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  1. Regulated degradation of the APC coactivator Cdc20

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    Robbins Jonathan A

    2010-09-01

    Full Text Available Abstract Background Cdc20 is a highly conserved activator of the anaphase-promoting complex (APC, promoting cell-cycle-regulated ubiquitination and proteolysis of a number of critical cell-cycle-regulatory targets including securin and mitotic cyclins. APC-Cdc20 activity is tightly regulated, and this regulation is likely important for accurate cell cycle control. One significant component of Cdc20 regulation is thought to be Cdc20 proteolysis. However, published literature suggests different mechanisms and requirements for Cdc20 proteolysis. The degree to which Cdc20 proteolysis is cell-cycle regulated, the dependence of Cdc20 proteolysis on Cdc20 destruction boxes (recognition sequences for APC-mediated ubiqutination, either by Cdc20 or by the related Cdh1 APC activator, and the need for APC itself for Cdc20 proteolysis all have been disputed to varying extents. In animals, Cdc20 proteolysis is thought to be mediated by Cdh1, contributing an intrinsic order of APC activation by Cdc20 and then by Cdh1. One report suggests a Cdh1 requirement for Cdc20 proteolysis in budding yeast; this idea has not been tested further. Results We characterized Cdc20 proteolysis using Cdc20 expressed from its endogenous locus; previous studies generally employed strongly overexpressed Cdc20, which can cause significant artifacts. We analyzed Cdc20 proteolysis with or without mutations in previously identified destruction box sequences, using varying methods of cell cycle synchronization, and in the presence or absence of Cdh1. Cdc20 instability is only partially dependent on destruction boxes. A much stronger dependence on Cdh1 for Cdc20 proteolysis was observed, but Cdh1-independent proteolysis was also clearly observed. Cdc20 proteolysis independent of both destruction boxes and Cdh1 was especially detectable around the G1/S transition; Cdh1-dependent proteolysis was most notable in late mitosis and G1. Conclusions Cdc20 proteolysis is under complex control

  2. Cdc20 mediates D-box-dependent degradation of Sp100

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    Wang, Ran; Li, Ke-min; Zhou, Cai-hong; Xue, Jing-lun [State Key Laboratory of Genetic Engineering, Institute of Genetics, School of Life Science, Fudan University, Shanghai (China); Ji, Chao-neng, E-mail: Chnji@fudan.edu.cn [State Key Laboratory of Genetic Engineering, Institute of Genetics, School of Life Science, Fudan University, Shanghai (China); Chen, Jin-zhong, E-mail: kingbellchen@fudan.edu.cn [State Key Laboratory of Genetic Engineering, Institute of Genetics, School of Life Science, Fudan University, Shanghai (China)

    2011-12-02

    Highlights: Black-Right-Pointing-Pointer Cdc20 is a co-activator of APC/C complex. Black-Right-Pointing-Pointer Cdc20 recruits Sp100 and mediates its degradation. Black-Right-Pointing-Pointer The D-box of Sp100 is required for Cdc20-mediated degradation. Black-Right-Pointing-Pointer Sp100 expresses consistently at both the mRNA and protein levels in cell cycle. -- Abstract: Cdc20 is a co-activator of the anaphase-promoting complex/cyclosome (APC/C complex), which recruits substrates at particular phases of the cell cycle and mediates their degradation. Sp100 is a PML-NB scaffold protein, which localizes to nuclear particles during interphase and disperses from them during mitosis, participates in viral resistance, transcriptional regulation, and apoptosis. However, its metabolism during the cell cycle has not yet been fully characterized. We found a putative D-box in Sp100 using the Eukaryotic Linear Motif (ELM) predictor database. The putative D-box of Sp100 was verified by mutational analysis. Overexpression of Cdc20 resulted in decreased levels of both endogenous Sp100 protein and overexpressed Sp100 mRNA in HEK 293 cells. Only an overexpressed D-box deletion mutant of Sp100 accumulated in HEK293 cells that also overexpressed Cdc20. Cdc20 knockdown by cdc20 specific siRNA resulted in increased Sp100 protein levels in cells. Furthermore, we discovered that the Cdc20 mediated degradation of Sp100 is diminished by the proteasome inhibitor MG132, which suggests that the ubiquitination pathway is involved in this process. However, unlike the other Cdc20 substrates, which display oscillating protein levels, the level of Sp100 protein remains constant throughout the cell cycle. Additionally, both overexpression and knockdown of endogenous Sp100 had no effect on the cell cycle. Our results suggested that sp100 is a novel substrate of Cdc20 and it is degraded by the ubiquitination pathway. The intact D-box of Sp100 was necessary for this process. These findings expand

  3. Cdc20 mediates D-box-dependent degradation of Sp100

    International Nuclear Information System (INIS)

    Wang, Ran; Li, Ke-min; Zhou, Cai-hong; Xue, Jing-lun; Ji, Chao-neng; Chen, Jin-zhong

    2011-01-01

    Highlights: ► Cdc20 is a co-activator of APC/C complex. ► Cdc20 recruits Sp100 and mediates its degradation. ► The D-box of Sp100 is required for Cdc20-mediated degradation. ► Sp100 expresses consistently at both the mRNA and protein levels in cell cycle. -- Abstract: Cdc20 is a co-activator of the anaphase-promoting complex/cyclosome (APC/C complex), which recruits substrates at particular phases of the cell cycle and mediates their degradation. Sp100 is a PML-NB scaffold protein, which localizes to nuclear particles during interphase and disperses from them during mitosis, participates in viral resistance, transcriptional regulation, and apoptosis. However, its metabolism during the cell cycle has not yet been fully characterized. We found a putative D-box in Sp100 using the Eukaryotic Linear Motif (ELM) predictor database. The putative D-box of Sp100 was verified by mutational analysis. Overexpression of Cdc20 resulted in decreased levels of both endogenous Sp100 protein and overexpressed Sp100 mRNA in HEK 293 cells. Only an overexpressed D-box deletion mutant of Sp100 accumulated in HEK293 cells that also overexpressed Cdc20. Cdc20 knockdown by cdc20 specific siRNA resulted in increased Sp100 protein levels in cells. Furthermore, we discovered that the Cdc20 mediated degradation of Sp100 is diminished by the proteasome inhibitor MG132, which suggests that the ubiquitination pathway is involved in this process. However, unlike the other Cdc20 substrates, which display oscillating protein levels, the level of Sp100 protein remains constant throughout the cell cycle. Additionally, both overexpression and knockdown of endogenous Sp100 had no effect on the cell cycle. Our results suggested that sp100 is a novel substrate of Cdc20 and it is degraded by the ubiquitination pathway. The intact D-box of Sp100 was necessary for this process. These findings expand our knowledge of both Sp100 and Cdc20 as well as their role in ubiquitination.

  4. Conserved CDC20 cell cycle functions are carried out by two of the five isoforms in Arabidopsis thaliana.

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    Zoltán Kevei

    Full Text Available The CDC20 and Cdh1/CCS52 proteins are substrate determinants and activators of the Anaphase Promoting Complex/Cyclosome (APC/C E3 ubiquitin ligase and as such they control the mitotic cell cycle by targeting the degradation of various cell cycle regulators. In yeasts and animals the main CDC20 function is the destruction of securin and mitotic cyclins. Plants have multiple CDC20 gene copies whose functions have not been explored yet. In Arabidopsis thaliana there are five CDC20 isoforms and here we aimed at defining their contribution to cell cycle regulation, substrate selectivity and plant development.Studying the gene structure and phylogeny of plant CDC20s, the expression of the five AtCDC20 gene copies and their interactions with the APC/C subunit APC10, the CCS52 proteins, components of the mitotic checkpoint complex (MCC and mitotic cyclin substrates, conserved CDC20 functions could be assigned for AtCDC20.1 and AtCDC20.2. The other three intron-less genes were silent and specific for Arabidopsis. We show that AtCDC20.1 and AtCDC20.2 are components of the MCC and interact with mitotic cyclins with unexpected specificity. AtCDC20.1 and AtCDC20.2 are expressed in meristems, organ primordia and AtCDC20.1 also in pollen grains and developing seeds. Knocking down both genes simultaneously by RNAi resulted in severe delay in plant development and male sterility. In these lines, the meristem size was reduced while the cell size and ploidy levels were unaffected indicating that the lower cell number and likely slowdown of the cell cycle are the cause of reduced plant growth.The intron-containing CDC20 gene copies provide conserved and redundant functions for cell cycle progression in plants and are required for meristem maintenance, plant growth and male gametophyte formation. The Arabidopsis-specific intron-less genes are possibly "retrogenes" and have hitherto undefined functions or are pseudogenes.

  5. Interphase APC/C-Cdc20 inhibition by cyclin A2-Cdk2 ensures efficient mitotic entry

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    Hein, Jamin B; Nilsson, Jakob

    2016-01-01

    Proper cell-cycle progression requires tight temporal control of the Anaphase Promoting Complex/Cyclosome (APC/C), a large ubiquitin ligase that is activated by one of two co-activators, Cdh1 or Cdc20. APC/C and Cdc20 are already present during interphase but APC/C-Cdc20 regulation during...... this window of the cell cycle, if any, is unknown. Here we show that cyclin A2-Cdk2 binds and phosphorylates Cdc20 in interphase and this inhibits APC/C-Cdc20 activity. Preventing Cdc20 phosphorylation results in pre-mature activation of the APC/C-Cdc20 and several substrates, including cyclin B1 and A2......, are destabilized which lengthens G2 and slows mitotic entry. Expressing non-degradable cyclin A2 but not cyclin B1 restores mitotic entry in these cells. We have thus uncovered a novel positive feedback loop centred on cyclin A2-Cdk2 inhibition of interphase APC/C-Cdc20 to allow further cyclin A2 accumulation...

  6. The PP2AB56 phosphatase promotes the association of Cdc20 with APC/C in mitosis.

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    Lee, Sun Joo; Rodriguez-Bravo, Veronica; Kim, Hyunjung; Datta, Sutirtha; Foley, Emily A

    2017-05-15

    PP2A comprising B56 regulatory subunit isoforms (PP2A B56 ) is a serine/threonine phosphatase essential for mitosis. At the kinetochore, PP2A B56 both stabilizes microtubule binding and promotes silencing of the spindle assembly checkpoint (SAC) through its association with the SAC protein BubR1. Cells depleted of the B56 regulatory subunits of PP2A are delayed in activation of Cdc20-containing APC/C (APC/C Cdc20 ), which is an essential step for mitotic exit. It has been hypothesized that this delay arises from increased production of the mitotic checkpoint complex (MCC), an APC/C Cdc20 inhibitor formed at unattached kinetochores through SAC signaling. In contrast to this prediction, we show that depletion of B56 subunits does not increase the amount or stability of the MCC. Rather, delays in APC/C Cdc20 activation in B56-depleted cells correlate with impaired Cdc20 binding to APC/C. Stimulation of APC/C Cdc20 assembly does not require binding between PP2A B56 and BubR1, and thus this contribution of PP2A B56 towards mitotic exit is distinct from its functions at kinetochores. PP2A B56 associates with APC/C constitutively in a BubR1-independent manner. A mitotic phosphorylation site on Cdc20, known to be a substrate of PP2A B56 , modulates APC/C Cdc20 assembly. These results elucidate the contributions of PP2A B56 towards completion of mitosis. © 2017. Published by The Company of Biologists Ltd.

  7. The internal Cdc20 binding site in BubR1 facilitates both spindle assembly checkpoint signalling and silencing

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    Lischetti, Tiziana; Zhang, Gang; Sedgwick, Garry G

    2014-01-01

    Improperly attached kinetochores activate the spindle assembly checkpoint (SAC) and by an unknown mechanism catalyse the binding of two checkpoint proteins, Mad2 and BubR1, to Cdc20 forming the mitotic checkpoint complex (MCC). Here, to address the functional role of Cdc20 kinetochore localization...... in the SAC, we delineate the molecular details of its interaction with kinetochores. We find that BubR1 recruits the bulk of Cdc20 to kinetochores through its internal Cdc20 binding domain (IC20BD). We show that preventing Cdc20 kinetochore localization by removal of the IC20BD has a limited effect...... on the SAC because the IC20BD is also required for efficient SAC silencing. Indeed, the IC20BD can disrupt the MCC providing a mechanism for its role in SAC silencing. We thus uncover an unexpected dual function of the second Cdc20 binding site in BubR1 in promoting both efficient SAC signalling and SAC...

  8. Cdc20 control of cell fate during prolonged mitotic arrest

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    Nilsson, Jakob

    2011-01-01

    The fate of cells arrested in mitosis by antimitotic compounds is complex but is influenced by competition between pathways promoting cell death and pathways promoting mitotic exit. As components of both of these pathways are regulated by Cdc20-dependent degradation, I hypothesize that variations...

  9. Nek2A destruction marks APC/C activation at the prophase-to-prometaphase transition by spindle-checkpoint-restricted Cdc20.

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    Boekhout, Michiel; Wolthuis, Rob

    2015-04-15

    Nek2 isoform A (Nek2A) is a presumed substrate of the anaphase-promoting complex/cyclosome containing Cdc20 (APC/C(Cdc20)). Nek2A, like cyclin A, is degraded in mitosis while the spindle checkpoint is active. Cyclin A prevents spindle checkpoint proteins from binding to Cdc20 and is recruited to the APC/C in prometaphase. We found that Nek2A and cyclin A avoid being stabilized by the spindle checkpoint in different ways. First, enhancing mitotic checkpoint complex (MCC) formation by nocodazole treatment inhibited the degradation of geminin and cyclin A, whereas Nek2A disappeared at a normal rate. Second, depleting Cdc20 effectively stabilized cyclin A but not Nek2A. Nevertheless, Nek2A destruction crucially depended on Cdc20 binding to the APC/C. Third, in contrast to cyclin A, Nek2A was recruited to the APC/C before the start of mitosis. Interestingly, the spindle checkpoint very effectively stabilized an APC/C-binding mutant of Nek2A, which required the Nek2A KEN box. Apparently, in cells, the spindle checkpoint primarily prevents Cdc20 from binding destruction motifs. Nek2A disappearance marks the prophase-to-prometaphase transition, when Cdc20, regardless of the spindle checkpoint, activates the APC/C. However, Mad2 depletion accelerated Nek2A destruction, showing that spindle checkpoint release further increases APC/C(Cdc20) catalytic activity. © 2015. Published by The Company of Biologists Ltd.

  10. Ubiquitination of Cdc20 by the APC occurs through an intramolecular mechanism

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    Foe, Ian T.; Foster, Scott A.; Cheung, Stephanie K.; DeLuca, Steven Z.; Morgan, David O.; Toczyski, David P.

    2012-01-01

    SUMMARY Background Cells control progression through late mitosis by regulating Cdc20 and Cdh1, the two mitotic activators of the Anaphase Promoting Complex (APC). The control of Cdc20 protein levels during the cell cycle is not well understood. Results Here, we demonstrate that Cdc20 is degraded in budding yeast by multiple APC-dependent mechanisms. We find that the majority of Cdc20 turnover does not involve a second activator molecule, but instead depends on in cis Cdc20 autoubiquitination while it is bound to its activator-binding site on the APC core. Unlike in trans ubiquitination of Cdc20 substrates, the APC ubiquitinates Cdc20 independent of APC activation by Cdc20’s C-box. Cdc20 turnover by this intramolecular mechanism is cell cycle-regulated, contributing to the decline in Cdc20 levels that occurs after anaphase. Interestingly, high substrate levels in vitro significantly reduce Cdc20 autoubiquitination. Conclusion We show here that Cdc20 fluctuates through the cell cycle via a distinct form of APC-mediated ubiquitination. This in cis autoubiquitination may preferentially occur in early anaphase, following depletion of Cdc20 substrates. This suggests that distinct mechanisms are able to target Cdc20 for ubiquitination at different points during the cell cycle. PMID:22079111

  11. Lysosomal proteolysis and autophagy require presenilin 1 and are disrupted by Alzheimer-related PS1 mutations.

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    Lee, Ju-Hyun; Yu, W Haung; Kumar, Asok; Lee, Sooyeon; Mohan, Panaiyur S; Peterhoff, Corrinne M; Wolfe, Devin M; Martinez-Vicente, Marta; Massey, Ashish C; Sovak, Guy; Uchiyama, Yasuo; Westaway, David; Cuervo, Ana Maria; Nixon, Ralph A

    2010-06-25

    Macroautophagy is a lysosomal degradative pathway essential for neuron survival. Here, we show that macroautophagy requires the Alzheimer's disease (AD)-related protein presenilin-1 (PS1). In PS1 null blastocysts, neurons from mice hypomorphic for PS1 or conditionally depleted of PS1, substrate proteolysis and autophagosome clearance during macroautophagy are prevented as a result of a selective impairment of autolysosome acidification and cathepsin activation. These deficits are caused by failed PS1-dependent targeting of the v-ATPase V0a1 subunit to lysosomes. N-glycosylation of the V0a1 subunit, essential for its efficient ER-to-lysosome delivery, requires the selective binding of PS1 holoprotein to the unglycosylated subunit and the Sec61alpha/oligosaccharyltransferase complex. PS1 mutations causing early-onset AD produce a similar lysosomal/autophagy phenotype in fibroblasts from AD patients. PS1 is therefore essential for v-ATPase targeting to lysosomes, lysosome acidification, and proteolysis during autophagy. Defective lysosomal proteolysis represents a basis for pathogenic protein accumulations and neuronal cell death in AD and suggests previously unidentified therapeutic targets.

  12. Driver or passenger effects of augmented c-Myc and Cdc20 in gliomagenesis.

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    Ji, Ping; Zhou, Xinhui; Liu, Qun; Fuller, Gregory N; Phillips, Lynette M; Zhang, Wei

    2016-04-26

    Cdc20 and c-Myc are commonly overexpressed in a broad spectrum of cancers, including glioblastoma (GBM). Despite this clear association, whether c-Myc and Cdc20 overexpression is a driver or passenger event in gliomagenesis remains unclear. Both c-Myc and Cdc20 induced the proliferation of primary glial progenitor cells. c-Myc also promoted the formation of soft agar anchorage-independent colonies. In the RCAS/Ntv-a glia-specific transgenic mouse model, c-Myc increased the GBM incidence from 19.1% to 47.4% by 12 weeks of age when combined with kRas and Akt3 in Ntv-a INK4a-ARF (also known as CDKN2A)-null mice. In contrast, Cdc20 decreased the GBM incidence from 19.1% to 9.1%. Moreover, cell differentiation was modulated by c-Myc in kRas/Akt3-induced GBM on the basis of Nestin/GFAP expression (glial progenitor cell differentiation), while Cdc20 had no effect on primary glial progenitor cell differentiation. We used glial progenitor cells from Ntv-a newborn mice to evaluate the role of c-Myc and Cdc20 in the proliferation and transformation of GBM in vitro and in vivo. We further determined whether c-Myc and Cdc20 have a driver or passenger role in GBM development using kRas/Akt3 signals in a RCAS/Ntv-a mouse model. These results suggest that the driver or passenger of oncogene signaling is dependent on cellular status. c-Myc is a driver when combined with kRas/Akt3 oncogenic signals in gliomagenesis, whereas Cdc20 overexpression is a passenger. Inhibition of cell differentiation of c-Myc may be a target for anti-glioma therapy.

  13. Fission Yeast Apc15 Stabilizes MCC-Cdc20-APC/C Complexes, Ensuring Efficient Cdc20 Ubiquitination and Checkpoint Arrest.

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    May, Karen M; Paldi, Flora; Hardwick, Kevin G

    2017-04-24

    During mitosis, cells must segregate the replicated copies of their genome to their daughter cells with extremely high fidelity. Segregation errors lead to an abnormal chromosome number (aneuploidy), which typically results in disease or cell death [1]. Chromosome segregation and anaphase onset are initiated through the action of the multi-subunit E3 ubiquitin ligase known as the anaphase-promoting complex or cyclosome (APC/C [2]). The APC/C is inhibited by the spindle checkpoint in the presence of kinetochore attachment defects [3, 4]. Here we demonstrate that two non-essential APC/C subunits (Apc14 and Apc15) regulate association of spindle checkpoint proteins, in the form of the mitotic checkpoint complex (MCC), with the APC/C. apc14Δ mutants display increased MCC association with the APC/C and are unable to silence the checkpoint efficiently. Conversely, apc15Δ mutants display reduced association between the MCC and APC/C, are defective in poly-ubiquitination of Cdc20, and are checkpoint defective. In vitro reconstitution studies have shown that human MCC-APC/C can contain two molecules of Cdc20 [5-7]. Using a yeast strain expressing two Cdc20 genes with different epitope tags, we show by co-immunoprecipitation that this is true in vivo. MCC binding to the second molecule of Cdc20 is mediated via the C-terminal KEN box in Mad3. Somewhat surprisingly, complexes containing both molecules of Cdc20 accumulate in apc15Δ cells, and the implications of this observation are discussed. Copyright © 2017 The Author(s). Published by Elsevier Ltd.. All rights reserved.

  14. Mechanisms controlling the temporal degradation of Nek2A and Kif18A by the APC/C-Cdc20 complex

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    Sedgwick, G.G.; Hayward, D.G.; Nilsson, J.

    2013-01-01

    The Anaphase Promoting Complex/Cyclosome (APC/C) in complex with its co-activator Cdc20 is responsible for targeting proteins for ubiquitin-mediated degradation during mitosis. The activity of APC/C-Cdc20 is inhibited during prometaphase by the Spindle Assembly Checkpoint (SAC) yet certain...... substrates escape this inhibition. Nek2A degradation during prometaphase depends on direct binding of Nek2A to the APC/C via a C-terminal MR dipeptide but whether this motif alone is sufficient is not clear. Here, we identify Kif18A as a novel APC/C-Cdc20 substrate and show that Kif18A degradation depends...... by the APC/C. Nek2A and the mitotic checkpoint complex (MCC) have an overlap in APC/C subunit requirements for binding and we propose that Nek2A binds with high affinity to apo-APC/C and is degraded by the pool of Cdc20 that avoids inhibition by the SAC....

