIAPP) 'amylin' by recombinant prohormone convertase enzymes, PC2 and PC3, in vitro.

    PubMed

    Higham, C E; Hull, R L; Lawrie, L; Shennan, K I; Morris, J F; Birch, N P; Docherty, K; Clark, A

    2000-08-01

    Islet amyloid polypeptide (IAPP), amylin, is the constituent peptide of pancreatic islet amyloid deposits which form in islets of Type 2 diabetic subjects. Human IAPP is synthesized as a 67-residue propeptide in islet beta-cells and colocalized with insulin in beta-cell granules. The mature 37-amino acid peptide is produced by proteolysis at pairs of basic residues at the C- and N-termini of the mature peptide. To determine the enzymes responsible for proteolysis and their activity at the potential cleavage sites, synthetic human proIAPP was incubated (0.5-16 h) with recombinant prohormone convertases, PC2 or PC3 at appropriate conditions of calcium and pH. The products were analysed by MS and HPLC. Proinsulin was used as a control and was cleaved by both recombinant enzymes resulting in intermediates. PC3 was active initially at the N-terminal-IAPP junction and later at the C-terminus, whereas initial PC2 activity was at the IAPP-C-terminal junction. Processing at the basic residues within the C-terminal flanking peptide rarely occurred. There was no evidence for substantial competition for the processing enzymes when the combined substrates proinsulin and proIAPP were incubated with both PC2 and PC3. As proinsulin cleavage is sequential in vivo (PC3 active at the B-chain-C-peptide junction, followed by PC2 at A chain-C-peptide junction), these data suggest that proteolysis of proIAPP and proinsulin is coincident in secretory granules and increased proinsulin secretion in diabetes could be accompanied by increased production of proIAPP.

  11. Estimated proinsulin processing activity of prohormone convertase (PC) 1/3 rather than PC2 is decreased in pancreatic β-cells of type 2 diabetic patients.

    PubMed

    Ozawa, Sachihiko; Katsuta, Hidenori; Suzuki, Kiyoshi; Takahashi, Kazuto; Tanaka, Toshiaki; Sumitani, Yoshikazu; Nishida, Susumu; Yoshimoto, Katsuhiko; Ishida, Hitoshi

    2014-01-01

    Type 2 diabetic (T2D) patients exhibit fasting relative hyperproinsulinemia owing to pancreatic β-cell dysfunction. To clarify the mechanism underlying this hyperproinsulinemic state, we evaluated the activities of the endopeptidases prohormone convertase (PC) 1/3 and PC2 in T2D patients. Fasting blood levels of intact proinsulin (IPI), total proinsulin (t-PI) and C-peptide were measured simultaneously, and intravenous glucagon loading was performed to investigate the dynamics of circulating proinsulin-related molecules released from pancreatic β-cells in 12 healthy volunteers and 18 T2D patients. Taking advantage of the 95% cross-reactivity between proinsulin and des-31,32-proinsulin (des-31,32-PI) with the human proinsulin radioimmunoassay kit used in this study, we estimated PC1/3 and PC2 activities using the following formulas: des-31,32-PI = (t-PI-IPI)/0.95; PC1/3 activity = des-31,32-PI/IPI; and PC2 activity = C-peptide/des-31,32-PI. C-peptide responses to glucagon were slightly lower among T2D patients. IPI and the IPI/C-peptide ratio were significantly higher in T2D patients (p<0.05 and p<0.01, respectively). There was no difference in des-31,32-PI levels or PC2 activity between the two groups. However, PC1/3 activity was significantly lower in T2D patients than in the control group (p<0.01). We propose that decreased activity of PC1/3 rather than PC2 in pancreatic β-cells is involved in the impaired proinsulin processing, resulting in elevated IPI levels in T2D patients.

  12. Implication of TNF-alpha convertase (TACE/ADAM17) in inducible nitric oxide synthase expression and inflammation in an experimental model of colitis.

    PubMed

    Colón, A L; Menchén, L A; Hurtado, O; De Cristóbal, J; Lizasoain, I; Leza, J C; Lorenzo, P; Moro, M A

    2001-12-21

    Tumour necrosis factor-alpha (TNF-alpha) is a pro-inflammatory cytokine which is shed in its soluble form by a disintegrin and metalloproteinase (ADAM) called TNF-alpha convertase (TACE; ADAM17). TNF-alpha plays a role in inflammatory bowel disease (IBD) and is involved in the expression of inducible nitric oxide synthase (iNOS) which has also been implicated in IBD. The study was designed to investigate whether colitis induced by trinitrobenzene sulphonic acid (TNBS) in rats produces an increase in TACE activity and/or expression and whether its pharmacological inhibition reduces TNF-alpha levels, iNOS expression and colonic damage in this model. TNBS (30 mg in 0.4 ml of 50% ethanol) was instilled into the colon of female Wistar rats. Saline or TACE inhibitor BB1101 (10 mg/kg/day) was administered intraperitoneally 5 days after TNBS instillation. On day 10, colons were removed and assessed for pathological score, myeloperoxidase (MPO), NO synthase (NOS), TACE enzymatic activity and protein levels, colonic TNF-alpha and NOx- levels. Instillation of TNBS caused an increase in TACE activity and expression and the release of TNF-alpha. TNBS also resulted in iNOS expression and colonic damage. BB1101 blocked TNBS-induced increase in TACE activity, TNF-alpha release and iNOS expression. Concomitantly, BB1101 ameliorated TNBS-induced colonic damage and inflammation. TNBS causes TNF-alpha release by an increase in TACE activity and expression and this results in the expression of iNOS and subsequent inflammation, suggesting that TACE inhibition may prove useful as a therapeutic means in IBD.

  13. PNA-encoded chemical libraries.

    PubMed

    Zambaldo, Claudio; Barluenga, Sofia; Winssinger, Nicolas

    2015-06-01

    Peptide nucleic acid (PNA)-encoded chemical libraries along with DNA-encoded libraries have provided a powerful new paradigm for library synthesis and ligand discovery. PNA-encoding stands out for its compatibility with standard solid phase synthesis and the technology has been used to prepare libraries of peptides, heterocycles and glycoconjugates. Different screening formats have now been reported including selection-based and microarray-based methods that have yielded specific ligands against diverse target classes including membrane receptors, lectins and challenging targets such as Hsp70.

  14. DNA sequences encoding osteoinductive products

    SciTech Connect

    Wang, E.A.; Wozney, J.M.; Rosen, V.

    1991-05-07

    This patent describes an isolated DNA sequence encoding an osteoinductive protein the DNA sequence comprising a coding sequence. It comprises: nucleotide No.1 through nucleotide No.387, nucleotide No.356 through nucleotide No.1543, nucleotide $402 through nucleotide No.1626, naturally occurring allelic sequences and equivalent degenerative codon sequences and sequences which hybridize to any of sequences under stringent hybridization conditions; and encode a protein characterized by the ability to induce the formation of bone and/or cartilage.

  15. Multidimensionally encoded magnetic resonance imaging.

    PubMed

    Lin, Fa-Hsuan

    2013-07-01

    Magnetic resonance imaging (MRI) typically achieves spatial encoding by measuring the projection of a q-dimensional object over q-dimensional spatial bases created by linear spatial encoding magnetic fields (SEMs). Recently, imaging strategies using nonlinear SEMs have demonstrated potential advantages for reconstructing images with higher spatiotemporal resolution and reducing peripheral nerve stimulation. In practice, nonlinear SEMs and linear SEMs can be used jointly to further improve the image reconstruction performance. Here, we propose the multidimensionally encoded (MDE) MRI to map a q-dimensional object onto a p-dimensional encoding space where p > q. MDE MRI is a theoretical framework linking imaging strategies using linear and nonlinear SEMs. Using a system of eight surface SEM coils with an eight-channel radiofrequency coil array, we demonstrate the five-dimensional MDE MRI for a two-dimensional object as a further generalization of PatLoc imaging and O-space imaging. We also present a method of optimizing spatial bases in MDE MRI. Results show that MDE MRI with a higher dimensional encoding space can reconstruct images more efficiently and with a smaller reconstruction error when the k-space sampling distribution and the number of samples are controlled. Copyright © 2012 Wiley Periodicals, Inc.

  16. Fly Photoreceptors Encode Phase Congruency

    PubMed Central

    Friederich, Uwe; Billings, Stephen A.; Hardie, Roger C.; Juusola, Mikko; Coca, Daniel

    2016-01-01

    More than five decades ago it was postulated that sensory neurons detect and selectively enhance behaviourally relevant features of natural signals. Although we now know that sensory neurons are tuned to efficiently encode natural stimuli, until now it was not clear what statistical features of the stimuli they encode and how. Here we reverse-engineer the neural code of Drosophila photoreceptors and show for the first time that photoreceptors exploit nonlinear dynamics to selectively enhance and encode phase-related features of temporal stimuli, such as local phase congruency, which are invariant to changes in illumination and contrast. We demonstrate that to mitigate for the inherent sensitivity to noise of the local phase congruency measure, the nonlinear coding mechanisms of the fly photoreceptors are tuned to suppress random phase signals, which explains why photoreceptor responses to naturalistic stimuli are significantly different from their responses to white noise stimuli. PMID:27336733

  17. Prohormone convertase 2 (PC2) null mice have increased mu opioid receptor levels accompanied by altered morphine-induced antinociception, tolerance and dependence.

    PubMed

    Lutfy, K; Parikh, D; Lee, D L; Liu, Y; Ferrini, M G; Hamid, A; Friedman, T C

    2016-08-04

    Chronic morphine treatment increases the levels of prohormone convertase 2 (PC2) in brain regions involved in nociception, tolerance and dependence. Thus, we tested if PC2 null mice exhibit altered morphine-induced antinociception, tolerance and dependence. PC2 null mice and their wild-type controls were tested for baseline hot plate latency, injected with morphine (1.25-10mg/kg) and tested for antinociception 30min later. For tolerance studies, mice were tested in the hot plate test before and 30min following morphine (5mg/kg) on day 1. Mice then received an additional dose so that the final dose of morphine was 10mg/kg on this day. On days 2-4, mice received additional doses of morphine (20, 40 and 80mg/kg on days 1, 2, 3, and 4, respectively). On day 5, mice were tested in the hot plate test before and 30min following morphine (5mg/kg). For withdrawal studies, mice were treated with the escalating doses of morphine (10, 20, 40 and 80mg/kg) for 4days, implanted with a morphine pellet on day 5 and 3 days later injected with naloxone (1mg/kg) and signs of withdrawal were recorded. Morphine dose-dependently induced antinociception and the magnitude of this response was greater in PC2 null mice. Tolerance to morphine was observed in wild-type mice and this phenomenon was blunted in PC2 null mice. Withdrawal signs were also reduced in PC2 null mice. Immunohistochemical studies showed up-regulation of the mu opioid receptor (MOP) protein expression in the periaqueductal gray area, ventral tegmental area, lateral hypothalamus, medial hypothalamus, nucleus accumbens, and somatosensory cortex in PC2 null mice. Likewise, naloxone specific binding was increased in the brains of these mice compared to their wild-type controls. The results suggest that the PC2-derived peptides may play a functional role in morphine-induced antinociception, tolerance and dependence. Alternatively, lack of opioid peptides led to up-regulation of the MOP and altered morphine

  18. Insights into the Effects of Complement Factor H on the Assembly and Decay of the Alternative Pathway C3 Proconvertase and C3 Convertase.

    PubMed

    Bettoni, Serena; Bresin, Elena; Remuzzi, Giuseppe; Noris, Marina; Donadelli, Roberta

    2016-04-08

    The activated fragment of C3 (C3b) and factor B form the C3 proconvertase (C3bB), which is cleaved by factor D to C3 convertase (C3bBb). Older studies (Conrad, D. H., Carlo, J. R., and Ruddy, S. (1978)J. Exp. Med.147, 1792-1805; Pangburn, M. K., and Müller-Eberhard, H. J. (1978)Proc. Natl. Acad. Sci. U.S.A.75, 2416-2420; Kazatchkine, M. D., Fearon, D. T., and Austen, K. F. (1979)J. Immunol.122, 75-81) indicated that the complement alternative pathway regulator factor H (FH) competes with factor B for C3b binding; however, the capability of FH to prevent C3bB assembly has not been formally investigated. Moreover, in the few published studies FH did not favor C3bB dissociation. Whether FH may affect C3bBb formation from C3bB is unknown. We set up user-friendly assays based on combined microplate/Western blotting techniques that specifically detect either C3bB or C3bBb, with the aim of investigating the effect of FH on C3bB assembly and decay and C3bBb formation and decay. We document that FH does not affect C3bB assembly, indicating that FH does not efficiently compete with factor B for C3b binding. We also found that FH does not dissociate C3bB. FH showed a strong C3bBb decay-accelerating activity, as reported previously, and also exerted an apparent inhibitory effect on C3bBb formation. The latter effect was not fully attributable to a rapid FH-mediated dissociation of C3bBb complexes, because blocking decay with properdin and C3 nephritic factor did not restore C3bBb formation. FH almost completely prevented release of the smaller cleavage subunit of FB (Ba), without modifying the amount of C3bB complexes, suggesting that FH inhibits the conversion of C3bB to C3bBb. Thus, the inhibitory effect of FH on C3bBb formation is likely the sum of inhibition of C3bB conversion to C3bBb and of C3bBb decay acceleration. Further studies are required to confirm these findings in physiological cell-based settings.

  19. Insights into the Effects of Complement Factor H on the Assembly and Decay of the Alternative Pathway C3 Proconvertase and C3 Convertase*

    PubMed Central

    Bettoni, Serena; Bresin, Elena; Remuzzi, Giuseppe; Noris, Marina; Donadelli, Roberta

    2016-01-01

    The activated fragment of C3 (C3b) and factor B form the C3 proconvertase (C3bB), which is cleaved by factor D to C3 convertase (C3bBb). Older studies (Conrad, D. H., Carlo, J. R., and Ruddy, S. (1978) J. Exp. Med. 147, 1792–1805; Pangburn, M. K., and Müller-Eberhard, H. J. (1978) Proc. Natl. Acad. Sci. U.S.A. 75, 2416–2420; Kazatchkine, M. D., Fearon, D. T., and Austen, K. F. (1979) J. Immunol. 122, 75–81) indicated that the complement alternative pathway regulator factor H (FH) competes with factor B for C3b binding; however, the capability of FH to prevent C3bB assembly has not been formally investigated. Moreover, in the few published studies FH did not favor C3bB dissociation. Whether FH may affect C3bBb formation from C3bB is unknown. We set up user-friendly assays based on combined microplate/Western blotting techniques that specifically detect either C3bB or C3bBb, with the aim of investigating the effect of FH on C3bB assembly and decay and C3bBb formation and decay. We document that FH does not affect C3bB assembly, indicating that FH does not efficiently compete with factor B for C3b binding. We also found that FH does not dissociate C3bB. FH showed a strong C3bBb decay-accelerating activity, as reported previously, and also exerted an apparent inhibitory effect on C3bBb formation. The latter effect was not fully attributable to a rapid FH-mediated dissociation of C3bBb complexes, because blocking decay with properdin and C3 nephritic factor did not restore C3bBb formation. FH almost completely prevented release of the smaller cleavage subunit of FB (Ba), without modifying the amount of C3bB complexes, suggesting that FH inhibits the conversion of C3bB to C3bBb. Thus, the inhibitory effect of FH on C3bBb formation is likely the sum of inhibition of C3bB conversion to C3bBb and of C3bBb decay acceleration. Further studies are required to confirm these findings in physiological cell-based settings. PMID:26903516

  20. Synaptic encoding of temporal contiguity

    PubMed Central

    Ostojic, Srdjan; Fusi, Stefano

    2013-01-01

    Often we need to perform tasks in an environment that changes stochastically. In these situations it is important to learn the statistics of sequences of events in order to predict the future and the outcome of our actions. The statistical description of many of these sequences can be reduced to the set of probabilities that a particular event follows another event (temporal contiguity). Under these conditions, it is important to encode and store in our memory these transition probabilities. Here we show that for a large class of synaptic plasticity models, the distribution of synaptic strengths encodes transitions probabilities. Specifically, when the synaptic dynamics depend on pairs of contiguous events and the synapses can remember multiple instances of the transitions, then the average synaptic weights are a monotonic function of the transition probabilities. The synaptic weights converge to the distribution encoding the probabilities also when the correlations between consecutive synaptic modifications are considered. We studied how this distribution depends on the number of synaptic states for a specific model of a multi-state synapse with hard bounds. In the case of bistable synapses, the average synaptic weights are a smooth function of the transition probabilities and the accuracy of the encoding depends on the learning rate. As the number of synaptic states increases, the average synaptic weights become a step function of the transition probabilities. We finally show that the information stored in the synaptic weights can be read out by a simple rate-based neural network. Our study shows that synapses encode transition probabilities under general assumptions and this indicates that temporal contiguity is likely to be encoded and harnessed in almost every neural circuit in the brain. PMID:23641210

  1. Holographically Encoded Volume Phase Masks

    DTIC Science & Technology

    2015-07-13

    yÞAg; (6) where à and B̃ are the Fourier transforms of A and B, respec- tively, and fx and fy are the spatial frequencies along the x and y axes...collimated and directed onto a reflecting spatial light modulator (SLM). Applying the SLM encoding technique by Arrizón et al.,42 the incident beam was...converted into the LP11 and LP21 HOMs. These modes were selected because their spatial phase pattern match the HPM with the encoded four-sector binary

  2. How Infants Encode Spatial Extent

    ERIC Educational Resources Information Center

    Duffy, Sean; Huttenlocher, Janellen; Levine, Susan; Duffy, Renee

    2005-01-01

    This study explores how infants encode an object's spatial extent. We habituated 6.5-month-old infants to a dowel inside a container and then tested whether they dishabituate to a change in absolute size when the relation between dowel and container is held constant (by altering the size of both container and dowel) and when the relation changes…

  3. Encoding Standards for Linguistic Corpora.

    ERIC Educational Resources Information Center

    Ide, Nancy

    The demand for extensive reusability of large language text collections for natural languages processing research requires development of standardized encoding formats. Such formats must be capable of representing different kinds of information across the spectrum of text types and languages, capable of representing different levels of…

  4. Encoding Ownership Types in Java

    NASA Astrophysics Data System (ADS)

    Cameron, Nicholas; Noble, James

    Ownership types systems organise the heap into a hierarchy which can be used to support encapsulation properties, effects, and invariants. Ownership types have many applications including parallelisation, concurrency, memory management, and security. In this paper, we show that several flavours and extensions of ownership types can be entirely encoded using the standard Java type system.

  5. Spectrally-encoded color imaging

    PubMed Central

    Kang, DongKyun; Yelin, Dvir; Bouma, Brett E.; Tearney, Guillermo J.

    2010-01-01

    Spectrally-encoded endoscopy (SEE) is a technique for ultraminiature endoscopy that encodes each spatial location on the sample with a different wavelength. One limitation of previous incarnations of SEE is that it inherently creates monochromatic images, since the spectral bandwidth is expended in the spatial encoding process. Here we present a spectrally-encoded imaging system that has color imaging capability. The new imaging system utilizes three distinct red, green, and blue spectral bands that are configured to illuminate the grating at different incident angles. By careful selection of the incident angles, the three spectral bands can be made to overlap on the sample. To demonstrate the method, a bench-top system was built, comprising a 2400-lpmm grating illuminated by three 525-μm-diameter beams with three different spectral bands. Each spectral band had a bandwidth of 75 nm, producing 189 resolvable points. A resolution target, color phantoms, and excised swine small intestine were imaged to validate the system's performance. The color SEE system showed qualitatively and quantitatively similar color imaging performance to that of a conventional digital camera. PMID:19688002

  6. Encoding information into precipitation structures

    NASA Astrophysics Data System (ADS)

    Martens, Kirsten; Bena, Ioana; Droz, Michel; Rácz, Zoltan

    2008-12-01

    Material design at submicron scales would be profoundly affected if the formation of precipitation patterns could be easily controlled. It would allow the direct building of bulk structures, in contrast to traditional techniques which consist of removing material in order to create patterns. Here, we discuss an extension of our recent proposal of using electrical currents to control precipitation bands which emerge in the wake of reaction fronts in A+ + B- → C reaction-diffusion processes. Our main result, based on simulating the reaction-diffusion-precipitation equations, is that the dynamics of the charged agents can be guided by an appropriately designed time-dependent electric current so that, in addition to the control of the band spacing, the width of the precipitation bands can also be tuned. This makes straightforward the encoding of information into precipitation patterns and, as an amusing example, we demonstrate the feasibility by showing how to encode a musical rhythm.

  7. Vector Encoding in Biochemical Networks

    NASA Astrophysics Data System (ADS)

    Potter, Garrett; Sun, Bo

    Encoding of environmental cues via biochemical signaling pathways is of vital importance in the transmission of information for cells in a network. The current literature assumes a single cell state is used to encode information, however, recent research suggests the optimal strategy utilizes a vector of cell states sampled at various time points. To elucidate the optimal sampling strategy for vector encoding, we take an information theoretic approach and determine the mutual information of the calcium signaling dynamics obtained from fibroblast cells perturbed with different concentrations of ATP. Specifically, we analyze the sampling strategies under the cases of fixed and non-fixed vector dimension as well as the efficiency of these strategies. Our results show that sampling with greater frequency is optimal in the case of non-fixed vector dimension but that, in general, a lower sampling frequency is best from both a fixed vector dimension and efficiency standpoint. Further, we find the use of a simple modified Ornstein-Uhlenbeck process as a model qualitatively captures many of our experimental results suggesting that sampling in biochemical networks is based on a few basic components.

  8. Flavoxobin, a serine protease from Trimeresurus flavoviridis (habu snake) venom, independently cleaves Arg726-Ser727 of human C3 and acts as a novel, heterologous C3 convertase

    PubMed Central

    Yamamoto, Chieko; Tsuru, Daisuke; Oda-Ueda, Naoko; Ohno, Motonori; Hattori, Shosaku; Kim, Sung-Teh

    2002-01-01

    We have recently shown that crude Trimeresurus flavoviridis (habu snake) venom has a strong capability for activating the human alternative complement system. To identify the active component, the crude venom was fractionated and purified by serial chromatography using Sephadex G-100, CM-cellulose C-52, diethylaminoethyl-Toyopearl 650M, and Butyl-Toyopearl, and the active fractions were evaluated by the C3a-releasing and soluble membrane attack complex-forming activities. Two peak fractions with the highest activities were detected after gel filtration and ion exchange chromatography, and the first fraction was purified to homogeneity. The homogeneous protein was examined for its N-terminal amino acid sequence by Edman degradation. The determined sequence of 25 amino acids completely coincided with that of a previously reported serine protease with coagulant activity, flavoxobin, purified from the same snake venom. To elucidate the molecular mechanism of the complement activation, the reactive products of the mixture of the purified human C3 and flavoxobin were examined by sodium dodecyl sulphate–polyacrylamide gel electrophoresis. The digesting pattern revealed that flavoxobin cleaves the α chain of the C3 molecule into two fragments. The N-terminal amino acid sequences for the remnant fragments of C3 disclosed that flavoxobin severs the human C3 at the Arg726-Ser727 site to form C3b and C3a the way C3bBb, the human alternative C3 convertase, does. In conclusion, flavoxobin acts as a novel, heterologous C3 convertase that independently cleaves human C3 and kick-starts the complement cascade. PMID:12225369

  9. Hall effect encoding of brushless dc motors

    NASA Technical Reports Server (NTRS)

    Berard, C. A.; Furia, T. J.; Goldberg, E. A.; Greene, R. C.

    1970-01-01

    Encoding mechanism integral to the motor and using the permanent magnets embedded in the rotor eliminates the need for external devices to encode information relating the position and velocity of the rotating member.

  10. NRZ Data Asymmetry Corrector and Convolutional Encoder

    NASA Technical Reports Server (NTRS)

    Pfiffner, H. J.

    1983-01-01

    Circuit compensates for timing, amplitude and symmetry perturbations. Data asymmetry corrector and convolutional encoder regenerate data and clock signals in spite of signal variations such as data or clock asymmetry, phase errors, and amplitude variations, then encode data for transmission.

  11. Novel optical encoder for harsh environments

    NASA Astrophysics Data System (ADS)

    Kress, Bernard; Mueller, Ulrich; Brac-de-la-Perriere, Vincent

    2014-09-01

    We are presenting a new optical encoder architecture for shaft encoding, both in incremental and absolute modes. This encoder is based on a diffractive optics technology platform. We have developed various disk based rotary diffractive encoders previously. This encoder is different in the way it is not a disk composed of successive gratings or computer generated holograms, but rather composed of a single element placed on the shaft. It is thus best suited for hollow shaft or end of shaft applications such as in encoder controlled electrical motors. This new architecture aims at solving some of the problems encountered with previous implementations of diffractive encoders such as disk wobble, disk to shaft centering and also encoding in harsh environments.

  12. Terahertz wavelength encoding compressive imaging

    NASA Astrophysics Data System (ADS)

    Zhang, Qiong; Wang, Xinke; Zhang, Yan

    2016-11-01

    Terahertz (THz) compressive imaging can obtain two dimensional image with a single or linear detector, which can overcome the bottleneck problem of lacking of THz two dimensional detectors. In this presentation, we propose a method to obtain two dimensional images using a linear detector. A plano-convex cylindrical lens is employed to perform Fourier transform and to encode one dimensional information of an object into wavelengths. After recording, both amplitude and phase information for different frequency at each pixel of the line detector are extracted, two dimensional image of the object can be reconstructed. Numerical simulation demonstrates the validity of the proposed method.

  13. Schematic driven layout of Reed Solomon encoders

    NASA Technical Reports Server (NTRS)

    Arave, Kari; Canaris, John; Miles, Lowell; Whitaker, Sterling

    1992-01-01

    Two Reed Solomon error correcting encoders are presented. Schematic driven layout tools were used to create the encoder layouts. Special consideration had to be given to the architecture and logic to provide scalability of the encoder designs. Knowledge gained from these projects was used to create a more flexible schematic driven layout system.

  14. Time Course of Grammatical Encoding in Agrammatism

    ERIC Educational Resources Information Center

    Lee, Jiyeon

    2011-01-01

    Producing a sentence involves encoding a preverbal message into a grammatical structure by retrieving lexical items and integrating them into a functional (semantic-to-grammatical) structure. Individuals with agrammatism are impaired in this grammatical encoding process. However, it is unclear what aspect of grammatical encoding is impaired and…

  15. Selection of DNA aptamers with two modified bases

    PubMed Central

    Gawande, Bharat N.; Rohloff, John C.; Carter, Jeffrey D.; von Carlowitz, Ira; Zhang, Chi; Schneider, Daniel J.; Janjic, Nebojsa

    2017-01-01

    The nucleobases comprising DNA and RNA aptamers provide considerably less chemical diversity than protein-based ligands, limiting their versatility. The introduction of novel functional groups at just one of the four bases in modified aptamers has recently led to dramatic improvement in the success rate of identifying nucleic acid ligands to protein targets. Here we explore the benefits of additional enhancement in physicochemical diversity by selecting modified DNA aptamers that contain amino-acid–like modifications on both pyrimidine bases. Using proprotein convertase subtilisin/kexin type 9 as a representative protein target, we identify specific pairwise combinations of modifications that result in higher affinity, metabolic stability, and inhibitory potency compared with aptamers with single modifications. Such doubly modified aptamers are also more likely to be encoded in shorter sequences and occupy nonoverlapping epitopes more frequently than aptamers with single modifications. These highly modified DNA aptamers have broad utility in research, diagnostic, and therapeutic applications. PMID:28265062

  16. Furin mediates enhanced production of fibrillogenic ABri peptides in familial British dementia.

    PubMed

    Kim, S H; Wang, R; Gordon, D J; Bass, J; Steiner, D F; Lynn, D G; Thinakaran, G; Meredith, S C; Sisodia, S S

    1999-11-01

    The genetic lesion underlying familial British dementia (FBD), an autosomal dominant neurodegenerative disorder, is a T-A transversion at the termination codon of the BRI gene. The mutant gene encodes BRI-L, the precursor of ABri peptides that accumulate in amyloid deposits in FBD brain. We now report that both BRI-L and its wild-type counterpart, BRI, were constitutively processed by the proprotein convertase, furin, resulting in the secretion of carboxyl-terminal peptides that encompass all or part of ABri. Elevated levels of peptides were generated from the mutant BRI precursor. Electron microscopic studies revealed that synthetic ABri peptides assembled into irregular, short fibrils. Collectively, our results support the view that enhanced furin-mediated processing of mutant BRI generates fibrillogenic peptides that initiate the pathogenesis of FBD.

  17. Selection of DNA aptamers with two modified bases.

    PubMed

    Gawande, Bharat N; Rohloff, John C; Carter, Jeffrey D; von Carlowitz, Ira; Zhang, Chi; Schneider, Daniel J; Janjic, Nebojsa

    2017-03-14

    The nucleobases comprising DNA and RNA aptamers provide considerably less chemical diversity than protein-based ligands, limiting their versatility. The introduction of novel functional groups at just one of the four bases in modified aptamers has recently led to dramatic improvement in the success rate of identifying nucleic acid ligands to protein targets. Here we explore the benefits of additional enhancement in physicochemical diversity by selecting modified DNA aptamers that contain amino-acid-like modifications on both pyrimidine bases. Using proprotein convertase subtilisin/kexin type 9 as a representative protein target, we identify specific pairwise combinations of modifications that result in higher affinity, metabolic stability, and inhibitory potency compared with aptamers with single modifications. Such doubly modified aptamers are also more likely to be encoded in shorter sequences and occupy nonoverlapping epitopes more frequently than aptamers with single modifications. These highly modified DNA aptamers have broad utility in research, diagnostic, and therapeutic applications.

  18. [Neurons that encode sound direction].

    PubMed

    Peña, J L

    In the auditory system, the inner ear breaks down complex signals into their spectral components, and encodes the amplitude and phase of each. In order to infer sound direction in space, a computation on each frequency component of the sound must be performed. Space specific neurons in the owl s inferior colliculus respond only to sounds coming from a particular direction and represent the results of this computation. The interaural time difference (ITD) and interaural level difference (ILD define the auditory space for the owl and are processed in separate neural pathways. The parallel pathways that process these cues merge in the external nucleus of the inferior colliculus where the space specific neurons are selective to combinations of ITD and ILD. How do inputs from the two sources interact to produce combination selectivity to ITD ILD pairs? A multiplication of postsynaptic potentials tuned to ITD and ILD can account for the subthreshold responses of these neurons to ITD ILD pairs. Examples of multiplication by neurons or neural circuits are scarce, but many computational models assume the existence of this basic operation. The owl s auditory system uses such operation to create a 2 dimensional map of auditory space. The map of space in the owl s auditory system shows important similarities with representations of space in the cerebral cortex and other sensory systems. In encoding space or other stimulus features, individual neurons appear to possess analogous functional properties related to the synthesis of high order receptive fields.

  19. PCSK9 — EDRN Public Portal

    Cancer.gov

    PCSK9 is a proprotein convertase belonging to the proteinase K subfamily of the secretory subtilase family. PCSK9 is synthesized as a soluble zymogen that undergoes autocatalytic intramolecular processing in the endoplasmic reticulum. It may function as a proprotein convertase, play a role in cholesterol homeostasis, and have a role in the differentiation of cortical neurons. PCSK9 activity is inhibited by EGTA. PCSK9 is expressed in neuro-epithelioma, colon carcinoma, hepatic and pancreatic cell lines, and in Schwann cells. Mutations in the PCSK9 gene have been associated with a third form of autosomal dominant familial hypercholesterolemia.

  20. Molecular mechanisms for protein-encoded inheritance.

    PubMed

    Wiltzius, Jed J W; Landau, Meytal; Nelson, Rebecca; Sawaya, Michael R; Apostol, Marcin I; Goldschmidt, Lukasz; Soriaga, Angela B; Cascio, Duilio; Rajashankar, Kanagalaghatta; Eisenberg, David

    2009-09-01

    In prion inheritance and transmission, strains are phenotypic variants encoded by protein 'conformations'. However, it is unclear how a protein conformation can be stable enough to endure transmission between cells or organisms. Here we describe new polymorphic crystal structures of segments of prion and other amyloid proteins, which offer two structural mechanisms for the encoding of prion strains. In packing polymorphism, prion strains are encoded by alternative packing arrangements (polymorphs) of beta-sheets formed by the same segment of a protein; in segmental polymorphism, prion strains are encoded by distinct beta-sheets built from different segments of a protein. Both forms of polymorphism can produce enduring conformations capable of encoding strains. These molecular mechanisms for transfer of protein-encoded information into prion strains share features with the familiar mechanism for transfer of nucleic acid-encoded information into microbial strains, including sequence specificity and recognition by noncovalent bonds.

  1. Molecular mechanisms for protein-encoded inheritance

    SciTech Connect

    Wiltzius, Jed J.W.; Landau, Meytal; Nelson, Rebecca; Sawaya, Michael R.; Apostol, Marcin I.; Goldschmidt, Lukasz; Soriaga, Angela B.; Cascio, Duilio; Rajashankar, Kanagalaghatta; Eisenberg, David

    2009-12-01

    In prion inheritance and transmission, strains are phenotypic variants encoded by protein 'conformations'. However, it is unclear how a protein conformation can be stable enough to endure transmission between cells or organisms. Here we describe new polymorphic crystal structures of segments of prion and other amyloid proteins, which offer two structural mechanisms for the encoding of prion strains. In packing polymorphism, prion strains are encoded by alternative packing arrangements (polymorphs) of {beta}-sheets formed by the same segment of a protein; in segmental polymorphism, prion strains are encoded by distinct {beta}-sheets built from different segments of a protein. Both forms of polymorphism can produce enduring conformations capable of encoding strains. These molecular mechanisms for transfer of protein-encoded information into prion strains share features with the familiar mechanism for transfer of nucleic acid-encoded information into microbial strains, including sequence specificity and recognition by noncovalent bonds.

  2. Photolithographic Encoding of Metal Complexes.

    PubMed

    Lang, Christiane; Bestgen, Sebastian; Welle, Alexander; Müller, Rouven; Roesky, Peter W; Barner-Kowollik, Christopher

    2015-10-12

    A platform technology for the creation of spatially resolved surfaces encoded with a monolayer consisting of different metal complexes was developed. The concept entails the light-triggered activation of a self- assembled monolayer (SAM) of UV-labile anchors, that is, phenacylsulfides, and the subsequent cycloaddition of selected diene-functionalized metal complexes at defined areas on the surface. The synthesis and characterization of the metal complexes for the UV-light assisted anchoring on the surface and a detailed study of a short-chain oligomer model system in solution confirm the high efficiency of the photoreaction. The hybrid materials obtained by this concept can potentially be utilized for the design of highly valuable catalytic or (opto-)electronic devices. © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  3. Engineering Genetically Encoded FRET Sensors

    PubMed Central

    Lindenburg, Laurens; Merkx, Maarten

    2014-01-01

    Förster Resonance Energy Transfer (FRET) between two fluorescent proteins can be exploited to create fully genetically encoded and thus subcellularly targetable sensors. FRET sensors report changes in energy transfer between a donor and an acceptor fluorescent protein that occur when an attached sensor domain undergoes a change in conformation in response to ligand binding. The design of sensitive FRET sensors remains challenging as there are few generally applicable design rules and each sensor must be optimized anew. In this review we discuss various strategies that address this shortcoming, including rational design approaches that exploit self-associating fluorescent domains and the directed evolution of FRET sensors using high-throughput screening. PMID:24991940

  4. Genetically Encoded Sensors for Metabolites

    PubMed Central

    Deuschle, Karen; Fehr, Marcus; Hilpert, Melanie; Lager, Ida; Lalonde, Sylvie; Looger, Loren L.; Okumoto, Sakiko; Persson, Jörgen; Schmidt, Anja; Frommer, Wolf B.

    2009-01-01

    Background Metabolomics, i.e., the multiparallel analysis of metabolite changes occurring in a cell or an organism, has become feasible with the development of highly efficient mass spectroscopic technologies. Functional genomics as a standard tool helped to identify the function of many of the genes that encode important transporters and metabolic enzymes over the past few years. Advanced expression systems and analysis technologies made it possible to study the biochemical properties of the corresponding proteins in great detail. We begin to understand the biological functions of the gene products by systematic analysis of mutants using systematic PTGS/RNAi, knockout and TILLING approaches. However, one crucial set of data especially relevant in the case of multicellular organisms is lacking: the knowledge of the spatial and temporal profiles of metabolite levels at cellular and subcellular levels. Methods We therefore developed genetically encoded nanosensors for several metabolites to provide a basic set of tools for the determination of cytosolic and subcellular metabolite levels in real time by using fluorescence microscopy. Results Prototypes of these sensors were successfully used in vitro and also in vivo, i.e., to measure sugar levels in fungal and animal cells. Conclusions One of the future goals will be to expand the set of sensors to a wider spectrum of substrates by using the natural spectrum of periplasmic binding proteins from bacteria and by computational design of proteins with altered binding pockets in conjunction with mutagenesis. This toolbox can then be applied for four-dimensional imaging of cells and tissues to elucidate the spatial and temporal distribution of metabolites as a discovery tool in functional genomics, as a tool for high-throughput, high-content screening for drugs, to test metabolic models, and to analyze the interplay of cells in a tissue or organ. PMID:15688353

  5. SnoVault and encodeD: A novel object-based storage system and applications to ENCODE metadata

    PubMed Central

    Podduturi, Nikhil R.; Glick, David I.; Baymuradov, Ulugbek K.; Malladi, Venkat S.; Chan, Esther T.; Davidson, Jean M.; Gabdank, Idan; Narayana, Aditi K.; Onate, Kathrina C.; Hilton, Jason; Ho, Marcus C.; Lee, Brian T.; Miyasato, Stuart R.; Dreszer, Timothy R.; Sloan, Cricket A.; Strattan, J. Seth; Tanaka, Forrest Y.; Hong, Eurie L.; Cherry, J. Michael

    2017-01-01

    The Encyclopedia of DNA elements (ENCODE) project is an ongoing collaborative effort to create a comprehensive catalog of functional elements initiated shortly after the completion of the Human Genome Project. The current database exceeds 6500 experiments across more than 450 cell lines and tissues using a wide array of experimental techniques to study the chromatin structure, regulatory and transcriptional landscape of the H. sapiens and M. musculus genomes. All ENCODE experimental data, metadata, and associated computational analyses are submitted to the ENCODE Data Coordination Center (DCC) for validation, tracking, storage, unified processing, and distribution to community resources and the scientific community. As the volume of data increases, the identification and organization of experimental details becomes increasingly intricate and demands careful curation. The ENCODE DCC has created a general purpose software system, known as SnoVault, that supports metadata and file submission, a database used for metadata storage, web pages for displaying the metadata and a robust API for querying the metadata. The software is fully open-source, code and installation instructions can be found at: http://github.com/ENCODE-DCC/snovault/ (for the generic database) and http://github.com/ENCODE-DCC/encoded/ to store genomic data in the manner of ENCODE. The core database engine, SnoVault (which is completely independent of ENCODE, genomic data, or bioinformatic data) has been released as a separate Python package. PMID:28403240

  6. SnoVault and encodeD: A novel object-based storage system and applications to ENCODE metadata.

    PubMed

    Hitz, Benjamin C; Rowe, Laurence D; Podduturi, Nikhil R; Glick, David I; Baymuradov, Ulugbek K; Malladi, Venkat S; Chan, Esther T; Davidson, Jean M; Gabdank, Idan; Narayana, Aditi K; Onate, Kathrina C; Hilton, Jason; Ho, Marcus C; Lee, Brian T; Miyasato, Stuart R; Dreszer, Timothy R; Sloan, Cricket A; Strattan, J Seth; Tanaka, Forrest Y; Hong, Eurie L; Cherry, J Michael

    2017-01-01

    The Encyclopedia of DNA elements (ENCODE) project is an ongoing collaborative effort to create a comprehensive catalog of functional elements initiated shortly after the completion of the Human Genome Project. The current database exceeds 6500 experiments across more than 450 cell lines and tissues using a wide array of experimental techniques to study the chromatin structure, regulatory and transcriptional landscape of the H. sapiens and M. musculus genomes. All ENCODE experimental data, metadata, and associated computational analyses are submitted to the ENCODE Data Coordination Center (DCC) for validation, tracking, storage, unified processing, and distribution to community resources and the scientific community. As the volume of data increases, the identification and organization of experimental details becomes increasingly intricate and demands careful curation. The ENCODE DCC has created a general purpose software system, known as SnoVault, that supports metadata and file submission, a database used for metadata storage, web pages for displaying the metadata and a robust API for querying the metadata. The software is fully open-source, code and installation instructions can be found at: http://github.com/ENCODE-DCC/snovault/ (for the generic database) and http://github.com/ENCODE-DCC/encoded/ to store genomic data in the manner of ENCODE. The core database engine, SnoVault (which is completely independent of ENCODE, genomic data, or bioinformatic data) has been released as a separate Python package.

  7. Evaluation of GOES encoder lamps

    NASA Technical Reports Server (NTRS)

    Viehmann, W.; Helmold, N.

    1983-01-01

    Aging characteristics and life expectancies of flight quality, tungsten filament, encoder lamps are similar to those of 'commercial' grade gas filled lamps of similar construction, filament material and filament temperature. The aging and final failure by filament burnout are caused by single crystal growth over large portions of the filament with the concomitant development of facets and notches resulting in reduction of cross section and mechanical weakening of the filament. The life expectancy of presently produced lamps is about one year at their nominal operating voltage of five volts dc. At 4.5 volts, it is about two years. These life times are considerably shorter, and the degradation rates of lamp current and light flux are considerably higher, than were observed in the laboratory and in orbit on lamps of the same type manufactured more than a decade ago. It is speculated that the filaments of these earlier lamps contained a crystallization retarding dopant, possibly thorium oxide. To obtain the desired life expectancy of or = to four years in present lamps, operating voltages of or = to four volts dc would be required.

  8. Multidimensional encoding of brain connectomes.

    PubMed

    Caiafa, Cesar F; Pestilli, Franco

    2017-09-13

    The ability to map brain networks in living individuals is fundamental in efforts to chart the relation between human behavior, health and disease. Advances in network neuroscience may benefit from developing new frameworks for mapping brain connectomes. We present a framework to encode structural brain connectomes and diffusion-weighted magnetic resonance (dMRI) data using multidimensional arrays. The framework integrates the relation between connectome nodes, edges, white matter fascicles and diffusion data. We demonstrate the utility of the framework for in vivo white matter mapping and anatomical computing by evaluating 1,490 connectomes, thirteen tractography methods, and three data sets. The framework dramatically reduces storage requirements for connectome evaluation methods, with up to 40x compression factors. Evaluation of multiple, diverse datasets demonstrates the importance of spatial resolution in dMRI. We measured large increases in connectome resolution as function of data spatial resolution (up to 52%). Moreover, we demonstrate that the framework allows performing anatomical manipulations on white matter tracts for statistical inference and to study the white matter geometrical organization. Finally, we provide open-source software implementing the method and data to reproduce the results.

  9. Novelty's effect on memory encoding.

    PubMed

    Rangel-Gomez, Mauricio; Janenaite, Sigita; Meeter, Martijn

    2015-07-01

    It is often thought that novelty benefits memory formation. However, support for this idea mostly comes from paradigms that are open to alternative explanations. In the present study we manipulated novelty in a word-learning task through task-irrelevant background images. These background images were either standard (presented repeatedly), or novel (presented only once). Two types of background images were used: Landscape pictures and fractals. EEG was also recorded during encoding. Contrary to the idea that novelty aids memory formation, memory performance was not affected by the novelty of the background. In the evoked response potentials, we found evidence of distracting effects of novelty: both the N1 and P3b components were smaller to words studied with novel backgrounds, and the amplitude of the N2b component correlated negatively with subsequent retrieval. We conclude that although evidence from other studies does suggest benefits on a longer time scale, novelty has no instantaneous benefits for learning. Copyright © 2015 Elsevier B.V. All rights reserved.

  10. Image encoding with triangulation wavelets

    NASA Astrophysics Data System (ADS)

    Hebert, D. J.; Kim, HyungJun

    1995-09-01

    We demonstrate some wavelet-based image processing applications of a class of simplicial grids arising in finite element computations and computer graphics. The cells of a triangular grid form the set of leaves of a binary tree and the nodes of a directed graph consisting of a single cycle. The leaf cycle of a uniform grid forms a pattern for pixel image scanning and for coherent computation of coefficients of splines and wavelets. A simple form of image encoding is accomplished with a 1D quadrature mirror filter whose coefficients represent an expansion of the image in terms of 2D Haar wavelets with triangular support. A combination the leaf cycle and an inherent quadtree structure allow efficient neighbor finding, grid refinement, tree pruning and storage. Pruning of the simplex tree yields a partially compressed image which requires no decoding, but rather may be rendered as a shaded triangulation. This structure and its generalization to n-dimensions form a convenient setting for wavelet analysis and computations based on simplicial grids.

  11. Molecular cloning and expression of prohormone convertases PC1 and PC2 in the pituitary gland of the bullfrog, Rana catesbeiana.

    PubMed

    Yaoi, Yuichi; Suzuki, Masakazu; Tomura, Hideaki; Kikuyama, Sakae; Tanaka, Shigeyasu

    2003-09-01

    We cloned cDNAs encoding PC1 and PC2 from a cDNA library constructed for the anterior pituitary gland of the bullfrog (Rana catesbeiana) and sequenced them. The bullfrog PC1 cDNA consisted of 2972 base pairs (bp) with an open reading frame of 2208 bp and encoded a protein of 736 amino acids, including a putative signal peptide of 26 amino acids. The protein showed a high homology to R. ridibunda PC1 (95.1%) and mammalian PC1 (72.6%). The bullfrog PC2 cDNA consisted of 2242 bp with an open reading frame of 1914 bp and encoded a protein of 638 amino acids, including a putative signal peptide of 23 amino acids. This protein showed a high homology to R. ridibunda PC2 (95.5%) and mammalian PC2 (84.8%). The catalytic triad of serine proteinases of the subtilisin family was found at Asp-168, His-209, and Ser-383 in the PC1 protein and at Asp-167, His-208, and Ser-384 in the PC2 protein. In situ hybridization staining revealed that PC2 mRNA was detected in corticotrope cells of the tadpoles, but not in those of the adults. In the adult, only PC1 mRNA was detected in the pars distalis but both PC1 and PC2 mRNAs were detected in the pars intermedia. The data also showed that PC1 mRNA was expressed in gonadotrope cells.

  12. Unconscious relational encoding depends on hippocampus.

    PubMed

    Duss, Simone B; Reber, Thomas P; Hänggi, Jürgen; Schwab, Simon; Wiest, Roland; Müri, René M; Brugger, Peter; Gutbrod, Klemens; Henke, Katharina

    2014-12-01

    Textbooks divide between human memory systems based on consciousness. Hippocampus is thought to support only conscious encoding, while neocortex supports both conscious and unconscious encoding. We tested whether processing modes, not consciousness, divide between memory systems in three neuroimaging experiments with 11 amnesic patients (mean age=45.55 years, standard deviation=8.74, range=23-60) and 11 matched healthy control subjects. Examined processing modes were single item versus relational encoding with only relational encoding hypothesized to depend on hippocampus. Participants encoded and later retrieved either single words or new relations between words. Consciousness of encoding was excluded by subliminal (invisible) word presentation. Amnesic patients and controls performed equally well on the single item task activating prefrontal cortex. But only the controls succeeded on the relational task activating the hippocampus, while amnesic patients failed as a group. Hence, unconscious relational encoding, but not unconscious single item encoding, depended on hippocampus. Yet, three patients performed normally on unconscious relational encoding in spite of amnesia capitalizing on spared hippocampal tissue and connections to language cortex. This pattern of results suggests that processing modes divide between memory systems, while consciousness divides between levels of function within a memory system. © The Author (2014). Published by Oxford University Press on behalf of the Guarantors of Brain.

  13. Unconscious relational encoding depends on hippocampus

    PubMed Central

    Duss, Simone B.; Reber, Thomas P.; Hänggi, Jürgen; Schwab, Simon; Wiest, Roland; Müri, René M.; Brugger, Peter; Gutbrod, Klemens

    2014-01-01

    Textbooks divide between human memory systems based on consciousness. Hippocampus is thought to support only conscious encoding, while neocortex supports both conscious and unconscious encoding. We tested whether processing modes, not consciousness, divide between memory systems in three neuroimaging experiments with 11 amnesic patients (mean age = 45.55 years, standard deviation = 8.74, range = 23–60) and 11 matched healthy control subjects. Examined processing modes were single item versus relational encoding with only relational encoding hypothesized to depend on hippocampus. Participants encoded and later retrieved either single words or new relations between words. Consciousness of encoding was excluded by subliminal (invisible) word presentation. Amnesic patients and controls performed equally well on the single item task activating prefrontal cortex. But only the controls succeeded on the relational task activating the hippocampus, while amnesic patients failed as a group. Hence, unconscious relational encoding, but not unconscious single item encoding, depended on hippocampus. Yet, three patients performed normally on unconscious relational encoding in spite of amnesia capitalizing on spared hippocampal tissue and connections to language cortex. This pattern of results suggests that processing modes divide between memory systems, while consciousness divides between levels of function within a memory system. PMID:25273998

  14. Encoders for block-circulant LDPC codes

    NASA Technical Reports Server (NTRS)

    Andrews, Kenneth; Dolinar, Sam; Thorpe, Jeremy

    2005-01-01

    In this paper, we present two encoding methods for block-circulant LDPC codes. The first is an iterative encoding method based on the erasure decoding algorithm, and the computations required are well organized due to the block-circulant structure of the parity check matrix. The second method uses block-circulant generator matrices, and the encoders are very similar to those for recursive convolutional codes. Some encoders of the second type have been implemented in a small Field Programmable Gate Array (FPGA) and operate at 100 Msymbols/second.

  15. Vibrational spectroscopic encoding of polystyrene-based resin beads: converting the encoding peaks into barcodes.

    PubMed

    Liu, Lie-Xiong; Huang, Zhen-Li; Zhao, Yuan-Di

    2005-12-01

    A detailed approach is described for the vibrational spectroscopic encoding of polystyrene-based resin beads by converting the infrared absorption peaks suitable for encoding (encoding peaks) into barcodes. Based on combining the FT-IR measurements and the quantum-chemical computations, the vibrational characteristics of p-tert-butylstyrene monomer, polystyrene and poly(p-tert-butylstyrene) resin beads are analyzed, which are helpful for the selection of encoding peaks. The vibrational spectroscopic encoding of polystyrene-based resin beads could be obtained by converting the wavenumber, intensity and full width at half maximum (FWHM) of the encoding peaks into barcodes automatically through a computer program designed in our laboratory.

  16. Functional analysis of four processing products from multiple precursors encoded by a lebocin-related gene from Manduca sexta

    PubMed Central

    Rayaprolu, Subrahmanyam; Wang, Yang; Kanost, Michael R.; Hartson, Steven; Jiang, Haobo

    2010-01-01

    Antimicrobial peptides (AMPs) are a crucial component of the natural immune system in insects. Five types of AMPs have been identified in the tobacco hornworm Manduca sexta, including attacin, cecropin, moricin, gloverin, and lebocin. Here we report the isolation of lebocin-related cDNA clones and antibacterial activity of their processed protein products. The seventeen cDNA sequences are composed of a constant 5′ end and a variable 3′ region containing 3∼16 copies of an 81-nucleotide repeat. The sequence of the corresponding gene isolated from a M. sexta genomic library and Southern blotting results indicated that the gene lacks introns and exists as a single copy in the genome. The genomic sequence contained 13 complete and one partial copy of the 81-nucleotide repeat. Northern blot analysis revealed multiple transcripts with major size differences. The mRNA level of M. sexta lebocin increased substantially in fat body after larvae had been injected with bacteria. The RXXR motifs in the protein sequences led us to postulate that the precursors are processed by an intracellular convertase to form four bioactive peptides. To test this hypothesis, we chemically synthesized the peptides and examined their antibacterial activity. Peptide 1 killed Gram-positive and Gram-negative bacteria. Peptide 2, similar in sequence to a Galleria mellonella AMP, did not affect the bacterial growth. Peptide 3 was inactive but peptide 3 with an extra Arg at the carboxyl terminus was active against E. coli at a high minimum inhibitory concentration. Peptide 4, encoded by the 81-bp repeat, was inactive in the antibacterial tests. The hypothesis that posttranslational processing of the precursor proteins produces multiple bioactive peptides for defense purposes was validated by identification of peptides 1, 2, and 3 from larval hemolymph via liquid chromatography and tandem mass spectrometry. Comparison with the orthologs from other lepidopteran insects indicates that the same

  17. Encoding and decoding in fMRI

    PubMed Central

    Naselaris, Thomas; Kay, Kendrick N.; Nishimoto, Shinji; Gallant, Jack L.

    2010-01-01

    Over the past decade fMRI researchers have developed increasingly sensitive techniques for analyzing the information represented in BOLD activity. The most popular of these techniques is linear classification, a simple technique for decoding information about experimental stimuli or tasks from patterns of activity across an array of voxels. A more recent development is the voxel-based encoding model, which describes the information about the stimulus or task that is represented in the activity of single voxels. Encoding and decoding are complementary operations: encoding uses stimuli to predict activity while decoding uses activity to predict information about stimuli. However, in practice these two operations are often confused, and their respective strengths and weaknesses have not been made clear. Here we use the concept of a linearizing feature space to clarify the relationship between encoding and decoding. We show that encoding and decoding operations can both be used to investigate some of the most common questions about how information is represented in the brain. However, focusing on encoding models offers two important advantages over decoding. First, an encoding model can in principle provide a complete functional description of a region of interest, while a decoding model can provide only a partial description. Second, while it is straightforward to derive an optimal decoding model from an encoding model it is much more difficult to derive an encoding model from a decoding model. We propose a systematic modeling approach that begins by estimating an encoding model for every voxel in a scan and ends by using the estimated encoding models to perform decoding. PMID:20691790

  18. Optoelectronic Shaft-Angle Encoder Tolerates Misalignments

    NASA Technical Reports Server (NTRS)

    Osborne, Eric P.

    1991-01-01

    Optoelectronic shaft-angle encoder measures angle of rotation of shaft with high precision while minimizing effects of eccentricity and other misalignments. Grooves on disk serve as reference marks to locate reading heads and measure increments of rotation of disk. Shaft-angle encoder, resembling optical compact-disk drive, includes two tracking heads illuminating grooves on disk and measures reflections from them.

  19. The Acquisition of Syntactically Encoded Evidentiality

    ERIC Educational Resources Information Center

    Rett, Jessica; Hyams, Nina

    2014-01-01

    This article presents several empirical studies of syntactically encoded evidentiality in English. The first part of our study consists of an adult online experiment that confirms claims in Asudeh & Toivonen (2012) that raised Perception Verb Similatives (PVSs; e.g. "John looks like he is sick") encode direct evidentiality. We then…

  20. DNA encoding a DNA repair protein

    DOEpatents

    Petrini, John H.; Morgan, William Francis; Maser, Richard Scott; Carney, James Patrick

    2006-08-15

    An isolated and purified DNA molecule encoding a DNA repair protein, p95, is provided, as is isolated and purified p95. Also provided are methods of detecting p95 and DNA encoding p95. The invention further provides p95 knock-out mice.

  1. Precision Digital Position Encoding For Resonant Scanners

    NASA Astrophysics Data System (ADS)

    Reich, Stanley; Tuchman, Israel

    1984-11-01

    Presented is a versatile precision digital encoding technique for mechanical resonant scanners. Described is it's application to electronic correction of the sinusoidal scan pattern traced. To contrast the flexibility of the electronic encoding technique a number of direct correction methods are described.

  2. The Acquisition of Syntactically Encoded Evidentiality

    ERIC Educational Resources Information Center

    Rett, Jessica; Hyams, Nina

    2014-01-01

    This article presents several empirical studies of syntactically encoded evidentiality in English. The first part of our study consists of an adult online experiment that confirms claims in Asudeh & Toivonen (2012) that raised Perception Verb Similatives (PVSs; e.g. "John looks like he is sick") encode direct evidentiality. We then…

  3. Experiments in encoding multilevel images as quadtrees

    NASA Technical Reports Server (NTRS)

    Lansing, Donald L.

    1987-01-01

    Image storage requirements for several encoding methods are investigated and the use of quadtrees with multigray level or multicolor images are explored. The results of encoding a variety of images having up to 256 gray levels using three schemes (full raster, runlength and quadtree) are presented. Although there is considerable literature on the use of quadtrees to store and manipulate binary images, their application to multilevel images is relatively undeveloped. The potential advantage of quadtree encoding is that an entire area with a uniform gray level may be encoded as a unit. A pointerless quadtree encoding scheme is described. Data are presented on the size of the quadtree required to encode selected images and on the relative storage requirements of the three encoding schemes. A segmentation scheme based on the statistical variation of gray levels within a quadtree quadrant is described. This parametric scheme may be used to control the storage required by an encoded image and to preprocess a scene for feature identification. Several sets of black and white and pseudocolor images obtained by varying the segmentation parameter are shown.

  4. The convertases furin and PC1 can both cleave the human immunodeficiency virus (HIV)-1 envelope glycoprotein gp160 into gp120 (HIV-1 SU) and gp41 (HIV-I TM).

    PubMed

    Decroly, E; Vandenbranden, M; Ruysschaert, J M; Cogniaux, J; Jacob, G S; Howard, S C; Marshall, G; Kompelli, A; Basak, A; Jean, F

    1994-04-22

    Intracellular proteolytic processing of human immunodeficiency virus envelope glycoprotein precursor (gp160) is an essential step for virus infectivity. Northern blot analysis provided evidence that furin and PC1, but not PC2, are expressed in the CD4+ human lymphoblastoid H9 cell line, suggesting the possible participation of these convertases in human immunodeficiency virus (HIV) gp160 proteolytic processing. Purified PC1 and furin cleaved specifically in vitro gp160 into gp120 (HIV-I SU) and gp41 (HIV-I TM). NH2-terminal sequence analysis of the produced gp41 (HIV-I TM) demonstrated that the cleavage occurred within the sequence Arg-Glu-Lys-Arg decreases Ala-Val-Gly-Ile, which is identical to the bond cleaved in vivo. Transition state analog peptides were designed and tested in vitro for their ability to inhibit the PC1- or furin-mediated gp160 cleavage. The best inhibitor was decanoyl-Arg-Lys-Arg-Arg-psi [CH2NH]-Phe-Leu-Gly-Phe-NH2.

  5. Recent advances on the encoding and selection methods of DNA-encoded chemical library.

    PubMed

    Shi, Bingbing; Zhou, Yu; Huang, Yiran; Zhang, Jianfu; Li, Xiaoyu

    2017-02-01

    DNA-encoded chemical library (DEL) has emerged as a powerful and versatile tool for ligand discovery in chemical biology research and in drug discovery. Encoding and selection methods are two of the most important technological aspects of DEL that can dictate the performance and utilities of DELs. In this digest, we have summarized recent advances on the encoding and selection strategies of DEL and also discussed the latest developments on DNA-encoded dynamic library, a new frontier in DEL research.

  6. Comparison of H.265/HEVC encoders

    NASA Astrophysics Data System (ADS)

    Trochimiuk, Maciej

    2016-09-01

    The H.265/HEVC is the state-of-the-art video compression standard, which allows the bitrate reduction up to 50% compared with its predecessor, H.264/AVC, maintaining equal perceptual video quality. The growth in coding efficiency was achieved by increasing the number of available intra- and inter-frame prediction features and improvements in existing ones, such as entropy encoding and filtering. Nevertheless, to achieve real-time performance of the encoder, simplifications in algorithm are inevitable. Some features and coding modes shall be skipped, to reduce time needed to evaluate modes forwarded to rate-distortion optimisation. Thus, the potential acceleration of the encoding process comes at the expense of coding efficiency. In this paper, a trade-off between video quality and encoding speed of various H.265/HEVC encoders is discussed.

  7. Industrial Applications Of Optical Shaft Encoders

    NASA Astrophysics Data System (ADS)

    Edmister, Brian W.

    1980-11-01

    The development of the microprocessor and mini-computer for industrial process control has made the optical shaft angle encoder a natural choice for a position feedback transducer. Many of these applications, however, require the encoder to operate reliably in extremely hostile environments. In response to this, the encoder manufacturer has been faced with reliability problems which fall into the following general categories: 1. Exposure to weather 2. Wide operating and storage temperature range 3. Exposure to corrosive chemicals 4. Severe shock and vibration 5. High electrical noise levels 6. Severe blows to encoder housing 7. Operation in explosive atmospheres Three of these applications expose the encoder to most of these environmental conditions: 1. A jack-up control position feedback for an offshore oil well drilling rig 2. A depth measurement system for oil well logging instrumentation 3. Elevation and azimuth feedback for a solar power plant heliostat

  8. Encoder: A Connectionist Model of How Learning to Visually Encode Fixated Text Images Improves Reading Fluency

    ERIC Educational Resources Information Center

    Martin, Gale L.

    2004-01-01

    This article proposes that visual encoding learning improves reading fluency by widening the span over which letters are recognized from a fixated text image so that fewer fixations are needed to cover a text line. Encoder is a connectionist model that learns to convert images like the fixated text images human readers encode into the…

  9. A model for visual memory encoding.

    PubMed

    Nenert, Rodolphe; Allendorfer, Jane B; Szaflarski, Jerzy P

    2014-01-01

    Memory encoding engages multiple concurrent and sequential processes. While the individual processes involved in successful encoding have been examined in many studies, a sequence of events and the importance of modules associated with memory encoding has not been established. For this reason, we sought to perform a comprehensive examination of the network for memory encoding using data driven methods and to determine the directionality of the information flow in order to build a viable model of visual memory encoding. Forty healthy controls ages 19-59 performed a visual scene encoding task. FMRI data were preprocessed using SPM8 and then processed using independent component analysis (ICA) with the reliability of the identified components confirmed using ICASSO as implemented in GIFT. The directionality of the information flow was examined using Granger causality analyses (GCA). All participants performed the fMRI task well above the chance level (>90% correct on both active and control conditions) and the post-fMRI testing recall revealed correct memory encoding at 86.33 ± 5.83%. ICA identified involvement of components of five different networks in the process of memory encoding, and the GCA allowed for the directionality of the information flow to be assessed, from visual cortex via ventral stream to the attention network and then to the default mode network (DMN). Two additional networks involved in this process were the cerebellar and the auditory-insular network. This study provides evidence that successful visual memory encoding is dependent on multiple modules that are part of other networks that are only indirectly related to the main process. This model may help to identify the node(s) of the network that are affected by a specific disease processes and explain the presence of memory encoding difficulties in patients in whom focal or global network dysfunction exists.