  15. APC/C-Cdc20 mediates deprotection of centromeric cohesin at meiosis II in yeast.

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

    2017-06-18

    Cells undergoing meiosis produce haploid gametes through one round of DNA replication followed by 2 rounds of chromosome segregation. This requires that cohesin complexes, which establish sister chromatid cohesion during S phase, are removed in a stepwise manner. At meiosis I, the separase protease triggers the segregation of homologous chromosomes by cleaving cohesin's Rec8 subunit on chromosome arms. Cohesin persists at centromeres because the PP2A phosphatase, recruited by the shugoshin protein, dephosphorylates Rec8 and thereby protects it from cleavage. While chromatids disjoin upon cleavage of centromeric Rec8 at meiosis II, it was unclear how and when centromeric Rec8 is liberated from its protector PP2A. One proposal is that bipolar spindle forces separate PP2A from Rec8 as cells enter metaphase II. We show here that sister centromere biorientation is not sufficient to "deprotect" Rec8 at meiosis II in yeast. Instead, our data suggest that the ubiquitin-ligase APC/C Cdc20 removes PP2A from centromeres by targeting for degradation the shugoshin Sgo1 and the kinase Mps1. This implies that Rec8 remains protected until entry into anaphase II when it is phosphorylated concurrently with the activation of separase. Here, we provide further support for this model and speculate on its relevance to mammalian oocytes.

  16. Parkin Regulates Mitosis and Genomic Stability through Cdc20/Cdh1.

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    Lee, Seung Baek; Kim, Jung Jin; Nam, Hyun-Ja; Gao, Bowen; Yin, Ping; Qin, Bo; Yi, Sang-Yeop; Ham, Hyoungjun; Evans, Debra; Kim, Sun-Hyun; Zhang, Jun; Deng, Min; Liu, Tongzheng; Zhang, Haoxing; Billadeau, Daniel D; Wang, Liewei; Giaime, Emilie; Shen, Jie; Pang, Yuan-Ping; Jen, Jin; van Deursen, Jan M; Lou, Zhenkun

    2015-10-01

    Mutations in the E3 ubiquitin ligase Parkin have been linked to familial Parkinson's disease. Parkin has also been implicated in mitosis through mechanisms that are unclear. Here we show that Parkin interacts with anaphase promoting complex/cyclosome (APC/C) coactivators Cdc20 and Cdh1 to mediate the degradation of several key mitotic regulators independent of APC/C. We demonstrate that ordered progression through mitosis is orchestrated by two distinct E3 ligases through the shared use of Cdc20 and Cdh1. Furthermore, Parkin is phosphorylated and activated by polo-like kinase 1 (Plk1) during mitosis. Parkin deficiency results in overexpression of its substrates, mitotic defects, genomic instability, and tumorigenesis. These results suggest that the Parkin-Cdc20/Cdh1 complex is an important regulator of mitosis. Copyright © 2015 Elsevier Inc. All rights reserved.

  17. Parkin Regulates Mitosis and Genomic Stability through Cdc20/Cdh1

    NARCIS (Netherlands)

    Lee, S.B.; Kim, J.J.; Nam, H.J.; Gao, B.; Yin, P.; Qin, B.; Yi, S.Y.; Ham, H.; Evans, D.; Kim, S.H.; Zhang, J.; Deng, M.; Liu, T.; Zhang, H.; Billadeau, D.D.; Wang, L.; Giaime, E.; Shen, J.; Pang, Y.P.; Jen, J.; Deursen, J.M.A. van; Lou, Z.

    2015-01-01

    Mutations in the E3 ubiquitin ligase Parkin have been linked to familial Parkinson's disease. Parkin has also been implicated in mitosis through mechanisms that are unclear. Here we show that Parkin interacts with anaphase promoting complex/cyclosome (APC/C) coactivators Cdc20 and Cdh1 to mediate

  18. Cdc20 is critical for meiosis I and fertility of female mice.

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    Fang Jin

    2010-09-01

    Full Text Available Chromosome missegregation in germ cells is an important cause of unexplained infertility, miscarriages, and congenital birth defects in humans. However, the molecular defects that lead to production of aneuploid gametes are largely unknown. Cdc20, the activating subunit of the anaphase-promoting complex/cyclosome (APC/C, initiates sister-chromatid separation by ordering the destruction of two key anaphase inhibitors, cyclin B1 and securin, at the transition from metaphase to anaphase. The physiological significance and full repertoire of functions of mammalian Cdc20 are unclear at present, mainly because of the essential nature of this protein in cell cycle progression. To bypass this problem we generated hypomorphic mice that express low amounts of Cdc20. These mice are healthy and have a normal lifespan, but females produce either no or very few offspring, despite normal folliculogenesis and fertilization rates. When mated with wild-type males, hypomorphic females yield nearly normal numbers of fertilized eggs, but as these embryos develop, they become malformed and rarely reach the blastocyst stage. In exploring the underlying mechanism, we uncover that the vast majority of these embryos have abnormal chromosome numbers, primarily due to chromosome lagging and chromosome misalignment during meiosis I in the oocyte. Furthermore, cyclin B1, cyclin A2, and securin are inefficiently degraded in metaphase I; and anaphase I onset is markedly delayed. These results demonstrate that the physiologically effective threshold level of Cdc20 is high for female meiosis I and identify Cdc20 hypomorphism as a mechanism for chromosome missegregation and formation of aneuploid gametes.

  19. The cellular response to vascular endothelial growth factors requires co-ordinated signal transduction, trafficking and proteolysis.

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    Smith, Gina A; Fearnley, Gareth W; Tomlinson, Darren C; Harrison, Michael A; Ponnambalam, Sreenivasan

    2015-08-18

    VEGFs (vascular endothelial growth factors) are a family of conserved disulfide-linked soluble secretory glycoproteins found in higher eukaryotes. VEGFs mediate a wide range of responses in different tissues including metabolic homoeostasis, cell proliferation, migration and tubulogenesis. Such responses are initiated by VEGF binding to soluble and membrane-bound VEGFRs (VEGF receptor tyrosine kinases) and co-receptors. VEGF and receptor splice isoform diversity further enhances complexity of membrane protein assembly and function in signal transduction pathways that control multiple cellular responses. Different signal transduction pathways are simultaneously activated by VEGFR-VEGF complexes with membrane trafficking along the endosome-lysosome network further modulating signal output from multiple enzymatic events associated with such pathways. Balancing VEGFR-VEGF signal transduction with trafficking and proteolysis is essential in controlling the intensity and duration of different intracellular signalling events. Dysfunction in VEGF-regulated signal transduction is important in chronic disease states including cancer, atherosclerosis and blindness. This family of growth factors and receptors is an important model system for understanding human disease pathology and developing new therapeutics for treating such ailments. © 2015 Authors.

  20. PPM1K Regulates Hematopoiesis and Leukemogenesis through CDC20-Mediated Ubiquitination of MEIS1 and p21

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    Xiaoye Liu

    2018-05-01

    Full Text Available Summary: In addition to acting as building blocks for biosynthesis, amino acids might serve as signaling regulators in various physiological and pathological processes. However, it remains unknown whether amino acid levels affect the activities of hematopoietic stem cells (HSCs. By using a genetically encoded fluorescent sensor of the intracellular levels of branched-chain amino acids (BCAAs, we could monitor the dynamics of BCAA metabolism in HSCs. A mitochondrial-targeted 2C-type Ser/Thr protein phosphatase (PPM1K promotes the catabolism of BCAAs to maintain MEIS1 and p21 levels by decreasing the ubiquitination-mediated degradation controlled by the E3 ubiquitin ligase CDC20. PPM1K deficiency led to a notable decrease in MEIS1/p21 signaling to reduce the glycolysis and quiescence of HSCs, followed by a severe impairment in repopulation activities. Moreover, the deletion of Ppm1k dramatically extended survival in a murine leukemia model. These findings will enhance the current understanding of nutrient signaling in metabolism and function of stem cells. : Liu et al. show that the dynamics of BCAA metabolism in hematopoietic stem cells (HSCs and leukemia-initiating cells (LICs can be monitored by a genetically encoded fluorescent sensor. PPM1K promotes BCAA catabolism and maintains the glycolysis and quiescence of HSCs/LICs through the downregulation of CDC20-mediated ubiquitination of MEIS1 and p21. Keywords: branched-chain amino acids, PPM1K, ubiquitination, CDC20, MEIS1/p21, hematopoietic stem cells, leukemia-initiating cells

  1. Spatiotemporally regulated proteolysis to dissect the role of vegetative proteins during Bacillus subtilis sporulation: cell-specific requirement of σH and σA.

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    Riley, Eammon P; Trinquier, Aude; Reilly, Madeline L; Durchon, Marine; Perera, Varahenage R; Pogliano, Kit; Lopez-Garrido, Javier

    2018-04-01

    Sporulation in Bacillus subtilis is a paradigm of bacterial development, which involves the interaction between a larger mother cell and a smaller forespore. The mother cell and the forespore activate different genetic programs, leading to the production of sporulation-specific proteins. A critical gap in our understanding of sporulation is how vegetative proteins, made before sporulation initiation, contribute to spore formation. Here we present a system, spatiotemporally regulated proteolysis (STRP), which enables the rapid, developmentally regulated degradation of target proteins, thereby providing a suitable method to dissect the cell- and developmental stage-specific role of vegetative proteins. STRP has been used to dissect the role of two major vegetative sigma factors, σ H and σ A , during sporulation. The results suggest that σ H is only required in predivisional cells, where it is essential for sporulation initiation, but that it is dispensable during subsequent steps of spore formation. However, evidence has been provided that σ A plays different roles in the mother cell, where it replenishes housekeeping functions, and in the forespore, where it plays an unexpected role in promoting spore germination and outgrowth. Altogether, the results demonstrate that STRP has the potential to provide a comprehensive molecular dissection of every stage of sporulation, germination and outgrowth. © 2018 John Wiley & Sons Ltd.

  2. Proteolysis in hyperthermophilic microorganisms

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    Donald E. Ward

    2002-01-01

    Full Text Available Proteases are found in every cell, where they recognize and break down unneeded or abnormal polypeptides or peptide-based nutrients within or outside the cell. Genome sequence data can be used to compare proteolytic enzyme inventories of different organisms as they relate to physiological needs for protein modification and hydrolysis. In this review, we exploit genome sequence data to compare hyperthermophilic microorganisms from the euryarchaeotal genus Pyrococcus, the crenarchaeote Sulfolobus solfataricus, and the bacterium Thermotoga maritima. An overview of the proteases in these organisms is given based on those proteases that have been characterized and on putative proteases that have been identified from genomic sequences, but have yet to be characterized. The analysis revealed both similarities and differences in the mechanisms utilized for proteolysis by each of these hyperthermophiles and indicated how these mechanisms relate to proteolysis in less thermophilic cells and organisms.

  3. Inhibition of Cell Survival by Curcumin Is Associated with Downregulation of Cell Division Cycle 20 (Cdc20) in Pancreatic Cancer Cells.

    Science.gov (United States)

    Zhang, Yu; Xue, Ying-Bo; Li, Hang; Qiu, Dong; Wang, Zhi-Wei; Tan, Shi-Sheng

    2017-02-04

    Pancreatic cancer is one of the most aggressive human tumors in the United States. Curcumin, a polyphenol derived from the Curcuma longa plant, has been reported to exert its antitumor activity in pancreatic cancer. However, the molecular mechanisms of curcumin-mediated tumor suppressive function have not been fully elucidated. In the current study, we explore whether curcumin exhibits its anti-cancer function through inhibition of oncoprotein cell division cycle 20 (Cdc20) in pancreatic cancer cells. We found that curcumin inhibited cell growth, enhanced apoptosis, induced cell cycle arrest and retarded cell invasion in pancreatic cancer cells. Moreover, we observed that curcumin significantly inhibited the expression of Cdc20 in pancreatic cancer cells. Furthermore, our results demonstrated that overexpression of Cdc20 enhanced cell proliferation and invasion, and abrogated the cytotoxic effects induced by curcumin in pancreatic cancer cells. Consistently, downregulation of Cdc20 promoted curcumin-mediated anti-tumor activity. Therefore, our findings indicated that inhibition of Cdc20 by curcumin could be useful for the treatment of pancreatic cancer patients.

  4. Mad2 binding to Mad1 and Cdc20, rather than oligomerization, is required for the spindle checkpoint

    DEFF Research Database (Denmark)

    Sironi, L; Melixetian, M; Faretta, M

    2001-01-01

    Mad2 is a key component of the spindle checkpoint, a device that controls the fidelity of chromosome segregation in mitosis. The ability of Mad2 to form oligomers in vitro has been correlated with its ability to block the cell cycle upon injection into Xenopus embryos. Here we show that Mad2 forms...

  5. Ganodermanontriol (GDNT) exerts its effect on growth and invasiveness of breast cancer cells through the down-regulation of CDC20 and uPA

    International Nuclear Information System (INIS)

    Jiang, Jiahua; Jedinak, Andrej; Sliva, Daniel

    2011-01-01

    Highlights: ► Ganodermanontriol (GDNT), a Ganoderma mushroom alcohol, inhibits growth of breast cancer cells. ► CDC20 is over-expressed in tumors but not in the tumor surrounding tissue in breast cancer patients. ► GDNT inhibits expression of CDC20 in breast cancer cells. ► GDNT inhibits cell adhesion, cell migration and cell invasion of breast cancer cells. ► GDNT inhibits secretion of uPA and down-regulates expression of uPAR in breast cancer cells. -- Abstract: Ganoderma lucidum is a medicinal mushroom that has been recognized by Traditional Chinese Medicine (TCM). Although some of the direct anticancer activities are attributed to the presence of triterpenes—ganoderic and lucidenic acids—the activity of other compounds remains elusive. Here we show that ganodermanontriol (GDNT), a Ganoderma alcohol, specifically suppressed proliferation (anchorage-dependent growth) and colony formation (anchorage-independent growth) of highly invasive human breast cancer cells MDA-MB-231. GDNT suppressed expression of the cell cycle regulatory protein CDC20, which is over-expressed in precancerous and breast cancer cells compared to normal mammary epithelial cells. Moreover, we found that CDC20 is over-expressed in tumors when compared to the tissue surrounding the tumor in specimens from breast cancer patients. GDNT also inhibited invasive behavior (cell adhesion, cell migration, and cell invasion) through the suppression of secretion of urokinase-plasminogen activator (uPA) and inhibited expression of uPA receptor. In conclusion, mushroom GDNT is a natural agent that has potential as a therapy for invasive breast cancers.

  6. Ganodermanontriol (GDNT) exerts its effect on growth and invasiveness of breast cancer cells through the down-regulation of CDC20 and uPA

    Energy Technology Data Exchange (ETDEWEB)

    Jiang, Jiahua; Jedinak, Andrej [Cancer Research Laboratory, Methodist Research Institute, Indiana University Health, Indianapolis, IN (United States); Sliva, Daniel, E-mail: dsliva@iuhealth.org [Cancer Research Laboratory, Methodist Research Institute, Indiana University Health, Indianapolis, IN (United States); Department of Medicine, School of Medicine, Indiana University, Indianapolis, IN (United States); Indiana University Simon Cancer Center, School of Medicine, Indiana University, Indianapolis, IN (United States)

    2011-11-18

    Highlights: Black-Right-Pointing-Pointer Ganodermanontriol (GDNT), a Ganoderma mushroom alcohol, inhibits growth of breast cancer cells. Black-Right-Pointing-Pointer CDC20 is over-expressed in tumors but not in the tumor surrounding tissue in breast cancer patients. Black-Right-Pointing-Pointer GDNT inhibits expression of CDC20 in breast cancer cells. Black-Right-Pointing-Pointer GDNT inhibits cell adhesion, cell migration and cell invasion of breast cancer cells. Black-Right-Pointing-Pointer GDNT inhibits secretion of uPA and down-regulates expression of uPAR in breast cancer cells. -- Abstract: Ganoderma lucidum is a medicinal mushroom that has been recognized by Traditional Chinese Medicine (TCM). Although some of the direct anticancer activities are attributed to the presence of triterpenes-ganoderic and lucidenic acids-the activity of other compounds remains elusive. Here we show that ganodermanontriol (GDNT), a Ganoderma alcohol, specifically suppressed proliferation (anchorage-dependent growth) and colony formation (anchorage-independent growth) of highly invasive human breast cancer cells MDA-MB-231. GDNT suppressed expression of the cell cycle regulatory protein CDC20, which is over-expressed in precancerous and breast cancer cells compared to normal mammary epithelial cells. Moreover, we found that CDC20 is over-expressed in tumors when compared to the tissue surrounding the tumor in specimens from breast cancer patients. GDNT also inhibited invasive behavior (cell adhesion, cell migration, and cell invasion) through the suppression of secretion of urokinase-plasminogen activator (uPA) and inhibited expression of uPA receptor. In conclusion, mushroom GDNT is a natural agent that has potential as a therapy for invasive breast cancers.

  7. Peptidomics for Studying Limited Proteolysis.

    Science.gov (United States)

    Tsuchiya, Takashi; Osaki, Tsukasa; Minamino, Naoto; Sasaki, Kazuki

    2015-11-06

    Limited proteolysis is a pivotal mechanism regulating protein functions. Identifying physiologically or pathophysiologically relevant cleavage sites helps to develop molecular tools that can be used for diagnostics or therapeutics. During proteolysis of secretory and membrane proteins, part of the cleaved protein is liberated and destined to undergo degradation but should retain original cleavage sites created by proteolytic enzymes. We profiled endogenous peptides accumulated for 4 h in media conditioned by primary cultured rat cardiac fibroblasts. A total of 3916 redundant peptide sequences from 94 secretory proteins and membrane proteins served to identify limited cleavage sites, both annotated and unannotated, for signal peptide or propeptide removal, peptide hormone processing, ectodomain shedding, and regulated intramembrane proteolysis. Incorrectly predicted signal cleavage sites are found in typical proteins such as extracellular matrix proteins and the peptide hormone precursor adrenomedullin ADM. The revealed signal peptide cleavage site for ADM was experimentally verified by identifying the major molecular form of flanking proadrenomedullin N-terminal peptide. We suggest that profiling of endogenous peptides, like transcriptome sequence reads, makes sense in regular cells such as fibroblasts and that peptidomics provides insight into proteolysis-regulated protein functions.

  8. 1-L-MT, an IDO inhibitor, prevented colitis-associated cancer by inducing CDC20 inhibition-mediated mitotic death of colon cancer cells.

    Science.gov (United States)

    Liu, Xiuting; Zhou, Wei; Zhang, Xin; Ding, Yang; Du, Qianming; Hu, Rong

    2018-04-01

    Indoleamine 2,3-dioxygenase 1 (IDO1), known as IDO, catabolizes tryptophan through kynurenine pathway, whose activity is correlated with impaired clinical outcome of colorectal cancer. Here we showed that 1-L-MT, a canonical IDO inhibitor, suppressed proliferation of human colorectal cancer cells through inducing mitotic death. Our results showed that inhibition of IDO decreased the transcription of CDC20, which resulted in G2/M cycle arrest of HCT-116 and HT-29. Furthermore, 1-L-MT induced mitochondria injuries and caused apoptotic cancer cells. Importantly, 1-L-MT protected mice from azoxymethane (AOM)/dextran sodium sulfate (DSS)-induced colon carcinogenesis, with reduced mortality, tumor number and size. What is more, IDO1-/- mice exhibited fewer tumor burdens and reduced proliferation in the neoplastic epithelium, while, 1-L-MT did not exhibit any further protective effects on IDO-/- mice, confirming the critical role of IDO and the protective effect of 1-L-MT-mediated IDO inhibition in CRC. Furthermore, 1-L-MT also alleviated CRC in Rag1-/- mice, demonstrating the modulatory effects of IDO independent of its role in modulating adaptive immunity. Taken together, our findings validated that the anti-proliferation effect of 1-L-MT in vitro and the prevention of CRC in vivo were through IDO-induced cell cycle disaster of colon cancer cells. Our results identified 1-L-MT as a promising candidate for the chemoprevention of CRC. © 2018 UICC.

  9. Regulation of proteolysis in Bacillus subtilis: effects of calcium ions and energy poisons

    International Nuclear Information System (INIS)

    O'Hara, M.B.; Hageman, J.H.

    1987-01-01

    Bacillus subtilis cells carry out extensive intracellular proteolysis (k = 0.15-0.23/h) during sporulation. Protein degradation was measured in cells growing in chemically defined sporulation medium, by following the release of [ 14 C]-leucine from the cells during spore formation. Sodium arsenate, carbonyl cyanide 3-chlorophenyl hydrazone, and sodium azide strongly inhibited proteolysis without altering cell viability greatly, which suggested that bulk proteolysis in B. subtilis is energy dependent. The authors have tested the hypothesis that the energy requirement may be for pumping in Ca 2+ . When [Ca 2+ ] was -6 , rates of proteolysis in sporulating cells were reduced 4-8 times that in cells in calcium ion- sufficient medium. Further, omission of Ca 2+ from the medium prevented the increase in the activity of the major intracellular serine protease. However, the presence of energy poisons in the media at levels which inhibited proteolysis, had no detectable effect on the uptake of by cells [ 45 Ca]. The authors concluded that B. subtilis cells required both metabolic energy and calcium ions for normal proteolysis

  10. Proteolysis and consistency of Meshanger cheese

    NARCIS (Netherlands)

    Jong, de L.

    1978-01-01

    Proteolysis in Meshanger cheese, estimated by quantitative polyacrylamide gel electrophoresis is discussed. The conversion of α s1 -casein was proportional to rennet concentration in the cheese. Changes in consistency, after a maximum, were correlated to breakdown of

  11. Protein trafficking and maturation regulate intramembrane proteolysis.

    Science.gov (United States)

    Morohashi, Yuichi; Tomita, Taisuke

    2013-12-01

    Intramembrane-cleaving proteases (I-CLiPs) are membrane embedded proteolytic enzymes. All substrates identified so far are also membrane proteins, involving a number of critical cellular signaling as well as human diseases. After synthesis and assembly at the endoplasmic reticulum, membrane proteins are exported to the Golgi apparatus and transported to their sites of action. A number of studies have revealed the importance of the intracellular membrane trafficking in i-CLiP-mediated intramembrane proteolysis, not only for limiting the unnecessary encounter between i-CLiPs and their substrate but also for their cleavage site preference. In this review, we will discuss recent advances in our understanding of how each i-CLiP proteolysis is regulated by intracellular vesicle trafficking. This article is part of a Special Issue entitled: Intramembrane Proteases. Copyright © 2013 Elsevier B.V. All rights reserved.