  10. Efficient reverse time migration with amplitude encoding

    NASA Astrophysics Data System (ADS)

    Hu, Jiangtao; Wang, Huazhong; Zhao, Lei; Shao, Yu; Wang, Meixia; Osen, Are

    2015-08-01

    Reverse time migration (RTM) is an accurate seismic imaging method for imaging the complex subsurface structure. Traditional common shot RTM suffers from low efficiency due to the large number of single shot gathers, especially for marine seismic data. Phase encoding is commonly used to reduce the computational cost of RTM. Phase encoding in the frequency domain is usually related to time shift in the time domain. Therefore, phase-encoding-based RTM needs time padding to avoid information loss which degrades the efficiency of the time-domain wavefield extrapolator. In this paper, an efficient time-domain RTM scheme based on the amplitude encoding is proposed. This scheme uses the orthogonal cosine basis as the encoding function, which has similar physical meaning to plane wave encoding (i.e. plane-wave components with different surface shooting angles). The proposed scheme can generate a qualified imaging result as well as common shot RTM but with less computational cost. Since this scheme does not need time padding, it is more efficient than the phase encoding schemes and can be conveniently implemented in the time domain. Numerical examples on the Sigsbee2a synthetic dataset demonstrate the feasibility of the proposed method.

  11. Encoding entanglement-assisted quantum stabilizer codes

    NASA Astrophysics Data System (ADS)

    Wang, Yun-Jiang; Bai, Bao-Ming; Li, Zhuo; Peng, Jin-Ye; Xiao, He-Ling

    2012-02-01

    We address the problem of encoding entanglement-assisted (EA) quantum error-correcting codes (QECCs) and of the corresponding complexity. We present an iterative algorithm from which a quantum circuit composed of CNOT, H, and S gates can be derived directly with complexity O(n2) to encode the qubits being sent. Moreover, we derive the number of each gate consumed in our algorithm according to which we can design EA QECCs with low encoding complexity. Another advantage brought by our algorithm is the easiness and efficiency of programming on classical computers.

  12. Pulse Vector-Excitation Speech Encoder

    NASA Technical Reports Server (NTRS)

    Davidson, Grant; Gersho, Allen

    1989-01-01

    Proposed pulse vector-excitation speech encoder (PVXC) encodes analog speech signals into digital representation for transmission or storage at rates below 5 kilobits per second. Produces high quality of reconstructed speech, but with less computation than required by comparable speech-encoding systems. Has some characteristics of multipulse linear predictive coding (MPLPC) and of code-excited linear prediction (CELP). System uses mathematical model of vocal tract in conjunction with set of excitation vectors and perceptually-based error criterion to synthesize natural-sounding speech.

  13. Programmable Pulse-Position-Modulation Encoder

    NASA Technical Reports Server (NTRS)

    Zhu, David; Farr, William

    2006-01-01

    A programmable pulse-position-modulation (PPM) encoder has been designed for use in testing an optical communication link. The encoder includes a programmable state machine and an electronic code book that can be updated to accommodate different PPM coding schemes. The encoder includes a field-programmable gate array (FPGA) that is programmed to step through the stored state machine and code book and that drives a custom high-speed serializer circuit board that is capable of generating subnanosecond pulses. The stored state machine and code book can be updated by means of a simple text interface through the serial port of a personal computer.

  14. VLSI Reed-Solomon Encoder With Interleaver

    NASA Technical Reports Server (NTRS)

    Hsu, In-Shek; Deutsch, L. J.; Truong, Trieu-Kie; Reed, I. S.

    1990-01-01

    Size, weight, and susceptibility to burst errors reduced. Encoding system built on single very-large-scale integrated (VLSI) circuit chip produces (255,223) Reed-Solomon (RS) code with programmable interleaving up to depth of 5. (225,223) RS encoder includes new remainder-and-interleaver unit providing programmable interleaving of code words. Remainder-and-interleaver unit contains shift registers and modulo-2 adders. Signals on "turn" and "no-turn" lines control depth of interleaving. Based on E. R. Berlekamp's bit-serial multiplication algorithm for (225,223) RS encoder over Galois Field (2 to the 8th power).

  15. Pulse Vector-Excitation Speech Encoder

    NASA Technical Reports Server (NTRS)

    Davidson, Grant; Gersho, Allen

    1989-01-01

    Proposed pulse vector-excitation speech encoder (PVXC) encodes analog speech signals into digital representation for transmission or storage at rates below 5 kilobits per second. Produces high quality of reconstructed speech, but with less computation than required by comparable speech-encoding systems. Has some characteristics of multipulse linear predictive coding (MPLPC) and of code-excited linear prediction (CELP). System uses mathematical model of vocal tract in conjunction with set of excitation vectors and perceptually-based error criterion to synthesize natural-sounding speech.

  16. Regulation of Pcsk6 expression during the preantral to antral follicle transition in mice: opposing roles of FSH and oocytes.

    PubMed

    Diaz, Francisco J; Sugiura, Koji; Eppig, John J

    2008-01-01

    Several secreted products of the TGFbeta superfamily have important roles during follicular development and are produced by both oocytes and somatic cells (granulosa and theca) in the follicle. The proprotein convertases are a family of seven known proteins that process TGFbeta ligands and other secreted products to their mature active form. The present study examined the regulation of steady-state levels of Pcsk6 mRNA, which encodes a convertase protein known to process members of the TGFbeta superfamily, during mouse follicular development. Pcsk6 mRNA and protein were expressed in preantral but not cumulus or mural granulosa cells. Pcsk6 mRNA levels in preantral granulosa cells were not regulated by growing oocytes of preantral follicles, but were elevated by FSH. Furthermore, Pcsk6 mRNA in preantral granulosa cells was potently suppressed by factor(s) secreted by fully grown oocytes from antral follicles, in part through SMAD2/3-mediated pathways. Oocytes acquired the ability to suppress the steady-state levels of Pcsk6 mRNA in granulosa cells during the preantral to antral follicle transition. Suppression of Pcsk6 mRNA by oocytes could reflect a change in the mechanism(s) regulating the activity of members of the TGFbeta superfamily.

  17. Association of low-frequency and rare coding-sequence variants with blood lipids and Coronary Heart Disease in 56,000 whites and blacks

    USDA-ARS?s Scientific Manuscript database

    Low-frequency coding DNA sequence variants in the proprotein convertase subtilisin/kexin type 9 gene (PCSK9) lower plasma low-density lipoprotein cholesterol (LDL-C), protect against risk of coronary heart disease (CHD), and have prompted the development of a new class of therapeutics. It is uncerta...

  18. Genetic variation at the PCSK9 locus, low density lipoproteins, response to pravastatin and coronary heart disease: results from PROSPER

    USDA-ARS?s Scientific Manuscript database

    Caucasian carriers of the T allele at R46L in the proprotein convertase subtilisin/kexin type 9 (PCSK9) locus have been reported to have 15% lower low-density lipoprotein (LDL) cholesterol (C) levels and 47% lower coronary heart disease (CHD) risk. Our objective was to examine two PCSK9 single nucle...

  19. Chemical Space of DNA-Encoded Libraries.

    PubMed

    Franzini, Raphael M; Randolph, Cassie

    2016-07-28

    In recent years, DNA-encoded chemical libraries (DECLs) have attracted considerable attention as a potential discovery tool in drug development. Screening encoded libraries may offer advantages over conventional hit discovery approaches and has the potential to complement such methods in pharmaceutical research. As a result of the increased application of encoded libraries in drug discovery, a growing number of hit compounds are emerging in scientific literature. In this review we evaluate reported encoded library-derived structures and identify general trends of these compounds in relation to library design parameters. We in particular emphasize the combinatorial nature of these libraries. Generally, the reported molecules demonstrate the ability of this technology to afford hits suitable for further lead development, and on the basis of them, we derive guidelines for DECL design.

  20. Optimal stimulus encoders for natural tasks

    PubMed Central

    Geisler, Wilson S.; Najemnik, Jiri; Ing, Almon D.

    2010-01-01

    Determining the features of natural stimuli that are most useful for specific natural tasks is critical for understanding perceptual systems. A new approach is described that involves finding the optimal encoder for the natural task of interest, given a relatively small population of noisy “neurons” between the encoder and decoder. The optimal encoder, which necessarily specifies the most useful features, is found by maximizing accuracy in the natural task, where the decoder is the Bayesian ideal observer operating on the population responses. The approach is illustrated for a patch identification task, where the goal is to identify patches of natural image, and for a foreground identification task, where the goal is to identify which side of a natural surface boundary belongs to the foreground object. The optimal features (receptive fields) are intuitive and perform well in the two tasks. The approach also provides insight into general principles of neural encoding and decoding. PMID:20055550

  1. A multifunctional rotary photoelectric encoder management system

    NASA Astrophysics Data System (ADS)

    Ye, Zunzhong; Ying, Yibin

    2005-11-01

    The rotary photoelectric encoder can be used in many fields, such as robot research, fruit assembly lines, and so on. If there have many photoelectric encoders in one system, it's difficult to manage them and acquire the right pulse number. So it's important to design a multifunctional management system. It includes a powerful microchip with high processing speed, assuring the acquisition precision of rotary pulse. It uses a special method to judge the rotary direction and will be competent for many occasions which rotary direction changes quickly. Considering encoder data transmission, the management system provides a serial port using RS-485 protocol to transmit current pulse data and rotary direction. It allows linking a maximum of 100 management systems using only two communication lines to up-systems and also configing the encoder counting pattern locally (using the keyboard) or remotely (through the computer).

  2. Cellobiohydrolase variants and polynucleotides encoding same

    DOEpatents

    Wogulis, Mark

    2013-09-24

    The present invention relates to variants of a parent cellobiohydrolase II. The present invention also relates to polynucleotides encoding the variants; nucleic acid constructs, vectors, and host cells comprising the polynucleotides; and methods of using the variants.

  3. Cellobiohydrolase variants and polynucleotides encoding same

    DOEpatents

    Wogulis, Mark

    2014-10-14

    The present invention relates to variants of a parent cellobiohydrolase II. The present invention also relates to polynucleotides encoding the variants; nucleic acid constructs, vectors, and host cells comprising the polynucleotides; and methods of using the variants.

  4. Cellobiohydrolase variants and polynucleotides encoding the same

    DOEpatents

    Wogulis, Mark

    2014-09-09

    The present invention relates to variants of a parent cellobiohydrolase. The present invention also relates to polynucleotides encoding the cellobiohydrolase variants; nucleic acid constructs, vectors, and host cells comprising the polynucleotides; and methods of using the cellobiohydrolase variants.

  5. Clustering of polarization-encoded images.

    PubMed

    Zallat, Jihad; Collet, Christophe; Takakura, Yoshitate

    2004-01-10

    Polarization-encoded imaging consists of the distributed measurements of polarization parameters for each pixel of an image. We address clustering of multidimensional polarization-encoded images. The spatial coherence of polarization information is considered. Two methods of analysis are proposed: polarization contrast enhancement and a more-sophisticated image-processing algorithm based on a Markovian model. The proposed algorithms are applied and validated with two different Mueller images acquired by a fully polarimetric imaging system.

  6. A Manual for Encoding Probability Distributions.

    DTIC Science & Technology

    1978-09-01

    summary of the most significant information contained in the report. If the report contains a significant bibliography or literature survey, mention it...probability distri- bution. Some terms in the literature that are used synonymously to Encoding: Assessment, Assignment (used for single events in this...sessions conducted as parts of practical decision analyses as well as on experimental evidence in the literature . Probability encoding can be applied

  7. Nucleotide sequences encoding a thermostable alkaline protease

    DOEpatents

    Wilson, D.B.; Lao, G.

    1998-01-06

    Nucleotide sequences, derived from a thermophilic actinomycete microorganism, which encode a thermostable alkaline protease are disclosed. Also disclosed are variants of the nucleotide sequences which encode a polypeptide having thermostable alkaline proteolytic activity. Recombinant thermostable alkaline protease or recombinant polypeptide may be obtained by culturing in a medium a host cell genetically engineered to contain and express a nucleotide sequence according to the present invention, and recovering the recombinant thermostable alkaline protease or recombinant polypeptide from the culture medium. 3 figs.

  8. Nucleotide sequences encoding a thermostable alkaline protease

    DOEpatents

    Wilson, David B.; Lao, Guifang

    1998-01-01

    Nucleotide sequences, derived from a thermophilic actinomycete microorganism, which encode a thermostable alkaline protease are disclosed. Also disclosed are variants of the nucleotide sequences which encode a polypeptide having thermostable alkaline proteolytic activity. Recombinant thermostable alkaline protease or recombinant polypeptide may be obtained by culturing in a medium a host cell genetically engineered to contain and express a nucleotide sequence according to the present invention, and recovering the recombinant thermostable alkaline protease or recombinant polypeptide from the culture medium.

  9. Using XML to encode TMA DES metadata

    PubMed Central

    Lyttleton, Oliver; Wright, Alexander; Treanor, Darren; Lewis, Paul

    2011-01-01

    Background: The Tissue Microarray Data Exchange Specification (TMA DES) is an XML specification for encoding TMA experiment data. While TMA DES data is encoded in XML, the files that describe its syntax, structure, and semantics are not. The DTD format is used to describe the syntax and structure of TMA DES, and the ISO 11179 format is used to define the semantics of TMA DES. However, XML Schema can be used in place of DTDs, and another XML encoded format, RDF, can be used in place of ISO 11179. Encoding all TMA DES data and metadata in XML would simplify the development and usage of programs which validate and parse TMA DES data. XML Schema has advantages over DTDs such as support for data types, and a more powerful means of specifying constraints on data values. An advantage of RDF encoded in XML over ISO 11179 is that XML defines rules for encoding data, whereas ISO 11179 does not. Materials and Methods: We created an XML Schema version of the TMA DES DTD. We wrote a program that converted ISO 11179 definitions to RDF encoded in XML, and used it to convert the TMA DES ISO 11179 definitions to RDF. Results: We validated a sample TMA DES XML file that was supplied with the publication that originally specified TMA DES using our XML Schema. We successfully validated the RDF produced by our ISO 11179 converter with the W3C RDF validation service. Conclusions: All TMA DES data could be encoded using XML, which simplifies its processing. XML Schema allows datatypes and valid value ranges to be specified for CDEs, which enables a wider range of error checking to be performed using XML Schemas than could be performed using DTDs. PMID:21969921

  10. Using XML to encode TMA DES metadata.

    PubMed

    Lyttleton, Oliver; Wright, Alexander; Treanor, Darren; Lewis, Paul

    2011-01-01

    The Tissue Microarray Data Exchange Specification (TMA DES) is an XML specification for encoding TMA experiment data. While TMA DES data is encoded in XML, the files that describe its syntax, structure, and semantics are not. The DTD format is used to describe the syntax and structure of TMA DES, and the ISO 11179 format is used to define the semantics of TMA DES. However, XML Schema can be used in place of DTDs, and another XML encoded format, RDF, can be used in place of ISO 11179. Encoding all TMA DES data and metadata in XML would simplify the development and usage of programs which validate and parse TMA DES data. XML Schema has advantages over DTDs such as support for data types, and a more powerful means of specifying constraints on data values. An advantage of RDF encoded in XML over ISO 11179 is that XML defines rules for encoding data, whereas ISO 11179 does not. We created an XML Schema version of the TMA DES DTD. We wrote a program that converted ISO 11179 definitions to RDF encoded in XML, and used it to convert the TMA DES ISO 11179 definitions to RDF. We validated a sample TMA DES XML file that was supplied with the publication that originally specified TMA DES using our XML Schema. We successfully validated the RDF produced by our ISO 11179 converter with the W3C RDF validation service. All TMA DES data could be encoded using XML, which simplifies its processing. XML Schema allows datatypes and valid value ranges to be specified for CDEs, which enables a wider range of error checking to be performed using XML Schemas than could be performed using DTDs.

  11. Neurally Encoding Time for Olfactory Navigation

    PubMed Central

    Park, In Jun; Hein, Andrew M.; Bobkov, Yuriy V.; Reidenbach, Matthew A.; Ache, Barry W.; Principe, Jose C.

    2016-01-01

    Accurately encoding time is one of the fundamental challenges faced by the nervous system in mediating behavior. We recently reported that some animals have a specialized population of rhythmically active neurons in their olfactory organs with the potential to peripherally encode temporal information about odor encounters. If these neurons do indeed encode the timing of odor arrivals, it should be possible to demonstrate that this capacity has some functional significance. Here we show how this sensory input can profoundly influence an animal’s ability to locate the source of odor cues in realistic turbulent environments—a common task faced by species that rely on olfactory cues for navigation. Using detailed data from a turbulent plume created in the laboratory, we reconstruct the spatiotemporal behavior of a real odor field. We use recurrence theory to show that information about position relative to the source of the odor plume is embedded in the timing between odor pulses. Then, using a parameterized computational model, we show how an animal can use populations of rhythmically active neurons to capture and encode this temporal information in real time, and use it to efficiently navigate to an odor source. Our results demonstrate that the capacity to accurately encode temporal information about sensory cues may be crucial for efficient olfactory navigation. More generally, our results suggest a mechanism for extracting and encoding temporal information from the sensory environment that could have broad utility for neural information processing. PMID:26730727

  12. With blood in the joint - what happens next? Could activation of a pro-inflammatory signalling axis leading to iRhom2/TNFα-convertase-dependent release of TNFα contribute to haemophilic arthropathy?

    PubMed

    Haxaire, C; Blobel, C P

    2014-05-01

    One of the main complications of haemophilia A is haemophilic arthropathy (HA), a debilitating disease with a significant negative impact on motility and quality of life. Despite major advances in the treatment of haemophilia A, many patients still suffer from HA. We wish to develop new treatments for HA, but must first better understand its causes. Our laboratory studies molecular scissors that release the pro-inflammatory cytokine tumour necrosis factor alpha (TNFα) from cells. TNFα is considered the 'fire alarm' of the body - it helps to fight infections, but can also cause diseases such as inflammatory arthritis. We know that the molecular scissors, called TNFα convertase (TACE), and its newly discovered regulator termed iRhom2 can be rapidly activated by small amounts of cytokines, growth factors, and pro-inflammatory mediators present in the blood. We hypothesize that the rapid activation of TACE could help explain one of the unsolved mysteries regarding the development of HA, which is how even small amounts of blood can provoke a persistent inflammatory response. We propose that once blood enters the joint, iRhom2 and TACE are activated to release TNFα and that this could promote the development of HA in a similar manner to that in which it promotes rheumatoid arthritis (RA). We are currently using immune cells stimulated with blood degradation products, and mouse models of HA, to test this hypothesis. If successful, our study could provide the rationale for testing anti-TNF antibodies, which are already used to treat RA, for the treatment of HA. In addition, they might uncover iRhom2 and TACE as attractive new candidate targets for the treatment of HA. © 2014 John Wiley & Sons Ltd.

  13. Ultraviolet B irradiation of the mouse eye induces pigmentation of the skin more strongly than does stress loading, by increasing the levels of prohormone convertase 2 and α-melanocyte-stimulating hormone.

    PubMed

    Hiramoto, K; Yamate, Y; Kobayashi, H; Ishii, M; Sato, E F; Inoue, M

    2013-01-01

    In previous studies, we made the unexpected finding that in mice, ultraviolet (UV)B irradiation of the eye increased the concentration of α-melanocyte-stimulating hormone (α-MSH) in plasma, and systemically stimulated epidermal melanocytes. To compare the extent of the pigmentation induced by social and restraint stress (which activate the hippocampus-pituitary system) with that induced by UVB irradiation. DBA/2 and sham-operated or hypophysectomized DBA/2 mice were subjected to local UVB exposure using a sunlamp directed at the eye, and two types of stress (social and restraint) were imposed. UVB irradiation of the eye or exposure to stress loading both increased the number of Dopa-positive melanocytes in the epidermis, and hypophysectomy strongly inhibited the UVB-induced and stress-induced stimulation of melanocytes. Irradiation of the eye caused a much greater increase in dopamine than did the stress load. Both UVB eye irradiation and stress increased the blood levels of α-MSH and adrenocorticotropic hormone (ACTH). In addition, the increase in plasma α-MSH was greater in animals subjected to UVB eye irradiation than in those subjected to stress loading, whereas the reverse occurred for plasma ACTH. UVB irradiation to the eye and stress loading increased the expression of prohormone convertase (PC)1/3 and PC2 in the pituitary gland. The increase in expression of pituitary PC2 was greater in animals subjected to UVB eye irradiation than to stress, whereas no difference was seen between the two groups for the increase in PC1/3. UVB eye irradiation exerts a stronger effect on pigmentation than stress loading, and is related to increased levels of α-MSH and PC2. © The Author(s). CED © 2012 British Association of Dermatologists.

  14. Multichannel Compressive Sensing MRI Using Noiselet Encoding

    PubMed Central

    Pawar, Kamlesh; Egan, Gary; Zhang, Jingxin

    2015-01-01

    The incoherence between measurement and sparsifying transform matrices and the restricted isometry property (RIP) of measurement matrix are two of the key factors in determining the performance of compressive sensing (CS). In CS-MRI, the randomly under-sampled Fourier matrix is used as the measurement matrix and the wavelet transform is usually used as sparsifying transform matrix. However, the incoherence between the randomly under-sampled Fourier matrix and the wavelet matrix is not optimal, which can deteriorate the performance of CS-MRI. Using the mathematical result that noiselets are maximally incoherent with wavelets, this paper introduces the noiselet unitary bases as the measurement matrix to improve the incoherence and RIP in CS-MRI. Based on an empirical RIP analysis that compares the multichannel noiselet and multichannel Fourier measurement matrices in CS-MRI, we propose a multichannel compressive sensing (MCS) framework to take the advantage of multichannel data acquisition used in MRI scanners. Simulations are presented in the MCS framework to compare the performance of noiselet encoding reconstructions and Fourier encoding reconstructions at different acceleration factors. The comparisons indicate that multichannel noiselet measurement matrix has better RIP than that of its Fourier counterpart, and that noiselet encoded MCS-MRI outperforms Fourier encoded MCS-MRI in preserving image resolution and can achieve higher acceleration factors. To demonstrate the feasibility of the proposed noiselet encoding scheme, a pulse sequences with tailored spatially selective RF excitation pulses was designed and implemented on a 3T scanner to acquire the data in the noiselet domain from a phantom and a human brain. The results indicate that noislet encoding preserves image resolution better than Fouirer encoding. PMID:25965548

  15. Multichannel compressive sensing MRI using noiselet encoding.

    PubMed

    Pawar, Kamlesh; Egan, Gary; Zhang, Jingxin

    2015-01-01

    The incoherence between measurement and sparsifying transform matrices and the restricted isometry property (RIP) of measurement matrix are two of the key factors in determining the performance of compressive sensing (CS). In CS-MRI, the randomly under-sampled Fourier matrix is used as the measurement matrix and the wavelet transform is usually used as sparsifying transform matrix. However, the incoherence between the randomly under-sampled Fourier matrix and the wavelet matrix is not optimal, which can deteriorate the performance of CS-MRI. Using the mathematical result that noiselets are maximally incoherent with wavelets, this paper introduces the noiselet unitary bases as the measurement matrix to improve the incoherence and RIP in CS-MRI. Based on an empirical RIP analysis that compares the multichannel noiselet and multichannel Fourier measurement matrices in CS-MRI, we propose a multichannel compressive sensing (MCS) framework to take the advantage of multichannel data acquisition used in MRI scanners. Simulations are presented in the MCS framework to compare the performance of noiselet encoding reconstructions and Fourier encoding reconstructions at different acceleration factors. The comparisons indicate that multichannel noiselet measurement matrix has better RIP than that of its Fourier counterpart, and that noiselet encoded MCS-MRI outperforms Fourier encoded MCS-MRI in preserving image resolution and can achieve higher acceleration factors. To demonstrate the feasibility of the proposed noiselet encoding scheme, a pulse sequences with tailored spatially selective RF excitation pulses was designed and implemented on a 3T scanner to acquire the data in the noiselet domain from a phantom and a human brain. The results indicate that noislet encoding preserves image resolution better than Fouirer encoding.

  16. ERP Correlates of Encoding Success and Encoding Selectivity in Attention Switching

    PubMed Central

    Yeung, Nick

    2016-01-01

    Long-term memory encoding depends critically on effective processing of incoming information. The degree to which participants engage in effective encoding can be indexed in electroencephalographic (EEG) data by studying event-related potential (ERP) subsequent memory effects. The current study investigated ERP correlates of memory success operationalised with two different measures—memory selectivity and global memory—to assess whether previously observed ERP subsequent memory effects reflect focused encoding of task-relevant information (memory selectivity), general encoding success (global memory), or both. Building on previous work, the present study combined an attention switching paradigm—in which participants were presented with compound object-word stimuli and switched between attending to the object or the word across trials—with a later recognition memory test for those stimuli, while recording their EEG. Our results provided clear evidence that subsequent memory effects resulted from selective attentional focusing and effective top-down control (memory selectivity) in contrast to more general encoding success effects (global memory). Further analyses addressed the question of whether successful encoding depended on similar control mechanisms to those involved in attention switching. Interestingly, differences in the ERP correlates of attention switching and successful encoding, particularly during the poststimulus period, indicated that variability in encoding success occurred independently of prestimulus demands for top-down cognitive control. These results suggest that while effects of selective attention and selective encoding co-occur behaviourally their ERP correlates are at least partly dissociable. PMID:27907075

  17. ERP Correlates of Encoding Success and Encoding Selectivity in Attention Switching.

    PubMed

    Richter, Franziska R; Yeung, Nick

    2016-01-01

    Long-term memory encoding depends critically on effective processing of incoming information. The degree to which participants engage in effective encoding can be indexed in electroencephalographic (EEG) data by studying event-related potential (ERP) subsequent memory effects. The current study investigated ERP correlates of memory success operationalised with two different measures-memory selectivity and global memory-to assess whether previously observed ERP subsequent memory effects reflect focused encoding of task-relevant information (memory selectivity), general encoding success (global memory), or both. Building on previous work, the present study combined an attention switching paradigm-in which participants were presented with compound object-word stimuli and switched between attending to the object or the word across trials-with a later recognition memory test for those stimuli, while recording their EEG. Our results provided clear evidence that subsequent memory effects resulted from selective attentional focusing and effective top-down control (memory selectivity) in contrast to more general encoding success effects (global memory). Further analyses addressed the question of whether successful encoding depended on similar control mechanisms to those involved in attention switching. Interestingly, differences in the ERP correlates of attention switching and successful encoding, particularly during the poststimulus period, indicated that variability in encoding success occurred independently of prestimulus demands for top-down cognitive control. These results suggest that while effects of selective attention and selective encoding co-occur behaviourally their ERP correlates are at least partly dissociable.

  18. An encyclopedia of mouse DNA elements (Mouse ENCODE).

    PubMed

    Stamatoyannopoulos, John A; Snyder, Michael; Hardison, Ross; Ren, Bing; Gingeras, Thomas; Gilbert, David M; Groudine, Mark; Bender, Michael; Kaul, Rajinder; Canfield, Theresa; Giste, Erica; Johnson, Audra; Zhang, Mia; Balasundaram, Gayathri; Byron, Rachel; Roach, Vaughan; Sabo, Peter J; Sandstrom, Richard; Stehling, A Sandra; Thurman, Robert E; Weissman, Sherman M; Cayting, Philip; Hariharan, Manoj; Lian, Jin; Cheng, Yong; Landt, Stephen G; Ma, Zhihai; Wold, Barbara J; Dekker, Job; Crawford, Gregory E; Keller, Cheryl A; Wu, Weisheng; Morrissey, Christopher; Kumar, Swathi A; Mishra, Tejaswini; Jain, Deepti; Byrska-Bishop, Marta; Blankenberg, Daniel; Lajoie, Bryan R; Jain, Gaurav; Sanyal, Amartya; Chen, Kaun-Bei; Denas, Olgert; Taylor, James; Blobel, Gerd A; Weiss, Mitchell J; Pimkin, Max; Deng, Wulan; Marinov, Georgi K; Williams, Brian A; Fisher-Aylor, Katherine I; Desalvo, Gilberto; Kiralusha, Anthony; Trout, Diane; Amrhein, Henry; Mortazavi, Ali; Edsall, Lee; McCleary, David; Kuan, Samantha; Shen, Yin; Yue, Feng; Ye, Zhen; Davis, Carrie A; Zaleski, Chris; Jha, Sonali; Xue, Chenghai; Dobin, Alex; Lin, Wei; Fastuca, Meagan; Wang, Huaien; Guigo, Roderic; Djebali, Sarah; Lagarde, Julien; Ryba, Tyrone; Sasaki, Takayo; Malladi, Venkat S; Cline, Melissa S; Kirkup, Vanessa M; Learned, Katrina; Rosenbloom, Kate R; Kent, W James; Feingold, Elise A; Good, Peter J; Pazin, Michael; Lowdon, Rebecca F; Adams, Leslie B

    2012-08-13

    To complement the human Encyclopedia of DNA Elements (ENCODE) project and to enable a broad range of mouse genomics efforts, the Mouse ENCODE Consortium is applying the same experimental pipelines developed for human ENCODE to annotate the mouse genome.

  19. The ENCODE Project at UC Santa Cruz.

    PubMed

    Thomas, Daryl J; Rosenbloom, Kate R; Clawson, Hiram; Hinrichs, Angie S; Trumbower, Heather; Raney, Brian J; Karolchik, Donna; Barber, Galt P; Harte, Rachel A; Hillman-Jackson, Jennifer; Kuhn, Robert M; Rhead, Brooke L; Smith, Kayla E; Thakkapallayil, Archana; Zweig, Ann S; Haussler, David; Kent, W James

    2007-01-01

    The goal of the Encyclopedia Of DNA Elements (ENCODE) Project is to identify all functional elements in the human genome. The pilot phase is for comparison of existing methods and for the development of new methods to rigorously analyze a defined 1% of the human genome sequence. Experimental datasets are focused on the origin of replication, DNase I hypersensitivity, chromatin immunoprecipitation, promoter function, gene structure, pseudogenes, non-protein-coding RNAs, transcribed RNAs, multiple sequence alignment and evolutionarily constrained elements. The ENCODE project at UCSC website (http://genome.ucsc.edu/ENCODE) is the primary portal for the sequence-based data produced as part of the ENCODE project. In the pilot phase of the project, over 30 labs provided experimental results for a total of 56 browser tracks supported by 385 database tables. The site provides researchers with a number of tools that allow them to visualize and analyze the data as well as download data for local analyses. This paper describes the portal to the data, highlights the data that has been made available, and presents the tools that have been developed within the ENCODE project. Access to the data and types of interactive analysis that are possible are illustrated through supplemental examples.

  20. An Information Theoretic Characterisation of Auditory Encoding

    PubMed Central

    Overath, Tobias; Cusack, Rhodri; Kumar, Sukhbinder; von Kriegstein, Katharina; Warren, Jason D; Grube, Manon; Carlyon, Robert P; Griffiths, Timothy D

    2007-01-01

    The entropy metric derived from information theory provides a means to quantify the amount of information transmitted in acoustic streams like speech or music. By systematically varying the entropy of pitch sequences, we sought brain areas where neural activity and energetic demands increase as a function of entropy. Such a relationship is predicted to occur in an efficient encoding mechanism that uses less computational resource when less information is present in the signal: we specifically tested the hypothesis that such a relationship is present in the planum temporale (PT). In two convergent functional MRI studies, we demonstrated this relationship in PT for encoding, while furthermore showing that a distributed fronto-parietal network for retrieval of acoustic information is independent of entropy. The results establish PT as an efficient neural engine that demands less computational resource to encode redundant signals than those with high information content. PMID:17958472

  1. Quantum repeater with continuous variable encoding

    NASA Astrophysics Data System (ADS)

    Li, Linshu; Albert, Victor V.; Michael, Marios; Muralidharan, Sreraman; Zou, Changling; Jiang, Liang

    2016-05-01

    Quantum communication enables faithful quantum state transfer between different parties and protocols for cryptographic purposes. However, quantum communication over long distances (>1000km) remains challenging due to optical channel attenuation. This calls for investigation on developing novel encoding schemes that correct photon loss errors efficiently. In this talk, we introduce the generalization of multi-component Schrödinger cat states and propose to encode quantum information in these cat states for ultrafast quantum repeaters. We detail the quantum error correction procedures at each repeater station and characterize the performance of this novel encoding scheme given practical imperfections, such as coupling loss. A comparison with other quantum error correcting codes for bosonic modes will be discussed.

  2. Noise level and MPEG-2 encoder statistics

    NASA Astrophysics Data System (ADS)

    Lee, Jungwoo

    1997-01-01

    Most software in the movie and broadcasting industries are still in analog film or tape format, which typically contains random noise that originated from film, CCD camera, and tape recording. The performance of the MPEG-2 encoder may be significantly degraded by the noise. It is also affected by the scene type that includes spatial and temporal activity. The statistical property of noise originating from camera and tape player is analyzed and the models for the two types of noise are developed. The relationship between the noise, the scene type, and encoder statistics of a number of MPEG-2 parameters such as motion vector magnitude, prediction error, and quant scale are discussed. This analysis is intended to be a tool for designing robust MPEG encoding algorithms such as preprocessing and rate control.

  3. Fatal attraction: cytomegalovirus-encoded chemokine homologs.

    PubMed

    Saederup, N; Mocarski, E S

    2002-01-01

    Members of the cytomegalovirus (CMV) subfamily of betaherpesviruses infecting primates and rodents encode divergent proteins with sequence characteristics and activities of chemokines, a class of small, secreted proteins that control leukocyte migration and trafficking behavior. Human CMV genes UL146 and UL147 encode proteins with sequence characteristics of CXC chemokines, whereas, murine CMV encodes a CC chemokine homolog (MCK-2). Human CMV UL146 encodes a neutrophil-attracting chemokine denoted viral CXC chemokine-1 (vCXCL1) that is as potent as host IL-8 and functions via the CXCR2 receptor, one of two human IL-8 receptors. Murine CMV MCK-2 is composed of a chemokine domain derived from open reading frame (ORF) m131 (and denoted MCK-1) as well as a domain derived from m129 that does not have sequence similarity to any known class of proteins. A synthetic version of murine CMV m131 (MCK-1) protein carries out many of the activities of a positive-acting chemokine, including transient release of intracellular calcium stores and cell adhesion of peritoneal macrophage populations. In the context of the viral genome and infection of the mouse host, the m131-m129 (MCK-2) gene product confers increased inflammation, higher levels of viremia, and higher titers of virus in salivary glands, consistent with a role in promoting dissemination by attracting an important mononuclear leukocyte population. Other characterized primate CMVs, but not other primate betaherpesviruses, encode gene products similar to human UL146 and UL147. Other characterized rodent CMVs encode a gene product similar to the murine CMV chemokine homolog, although not as a spliced gene product. Thus chemokines, like viral proteins that downmodulate MHC class I expression or have sequence homology to host MHC class I proteins, have evolved in primate and rodent CMVs to carry out an analogous set of immunomodulatory functions during infection of the host even though they arise from distinct origins.

  4. Vector Adaptive/Predictive Encoding Of Speech

    NASA Technical Reports Server (NTRS)

    Chen, Juin-Hwey; Gersho, Allen

    1989-01-01

    Vector adaptive/predictive technique for digital encoding of speech signals yields decoded speech of very good quality after transmission at coding rate of 9.6 kb/s and of reasonably good quality at 4.8 kb/s. Requires 3 to 4 million multiplications and additions per second. Combines advantages of adaptive/predictive coding, and code-excited linear prediction, yielding speech of high quality but requires 600 million multiplications and additions per second at encoding rate of 4.8 kb/s. Vector adaptive/predictive coding technique bridges gaps in performance and complexity between adaptive/predictive coding and code-excited linear prediction.

  5. Vector Adaptive/Predictive Encoding Of Speech

    NASA Technical Reports Server (NTRS)

    Chen, Juin-Hwey; Gersho, Allen

    1989-01-01

    Vector adaptive/predictive technique for digital encoding of speech signals yields decoded speech of very good quality after transmission at coding rate of 9.6 kb/s and of reasonably good quality at 4.8 kb/s. Requires 3 to 4 million multiplications and additions per second. Combines advantages of adaptive/predictive coding, and code-excited linear prediction, yielding speech of high quality but requires 600 million multiplications and additions per second at encoding rate of 4.8 kb/s. Vector adaptive/predictive coding technique bridges gaps in performance and complexity between adaptive/predictive coding and code-excited linear prediction.

  6. Matrix Encoding For Correction Of Errors

    NASA Technical Reports Server (NTRS)

    Dotson, Ronald S.

    1991-01-01

    Method of matrix encoding and associated decoding provides for correction of errors in digital data recorded on magnetic tape. Intended specifically for use with commercial control circuit board and associated software making it possible to use video cassette recorder as backup for hard-disk memory of personal computer.

  7. Retrieval during Learning Facilitates Subsequent Memory Encoding

    ERIC Educational Resources Information Center

    Pastotter, Bernhard; Schicker, Sabine; Niedernhuber, Julia; Bauml, Karl-Heinz T.

    2011-01-01

    In multiple-list learning, retrieval during learning has been suggested to improve recall of the single lists by enhancing list discrimination and, at test, reducing interference. Using electrophysiological, oscillatory measures of brain activity, we examined to what extent retrieval during learning facilitates list encoding. Subjects studied 5…

  8. Young Children's Automatic Encoding of Social Categories

    ERIC Educational Resources Information Center

    Weisman, Kara; Johnson, Marissa V.; Shutts, Kristin

    2015-01-01

    The present research investigated young children's automatic encoding of two social categories that are highly relevant to adults: gender and race. Three- to 6-year-old participants learned facts about unfamiliar target children who varied in either gender or race and were asked to remember which facts went with which targets. When participants…

  9. Encoding and Retrieval During Bimanual Rhythmic Coordination

    ERIC Educational Resources Information Center

    Shockley, Kevin; Turvey, Michael T.

    2005-01-01

    In 2 experiments, bimanual 1:1 rhythmic coordination was performed concurrently with encoding or retrieval of word lists. Effects of divided attention (DA) on coordination were indexed by changes in mean relative phase and recurrence measures of shared activity between the 2 limbs. Effects of DA on memory were indexed by deficits in recall…

  10. Recombinant DNA encoding a desulfurization biocatalyst

    DOEpatents

    Rambosek, John; Piddington, Chris S.; Kovacevich, Brian R.; Young, Kevin D.; Denome, Sylvia A.

    1994-01-01

    This invention relates to a recombinant DNA molecule containing a gene or genes which encode a biocatalyst capable of desulfurizing a fossil fuel which contains organic sulfur molecules. For example, the present invention encompasses a recombinant DNA molecule containing a gene or genes of a strain of Rhodococcus rhodochrous.

  11. An extraordinary retrotransposon family encoding dual endonucleases

    PubMed Central

    Kojima, Kenji K.; Fujiwara, Haruhiko

    2005-01-01

    Retrotransposons commonly encode a reverse transcriptase (RT), but other functional domains are variable. The acquisition of new domains is the dominant evolutionary force that brings structural variety to retrotransposons. Non-long-terminal-repeat (non-LTR) retrotransposons are classified into two groups by their structure. Early branched non-LTR retrotransposons encode a restriction-like endonuclease (RLE), and recently branched non-LTR retrotransposons encode an apurinic/apyrimidinic endonuclease-like endonuclease (APE). In this study, we report a novel non-LTR retrotransposon family Dualen, identified from the Chlamydomonas reinhardtii genome. Dualen encodes two endonucleases, RLE and APE, with RT, ribonuclease H, and cysteine protease. Phylogenetic analyses of the RT domains revealed that Dualen is positioned at the midpoint between the early-branched and the recently branched groups. In the APE tree, Dualen was branched earlier than the I group and the Jockey group. The ribonuclease H domains among the Dualen family and other non-LTR retrotransposons are monophyletic. Phylogenies of three domains revealed the monophyly of the Dualen family members. The domain structure and the phylogeny of each domain imply that Dualen is a retrotransposon conserving the domain structure just after the acquisition of APE. From these observations, we discuss the evolution of domain structure of non-LTR retrotransposons. PMID:16077010

  12. Retrieval during Learning Facilitates Subsequent Memory Encoding

    ERIC Educational Resources Information Center

    Pastotter, Bernhard; Schicker, Sabine; Niedernhuber, Julia; Bauml, Karl-Heinz T.

    2011-01-01

    In multiple-list learning, retrieval during learning has been suggested to improve recall of the single lists by enhancing list discrimination and, at test, reducing interference. Using electrophysiological, oscillatory measures of brain activity, we examined to what extent retrieval during learning facilitates list encoding. Subjects studied 5…

  13. Gene encoding acetyl-coenzyme A carboxylase

    DOEpatents

    Roessler, Paul G.; Ohlrogge, John B.

    1996-01-01

    A DNA encoding an acetyl-coenzyme A carboxylase (ACCase) from a photosynthetic organism and functional derivatives thereof which are resistant to inhibition from certain herbicides. This gene can be placed in organisms to increase their fatty acid content or to render them resistant to certain herbicides.

  14. Gene encoding acetyl-coenzyme A carboxylase

    SciTech Connect

    Roessler, P.G.; Ohlrogge, J.B.

    1996-09-24

    A DNA encoding an acetyl-coenzyme A carboxylase (ACCase) from a photosynthetic organism and functional derivatives are disclosed which are resistant to inhibition from certain herbicides. This gene can be placed in organisms to increase their fatty acid content or to render them resistant to certain herbicides. 5 figs.

  15. Encoding of Others' Beliefs without Overt Instruction

    ERIC Educational Resources Information Center

    Cohen, Adam S.; German, Tamsin C.

    2009-01-01

    Under what conditions do people automatically encode and track the mental states of others? A recent investigation showed that when subjects are instructed to track the location of an object but are not instructed to track a belief about that location in a non-verbal false-belief task, they respond more slowly to questions about an agent's belief,…

  16. Design Primer for Reed-Solomon Encoders

    NASA Technical Reports Server (NTRS)

    Perlman, M.; Lee, J. J.

    1985-01-01

    Design and operation of Reed-Solomon (RS) encoders discussed in document prepared as instruction manual for computer designers and others in dataprocessing field. Conventional and Berlekamp architectures compared. Engineers who equip computer memory chips with burst-error and dropout detection and correction find report especially useful.

  17. Design Primer for Reed-Solomon Encoders

    NASA Technical Reports Server (NTRS)

    Perlman, M.; Lee, J. J.

    1985-01-01

    Design and operation of Reed-Solomon (RS) encoders discussed in document prepared as instruction manual for computer designers and others in dataprocessing field. Conventional and Berlekamp architectures compared. Engineers who equip computer memory chips with burst-error and dropout detection and correction find report especially useful.

  18. Recombinant DNA encoding a desulfurization biocatalyst

    DOEpatents

    Rambosek, J.; Piddington, C.S.; Kovacevich, B.R.; Young, K.D.; Denome, S.A.

    1994-10-18

    This invention relates to a recombinant DNA molecule containing a gene or genes which encode a biocatalyst capable of desulfurizing a fossil fuel which contains organic sulfur molecules. For example, the present invention encompasses a recombinant DNA molecule containing a gene or genes of a strain of Rhodococcus rhodochrous. 13 figs.

  19. Gene encoding acetyl-coenzyme A carboxylase

    DOEpatents

    Roessler, P.G.; Ohlrogge, J.B.

    1996-09-24

    A DNA encoding an acetyl-coenzyme A carboxylase (ACCase) from a photosynthetic organism and functional derivatives are disclosed which are resistant to inhibition from certain herbicides. This gene can be placed in organisms to increase their fatty acid content or to render them resistant to certain herbicides. 5 figs.

  20. Encoders for block-circulant LDPC codes

    NASA Technical Reports Server (NTRS)

    Divsalar, Dariush (Inventor); Abbasfar, Aliazam (Inventor); Jones, Christopher R. (Inventor); Dolinar, Samuel J. (Inventor); Thorpe, Jeremy C. (Inventor); Andrews, Kenneth S. (Inventor); Yao, Kung (Inventor)

    2009-01-01

    Methods and apparatus to encode message input symbols in accordance with an accumulate-repeat-accumulate code with repetition three or four are disclosed. Block circulant matrices are used. A first method and apparatus make use of the block-circulant structure of the parity check matrix. A second method and apparatus use block-circulant generator matrices.

  1. Young Children's Automatic Encoding of Social Categories

    ERIC Educational Resources Information Center

    Weisman, Kara; Johnson, Marissa V.; Shutts, Kristin

    2015-01-01

    The present research investigated young children's automatic encoding of two social categories that are highly relevant to adults: gender and race. Three- to 6-year-old participants learned facts about unfamiliar target children who varied in either gender or race and were asked to remember which facts went with which targets. When participants…

  2. 47 CFR 11.32 - EAS Encoder.

    Code of Federal Regulations, 2013 CFR

    2013-10-01

    ... used for audio messages and at least one input port used for data messages. (3) Outputs. The encoder shall have at least one audio output port and at least one data output port. (4) Calibration. EAS... that complies with the following: (i) Tone Frequencies. The audio tones shall have fundamental...

  3. 47 CFR 11.32 - EAS Encoder.

    Code of Federal Regulations, 2014 CFR

    2014-10-01

    ... used for audio messages and at least one input port used for data messages. (3) Outputs. The encoder shall have at least one audio output port and at least one data output port. (4) Calibration. EAS... that complies with the following: (i) Tone Frequencies. The audio tones shall have fundamental...

  4. 47 CFR 11.32 - EAS Encoder.

    Code of Federal Regulations, 2012 CFR

    2012-10-01

    ... used for audio messages and at least one input port used for data messages. (3) Outputs. The encoder shall have at least one audio output port and at least one data output port. (4) Calibration. EAS... that complies with the following: (i) Tone Frequencies. The audio tones shall have fundamental...

  5. Comparative Analysis of Chromosome-Encoded Microcins

    PubMed Central

    Poey, María Eloisa; Azpiroz, María F.; Laviña, Magela

    2006-01-01

    Microcins are ribosomally synthesized peptide antibiotics that are produced by enterobacterial strains. Although the first studies concentrated on plasmid-encoded activities, in the last years three chromosome-encoded microcins have been described: H47, E492, and M. Here, a new microcin, I47, is presented as a fourth member of this group. Common features exhibited by chromosome-encoded microcins were searched for. The comparison of the genetic clusters responsible for microcin production revealed a preserved general scheme. The clusters essentially comprise a pair of activity-immunity genes which determine antibiotic specificity and a set of microcin maturation and secretion genes which are invariably present and whose protein products are highly homologous among the different producing strains. A strict functional relationship between the maturation and secretion pathways of microcins H47, I47, and E492 was demonstrated through genetic analyses, which included heterologous complementation assays. The peptide precursors of these microcins share a maturation process which implies the addition of a catecholate siderophore of the salmochelin type. Microcins thus acquire the ability to enter gram-negative cells through the catechol receptors. In addition, they employ a common mode of secretion to reach the external milieu by means of a type I export apparatus. The results presented herein lead us to propose that chromosome-encoded microcins constitute a defined subgroup of peptide antibiotics which are strictly related by their modes of synthesis, secretion, and uptake. PMID:16569859

  6. How Attention Modulates Encoding of Dynamic Stimuli

    PubMed Central

    Oren, Noga; Shapira-Lichter, Irit; Lerner, Yulia; Tarrasch, Ricardo; Hendler, Talma; Giladi, Nir; Ash, Elissa L.

    2016-01-01

    When encoding a real-life, continuous stimulus, the same neural circuits support processing and integration of prior as well as new incoming information. This ongoing interplay is modulated by attention, and is evident in regions such as the prefrontal cortex section of the task positive network (TPN), and in the posterior cingulate cortex (PCC), a hub of the default mode network (DMN). Yet the exact nature of such modulation is still unclear. To investigate this issue, we utilized an fMRI task that employed movies as the encoded stimuli and manipulated attentional load via an easy or hard secondary task that was performed simultaneously with encoding. Results showed increased intersubject correlation (inter-SC) levels when encoding movies in a condition of high, as compared to low attentional load. This was evident in bilateral ventrolateral and dorsomedial prefrontal cortices and the dorsal PCC (dPCC). These regions became more attuned to the combination of the movie and the secondary task as the attentional demand of the latter increased. Activation analyses revealed that at higher load the prefrontal TPN regions were more activated, whereas the dPCC was more deactivated. Attentional load also influenced connectivity within and between the networks. At high load the dPCC was anti-correlated to the prefrontal regions, which were more functionally coherent amongst themselves. Finally and critically, greater inter-SC in the dPCC at high load during encoding predicted lower memory strength when that information was retrieved. This association between inter-SC levels and memory strength suggest that as attentional demands increased, the dPCC was more attuned to the secondary task at the expense of the encoded stimulus, thus weakening memory for the encoded stimulus. Together, our findings show that attentional load modulated the function of core TPN and DMN regions. Furthermore, the observed relationship between memory strength and the modulation of the dPCC points

  7. JPEG 2000 Encoding with Perceptual Distortion Control

    NASA Technical Reports Server (NTRS)

    Watson, Andrew B.; Liu, Zhen; Karam, Lina J.

    2008-01-01

    An alternative approach has been devised for encoding image data in compliance with JPEG 2000, the most recent still-image data-compression standard of the Joint Photographic Experts Group. Heretofore, JPEG 2000 encoding has been implemented by several related schemes classified as rate-based distortion-minimization encoding. In each of these schemes, the end user specifies a desired bit rate and the encoding algorithm strives to attain that rate while minimizing a mean squared error (MSE). While rate-based distortion minimization is appropriate for transmitting data over a limited-bandwidth channel, it is not the best approach for applications in which the perceptual quality of reconstructed images is a major consideration. A better approach for such applications is the present alternative one, denoted perceptual distortion control, in which the encoding algorithm strives to compress data to the lowest bit rate that yields at least a specified level of perceptual image quality. Some additional background information on JPEG 2000 is prerequisite to a meaningful summary of JPEG encoding with perceptual distortion control. The JPEG 2000 encoding process includes two subprocesses known as tier-1 and tier-2 coding. In order to minimize the MSE for the desired bit rate, a rate-distortion- optimization subprocess is introduced between the tier-1 and tier-2 subprocesses. In tier-1 coding, each coding block is independently bit-plane coded from the most-significant-bit (MSB) plane to the least-significant-bit (LSB) plane, using three coding passes (except for the MSB plane, which is coded using only one "clean up" coding pass). For M bit planes, this subprocess involves a total number of (3M - 2) coding passes. An embedded bit stream is then generated for each coding block. Information on the reduction in distortion and the increase in the bit rate associated with each coding pass is collected. This information is then used in a rate-control procedure to determine the

  8. Encoding Orientation and the Remembering of Schizophrenic Young Adults

    ERIC Educational Resources Information Center

    Koh, Soon D.; Peterson, Rolf A.

    1978-01-01

    This research examines different types of encoding strategies, in addition to semantic and organizational encodings, and their effects on schizophrenics' remembering. Based on Craik and Lockhart (1972), i.e., memory performance is a function of depth of encoding processing, this analysis compares schizophrenics' encoding processing with that of…

  9. Amygdala neurons differentially encode motivation and reinforcement.

    PubMed

    Tye, Kay M; Janak, Patricia H

    2007-04-11

    Lesion studies demonstrate that the basolateral amygdala complex (BLA) is important for assigning motivational significance to sensory stimuli, but little is known about how this information is encoded. We used in vivo electrophysiology procedures to investigate how the amygdala encodes motivating and reinforcing properties of cues that induce reinstatement of reward-seeking behavior. Two groups of rats were trained to respond to a sucrose reward. The "paired" group was trained with a reward-predictive cue, whereas the "unpaired" group was trained with a randomly presented cue. Both groups underwent identical extinction and reinstatement procedures during which the reward was withheld. The proportion of neurons that were phasically cue responsive during reinstatement was significantly higher in the paired group (46 of 100) than in the unpaired group (8 of 112). Cues that induce reward-seeking behavior can do so by acting as incentives or reinforcers. Distinct populations of neurons responded to the cue in trials in which the cue acted as an incentive, triggering a motivated reward-seeking state, or as a reinforcer, supporting continued instrumental responding. The incentive motivation-encoding population of neurons (34 of 46 cue-responsive neurons; 74%) extinguished in temporal agreement with a decrease in the rate of instrumental responding. The conditioned reinforcement-encoding population of neurons (12 of 46 cue-responsive neurons; 26%) maintained their response for the duration of cue-reinforced instrumental responding. These data demonstrate that separate populations of cue-responsive neurons in the BLA encode the motivating or reinforcing properties of a cue previously associated with a reward.

  10. Effortful Retrieval Reduces Hippocampal Activity and Impairs Incidental Encoding

    PubMed Central

    Reas, Emilie T.; Brewer, James B.

    2014-01-01

    Functional imaging studies frequently report that the hippocampus is engaged by successful episodic memory retrieval. However, considering that concurrent encoding of the background environment occurs during retrieval and influences medial temporal lobe activity, it is plausible that hippocampal encoding functions are reduced with increased attentional engagement during effortful retrieval. Expanding upon evidence that retrieval efforts suppress activity in hippocampal regions implicated in encoding, this study examines the influence of retrieval effort on encoding performance and the interactive effects of encoding and retrieval on hippocampal and neocortical activity. Functional magnetic resonance imaging was conducted while subjects performed a word recognition task with incidental picture encoding. Both lower memory strength and increased search duration were associated with encoding failure and reduced hippocampal and default network activity. Activity in the anterior hippocampus tracked encoding, which was more strongly deactivated when incidental encoding was unsuccessful. These findings highlight potential contributions from background encoding processes to hippocampal activations during neuroimaging studies of episodic memory retrieval. PMID:23378272

  11. (E)-2(R)-[1(S)-(Hydroxycarbamoyl)-4-phenyl-3-butenyl]-2'-isobutyl-2'-(methanesulfonyl)-4-methylvalerohydrazide (Ro 32-7315), a selective and orally active inhibitor of tumor necrosis factor-alpha convertase.

    PubMed

    Beck, G; Bottomley, G; Bradshaw, D; Brewster, M; Broadhurst, M; Devos, R; Hill, C; Johnson, W; Kim, H-J; Kirtland, S; Kneer, J; Lad, N; Mackenzie, R; Martin, R; Nixon, J; Price, G; Rodwell, A; Rose, F; Tang, J-P; Walter, D S; Wilson, K; Worth, E

    2002-07-01

    Tumor necrosis factor-alpha (TNF-alpha), a cytokine secreted by inflammatory cells, has been implicated in several inflammatory disease states. (E)-2(R)-[1(S)-(Hydroxycarbamoyl)-4-phenyl-3-butenyl]-2'-isobutyl-2'-(methanesulfonyl)-4-methylvalerohydrazide (Ro 32-7315), is a potent, orally active inhibitor of the TNF-alpha convertase (TACE), an enzyme responsible for proteolytic cleavage of the membrane bound precursor, pro-TNF-alpha. Ro 32-7315 inhibited a recombinant form of TACE (IC(50) = 5.2 nM) with selectivity over related matrix metalloproteinases. In a cellular assay system, THP-1 cell line, and in human and rat whole blood, Ro 32-7315 significantly reduced lipopolysaccharide (LPS)-induced TNF-alpha release with IC(50) values of 350 +/- 14 nM (n = 5), 2.4 +/- 0.5 microM (n = 5), and 110 +/- 18 nM (n = 5), respectively. Oral administration of Ro 32-7315 to Wistar rats caused a dose-dependent inhibition of LPS-induced release of systemic TNF-alpha with an ED(50) of 25 mg/kg. Treatment (days 0-14) of Allen and Hamburys hooded rats with Ro 32-7315 (2.5, 5, 10, and 20 mg/kg, i.p., twice daily) significantly reduced adjuvant-induced secondary paw swelling (42, 71, 83, and 93%, respectively) as compared with the vehicle group. In the Ro 32-7315-treated group, the reduced paw swelling was associated with improved lesion score and joint mobility. Furthermore, in a placebo-controlled, single-dose study, Ro 32-7315 given orally (450 mg) significantly suppressed ex vivo, LPS-induced TNF-alpha release in the whole-blood samples taken from healthy male and female volunteers (mean inhibition of 42% over a 4-h duration, n = 6). These data collectively support the potential use of such a compound for the oral treatment of inflammatory disorders.

  12. Implement of FGS video encoding based on H.264

    NASA Astrophysics Data System (ADS)

    Lin, Qiwei; Feng, Gui

    2007-01-01

    In H.264 video coding standard, the combination encoding frame was adopted. It introduces some new algorithms, and modifies several aspects of the encoding scheme. So the encoding scheme improves the encoding efficiency obviously. But the H.264 standard is not supporting FGS encoding. So a H.264 based self-adaptive FGS (Fine Granular Scalable)(H.264-FGS) encoding scheme is proposed in this paper. In this encoding scheme, the base layer of encoder is keeping H.264 encoder architecture, which consists of the motion estimation, motion compensation, intra predictive, integer transformation, loop filtering, content based arithmetic encoding, and etc. In the base layer generated block we obtain base code flux of FGS. Subtracting the original image from the reconstruction image of the base layer, we get the residual error. Then after the DCT transform and the variable length encoding compresses, we obtain the enhanced code flux of FGS. Compared with the original MPEG-4 FGS encoding scheme, the proposed FGS encoding scheme has the feature of increasing encoding efficiency by 1~3 dB and keep the all properties that MPEG-4 FGS encoding technology provided.

  13. Genetically Encoded Voltage Indicators in Circulation Research

    PubMed Central

    Kaestner, Lars; Tian, Qinghai; Kaiser, Elisabeth; Xian, Wenying; Müller, Andreas; Oberhofer, Martin; Ruppenthal, Sandra; Sinnecker, Daniel; Tsutsui, Hidekazu; Miyawaki, Atsushi; Moretti, Alessandra; Lipp, Peter

    2015-01-01

    Membrane potentials display the cellular status of non-excitable cells and mediate communication between excitable cells via action potentials. The use of genetically encoded biosensors employing fluorescent proteins allows a non-invasive biocompatible way to read out the membrane potential in cardiac myocytes and other cells of the circulation system. Although the approaches to design such biosensors date back to the time when the first fluorescent-protein based Förster Resonance Energy Transfer (FRET) sensors were constructed, it took 15 years before reliable sensors became readily available. Here, we review different developments of genetically encoded membrane potential sensors. Furthermore, it is shown how such sensors can be used in pharmacological screening applications as well as in circulation related basic biomedical research. Potentials and limitations will be discussed and perspectives of possible future developments will be provided. PMID:26370981

  14. Asymmetric synthesis using chiral-encoded metal

    NASA Astrophysics Data System (ADS)

    Yutthalekha, Thittaya; Wattanakit, Chularat; Lapeyre, Veronique; Nokbin, Somkiat; Warakulwit, Chompunuch; Limtrakul, Jumras; Kuhn, Alexander

    2016-08-01

    The synthesis of chiral compounds is of crucial importance in many areas of society and science, including medicine, biology, chemistry, biotechnology and agriculture. Thus, there is a fundamental interest in developing new approaches for the selective production of enantiomers. Here we report the use of mesoporous metal structures with encoded geometric chiral information for inducing asymmetry in the electrochemical synthesis of mandelic acid as a model molecule. The chiral-encoded mesoporous metal, obtained by the electrochemical reduction of platinum salts in the presence of a liquid crystal phase and the chiral template molecule, perfectly retains the chiral information after removal of the template. Starting from a prochiral compound we demonstrate enantiomeric excess of the (R)-enantiomer when using (R)-imprinted electrodes and vice versa for the (S)-imprinted ones. Moreover, changing the amount of chiral cavities in the material allows tuning the enantioselectivity.

  15. Asymmetric synthesis using chiral-encoded metal

    PubMed Central

    Yutthalekha, Thittaya; Wattanakit, Chularat; Lapeyre, Veronique; Nokbin, Somkiat; Warakulwit, Chompunuch; Limtrakul, Jumras; Kuhn, Alexander

    2016-01-01

    The synthesis of chiral compounds is of crucial importance in many areas of society and science, including medicine, biology, chemistry, biotechnology and agriculture. Thus, there is a fundamental interest in developing new approaches for the selective production of enantiomers. Here we report the use of mesoporous metal structures with encoded geometric chiral information for inducing asymmetry in the electrochemical synthesis of mandelic acid as a model molecule. The chiral-encoded mesoporous metal, obtained by the electrochemical reduction of platinum salts in the presence of a liquid crystal phase and the chiral template molecule, perfectly retains the chiral information after removal of the template. Starting from a prochiral compound we demonstrate enantiomeric excess of the (R)-enantiomer when using (R)-imprinted electrodes and vice versa for the (S)-imprinted ones. Moreover, changing the amount of chiral cavities in the material allows tuning the enantioselectivity. PMID:27562028

  16. DNA-Encoded Dynamic Combinatorial Chemical Libraries.

    PubMed

    Reddavide, Francesco V; Lin, Weilin; Lehnert, Sarah; Zhang, Yixin

    2015-06-26

    Dynamic combinatorial chemistry (DCC) explores the thermodynamic equilibrium of reversible reactions. Its application in the discovery of protein binders is largely limited by difficulties in the analysis of complex reaction mixtures. DNA-encoded chemical library (DECL) technology allows the selection of binders from a mixture of up to billions of different compounds; however, experimental results often show low a signal-to-noise ratio and poor correlation between enrichment factor and binding affinity. Herein we describe the design and application of DNA-encoded dynamic combinatorial chemical libraries (EDCCLs). Our experiments have shown that the EDCCL approach can be used not only to convert monovalent binders into high-affinity bivalent binders, but also to cause remarkably enhanced enrichment of potent bivalent binders by driving their in situ synthesis. We also demonstrate the application of EDCCLs in DNA-templated chemical reactions.