  12. Plant senescence and proteolysis: two processes with one destiny.

    Science.gov (United States)

    Diaz-Mendoza, Mercedes; Velasco-Arroyo, Blanca; Santamaria, M Estrella; González-Melendi, Pablo; Martinez, Manuel; Diaz, Isabel

    2016-01-01

    Senescence-associated proteolysis in plants is a complex and controlled process, essential for mobilization of nutrients from old or stressed tissues, mainly leaves, to growing or sink organs. Protein breakdown in senescing leaves involves many plastidial and nuclear proteases, regulators, different subcellular locations and dynamic protein traffic to ensure the complete transformation of proteins of high molecular weight into transportable and useful hydrolysed products. Protease activities are strictly regulated by specific inhibitors and through the activation of zymogens to develop their proteolytic activity at the right place and at the proper time. All these events associated with senescence have deep effects on the relocation of nutrients and as a consequence, on grain quality and crop yield. Thus, it can be considered that nutrient recycling is the common destiny of two processes, plant senescence and, proteolysis. This review article covers the most recent findings about leaf senescence features mediated by abiotic and biotic stresses as well as the participants and steps required in this physiological process, paying special attention to C1A cysteine proteases, their specific inhibitors, known as cystatins, and their potential targets, particularly the chloroplastic proteins as source for nitrogen recycling.

  13. Taking a position on intramembrane proteolysis.

    Science.gov (United States)

    Lemieux, M Joanne

    2018-03-30

    Decades of work have contributed to our in-depth mechanistic understanding of soluble proteases, but much less is known about the catalytic mechanism of intramembrane proteolysis due to inherent difficulties in both preparing and analyzing integral membrane enzymes and transmembrane substrates. New work from Naing et al. tackles this challenge by examining the catalytic parameters of an aspartyl intramembrane protease homologous to the enzyme that cleaves amyloid precursor protein, finding that both chemistry and register contribute to specificity in substrate cleavage. © 2018 Joanne Lemieux.

  14. Ubiquitin-mediated proteolysis in Xenopus extract.

    Science.gov (United States)

    McDowell, Gary S; Philpott, Anna

    2016-01-01

    The small protein modifier, ubiquitin, can be covalently attached to proteins in the process of ubiquitylation, resulting in a variety of functional outcomes. In particular, the most commonly-associated and well-studied fate for proteins modified with ubiquitin is their ultimate destruction: degradation by the 26S proteasome via the ubiquitin-proteasome system, or digestion in lysosomes by proteolytic enzymes. From the earliest days of ubiquitylation research, a reliable and versatile "cell-in-a-test-tube" system has been employed in the form of cytoplasmic extracts from the eggs and embryos of the frog Xenopus laevis. Biochemical studies of ubiquitin and protein degradation using this system have led to significant advances particularly in the study of ubiquitin-mediated proteolysis, while the versatility of Xenopus as a developmental model has allowed investigation of the in vivo consequences of ubiquitylation. Here we describe the use and history of Xenopus extract in the study of ubiquitin-mediated protein degradation, and highlight the versatility of this system that has been exploited to uncover mechanisms and consequences of ubiquitylation and proteolysis.

  15. Rca1 inhibits APC-Cdh1(Fzr) and is required to prevent cyclin degradation in G2.

    Science.gov (United States)

    Grosskortenhaus, Ruth; Sprenger, Frank

    2002-01-01

    We demonstrate that Rca1 is an essential inhibitor of the anaphase-promoting complex/cyclosome (APC) in Drosophila. APC activity is restricted to mitotic stages and G1 by its activators Cdc20-Fizzy (Cdc20(Fzy)) and Cdh1-Fizzy-related (Cdh1(Fzr)), respectively. In rca1 mutants, cyclins are degraded prematurely in G2 by APC-Cdh1(Fzr)-dependent proteolysis, and cells fail to execute mitosis. Overexpression of Cdh1(Fzr) mimics the rca1 phenotype, and coexpression of Rca1 blocks this Cdh1(Fzr) function. We show that Rca1 and Cdh1(Fzr) are in a complex that also includes the APC component Cdc27. Previous studies have shown that phosphorylation of Cdh1 prevents its interaction with the APC. Our data reveal a different mode of APC regulation by Rca1 at the G2 stage, when low Cdk activity is unable to inhibit Cdh1(Fzr) interaction.

  16. Functional Imaging of Proteolysis: Stromal and Inflammatory Cells Increase Tumor Proteolysis

    Directory of Open Access Journals (Sweden)

    Mansoureh Sameni

    2003-07-01

    Full Text Available The underlying basement membrane is degraded during progression of breast and colon carcinoma. Thus, we imaged degradation of a quenched fluorescent derivative of basement membrane type IV collagen (DQ-collagen IV by living human breast and colon tumor spheroids. Proteolysis of DQ-collagen IV by HCT 116 and HKh-2 human colon tumor spheroids was both intracellular and pericellular. In contrast, proteolysis of DQ-collagen IV by BT20 human breast tumor spheroids was pericellular. As stromal elements can contribute to proteolytic activities associated with tumors, we also examined degradation of DQ-collagen IV by human monocytes/macrophages and colon and breast fibroblasts. Fibroblasts themselves exhibited a modest amount of pericellular degradation. Degradation was increased 4–17-fold in cocultures of fibroblasts and tumor cells as compared to either cell type alone. Inhibitors of matrix metalloproteinases, plasmin, and the cysteine protease, cathepsin B, all reduced degradation in the cocultures. Monocytes did not degrade DQ-collagen IV; however, macrophages degraded DQ-collagen IV intracellularly. In coculture of tumor cells, fibroblasts, and macrophages, degradation of DQ-collagen IV was further increased. Imaging of living tumor and stromal cells has, thus, allowed us to establish that tumor proteolysis occurs pericellularly and intracellularly and that tumor, stromal, and inflammatory cells all contribute to degradative processes.

  17. Allosteric regulation of rhomboid intramembrane proteolysis.

    Science.gov (United States)

    Arutyunova, Elena; Panwar, Pankaj; Skiba, Pauline M; Gale, Nicola; Mak, Michelle W; Lemieux, M Joanne

    2014-09-01

    Proteolysis within the lipid bilayer is poorly understood, in particular the regulation of substrate cleavage. Rhomboids are a family of ubiquitous intramembrane serine proteases that harbour a buried active site and are known to cleave transmembrane substrates with broad specificity. In vitro gel and Förster resonance energy transfer (FRET)-based kinetic assays were developed to analyse cleavage of the transmembrane substrate psTatA (TatA from Providencia stuartii). We demonstrate significant differences in catalytic efficiency (kcat/K0.5) values for transmembrane substrate psTatA (TatA from Providencia stuartii) cleavage for three rhomboids: AarA from P. stuartii, ecGlpG from Escherichia coli and hiGlpG from Haemophilus influenzae demonstrating that rhomboids specifically recognize this substrate. Furthermore, binding of psTatA occurs with positive cooperativity. Competitive binding studies reveal an exosite-mediated mode of substrate binding, indicating allostery plays a role in substrate catalysis. We reveal that exosite formation is dependent on the oligomeric state of rhomboids, and when dimers are dissociated, allosteric substrate activation is not observed. We present a novel mechanism for specific substrate cleavage involving several dynamic processes including positive cooperativity and homotropic allostery for this interesting class of intramembrane proteases. © 2014 The Authors.

  18. Regulatory Proteolysis in Arabidopsis-Pathogen Interactions.

    Science.gov (United States)

    Pogány, Miklós; Dankó, Tamás; Kámán-Tóth, Evelin; Schwarczinger, Ildikó; Bozsó, Zoltán

    2015-09-24

    Approximately two and a half percent of protein coding genes in Arabidopsis encode enzymes with known or putative proteolytic activity. Proteases possess not only common housekeeping functions by recycling nonfunctional proteins. By irreversibly cleaving other proteins, they regulate crucial developmental processes and control responses to environmental changes. Regulatory proteolysis is also indispensable in interactions between plants and their microbial pathogens. Proteolytic cleavage is simultaneously used both by plant cells, to recognize and inactivate invading pathogens, and by microbes, to overcome the immune system of the plant and successfully colonize host cells. In this review, we present available results on the group of proteases in the model plant Arabidopsis thaliana whose functions in microbial pathogenesis were confirmed. Pathogen-derived proteolytic factors are also discussed when they are involved in the cleavage of host metabolites. Considering the wealth of review papers available in the field of the ubiquitin-26S proteasome system results on the ubiquitin cascade are not presented. Arabidopsis and its pathogens are conferred with abundant sets of proteases. This review compiles a list of those that are apparently involved in an interaction between the plant and its pathogens, also presenting their molecular partners when available.

  19. The ATPase and protease domains of yeast mitochondrial Lon : Roles in proteolysis and respiration-dependent growth

    NARCIS (Netherlands)

    van Dijl, JM; Kutejova, E; Suda, K; Perecko, D; Schatz, G; Suzuki, CK

    1998-01-01

    The ATP-dependent Lon protease of Saccharomyces cerevisiae mitochondria is required for selective proteolysis in the matrix, maintenance of mitochondrial DNA, and respiration-dependent growth. Lon may also possess a chaperone-like function that facilitates protein degradation and protein-complex

  20. Complementary methods for the identification of substrates of proteolysis.

    Science.gov (United States)

    Pham, Victoria C; Anania, Veronica G; Phung, Qui T; Lill, Jennie R

    2014-01-01

    Proteolysis describes the cleavage of proteins into smaller components, which in vivo occurs typically to either activate or impair the functionality of cellular proteins. Proteolysis can occur during cellular homeostasis or can be induced due to external stress stimuli such as heat, biological or chemical insult, and is mediated by the activity of cellular enzymes, namely, proteases. Proteolytic cleavage of proteins can influence protein activation by exposing an active site or disrupting inhibitor binding. Conversely, proteolytic cleavage of many proteins has also been shown to lead to protein degradation resulting in inactivation of the substrate. Thousands of proteolytic events are known to take place in regulated cellular processes such as apoptosis and pyroptosis, however, their individual contribution to these processes remains poorly understood. Additionally, many cellular homeostatic processes are regulated by proteolytic events, however, in some cases, few proteolytic substrates have been identified. To gain further insight into the mechanism of action of these cellular processes, and to characterize biomarkers of cell death and other pathological indications, it is imperative to utilize a complete arsenal of tools for studying proteolysis events in vivo and in vitro. In this chapter, we focus on alternative methodologies to N-terminomics for profiling substrates of proteolysis and describe an additional suite of tools including orthogonal biophysical separation techniques such as COFRADIC or GASSP, and affinity capture tools that can enrich for newly formed C-termini (C-terminomics) generated as a result of caspase-mediated proteolysis. © 2014 Elsevier Inc. All rights reserved.

  1. Effects of proteolysis on the adenosinetriphosphatase activities of thymus myosin

    International Nuclear Information System (INIS)

    Vu, N.D.; Wagner, P.D.

    1987-01-01

    Limited proteolysis was used to identify regions on the heavy chains of calf thymus myosin which may be involved in ATP and actin binding. Assignments of the various proteolytic fragments to different parts of the myosin heavy chain were based on solubility, gel filtration, electron microscopy, and binding of 32 P-labeled regulatory light chains. Chymotrypsin rapidly cleaved within the head of thymus myosin to give a 70,000-dalton N-terminal fragment and a 140,000-dalton C-terminal fragment. These two fragments did not dissociate under nondenaturing conditions. Cleavage within the myosin tail to give heavy meromyosin occurred more slowly. Cleavage at the site 70,000 daltons from the N-terminus of the heavy chain caused about a 30-fold decrease in the actin concentration required to achieve half-maximal stimulation of the magnesium-adenosinetriphosphatase (Mg-ATPase) activity of unphosphorylated thymus myosin. The actin-activated ATPase activity of this digested myosin was only slightly affected by light chain phosphorylation. Actin inhibited the cleavage at this site by chymotrypsin. In the presence of ATP, chymotrypsin rapidly cleaved the thymus myosin heavy chain at an additional site about 4000 daltons from the N-terminus. Cleavage at this site caused a 2-fold increase in the ethylenediaminetetraacetic acid-ATPase activity and 3-fold decreases in the Ca 2+ - and Mg-ATPase activities of thymus myosin. Thus, cleavage at the N-terminus of thymus myosin was affected by ATP, and this cleavage altered ATPase activity. Papain cleaved the thymus myosin heavy chain about 94,000 daltons from the N-terminus to give subfragment 1. Although this subfragment 1 contained intact light chains, its actin-activated ATPase activity was not affected by light chain phosphorylation

  2. The effect of different packaging materials on proteolysis, sensory ...

    African Journals Online (AJOL)

    In this study, tulum cheese was manufactured using raw ewe's milk and was ripened in goat's skin and plastic bags. The effect of ripening materials (skin bag or plastic) on proteolysis was investigated during 120 days of ripening. In addition, sensory scores of the cheeses were assessed at the 90th and 120th days.

  3. Physiological regulation of epithelial sodium channel by proteolysis

    DEFF Research Database (Denmark)

    Svenningsen, Per; Friis, Ulla G; Bistrup, Claus

    2011-01-01

    PURPOSE OF REVIEW: Activation of epithelial sodium channel (ENaC) by proteolysis appears to be relevant for day-to-day physiological regulation of channel activity in kidney and other epithelial tissues. Pathophysiogical, proteolytic activation of ENaC in kidney has been demonstrated in proteinuric...

  4. Perlecan and the Blood-Brain Barrier: Beneficial Proteolysis?

    Directory of Open Access Journals (Sweden)

    Jill eRoberts

    2012-08-01

    Full Text Available The cerebral microvasculature is important for maintaining brain homeostasis. This is achieved via the blood-brain barrier (BBB, composed of endothelial cells with specialized tight junctions, astrocytes and a basement membrane. Prominent components of the basement membrane extracellular matrix (ECM include fibronectin, laminin, collagen IV and perlecan, all of which regulate cellular processes via signal transduction through various cell membrane bound ECM receptors. Expression and proteolysis of these ECM components can be rapidly altered during pathological states of the central nervous system. In particular, proteolysis of perlecan, a heparan sulfate proteoglycan, occurs within hours following ischemia induced by experimental stroke. Proteolysis of ECM components following stroke results in the degradation of the basement membrane and further disruption of the BBB. While it is clear that such proteolysis has negative consequences for the BBB, we propose that it also may lead to generation of ECM protein fragments, including the C-terminal domain V (DV of perlecan, that potentially have a positive influence on other aspects of CNS health. Indeed, perlecan DV has been shown to be persistently generated after stroke and beneficial as a neuroprotective molecule and promoter of post-stroke brain repair. This mini-review will discuss beneficial roles of perlecan protein fragment generation within the brain during stroke.

  5. Chemotherapy inhibits skeletal muscle ubiquitin-proteasome-dependent proteolysis.

    Science.gov (United States)

    Tilignac, Thomas; Temparis, Sandrine; Combaret, Lydie; Taillandier, Daniel; Pouch, Marie-Noëlle; Cervek, Matjaz; Cardenas, Diana M; Le Bricon, Thierry; Debiton, Eric; Samuels, Susan E; Madelmont, Jean-Claude; Attaix, Didier

    2002-05-15

    Chemotherapy has cachectic effects, but it is unknown whether cytostatic agents alter skeletal muscle proteolysis. We hypothesized that chemotherapy-induced alterations in protein synthesis should result in the increased incidence of abnormal proteins, which in turn should stimulate ubiquitin-proteasome-dependent proteolysis. The effects of the nitrosourea cystemustine were investigated in skeletal muscles from both healthy and colon 26 adenocarcinoma-bearing mice, an appropriate model for testing the impact of cytostatic agents. Muscle wasting was seen in both groups of mice 4 days after a single cystemustine injection, and the drug further increased the loss of muscle proteins already apparent in tumor-bearing animals. Cystemustine cured the tumor-bearing mice with 100% efficacy. Surprisingly, within 11 days of treatment, rates of muscle proteolysis progressively decreased below basal levels observed in healthy control mice and contributed to the cessation of muscle wasting. Proteasome-dependent proteolysis was inhibited by mechanisms that include reduced mRNA levels for 20S and 26S proteasome subunits, decreased protein levels of 20S proteasome subunits and the S14 non-ATPase subunit of the 26S proteasome, and impaired chymotrypsin- and trypsin-like activities of the enzyme. A combination of cisplatin and ifosfamide, two drugs that are widely used in the treatment of cancer patients, also depressed the expression of proteasomal subunits in muscles from rats bearing the MatB adenocarcinoma below basal levels. Thus, a down-regulation of ubiquitin-proteasome-dependent proteolysis is observed with various cytostatic agents and contributes to reverse the chemotherapy-induced muscle wasting.

  6. Cell cycle- and cell growth-regulated proteolysis of mammalian CDC6 is dependent on APC-CDH1

    DEFF Research Database (Denmark)

    Petersen, B O; Wagener, C; Marinoni, F

    2000-01-01

    is targeted for ubiquitin-mediated proteolysis by the anaphase promoting complex (APC)/cyclosome in G(1). A combination of point mutations in the destruction box and KEN-box motifs in CDC6 stabilizes the protein in G(1) and in quiescent cells. Furthermore, APC, in association with CDH1, ubiquitinates CDC6...... in vitro, and both APC and CDH1 are required and limiting for CDC6 proteolysis in vivo. Although a stable mutant of CDC6 is biologically active, overexpression of this mutant or wild-type CDC6 is not sufficient to induce multiple rounds of DNA replication in the same cell cycle. The APC-CDH1-dependent...

  7. Proteolysis of proBDNF is a key regulator in the formation of memory.

    Directory of Open Access Journals (Sweden)

    Philip Barnes

    2008-09-01

    Full Text Available It is essential to understand the molecular processes underlying long-term memory to provide therapeutic targets of aberrant memory that produce pathological behaviour in humans. Under conditions of recall, fully-consolidated memories can undergo reconsolidation or extinction. These retrieval-mediated memory processes may rely on distinct molecular processes. The cellular mechanisms initiating the signature molecular events are not known. Using infusions of protein synthesis inhibitors, antisense oligonucleotide targeting brain-derived neurotrophic factor (BDNF mRNA or tPA-STOP (an inhibitor of the proteolysis of BDNF protein into the hippocampus of the awake rat, we show that acquisition and extinction of contextual fear memory depended on the increased and decreased proteolysis of proBDNF (precursor BDNF in the hippocampus, respectively. Conditions of retrieval that are known to initiate the reconsolidation of contextual fear memory, a BDNF-independent memory process, were not correlated with altered proBDNF cleavage. Thus, the processing of BDNF was associated with the acquisition of new information and the updating of information about a salient stimulus. Furthermore, the differential requirement for the processing of proBDNF by tPA in distinct memory processes suggest that the molecular events actively engaged to support the storage and/or the successful retrieval of memory depends on the integration of ongoing experience with past learning.

  8. The use of in situ proteolysis in the crystallization of murine CstF-77

    International Nuclear Information System (INIS)

    Bai, Yun; Auperin, Thierry C.; Tong, Liang

    2007-01-01

    In situ proteolysis with fungal protease or subtilisin is crucial for the crystallization of murine CstF-77. The cleavage-stimulation factor (CstF) is required for the cleavage of the 3′-end of messenger RNA precursors in eukaryotes. During structure determination of the 77 kDa subunit of the murine CstF complex (CstF-77), it was serendipitously discovered that a solution infected by a fungus was crucial for the crystallization of this protein. CstF-77 was partially proteolyzed during crystallization; this was very likely to have been catalyzed by a protease secreted by the fungus. It was found that the fungal protease can be replaced by subtilisin and this in situ proteolysis protocol produced crystals of sufficient size for structural studies. After an extensive search, it was found that 55% glucose can be used as a cryoprotectant while maintaining the diffraction quality of the crystals; most other commonly used cryoprotectants were detrimental to the diffraction quality

  9. Excess alpha chains are lost from beta-thalassemic reticulocytes by proteolysis

    International Nuclear Information System (INIS)

    Testa, U.; Hinard, N.; Beuzard, Y.; Tsapis, A.; Galacteros, F.; Thomopoulos, P.; Rosa, J.

    1981-01-01

    During incubation of reticulocytes from patients with beta-thalassemia, after labeling of the hemoglobin with radioactive amino acids, the excess alpha chains are gradually lost from the cells. The aim of this study was to investigate the mechanism of this phenomenon. A system was developed in which reticulocytes from beta-thalassemia patients are labeled with [3H]leucine, washed several times in nonradioactive medium, and then incubated in the same medium containing puromycin added in order to stop further protein synthesis. The results have clearly shown that excess alpha chains are gradually degraded by proteolysis. N-ethylmaleimide or epsilon-aminocaproic acid inhibited the proteolysis of free alpha chains. The addition of either ATP or hemin did not change the rate of alpha chain degradation. The time required to degrade 50% of the pool of free alpha chains was directly dependent on the initial value of this pool. This finding suggests the absence of a significant individual variation in the ability to proteolyse free alpha chains

  10. Complex regulation controls Neurogenin3 proteolysis

    Directory of Open Access Journals (Sweden)

    Ryan Roark

    2012-10-01

    The ubiquitin proteasome system (UPS is known to be responsible for the rapid turnover of many transcription factors, where half-life is held to be critical for regulation of transcriptional activity. However, the stability of key transcriptional regulators of development is often very poorly characterised. Neurogenin 3 (Ngn3 is a basic helix–loop–helix transcription factor that plays a central role in specification and differentiation of endocrine cells of the pancreas and gut, as well as spermatogonia and regions of the brain. Here we demonstrate that Ngn3 protein stability is regulated by the ubiquitin proteasome system and that Ngn3 can be ubiquitylated on lysines, the N-terminus and, highly unusually, on non-canonical residues including cysteines and serines/threonines. Rapid turnover of Ngn3 is regulated both by binding to its heterodimeric partner E protein and by the presence of cdk inhibitors. We show that protein half-life does appear to regulate the activity of Ngn3 in vivo, but, unlike the related transcription factor c-myc, ubiquitylation on canonical sites is not a requirement for transcriptional activity of Ngn3. Hence, we characterise an important new level of Ngn3 post-translational control, which may regulate its transcriptional activity.