  17. Storing data encoded DNA in living organisms

    DOEpatents

    Wong; Pak C. , Wong; Kwong K. , Foote; Harlan P.

    2006-06-06

    Current technologies allow the generation of artificial DNA molecules and/or the ability to alter the DNA sequences of existing DNA molecules. With a careful coding scheme and arrangement, it is possible to encode important information as an artificial DNA strand and store it in a living host safely and permanently. This inventive technology can be used to identify origins and protect R&D investments. It can also be used in environmental research to track generations of organisms and observe the ecological impact of pollutants. Today, there are microorganisms that can survive under extreme conditions. As well, it is advantageous to consider multicellular organisms as hosts for stored information. These living organisms can provide as memory housing and protection for stored data or information. The present invention provides well for data storage in a living organism wherein at least one DNA sequence is encoded to represent data and incorporated into a living organism.

  18. Temporal information encoding in dynamic memristive devices

    SciTech Connect

    Ma, Wen; Chen, Lin; Du, Chao; Lu, Wei D.

    2015-11-09

    We show temporal and frequency information can be effectively encoded in memristive devices with inherent short-term dynamics. Ag/Ag{sub 2}S/Pd based memristive devices with low programming voltage (∼100 mV) were fabricated and tested. At weak programming conditions, the devices exhibit inherent decay due to spontaneous diffusion of the Ag atoms. When the devices were subjected to pulse train inputs emulating different spiking patterns, the switching probability distribution function diverges from the standard Poisson distribution and evolves according to the input pattern. The experimentally observed switching probability distributions and the associated cumulative probability functions can be well-explained using a model accounting for the short-term decay effects. Such devices offer an intriguing opportunity to directly encode neural signals for neural information storage and analysis.

  19. Nucleic acid compositions and the encoding proteins

    DOEpatents

    Preston, III, James F.; Chow, Virginia; Nong, Guang; Rice, John D.; St. John, Franz J.

    2014-09-02

    The subject invention provides at least one nucleic acid sequence encoding an aldouronate-utilization regulon isolated from Paenibacillus sp. strain JDR-2, a bacterium which efficiently utilizes xylan and metabolizes aldouronates (methylglucuronoxylosaccharides). The subject invention also provides a means for providing a coordinately regulated process in which xylan depolymerization and product assimilation are coupled in Paenibacillus sp. strain JDR-2 to provide a favorable system for the conversion of lignocellulosic biomass to biobased products. Additionally, the nucleic acid sequences encoding the aldouronate-utilization regulon can be used to transform other bacteria to form organisms capable of producing a desired product (e.g., ethanol, 1-butanol, acetoin, 2,3-butanediol, 1,3-propanediol, succinate, lactate, acetate, malate or alanine) from lignocellulosic biomass.

  20. Determination of Laser Tracker Angle Encoder Errors

    NASA Astrophysics Data System (ADS)

    Nasr, Karim M.; Hughes, Ben; Forbes, Alistair; Lewis, Andrew

    2014-08-01

    Errors in the angle encoders of a laser tracker may potentially produce large errors in long range coordinate measurements. To determine the azimuth angle encoder errors and verify their values stored in the tracker's internal error map, several methodologies were evaluated, differing in complexity, measurement time and the need for specialised measuring equipment. These methodologies are: an artefact-based technique developed by NIST; a multi-target network technique developed by NPL; and the classical precision angular indexing table technique. It is shown that the three methodologies agree within their respective measurement uncertainties and that the NPL technique has the advantages of a short measurement time and no reliance on specialised measurement equipment or artefacts.

  1. Encoded Dynamical Recoupling with Shaped Pulses

    NASA Astrophysics Data System (ADS)

    Li, Yunfan; Lidar, Daniel A.; Pryadko, Leonid P.

    2008-03-01

    Encoded Dynamical Recoupling is a passive error correction techique which can be used to enhance the performance of a quantum error correction code (QECC) against low-frequency component of the thermal bath. The elements of the stabilizer group are used in the decoupling cycle which makes the encoded logic operations fault-tolerant. We studied the effectiveness of this techique both analytically and numerically for several three- and five-qubit codes, with decoupling sequences utilizing either Gaussian or self-refocusing pulse shapes. When logic pulses are intercalated between the decoupling cycles, the technique may be very effective in cancelling constant perturbation terms, but its performance is much weaker against a time-dependent perturbation simulated as a classical correlated noise. The decoupling accuracy can be substantially improved if logic is applied slowly and concurrently with the decoupling, so that a certain adiabaticity condition is satisfied.

  2. Polarization encoding for optical security systems

    NASA Astrophysics Data System (ADS)

    Javidi, Bahram; Nomura, Takanori

    1999-10-01

    An idea for security verification of identification such as passports, credit cards, and others in terms of difficulty of reproduction is proposed. The polarization information is used for security verification. As the state of the polarization encoded cannot be seen by an ordinary intensity sensitive device such as a CCD camera, it cannot be copied. For optical validation system, a joint transform correlator is used. Computer simulations and optical experimental results are shown to confirm our proposed method.

  3. Gene encoding herbicide safener binding protein

    SciTech Connect

    Walton, J.D.; Scott-Craig, J.S.

    1999-10-26

    The cDNA encoding safener binding protein (SafBP), also referred to as SBP1, is presented. The deduced amino acid sequence is provided. Methods of making and using SBP1 and SafBP to alter a plant's sensitivity to certain herbicides or a plant's responsiveness to certain safeners are also provided, as well as expression vectors, transgenic plants or other organisms transfected with vectors and seeds from the plants.

  4. Population Encoding With Hodgkin–Huxley Neurons

    PubMed Central

    Lazar, Aurel A.

    2013-01-01

    The recovery of (weak) stimuli encoded with a population of Hodgkin–Huxley neurons is investigated. In the absence of a stimulus, the Hodgkin–Huxley neurons are assumed to be tonically spiking. The methodology employed calls for 1) finding an input–output (I/O) equivalent description of the Hodgkin–Huxley neuron and 2) devising a recovery algorithm for stimuli encoded with the I/O equivalent neuron(s). A Hodgkin–Huxley neuron with multiplicative coupling is I/O equivalent with an Integrate-and-Fire neuron with a variable threshold sequence. For bandlimited stimuli a perfect recovery of the stimulus can be achieved provided that a Nyquist-type rate condition is satisfied. A Hodgkin–Huxley neuron with additive coupling and deterministic conductances is first-order I/O equivalent with a Project-Integrate-and-Fire neuron that integrates a projection of the stimulus on the phase response curve. The stimulus recovery is formulated as a spline interpolation problem in the space of finite length bounded energy signals. A Hodgkin–Huxley neuron with additive coupling and stochastic conductances is shown to be first-order I/O equivalent with a Project-Integrate-and-Fire neuron with random thresholds. For stimuli modeled as elements of Sobolev spaces the reconstruction algorithm minimizes a regularized quadratic optimality criterion. Finally, all previous recovery results of stimuli encoded with Hodgkin–Huxley neurons with multiplicative and additive coupling, and deterministic and stochastic conductances are extended to stimuli encoded with a population of Hodgkin–Huxley neurons. PMID:24194625

  5. Population Encoding With Hodgkin-Huxley Neurons.

    PubMed

    Lazar, Aurel A

    2010-02-01

    The recovery of (weak) stimuli encoded with a population of Hodgkin-Huxley neurons is investigated. In the absence of a stimulus, the Hodgkin-Huxley neurons are assumed to be tonically spiking. The methodology employed calls for 1) finding an input-output (I/O) equivalent description of the Hodgkin-Huxley neuron and 2) devising a recovery algorithm for stimuli encoded with the I/O equivalent neuron(s). A Hodgkin-Huxley neuron with multiplicative coupling is I/O equivalent with an Integrate-and-Fire neuron with a variable threshold sequence. For bandlimited stimuli a perfect recovery of the stimulus can be achieved provided that a Nyquist-type rate condition is satisfied. A Hodgkin-Huxley neuron with additive coupling and deterministic conductances is first-order I/O equivalent with a Project-Integrate-and-Fire neuron that integrates a projection of the stimulus on the phase response curve. The stimulus recovery is formulated as a spline interpolation problem in the space of finite length bounded energy signals. A Hodgkin-Huxley neuron with additive coupling and stochastic conductances is shown to be first-order I/O equivalent with a Project-Integrate-and-Fire neuron with random thresholds. For stimuli modeled as elements of Sobolev spaces the reconstruction algorithm minimizes a regularized quadratic optimality criterion. Finally, all previous recovery results of stimuli encoded with Hodgkin-Huxley neurons with multiplicative and additive coupling, and deterministic and stochastic conductances are extended to stimuli encoded with a population of Hodgkin-Huxley neurons.

  6. Absolute Position Encoders With Vertical Image Binning

    NASA Technical Reports Server (NTRS)

    Leviton, Douglas B.

    2005-01-01

    Improved optoelectronic patternrecognition encoders that measure rotary and linear 1-dimensional positions at conversion rates (numbers of readings per unit time) exceeding 20 kHz have been invented. Heretofore, optoelectronic pattern-recognition absoluteposition encoders have been limited to conversion rates <15 Hz -- too low for emerging industrial applications in which conversion rates ranging from 1 kHz to as much as 100 kHz are required. The high conversion rates of the improved encoders are made possible, in part, by use of vertically compressible or binnable (as described below) scale patterns in combination with modified readout sequences of the image sensors [charge-coupled devices (CCDs)] used to read the scale patterns. The modified readout sequences and the processing of the images thus read out are amenable to implementation by use of modern, high-speed, ultra-compact microprocessors and digital signal processors or field-programmable gate arrays. This combination of improvements makes it possible to greatly increase conversion rates through substantial reductions in all three components of conversion time: exposure time, image-readout time, and image-processing time.

  7. Genetically Encoded Voltage Indicators: Opportunities and Challenges.

    PubMed

    Yang, Helen H; St-Pierre, François

    2016-09-28

    A longstanding goal in neuroscience is to understand how spatiotemporal patterns of neuronal electrical activity underlie brain function, from sensory representations to decision making. An emerging technology for monitoring electrical dynamics, voltage imaging using genetically encoded voltage indicators (GEVIs), couples the power of genetics with the advantages of light. Here, we review the properties that determine indicator performance and applicability, discussing both recent progress and technical limitations. We then consider GEVI applications, highlighting studies that have already deployed GEVIs for biological discovery. We also examine which classes of biological questions GEVIs are primed to address and which ones are beyond their current capabilities. As GEVIs are further developed, we anticipate that they will become more broadly used by the neuroscience community to eavesdrop on brain activity with unprecedented spatiotemporal resolution. Genetically encoded voltage indicators are engineered light-emitting protein sensors that typically report neuronal voltage dynamics as changes in brightness. In this review, we systematically discuss the current state of this emerging method, considering both its advantages and limitations for imaging neural activity. We also present recent applications of this technology and discuss what is feasible now and what we anticipate will become possible with future indicator development. This review will inform neuroscientists of recent progress in the field and help potential users critically evaluate the suitability of genetically encoded voltage indicator imaging to answer their specific biological questions. Copyright © 2016 the authors 0270-6474/16/369977-13$15.00/0.

  8. Task unrelated thought whilst encoding information.

    PubMed

    Smallwood, Jonathan M; Baracaia, Simona F; Lowe, Michelle; Obonsawin, Marc

    2003-09-01

    Task unrelated thought (TUT) refers to thought directed away from the current situation, for example a daydream. Three experiments were conducted on healthy participants, with two broad aims. First, to contrast distributed and encapsulated views of cognition by comparing the encoding of categorical and random lists of words (Experiments One and Two). Second, to examine the consequences of experiencing TUT during study on the subsequent retrieval of information (Experiments One, Two, and Three). Experiments One and Two demonstrated lower levels of TUT and higher levels of word-fragment completion whilst encoding categorical relative to random stimuli, supporting the role of a distributed resource in the maintenance of TUT. In addition the results of all three experiments suggested that experiencing TUT during study had a measurable effect on subsequent retrieval. TUT was associated with increased frequency of false alarms at retrieval (Experiment One). In the subsequent experiments TUT was associated with no advantage to retrieval based on recollection, by manipulating instructions at encoding (Experiment Two), and/or at retrieval (Experiment Three). The implications of the results of all three experiments are discussed in terms of recent accounts of memory retrieval and conscious awareness.

  9. Nucleic acids encoding human trithorax protein

    DOEpatents

    Evans, Glen A.; Djabali, Malek; Selleri, Licia; Parry, Pauline

    2001-01-01

    In accordance with the present invention, there is provided an isolated peptide having the characteristics of human trithorax protein (as well as DNA encoding same, antisense DNA derived therefrom and antagonists therefor). The invention peptide is characterized by having a DNA binding domain comprising multiple zinc fingers and at least 40% amino acid identity with respect to the DNA binding domain of Drosophila trithorax protein and at least 70% conserved sequence with respect to the DNA binding domain of Drosophila trithorax protein, and wherein said peptide is encoded by a gene located at chromosome 11 of the human genome at q23. Also provided are methods for the treatment of subject(s) suffering from immunodeficiency, developmental abnormality, inherited disease, or cancer by administering to said subject a therapeutically effective amount of one of the above-described agents (i.e., peptide, antagonist therefor, DNA encoding said peptide or antisense DNA derived therefrom). Also provided is a method for the diagnosis, in a subject, of immunodeficiency, developmental abnormality, inherited disease, or cancer associated with disruption of chromosome 11 at q23.

  10. Virus-encoded microRNAs

    PubMed Central

    Grundhoff, Adam; Sullivan, Christopher S.

    2011-01-01

    microRNAs (miRNAs) are the subject of enormous interest. They are small non-coding RNAs that play a regulatory role in numerous and diverse cellular processes such as immune function, apoptosis and tumorigenesis. Several virus families have been shown to encode miRNAs, and an appreciation for their roles in the viral infectious cycle continues to grow. Despite the identification of numerous (>225) viral miRNAs, an in depth functional understanding of most virus-encoded miRNAs is lacking. Here we focus on a few viral miRNAs with well-defined functions. We use these examples to extrapolate general themes of viral miRNA activities including autoregulation of gene expression, avoidance of host defenses, and a likely important role in maintaining latent and persistent infections. We hypothesize that although the molecular mechanisms and machinery are similar, the majority of viral miRNAs may utilize a target strategy that differs from host miRNAs. That is, many viral miRNAs may have evolved to regulate viral-encoded transcripts or networks of host genes that are unique to viral miRNAs. Included in this latter category are a likely abundant class of viral miRNAs that may regulate only one or a few principal host genes. Key steps forward for the field are discussed, including the need for additional functional studies that utilize surgical viral miRNA mutants combined with relevant models of infection. PMID:21277611

  11. Plasmid-Encoded Iron Uptake Systems.

    PubMed

    Di Lorenzo, Manuela; Stork, Michiel

    2014-12-01

    Plasmids confer genetic information that benefits the bacterial cells containing them. In pathogenic bacteria, plasmids often harbor virulence determinants that enhance the pathogenicity of the bacterium. The ability to acquire iron in environments where it is limited, for instance the eukaryotic host, is a critical factor for bacterial growth. To acquire iron, bacteria have evolved specific iron uptake mechanisms. These systems are often chromosomally encoded, while those that are plasmid-encoded are rare. Two main plasmid types, ColV and pJM1, have been shown to harbor determinants that increase virulence by providing the cell with essential iron for growth. It is clear that these two plasmid groups evolved independently from each other since they do not share similarities either in the plasmid backbones or in the iron uptake systems they harbor. The siderophores aerobactin and salmochelin that are found on ColV plasmids fall in the hydroxamate and catechol group, respectively, whereas both functional groups are present in the anguibactin siderophore, the only iron uptake system found on pJM1-type plasmids. Besides siderophore-mediated iron uptake, ColV plasmids carry additional genes involved in iron metabolism. These systems include ABC transporters, hemolysins, and a hemoglobin protease. ColV- and pJM1-like plasmids have been shown to confer virulence to their bacterial host, and this trait can be completely ascribed to their encoded iron uptake systems.

  12. Dual-channel spectrally encoded endoscopic probe.

    PubMed

    Engel, Guy; Genish, Hadar; Rosenbluh, Michael; Yelin, Dvir

    2012-08-01

    High quality imaging through sub-millimeter endoscopic probes provides clinicians with valuable diagnostics capabilities in hard to reach locations within the body. Spectrally encoded endoscopy (SEE) has been shown promising for such task; however, challenging probe fabrication and high speckle noise had prevented its testing in in vivo studies. Here we demonstrate a novel miniature SEE probe which incorporates some of the recent progress in spectrally encoded technology into a compact and robust endoscopic system. A high-quality miniature diffraction grating was fabricated using automated femtosecond laser cutting from a large bulk grating. Using one spectrally encoded channel for imaging and a separate channel for incoherent illumination, the new system has large depth of field, negligible back reflections and well controlled speckle noise which depends on the core diameter of the illumination fiber. Moreover, by using a larger imaging channel, higher groove density grating, shorter wavelength and broader spectrum, the new endoscopic system now allow significant improvements in almost all imaging parameter compared to previous systems, through an ultra-miniature endoscopic probe.

  13. Genetically Encoded Voltage Indicators: Opportunities and Challenges

    PubMed Central

    Yang, Helen H.

    2016-01-01

    A longstanding goal in neuroscience is to understand how spatiotemporal patterns of neuronal electrical activity underlie brain function, from sensory representations to decision making. An emerging technology for monitoring electrical dynamics, voltage imaging using genetically encoded voltage indicators (GEVIs), couples the power of genetics with the advantages of light. Here, we review the properties that determine indicator performance and applicability, discussing both recent progress and technical limitations. We then consider GEVI applications, highlighting studies that have already deployed GEVIs for biological discovery. We also examine which classes of biological questions GEVIs are primed to address and which ones are beyond their current capabilities. As GEVIs are further developed, we anticipate that they will become more broadly used by the neuroscience community to eavesdrop on brain activity with unprecedented spatiotemporal resolution. SIGNIFICANCE STATEMENT Genetically encoded voltage indicators are engineered light-emitting protein sensors that typically report neuronal voltage dynamics as changes in brightness. In this review, we systematically discuss the current state of this emerging method, considering both its advantages and limitations for imaging neural activity. We also present recent applications of this technology and discuss what is feasible now and what we anticipate will become possible with future indicator development. This review will inform neuroscientists of recent progress in the field and help potential users critically evaluate the suitability of genetically encoded voltage indicator imaging to answer their specific biological questions. PMID:27683896

  14. Neural signals encoding shifts in beliefs

    PubMed Central

    Schwartenbeck, Philipp; FitzGerald, Thomas H.B.; Dolan, Ray

    2016-01-01

    Dopamine is implicated in a diverse range of cognitive functions including cognitive flexibility, task switching, signalling novel or unexpected stimuli as well as advance information. There is also longstanding line of thought that links dopamine with belief formation and, crucially, aberrant belief formation in psychosis. Integrating these strands of evidence would suggest that dopamine plays a central role in belief updating and more specifically in encoding of meaningful information content in observations. The precise nature of this relationship has remained unclear. To directly address this question we developed a paradigm that allowed us to decompose two distinct types of information content, information-theoretic surprise that reflects the unexpectedness of an observation, and epistemic value that induces shifts in beliefs or, more formally, Bayesian surprise. Using functional magnetic-resonance imaging in humans we show that dopamine-rich midbrain regions encode shifts in beliefs whereas surprise is encoded in prefrontal regions, including the pre-supplementary motor area and dorsal cingulate cortex. By linking putative dopaminergic activity to belief updating these data provide a link to false belief formation that characterises hyperdopaminergic states associated with idiopathic and drug induced psychosis. PMID:26520774

  15. Complementary attentional components of successful memory encoding

    PubMed Central

    Turk-Browne, Nicholas B.; Golomb, Julie D.; Chun, Marvin M.

    2012-01-01

    Attention during encoding improves later memory, but how this happens is poorly understood. To investigate the role of attention in memory formation, we combined a variant of a spatial attention cuing task with a subsequent memory fMRI design. Scene stimuli were presented in the periphery to either the left or right of fixation, preceded by a central face cue whose gaze oriented attention to the probable location of the scene. We contrasted activity for scenes appearing in cued versus uncued locations to identify: (1) regions where cuing facilitated processing, and (2) regions involved in reorienting. We then tested how activity in these facilitation and reorienting regions of interest predicted subsequent long-term memory for individual scenes. In facilitation regions such as parahippocampal cortex, greater activity during encoding predicted memory success. In reorienting regions such as right temporoparietal junction, greater activity during encoding predicted memory failure. We interpret these results as evidence that memory formation benefits from attentional facilitation of perceptual processing combined with suppression of the ventral attention network to prevent reorienting to distractors. PMID:23108276

  16. Dual-channel spectrally encoded endoscopic probe

    PubMed Central

    Engel, Guy; Genish, Hadar; Rosenbluh, Michael; Yelin, Dvir

    2012-01-01

    High quality imaging through sub-millimeter endoscopic probes provides clinicians with valuable diagnostics capabilities in hard to reach locations within the body. Spectrally encoded endoscopy (SEE) has been shown promising for such task; however, challenging probe fabrication and high speckle noise had prevented its testing in in vivo studies. Here we demonstrate a novel miniature SEE probe which incorporates some of the recent progress in spectrally encoded technology into a compact and robust endoscopic system. A high-quality miniature diffraction grating was fabricated using automated femtosecond laser cutting from a large bulk grating. Using one spectrally encoded channel for imaging and a separate channel for incoherent illumination, the new system has large depth of field, negligible back reflections and well controlled speckle noise which depends on the core diameter of the illumination fiber. Moreover, by using a larger imaging channel, higher groove density grating, shorter wavelength and broader spectrum, the new endoscopic system now allow significant improvements in almost all imaging parameter compared to previous systems, through an ultra-miniature endoscopic probe. PMID:22876349

  17. Encoding and decoding messages with chaotic lasers

    SciTech Connect

    Alsing, P.M.; Gavrielides, A.; Kovanis, V.; Roy, R.; Thornburg, K.S. Jr.

    1997-12-01

    We investigate the structure of the strange attractor of a chaotic loss-modulated solid-state laser utilizing return maps based on a combination of intensity maxima and interspike intervals, as opposed to those utilizing Poincar{acute e} sections defined by the intensity maxima of the laser ({dot I}=0,{umlt I}{lt}0) alone. We find both experimentally and numerically that a simple, intrinsic relationship exists between an intensity maximum and the pair of preceding and succeeding interspike intervals. In addition, we numerically investigate encoding messages on the output of a chaotic transmitter laser and its subsequent decoding by a similar receiver laser. By exploiting the relationship between the intensity maxima and the interspike intervals, we demonstrate that the method utilized to encode the message is vital to the system{close_quote}s ability to hide the signal from unwanted deciphering. In this work alternative methods are studied in order to encode messages by modulating the magnitude of pumping of the transmitter laser and also by driving its loss modulation with more than one frequency. {copyright} {ital 1997} {ital The American Physical Society}

  18. Digital raster timing encoder-decoder

    NASA Astrophysics Data System (ADS)

    Gennetten, E. W.; Gomez, D. A.

    1984-06-01

    The invention presents a digital raster timing encoder/decoder system wherein television raster synchronization pulses and other timing pulses and information data are generated. The invention comprises a digital clock source having coding means which combined with the clock source encodes preselected digital signals containing information on raster synchronization control and timing, and also any other information data that is desired. The encoded signal output is transmitted either directly or via some special transmission link, for example by use of a Manchester Decoder clock multiplier system, to a receiving and decoding means. The received signal is decoded to create timing signals for operation control of the video television scanning system, and to read out other desired digital data that has been also transmitted within the signal. The system takes advantage of the fact that control signals of conventional synchronization pulses contain large deadband areas within which additional data information may be placed. Such additional digital information may be used to add other control pulse capability, or to add information transmission capability. A principle feature of the system is that only a single channel is needed to transmit synchronization pulses and any additional timing pulses or information of interest.

  19. Encoded libraries of chemically modified peptides.

    PubMed

    Heinis, Christian; Winter, Greg

    2015-06-01

    The use of powerful technologies for generating and screening DNA-encoded protein libraries has helped drive the development of proteins as pharmaceutical ligands. However the development of peptides as pharmaceutical ligands has been more limited. Although encoded peptide libraries are typically several orders of magnitude larger than classical chemical libraries, can be more readily screened, and can give rise to higher affinity ligands, their use as pharmaceutical ligands is limited by their intrinsic properties. Two of the intrinsic limitations include the rotational flexibility of the peptide backbone and the limited number (20) of natural amino acids. However these limitations can be overcome by use of chemical modification. For example, the libraries can be modified to introduce topological constraints such as cyclization linkers, or to introduce new chemical entities such as small molecule ligands, fluorophores and photo-switchable compounds. This article reviews the chemistry involved, the properties of the peptide ligands, and the new opportunities offered by chemical modification of DNA-encoded peptide libraries.

  20. Architecture for VLSI design of Reed-Solomon encoders

    NASA Technical Reports Server (NTRS)

    Liu, K. Y.

    1981-01-01

    The logic structure of a universal VLSI chip called the symbol-slice Reed-Solomon (RS) encoder chip is discussed. An RS encoder can be constructed by cascading and properly interconnecting a group of such VLSI chips. As a design example, it is shown that a (255,223) RD encoder requiring around 40 discrete CMOS ICs may be replaced by an RS encoder consisting of four identical interconnected VLSI RS encoder chips. Besides the size advantage, the VLSI RS encoder also has the potential advantages of requiring less power and having a higher reliability.

  1. The Large Binocular Telescope azimuth and elevation encoder system

    NASA Astrophysics Data System (ADS)

    Ashby, David S.; Sargent, Tom; Cox, Dan; Rosato, Jerry; Brynnel, Joar G.

    2008-08-01

    A typical high-resolution encoder interpolator relies on careful mechanical alignment of the encoder read-heads and tight electrical tolerances of the signal processing electronics to ensure linearity. As the interpolation factor increases, maintaining these tight mechanical and electrical tolerances becomes impractical. The Large Binocular Telescope (LBT) is designed to utilize strip-type encoders on the main axes. Because of the very large scale of the telescope, the accumulative length of the azimuth and elevation encoder strips exceeds 80 meters, making optical tape prohibitively expensive. Consequently, the designers of the LBT incorporated the far less expensive Farrand Controls Inductosyn® linear strip encoder to encode the positions of the main axes and the instrument rotators. Since the cycle pitch of these encoders is very large compared to that of optical strip encoders, the interpolation factor must also be large in order to achieve the 0.005 arcsecond encoder resolution as specified. The authors present a description of the innovative DSP-based hardware / software solution that adaptively characterizes and removes common systematic cycle-to-cycle encoder interpolation errors. These errors can be caused by mechanical misalignment, encoder manufacturing flaws, variations in electrical gain, signal offset or cross-coupling of the encoder signals. Simulation data are presented to illustrate the performance of the interpolation algorithm, and telemetry data are presented to demonstrate the actual performance of the LBT main-axis encoder system.

  2. ChIP-seq guidelines and practices of the ENCODE and modENCODE consortia

    PubMed Central

    Landt, Stephen G.; Marinov, Georgi K.; Kundaje, Anshul; Kheradpour, Pouya; Pauli, Florencia; Batzoglou, Serafim; Bernstein, Bradley E.; Bickel, Peter; Brown, James B.; Cayting, Philip; Chen, Yiwen; DeSalvo, Gilberto; Epstein, Charles; Fisher-Aylor, Katherine I.; Euskirchen, Ghia; Gerstein, Mark; Gertz, Jason; Hartemink, Alexander J.; Hoffman, Michael M.; Iyer, Vishwanath R.; Jung, Youngsook L.; Karmakar, Subhradip; Kellis, Manolis; Kharchenko, Peter V.; Li, Qunhua; Liu, Tao; Liu, X. Shirley; Ma, Lijia; Milosavljevic, Aleksandar; Myers, Richard M.; Park, Peter J.; Pazin, Michael J.; Perry, Marc D.; Raha, Debasish; Reddy, Timothy E.; Rozowsky, Joel; Shoresh, Noam; Sidow, Arend; Slattery, Matthew; Stamatoyannopoulos, John A.; Tolstorukov, Michael Y.; White, Kevin P.; Xi, Simon; Farnham, Peggy J.; Lieb, Jason D.; Wold, Barbara J.; Snyder, Michael

    2012-01-01

    Chromatin immunoprecipitation (ChIP) followed by high-throughput DNA sequencing (ChIP-seq) has become a valuable and widely used approach for mapping the genomic location of transcription-factor binding and histone modifications in living cells. Despite its widespread use, there are considerable differences in how these experiments are conducted, how the results are scored and evaluated for quality, and how the data and metadata are archived for public use. These practices affect the quality and utility of any global ChIP experiment. Through our experience in performing ChIP-seq experiments, the ENCODE and modENCODE consortia have developed a set of working standards and guidelines for ChIP experiments that are updated routinely. The current guidelines address antibody validation, experimental replication, sequencing depth, data and metadata reporting, and data quality assessment. We discuss how ChIP quality, assessed in these ways, affects different uses of ChIP-seq data. All data sets used in the analysis have been deposited for public viewing and downloading at the ENCODE (http://encodeproject.org/ENCODE/) and modENCODE (http://www.modencode.org/) portals. PMID:22955991

  3. Automatic medical encoding with SNOMED categories.

    PubMed

    Ruch, Patrick; Gobeill, Julien; Lovis, Christian; Geissbühler, Antoine

    2008-10-27

    In this paper, we describe the design and preliminary evaluation of a new type of tools to speed up the encoding of episodes of care using the SNOMED CT terminology. The proposed system can be used either as a search tool to browse the terminology or as a categorization tool to support automatic annotation of textual contents with SNOMED concepts. The general strategy is similar for both tools and is based on the fusion of two complementary retrieval strategies with thesaural resources. The first classification module uses a traditional vector-space retrieval engine which has been fine-tuned for the task, while the second classifier is based on regular variations of the term list. For evaluating the system, we use a sample of MEDLINE. SNOMED CT categories have been restricted to Medical Subject Headings (MeSH) using the SNOMED-MeSH mapping provided by the UMLS (version 2006). Consistent with previous investigations applied on biomedical terminologies, our results show that performances of the hybrid system are significantly improved as compared to each single module. For top returned concepts, a precision at high ranks (P0) of more than 80% is observed. In addition, a manual and qualitative evaluation on a dozen of MEDLINE abstracts suggests that SNOMED CT could represent an improvement compared to existing medical terminologies such as MeSH. Although the precision of the SNOMED categorizer seems sufficient to help professional encoders, it is concluded that clinical benchmarks as well as usability studies are needed to assess the impact of our SNOMED encoding method in real settings. AVAILABILITIES: The system is available for research purposes on: http://eagl.unige.ch/SNOCat.

  4. Plasmid-encoded trimethoprim resistance in staphylococci.

    PubMed Central

    Archer, G L; Coughter, J P; Johnston, J L

    1986-01-01

    High-level (greater than 1,000 micrograms/ml) resistance to the antimicrobial agent trimethoprim was found in 17 of 101 (17%) coagulase-negative staphylococci and 5 of 51 (10%) Staphylococcus aureus from a number of different hospitals in the United States. Resistance was plasmid encoded and could be transferred by conjugation in 4 of the 17 (24%) Tpr coagulase-negative staphylococci and 3 of the 5 (60%) Tpr S. aureus. A 1.2-kilobase segment of plasmid DNA from one of the plasmids (pG01) was cloned on a high-copy-number vector in Escherichia coli and expressed high-level Tpr (MIC, 1,025 micrograms/ml) in the gram-negative host. In situ filter hybridization demonstrated homology between the cloned Tpr gene probe and plasmid DNA from each conjugative Tpr plasmid, a single nonconjugative plasmid from a United States Staphylococcus epidermidis isolate, a nonconjugative plasmid from an Australian methicillin-resistant S. aureus isolate, and chromosomal DNA from three Tpr S. epidermidis isolates that did not contain any plasmid DNA that was homologous with the probe. No homology was seen between the probe and staphylococcal plasmids not mediating Tpr, plasmid DNA from 12 Tpr S. epidermidis isolates not transferring Tpr by conjugation, or plasmid-encoded Tpr genes derived from gram-negative bacteria. Plasmid-encoded Tpr appears to be a relatively new gene in staphylococci and, because it can be transferred by conjugation, could become more prevalent in nonsocomial isolates. Images PMID:3729338

  5. Fast acceleration-encoded magnetic resonance imaging.

    PubMed

    Forster, J; Sieverding, L; Breuer, J; Lutz, O; Schick, F

    2001-01-01

    Direct acceleration imaging with high spatial resolution was implemented and tested. The well-known principle of phase encoding motion components was applied. Suitable gradient switching provides a signal phase shift proportional to the acceleration perpendicular to the slice in the first scan of the sequences. An additional scan serving as a reference was recorded for compensation of phase effects due to magnetic field inhomogeneities. The first scan compensated for phase shifts from undesired first- and second-order motions; the second scan was completely insensitive to velocity and acceleration in all directions. Advantages of the proposed two-step technique compared to former approaches with Fourier acceleration encoding (with several phase encoding steps) are relatively short echo times and short total measuring times. On the other hand, the new approach does not allow us to assess the velocity or acceleration spectrum simultaneously. The capabilities of the sequences were tested on a modern 1.5 T whole body MR unit providing relatively high gradient amplitudes (25 mT/m) and short rise times (600 micros to maximum amplitude). The results from a mechanical acceleration phantom showed a standard deviation of 0.3 m/s2 in sequences with an acceleration range between -12 and 12 m/s2. This range covers the expected maximum acceleration in the human aorta of 10 m/s2. Further tests were performed on a stenosis phantom with a variable volume flow rate to assess the flow characteristics and possible displacement artifacts of the sequences. Preliminary examinations of volunteers demonstrate the potential applicability of the technique in vivo.

  6. Evaluating standard terminologies for encoding allergy information

    PubMed Central

    Goss, Foster R; Zhou, Li; Plasek, Joseph M; Broverman, Carol; Robinson, George; Middleton, Blackford; Rocha, Roberto A

    2013-01-01

    Objective Allergy documentation and exchange are vital to ensuring patient safety. This study aims to analyze and compare various existing standard terminologies for representing allergy information. Methods Five terminologies were identified, including the Systemized Nomenclature of Medical Clinical Terms (SNOMED CT), National Drug File–Reference Terminology (NDF-RT), Medication Dictionary for Regulatory Activities (MedDRA), Unique Ingredient Identifier (UNII), and RxNorm. A qualitative analysis was conducted to compare desirable characteristics of each terminology, including content coverage, concept orientation, formal definitions, multiple granularities, vocabulary structure, subset capability, and maintainability. A quantitative analysis was also performed to compare the content coverage of each terminology for (1) common food, drug, and environmental allergens and (2) descriptive concepts for common drug allergies, adverse reactions (AR), and no known allergies. Results Our qualitative results show that SNOMED CT fulfilled the greatest number of desirable characteristics, followed by NDF-RT, RxNorm, UNII, and MedDRA. Our quantitative results demonstrate that RxNorm had the highest concept coverage for representing drug allergens, followed by UNII, SNOMED CT, NDF-RT, and MedDRA. For food and environmental allergens, UNII demonstrated the highest concept coverage, followed by SNOMED CT. For representing descriptive allergy concepts and adverse reactions, SNOMED CT and NDF-RT showed the highest coverage. Only SNOMED CT was capable of representing unique concepts for encoding no known allergies. Conclusions The proper terminology for encoding a patient's allergy is complex, as multiple elements need to be captured to form a fully structured clinical finding. Our results suggest that while gaps still exist, a combination of SNOMED CT and RxNorm can satisfy most criteria for encoding common allergies and provide sufficient content coverage. PMID:23396542

  7. Using the ENCODE Resource for Functional Annotation of Genetic Variants.

    PubMed

    Pazin, Michael J

    2015-03-11

    This article illustrates the use of the Encyclopedia of DNA Elements (ENCODE) resource to generate or refine hypotheses from genomic data on disease and other phenotypic traits. First, the goals and history of ENCODE and related epigenomics projects are reviewed. Second, the rationale for ENCODE and the major data types used by ENCODE are briefly described, as are some standard heuristics for their interpretation. Third, the use of the ENCODE resource is examined. Standard use cases for ENCODE, accessing the ENCODE resource, and accessing data from related projects are discussed. Although the focus of this article is the use of ENCODE data, some of the same approaches can be used with data from other projects.

  8. Mouse redox histology using genetically encoded probes.

    PubMed

    Fujikawa, Yuuta; Roma, Leticia P; Sobotta, Mirko C; Rose, Adam J; Diaz, Mauricio Berriel; Locatelli, Giuseppe; Breckwoldt, Michael O; Misgeld, Thomas; Kerschensteiner, Martin; Herzig, Stephan; Müller-Decker, Karin; Dick, Tobias P

    2016-03-15

    Mapping the in vivo distribution of endogenous oxidants in animal tissues is of substantial biomedical interest. Numerous health-related factors, including diet, physical activity, infection, aging, toxins, or pharmacological intervention, may cause redox changes. Tools are needed to pinpoint redox state changes to particular organs, tissues, cell types, and subcellular organelles. We describe a procedure that preserves the in vivo redox state of genetically encoded redox biosensors within histological tissue sections, thus providing "redox maps" for any tissue and comparison of interest. We demonstrate the utility of the technique by visualizing endogenous redox differences and changes in the context of tumor growth, inflammation, embryonic development, and nutrient starvation.

  9. Wavelet encoding and variable resolution progressive transmission

    NASA Technical Reports Server (NTRS)

    Blanford, Ronald P.

    1993-01-01

    Progressive transmission is a method of transmitting and displaying imagery in stages of successively improving quality. The subsampled lowpass image representations generated by a wavelet transformation suit this purpose well, but for best results the order of presentation is critical. Candidate data for transmission are best selected using dynamic prioritization criteria generated from image contents and viewer guidance. We show that wavelets are not only suitable but superior when used to encode data for progressive transmission at non-uniform resolutions. This application does not preclude additional compression using quantization of highpass coefficients, which to the contrary results in superior image approximations at low data rates.

  10. Spatially encoded multiple-quantum excitation.

    PubMed

    Ridge, Clark D; Borvayeh, Leila; Walls, Jamie D

    2013-05-28

    In this work, we present a simple method to spatially encode the transition frequencies of nuclear spin transitions and to read out these frequencies within a single scan. The experiment works by combining pulsed field gradients with an excitation sequence that selectively excites spin transitions within certain sample regions. After the initial excitation, imaging the resulting ẑ-magnetization is used to determine the locations where the excitations occurred, from which the corresponding transition frequencies are determined. Simple experimental demonstrations of this technique on one- and two-spin systems are presented.

  11. Space Qualified High Speed Reed Solomon Encoder

    NASA Technical Reports Server (NTRS)

    Gambles, Jody W.; Winkert, Tom

    1993-01-01

    This paper reports a Class S CCSDS recommendation Reed Solomon encoder circuit baselined for several NASA programs. The chip is fabricated using United Technologies Microelectronics Center's UTE-R radiation-hardened gate array family, contains 64,000 p-n transistor pairs, and operates at a sustained output data rate of 200 MBits/s. The chip features a pin selectable message interleave depth of from 1 to 8 and supports output block lengths of 33 to 255 bytes. The UTE-R process is reported to produce parts that are radiation hardened to 16 Rads (Si) total dose and 1.0(exp -10) errors/bit-day.

  12. Subband image encoder using discrete wavelet transform

    NASA Astrophysics Data System (ADS)

    Seong, Hae Kyung; Rhee, Kang Hyeon

    2004-03-01

    Introduction of digital communication network such as Integrated Services Digital Networks (ISDN) and digital storage media have rapidly developed. Due to a large amount of image data, compression is the key techniques in still image and video using digital signal processing for transmitting and storing. Digital image compression provides solutions for various image applications that represent digital image requiring a large amount of data. In this paper, the proposed DWT (Discrete Wavelet Transform) filter bank is consisted of simple architecture, but it is efficiently designed that a user obtains a wanted compression rate as only input parameter. If it is implemented by FPGA chip, the designed encoder operates in 12 MHz.

  13. Digitally encoded all-optical sensor multiplexing

    NASA Astrophysics Data System (ADS)

    Pervez, Anjum

    1992-01-01

    A digital, all-optical temperature sensor design concept based on optical sampling and digital encoding is presented. The proposed sensor generates 2M binary digital codewords of length M bits. The codewords are generated serially and, therefore, only a single output fiber line is required. A multiplexing scheme, which minimizes the power requirement per sensor array and facilitates a cost-effective digit regeneration for remote monitoring over long distance, is presented. The sensor arrays are used as building blocks to configure large scale sensor networks based on LAN topologies.

  14. Flow cytometry using spectrally encoded confocal microscopy.

    PubMed

    Golan, Lior; Yelin, Dvir

    2010-07-01

    Flow cytometry techniques often rely on detecting fluorescence from single cells flowing through the cross section of a laser beam, providing invaluable information on vast numbers of cells. Such techniques, however, are often limited in their ability to resolve clusters of cells or parallel cell flow through large vessels. We present a confocal imaging technique that images unstained cells flowing in parallel through a wide channel, using spectrally encoded reflectance confocal microscopy that does not require mechanical scanning. Images of red blood cells from our system are compared to conventional transmission microscopy, and imaging of flowing red blood cells in vitro is experimentally demonstrated.

  15. Polynucleotides encoding TRF1 binding proteins

    DOEpatents

    Campisi, Judith; Kim, Sahn-Ho

    2002-01-01

    The present invention provides a novel telomere associated protein (Trf1-interacting nuclear protein 2 "Tin2") that hinders the binding of Trf1 to its specific telomere repeat sequence and mediates the formation of a Tin2-Trf1-telomeric DNA complex that limits telomerase access to the telomere. Also included are the corresponding nucleic acids that encode the Tin2 of the present invention, as well as mutants of Tin2. Methods of making, purifying and using Tin2 of the present invention are described. In addition, drug screening assays to identify drugs that mimic and/or complement the effect of Tin2 are presented.

  16. Rapidly-Indexing Incremental-Angle Encoder

    NASA Technical Reports Server (NTRS)

    Christon, Philip R.; Meyer, Wallace W.

    1989-01-01

    Optoelectronic system measures relative angular position of shaft or other device to be turned, also measures absolute angular position after device turned through small angle. Relative angular position measured with fine resolution by optoelectronically counting finely- and uniformly-spaced light and dark areas on encoder disk as disk turns past position-sensing device. Also includes track containing coarsely- and nonuniformly-spaced light and dark areas, angular widths varying in proportion to absolute angular position. This second track provides gating and indexing signal.

  17. Auditory location as an encoding dimension.

    PubMed

    Weeks, R A

    1975-05-01

    In two experiments, subjects were given five successive short-term memory tests. In Experiment 1, recall was not significantly facilitated when memory material in the final test was delivered to the ear opposite to the one that received the memory material in the four preceding tests. In Experiment 2, events were presented from two differentially located speakers rather than through headphones. A shift across speakers on the final test did produce proactive interference release. These findings suggest spatial location as a potential encoding dimension of verbal material.

  18. The ENCODE (ENCyclopedia Of DNA Elements) Project.

    PubMed

    2004-10-22

    The ENCyclopedia Of DNA Elements (ENCODE) Project aims to identify all functional elements in the human genome sequence. The pilot phase of the Project is focused on a specified 30 megabases (approximately 1%) of the human genome sequence and is organized as an international consortium of computational and laboratory-based scientists working to develop and apply high-throughput approaches for detecting all sequence elements that confer biological function. The results of this pilot phase will guide future efforts to analyze the entire human genome.

  19. A space-compatible angular contact encoder

    NASA Astrophysics Data System (ADS)

    Flew, A. R.

    1985-12-01

    An electromechanical digital position encoder, based on existing commercial technology, for spacecraft applications is described. The device contains electrical wiping contacts, a gear mechanism, and plain and rolling-element bearings all operating without wet lubrication. Designed to function continuously for 10 years, certain contacts will perform in excess of 20 million cycles. To investigate the performance of these contacts, automatic test equipment was designed to monitor the accuracy of 8192 separate output conditions on a regular basis throughout an accelerated-life thermal vacuum test. The equipment also checks for missing bits and edge noise and logs any errors that are found.

  20. Image compression based on GPU encoding

    NASA Astrophysics Data System (ADS)

    Bai, Zhaofeng; Qiu, Yuehong

    2015-07-01

    With the rapid development of digital technology, the data increased greatly in both static image and dynamic video image. It is noticeable how to decrease the redundant data in order to save or transmit information more efficiently. So the research on image compression becomes more and more important. Using GPU to achieve higher compression ratio has superiority in interactive remote visualization. Contrast to CPU, GPU may be a good way to accelerate the image compression. Currently, GPU of NIVIDIA has evolved into the eighth generation, which increasingly dominates the high-powered general purpose computer field. This paper explains the way of GPU encoding image. Some experiment results are also presented.

  1. Gene encoding herbicide safener binding protein

    DOEpatents

    Walton, Jonathan D.; Scott-Craig, John S.

    1999-01-01

    The cDNA encoding safener binding protein (SafBP), also referred to as SBP1, is set forth in FIG. 5 and SEQ ID No. 1. The deduced amino acid sequence is provided in FIG. 5 and SEQ ID No. 2. Methods of making and using SBP1 and SafBP to alter a plant's sensitivity to certain herbicides or a plant's responsiveness to certain safeners are also provided, as well as expression vectors, transgenic plants or other organisms transfected with said vectors and seeds from said plants.

  2. Optical Pseudocolor Encoding Of Gray-Scale Image

    NASA Technical Reports Server (NTRS)

    Chao, Tien-Hsin; Liu, Hua-Kuang

    1990-01-01

    Optical encoding much faster than digital electronic encoding. In optical pseudocolor-encoding apparatus brightness modulation in image from television camera transformed into polarization modulation in LCTV, and then into pseudocolor modulation in image on projection screen. Advantageous for such purposes as thermography, inspection of circuit boards, mammography, and mapping.

  3. An encyclopedia of mouse DNA elements (Mouse ENCODE)

    PubMed Central

    2012-01-01

    To complement the human Encyclopedia of DNA Elements (ENCODE) project and to enable a broad range of mouse genomics efforts, the Mouse ENCODE Consortium is applying the same experimental pipelines developed for human ENCODE to annotate the mouse genome. PMID:22889292

  4. Socialization Processes in Encoding and Decoding: Learning Effective Nonverbal Behavior.

    ERIC Educational Resources Information Center

    Feldman, Robert S.; Coats, Erik

    This study examined the relationship of nonverbal encoding and decoding skills to the level of exposure to television. Subjects were children in second through sixth grade. Three nonverbal skills (decoding, spontaneous encoding, and posed encoding) were assessed for each of five emotions: anger, disgust, fear or surprise, happiness, and sadness.…

  5. Self-encoding resin beads of combinatorial library screening

    NASA Astrophysics Data System (ADS)

    Lei, Du; Zhao, Yuandi; Cheng, Tongsheng; Zeng, Shaoqun; Luo, Qingming

    2003-07-01

    The latest self-encoding resin bead is a novel technology for solid phase synthesis combinatorial library screening. A new encode-positional deconvolution strategy which was based on that technology been illustrated compared with positional scanning and iterative strategies. The self-encoding resin beads technology provides an efficient method for improving the high-throughput screening of combinatorial library.

  6. A genetic linkage map of mouse chromosome 2 extending from thrombospondin to paired box gene 1, including the H3 minor histocompatibility complex

    SciTech Connect

    Zuberi, A.R.; Nguyen, H.Q.; Auman, H.J.

    1996-04-01

    The classical minor histocompatibility 3(H3) locus was originally defined by the phenotype of skin graft rejection, which is complex genetic trait. H3 is now known to be a gene complex comprised of a minimum of two functionally interdependent alloantigen-encoding loci, H3a and H3b. H3a encodes a peptide recognized by cytotoxic T cells, and H3b encodes a peptide that stimulates helper T cells. The H3 complex also contains the {beta}{sub 2}-microglobulin gene (B2m), and polymorphisms in B2m contribute to the tissue rejection phenotype. We describe a high-density genetic linkage map of a 16-cM region of mouse Chromosome 2 from thromospondin (Thbs1) to paired box gene 1 (Pax1). This genetic map includes H3a, H3b, and B2m. Other genes and anonymous loci have also been placed on the map. H3a maps between D2Mit444 and B2m in close vicinity to several known genes. H3b maps 12 cM distal to H3a, and the proprotein convertase subtilisin/kexin type 2 gene (Pcsk2; formerly Nec2) cosegregates with H3b in a high-resolution backcross panel. The H3 complex spans a region that shows conserved synteny to human chromosomes 15q, 2q, and 20p. 59 refs., 4 figs., 1 tab.

  7. Area MT Encodes Three-Dimensional Motion

    PubMed Central

    Huk, Alexander C.; Cormack, Lawrence K.; Kohn, Adam

    2014-01-01

    We use visual information to determine our dynamic relationship with other objects in a three-dimensional (3D) world. Despite decades of work on visual motion processing, it remains unclear how 3D directions—trajectories that include motion toward or away from the observer—are represented and processed in visual cortex. Area MT is heavily implicated in processing visual motion and depth, yet previous work has found little evidence for 3D direction sensitivity per se. Here we use a rich ensemble of binocular motion stimuli to reveal that most neurons in area MT of the anesthetized macaque encode 3D motion information. This tuning for 3D motion arises from multiple mechanisms, including different motion preferences in the two eyes and a nonlinear interaction of these signals when both eyes are stimulated. Using a novel method for functional binocular alignment, we were able to rule out contributions of static disparity tuning to the 3D motion tuning we observed. We propose that a primary function of MT is to encode 3D motion, critical for judging the movement of objects in dynamic real-world environments. PMID:25411482

  8. Optical Security System with Fourier Plane encoding.

    PubMed

    Javidi, B; Ahouzi, E

    1998-09-10

    We propose a new technique for security verification of personal documents and other forms of personal identifications such as ID cards, passports, or credit cards. In this technique a primary pattern that might be a phase-encoded image is convolved by a random code. The information is phase encoded on the personal document. Therefore the information cannot be reproduced by an intensity detector such as a CCD camera. An optical processor based on the nonlinear joint transform correlator is used to perform the verification and the validation of documents with this technique. By verification of the biometrics information and the random code simultaneously, the proposed optical system determines whether a card is authentic or is being used by an authorized person. We tested the performance of the optical system for security and validation in the presence of input noise and in the presence of distortion of the information on the card. The performance of the proposed method is evaluated by use of a number of metrics. Statistical analysis of the system is performed to investigate the noise tolerance and the discrimination against false inputs for security verification.

  9. Optical Security System with Fourier Plane encoding

    NASA Astrophysics Data System (ADS)

    Javidi, Bahram; Ahouzi, Esmail

    1998-09-01

    We propose a new technique for security verification of personal documents and other forms of personal identifications such as ID cards, passports, or credit cards. In this technique a primary pattern that might be a phase-encoded image is convolved by a random code. The information is phase encoded on the personal document. Therefore the information cannot be reproduced by an intensity detector such as a CCD camera. An optical processor based on the nonlinear joint transform correlator is used to perform the verification and the validation of documents with this technique. By verification of the biometrics information and the random code simultaneously, the proposed optical system determines whether a card is authentic or is being used by an authorized person. We tested the performance of the optical system for security and validation in the presence of input noise and in the presence of distortion of the information on the card. The performance of the proposed method is evaluated by use of a number of metrics. Statistical analysis of the system is performed to investigate the noise tolerance and the discrimination against false inputs for security verification.

  10. Encoded multisite two-photon microscopy.

    PubMed

    Ducros, Mathieu; Goulam Houssen, Yannick; Bradley, Jonathan; de Sars, Vincent; Charpak, Serge

    2013-08-06

    The advent of scanning two-photon microscopy (2PM) has created a fertile new avenue for noninvasive investigation of brain activity in depth. One principal weakness of this method, however, lies with the limit of scanning speed, which makes optical interrogation of action potential-like activity in a neuronal network problematic. Encoded multisite two-photon microscopy (eMS2PM), a scanless method that allows simultaneous imaging of multiple targets in depth with high temporal resolution, addresses this drawback. eMS2PM uses a liquid crystal spatial light modulator to split a high-power femto-laser beam into multiple subbeams. To distinguish them, a digital micromirror device encodes each subbeam with a specific binary amplitude modulation sequence. Fluorescence signals from all independently targeted sites are then collected simultaneously onto a single photodetector and site-specifically decoded. We demonstrate that eMS2PM can be used to image spike-like voltage transients in cultured cells and fluorescence transients (calcium signals in neurons and red blood cells in capillaries from the cortex) in depth in vivo. These results establish eMS2PM as a unique method for simultaneous acquisition of neuronal network activity.

  11. Genetically Encoded Libraries of Nonstandard Peptides

    PubMed Central

    Kawakami, Takashi; Murakami, Hiroshi

    2012-01-01

    The presence of a nonproteinogenic moiety in a nonstandard peptide often improves the biological properties of the peptide. Non-standard peptide libraries are therefore used to obtain valuable molecules for biological, therapeutic, and diagnostic applications. Highly diverse non-standard peptide libraries can be generated by chemically or enzymatically modifying standard peptide libraries synthesized by the ribosomal machinery, using posttranslational modifications. Alternatively, strategies for encoding non-proteinogenic amino acids into the genetic code have been developed for the direct ribosomal synthesis of non-standard peptide libraries. In the strategies for genetic code expansion, non-proteinogenic amino acids are assigned to the nonsense codons or 4-base codons in order to add these amino acids to the universal genetic code. In contrast, in the strategies for genetic code reprogramming, some proteinogenic amino acids are erased from the genetic code and non-proteinogenic amino acids are reassigned to the blank codons. Here, we discuss the generation of genetically encoded non-standard peptide libraries using these strategies and also review recent applications of these libraries to the selection of functional non-standard peptides. PMID:23097693

  12. V123 BEAM SYNCHRONOUS ENCODER MODULE.

    SciTech Connect

    KERNER,T.; CONKLING,C.R.; OERTER,B.

    1999-03-29

    The V123 Synchronous Encoder Module transmits events to distributed trigger modules and embedded decoders around the RHIC rings where they are used to provide beam instrumentation triggers [1,2,3]. The RHIC beam synchronous event link hardware is mainly comprised of three VMEbus board designs, the central input modules (V201), and encoder modules (V123), and the distributed trigger modules (V124). Two beam synchronous links, one for each ring, are distributed via fiber optics and fanned out via twisted wire pair cables. The V123 synchronizes with the RF system clock derived from the beam bucket frequency and a revolution fiducial pulse. The RF system clock is used to create the beam synchronous event link carrier and events are synchronized with the rotation fiducial. A low jitter RF clock is later recovered from this carrier by phase lock loops in the trigger modules. Prioritized hardware and software triggers fill up to 15 beam event code transmission slots per revolution while tracking the ramping RF acceleration frequency and storage frequency. The revolution fiducial event is always the first event transmitted which is used to synchronize the firing of the abort kicker and to locate the first bucket for decoders distributed about the ring.

  13. The First Gene-encoded Amphibian Neurotoxin*

    PubMed Central

    You, Dewen; Hong, Jing; Rong, Mingqiang; Yu, Haining; Liang, Songping; Ma, Yufang; Yang, Hailong; Wu, Jing; Lin, Donghai; Lai, Ren

    2009-01-01

    Many gene-encoded neurotoxins with various functions have been discovered in fish, reptiles, and mammals. A novel 60-residue neurotoxin peptide (anntoxin) that inhibited tetrodotoxin-sensitive (TTX-S) voltage-gated sodium channel (VGSC) was purified and characterized from the skin secretions of the tree frog Hyla annectans (Jerdon). This is the first gene-encoded neurotoxin found in amphibians. The IC50 of anntoxin for the TTX-S channel was about 3.4 μm. Anntoxin shares sequence homology with Kunitz-type toxins but contains only two of three highly conserved cysteine bridges, which are typically found in these small, basic neurotoxin modules, i.e. snake dendrotoxins. Anntoxin showed an inhibitory ability against trypsin with an inhibitory constant (Ki) of 0.025 μm. Anntoxin was distributed in skin, brain, stomach, and liver with a concentration of 25, 7, 3, and 2 μg/g wet tissue, respectively. H. annectans lives on trees or other plants for its entire life cycle, and its skin contains the largest amount of anntoxin, which possibly helps defend against various aggressors or predators. A low dose of anntoxin was found to induce lethal toxicity for several potential predators, including the insect, snake, bird, and mouse. The tissue distribution and functional properties of the current toxin may provide insights into the ecological adaptation of tree-living amphibians. PMID:19535333

  14. Directed forgetting benefits motor sequence encoding.

    PubMed

    Tempel, Tobias; Frings, Christian

    2016-04-01

    Two experiments investigated directed forgetting of newly learned motor sequences. Concurrently with the list method of directed forgetting, participants successively learned two lists of motor sequences. Each sequence consisted of four consecutive finger movements. After a short distractor task, a recall test was given. Both experiments compared a forget group that was instructed to forget list-1 items with a remember group not receiving a forget instruction. We found that the instruction to forget list 1 enhanced recall of subsequently learned motor sequences. This benefit of directed forgetting occurred independently of costs for list 1. A mediation analysis showed that the encoding accuracy of list 2 was a mediator of the recall benefit, that is, the more accurate execution of motor sequences of list 2 after receiving a forget instruction for list 1 accounted for better recall of list 2. Thus, the adaptation of the list method to motor action provided more direct evidence on the effect of directed forgetting on subsequent learning. The results corroborate the assumption of a reset of encoding as a consequence of directed forgetting.

  15. Encoded multisite two-photon microscopy

    PubMed Central

    Ducros, Mathieu; Houssen, Yannick Goulam; Bradley, Jonathan; de Sars, Vincent; Charpak, Serge

    2013-01-01

    The advent of scanning two-photon microscopy (2PM) has created a fertile new avenue for noninvasive investigation of brain activity in depth. One principal weakness of this method, however, lies with the limit of scanning speed, which makes optical interrogation of action potential-like activity in a neuronal network problematic. Encoded multisite two-photon microscopy (eMS2PM), a scanless method that allows simultaneous imaging of multiple targets in depth with high temporal resolution, addresses this drawback. eMS2PM uses a liquid crystal spatial light modulator to split a high-power femto-laser beam into multiple subbeams. To distinguish them, a digital micromirror device encodes each subbeam with a specific binary amplitude modulation sequence. Fluorescence signals from all independently targeted sites are then collected simultaneously onto a single photodetector and site-specifically decoded. We demonstrate that eMS2PM can be used to image spike-like voltage transients in cultured cells and fluorescence transients (calcium signals in neurons and red blood cells in capillaries from the cortex) in depth in vivo. These results establish eMS2PM as a unique method for simultaneous acquisition of neuronal network activity. PMID:23798397

  16. Fast Huffman encoding algorithms in MPEG-4 advanced audio coding

    NASA Astrophysics Data System (ADS)

    Brzuchalski, Grzegorz

    2014-11-01

    This paper addresses the optimisation problem of Huffman encoding in MPEG-4 Advanced Audio Coding stan- dard. At first, the Huffman encoding problem and the need of encoding two side info parameters scale factor and Huffman codebook are presented. Next, Two Loop Search, Maximum Noise Mask Ratio and Trellis Based algorithms of bit allocation are briefly described. Further, Huffman encoding optimisation are shown. New methods try to check and change scale factor bands as little as possible to estimate bitrate cost or its change. Finally, the complexity of old and new methods is calculated, compared and measured time of encoding is given.

  17. Architecture for VLSI design of Reed-Solomon encoders

    NASA Technical Reports Server (NTRS)

    Liu, K. Y.

    1982-01-01

    A description is given of the logic structure of the universal VLSI symbol-slice Reed-Solomon (RS) encoder chip, from a group of which an RS encoder may be constructed through cascading and proper interconnection. As a design example, it is shown that an RS encoder presently requiring approximately 40 discrete CMOS ICs may be replaced by an RS encoder consisting of four identical, interconnected VLSI RS encoder chips, offering in addition to greater compactness both a lower power requirement and greater reliability.

  18. Negative base encoding in optical linear algebra processors

    NASA Technical Reports Server (NTRS)

    Perlee, C.; Casasent, D.

    1986-01-01

    In the digital multiplication by analog convolution algorithm, the bits of two encoded numbers are convolved to form the product of the two numbers in mixed binary representation; this output can be easily converted to binary. Attention is presently given to negative base encoding, treating base -2 initially, and then showing that the negative base system can be readily extended to any radix. In general, negative base encoding in optical linear algebra processors represents a more efficient technique than either sign magnitude or 2's complement encoding, when the additions of digitally encoded products are performed in parallel.

  19. Negative base encoding in optical linear algebra processors

    NASA Technical Reports Server (NTRS)

    Perlee, C.; Casasent, D.

    1986-01-01

    In the digital multiplication by analog convolution algorithm, the bits of two encoded numbers are convolved to form the product of the two numbers in mixed binary representation; this output can be easily converted to binary. Attention is presently given to negative base encoding, treating base -2 initially, and then showing that the negative base system can be readily extended to any radix. In general, negative base encoding in optical linear algebra processors represents a more efficient technique than either sign magnitude or 2's complement encoding, when the additions of digitally encoded products are performed in parallel.