  11. Inhibitors of the proteasome reduce the accelerated proteolysis in atrophying rat skeletal muscles.

    Science.gov (United States)

    Tawa, N E; Odessey, R; Goldberg, A L

    1997-07-01

    Several observations have suggested that the enhanced proteolysis and atrophy of skeletal muscle in various pathological states is due primarily to activation of the ubiquitin-proteasome pathway. To test this idea, we investigated whether peptide aldehyde inhibitors of the proteasome, N-acetyl-leucyl-leucyl-norleucinal (LLN), or the more potent CBZ-leucyl-leucyl-leucinal (MG132) suppressed proteolysis in incubated rat skeletal muscles. These agents (e.g., MG132 at 10 microM) inhibited nonlysosomal protein breakdown by up to 50% (P protein synthesis or amino acid pools, but improved overall protein balance in the muscle. Upon treatment with MG132, ubiquitin-conjugated proteins accumulated in the muscle. The inhibition of muscle proteolysis correlated with efficacy against the proteasome, although these agents could also inhibit calpain-dependent proteolysis induced with Ca2+. These inhibitors had much larger effects on proteolysis in atrophying muscles than in controls. In the denervated soleus undergoing atrophy, the increase in ATP-dependent proteolysis was reduced 70% by MG132 (P muscle proteolysis induced by administering thyroid hormones was reduced 40-70% by the inhibitors. Finally, in rats made septic by cecal puncture, the increase in muscle proteolysis was completely blocked by MG132. Thus, the enhanced proteolysis in many catabolic states (including denervation, hyperthyroidism, and sepsis) is due to a proteasome-dependent pathway, and inhibition of proteasome function may be a useful approach to reduce muscle wasting.

  12. A model of proteolysis and amino acid biosynthesis for Lactobacillus delbrueckii subsp. bulgaricus in whey.

    Science.gov (United States)

    Liu, Enuo; Zheng, Huajun; Hao, Pei; Konno, Tomonobu; Yu, Yao; Kume, Hisae; Oda, Munehiro; Ji, Zai-Si

    2012-12-01

    Lactobacillus delbrueckii subsp. bulgaricus 2038 (L. bulgaricus 2038) is a bacterium that is used as a starter for dairy products by Meiji Co., Ltd of Japan. Culturing L. bulgaricus 2038 with whey as the sole nitrogen source results in a shorter lag phase than other milk proteins under the same conditions (carbon source, minerals, and vitamins). Microarray results of gene expression revealed characteristics of amino acid anabolism with whey as the nitrogen source and established a model of proteolysis and amino acid biosynthesis for L. bulgaricus. Whey peptides and free amino acids are readily metabolized, enabling rapid entry into the logarithmic growth phase. The oligopeptide transport system is the primary pathway for obtaining amino acids. Amino acid biosynthesis maintains the balance between amino acids required for cell growth and the amount obtained from environment. The interconversion of amino acids is also important for L. bulgaricus 2038 growth.

  13. Phenylbutyric acid rescues endoplasmic reticulum stress-induced suppression of APP proteolysis and prevents apoptosis in neuronal cells.

    Directory of Open Access Journals (Sweden)

    Jesse C Wiley

    Full Text Available BACKGROUND: The familial and sporadic forms of Alzheimer's disease (AD have an identical pathology with a severe disparity in the time of onset [1]. The pathological similarity suggests that epigenetic processes may phenocopy the Familial Alzheimer's disease (FAD mutations within sporadic AD. Numerous groups have demonstrated that FAD mutations in presenilin result in 'loss of function' of gamma-secretase mediated APP cleavage [2], [3], [4], [5]. Accordingly, ER stress is prominent within the pathologically impacted brain regions in AD patients [6] and is reported to inhibit APP trafficking through the secretory pathway [7], [8]. As the maturation of APP and the cleaving secretases requires trafficking through the secretory pathway [9], [10], [11], we hypothesized that ER stress may block trafficking requisite for normal levels of APP cleavage and that the small molecular chaperone 4-phenylbutyrate (PBA may rescue the proteolytic deficit. METHODOLOGY/PRINCIPAL FINDINGS: The APP-Gal4VP16/Gal4-reporter screen was stably incorporated into neuroblastoma cells in order to assay gamma-secretase mediated APP proteolysis under normal and pharmacologically induced ER stress conditions. Three unrelated pharmacological agents (tunicamycin, thapsigargin and brefeldin A all repressed APP proteolysis in parallel with activation of unfolded protein response (UPR signaling-a biochemical marker of ER stress. Co-treatment of the gamma-secretase reporter cells with PBA blocked the repressive effects of tunicamycin and thapsigargin upon APP proteolysis, UPR activation, and apoptosis. In unstressed cells, PBA stimulated gamma-secretase mediated cleavage of APP by 8-10 fold, in the absence of any significant effects upon amyloid production, by promoting APP trafficking through the secretory pathway and the stimulation of the non-pathogenic alpha/gamma-cleavage. CONCLUSIONS/SIGNIFICANCE: ER stress represses gamma-secretase mediated APP proteolysis, which replicates

  14. Estrogen receptor of primary breast cancers: evidence for intracellular proteolysis

    International Nuclear Information System (INIS)

    Maaroufi, Younes; Lacroix, Marc; Lespagnard, Laurence; Journé, Fabrice; Larsimont, Denis; Leclercq, Guy

    2000-01-01

    Iodinated oestradiol-labeled oestrogen receptor (ER) isoforms devoid of amino-terminal ABC domains represent about two-thirds of the whole receptor population detected in cytosol samples from human breast cancers. This high frequency could not be ascribed to the expression of truncated mRNAs, or to the proteolysis of the native ER peptide at the time of homogenization or assay, suggesting an intracellular proteolysis. Free amino-terminal and ligand-binding domains maintained together within oligomeric structure(s); increase of ionic strength separated them. The amino-terminal region was consistently detected in the cell nucleus by specific immunohistochemistry leading to the concept of a potential intranuclear association between ER cleavage products and/or other regulatory proteins. We previously reported that about two-thirds of [ 125 I]oestradiol-labelled cytosolic ERs from breast cancer samples eluted as low-molecular-weight isoforms (≤ 37 kDa, size-exclusion fast pressure liquid chromatography [FPLC]). These isoforms failed to adsorb strongly to hydroxylapatite at high ionic strength, a property that was ascribed to receptors devoid of amino-terminal ABC domains. In view of recent data concerning intracellular proteolysis of several transcriptional regulators, the possibility of such behaviour for ER was assessed. The clinical significance of ER measurement in breast cancer cytosols is well established; approximately 50% of ER-positive cases respond to endocrine therapy. Whether such a poor correlation is related to a high proportion of cleaved ER is a question of prime importance. Failure of routine ER assays to discriminate between full-length and cleaved receptors led us to develop an oestradiol-binding assay based on hydroxylapatite adsorption. The aims of the present study were to demonstrate that hydroxylapatite adsorption assay easily identifies cleaved cytosolic ER forms and to assess the origin of such ER forms. Breast cancer cytosols classified as

  15. Enzymatically structured emulsions in simulated gastrointestinal environment: impact on interfacial proteolysis and diffusion in intestinal mucus.

    Science.gov (United States)

    Macierzanka, Adam; Böttger, Franziska; Rigby, Neil M; Lille, Martina; Poutanen, Kaisa; Mills, E N Clare; Mackie, Alan R

    2012-12-18

    Fundamental knowledge of physicochemical interactions in the gastrointestinal environment is required in order to support rational designing of protein-stabilized colloidal food and pharmaceutical delivery systems with controlled behavior. In this paper, we report on the colloidal behavior of emulsions stabilized with the milk protein sodium caseinate (Na-Cas), and exposed to conditions simulating the human upper gastrointestinal tract. In particular, we looked at how the kinetics of proteolysis was affected by adsorption to an oil-water interface in emulsion and whether the proteolysis and the emulsion stability could be manipulated by enzymatic structuring of the interface. After cross-linking with the enzyme transglutaminase, the protein was digested with use of an in vitro model of gastro-duodenal proteolysis in the presence or absence of physiologically relevant surfactants (phosphatidylcholine, PC; bile salts, BS). Significant differences were found between the rates of digestion of Na-Cas cross-linked in emulsion (adsorbed protein) and in solution. In emulsion, the digestion of a population of polypeptides of M(r) ca. 50-100 kDa was significantly retarded through the gastric digestion. The persistent interfacial polypeptides maintained the original emulsion droplet size and prevented the system from phase separating. Rapid pepsinolysis of adsorbed, non-cross-linked Na-Cas and its displacement by PC led to emulsion destabilization. These results suggest that structuring of emulsions by enzymatic cross-linking of the interfacial protein may affect the phase behavior of emulsion in the stomach and the gastric digestion rate in vivo. Measurements of ζ-potential revealed that BS displaced the remaining protein from the oil droplets during the simulated duodenal phase of digestion. Diffusion of the postdigestion emulsion droplets through ex vivo porcine intestinal mucus was only significant in the presence of BS due to the high negative charge these

  16. Laser-light backscattering response to water content and proteolysis in dry-cured ham

    DEFF Research Database (Denmark)

    Fulladosa, E.; Rubio-Celorio, M.; Skytte, Jacob Lercke

    2017-01-01

    on the acquisition conditions used. Laser backscattering was influenced by both dryness and proteolysis intensity showing an average light intensity decrease of 0.2 when decreasing water content (1% weight loss) and increasing proteolysis (equivalent to one-hour enzyme action). However, a decrease of scattering area...... was only detected when the water content was decreased (618 mm(2) per 1% weight loss). Changes on scattering of light profiles were only observed when the water content changed. Although there is a good correlation between water content and LBI parameters when analysing commercial samples, proteolysis...... of laser incidence) and to analyse the laser-light backscattering changes caused by additional hot air drying and proteolysis of dry-cured ham slices. The feasibility of the technology to determine water content and proteolysis (which is related to textural characteristics) of commercial sliced dry...

  17. Proteolysis targeting peptide (PROTAP) strategy for protein ubiquitination and degradation.

    Science.gov (United States)

    Zheng, Jing; Tan, Chunyan; Xue, Pengcheng; Cao, Jiakun; Liu, Feng; Tan, Ying; Jiang, Yuyang

    2016-02-19

    Ubiquitination proteasome pathway (UPP) is the most important and selective way to degrade proteins in vivo. Here, a novel proteolysis targeting peptide (PROTAP) strategy, composed of a target protein binding peptide, a linker and a ubiquitin E3 ligase recognition peptide, was designed to recruit both target protein and E3 ligase and then induce polyubiquitination and degradation of the target protein through UPP. In our study, the PROTAP strategy was proved to be a general method with high specificity using Bcl-xL protein as model target in vitro and in cells, which indicates that the strategy has great potential for in vivo application. Copyright © 2016 Elsevier Inc. All rights reserved.

  18. The Degradome database: mammalian proteases and diseases of proteolysis.

    Science.gov (United States)

    Quesada, Víctor; Ordóñez, Gonzalo R; Sánchez, Luis M; Puente, Xose S; López-Otín, Carlos

    2009-01-01

    The degradome is defined as the complete set of proteases present in an organism. The recent availability of whole genomic sequences from multiple organisms has led us to predict the contents of the degradomes of several mammalian species. To ensure the fidelity of these predictions, our methods have included manual curation of individual sequences and, when necessary, direct cloning and sequencing experiments. The results of these studies in human, chimpanzee, mouse and rat have been incorporated into the Degradome database, which can be accessed through a web interface at http://degradome.uniovi.es. The annotations about each individual protease can be retrieved by browsing catalytic classes and families or by searching specific terms. This web site also provides detailed information about genetic diseases of proteolysis, a growing field of great importance for multiple users. Finally, the user can find additional information about protease structures, protease inhibitors, ancillary domains of proteases and differences between mammalian degradomes.

  19. Conformational changes in DNA gyrase revealed by limited proteolysis

    DEFF Research Database (Denmark)

    Kampranis, S C; Maxwell, A

    1998-01-01

    We have used limited proteolysis to identify conformational changes in DNA gyrase. Gyrase exhibits a proteolytic fingerprint dominated by two fragments, one of approximately 62 kDa, deriving from the A protein, and another of approximately 25 kDa from the B protein. Quinolone binding to the enzyme......-DNA intermediate by calcium ions does not reveal any protection, suggesting that the quinolone-induced conformational change is different from an "open-gate" state of the enzyme. A quinolone-resistant mutant of gyrase fails to give the characteristic quinolone-associated proteolytic signature. The ATP...... does not prevent dimerization since incubation of the enzyme-DNA complex with both ADPNP and quinolones gives rise to a complex whose proteolytic pattern retains the characteristic signature of dimerization but has lost the quinolone-induced protection. As a result, the quinolone-gyrase complex can...

  20. Age-related changes in factor VII proteolysis in vivo.

    Science.gov (United States)

    Ofosu, F A; Craven, S; Dewar, L; Anvari, N; Andrew, M; Blajchman, M A

    1996-08-01

    Previous studies have reported that pre-operative plasmas of patients over the age of 40 years who developed post-operative deep vein thrombosis (DVT) had approximately twice the amount of proteolysed factor VII found in plasmas of patients in whom prophylaxis with heparin or low M(r) heparin was successful. These and other studies also reported higher concentrations of thrombin-antithrombin III in pre- and post-operative plasmas of patients who developed post-operative thrombosis than in plasmas of patients in whom prophylaxis was successful. Whether the extent of factor VII proteolysis seen in the patients who developed post-operative DVT is related to the severity of their disease or age is not known. This report investigated age-related changes in the concentrations of total factor VII protein, factor VII zymogen, factor VIIa, tissue factor pathway inhibitor, thrombin-antithrombin III, and prothrombin fragment 1 + 2 in normal plasmas and the relationships between these parameters. With the exception of thrombin-antithrombin III, statistically significant increases in the concentrations of these parameters with age were found. Additionally, the differences between the concentrations of total factor VII protein and factor VII zymogen, an index factor VII proteolysis in vivo, were statistically significant only for individuals over age 40. Using linear regression analysis, a significant correlation was found to exist between the concentrations of plasma factor VIIa and prothrombin fragment 1 + 2. Since factor VIIa-tissue factor probably initiates coagulation in vivo, we hypothesize that the elevated plasma factor VIIa (reflecting a less tightly regulated tissue factor activity and therefore increased thrombin production in vivo) accounts for the high risk for post-operative thrombosis seen in individuals over the age of 40.

  1. Proteolysis in model Portuguese cheeses: Effects of rennet and starter culture.

    Science.gov (United States)

    Pereira, Cláudia I; Gomes, Eliza O; Gomes, Ana M P; Malcata, F Xavier

    2008-06-01

    To shed further light onto the mechanisms of proteolysis that prevail throughout ripening of Portuguese cheeses, model cheeses were manufactured from bovine milk, following as much as possible traditional manufacture practices - using either animal or plant rennet. The individual role upon proteolysis of two (wild) strains of lactic acid bacteria - viz. Lactococcus lactis and Lactobacillus brevis, which are normally found to high viable numbers in said cheeses, was also considered, either as single or mixed cultures. Our experimental results confirmed the influence of rennet on the proteolysis extent, but not on proteolysis depth. On the other hand, the aforementioned strains clearly improved release of medium- and small-sized peptides, and contributed as well to the free amino acid pool in cheese. Copyright © 2007 Elsevier Ltd. All rights reserved.

  2. Effects of natural plant tenderizers on proteolysis and texture of dry ...

    African Journals Online (AJOL)

    , and overall acceptability. From these results, it is shown that those enzymes as a raw plant juices could be used as tenderizers in dry sausage production. Keywords: Dry sausages, wild boar meat, plant enzymes, proteolysis, texture, sensory ...

  3. Pre-cure freezing affects proteolysis in dry-cured hams.

    Science.gov (United States)

    Bañón, S; Cayuela, J M; Granados, M V; Garrido, M D

    1999-01-01

    Several parameters (sodium chloride, moisture, intramuscular fat, total nitrogen, non-protein nitrogen, white precipitates, free tyrosine, L* a* b* values and acceptability) related with proteolysis during the curing were compared in dry-cured hams manufactured from refrigerated and frozen/thawed raw material. Pre-cure freezing increased the proteolysis levels significantly (pcured meat, although it does not significantly affect the sensory quality of the dry-cured ham.

  4. Inhibitors of the proteasome reduce the accelerated proteolysis in atrophying rat skeletal muscles.

    OpenAIRE

    Tawa, N E; Odessey, R; Goldberg, A L

    1997-01-01

    Several observations have suggested that the enhanced proteolysis and atrophy of skeletal muscle in various pathological states is due primarily to activation of the ubiquitin-proteasome pathway. To test this idea, we investigated whether peptide aldehyde inhibitors of the proteasome, N-acetyl-leucyl-leucyl-norleucinal (LLN), or the more potent CBZ-leucyl-leucyl-leucinal (MG132) suppressed proteolysis in incubated rat skeletal muscles. These agents (e.g., MG132 at 10 microM) inhibited nonlyso...

  5. 11S Storage globulin from pumpkin seeds: regularities of proteolysis by papain.

    Science.gov (United States)

    Rudakova, A S; Rudakov, S V; Kakhovskaya, I A; Shutov, A D

    2014-08-01

    Limited proteolysis of the α- and β-chains and deep cleavage of the αβ-subunits by the cooperative (one-by-one) mechanism was observed in the course of papain hydrolysis of cucurbitin, an 11S storage globulin from seeds of the pumpkin Cucurbita maxima. An independent analysis of the kinetics of the limited and cooperative proteolyses revealed that the reaction occurs in two successive steps. In the first step, limited proteolysis consisting of detachments of short terminal peptides from the α- and β-chains was observed. The cooperative proteolysis, which occurs as a pseudo-first order reaction, started at the second step. Therefore, the limited proteolysis at the first step plays a regulatory role, impacting the rate of deep degradation of cucurbitin molecules by the cooperative mechanism. Structural alterations of cucurbitin induced by limited proteolysis are suggested to generate its susceptibility to cooperative proteolysis. These alterations are tentatively discussed on the basis of the tertiary structure of the cucurbitin subunit pdb|2EVX in comparison with previously obtained data on features of degradation of soybean 11S globulin hydrolyzed by papain.

  6. Sequence analysis of PROTEOLYSIS 6 from Solanum lycopersicum

    Science.gov (United States)

    Roslan, Nur Farhana; Chew, Bee Lyn; Goh, Hoe-Han; Isa, Nurulhikma Md

    2018-04-01

    The N-end rule pathway is a protein degradation pathway that relates the protein half-life with the identity of its N-terminal residues. A destabilizing N-terminal residues is created by enzymatic reaction or chemical modifications. This destabilized substrate will be recognized by PROTEOLYSIS 6 (PRT6) protein, which encodes an E3 ligase enzyme and resulted in substrate degradation by proteasome. PRT6 has been studied in Arabidopsis thaliana and barley but not yet been studied in fleshy fruit plants. Hence, this study was carried out in tomato that is known as the model for fleshy fruit plants. BLASTX analysis identified that Solyc09g010830 which encodes for a PRT6 gene in tomato based on its sequence similarity with PRT6 in A. thaliana. In silico gene expression analysis shows that PRT6 gene was highly expressed in tomato fruits breaker +5. Co-expression analysis shows that PRT6 may not only involved in abiotic stresses but also in biotic stresses. The objective is to analyze the sequence and characterize PRT6 gene in tomato.

  7. Acid-Mediated Tumor Proteolysis: Contribution of Cysteine Cathepsins

    Directory of Open Access Journals (Sweden)

    Jennifer M Rothberg

    2013-10-01

    Full Text Available One of the noncellular microenvironmental factors that contribute to malignancy of solid tumors is acidic peritumoral pH. We have previously demonstrated that extracellular acidosis leads to localization of the cysteine pro-tease cathepsin B on the tumor cell membrane and its secretion. The objective of the present study was to determine if an acidic extracellular pH such as that observed in vivo (i.e., pHe 6.8 affects the activity of proteases, e.g., cathepsin B, that contribute to degradation of collagen IV by tumor cells when grown in biologically relevant three-dimensional (3D cultures. For these studies, we used 1 3D reconstituted basement membrane overlay cultures of human carcinomas, 2 live cell imaging assays to assess proteolysis, and 3 in vivo imaging of active tumor proteases. At pHe 6.8, there were increases in pericellular active cysteine cathepsins and in degradation of dye-quenched collagen IV, which was partially blocked by a cathepsin B inhibitor. Imaging probes for active cysteine cathepsins localized to tumors in vivo. The amount of bound probe decreased in tumors in bicarbonate-treated mice, a treatment previously shown to increase peritumoral pHe and reduce local invasion of the tumors. Our results are consistent with the acid-mediated invasion hypothesis and with a role for cathepsin B in promoting degradation of a basement membrane protein substrate, i.e., type IV collagen, in an acidic peritumoral environment.

  8. Proteolysis of truncated hemolysin A yields a stable dimerization interface

    Energy Technology Data Exchange (ETDEWEB)

    Novak, Walter R.P.; Bhattacharyya, Basudeb; Grilley, Daniel P.; Weaver, Todd M. (Wabash); (UW)

    2017-02-21

    Wild-type and variant forms of HpmA265 (truncated hemolysin A) fromProteus mirabilisreveal a right-handed, parallel β-helix capped and flanked by segments of antiparallel β-strands. The low-salt crystal structures form a dimeric structureviathe implementation of on-edge main-chain hydrogen bonds donated by residues 243–263 of adjacent monomers. Surprisingly, in the high-salt structures of two variants, Y134A and Q125A-Y134A, a new dimeric interface is formedviamain-chain hydrogen bonds donated by residues 203–215 of adjacent monomers, and a previously unobserved tetramer is formed. In addition, an eight-stranded antiparallel β-sheet is formed from the flap regions of crystallographically related monomers in the high-salt structures. This new interface is possible owing to additional proteolysis of these variants after Tyr240. The interface formed in the high-salt crystal forms of hemolysin A variants may mimic the on-edge β-strand positioning used in template-assisted hemolytic activity.

  9. Imaging Proteolysis by Living Human Breast Cancer Cells

    Directory of Open Access Journals (Sweden)

    Mansoureh Sameni

    2000-01-01

    Full Text Available Malignant progression is accompanied by degradation of extracellular matrix proteins. Here we describe a novel confocal assay in which we can observe proteolysis by living human breast cancer cells (BT20 and BT549 through the use of quenchedfluorescent protein substrates. Degradation thus was imaged, by confocal optical sectioning, as an accumulation of fluorescent products. With the BT20 cells, fluorescence was localized to pericellular focal areas that coincide with pits in the underlying matrix. In contrast, fluorescence was localized to intracellular vesicles in the BT549 cells, vesicles that also label for lysosomal markers. Neither intracellular nor pericellular fluorescence was observed in the BT549 cells in the presence of cytochalasin B, suggesting that degradation occurred intracellularly and was dependent on endocytic uptake of substrate. In the presence of a cathepsin 13-selective cysteine protease inhibitor, intracellular fluorescence was decreased ~90% and pericellular fluorescence decreased 67% to 96%, depending on the protein substrate. Matrix metallo protease inhibitors reduced pericellular fluorescence ~50%, i.e., comparably to a serine and a broad spectrum cysteine protease inhibitor. Our results suggest that: 1 a proteolytic cascade participates in pericellular digestion of matrix proteins by living human breast cancer cells, and 2 the cysteine protease cathepsin B participates in both pericellular and intracellular digestion of matrix proteins by living human breast cancer cells.

  10. Stable MCC binding to the APC/C is required for a functional spindle assembly checkpoint

    DEFF Research Database (Denmark)

    Hein, Jamin B; Nilsson, Jakob

    2014-01-01

    stably to the APC/C. Whether MCC formation per se is sufficient for a functional SAC or MCC association with the APC/C is required remains unclear. Here, we analyze the role of two conserved motifs in Cdc20, IR and C-Box, in binding of the MCC to the APC/C. Mutants in both motifs assemble the MCC....../C is critical for a functional SAC....

  11. Molecular characterization of a proteolysis-resistant lipase from Bacillus pumilus SG2

    Directory of Open Access Journals (Sweden)

    R. Sangeetha

    2014-06-01

    Full Text Available Proteolysis-resistant lipases can be well exploited by industrial processes which employ both lipase and protease as biocatalysts. A proteolysis resistant lipase from Bacillus pumilus SG2 was isolated, purified and characterized earlier. The lipase was resistant to native and commercial proteases. In the present work, we have characterized the lip gene which encodes the proteolysis-resistant lipase from Bacillus pumilus SG2. The parameters and structural details of lipase were analysed. The lip gene consisted of 650 bp. The experimental molecular weight of SG2 lipase was nearly double that of its theoretical molecular weight, thus suggesting the existence of the functional lipase as a covalent dimer. The proteolytic cleavage sites of the lipase would have been made inaccessible by dimerisation, thus rendering the lipase resistant to protease.

  12. A SIMPLE FLUORESCENT LABELING METHOD FOR STUDIES OF PROTEIN OXIDATION, PROTEIN MODIFICATION, AND PROTEOLYSIS

    Science.gov (United States)

    Pickering, Andrew. M.; Davies, Kelvin. J. A.

    2014-01-01

    Proteins are sensitive to oxidation, and oxidized proteins are excellent substrates for degradation by proteolytic enzymes such as the Proteasome and the mitochondrial Lon protease. Protein labeling is required for studies of protein turnover. Unfortunately, most labeling techniques involve 3H or 14C methylation which is expensive, exposes researchers to radioactivity, generates large amounts of radioactive waste, and allows only single-point assays because samples require acid-precipitation. Alternative labeling methods, have largely proven unsuitable, either because the probe itself is modified by the oxidant(s) being studied, or because the alternative labeling techniques are too complex or too costly for routine use. What is needed is a simple, quick, and cheap labeling technique that uses a non-radioactive marker, that binds strongly to proteins, is resistant to oxidative modification, and emits a strong signal. We have devised a new reductive method for labeling free carboxyl groups of proteins with the small fluorophore 7-amino-4-methycoumarin (AMC). When bound to target proteins, AMC fluoresces very weakly but when AMC is released by proteinases, proteases, or peptidases, it fluoresces strongly. Thus, without acid-precipitation, the proteolysis of any target protein can be studied continuously, in multiwell plates. In direct comparisons, 3H-labeled proteins and AMC-labeled proteins exhibited essentially identical degradation patterns during incubation with trypsin, cell extracts, and purified proteasome. AMC-labeled proteins are well-suited to study increased proteolytic susceptibility following protein modification, since the AMC-protein bond is resistant to oxidizing agents such as hydrogen peroxide and peroxynitrite, and is stable over time and to extremes of pH, temperature (even boiling), freeze-thawing, mercaptoethanol, and methanol. PMID:21988844

  13. The cachectic mediator proteolysis inducing factor activates NF-kappaB and STAT3 in human Kupffer cells and monocytes

    NARCIS (Netherlands)

    Watchorn, T.M.; Dowidar, N.; Dejong, C.H.; Waddell, I.D.; Garden, O.J.; Ross, J.A.

    2005-01-01

    A novel proteoglycan, proteolysis inducing factor (PIF), is capable of inducing muscle proteolysis during the process of cancer cachexia, and of inducing an acute phase response in human hepatocytes. We investigated whether PIF is able to activate pro-inflammatory pathways in human Kupffer cells,

  14. Metal ion release from metallothioneins: proteolysis as an alternative to oxidation.

    Science.gov (United States)

    Peroza, Estevão A; dos Santos Cabral, Augusto; Wan, Xiaoqiong; Freisinger, Eva

    2013-09-01

    Metallothioneins (MTs) are among others involved in the cellular regulation of essential Zn(II) and Cu(I) ions. However, the high binding affinity of these proteins requires additional factors to promote metal ion release under physiological conditions. The mechanisms and efficiencies of these processes leave many open questions. We report here a comprehensive analysis of the Zn(II)-release properties of various MTs with special focus on members of the four main subfamilies of plant MTs. Zn(II) competition experiments with the metal ion chelator 4-(2-pyridylazo)resorcinol (PAR) in the presence of the cellular redox pair glutathione (GSH)/glutathione disulfide (GSSG) show that plant MTs from the subfamilies MT1, MT2, and MT3 are remarkably more affected by oxidative stress than those from the Ec subfamily and the well-characterized human MT2 form. In addition, we evaluated proteolytic digestion with trypsin and proteinase K as an alternative mechanism for selective promotion of metal ion release from MTs. Also here the observed percentage of liberated metal ions depends strongly on the MT form evaluated. Closer evaluation of the data additionally allowed deducing the thermodynamic and kinetic properties of the Zn(II) release processes. The Cu(I)-form of chickpea MT2 was used to exemplify that both oxidation and proteolysis are also effective ways to increase the transfer of copper ions to other molecules. Zn(II) release experiments with the individual metal-binding domains of Ec-1 from wheat grain reveal distinct differences from the full-length protein. This triggers the question about the roles of the long cysteine-free peptide stretches typical for plant MTs.

  15. Energy and calcium ion dependence of proteolysis during sporulation of Bacillus subtilis cells

    International Nuclear Information System (INIS)

    O'Hara, M.B.; Hageman, J.H.

    1990-01-01

    The authors have shown, with an optimized [ 14 C]leucine-labeling and chasing procedure, that intracellular protein degradation in sporulating cells of Bacillus subtilis 168 (trpC2) is apparently energy dependent. Sodium arsenate, sodium azide, carbonyl cyanide m-chlorophenylhydrozone, and N,N'-dicyclohexylcarbodiimide, at levels which did not induce appreciable lysis (≤ 10%) over 10-h periods of sporulation, inhibited intracellular proteolysis by 13 to 93%. Exponentially growing cells acquired arsenate resistance. In contrast to earlier reports, the authors found that chloramphenicol strongly inhibited proteolysis even when added 6 h into the sporulation process. Restricting the calcium ion concentration in the medium had no effect on rates or extent of vegetative growth, strongly inhibited sporulation, and inhibited rates of proteolysis by 60% or more. Inhibitors of energy metabolism, at the same levels which inhibited proteolysis, did not affect the rate or degree of uptake of Ca 2+ by cells. Restricting the Ca 2+ concentration in the medium reduced by threefold of the specific activity in cells of the major intracellular serine proteinase after 12 h of sporulation. finally, cells of a mutant of B. subtilis bearing an insertionally inactivated gene for the Ca 2+ -dependent intracellular proteinase-1 degraded protein in chemically defined sporulation medium at a rate indistinguishable from that of the wild-type cells for period of 8 h

  16. A primary study on texture modification and proteolysis of mao-tofu ...

    African Journals Online (AJOL)

    Proteolysis occurred during fermentation was evaluated by SDS-PAGE and chemical analysis. Results from Texture Profile Analysis showed that adhesiveness of mao-tofu had an increase trend while hardness, cohesiveness and springiness had a decrease trend as fermentation progressed. SEM analysis showed that the ...

  17. Characterization of linear forms of the circular enterocin AS-48 obtained by limited proteolysis

    NARCIS (Netherlands)

    Montalbán-López, Manuel; Spolaore, Barbara; Pinato, Odra; Martínez-Bueno, Manuel; Valdivia, Eva; Maqueda, Mercedes; Fontana, Angelo

    2008-01-01

    AS-48 is a 70-residue circular peptide from Enterococcus faecalis with a broad antibacterial activity. Here, we produced by limited proteolysis a protein species carrying a single nicking and fragments of 55 and 38 residues. Nicked AS-48 showed a lower helicity by far-ultraviolet circular dichroism

  18. Immobilization of trypsin on miniature incandescent bulbs for infrared-assisted proteolysis

    Energy Technology Data Exchange (ETDEWEB)

    Ge, Huimin; Bao, Huimin; Zhang, Luyan; Chen, Gang, E-mail: gangchen@fudan.edu.cn

    2014-10-03

    Highlights: • Trypsin was immobilized on miniature incandescent bulbs via chitosan coating. • The bulbs acted as enzymatic reactors and the generators of infrared radiation. • The bulb bioreactors were successfully employed in infrared-assisted proteolysis. • The proteolysis could accomplish within 5 min with high sequence coverages. - Abstract: A novel efficient proteolysis approach was developed based on trypsin-immobilized miniature incandescent bulbs and infrared (IR) radiation. Trypsin was covalently immobilized in the chitosan coating on the outer surface of miniature incandescent bulbs with the aid of glutaraldehyde. When an illuminated enzyme-immobilized bulb was immersed in protein solution, the emitted IR radiation could trigger and accelerate heterogeneous protein digestion. The feasibility and performance of the novel proteolysis approach were demonstrated by the digestion of hemoglobin (HEM), cytochrome c (Cyt-c), lysozyme (LYS), and ovalbumin (OVA) and the digestion time was significantly reduced to 5 min. The obtained digests were identified by MALDI-TOF-MS with the sequence coverages of 91%, 77%, 80%, and 52% for HEM, Cyt-c, LYS, and OVA (200 ng μL{sup −1} each), respectively. The suitability of the prepared bulb bioreactors to complex proteins was demonstrated by digesting human serum.

  19. Proteolysis produced within biofilms of bacterial isolates from raw milk tankers.

    Science.gov (United States)

    Teh, Koon Hoong; Flint, Steve; Palmer, Jon; Andrewes, Paul; Bremer, Phil; Lindsay, Denise

    2012-06-15

    In this study, six bacterial isolates that produced thermo-resistant enzymes isolated from the internal surfaces of raw milk tankers were evaluated for their ability to produce proteolysis within either single culture biofilms or co-culture biofilms. Biofilms were formed in an in vitro model system that simulated the upper internal surface of a raw milk tanker during a typical summer's day of milk collection in New Zealand. The bacterial isolates were further evaluated for their ability to form biofilms at 25, 30 and 37°C. Mutual and competitive effects were observed in some of the co-culture biofilms, with all isolates being able to form biofilms in either single culture or co-culture at the three temperatures. The proteolysis was also evaluated in both biofilms and corresponding planktonic cultures. The proteolysis per cell decreased as the temperature of incubation (20-37°C) increased. Furthermore, mutualistic interactions in terms of proteolysis were observed when cultures were grown as co-culture biofilms. This is the first study to show that proteolytic enzymes can be produced in biofilms on the internal surfaces of raw milk tankers. This has important implications for the cleaning and the temperature control of raw milk transport tankers. Copyright © 2012 Elsevier B.V. All rights reserved.

  20. A novel Meloidogyne graminicola effector, MgGPP, is secreted into host cells and undergoes glycosylation in concert with proteolysis to suppress plant defenses and promote parasitism.

    Directory of Open Access Journals (Sweden)

    Jiansong Chen

    2017-04-01

    Full Text Available Plant pathogen effectors can recruit the host post-translational machinery to mediate their post-translational modification (PTM and regulate their activity to facilitate parasitism, but few studies have focused on this phenomenon in the field of plant-parasitic nematodes. In this study, we show that the plant-parasitic nematode Meloidogyne graminicola has evolved a novel effector, MgGPP, that is exclusively expressed within the nematode subventral esophageal gland cells and up-regulated in the early parasitic stage of M. graminicola. The effector MgGPP plays a role in nematode parasitism. Transgenic rice lines expressing MgGPP become significantly more susceptible to M. graminicola infection than wild-type control plants, and conversely, in planta, the silencing of MgGPP through RNAi technology substantially increases the resistance of rice to M. graminicola. Significantly, we show that MgGPP is secreted into host plants and targeted to the ER, where the N-glycosylation and C-terminal proteolysis of MgGPP occur. C-terminal proteolysis promotes MgGPP to leave the ER, after which it is transported to the nucleus. In addition, N-glycosylation of MgGPP is required for suppressing the host response. The research data provide an intriguing example of in planta glycosylation in concert with proteolysis of a pathogen effector, which depict a novel mechanism by which parasitic nematodes could subjugate plant immunity and promote parasitism and may present a promising target for developing new strategies against nematode infections.

  1. Peptides Displayed as High Density Brush Polymers Resist Proteolysis and Retain Bioactivity

    Science.gov (United States)

    2015-01-01

    We describe a strategy for rendering peptides resistant to proteolysis by formulating them as high-density brush polymers. The utility of this approach is demonstrated by polymerizing well-established cell-penetrating peptides (CPPs) and showing that the resulting polymers are not only resistant to proteolysis but also maintain their ability to enter cells. The scope of this design concept is explored by studying the proteolytic resistance of brush polymers composed of peptides that are substrates for either thrombin or a metalloprotease. Finally, we demonstrate that the proteolytic susceptibility of peptide brush polymers can be tuned by adjusting the density of the polymer brush and offer in silico models to rationalize this finding. We contend that this strategy offers a plausible method of preparing peptides for in vivo use, where rapid digestion by proteases has traditionally restricted their utility. PMID:25314576

  2. Autophagic dysfunction in a lysosomal storage disorder due to impaired proteolysis

    OpenAIRE

    Elrick, Matthew J.; Lieberman, Andrew P.

    2013-01-01

    Alterations in macroautophagy (hereafter referred to as “autophagy”) are a common feature of lysosomal storage disorders, and have been hypothesized to play a major role in the pathogenesis of these diseases. We have recently reported multiple defects in autophagy contributing to the lysosomal storage disorder Niemann-Pick type C (NPC). These include increased formation of autophagosomes, slowed turnover of autophagosomes secondary to impaired lysosomal proteolysis, and delivery of stored lip...

  3. Extracellular proteolysis in structural and functional plasticity of mossy fiber synapses in hippocampus

    Directory of Open Access Journals (Sweden)

    Grzegorz eWiera

    2015-11-01

    Full Text Available Brain is continuously altered in response to experience and environmental changes. One of the underlying mechanisms is synaptic plasticity, which is manifested by modification of synapse structure and function. It is becoming clear that regulated extracellular proteolysis plays a pivotal role in the structural and functional remodeling of synapses during brain development, learning and memory formation. Clearly, plasticity mechanisms may substantially differ between projections. Mossy fiber synapses onto CA3 pyramidal cells display several unique functional features, including pronounced short-term facilitation, a presynaptically expressed LTP that is independent of NMDAR activation, and NMDA-dependent metaplasticity. Moreover, structural plasticity at mossy fiber synapses ranges from the reorganization of projection topology after hippocampus-dependent learning, through intrinsically different dynamic properties of synaptic boutons to pre- and postsynaptic structural changes accompanying LTP induction. Although concomitant functional and structural plasticity in this pathway strongly suggests a role of extracellular proteolysis, its impact only starts to be investigated in this projection. In the present report, we review the role of extracellular proteolysis in various aspects of synaptic plasticity in hippocampal mossy fiber synapses. A growing body of evidence demonstrates that among perisynaptic proteases, tPA/plasmin system, β-site amyloid precursor protein-cleaving enzyme 1 (BACE1 and metalloproteinases play a crucial role in shaping plastic changes in this projection. We discuss recent advances and emerging hypotheses on the roles of proteases in mechanisms underlying mossy fiber target specific synaptic plasticity and memory formation.

  4. Collagen fibril architecture, domain organization, and triple-helical conformation govern its proteolysis.

    Science.gov (United States)

    Perumal, Shiamalee; Antipova, Olga; Orgel, Joseph P R O

    2008-02-26

    We describe the molecular structure of the collagen fibril and how it affects collagen proteolysis or "collagenolysis." The fibril-forming collagens are major components of all mammalian connective tissues, providing the structural and organizational framework for skin, blood vessels, bone, tendon, and other tissues. The triple helix of the collagen molecule is resistant to most proteinases, and the matrix metalloproteinases that do proteolyze collagen are affected by the architecture of collagen fibrils, which are notably more resistant to collagenolysis than lone collagen monomers. Until now, there has been no molecular explanation for this. Full or limited proteolysis of the collagen fibril is known to be a key process in normal growth, development, repair, and cell differentiation, and in cancerous tumor progression and heart disease. Peptide fragments generated by collagenolysis, and the conformation of exposed sites on the fibril as a result of limited proteolysis, regulate these processes and that of cellular attachment, but it is not known how or why. Using computational and molecular visualization methods, we found that the arrangement of collagen monomers in the fibril (its architecture) protects areas vulnerable to collagenolysis and strictly governs the process. This in turn affects the accessibility of a cell interaction site located near the cleavage region. Our observations suggest that the C-terminal telopeptide must be proteolyzed before collagenase can gain access to the cleavage site. Collagenase then binds to the substrate's "interaction domain," which facilitates the triple-helix unwinding/dissociation function of the enzyme before collagenolysis.

  5. New concepts in molecular imaging: non-invasive MRI spotting of proteolysis using an Overhauser effect switch.

    Directory of Open Access Journals (Sweden)

    Philippe Mellet

    Full Text Available Proteolysis, involved in many processes in living organisms, is tightly regulated in space and time under physiological conditions. However deregulation can occur with local persistent proteolytic activities, e.g. in inflammation, cystic fibrosis, tumors, or pancreatitis. Furthermore, little is known about the role of many proteases, hence there is a need of new imaging methods to visualize specifically normal or disease-related proteolysis in intact bodies.In this paper, a new concept for non invasive proteolysis imaging is proposed. Overhauser-enhanced Magnetic Resonance Imaging (OMRI at 0.2 Tesla was used to monitor the enzymatic hydrolysis of a nitroxide-labeled protein. In vitro, image intensity switched from 1 to 25 upon proteolysis due to the associated decrease in the motional correlation time of the substrate. The OMRI experimental device used in this study is consistent with protease imaging in mice at 0.2 T without significant heating. Simulations show that this enzymatic-driven OMRI signal switch can be obtained at lower frequencies suitable for larger animals or humans.The method is highly sensitive and makes possible proteolysis imaging in three dimensions with a good spatial resolution. Any protease could be targeted specifically through the use of taylor-made cleavable macromolecules. At short term OMRI of proteolysis may be applied to basic research as well as to evaluate therapeutic treatments in small animal models of experimental diseases.