  20. Improvement of protein secondary structure prediction using binary word encoding.

    PubMed

    Kawabata, T; Doi, J

    1997-01-01

    We propose a binary word encoding to improve the protein secondary structure prediction. A binary word encoding encodes a local amino acid sequence to a binary word, which consists of 0 or 1. We use an encoding function to map an amino acid to 0 or 1. Using the binary word encoding, we can statistically extract the multiresidue information, which depends on more than one residue. We combine the binary word encoding with the GOR method, its modified version, which shows better accuracy, and the neural network method. The binary word encoding improves the accuracy of GOR by 2.8%. We obtain similar improvement when we combine this with the modified GOR method and the neural network method. When we use multiple sequence alignment data, the binary word encoding similarly improves the accuracy. The accuracy of our best combined method is 68.2%. In this paper, we only show improvement of the GOR and neural network method, we cannot say that the encoding improves the other methods. But the improvement by the encoding suggests that the multiresidue interaction affects the formation of secondary structure. In addition, we find that the optimal encoding function obtained by the simulated annealing method relates to nonpolarity. This means that nonpolarity is important to the multiresidue interaction.

  1. A Multi-Encoding Approach for LTL Symbolic Satisfiability Checking

    NASA Technical Reports Server (NTRS)

    Rozier, Kristin Y.; Vardi, Moshe Y.

    2011-01-01

    Formal behavioral specifications written early in the system-design process and communicated across all design phases have been shown to increase the efficiency, consistency, and quality of the system under development. To prevent introducing design or verification errors, it is crucial to test specifications for satisfiability. Our focus here is on specifications expressed in linear temporal logic (LTL). We introduce a novel encoding of symbolic transition-based Buchi automata and a novel, "sloppy," transition encoding, both of which result in improved scalability. We also define novel BDD variable orders based on tree decomposition of formula parse trees. We describe and extensively test a new multi-encoding approach utilizing these novel encoding techniques to create 30 encoding variations. We show that our novel encodings translate to significant, sometimes exponential, improvement over the current standard encoding for symbolic LTL satisfiability checking.

  2. Hierarchical encoding of behavior: translating perception into action.

    PubMed

    Hard, Bridgette Martin; Lozano, Sandra C; Tversky, Barbara

    2006-11-01

    People encode goal-directed behaviors, such as assembling an object, by segmenting them into discrete actions, organized as goal-subgoal hierarchies. Does hierarchical encoding contribute to observational learning? Participants in 3 experiments segmented an object assembly task into coarse and fine units of action and later performed it themselves. Hierarchical encoding, measured by segmentation patterns, correlated with more accurate and more hierarchically structured performance of the later assembly task. Furthermore, hierarchical encoding increased when participants (a) segmented coarse units first, (b) explicitly looked for hierarchical structure, and (c) described actions while segmenting them. Improving hierarchical encoding always led to improvements in learning, as well as a surprising shift toward encoding and executing actions from the actor's spatial perspective instead of the participants' own. Hierarchical encoding facilitates observational learning by organizing perceived actions into a representation that can serve as an action plan.

  3. Optically coupled digital altitude encoder for general aviation altimeters

    NASA Technical Reports Server (NTRS)

    Bryant, F. R.

    1975-01-01

    An optically coupled pressure altitude encoder which can be incorporated into commercially available inexpensive general aviation altimeters was successfully developed. The encoding of pressure altitude is accomplished in 100-ft (30.48-m) increments from -1000 to 20,000ft (-304.8 to 6096 m). The prototype encoders were retrofitted into two different internal altimeter configurations. A prototype encoder was checked for accuracy of transition points and environmental effects. Each altimeter configuration, with the encoder incorporated, was laboratory tested for performance and was subsequently flight-tested over the specified altitude range. With few exceptions, the assembled altimeter-encoder met aeronautical standards for altimeters and encoders. Design changes are suggested to improve performance to meet required standards consistently.

  4. DNA-Encoded Solid-Phase Synthesis: Encoding Language Design and Complex Oligomer Library Synthesis

    PubMed Central

    2015-01-01

    The promise of exploiting combinatorial synthesis for small molecule discovery remains unfulfilled due primarily to the “structure elucidation problem”: the back-end mass spectrometric analysis that significantly restricts one-bead-one-compound (OBOC) library complexity. The very molecular features that confer binding potency and specificity, such as stereochemistry, regiochemistry, and scaffold rigidity, are conspicuously absent from most libraries because isomerism introduces mass redundancy and diverse scaffolds yield uninterpretable MS fragmentation. Here we present DNA-encoded solid-phase synthesis (DESPS), comprising parallel compound synthesis in organic solvent and aqueous enzymatic ligation of unprotected encoding dsDNA oligonucleotides. Computational encoding language design yielded 148 thermodynamically optimized sequences with Hamming string distance ≥ 3 and total read length <100 bases for facile sequencing. Ligation is efficient (70% yield), specific, and directional over 6 encoding positions. A series of isomers served as a testbed for DESPS’s utility in split-and-pool diversification. Single-bead quantitative PCR detected 9 × 104 molecules/bead and sequencing allowed for elucidation of each compound’s synthetic history. We applied DESPS to the combinatorial synthesis of a 75 645-member OBOC library containing scaffold, stereochemical and regiochemical diversity using mixed-scale resin (160-μm quality control beads and 10-μm screening beads). Tandem DNA sequencing/MALDI-TOF MS analysis of 19 quality control beads showed excellent agreement (<1 ppt) between DNA sequence-predicted mass and the observed mass. DESPS synergistically unites the advantages of solid-phase synthesis and DNA encoding, enabling single-bead structural elucidation of complex compounds and synthesis using reactions normally considered incompatible with unprotected DNA. The widespread availability of inexpensive oligonucleotide synthesis, enzymes, DNA sequencing, and

  5. DNA-Encoded Solid-Phase Synthesis: Encoding Language Design and Complex Oligomer Library Synthesis.

    PubMed

    MacConnell, Andrew B; McEnaney, Patrick J; Cavett, Valerie J; Paegel, Brian M

    2015-09-14

    The promise of exploiting combinatorial synthesis for small molecule discovery remains unfulfilled due primarily to the "structure elucidation problem": the back-end mass spectrometric analysis that significantly restricts one-bead-one-compound (OBOC) library complexity. The very molecular features that confer binding potency and specificity, such as stereochemistry, regiochemistry, and scaffold rigidity, are conspicuously absent from most libraries because isomerism introduces mass redundancy and diverse scaffolds yield uninterpretable MS fragmentation. Here we present DNA-encoded solid-phase synthesis (DESPS), comprising parallel compound synthesis in organic solvent and aqueous enzymatic ligation of unprotected encoding dsDNA oligonucleotides. Computational encoding language design yielded 148 thermodynamically optimized sequences with Hamming string distance ≥ 3 and total read length <100 bases for facile sequencing. Ligation is efficient (70% yield), specific, and directional over 6 encoding positions. A series of isomers served as a testbed for DESPS's utility in split-and-pool diversification. Single-bead quantitative PCR detected 9 × 10(4) molecules/bead and sequencing allowed for elucidation of each compound's synthetic history. We applied DESPS to the combinatorial synthesis of a 75,645-member OBOC library containing scaffold, stereochemical and regiochemical diversity using mixed-scale resin (160-μm quality control beads and 10-μm screening beads). Tandem DNA sequencing/MALDI-TOF MS analysis of 19 quality control beads showed excellent agreement (<1 ppt) between DNA sequence-predicted mass and the observed mass. DESPS synergistically unites the advantages of solid-phase synthesis and DNA encoding, enabling single-bead structural elucidation of complex compounds and synthesis using reactions normally considered incompatible with unprotected DNA. The widespread availability of inexpensive oligonucleotide synthesis, enzymes, DNA sequencing, and PCR

  6. Genetically encoded sensors for calcium and zinc

    NASA Astrophysics Data System (ADS)

    Palmer, Amy E.; Dittmer, Philip; McCombs, Janet E.

    2008-02-01

    Our lab focuses on developing fluorescent biosensors based on fluorescence resonance energy transfer (FRET) so that we can monitor signaling ions in living cells. These sensors are comprised of two fluorescent proteins and a sensing domain that undergoes a conformational change upon binding the target ligand. These sensors can be genetically encoded and hence incorporated into cells by transgenic technologies. Here we discuss the latest developments in our efforts to reengineer calcium sensors as well as develop new sensors for zinc. In these efforts we employ a combination of naturally occurring calcium and zinc binding domains, combined with protein engineering. We are also developing new methodologies to screen and sort sensor libraries using optically-integrated microfluidic devices. Thus far, we have targeted sensors to the ER, mitochondria, Golgi, nucleus, and plasma membrane in order to examine the spatial heterogeneity and localization of signaling processes.

  7. Encoding and decoding time in neural development.

    PubMed

    Toma, Kenichi; Wang, Tien-Cheng; Hanashima, Carina

    2016-01-01

    The development of a multicellular organism involves time-dependent changes in molecular and cellular states; therefore 'time' is an indispensable mathematical parameter of ontogenesis. Regardless of their inextricable relationship, there is a limited number of events for which the output of developmental phenomena primarily uses temporal cues that are generated through multilevel interactions between molecules, cells, and tissues. In this review, we focus on neural stem cells, which serve as a faithful decoder of temporal cues to transmit biological information and generate specific output in the developing nervous system. We further explore the identity of the temporal information that is encoded in neural development, and how this information is decoded into various cellular fate decisions. © 2016 The Authors Development, Growth & Differentiation published by John Wiley & Sons Australia, Ltd on behalf of Japanese Society of Developmental Biologists.

  8. Schistosome satellite DNA encodes active hammerhead ribozymes.

    PubMed

    Ferbeyre, G; Smith, J M; Cedergren, R

    1998-07-01

    Using a computer program designed to search for RNA structural motifs in sequence databases, we have found a hammerhead ribozyme domain encoded in the Smalpha repetitive DNA of Schistosoma mansoni. Transcripts of these repeats are expressed as long multimeric precursor RNAs that cleave in vitro and in vivo into unit-length fragments. This RNA domain is able to engage in both cis and trans cleavage typical of the hammerhead ribozyme. Further computer analysis of S. mansoni DNA identified a potential trans cleavage site in the gene coding for a synaptobrevin-like protein, and RNA transcribed from this gene was efficiently cleaved by the Smalpha ribozyme in vitro. Similar families of repeats containing the hammerhead domain were found in the closely related Schistosoma haematobium and Schistosomatium douthitti species but were not present in Schistosoma japonicum or Heterobilharzia americana, suggesting that the hammerhead domain was not acquired from a common schistosome ancestor.

  9. Designing and encoding models for synthetic biology

    PubMed Central

    Endler, Lukas; Rodriguez, Nicolas; Juty, Nick; Chelliah, Vijayalakshmi; Laibe, Camille; Li, Chen; Le Novère, Nicolas

    2009-01-01

    A key component of any synthetic biology effort is the use of quantitative models. These models and their corresponding simulations allow optimization of a system design, as well as guiding their subsequent analysis. Once a domain mostly reserved for experts, dynamical modelling of gene regulatory and reaction networks has been an area of growth over the last decade. There has been a concomitant increase in the number of software tools and standards, thereby facilitating model exchange and reuse. We give here an overview of the model creation and analysis processes as well as some software tools in common use. Using markup language to encode the model and associated annotation, we describe the mining of components, their integration in relational models, formularization and parametrization. Evaluation of simulation results and validation of the model close the systems biology ‘loop’. PMID:19364720

  10. Ribosomally encoded cyclic peptide toxins from mushrooms.

    PubMed

    Walton, Jonathan D; Luo, Hong; Hallen-Adams, Heather

    2012-01-01

    The cyclic peptide toxins of poisonous Amanita mushrooms are chemically unique among known natural products. Furthermore, they differ from other fungal cyclic peptides in being synthesized on ribosomes instead of by nonribosomal peptide synthetases. Because of their novel structures and biogenic origins, elucidation of the biosynthetic pathway of the Amanita cyclic peptides presents both challenges and opportunities. In particular, a full understanding of the pathway should lead to the ability to direct synthesis of a large number of novel cyclic peptides based on the Amanita toxin scaffold by genetic engineering of the encoding genes. Here, we highlight some of the principal methods for working with the Amanita cyclic peptides and the known steps in their biosynthesis.

  11. Visually lossless encoding for JPEG2000.

    PubMed

    Oh, Han; Bilgin, Ali; Marcellin, Michael W

    2013-01-01

    Due to exponential growth in image sizes, visually lossless coding is increasingly being considered as an alternative to numerically lossless coding, which has limited compression ratios. This paper presents a method of encoding color images in a visually lossless manner using JPEG2000. In order to hide coding artifacts caused by quantization, visibility thresholds (VTs) are measured and used for quantization of subband signals in JPEG2000. The VTs are experimentally determined from statistically modeled quantization distortion, which is based on the distribution of wavelet coefficients and the dead-zone quantizer of JPEG2000. The resulting VTs are adjusted for locally changing backgrounds through a visual masking model, and then used to determine the minimum number of coding passes to be included in the final codestream for visually lossless quality under the desired viewing conditions. Codestreams produced by this scheme are fully JPEG2000 Part-I compliant.

  12. Ultrasonically encoded photoacoustic flowgraphy in biological tissue.

    PubMed

    Wang, Lidai; Xia, Jun; Yao, Junjie; Maslov, Konstantin I; Wang, Lihong V

    2013-11-15

    Blood flow speed is an important functional parameter. Doppler ultrasound flowmetry lacks sufficient sensitivity to slow blood flow (several to tens of millimeters per second) in deep tissue. To address this challenge, we developed ultrasonically encoded photoacoustic flowgraphy combining ultrasonic thermal tagging with photoacoustic imaging. Focused ultrasound generates a confined heat source in acoustically absorptive fluid. Thermal waves propagate with the flow and are directly visualized in pseudo color using photoacoustic computed tomography. The Doppler shift is employed to calculate the flow speed. This method requires only acoustic and optical absorption, and thus is applicable to continuous fluid. A blood flow speed as low as 0.24  mm·s(-1)} was successfully measured. Deep blood flow imaging was experimentally demonstrated under 5-mm-thick chicken breast tissue.

  13. Ultrathin nonlinear metasurface for optical image encoding.

    PubMed

    Walter, Felicitas; Li, Guixin; Meier, Cedrik; Zhang, Shuang; Zentgraf, Thomas

    2017-04-14

    Security of optical information is of great importance in modern society. Many cryptography techniques based on classical and quantum optics have been widely explored in the linear optical regime. Nonlinear optical encryption, in which encoding and decoding involve nonlinear frequency conversions, represents a new strategy for securing optical information. Here, we demonstrate that an ultrathin nonlinear photonic metasurface, consisting of meta-atoms with three-fold rotational symmetry, can be used to hide optical images under illumination with a fundamental wave. However, the hidden image can be read out from second harmonic generation (SHG) waves. This is achieved by controlling the destructive and constructive interferences of SHG waves from two neighboring meta-atoms. In addition, we apply this concept to obtain grey-scale SHG imaging. Nonlinear metasurfaces based on space variant optical interference open new avenues for multi-level image encryption, anti-counterfeiting and background free image reconstruction.

  14. Photoelectrical encoder employing an optical grating

    SciTech Connect

    Kabaya, Y.

    1985-02-12

    A photoelectrical encoder is disclosed wherein a physical quantity is detected from brightness obtained by moving a first and a second scale. Each scale is provided thereon with an optical grating relative to each other. The grating in one of the scales is constructed such that a first signal lead-out material layer made of a light shielding conductive material, a PN semiconductor layer for converting light into electricity, and a second signal lead-out material layer made of a light transmitting conductive material are laminated on a light transmitting base member to form a narrow belt-shaped light receiving portion and a plurality of narrow belt-shaped light receiving portions arranged at regular pitches. Against the light from the light transmitting base member, the light receiving portions function as light shielding slits, and intervals between the light receiving portions are formed into light transmitting slits.

  15. Brain Circuits Encoding Reward from Pain Relief

    PubMed Central

    Navratilova, Edita; Atcherley, Christopher; Porreca, Frank

    2015-01-01

    Relief from pain in humans is rewarding and pleasurable. Primary rewards, or reward predictive cues, are encoded in brain reward/motivational circuits. While considerable advances have been made in our understanding of reward circuits underlying positive reinforcement, less is known about the circuits underlying the hedonic and reinforcing actions of pain relief. We review findings from electrophysiological, neuroimaging and behavioral studies supporting the concept that the rewarding effect of pain relief requires opioid signaling in the anterior cingulate cortex, activation of midbrain dopamine neurons and release of dopamine in the nucleus accumbens. Understanding of circuits that govern the reward of pain relief may allow the discovery of more effective and satisfying therapies for patients with acute and chronic pain. PMID:26603560

  16. Ultrasonically Encoded Photoacoustic Flowgraphy in Biological Tissue

    NASA Astrophysics Data System (ADS)

    Wang, Lidai; Xia, Jun; Yao, Junjie; Maslov, Konstantin I.; Wang, Lihong V.

    2013-11-01

    Blood flow speed is an important functional parameter. Doppler ultrasound flowmetry lacks sufficient sensitivity to slow blood flow (several to tens of millimeters per second) in deep tissue. To address this challenge, we developed ultrasonically encoded photoacoustic flowgraphy combining ultrasonic thermal tagging with photoacoustic imaging. Focused ultrasound generates a confined heat source in acoustically absorptive fluid. Thermal waves propagate with the flow and are directly visualized in pseudo color using photoacoustic computed tomography. The Doppler shift is employed to calculate the flow speed. This method requires only acoustic and optical absorption, and thus is applicable to continuous fluid. A blood flow speed as low as 0.24mm·s-1 was successfully measured. Deep blood flow imaging was experimentally demonstrated under 5-mm-thick chicken breast tissue.

  17. Encoding and Decoding Models in Cognitive Electrophysiology.

    PubMed

    Holdgraf, Christopher R; Rieger, Jochem W; Micheli, Cristiano; Martin, Stephanie; Knight, Robert T; Theunissen, Frederic E

    2017-01-01

    Cognitive neuroscience has seen rapid growth in the size and complexity of data recorded from the human brain as well as in the computational tools available to analyze this data. This data explosion has resulted in an increased use of multivariate, model-based methods for asking neuroscience questions, allowing scientists to investigate multiple hypotheses with a single dataset, to use complex, time-varying stimuli, and to study the human brain under more naturalistic conditions. These tools come in the form of "Encoding" models, in which stimulus features are used to model brain activity, and "Decoding" models, in which neural features are used to generated a stimulus output. Here we review the current state of encoding and decoding models in cognitive electrophysiology and provide a practical guide toward conducting experiments and analyses in this emerging field. Our examples focus on using linear models in the study of human language and audition. We show how to calculate auditory receptive fields from natural sounds as well as how to decode neural recordings to predict speech. The paper aims to be a useful tutorial to these approaches, and a practical introduction to using machine learning and applied statistics to build models of neural activity. The data analytic approaches we discuss may also be applied to other sensory modalities, motor systems, and cognitive systems, and we cover some examples in these areas. In addition, a collection of Jupyter notebooks is publicly available as a complement to the material covered in this paper, providing code examples and tutorials for predictive modeling in python. The aim is to provide a practical understanding of predictive modeling of human brain data and to propose best-practices in conducting these analyses.

  18. The gene encoding proopiomelanocortin in the dog.

    PubMed

    Mol, J A; van Mansfeld, A D; Kwant, M M; van Wolferen, M; Rothuizen, J

    1991-01-01

    The regulation of the synthesis of ACTH in the dog is of interest for studies of the physiology of the pituitary-adrenocortical axis as well as for studies of the pathogenesis of pituitary-dependent hyperadrenocorticism. Despite this broad interest the nucleotide sequence encoding ACTH and its precursor proopiomelanocortin (POMC) is not known, nor is it clear whether there are differences in POMC mRNA from the anterior lobe or the intermediate lobe of the normal pituitary or from pituitary tumours causing ACTH excess. Following the preparation of a cDNA library from the canine intermediate lobe of the pituitary gland, the part of the mRNA that is translated into the proopiomelanocortin prohormone was amplified using a polymerase chain reaction. Sequence analysis revealed the highest homology with the porcine mRNA sequence. Translation in a single reading frame revealed highly homologous areas in the amino-terminal, carboxy-terminal, and ACTH part of the prohormone, whereas a high diversity was noticed at the sequence preceding ACTH and the beginning of beta-lipotropin. Northern blot analysis disclosed the presence of a POMC mRNA of approximately 1300 nucleotides. There were no size differences between the anterior lobe, intermediate lobe, and pituitary tumour derived POMC mRNA. The highest expression levels of POMC mRNA as related to the expression of the gene encoding glyceraldehyde-3-phosphate dehydrogenase were found in the intermediate lobe of the canine pituitary gland. It is concluded that excessive production of ACTH by pituitary tumours is not caused by relatively high expression levels or alterations in the size of mRNA.

  19. Miniature Grating for Spectrally-Encoded Endoscopy

    PubMed Central

    Kang, Dongkyun; Martinez, Ramses V.; Whitesides, George M.

    2013-01-01

    Spectrally-encoded endoscopy (SEE) is an ultraminiature endoscopy technology that acquires high-definition images of internal organs through a sub-mm endoscopic probe. In SEE, a grating at the tip of the imaging optics diffracts the broadband light into multiple beams, where each beam with a distinctive wavelength is illuminated on a unique transverse location of the tissue. By encoding one transverse coordinate with the wavelength, SEE can image a line of the tissue at a time without using any beam scanning devices. This feature of the SEE technology allows the SEE probe to be miniaturized to sub-mm dimensions. While previous studies have shown that SEE has the potential to be utilized for various clinical imaging applications, the translation of SEE for medicine has been hampered by challenges in fabricating the miniature grating inherent to SEE probes. This paper describes a new fabrication method for SEE probes. The new method uses a soft lithographic approach to pattern a high-aspect-ratio grating at the tip of the miniature imaging optics. Using this technique, we have constructed a 500-μm-diameter SEE probe. The miniature grating at the tip of the probe had a measured diffraction efficiency of 75%. The new SEE probe was used to image a human finger and formalin fixed mouse embryos, demonstrating the capability of this device to visualize key anatomic features of tissues with high image contrast. In addition to providing high quality imaging SEE optics, the soft lithography method allows cost-effective and reliable fabrication of these miniature endoscopes, which will facilitate the clinical translation of SEE technology. PMID:23503940

  20. Encoding continuous spatial phenomena in GML

    NASA Astrophysics Data System (ADS)

    de Vries, M. E.; Ledoux, H.

    2009-04-01

    In the discussion about how to model and encode geographic information two meta-models of space exist: the 'object' view and the 'field' view. This difference in conceptual view is also reflected in different data models and encoding formats. Among GIS practitioners, ‘fields' (or ‘coverages') are being used almost exclusively in 2D, while in the geoscience community 3D and higher-dimensional fields are widely used. (Note that the dimensions in oceanographic/atmospheric coverages are not necessarily spatial dimensions, as any parameters (e.g. temperature of the air, or density of water) can be considered a dimension.) While standardisation work in ISO and OGC has led to agreement on how to best encode discrete spatial objects, for the modelling and encoding of continuous ‘fields' there are still a number of open issues. In the presentation we will shortly discuss the current standards related to fields, and look at their shortcomings and potential. In ISO 19123 for example a distinction is made between discrete and continuous coverages, but the difference is not very clear and hard to capture for implementers. As far as encoding is concerned: GML 3.x (ISO 19136) has a discrete coverage data type, but no continuous coverage type. We will then present an alternative solution to model fields, and show how it can be implemented using some parts of GML, but not the ISO/GML coverage type. This alternative data model for fields permits us to represent fields in 2D and 3D, although conceptually it can be easily extended to higher dimensions. Unlike current standards where there is a distinction between discrete and continuous fields/coverages, we argue that a field should always have one - and only one! - value for a given attribute at every location in the spatial domain (be this domain the surface of the Earth, a 3D volume, or even a 4D spatio-temporal hypercube). The principal idea behind the proposed model is that two things are needed to have a coverage: 1. a set

  1. Modular verification of chemical reaction network encodings via serializability analysis

    PubMed Central

    Lakin, Matthew R.; Stefanovic, Darko; Phillips, Andrew

    2015-01-01

    Chemical reaction networks are a powerful means of specifying the intended behaviour of synthetic biochemical systems. A high-level formal specification, expressed as a chemical reaction network, may be compiled into a lower-level encoding, which can be directly implemented in wet chemistry and may itself be expressed as a chemical reaction network. Here we present conditions under which a lower-level encoding correctly emulates the sequential dynamics of a high-level chemical reaction network. We require that encodings are transactional, such that their execution is divided by a “commit reaction” that irreversibly separates the reactant-consuming phase of the encoding from the product-generating phase. We also impose restrictions on the sharing of species between reaction encodings, based on a notion of “extra tolerance”, which defines species that may be shared between encodings without enabling unwanted reactions. Our notion of correctness is serializability of interleaved reaction encodings, and if all reaction encodings satisfy our correctness properties then we can infer that the global dynamics of the system are correct. This allows us to infer correctness of any system constructed using verified encodings. As an example, we show how this approach may be used to verify two- and four-domain DNA strand displacement encodings of chemical reaction networks, and we generalize our result to the limit where the populations of helper species are unlimited. PMID:27325906

  2. Modular verification of chemical reaction network encodings via serializability analysis.

    PubMed

    Lakin, Matthew R; Stefanovic, Darko; Phillips, Andrew

    2016-06-13

    Chemical reaction networks are a powerful means of specifying the intended behaviour of synthetic biochemical systems. A high-level formal specification, expressed as a chemical reaction network, may be compiled into a lower-level encoding, which can be directly implemented in wet chemistry and may itself be expressed as a chemical reaction network. Here we present conditions under which a lower-level encoding correctly emulates the sequential dynamics of a high-level chemical reaction network. We require that encodings are transactional, such that their execution is divided by a "commit reaction" that irreversibly separates the reactant-consuming phase of the encoding from the product-generating phase. We also impose restrictions on the sharing of species between reaction encodings, based on a notion of "extra tolerance", which defines species that may be shared between encodings without enabling unwanted reactions. Our notion of correctness is serializability of interleaved reaction encodings, and if all reaction encodings satisfy our correctness properties then we can infer that the global dynamics of the system are correct. This allows us to infer correctness of any system constructed using verified encodings. As an example, we show how this approach may be used to verify two- and four-domain DNA strand displacement encodings of chemical reaction networks, and we generalize our result to the limit where the populations of helper species are unlimited.

  3. New Ultra-High Sensitivity, Absolute, Linear, and Rotary Encoders

    NASA Technical Reports Server (NTRS)

    Leviton, Douglas B.

    1998-01-01

    Several new types of absolute optical encoders of both rotary and linear function are discussed. The means for encoding are complete departures from conventional optical encoders and offer advantages of compact form, immunity to damage-induced dropouts of position information, and about an order of magnitude higher sensitivity over what is commercially available. Rotary versions have sensitivity from 0.02 arcseconds down to 0.003 arcsecond while linear models have sensitivity of 10 nm.

  4. Aging affects neural precision of speech encoding

    PubMed Central

    Anderson, Samira; Parbery-Clark, Alexandra; White-Schwoch, Travis; Kraus, Nina

    2012-01-01

    Older adults frequently report they can hear what is said but cannot understand the meaning, especially in noise. This difficulty may arise from the inability to process rapidly changing elements of speech. Aging is accompanied by a general slowing of neural processing and decreased neural inhibition, both of which likely interfere with temporal processing in auditory and other sensory domains. Age-related reductions in inhibitory neurotransmitter levels and delayed neural recovery can contribute to decreases in the auditory system’s temporal precision. Decreased precision may lead to neural timing delays, reductions in neural response magnitude, and a disadvantage in processing the rapid acoustic changes in speech. The auditory brainstem response (ABR), a scalp-recorded electrical potential, is known for its ability to capture precise neural synchrony within subcortical auditory nuclei; therefore, we hypothesized that a loss of temporal precision results in subcortical timing delays and decreases in response consistency and magnitude. To assess this hypothesis, we recorded ABRs to the speech syllable /da/ in normal hearing younger (ages 18 to 30) and older adult humans (60 to 67). Older adults had delayed ABRs, especially in response to the rapidly changing formant transition, and greater response variability. We also found that older adults had decreased phase locking and smaller response magnitudes than younger adults. Taken together, our results support the theory that older adults have a loss of temporal precision in subcortical encoding of sound, which may account, at least in part, for their difficulties with speech perception. PMID:23055485

  5. Encoding pitch contours using current steering.

    PubMed

    Luo, Xin; Landsberger, David M; Padilla, Monica; Srinivasan, Arthi G

    2010-09-01

    This study investigated cochlear implant (CI) users' ability to perceive pitch cues from time-varying virtual channels (VCs) to identify pitch contours. Seven CI users were tested on apical, medial, and basal electrode pairs with stimulus durations from 100 to 1000 ms. In one stimulus set, 9 pitch contours were created by steering current between the component electrodes and the VC halfway between the electrodes. Another stimulus set only contained 3 pitch contours (flat, falling, and rising). VC discrimination was also tested on the same electrodes. The total current level of dual-electrode stimuli was linearly interpolated between those of single-electrode stimuli to minimize loudness changes. The results showed that pitch contour identification (PCI) scores were similar across electrode locations, and significantly improved at longer durations. For durations longer than 300 ms, 2 subjects had nearly perfect 9-contour identification, and 5 subjects perfectly identified the 3 basic contours. Both PCI and VC discrimination varied greatly across subjects. Cumulative d(') values for VC discrimination were significantly correlated with 100-, 200-, and 500-ms PCI scores. These results verify the feasibility of encoding pitch contours using current steering, and suggest that identification of such pitch contours strongly relies on CI users' sensitivity to VCs.

  6. Categorical encoding of color in the brain

    PubMed Central

    Bird, Chris M.; Berens, Samuel C.; Horner, Aidan J.; Franklin, Anna

    2014-01-01

    The areas of the brain that encode color categorically have not yet been reliably identified. Here, we used functional MRI adaptation to identify neuronal populations that represent color categories irrespective of metric differences in color. Two colors were successively presented within a block of trials. The two colors were either from the same or different categories (e.g., “blue 1 and blue 2” or “blue 1 and green 1”), and the size of the hue difference was varied. Participants performed a target detection task unrelated to the difference in color. In the middle frontal gyrus of both hemispheres and to a lesser extent, the cerebellum, blood-oxygen level-dependent response was greater for colors from different categories relative to colors from the same category. Importantly, activation in these regions was not modulated by the size of the hue difference, suggesting that neurons in these regions represent color categorically, regardless of metric color difference. Representational similarity analyses, which investigated the similarity of the pattern of activity across local groups of voxels, identified other regions of the brain (including the visual cortex), which responded to metric but not categorical color differences. Therefore, categorical and metric hue differences appear to be coded in qualitatively different ways and in different brain regions. These findings have implications for the long-standing debate on the origin and nature of color categories, and also further our understanding of how color is processed by the brain. PMID:24591602

  7. Comparative genomics of Shiga toxin encoding bacteriophages

    PubMed Central

    2012-01-01

    Background Stx bacteriophages are responsible for driving the dissemination of Stx toxin genes (stx) across their bacterial host range. Lysogens carrying Stx phages can cause severe, life-threatening disease and Stx toxin is an integral virulence factor. The Stx-bacteriophage vB_EcoP-24B, commonly referred to as Ф24B, is capable of multiply infecting a single bacterial host cell at a high frequency, with secondary infection increasing the rate at which subsequent bacteriophage infections can occur. This is biologically unusual, therefore determining the genomic content and context of Ф24B compared to other lambdoid Stx phages is important to understanding the factors controlling this phenomenon and determining whether they occur in other Stx phages. Results The genome of the Stx2 encoding phage, Ф24B was sequenced and annotated. The genomic organisation and general features are similar to other sequenced Stx bacteriophages induced from Enterohaemorrhagic Escherichia coli (EHEC), however Ф24B possesses significant regions of heterogeneity, with implications for phage biology and behaviour. The Ф24B genome was compared to other sequenced Stx phages and the archetypal lambdoid phage, lambda, using the Circos genome comparison tool and a PCR-based multi-loci comparison system. Conclusions The data support the hypothesis that Stx phages are mosaic, and recombination events between the host, phages and their remnants within the same infected bacterial cell will continue to drive the evolution of Stx phage variants and the subsequent dissemination of shigatoxigenic potential. PMID:22799768

  8. Genes Encoding Enzymes Involved in Ethanol Metabolism

    PubMed Central

    Hurley, Thomas D.; Edenberg, Howard J.