  6. Dynamic assembly, localization and proteolysis of the Bacillus subtilis SMC complex

    Directory of Open Access Journals (Sweden)

    Rinn Cornelia

    2005-06-01

    Full Text Available Abstract Background SMC proteins are key components of several protein complexes that perform vital tasks in different chromosome dynamics. Bacterial SMC forms a complex with ScpA and ScpB that is essential for chromosome arrangement and segregation. The complex localizes to discrete centres on the nucleoids that during most of the time of the cell cycle localize in a bipolar manner. The complex binds to DNA and condenses DNA in an as yet unknown manner. Results We show that in vitro, ScpA and ScpB form different complexes with each other, among which the level of the putative 2 ScpA/4 ScpB complex showed a pronounced decrease in level upon addition of SMC protein. Different mutations of the ATPase-binding pocket of SMC reduced, but did not abolish interaction of mutant SMC with ScpA and ScpB. The loss of SMC ATPase activity led to a loss of function in vivo, and abolished proper localization of the SMC complex. The formation of bipolar SMC centres was also lost after repression of gyrase activity, and was abnormal during inhibition of replication, resulting in single central clusters. Resumption of replication quickly re-established bipolar SMC centres, showing that proper localization depends on ongoing replication. We also found that the SMC protein is subject to induced proteolysis, most strikingly as cells enter stationary phase, which is partly achieved by ClpX and LonA proteases. Atomic force microscopy revealed the existence of high order rosette-like SMC structures in vitro, which might explain the formation of the SMC centres in vivo. Conclusion Our data suggest that a ScpA/ScpB sub-complex is directly recruited into the SMC complex. This process does not require SMC ATPase activity, which, however, appears to facilitate loading of ScpA and ScpB. Thus, the activity of SMC could be regulated through binding and release of ScpA and ScpB, which has been shown to affect SMC ATPase activity. The proper bipolar localization of the SMC

  7. Metabolic and morphological alterations induced by proteolysis-inducing factor from Walker tumour-bearing rats in C2C12 myotubes

    International Nuclear Information System (INIS)

    Yano, Claudia L; Ventrucci, Gislaine; Field, William N; Tisdale, Michael J; Gomes-Marcondes, Maria Cristina C

    2008-01-01

    Patients with advanced cancer suffer from cachexia, which is characterised by a marked weight loss, and is invariably associated with the presence of tumoral and humoral factors which are mainly responsible for the depletion of fat stores and muscular tissue. In this work, we used cytotoxicity and enzymatic assays and morphological analysis to examine the effects of a proteolysis-inducing factor (PIF)-like molecule purified from ascitic fluid of Walker tumour-bearing rats (WF), which has been suggested to be responsible for muscle atrophy, on cultured C 2 C 12 muscle cells. WF decreased the viability of C 2 C 12 myotubes, especially at concentrations of 20–25 μg.mL -1 . There was an increase in the content of the pro-oxidant malondialdehyde, and a decrease in antioxidant enzyme activity. Myotubes protein synthesis decreased and protein degradation increased together with an enhanced in the chymotrypsin-like enzyme activity, a measure of functional proteasome activity, after treatment with WF. Morphological alterations such as cell retraction and the presence of numerous cells in suspension were observed, particularly at high WF concentrations. These results indicate that WF has similar effects to those of proteolysis-inducing factor, but is less potent than the latter. Further studies are required to determine the precise role of WF in this experimental model

  8. Metabolic and morphological alterations induced by proteolysis-inducing factor from Walker tumour-bearing rats in C2C12 myotubes

    Directory of Open Access Journals (Sweden)

    Tisdale Michael J

    2008-01-01

    Full Text Available Abstract Background Patients with advanced cancer suffer from cachexia, which is characterised by a marked weight loss, and is invariably associated with the presence of tumoral and humoral factors which are mainly responsible for the depletion of fat stores and muscular tissue. Methods In this work, we used cytotoxicity and enzymatic assays and morphological analysis to examine the effects of a proteolysis-inducing factor (PIF-like molecule purified from ascitic fluid of Walker tumour-bearing rats (WF, which has been suggested to be responsible for muscle atrophy, on cultured C2C12 muscle cells. Results WF decreased the viability of C2C12 myotubes, especially at concentrations of 20–25 μg.mL-1. There was an increase in the content of the pro-oxidant malondialdehyde, and a decrease in antioxidant enzyme activity. Myotubes protein synthesis decreased and protein degradation increased together with an enhanced in the chymotrypsin-like enzyme activity, a measure of functional proteasome activity, after treatment with WF. Morphological alterations such as cell retraction and the presence of numerous cells in suspension were observed, particularly at high WF concentrations. Conclusion These results indicate that WF has similar effects to those of proteolysis-inducing factor, but is less potent than the latter. Further studies are required to determine the precise role of WF in this experimental model.

  9. Metabolic and morphological alterations induced by proteolysis-inducing factor from Walker tumour-bearing rats in C{sub 2}C{sub 12} myotubes

    Energy Technology Data Exchange (ETDEWEB)

    Yano, Claudia L; Ventrucci, Gislaine [Departamento de Fisiologia e Biofísica, Instituto de Biologia, Universidade Estadual de Campinas (UNICAMP), CP 6109, 13083-970, Campinas, São Paulo (Brazil); Field, William N; Tisdale, Michael J [Cancer Research Laboratory, Pharmaceutical Sciences Research Institute, Aston University, Birmingham, B4 7ET (United Kingdom); Gomes-Marcondes, Maria Cristina C [Departamento de Fisiologia e Biofísica, Instituto de Biologia, Universidade Estadual de Campinas (UNICAMP), CP 6109, 13083-970, Campinas, São Paulo (Brazil)

    2008-01-28

    Patients with advanced cancer suffer from cachexia, which is characterised by a marked weight loss, and is invariably associated with the presence of tumoral and humoral factors which are mainly responsible for the depletion of fat stores and muscular tissue. In this work, we used cytotoxicity and enzymatic assays and morphological analysis to examine the effects of a proteolysis-inducing factor (PIF)-like molecule purified from ascitic fluid of Walker tumour-bearing rats (WF), which has been suggested to be responsible for muscle atrophy, on cultured C{sub 2}C{sub 12} muscle cells. WF decreased the viability of C{sub 2}C{sub 12} myotubes, especially at concentrations of 20–25 μg.mL{sup -1}. There was an increase in the content of the pro-oxidant malondialdehyde, and a decrease in antioxidant enzyme activity. Myotubes protein synthesis decreased and protein degradation increased together with an enhanced in the chymotrypsin-like enzyme activity, a measure of functional proteasome activity, after treatment with WF. Morphological alterations such as cell retraction and the presence of numerous cells in suspension were observed, particularly at high WF concentrations. These results indicate that WF has similar effects to those of proteolysis-inducing factor, but is less potent than the latter. Further studies are required to determine the precise role of WF in this experimental model.

  10. The lumbrical muscle: a novel in situ system to evaluate adult skeletal muscle proteolysis and anticatabolic drugs for therapeutic purposes.

    Science.gov (United States)

    Bergantin, Leandro Bueno; Figueiredo, Leonardo Bruno; Godinho, Rosely Oliveira

    2011-12-01

    The molecular regulation of skeletal muscle proteolysis and the pharmacological screening of anticatabolic drugs have been addressed by measuring tyrosine release from prepubertal rat skeletal muscles, which are thin enough to allow adequate in vitro diffusion of oxygen and substrates. However, the use of muscle at accelerated prepubertal growth has limited the analysis of adult muscle proteolysis or that associated with aging and neurodegenerative diseases. Here we established the adult rat lumbrical muscle (4/hindpaw; 8/rat) as a new in situ experimental model for dynamic measurement of skeletal muscle proteolysis. By incubating lumbrical muscles attached to their individual metatarsal bones in Tyrode solution, we showed that the muscle proteolysis rate of adult and aged rats (3-4 to 24 mo old) is 45-25% of that in prepubertal animals (1 mo old), which makes questionable the usual extrapolation of proteolysis from prepubertal to adult/senile muscles. While acute mechanical injury or 1- to 7-day denervation increased tyrosine release from adult lumbrical muscle by up to 60%, it was reduced by 20-28% after 2-h incubation with β-adrenoceptor agonists, forskolin or phosphodiesterase inhibitor IBMX. Using inhibitors of 26S-proteasome (MG132), lysosome (methylamine), or calpain (E64/leupeptin) systems, we showed that ubiquitin-proteasome is accountable for 40-50% of total lumbrical proteolysis of adult, middle-aged, and aged rats. In conclusion, the lumbrical model allows the analysis of muscle proteolysis rate from prepubertal to senile rats. By permitting eight simultaneous matched measurements per rat, the new model improves similar protocols performed in paired extensor digitorum longus (EDL) muscles from prepubertal rats, optimizing the pharmacological screening of drugs for anticatabolic purposes.

  11. Transthyretin neuroprotection in Alzheimer's disease is dependent on proteolysis.

    Science.gov (United States)

    Silva, Catarina S; Eira, Jessica; Ribeiro, Carlos A; Oliveira, Ângela; Sousa, Mónica M; Cardoso, Isabel; Liz, Márcia A

    2017-11-01

    The deposition of amyloid β peptide (Aβ) in the hippocampus is one of the major hallmarks of Alzheimer's disease, a neurodegenerative disorder characterized by memory loss and cognitive impairment. The modulation of Aβ levels in the brain results from an equilibrium between its production from the amyloid precursor protein and removal by amyloid clearance proteins, which might occur via enzymatic (Aβ-degrading enzymes) or nonenzymatic (binding/transport proteins) reactions. Transthyretin (TTR) is one of the major Aβ-binding proteins acting as a neuroprotector in AD. In addition, TTR cleaves Aβ peptide in vitro. In this work, we show that proteolytically active TTR, and not the inactive form of the protein, impacts on Aβ fibrillogenesis, degrades neuronal-secreted Aβ, and reduces Aβ-induced toxicity in hippocampal neurons. Our data demonstrate that TTR proteolytic activity is required for the neuroprotective effect of the protein constituting a putative novel therapeutic target for AD. Copyright © 2017 Elsevier Inc. All rights reserved.

  12. cAMP-dependent proteolysis of GATA-6 is linked to JNK-signaling pathway

    Energy Technology Data Exchange (ETDEWEB)

    Ushijima, Hironori [Department of Molecular Biology, School of Pharmacy, Iwate Medical University, 2-1-1, Nishitokuta, Yahaba, Shiwagun, Iwate 028-3694 (Japan); Maeda, Masatomo, E-mail: mmaeda@iwate-med.ac.jp [Department of Molecular Biology, School of Pharmacy, Iwate Medical University, 2-1-1, Nishitokuta, Yahaba, Shiwagun, Iwate 028-3694 (Japan)

    2012-07-13

    Highlights: Black-Right-Pointing-Pointer A JNK inhibitor SP600125 inhibited cAMP-dependent proteolysis of GATA-6. Black-Right-Pointing-Pointer Effect of a JNK activator anisomycin on the proteolysis was examined. Black-Right-Pointing-Pointer Anisomycin stimulated the export of nuclear GATA-6 into the cytoplasm. Black-Right-Pointing-Pointer JNK activated the CRM1 mediated nuclear export of GATA-6. Black-Right-Pointing-Pointer JNK further stimulated slowly the degradation of GATA-6 by cytoplasmic proteasomes. -- Abstract: A JNK inhibitor SP600125 inhibited cAMP-dependent proteolysis of GATA-6 by proteasomes around its IC50. We further examined the effects of SP600125 on the degradation of GATA-6 in detail, since an activator of JNK (anisomycin) is available. Interestingly, anisomycin immediately stimulated the export of nuclear GATA-6 into the cytoplasm, and then the cytoplasmic content of GATA-6 decreased slowly through degradation by proteasomes. Such an effect of anisomycin was inhibited by SP600125, indicating that the observed phenomenon might be linked to the JNK signaling pathway. The inhibitory effect of SP600125 could not be ascribed to the inhibition of PKA, since phosphorylation of CREB occurred in the presence of dbcAMP and SP600125. The nuclear export of GATA-6 was inhibited by leptomycin B, suggesting that CRM1-mediated export could be activated by anisomycin. Furthermore, it seems likely that the JNK activated by anisomycin may stimulate not only the nuclear export of GATA-6 through CRM1 but also the degradation of GATA-6 by cytoplasmic proteasomes. In contrast, A-kinase might activate only the latter process through JNK.

  13. Use of high-gradient magnetic fishing for reducing proteolysis during fermentation

    DEFF Research Database (Denmark)

    Maury, Trine Lütken; Ottow, Kim Ekelund; Brask, Jesper

    2012-01-01

    Proteolysis during fermentation may have a severe impact on the yield and quality of a secreted product. In the current study, we demonstrate the use of high-gradient magnetic fishing (HGMF) as an efficient alternative to the more conventional methods of preventing proteolytic degradation....... Bacitracin-linked magnetic affinity adsorbents were employed directly in a fermenter during Bacillus licheniformis cultivation to remove trace amounts of unwanted proteases. The constructed magnetic adsorbents had excellent, highly specific binding characteristics in the fermentation broth (K(d) = 1...

  14. Autophagic dysfunction in a lysosomal storage disorder due to impaired proteolysis.

    Science.gov (United States)

    Elrick, Matthew J; Lieberman, Andrew P

    2013-02-01

    Alterations in macroautophagy (hereafter referred to as "autophagy") are a common feature of lysosomal storage disorders, and have been hypothesized to play a major role in the pathogenesis of these diseases. We have recently reported multiple defects in autophagy contributing to the lysosomal storage disorder Niemann-Pick type C (NPC). These include increased formation of autophagosomes, slowed turnover of autophagosomes secondary to impaired lysosomal proteolysis, and delivery of stored lipids to the lysosome via autophagy. The study summarized here describes novel methods for the interrogation of individual stages of the autophagic pathway, and suggests mechanisms by which lipid storage may result in broader lysosomal dysfunction.

  15. Histopathological growth pattern, proteolysis and angiogenesis in chemonaive patients resected for multiple colorectal liver metastases

    DEFF Research Database (Denmark)

    Eefsen, Rikke Løvendahl; Van den Eynden, Gert G; Høyer-Hansen, Gunilla

    2012-01-01

    The purpose of this study was to characterise growth patterns, proteolysis, and angiogenesis in colorectal liver metastases from chemonaive patients with multiple liver metastases. Twenty-four patients were included in the study, resected for a median of 2.6 metastases. The growth pattern......-type plasminogen activator receptor (P = 0.0008). Angiogenesis was most pronounced in metastases with a pushing growth pattern in comparison to those with desmoplastic (P = 0.0007) and replacement growth pattern (P = 0.021). Although a minor fraction of the patients harboured metastases with different growth...

  16. Overexpression of the essential Sis1 chaperone reduces TDP-43 effects on toxicity and proteolysis

    Science.gov (United States)

    Park, Sei-Kyoung; Hong, Joo Y.; Arslan, Fatih; Tietsort, Alex; Tank, Elizabeth M. H.; Li, Xingli

    2017-01-01

    Amyotrophic lateral sclerosis (ALS) is a devastating neurodegenerative disease characterized by selective loss of motor neurons with inclusions frequently containing the RNA/DNA binding protein TDP-43. Using a yeast model of ALS exhibiting TDP-43 dependent toxicity, we now show that TDP-43 overexpression dramatically alters cell shape and reduces ubiquitin dependent proteolysis of a reporter construct. Furthermore, we show that an excess of the Hsp40 chaperone, Sis1, reduced TDP-43’s effect on toxicity, cell shape and proteolysis. The strength of these effects was influenced by the presence of the endogenous yeast prion, [PIN+]. Although overexpression of Sis1 altered the TDP-43 aggregation pattern, we did not detect physical association of Sis1 with TDP-43, suggesting the possibility of indirect effects on TDP-43 aggregation. Furthermore, overexpression of the mammalian Sis1 homologue, DNAJB1, relieves TDP-43 mediated toxicity in primary rodent cortical neurons, suggesting that Sis1 and its homologues may have neuroprotective effects in ALS. PMID:28531192

  17. Proteolysis of Sardine (Sardina pilchardus and Anchovy (Stolephorus commersonii by Commercial Enzymes in Saline Solutions

    Directory of Open Access Journals (Sweden)

    Chau Minh Le

    2015-01-01

    Full Text Available Fish sauce production is a very long process and there is a great interest in shortening it. Among the different strategies to speed up this process, the addition of external proteases could be a solution. This study focuses on the eff ect of two commercial enzymes (Protamex and Protex 51FP on the proteolysis of two fish species traditionally converted into fish sauce: sardine and anchovy, by comparison with classical autolysis. Hydrolysis reactions were conducted with fresh fish at a temperature of 30 °C and under different saline conditions (from 0 to 30 % NaCl. Hydrolysis degree and liquefaction of the raw material were used to follow the process. As expected, the proteolysis decreased with increasing amount of salt. Regarding the fi sh species, higher rate of liquefaction and higher hydrolysis degree were obtained with anchovy. Between the two proteases, Protex 51FP gave better results with both fi sh types. This study demonstrates that the addition of commercial proteases could be helpful for the liquefaction of fi sh and cleavage of peptide bonds that occur during fi sh sauce production and thus speed up the production process.

  18. The role of extracellular proteolysis in synaptic plasticity of the central nervous system 

    Directory of Open Access Journals (Sweden)

    Anna Konopka

    2012-11-01

    Full Text Available The extracellular matrix (ECM of the central nervous system has a specific structure and protein composition that are different from those in other organs. Today we know that the ECM not only provides physical scaffolding for the neurons and glia, but also actively modifies their functions. Over the last two decades, a growing body of research evidence has been collected, suggesting an important role of ECM proteolysis in synaptic plasticity of the brain. So far the majority of data concern two large families of proteases: the serine proteases and the matrix metalloproteinases. The members of these families are localized at the synapses, and are secreted into the extracellular space in an activity-dependent manner. The proteases remodel the local environment as well as influencing synapse structure and function. The structural modifications induced by proteases include shape and size changes, as well as synapse elimination, and synaptogenesis. The functional changes include modifications of receptor function in the postsynaptic part of the synapse, as well as the potentiation or depression of neurotransmitter secretion by the presynaptic site. The present review summarizes the current view on the role of extracellular proteolysis in the physiological synaptic plasticity underlying the phenomena of learning and memory, as well as in the pathological plasticity occurring during epileptogenesis or development of drug addiction. 

  19. Increased intracellular proteolysis reduces disease severity in an ER stress-associated dwarfism.

    Science.gov (United States)

    Mullan, Lorna A; Mularczyk, Ewa J; Kung, Louise H; Forouhan, Mitra; Wragg, Jordan Ma; Goodacre, Royston; Bateman, John F; Swanton, Eileithyia; Briggs, Michael D; Boot-Handford, Raymond P

    2017-10-02

    The short-limbed dwarfism metaphyseal chondrodysplasia type Schmid (MCDS) is linked to mutations in type X collagen, which increase ER stress by inducing misfolding of the mutant protein and subsequently disrupting hypertrophic chondrocyte differentiation. Here, we show that carbamazepine (CBZ), an autophagy-stimulating drug that is clinically approved for the treatment of seizures and bipolar disease, reduced the ER stress induced by 4 different MCDS-causing mutant forms of collagen X in human cell culture. Depending on the nature of the mutation, CBZ application stimulated proteolysis of misfolded collagen X by either autophagy or proteasomal degradation, thereby reducing intracellular accumulation of mutant collagen. In MCDS mice expressing the Col10a1.pN617K mutation, CBZ reduced the MCDS-associated expansion of the growth plate hypertrophic zone, attenuated enhanced expression of ER stress markers such as Bip and Atf4, increased bone growth, and reduced skeletal dysplasia. CBZ produced these beneficial effects by reducing the MCDS-associated abnormalities in hypertrophic chondrocyte differentiation. Stimulation of intracellular proteolysis using CBZ treatment may therefore be a clinically viable way of treating the ER stress-associated dwarfism MCDS.

  20. Bystander protein protects potential vaccine-targeting ligands against intestinal proteolysis.

    Science.gov (United States)

    Reuter, Fabian; Bade, Steffen; Hirst, Timothy R; Frey, Andreas

    2009-07-20

    Endowing mucosal vaccines with ligands that target antigen to mucosal lymphoid tissues may improve immunization efficacy provided that the ligands withstand the proteolytic environment of the gastro-intestinal tract until they reach their destination. Our aim was to investigate whether and how three renowned ligands - Ulex europaeus agglutinin I and the B subunits of cholera toxin and E. coli heat-labile enterotoxin - master this challenge. We assessed the digestive power of natural murine intestinal fluid (natIF) using assays for trypsin, chymotrypsin and pancreatic elastase along with a test for nonspecific proteolysis. The natIF was compared with simulated murine intestinal fluid (simIF) that resembled the trypsin, chymotrypsin and elastase activities of its natural counterpart but lacked or contained albumins as additional protease substrates. The ligands were exposed to the digestive fluids and degradation was determined. The studies revealed that (i) the three pancreatic endoproteases constitute only one third of the total protease activity of natIF and (ii) the ligands resist proteolysis in natIF and protein-enriched simIF over 3 h but (iii) are partially destroyed in simIF that lacks additional protease substrate. We assume that the proteins of natIF are preferred substrates for the intestinal proteases and thus can protect vaccine-targeting ligands from destruction.

  1. Calpain-mediated proteolysis of tropomodulin isoforms leads to thin filament elongation in dystrophic skeletal muscle.

    Science.gov (United States)

    Gokhin, David S; Tierney, Matthew T; Sui, Zhenhua; Sacco, Alessandra; Fowler, Velia M

    2014-03-01

    Duchenne muscular dystrophy (DMD) induces sarcolemmal mechanical instability and rupture, hyperactivity of intracellular calpains, and proteolytic breakdown of muscle structural proteins. Here we identify the two sarcomeric tropomodulin (Tmod) isoforms, Tmod1 and Tmod4, as novel proteolytic targets of m-calpain, with Tmod1 exhibiting ∼10-fold greater sensitivity to calpain-mediated cleavage than Tmod4 in situ. In mdx mice, increased m-calpain levels in dystrophic soleus muscle are associated with loss of Tmod1 from the thin filament pointed ends, resulting in ∼11% increase in thin filament lengths. In mdx/mTR mice, a more severe model of DMD, Tmod1 disappears from the thin filament pointed ends in both tibialis anterior (TA) and soleus muscles, whereas Tmod4 additionally disappears from soleus muscle, resulting in thin filament length increases of ∼10 and ∼12% in TA and soleus muscles, respectively. In both mdx and mdx/mTR mice, both TA and soleus muscles exhibit normal localization of α-actinin, the nebulin M1M2M3 domain, Tmod3, and cytoplasmic γ-actin, indicating that m-calpain does not cause wholesale proteolysis of other sarcomeric and actin cytoskeletal proteins in dystrophic skeletal muscle. These results implicate Tmod proteolysis and resultant thin filament length misspecification as novel mechanisms that may contribute to DMD pathology, affecting muscles in a use- and disease severity-dependent manner.

  2. Hepatic steatosis inhibits autophagic proteolysis via impairment of autophagosomal acidification and cathepsin expression

    International Nuclear Information System (INIS)

    Inami, Yoshihiro; Yamashina, Shunhei; Izumi, Kousuke; Ueno, Takashi; Tanida, Isei; Ikejima, Kenichi; Watanabe, Sumio

    2011-01-01

    Highlights: → Acidification of autophagosome was blunted in steatotic hepatocytes. → Hepatic steatosis did not disturb fusion of isolated autophagosome and lysosome. → Proteinase activity of cathepsin B and L in autolysosomes was inhibited by steatosis. → Hepatic expression of cathepsin B and L was suppressed by steatosis. -- Abstract: Autophagy, one of protein degradation system, contributes to maintain cellular homeostasis and cell defense. Recently, some evidences indicated that autophagy and lipid metabolism are interrelated. Here, we demonstrate that hepatic steatosis impairs autophagic proteolysis. Though accumulation of autophagosome is observed in hepatocytes from ob/ob mice, expression of p62 was augmented in liver from ob/ob mice more than control mice. Moreover, degradation of the long-lived protein leucine was significantly suppressed in hepatocytes isolated from ob/ob mice. More than 80% of autophagosomes were stained by LysoTracker Red (LTR) in hepatocytes from control mice; however, rate of LTR-stained autophagosomes in hepatocytes were suppressed in ob/ob mice. On the other hand, clearance of autolysosomes loaded with LTR was blunted in hepatocytes from ob/ob mice. Although fusion of isolated autophagosome and lysosome was not disturbed, proteinase activity of cathepsin B and L in autolysosomes and cathepsin B and L expression of liver were suppressed in ob/ob mice. These results indicate that lipid accumulation blunts autophagic proteolysis via impairment of autophagosomal acidification and cathepsin expression.

  3. Hepatic steatosis inhibits autophagic proteolysis via impairment of autophagosomal acidification and cathepsin expression

    Energy Technology Data Exchange (ETDEWEB)

    Inami, Yoshihiro [Department of Gastroenterology, Juntendo University School of Medicine, Hongo 2-1-1, Bunkyo-ku, Tokyo 113-8421 (Japan); Yamashina, Shunhei, E-mail: syamashi@juntendo.ac.jp [Department of Gastroenterology, Juntendo University School of Medicine, Hongo 2-1-1, Bunkyo-ku, Tokyo 113-8421 (Japan); Izumi, Kousuke [Department of Gastroenterology, Juntendo University School of Medicine, Hongo 2-1-1, Bunkyo-ku, Tokyo 113-8421 (Japan); Ueno, Takashi [Department of Biochemistry, Juntendo University School of Medicine, Hongo 2-1-1, Bunkyo-ku, Tokyo 113-8421 (Japan); Tanida, Isei [Department of Biochemistry and Cell Biology, Laboratory of Biomembranes, National Institute of Infectious Disease, Toyama 1-23-1, Shinjuku-ku, Tokyo 162-8640 (Japan); Ikejima, Kenichi; Watanabe, Sumio [Department of Gastroenterology, Juntendo University School of Medicine, Hongo 2-1-1, Bunkyo-ku, Tokyo 113-8421 (Japan)

    2011-09-09

    Highlights: {yields} Acidification of autophagosome was blunted in steatotic hepatocytes. {yields} Hepatic steatosis did not disturb fusion of isolated autophagosome and lysosome. {yields} Proteinase activity of cathepsin B and L in autolysosomes was inhibited by steatosis. {yields} Hepatic expression of cathepsin B and L was suppressed by steatosis. -- Abstract: Autophagy, one of protein degradation system, contributes to maintain cellular homeostasis and cell defense. Recently, some evidences indicated that autophagy and lipid metabolism are interrelated. Here, we demonstrate that hepatic steatosis impairs autophagic proteolysis. Though accumulation of autophagosome is observed in hepatocytes from ob/ob mice, expression of p62 was augmented in liver from ob/ob mice more than control mice. Moreover, degradation of the long-lived protein leucine was significantly suppressed in hepatocytes isolated from ob/ob mice. More than 80% of autophagosomes were stained by LysoTracker Red (LTR) in hepatocytes from control mice; however, rate of LTR-stained autophagosomes in hepatocytes were suppressed in ob/ob mice. On the other hand, clearance of autolysosomes loaded with LTR was blunted in hepatocytes from ob/ob mice. Although fusion of isolated autophagosome and lysosome was not disturbed, proteinase activity of cathepsin B and L in autolysosomes and cathepsin B and L expression of liver were suppressed in ob/ob mice. These results indicate that lipid accumulation blunts autophagic proteolysis via impairment of autophagosomal acidification and cathepsin expression.

  4. Impact of microencapsulated peptidase (Aspergillus oryzae) on cheddar cheese proteolysis and its biologically active peptide profile.

    Science.gov (United States)

    Seneweera, Saman; Kailasapathy, Kaila

    2011-07-01

    We investigated the delivery of calcium-alginate encapsulated peptidase (Flavourzyme(®), Aspergillus oryzae) on proteolysis of Cheddar cheese. Physical and chemical characteristics such as moisture, pH and fat content were measured, and no differences were found between control and experimental cheese at day 0. SDS-PAGE analysis clearly showed that proteolysis of α and k casein was significantly accelerated after three months of maturity in the experimental cheese. A large number of low molecular weight peptides were found in the water soluble fraction of the experimental cheeses and some of these peptides were new. N-terminal amino acid sequence analysis identified these as P(1), Leu-Thu-Glu; P(3), Asp-Val-Pro-Ser-Glu) and relatively abundant stable peptides P(2), P(4), Arg-Pro-Lys-His-Pro-Ile; P(5), Arg-Pro-Lys-His-Pro-Ile-Lys and P(6). These peptides were mainly originated from αs1-CN and β-CN. Three of the identified peptides (P(1), P(2), P(3) and P(4)) are known to biologically active and P(1) and P(3) were only present in experimental cheese suggesting that experimental cheese has improved health benefits.

  5. Proteolysis inside the membrane is a rate-governed reaction not driven by substrate affinity.

    Science.gov (United States)

    Dickey, Seth W; Baker, Rosanna P; Cho, Sangwoo; Urban, Siniša

    2013-12-05

    Enzymatic cleavage of transmembrane anchors to release proteins from the membrane controls diverse signaling pathways and is implicated in more than a dozen diseases. How catalysis works within the viscous, water-excluding, two-dimensional membrane is unknown. We developed an inducible reconstitution system to interrogate rhomboid proteolysis quantitatively within the membrane in real time. Remarkably, rhomboid proteases displayed no physiological affinity for substrates (K(d) ~190 μM/0.1 mol%). Instead, ~10,000-fold differences in proteolytic efficiency with substrate mutants and diverse rhomboid proteases were reflected in k(cat) values alone. Analysis of gate-open mutant and solvent isotope effects revealed that substrate gating, not hydrolysis, is rate limiting. Ultimately, a single proteolytic event within the membrane normally takes minutes. Rhomboid intramembrane proteolysis is thus a slow, kinetically controlled reaction not driven by transmembrane protein-protein affinity. These properties are unlike those of other studied proteases or membrane proteins but are strikingly reminiscent of one subset of DNA-repair enzymes, raising important mechanistic and drug-design implications. Copyright © 2013 Elsevier Inc. All rights reserved.

  6. Chemical and proteolysis-derived changes during long-term storage of lactose-hydrolyzed ultrahigh-temperature (UHT) milk.

    Science.gov (United States)

    Jansson, Therese; Jensen, Hanne B; Sundekilde, Ulrik K; Clausen, Morten R; Eggers, Nina; Larsen, Lotte B; Ray, Colin; Andersen, Henrik J; Bertram, Hanne C

    2014-11-19

    Proteolytic activity in milk may release bitter-tasting peptides and generate free amino terminals that react with carbohydrates, which initiate Maillard reaction. Ultrahigh temperature (UHT) heat treatment inactivates the majority of proteolytic enzymes in milk. In lactose-hydrolyzed milk a β-galactosidase preparation is applied to the milk after heat treatment, which has proteolytic side activities that may induce quality deterioration of long-term-stored milk. In the present study proteolysis, glycation, and volatile compound formation were investigated in conventional (100% lactose), filtered (60% lactose), and lactose-hydrolyzed (<1% lactose) UHT milk using reverse phase high-pressure liquid chromatography-mass spectrometry, proton nuclear magnetic resonance, and gas chromatography-mass spectrometry. Proteolysis was observed in all milk types. However, the degree of proteolysis was significantly higher in the lactose-hydrolyzed milk compared to the conventional and filtered milk. The proteins most prone to proteolysis were β-CN and αs1-CN, which were clearly hydrolyzed after approximately 90 days of storage in the lactose-hydrolyzed milk.

  7. MBTPS2 mutations cause defective regulated intramembrane proteolysis in X-linked osteogenesis imperfecta

    Science.gov (United States)

    Lindert, Uschi; Cabral, Wayne A.; Ausavarat, Surasawadee; Tongkobpetch, Siraprapa; Ludin, Katja; Barnes, Aileen M.; Yeetong, Patra; Weis, Maryann; Krabichler, Birgit; Srichomthong, Chalurmpon; Makareeva, Elena N.; Janecke, Andreas R.; Leikin, Sergey; Röthlisberger, Benno; Rohrbach, Marianne; Kennerknecht, Ingo; Eyre, David R.; Suphapeetiporn, Kanya; Giunta, Cecilia; Marini, Joan C.; Shotelersuk, Vorasuk

    2016-01-01

    Osteogenesis imperfecta (OI) is a collagen-related bone dysplasia. We identified an X-linked recessive form of OI caused by defects in MBTPS2, which encodes site-2 metalloprotease (S2P). MBTPS2 missense mutations in two independent kindreds with moderate/severe OI cause substitutions at highly conserved S2P residues. Mutant S2P has normal stability, but impaired functioning in regulated intramembrane proteolysis (RIP) of OASIS, ATF6 and SREBP transcription factors, consistent with decreased proband secretion of type I collagen. Further, hydroxylation of the collagen lysine residue (K87) critical for crosslinking is reduced in proband bone tissue, consistent with decreased lysyl hydroxylase 1 in proband osteoblasts. Reduced collagen crosslinks presumptively undermine bone strength. Also, proband osteoblasts have broadly defective differentiation. These mutations provide evidence that RIP plays a fundamental role in normal bone development. PMID:27380894

  8. VEGFR2 Trafficking, Signaling and Proteolysis is Regulated by the Ubiquitin Isopeptidase USP8.

    Science.gov (United States)

    Smith, Gina A; Fearnley, Gareth W; Abdul-Zani, Izma; Wheatcroft, Stephen B; Tomlinson, Darren C; Harrison, Michael A; Ponnambalam, Sreenivasan

    2016-01-01

    Vascular endothelial growth factor A (VEGF-A) regulates many aspects of vascular function. VEGF-A binding to vascular endothelial growth factor receptor 2 (VEGFR2) stimulates endothelial signal transduction and regulates multiple cellular responses. Activated VEGFR2 undergoes ubiquitination but the enzymes that regulate this post-translational modification are unclear. In this study, the de-ubiquitinating enzyme, USP8, is shown to regulate VEGFR2 trafficking, de-ubiquitination, proteolysis and signal transduction. USP8-depleted endothelial cells displayed altered VEGFR2 ubiquitination and production of a unique VEGFR2 extracellular domain proteolytic fragment caused by VEGFR2 accumulation in the endosome-lysosome system. In addition, perturbed VEGFR2 trafficking impaired VEGF-A-stimulated signal transduction in USP8-depleted cells. Thus, regulation of VEGFR2 ubiquitination and de-ubiquitination has important consequences for the endothelial cell response and vascular physiology. © 2015 The Authors. Traffic published by John Wiley & Sons Ltd.

  9. Cross-system excision of chaperone-mediated proteolysis in chaperone-assisted recombinant protein production

    Science.gov (United States)

    Martínez-Alonso, Mónica; Villaverde, Antonio

    2010-01-01

    Main Escherichia coli cytosolic chaperones such as DnaK are key components of the control quality network designed to minimize the prevalence of polypeptides with aberrant conformations. This is achieved by both favoring refolding activities but also stimulating proteolytic degradation of folding reluctant species. This last activity is responsible for the decrease of the proteolytic stability of recombinant proteins when co-produced along with DnaK, where an increase in solubility might be associated to a decrease in protein yield. However, when DnaK and its co-chaperone DnaJ are co-produced in cultured insect cells or whole insect larvae (and expectedly, in other heterologous hosts), only positive, folding-related effects of these chaperones are observed, in absence of proteolysis-mediated reduction of recombinant protein yield. PMID:21326941

  10. Implications of the Maillard reaction on bovine alpha-lactalbumin and its proteolysis during in vitro infant digestion.

    Science.gov (United States)

    Joubran, Yousef; Moscovici, Alice; Portmann, Reto; Lesmes, Uri

    2017-06-21

    This study investigated the functionality and digestibility of Maillard reaction products (MRPs) of alpha-lactalbumin (α-la), a major whey protein and component of infant formulas. The impact of different carbohydrates (glucose, galactose or galacto-oligosaccharides (GOS)) and heating duration was studied. SDS-PAGE, UV and color measurements monitored reaction extent, which varied between carbohydrates whereby galactose reacted more readily than glucose. Surface hydrophobicity and antioxidant capacity were found to be significantly (p Maillard conjugation, with GOS-based MRPs elevating antioxidant capacity ∼50-fold compared to α-la. In addition, the digestive proteolysis of MRPs was evaluated using an infant in vitro gastro-duodenal model. SDS-PAGE analyses of digesta revealed Maillard conjugation generally increased α-la's susceptibility to proteolysis. Interestingly, GOS-based MRPs presented an optimization challenge, since heating for 12 h delayed proteolysis, while extended heating resulted in the highest susceptibility to proteolysis. Proteomic analyses further demonstrated the differences in enzymatic cleavage patterns and helped identify bioactive peptides rendered bioaccessible during the digestion of α-la or its MRPs. Bioinformatic mining of the proteomic data using PeptideRanker also gave rise to two potentially novel bioactive peptides, FQINNKIW and GINYWLAHKALCS. Finally, antioxidant capacity of luminal contents, measured by DPPH, revealed Maillard conjugation increased the antioxidant capacity of both gastric and duodenal digesta. Overall, this work draws a link between the Maillard reaction, digestive proteolysis and the bioaccessibility of bioactive peptides and antioxidant species in the infant alimentary canal. This could help rationally process infant formulas towards improved nutritional and extra-nutritional benefits.

  11. Proteolysis of bovine beta-lactoglobulin during thermal treatment in subdenaturing conditions highlights some structural features of the temperature-modified protein and yields fragments with low immunoreactivity

    DEFF Research Database (Denmark)

    Iametti, S.; Rasmussen, P.; Frøkiær, Hanne

    2002-01-01

    Bovine beta-lactoglobulin was hydrolyzed with trypsin or chymotrypsin in the course of heat treatment at 55, 60 and 65 C at neutral pH. At these temperatures beta-lactoglobulin undergoes significant but reversible structural changes. In the conditions used in the present study, beta......-lactoglobulin was virtually insensitive to proteolysis by either enzyme at room temperature, but underwent extensive proteolysis when either protease was present during the heat treatment. High-temperature proteolysis occurs in a progressive manner. Mass spectrometry analysis of some large-sized breakdown intermediates...

  12. Discovery of Anti-Hypertensive Oligopeptides from Adlay Based on In Silico Proteolysis and Virtual Screening

    Directory of Open Access Journals (Sweden)

    Liansheng Qiao

    2016-12-01

    Full Text Available Adlay (Coix larchryma-jobi L. was the commonly used Traditional Chinese Medicine (TCM with high content of seed storage protein. The hydrolyzed bioactive oligopeptides of adlay have been proven to be anti-hypertensive effective components. However, the structures and anti-hypertensive mechanism of bioactive oligopeptides from adlay were not clear. To discover the definite anti-hypertensive oligopeptides from adlay, in silico proteolysis and virtual screening were implemented to obtain potential oligopeptides, which were further identified by biochemistry assay and molecular dynamics simulation. In this paper, ten sequences of adlay prolamins were collected and in silico hydrolyzed to construct the oligopeptide library with 134 oligopeptides. This library was reverse screened by anti-hypertensive pharmacophore database, which was constructed by our research team and contained ten anti-hypertensive targets. Angiotensin-I converting enzyme (ACE was identified as the main potential target for the anti-hypertensive activity of adlay oligopeptides. Three crystal structures of ACE were utilized for docking studies and 19 oligopeptides were finally identified with potential ACE inhibitory activity. According to mapping features and evaluation indexes of pharmacophore and docking, three oligopeptides were selected for biochemistry assay. An oligopeptide sequence, NPATY (IC50 = 61.88 ± 2.77 µM, was identified as the ACE inhibitor by reverse-phase high performance liquid chromatography (RP-HPLC assay. Molecular dynamics simulation of NPATY was further utilized to analyze interactive bonds and key residues. ALA354 was identified as a key residue of ACE inhibitors. Hydrophobic effect of VAL518 and electrostatic effects of HIS383, HIS387, HIS513 and Zn2+ were also regarded as playing a key role in inhibiting ACE activities. This study provides a research strategy to explore the pharmacological mechanism of Traditional Chinese Medicine (TCM proteins based on

  13. Effect of pulp preconditioning on acidification, proteolysis, sugars and free fatty acids concentration during fermentation of cocoa (Theobroma cacao) beans.

    Science.gov (United States)

    Afoakwa, Emmanuel Ohene; Quao, Jennifer; Budu, Agnes Simpson; Takrama, Jemmy; Saalia, Firibu Kwesi

    2011-11-01

    Changes in acidification, proteolysis, sugars and free fatty acids (FFAs) concentrations of Ghanaian cocoa beans as affected by pulp preconditioning (pod storage or PS) and fermentation were investigated. Non-volatile acidity, pH, proteolysis, sugars (total, reducing and non-reducing) and FFAs concentrations were analysed using standard methods. Increasing PS consistently decreased the non-volatile acidity with concomitant increase in pH during fermentation of the beans. Fermentation decreased the pH of the unstored beans from 6.7 to 4.9 within the first 4 days and then increased slightly again to 5.3 by the sixth day. Protein, total sugars and non-reducing sugars decreased significantly (p cocoa beans was largely affected by fermentation than by PS.

  14. Perennial peanut (Arachis glabrata Benth.) contains polyphenol oxidase (PPO) and PPO substrates that can reduce post-harvest proteolysis.

    Science.gov (United States)

    Sullivan, Michael L; Foster, Jamie L

    2013-08-15

    Studies of perennial peanut (Arachis glabrata Benth.) suggest its hay and haylage have greater levels of rumen undegraded protein (RUP) than other legume forages such as alfalfa (Medicago sativa L.). Greater RUP can result in more efficient nitrogen utilization by ruminant animals with positive economic and environmental effects. We sought to determine whether, like red clover (Trifolium pretense L.), perennial peanut contains polyphenol oxidase (PPO) and PPO substrates that might be responsible for increased RUP. Perennial peanut extracts contain immunologically detectible PPO protein and high levels of PPO activity (>100 nkatal mg(-1) protein). Addition of caffeic acid (PPO substrate) to perennial peanut extracts depleted of endogenous substrates reduced proteolysis by 90%. Addition of phenolics prepared from perennial peanut leaves to extracts of either transgenic PPO-expressing or control (non-expressing) alfalfa showed peanut phenolics could reduce proteolysis >70% in a PPO-dependent manner. Two abundant likely PPO substrates are present in perennial peanut leaves including caftaric acid. Perennial peanut contains PPO and PPO substrates that together are capable of inhibiting post-harvest proteolysis, suggesting a possible mechanism for increased RUP in this forage. Research related to optimizing the PPO system in other forage crops will likely be applicable to perennial peanut. Published 2013. This article is a U.S. Government work and is in the public domain in the USA.

  15. Granzyme B-dependent proteolysis acts as a switch to enhance the proinflammatory activity of IL-1α.

    LENUS (Irish Health Repository)

    Afonina, Inna S

    2011-10-21

    Granzyme B is a cytotoxic lymphocyte-derived protease that plays a central role in promoting apoptosis of virus-infected target cells, through direct proteolysis and activation of constituents of the cell death machinery. However, previous studies have also implicated granzymes A and B in the production of proinflammatory cytokines, via a mechanism that remains undefined. Here we show that IL-1α is a substrate for granzyme B and that proteolysis potently enhanced the biological activity of this cytokine in vitro as well as in vivo. Consistent with this, compared with full-length IL-1α, granzyme B-processed IL-1α exhibited more potent activity as an immunoadjuvant in vivo. Furthermore, proteolysis of IL-1α within the same region, by proteases such as calpain and elastase, was also found to enhance its biological potency. Thus, IL-1α processing by multiple immune-related proteases, including granzyme B, acts as a switch to enhance the proinflammatory properties of this cytokine.

  16. Yak milk casein as potential precursor of angiotensin I-converting enzyme inhibitory peptides based on in silico proteolysis.

    Science.gov (United States)

    Lin, Kai; Zhang, Lan-Wei; Han, Xue; Xin, Liang; Meng, Zhao-Xu; Gong, Pi-Min; Cheng, Da-You

    2018-07-15

    Yak milk casein was selected as a potential precursor of bioactive peptides based on in silico analysis. Most notable among these are the angiotensin I-converting enzyme (ACE) inhibitory peptides. First, yak milk casein has high homology with cow milk casein by homologous analysis. The potential of yak milk casein for the releasing bioactive peptides was evaluated by determining the frequency of occurrence of fragments with a given activity. Through the BIOPEP database analysis, there are many bioactive peptides in yak milk casein sequences. Then, an in silico proteolysis using single or combined enzymes to obtained ACE inhibitory peptides was investigated. Cytotoxicity analysis using the online toxic prediction tool ToxinPred revealed that all in silico proteolysis derived ACE inhibitory peptides are non-cytotoxic. Overall, the present study highlights a in silico proteolysis approach to assist the yak milk casein releasing ACE inhibitory peptides and provides a guidance for the actual hydrolysis of proteins for the production of bioactive peptides. Copyright © 2018 Elsevier Ltd. All rights reserved.

  17. [Pharmacological mechanism analysis of oligopeptide from Pinctada fucata based on in silico proteolysis and protein interaction network].