    2012-01-01

    The effects of beverage alcohol (ethanol) on the body are determined largely by the rate at which it and its main breakdown product, acetaldehyde, are metabolized after consumption. The main metabolic pathway for ethanol involves the enzymes alcohol dehydrogenase (ADH) and aldehyde dehydrogenase (ALDH). Seven different ADHs and three different ALDHs that metabolize ethanol have been identified. The genes encoding these enzymes exist in different variants (i.e., alleles), many of which differ by a single DNA building block (i.e., single nucleotide polymorphisms [SNPs]). Some of these SNPs result in enzymes with altered kinetic properties. For example, certain ADH1B and ADH1C variants that are commonly found in East Asian populations lead to more rapid ethanol breakdown and acetaldehyde accumulation in the body. Because acetaldehyde has harmful effects on the body, people carrying these alleles are less likely to drink and have a lower risk of alcohol dependence. Likewise, an ALDH2 variant with reduced activity results in acetaldehyde buildup and also has a protective effect against alcoholism. In addition to affecting drinking behaviors and risk for alcoholism, ADH and ALDH alleles impact the risk for esophageal cancer. PMID:23134050

  9. Dynamical encoding of looming, receding, and focussing

    NASA Astrophysics Data System (ADS)

    Longtin, Andre; Clarke, Stephen Elisha; Maler, Leonard; CenterNeural Dynamics Collaboration

    This talk will discuss a non-conventional neural coding task that may apply more broadly to many senses in higher vertebrates. We ask whether and how a non-visual sensory system can focus on an object. We present recent experimental and modeling work that shows how the early sensory circuitry of electric sense can perform such neuronal focusing that is manifested behaviorally. This sense is the main one used by weakly electric fish to navigate, locate prey and communicate in the murky waters of their natural habitat. We show that there is a distance at which the Fisher information of a neuron's response to a looming and receding object is maximized, and that this distance corresponds to a behaviorally relevant one chosen by these animals. Strikingly, this maximum occurs at a bifurcation between tonic firing and bursting. We further discuss how the invariance of this distance to signal attributes can arise, a process that first involves power-law spike frequency adaptation. The talk will also highlight the importance of expanding the classic dual neural encoding of contrast using ON and OFF cells in the context of looming and receding stimuli. The authors acknowledge support from CIHR and NSERC.

  10. EEG Correlates of Relative Motion Encoding.

    PubMed

    Thunell, Evelina; Plomp, Gijs; Ögmen, Haluk; Herzog, Michael H

    2016-03-01

    A large portion of the visual cortex is organized retinotopically, but perception is usually non-retinotopic. For example, a reflector on the spoke of a bicycle wheel appears to move on a circular or prolate cycloidal orbit as the bicycle moves forward, while in fact it traces out a curtate cycloidal trajectory. The moving bicycle serves as a non-retinotopic reference system to which the motion of the reflector is anchored. To study the neural correlates of non-retinotopic motion processing, we used the Ternus-Pikler display, where retinotopic processing in a stationary reference system is contrasted against non-retinotopic processing in a moving one. Using high-density EEG, we found similar brain responses for both retinotopic and non-retinotopic rotational apparent motion from the earliest evoked peak (around 120 ms) and throughout the rest of the visual processing, but only minor correlates of the motion of the reference system itself (mainly around 100-120 ms). We suggest that the visual system efficiently discounts the motion of the reference system from early on, allowing a largely reference system independent encoding of the motion of object parts.

  11. Peafowl antipredator calls encode information about signalers.

    PubMed

    Yorzinski, Jessica L

    2014-02-01

    Animals emit vocalizations that convey information about external events. Many of these vocalizations, including those emitted in response to predators, also encode information about the individual that produced the call. The relationship between acoustic features of antipredator calls and information relating to signalers (including sex, identity, body size, and social rank) were examined in peafowl (Pavo cristatus). The "bu-girk" antipredator calls of male and female peafowl were recorded and 20 acoustic parameters were automatically extracted from each call. Both the bu and girk elements of the antipredator call were individually distinctive and calls were classified to the correct signaler with over 90% and 70% accuracy in females and males, respectively. Females produced calls with a higher fundamental frequency (F0) than males. In both females and males, body size was negatively correlated with F0. In addition, peahen rank was related to the duration, end mean frequency, and start harmonicity of the bu element. Peafowl antipredator calls contain detailed information about the signaler and can potentially be used by receivers to respond to dangerous situations.

  12. Neural encoding and retrieval of sound sequences.

    PubMed

    Rauschecker, Josef P

    2005-12-01

    Although considerable progress has been made recently in our understanding of the coding of complex sounds in the cerebral cortex, the processing and storage of tone sequences is still poorly understood. We have used functional magnetic resonance imaging to identify brain mechanisms involved in the encoding and retrieval of melodies by studying the anticipation of familiar music. The results suggest a specific role for each of the following brain structures: the anterior part of the right superior temporal cortex, the right inferior frontal cortex and anterior insula, the left anterior prefrontal cortex, the lateral cerebellum, and the anterior cingulate. In a separate study, we investigated single-neuron responses in the auditory cortex of awake behaving monkeys to alternating tone sequences that in humans evoke the perception of "streaming." Depending on the frequency separation between the tones, an initial single stream may segregate into two streams after a build-up period of several seconds. The neural responses in the monkeys' primary auditory cortex (A1) mirror the psychophysical time course extremely well, suggesting that habituation within A1 may be one reason for stream segregation. However, the higher auditory and prefrontal areas found to be activated by musical melodies are expected to interact with primary areas in both bottom-up and top-down fashion to bring about the perceptual organization of sound sequences.

  13. Oligonucleotide and Long Polymeric DNA Encoding

    SciTech Connect

    Miller, E; Mariella Jr., R P; Christian, A T; Gardner, S N; Williams, J M

    2003-11-24

    This report summarizes the work done at Lawrence Livermore National Laboratory for the Oligonucleotide and Long Polymeric DNA Encoding project, part of the Microelectronic Bioprocesses Program at DARPA. The goal of the project was to develop a process by which long (circa 10,000 base-pair) synthetic DNA molecules could be synthesized in a timely and economic manner. During construction of the long molecule, errors in DNA sequence occur during hybridization and/or the subsequent enzymatic process. The work done on this project has resulted in a novel synthesis scheme that we call the parallel pyramid synthesis protocol, the development of a suit of computational tools to minimize and quantify errors in the synthesized DNA sequence, and experimental proof of this technique. The modeling consists of three interrelated modules: the bioinformatics code which determines the specifics of parallel pyramid synthesis for a given chain of long DNA, the thermodynamics code which tracks the products of DNA hybridization and polymerase extension during the later steps in the process, and the kinetics model which examines the temporal and spatial processes during one thermocycle. Most importantly, we conducted the first successful syntheses of a gene using small starting oligomers (tetramers). The synthesized sequence, 813 base pairs long, contained a 725 base pair gene, modified green fluorescent protein (mGFP), which has been shown to be a functional gene by cloning into cells and observing its green fluorescent product.

  14. New insights into cochlear sound encoding

    PubMed Central

    Moser, Tobias; Vogl, Christian

    2016-01-01

    The inner ear uses specialized synapses to indefatigably transmit sound information from hair cells to spiral ganglion neurons at high rates with submillisecond precision. The emerging view is that hair cell synapses achieve their demanding function by employing an unconventional presynaptic molecular composition. Hair cell active zones hold the synaptic ribbon, an electron-dense projection made primarily of RIBEYE, which tethers a halo of synaptic vesicles and is thought to enable a large readily releasable pool of vesicles and to contribute to its rapid replenishment. Another important presynaptic player is otoferlin, coded by a deafness gene, which assumes a multi-faceted role in vesicular exocytosis and, when disrupted, causes auditory synaptopathy. A functional peculiarity of hair cell synapses is the massive heterogeneity in the sizes and shapes of excitatory postsynaptic currents. Currently, there is controversy as to whether this reflects multiquantal release with a variable extent of synchronization or uniquantal release through a dynamic fusion pore. Another important question in the field has been the precise mechanisms of coupling presynaptic Ca 2+ channels and vesicular Ca 2+ sensors. This commentary provides an update on the current understanding of sound encoding in the cochlea with a focus on presynaptic mechanisms. PMID:27635230

  15. Categorical encoding of color in the brain.

    PubMed

    Bird, Chris M; Berens, Samuel C; Horner, Aidan J; Franklin, Anna

    2014-03-25

    The areas of the brain that encode color categorically have not yet been reliably identified. Here, we used functional MRI adaptation to identify neuronal populations that represent color categories irrespective of metric differences in color. Two colors were successively presented within a block of trials. The two colors were either from the same or different categories (e.g., "blue 1 and blue 2" or "blue 1 and green 1"), and the size of the hue difference was varied. Participants performed a target detection task unrelated to the difference in color. In the middle frontal gyrus of both hemispheres and to a lesser extent, the cerebellum, blood-oxygen level-dependent response was greater for colors from different categories relative to colors from the same category. Importantly, activation in these regions was not modulated by the size of the hue difference, suggesting that neurons in these regions represent color categorically, regardless of metric color difference. Representational similarity analyses, which investigated the similarity of the pattern of activity across local groups of voxels, identified other regions of the brain (including the visual cortex), which responded to metric but not categorical color differences. Therefore, categorical and metric hue differences appear to be coded in qualitatively different ways and in different brain regions. These findings have implications for the long-standing debate on the origin and nature of color categories, and also further our understanding of how color is processed by the brain.

  16. Retinal Encoding of Ultrabrief Shape Recognition Cues

    PubMed Central

    Greene, Ernest

    2007-01-01

    Shape encoding mechanisms can be probed by the sequential brief display of dots that mark the boundary of the shape, and delays of less that a millisecond between successive dots can impair recognition. It is not entirely clear whether this is accomplished by preserving stimulus timing in the signal being sent to the brain, or calls for a retinal binding mechanism. Two experiments manipulated the degree of simultaneity among and within dot pairs, requiring also that the pair members be in the same half of the visual field or on opposite halves, i.e., across the midline from one another. Recognition performance was impaired the same for these two conditions. The results make it likely that simultaneity of cues is being registered within the retina. A potential mechanism is suggested, calling for linkage of stimulated sites through activation of PA1 cells. A third experiment confirmed a prior finding that the overall level of recognition deficit is partly a function of display-set size, and affirmed submillisecond resolution in binding dot pairs into effective shape-recognition cues. PMID:17849001

  17. Improved reader for magnetically-encoded ID cards

    NASA Technical Reports Server (NTRS)

    Wu, T. T.

    1979-01-01

    Hybrid demodulator in electronic card reader for magnetically encoded identification cards, accommodates variations in insertion speeds, yet is simpler and less expensive than equivalent all-digital circuits.

  18. Optical position encoder based on four-section diffraction grating

    NASA Astrophysics Data System (ADS)

    Zherdev, A. Y.; Odinokov, S. B.; Lushnikov, D. S.; Markin, V. V.; Gurylev, O. A.; Shishova, M. V.

    2017-05-01

    Optical position encoder consists of movable coding grating and fixed analyzing grating. Light passing and diffracting through these two gratings creates interference signal on optical detector. Decoding of interference signal phase allows to determinate current position. Known optical position encoders use several accurate adjusted optical channels and detectors to gather several signals with different phase for higher encoder accuracy. We propose to use one optical channel with several-section analyzing diffraction grating for this purpose to simplify optical scheme and adjusting requirements. Optical scheme of position encoder based on four-section analyzing diffraction grating is developed and described in this paper.

  19. Encoding complex values using two DLP spatial light modulators

    NASA Astrophysics Data System (ADS)

    Becker, Michael F.; Wu, Sih-Ying; Liang, Jinyang

    2013-03-01

    We present a method to encode complex values into three or four quantized complex values for wavefront modulation using two digital micromirror devices (DMDs). This encoding offers advantages to eliminate the twin image or suppress the zero order diffraction as well to improve hologram fidelity. The optical architecture utilizes a Michelson interferometer with a DMD in Littrow configuration replacing the mirrors to combine the two holograms with the desired phase shift. System performance was examined using numerical simulations and experimental measurements to explore different encoding methods for hologram reconstruction. Both ZOD and conjugate image suppression were demonstrated for different encoding schemes.

  20. Apoferritin-Templated Synthesis of Encoded Metallic Phosphate Nanoparticle Tags

    SciTech Connect

    Liu, Guodong; Wu, Hong; Dohnalkova, Alice; Lin, Yuehe

    2007-07-31

    Encoded metallic-phosphate nanoparticle tags, with distinct encoding patterns, have been prepared using an apoferritin template. A center-cavity structure as well as the disassociation and reconstructive characteristics of apoferritin at different pH environments provide a facile route for preparing such encoded nanoparticle tags. Encapsulation and diffusion approaches have been investigated during the preparation. The encapsulation approach, which is based on the dissociation and reconstruction of apoferritin at different pHs, exhibits an effective route to prepare such encoded metallic-phosphate nanoparticle tags. The compositionally encoded nanoparticle tag leads to a high coding capacity with a large number of distinguishable voltammetric signals, reflecting the predetermined composition of the metal mixture solution (and hence the nanoparticle composition). Releasing the metal components from the nanoparticle tags at pH 4.6 acetate buffer avoids harsh dissolution conditions, such as strong acids. Such a synthesis of encoded nanoparticle tags, including single-component and compositionally encoded nanoparticle tags, is substantially simple, fast, and convenient compared to that of encoded metal nanowires and semiconductor nanoparticle (CdS, PbS, and ZnS) incorporated polystyrene beads. The encoded metallic-phosphate nanoparticle tags thus show great promise for bioanalytical or product-tracking/identification/protection applications.

  1. Novel encoding methods for DNA-templated chemical libraries.

    PubMed

    Li, Gang; Zheng, Wenlu; Liu, Ying; Li, Xiaoyu

    2015-06-01

    Among various types of DNA-encoded chemical libraries, DNA-templated library takes advantage of the sequence-specificity of DNA hybridization, enabling not only highly effective DNA-templated chemical reactions, but also high fidelity in library encoding. This brief review summarizes recent advances that have been made on the encoding strategies for DNA-templated libraries, and it also highlights their respective advantages and limitations for the preparation of DNA-encoded libraries. Copyright © 2015 Elsevier Ltd. All rights reserved.

  2. Two-layer contractive encodings for learning stable nonlinear features.

    PubMed

    Schulz, Hannes; Cho, Kyunghyun; Raiko, Tapani; Behnke, Sven

    2015-04-01

    Unsupervised learning of feature hierarchies is often a good strategy to initialize deep architectures for supervised learning. Most existing deep learning methods build these feature hierarchies layer by layer in a greedy fashion using either auto-encoders or restricted Boltzmann machines. Both yield encoders which compute linear projections of input followed by a smooth thresholding function. In this work, we demonstrate that these encoders fail to find stable features when the required computation is in the exclusive-or class. To overcome this limitation, we propose a two-layer encoder which is less restricted in the type of features it can learn. The proposed encoder is regularized by an extension of previous work on contractive regularization. This proposed two-layer contractive encoder potentially poses a more difficult optimization problem, and we further propose to linearly transform hidden neurons of the encoder to make learning easier. We demonstrate the advantages of the two-layer encoders qualitatively on artificially constructed datasets as well as commonly used benchmark datasets. We also conduct experiments on a semi-supervised learning task and show the benefits of the proposed two-layer encoders trained with the linear transformation of perceptrons.

  3. Double image encryption based on phase-amplitude mixed encoding and multistage phase encoding in gyrator transform domains

    NASA Astrophysics Data System (ADS)

    Wang, Qu; Guo, Qing; Lei, Liang

    2013-06-01

    We present a novel method for double image encryption that is based on amplitude-phase mixed encoding and multistage random phase encoding in gyrator transform (GT) domains. In the amplitude-phase mixed encoding operation, a random binary distribution matrix is defined to mixed encode two primitive images to a single complex-valued image, which is then encrypted into a stationary white noise distribution by the multistage phase encoding with GTs. Compared with the earlier methods that uses fully phase encoding, the proposed method reduces the difference between two primitive images in key space and sensitivity to the GT orders. The primitive images can be recovered exactly by applying correct keys with initial conditions of chaotic system, the GT orders and the pixel scrambling operation. Numerical simulations demonstrate that the proposed scheme has considerably high security level and certain robustness against data loss and noise disturbance.

  4. Encoding frequency contrast in primate auditory cortex

    PubMed Central

    Scott, Brian H.; Semple, Malcolm N.

    2014-01-01

    Changes in amplitude and frequency jointly determine much of the communicative significance of complex acoustic signals, including human speech. We have previously described responses of neurons in the core auditory cortex of awake rhesus macaques to sinusoidal amplitude modulation (SAM) signals. Here we report a complementary study of sinusoidal frequency modulation (SFM) in the same neurons. Responses to SFM were analogous to SAM responses in that changes in multiple parameters defining SFM stimuli (e.g., modulation frequency, modulation depth, carrier frequency) were robustly encoded in the temporal dynamics of the spike trains. For example, changes in the carrier frequency produced highly reproducible changes in shapes of the modulation period histogram, consistent with the notion that the instantaneous probability of discharge mirrors the moment-by-moment spectrum at low modulation rates. The upper limit for phase locking was similar across SAM and SFM within neurons, suggesting shared biophysical constraints on temporal processing. Using spike train classification methods, we found that neural thresholds for modulation depth discrimination are typically far lower than would be predicted from frequency tuning to static tones. This “dynamic hyperacuity” suggests a substantial central enhancement of the neural representation of frequency changes relative to the auditory periphery. Spike timing information was superior to average rate information when discriminating among SFM signals, and even when discriminating among static tones varying in frequency. This finding held even when differences in total spike count across stimuli were normalized, indicating both the primacy and generality of temporal response dynamics in cortical auditory processing. PMID:24598525

  5. What physics is encoded in Maxwell's equations?

    NASA Astrophysics Data System (ADS)

    Kosyakov, B. P.

    2005-08-01

    We reconstruct Maxwell's equations showing that a major part of the information encoded in them is taken from topological properties of spacetime, and the residual information, divorced from geometry, which represents the physical contents of electrodynamics, %these equations, translates into four assumptions:(i) locality; (ii) linearity; %of the dynamical law; (iii) identity of the charge-source and the charge-coupling; and (iv) lack of magnetic monopoles. However, a closer inspection of symmetries peculiar to electrodynamics shows that these assumptions may have much to do with geometry. Maxwell's equations tell us that we live in a three-dimensional space with trivial (Euclidean) topology; time is a one-dimensional unidirectional and noncompact continuum; and spacetime is endowed with a light cone structure readable in the conformal invariance of electrodynamics. Our geometric feelings relate to the fact that Maxwell's equations are built in our brain, hence our space and time orientation, our visualization and imagination capabilities are ensured by perpetual instinctive processes of solving Maxwell's equations. People are usually agree in their observations of angle relations, for example, a right angle is never confused with an angle slightly different from right. By contrast, we may disagree in metric issues, say, a colour-blind person finds the light wave lengths quite different from those found by a man with normal vision. This lends support to the view that conformal invariance of Maxwell's equations is responsible for producing our notion of space. Assuming that our geometric intuition is guided by our innate realization of electrodynamical laws, some abnormal mental phenomena, such as clairvoyance, may have a rational explanation.

  6. Genetically Encoded Protein Sensors of Membrane Potential.

    PubMed

    Storace, Douglas; Rad, Masoud Sepehri; Han, Zhou; Jin, Lei; Cohen, Lawrence B; Hughes, Thom; Baker, Bradley J; Sung, Uhna

    2015-01-01

    Organic voltage-sensitive dyes offer very high spatial and temporal resolution for imaging neuronal function. However these dyes suffer from the drawbacks of non-specificity of cell staining and low accessibility of the dye to some cell types. Further progress in imaging activity is expected from the development of genetically encoded fluorescent sensors of membrane potential. Cell type specificity of expression of these fluorescent protein (FP) voltage sensors can be obtained via several different mechanisms. One is cell type specificity of infection by individual virus subtypes. A second mechanism is specificity of promoter expression in individual cell types. A third, depends on the offspring of transgenic animals with cell type specific expression of cre recombinase mated with an animal that has the DNA for the FP voltage sensor in all of its cells but its expression is dependent on the recombinase activity. Challenges remain. First, the response time constants of many of the new FP voltage sensors are slower (2-10 ms) than those of organic dyes. This results in a relatively small fractional fluorescence change, ΔF/F, for action potentials. Second, the largest signal presently available is only ~40% for a 100 mV depolarization and many of the new probes have signals that are substantially smaller. Large signals are especially important when attempting to detect fast events because the shorter measurement interval results in a relatively small number of detected photons and therefore a relatively large shot noise (see Chap. 1). Another kind of challenge has occurred when attempts were made to transition from one species to another or from one cell type to another or from cell culture to in vivo measurements.Several laboratories have recently described a number of novel FP voltage sensors. Here we attempt to critically review the current status of these developments in terms of signal size, time course, and in vivo function.

  7. Electrocorticogram encoding of upper extremity movement duration.

    PubMed

    Wang, Po T; King, Christine E; McCrimmon, Colin M; Shaw, Susan J; Millett, David E; Liu, Charles Y; Chui, Luis A; Nenadic, Zoran; Do, An H

    2014-01-01

    Electrocorticogram (ECoG) is a promising long-term signal acquisition platform for brain-computer interface (BCI) systems such as upper extremity prostheses. Several studies have demonstrated decoding of arm and finger trajectories from ECoG high-gamma band (80-160 Hz) signals. In this study, we systematically vary the velocity of three elementary movement types (pincer grasp, elbow and shoulder flexion/extension) to test whether the high-gamma band encodes for the entirety of the movements, or merely the movement onset. To this end, linear regression models were created for the durations and amplitudes of high-gamma power bursts and velocity deflections. One subject with 8×8 high-density ECoG grid (4 mm center-to-center electrode spacing) participated in the experiment. The results of the regression models indicated that the power burst durations varied directly with the movement durations (e.g. R(2)=0.71 and slope=1.0 s/s for elbow). The persistence of power bursts for the duration of the movement suggests that the primary motor cortex (M1) is likely active for the entire duration of a movement, instead of providing a marker for the movement onset. On the other hand, the amplitudes were less co-varied. Furthermore, the electrodes of maximum R(2) conformed to somatotopic arrangement of the brain. Also, electrodes responsible for flexion and extension movements could be resolved on the high-density grid. In summary, these findings suggest that M1 may be directly responsible for activating the individual muscle motor units, and future BCI may be able to utilize them for better control of prostheses.

  8. Neutron encoded labeling for peptide identification.

    PubMed

    Rose, Christopher M; Merrill, Anna E; Bailey, Derek J; Hebert, Alexander S; Westphall, Michael S; Coon, Joshua J

    2013-05-21

    Metabolic labeling of cells using heavy amino acids is most commonly used for relative quantitation; however, partner mass shifts also detail the number of heavy amino acids contained within the precursor species. Here, we use a recently developed metabolic labeling technique, NeuCode (neutron encoding) stable isotope labeling with amino acids in cell culture (SILAC), which produces precursor partners spaced ~40 mDa apart to enable amino acid counting. We implement large scale counting of amino acids through a program, "Amino Acid Counter", which determines the most likely combination of amino acids within a precursor based on NeuCode SILAC partner spacing and filters candidate peptide sequences during a database search using this information. Counting the number of lysine residues for precursors selected for MS/MS decreases the median number of candidate sequences from 44 to 14 as compared to an accurate mass search alone (20 ppm). Furthermore, the ability to co-isolate and fragment NeuCode SILAC partners enables counting of lysines in product ions, and when the information is used, the median number of candidates is reduced to 7. We then demonstrate counting leucine in addition to lysine results in a 6-fold decrease in search space, 43 to 7, when compared to an accurate mass search. We use this scheme to analyze a nanoLC-MS/MS experiment and demonstrate that accurate mass plus lysine and leucine counting reduces the number of candidate sequences to one for ~20% of all precursors selected, demonstrating an ability to identify precursors without MS/MS analysis.

  9. Neutron Encoded Labeling for Peptide Identification

    PubMed Central

    Rose, Christopher M.; Merrill, Anna E.; Bailey, Derek J.; Hebert, Alexander S.; Westphall, Michael S.; Coon, Joshua J.

    2013-01-01

    Metabolic labeling of cells using heavy amino acids is most commonly used for relative quantitation; however, partner mass shifts also detail the number of heavy amino acids contained within the precursor species. Here, we use a recently developed metabolic labeling technique, NeuCode (neutron encoding) stable isotope labeling with amino acids in cell culture (SILAC), which produces precursor partners spaced ~40 mDa apart to enable amino acid counting. We implement large scale counting of amino acids through a program, “Amino Acid Counter”, which determines the most likely combination of amino acids within a precursor based on NeuCode SILAC partner spacing and filters candidate peptide sequences during a database search using this information. Counting the number of lysine residues for precursors selected for MS/MS decreases the median number of candidate sequences from 44 to 14 as compared to an accurate mass search alone (20 ppm). Furthermore, the ability to co-isolate and fragment NeuCode SILAC partners enables counting of lysines in product ions, and when the information is used, the median number of candidates is reduced to 7. We then demonstrate counting leucine in addition to lysine results in a 6-fold decrease in search space, 43 to 7, when compared to an accurate mass search. We use this scheme to analyze a nanoLC-MS/MS experiment and demonstrate that accurate mass plus lysine and leucine counting reduces the number of candidate sequences to one for ~20% of all precursors selected, demonstrating an ability to identify precursors without MS/MS analysis. PMID:23638792

  10. Encoding of human action in Broca's area.

    PubMed

    Fazio, Patrik; Cantagallo, Anna; Craighero, Laila; D'Ausilio, Alessandro; Roy, Alice C; Pozzo, Thierry; Calzolari, Ferdinando; Granieri, Enrico; Fadiga, Luciano

    2009-07-01

    Broca's area has been considered, for over a century, as the brain centre responsible for speech production. Modern neuroimaging and neuropsychological evidence have suggested a wider functional role is played by this area. In addition to the evidence that it is involved in syntactical analysis, mathematical calculation and music processing, it has recently been shown that Broca's area may play some role in language comprehension and, more generally, in understanding actions of other individuals. As shown by functional magnetic resonance imaging, Broca's area is one of the cortical areas activated by hand/mouth action observation and it has been proposed that it may form a crucial node of a human mirror-neuron system. If, on the one hand, neuroimaging studies use a correlational approach which cannot offer a final proof for such claims, available neuropsychological data fail to offer a conclusive demonstration for two main reasons: (i) they use tasks taxing both language and action systems; and (ii) they rarely consider the possibility that Broca's aphasics may also be affected by some form of apraxia. We administered a novel action comprehension test--with almost no linguistic requirements--on selected frontal aphasic patients lacking apraxic symptoms. Patients, as well as matched controls, were shown short movies of human actions or of physical events. Their task consisted of ordering, in a temporal sequence, four pictures taken from each movie and randomly presented on the computer screen. Patient's performance showed a specific dissociation in their ability to re-order pictures of human actions (impaired) with respect to physical events (spared). Our study provides a demonstration that frontal aphasics, not affected by apraxia, are specifically impaired in their capability to correctly encode observed human actions.

  11. Stress as a mnemonic filter: Interactions between medial temporal lobe encoding processes and post-encoding stress.

    PubMed

    Ritchey, Maureen; McCullough, Andrew M; Ranganath, Charan; Yonelinas, Andrew P

    2017-01-01

    Acute stress has been shown to modulate memory for recently learned information, an effect attributed to the influence of stress hormones on medial temporal lobe (MTL) consolidation processes. However, little is known about which memories will be affected when stress follows encoding. One possibility is that stress interacts with encoding processes to selectively protect memories that had elicited responses in the hippocampus and amygdala, two MTL structures important for memory formation. There is limited evidence for interactions between encoding processes and consolidation effects in humans, but recent studies of consolidation in rodents have emphasized the importance of encoding "tags" for determining the impact of consolidation manipulations on memory. Here, we used functional magnetic resonance imaging in humans to test the hypothesis that the effects of post-encoding stress depend on MTL processes observed during encoding. We found that changes in stress hormone levels were associated with an increase in the contingency of memory outcomes on hippocampal and amygdala encoding responses. That is, for participants showing high cortisol reactivity, memories became more dependent on MTL activity observed during encoding, thereby shifting the distribution of recollected events toward those that had elicited relatively high activation. Surprisingly, this effect was generally larger for neutral, compared to emotionally negative, memories. The results suggest that stress does not uniformly enhance memory, but instead selectively preserves memories tagged during encoding, effectively acting as mnemonic filter. © 2016 Wiley Periodicals, Inc.

  12. Polypeptides having laccase activity and polynucleotides encoding same

    DOEpatents

    Liu, Ye; Tang, Lan; Duan, Junxin; Zhang, Yu

    2017-08-22

    The present invention relates to isolated polypeptides having laccase activity and polynucleotides encoding the polypeptides and polynucleotides encoding the polypeptides. The invention also relates to nucleic acid constructs, vectors, and host cells comprising the polynucleotides as well as methods of producing and using the polypeptides.

  13. Hierarchical Encoding of Behavior: Translating Perception into Action

    ERIC Educational Resources Information Center

    Hard, Bridgette Martin; Lozano, Sandra C.; Tversky, Barbara

    2006-01-01

    People encode goal-directed behaviors, such as assembling an object, by segmenting them into discrete actions, organized as goal-subgoal hierarchies. Does hierarchical encoding contribute to observational learning? Participants in 3 experiments segmented an object assembly task into coarse and fine units of action and later performed it…

  14. Multiple channel secure communication using chaotic system encoding

    SciTech Connect

    Miller, S.L.

    1996-12-31

    fA new method to encrypt signals using chaotic systems has been developed that offers benefits over conventional chaotic encryption methods. The method simultaneously encodes multiple plaintext streams using a chaotic system; a key is required to extract the plaintext from the chaotic cipertext. A working prototype demonstrates feasibility of the method by simultaneously encoding and decoding multiple audio signals using electrical circuits.

  15. Functional Neuroimaging of Self-Referential Encoding with Age

    ERIC Educational Resources Information Center