    Science.gov (United States)

    Chen, Yan-Kun; Qiao, Lian-Sheng; Huo, Xiao-Qian; Zhang, Xu; Han, Na; Zhang, Yan-Ling

    2017-09-01

    Pinctada fucata oligopeptide is one of key pharmaceutical effective constituents of P. fucata. It is significant to analyze its pharmacological effect and mechanism. This study aims to discover the potential oligopeptides from P. fucata and analyze the mechanism of P. fucata oligopeptide based on in silico technologies and protein interaction network(PIN). First, main protein sequences of P. fucata were collected, and oligopeptides were obtained using in silico gastrointestinal tract proteolysis. Then, key potential targets of P. fucata oligopeptides were obtained through pharmacophore screening. The protein-protein interaction(PPI) of targets was achieved and implemented to construct PIN and analyze the mechanism of P. fucata oligopeptides. P. fucata oligopeptide database was constructed based on in silico technologies, including 458 oligopeptides. Twelve modules were identified from PIN by a graph theoretic clustering algorithm Molecular Complex Detection(MCODE) and analyzed by Gene ontology(GO) enrichment. The results indicated that P. fucata oligopeptides have an effect in treating neurological diseases, such as Alzheimer's disease. In silico proteolysis could be used to analyze the protein sequences of traditional Chinese medicine(TCM). According to the combination of in silico proteolysis and PIN, the biological activity of oligopeptides could be interpreted rapidly based on the known TCM protein sequence. The study provides the methodology basis for rapidly and efficiently implementing the mechanism analysis of TCM oligopeptides. Copyright© by the Chinese Pharmaceutical Association.

  18. Involvement of μ/m-calpain in the proteolysis and meat quality changes during postmortem storage of chicken breast muscle.

    Science.gov (United States)

    Zhao, Liang; Xing, Tong; Huang, Jichao; Qiao, Yan; Chen, Yulian; Huang, Ming

    2018-02-01

    The objective of this study was to investigate the role of calpain isotypes, especially poultry-specific μ/m-calpain in the proteolysis and meat quality changes of chicken breast muscle during postmortem storage. Calpain activity was detected by casein zymography, while the degradation of titin, desmin and Troponin-T was analyzed by sodium dodecyl sulfate - polyacrylamide gel electrophoresis and western blot. Meat quality indicators such as water holding capacity and tenderness were also studied. The correlation analysis between calpain activity, proteolysis and the changes in meat quality indicators indicated that there were strong correlations for μ-calpain during the first 12 h of storage, while such strong correlations for μ/m-calpain were only found in samples stored from 12 h to 7 days. Our study suggested that μ-calpain played a major role in meat quality changes while μ/m-calpain could also be involved but played a limited role in the proteolysis and meat quality changes during 12 h to 7 days postmortem storage of chicken breast muscle. © 2017 Japanese Society of Animal Science.

  19. Proteolysis of milk fat globule membrane proteins during in vitro gastric digestion of milk.

    Science.gov (United States)

    Ye, A; Cui, J; Singh, H

    2011-06-01

    The influence of gastric proteolysis on the physicochemical characteristics of milk fat globules and the proteins of the milk fat globule membrane (MFGM) in raw milk and cream was examined in vitro in simulated gastric fluid (SGF) containing various pepsin concentrations at pH 1.6 for up to 2h. Apparent flocculation of the milk fat globules occurred in raw milk samples incubated in SGF containing pepsin, but no coalescence was observed in either raw milk samples or cream samples. The changes in the particle size of the fat globules as a result of the flocculation were dependent on the pepsin concentration. Correspondingly, the physical characteristics of the fat globules and the composition of the MFGM proteins in raw milk changed during incubation in SGF containing pepsin. The major MFGM proteins were hydrolyzed at different rates by the pepsin in the SGF; butyrophilin was more resistant than xanthine oxidase, PAS 6, or PAS 7. Peptides with various molecular weights, which altered with the time of incubation and the pepsin concentration, were present at the surfaces of the fat globules. Copyright © 2011 American Dairy Science Association. Published by Elsevier Inc. All rights reserved.

  20. Rapid and efficient proteolysis through laser-assisted immobilized enzyme reactors.

    Science.gov (United States)

    Zhang, Peng; Gao, Mingxia; Zhu, Shaochun; Lei, Jie; Zhang, Xiangmin

    2011-11-25

    In this report, laser radiation (808nm) for the first time was employed to enhance the efficiency of proteolysis through immobilized enzyme reactor (IMER). IMER based monolithic support was prepared in the fused-silica capillary via a simple two-step procedure including acryloylation on trypsin surface and in situ aqueous polymerization/immobilization. The feasibility and high efficiency of the laser-assisted IMER were demonstrated by the digestion of bovine serum albumin (BSA), cytochrome c (Cyt-c) and β-casein. The digestion process was achieved in 60s. The peptides were identified by MALDI-TOF-MS, yielding the sequence coverage of 33% for BSA, 73% for Cyt-c and 22% for β-casein. The comparisons between the in-solution digestion and on IMER reaction with/without laser assistance were made. To further confirm its efficiency in proteome analysis, the laser-assisted IMER was also applied to the analysis of one fraction of human serum sample through two-dimensional (2-D) separation of strong anion exchange/reversed-phase liquid chromatography (SAX/RPLC). After a database search, 49 unique peptides corresponding to 5 proteins were identified. The results showed that the laser-assisted IMER provides a promising platform for the high-throughput protein identification. Copyright © 2011 Elsevier B.V. All rights reserved.

  1. PMA Induces SnoN Proteolysis and CD61 Expression through an Autocrine Mechanism

    Science.gov (United States)

    Li, Chonghua; Peart, Natoya; Xuan, Zhenyu; Lewis, Dorothy E; Xia, Yang; Jin, Jianping

    2014-01-01

    Phorbol-12-myristate-13-acetate, also called PMA, is a small molecule that activates protein kinase C and functions to differentiate hematologic lineage cells. However, the mechanism of PMA-induced cellular differentiation is not fully understood. We found that PMA triggers global enhancement of protein ubiquitination in K562, a myelogenous leukemia cell line and one of the enhanced-ubiquitination targets is SnoN, an inhibitor of the Smad signaling pathway. Our data indicated that PMA stimulated the production of Activin A, a cytokine of the TGF-β family. Activin A then activated the phosphorylation of both Smad2 and Smad3. In consequence, SnoN is ubiquitinated by the APCCdh1 ubiquitin ligase with the help of phosphorylated Smad2. Furthermore, we found that SnoN proteolysis is important for the expression of CD61, a marker of megakaryocyte. These results indicate that protein ubiquitination promotes megakaryopoiesis via degrading SnoN, an inhibitor of CD61 expression, strengths the roles of ubiquitination in cellular differentiation. PMID:24637302

  2. Structural Basis for Regulated Proteolysis by the α-Secretase ADAM10.

    Science.gov (United States)

    Seegar, Tom C M; Killingsworth, Lauren B; Saha, Nayanendu; Meyer, Peter A; Patra, Dhabaleswar; Zimmerman, Brandon; Janes, Peter W; Rubinstein, Eric; Nikolov, Dimitar B; Skiniotis, Georgios; Kruse, Andrew C; Blacklow, Stephen C

    2017-12-14

    Cleavage of membrane-anchored proteins by ADAM (a disintegrin and metalloproteinase) endopeptidases plays a key role in a wide variety of biological signal transduction and protein turnover processes. Among ADAM family members, ADAM10 stands out as particularly important because it is both responsible for regulated proteolysis of Notch receptors and catalyzes the non-amyloidogenic α-secretase cleavage of the Alzheimer's precursor protein (APP). We present here the X-ray crystal structure of the ADAM10 ectodomain, which, together with biochemical and cellular studies, reveals how access to the enzyme active site is regulated. The enzyme adopts an unanticipated architecture in which the C-terminal cysteine-rich domain partially occludes the enzyme active site, preventing unfettered substrate access. Binding of a modulatory antibody to the cysteine-rich domain liberates the catalytic domain from autoinhibition, enhancing enzymatic activity toward a peptide substrate. Together, these studies reveal a mechanism for regulation of ADAM activity and offer a roadmap for its modulation. Copyright © 2017 Elsevier Inc. All rights reserved.

  3. Preventing mutant huntingtin proteolysis and intermittent fasting promote autophagy in models of Huntington disease.

    Science.gov (United States)

    Ehrnhoefer, Dagmar E; Martin, Dale D O; Schmidt, Mandi E; Qiu, Xiaofan; Ladha, Safia; Caron, Nicholas S; Skotte, Niels H; Nguyen, Yen T N; Vaid, Kuljeet; Southwell, Amber L; Engemann, Sabine; Franciosi, Sonia; Hayden, Michael R

    2018-03-06

    Huntington disease (HD) is caused by the expression of mutant huntingtin (mHTT) bearing a polyglutamine expansion. In HD, mHTT accumulation is accompanied by a dysfunction in basal autophagy, which manifests as specific defects in cargo loading during selective autophagy. Here we show that the expression of mHTT resistant to proteolysis at the caspase cleavage site D586 (C6R mHTT) increases autophagy, which may be due to its increased binding to the autophagy adapter p62. This is accompanied by faster degradation of C6R mHTT in vitro and a lack of mHTT accumulation the C6R mouse model with age. These findings may explain the previously observed neuroprotective properties of C6R mHTT. As the C6R mutation cannot be easily translated into a therapeutic approach, we show that a scheduled feeding paradigm is sufficient to lower mHTT levels in YAC128 mice expressing cleavable mHTT. This is consistent with a previous model, where the presence of cleavable mHTT impairs basal autophagy, while fasting-induced autophagy remains functional. In HD, mHTT clearance and autophagy may become increasingly impaired as a function of age and disease stage, because of gradually increased activity of mHTT-processing enzymes. Our findings imply that mHTT clearance could be enhanced by a regulated dietary schedule that promotes autophagy.

  4. Activation-induced proteolysis of cytoplasmic domain of zeta in T cell receptors and Fc receptors.

    Science.gov (United States)

    Taupin, J L; Anderson, P

    1994-12-01

    The CD3-T cell receptor (TCR) complex on T cells and the Fc gamma receptor type III (Fc gamma RIII)-zeta-gamma complex on natural killer cells are functionally analogous activation receptors that associate with a family of disulfide-linked dimers composed of the related subunits zeta and gamma. Immunochemical analysis of receptor complexes separated on two-dimensional diagonal gels allowed the identification of a previously uncharacterized zeta-p14 heterodimer. zeta-p14 is a component of both CD3-TCR and Fc gamma RIII-zeta-gamma. Peptide mapping analysis shows that p14 is structurally related to zeta, suggesting that it is either: (i) derived from zeta proteolytically or (ii) the product of an alternatively spliced mRNA. The observation that COS cells transformed with a cDNA encoding zeta express zeta-p14 supports the former possibility. The expression of CD3-TCR complexes including zeta-p14 increases following activation with phorbol 12-myristate 13-acetate or concanavalin A, suggesting that proteolysis of zeta may contribute to receptor modulation or desensitization.

  5. Locked and proteolysis-based transcription activator-like effector (TALE) regulation.

    Science.gov (United States)

    Lonzarić, Jan; Lebar, Tina; Majerle, Andreja; Manček-Keber, Mateja; Jerala, Roman

    2016-02-18

    Development of orthogonal, designable and adjustable transcriptional regulators is an important goal of synthetic biology. Their activity has been typically modulated through stimulus-induced oligomerization or interaction between the DNA-binding and activation/repression domain. We exploited a feature of the designable Transcription activator-like effector (TALE) DNA-binding domain that it winds around the DNA which allows to topologically prevent it from binding by intramolecular cyclization. This new approach was investigated through noncovalent ligand-induced cyclization or through a covalent split intein cyclization strategy, where the topological inhibition of DNA binding by cyclization and its restoration by a proteolytic release of the topologic constraint was expected. We show that locked TALEs indeed have diminished DNA binding and regain full transcriptional activity by stimulation with the rapamycin ligand or site-specific proteolysis of the peptide linker, with much higher level of activation than rapamycin-induced heterodimerization. Additionally, we demonstrated reversibility, activation of genomic targets and implemented logic gates based on combinations of protein cyclization, proteolytic cleavage and ligand-induced dimerization, where the strongest fold induction was achieved by the proteolytic cleavage of a repression domain from a linear TALE. © The Author(s) 2016. Published by Oxford University Press on behalf of Nucleic Acids Research.

  6. Region-specific proteolysis differentially regulates type 1 inositol 1,4,5-trisphosphate receptor activity.

    Science.gov (United States)

    Wang, Liwei; Wagner, Larry E; Alzayady, Kamil J; Yule, David I

    2017-07-14

    The inositol 1,4,5 trisphosphate receptor (IP 3 R) is an intracellular Ca 2+ release channel expressed predominately on the membranes of the endoplasmic reticulum. IP 3 R1 can be cleaved by caspase or calpain into at least two receptor fragments. However, the functional consequences of receptor fragmentation are poorly understood. Our previous work has demonstrated that IP 3 R1 channels, formed following either enzymatic fragmentation or expression of the corresponding complementary polypeptide chains, retain tetrameric architecture and are still activated by IP 3 binding despite the loss of peptide continuity. In this study, we demonstrate that region-specific receptor fragmentation modifies channel regulation. Specifically, the agonist-evoked temporal Ca 2+ release profile and protein kinase A modulation of Ca 2+ release are markedly altered. Moreover, we also demonstrate that activation of fragmented IP 3 R1 can result in a distinct functional outcome. Our work suggests that proteolysis of IP 3 R1 may represent a novel form of modulation of IP 3 R1 channel function and increases the repertoire of Ca 2+ signals achievable through this channel. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

  7. VEGF-A isoforms program differential VEGFR2 signal transduction, trafficking and proteolysis

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    Gareth W. Fearnley

    2016-05-01

    Full Text Available Vascular endothelial growth factor A (VEGF-A binding to the receptor tyrosine kinase VEGFR2 triggers multiple signal transduction pathways, which regulate endothelial cell responses that control vascular development. Multiple isoforms of VEGF-A can elicit differential signal transduction and endothelial responses. However, it is unclear how such cellular responses are controlled by isoform-specific VEGF-A–VEGFR2 complexes. Increasingly, there is the realization that the membrane trafficking of receptor–ligand complexes influences signal transduction and protein turnover. By building on these concepts, our study shows for the first time that three different VEGF-A isoforms (VEGF-A165, VEGF-A121 and VEGF-A145 promote distinct patterns of VEGFR2 endocytosis for delivery into early endosomes. This differential VEGFR2 endocytosis and trafficking is linked to VEGF-A isoform-specific signal transduction events. Disruption of clathrin-dependent endocytosis blocked VEGF-A isoform-specific VEGFR2 activation, signal transduction and caused substantial depletion in membrane-bound VEGFR1 and VEGFR2 levels. Furthermore, such VEGF-A isoforms promoted differential patterns of VEGFR2 ubiquitylation, proteolysis and terminal degradation. Our study now provides novel insights into how different VEGF-A isoforms can bind the same receptor tyrosine kinase and elicit diverse cellular outcomes.

  8. VEGF-A isoforms program differential VEGFR2 signal transduction, trafficking and proteolysis.

    Science.gov (United States)

    Fearnley, Gareth W; Smith, Gina A; Abdul-Zani, Izma; Yuldasheva, Nadira; Mughal, Nadeem A; Homer-Vanniasinkam, Shervanthi; Kearney, Mark T; Zachary, Ian C; Tomlinson, Darren C; Harrison, Michael A; Wheatcroft, Stephen B; Ponnambalam, Sreenivasan

    2016-05-15

    Vascular endothelial growth factor A (VEGF-A) binding to the receptor tyrosine kinase VEGFR2 triggers multiple signal transduction pathways, which regulate endothelial cell responses that control vascular development. Multiple isoforms of VEGF-A can elicit differential signal transduction and endothelial responses. However, it is unclear how such cellular responses are controlled by isoform-specific VEGF-A-VEGFR2 complexes. Increasingly, there is the realization that the membrane trafficking of receptor-ligand complexes influences signal transduction and protein turnover. By building on these concepts, our study shows for the first time that three different VEGF-A isoforms (VEGF-A165, VEGF-A121 and VEGF-A145) promote distinct patterns of VEGFR2 endocytosis for delivery into early endosomes. This differential VEGFR2 endocytosis and trafficking is linked to VEGF-A isoform-specific signal transduction events. Disruption of clathrin-dependent endocytosis blocked VEGF-A isoform-specific VEGFR2 activation, signal transduction and caused substantial depletion in membrane-bound VEGFR1 and VEGFR2 levels. Furthermore, such VEGF-A isoforms promoted differential patterns of VEGFR2 ubiquitylation, proteolysis and terminal degradation. Our study now provides novel insights into how different VEGF-A isoforms can bind the same receptor tyrosine kinase and elicit diverse cellular outcomes. © 2016. Published by The Company of Biologists Ltd.

  9. Quantitative proteomics and terminomics to elucidate the role of ubiquitination and proteolysis in adaptive immunity.

    Science.gov (United States)

    Klein, Theo; Viner, Rosa I; Overall, Christopher M

    2016-10-28

    Adaptive immunity is the specialized defence mechanism in vertebrates that evolved to eliminate pathogens. Specialized lymphocytes recognize specific protein epitopes through antigen receptors to mount potent immune responses, many of which are initiated by nuclear factor-kappa B activation and gene transcription. Most, if not all, pathways in adaptive immunity are further regulated by post-translational modification (PTM) of signalling proteins, e.g. phosphorylation, citrullination, ubiquitination and proteolytic processing. The importance of PTMs is reflected by genetic or acquired defects in these pathways that lead to a dysfunctional immune response. Here we discuss the state of the art in targeted proteomics and systems biology approaches to dissect the PTM landscape specifically regarding ubiquitination and proteolysis in B- and T-cell activation. Recent advances have occurred in methods for specific enrichment and targeted quantitation. Together with improved instrument sensitivity, these advances enable the accurate analysis of often rare PTM events that are opaque to conventional proteomics approaches, now rendering in-depth analysis and pathway dissection possible. We discuss published approaches, including as a case study the profiling of the N-terminome of lymphocytes of a rare patient with a genetic defect in the paracaspase protease MALT1, a key regulator protease in antigen-driven signalling, which was manifested by elevated linear ubiquitination.This article is part of the themed issue 'Quantitative mass spectrometry'. © 2016 The Authors.

  10. Matriptase activation connects tissue factor-dependent coagulation initiation to epithelial proteolysis and signaling.

    Science.gov (United States)

    Le Gall, Sylvain M; Szabo, Roman; Lee, Melody; Kirchhofer, Daniel; Craik, Charles S; Bugge, Thomas H; Camerer, Eric

    2016-06-23

    The coagulation cascade is designed to sense tissue injury by physical separation of the membrane-anchored cofactor tissue factor (TF) from inactive precursors of coagulation proteases circulating in plasma. Once TF on epithelial and other extravascular cells is exposed to plasma, sequential activation of coagulation proteases coordinates hemostasis and contributes to host defense and tissue repair. Membrane-anchored serine proteases (MASPs) play critical roles in the development and homeostasis of epithelial barrier tissues; how MASPs are activated in mature epithelia is unknown. We here report that proteases of the extrinsic pathway of blood coagulation transactivate the MASP matriptase, thus connecting coagulation initiation to epithelial proteolysis and signaling. Exposure of TF-expressing cells to factors (F) VIIa and Xa triggered the conversion of latent pro-matriptase to an active protease, which in turn cleaved the pericellular substrates protease-activated receptor-2 (PAR2) and pro-urokinase. An activation pathway-selective PAR2 mutant resistant to direct cleavage by TF:FVIIa and FXa was activated by these proteases when cells co-expressed pro-matriptase, and matriptase transactivation was necessary for efficient cleavage and activation of wild-type PAR2 by physiological concentrations of TF:FVIIa and FXa. The coagulation initiation complex induced rapid and prolonged enhancement of the barrier function of epithelial monolayers that was dependent on matriptase transactivation and PAR2 signaling. These observations suggest that the coagulation cascade engages matriptase to help coordinate epithelial defense and repair programs after injury or infection, and that matriptase may contribute to TF-driven pathogenesis in cancer and inflammation.

  11. Protein translation, proteolysis and autophagy in human skeletal muscle atrophy after spinal cord injury.

    Science.gov (United States)

    Lundell, L S; Savikj, M; Kostovski, E; Iversen, P O; Zierath, J R; Krook, A; Chibalin, A V; Widegren, U

    2018-02-08

    Spinal cord injury-induced loss of skeletal muscle mass does not progress linearly. In humans, peak muscle loss occurs during the first 6 weeks postinjury, and gradually continues thereafter. The aim of this study was to delineate the regulatory events underlying skeletal muscle atrophy during the first year following spinal cord injury. Key translational, autophagic and proteolytic proteins were analysed by immunoblotting of human vastus lateralis muscle obtained 1, 3 and 12 months following spinal cord injury. Age-matched able-bodied control subjects were also studied. Several downstream targets of Akt signalling decreased after spinal cord injury in skeletal muscle, without changes in resting Akt Ser 473 and Akt Thr 308 phosphorylation or total Akt protein. Abundance of mTOR protein and mTOR Ser 2448 phosphorylation, as well as FOXO1 Ser 256 phosphorylation and FOXO3 protein, decreased in response to spinal cord injury, coincident with attenuated protein abundance of E3 ubiquitin ligases, MuRF1 and MAFbx. S6 protein and Ser 235/236 phosphorylation, as well as 4E-BP1 Thr 37/46 phosphorylation, increased transiently after spinal cord injury, indicating higher levels of protein translation early after injury. Protein abundance of LC3-I and LC3-II decreased 3 months postinjury as compared with 1 month postinjury, but not compared to able-bodied control subjects, indicating lower levels of autophagy. Proteins regulating proteasomal degradation were stably increased in response to spinal cord injury. Together, these data provide indirect evidence suggesting that protein translation and autophagy transiently increase, while whole proteolysis remains stably higher in skeletal muscle within the first year after spinal cord injury. © 2018 Scandinavian Physiological Society. Published by John Wiley & Sons Ltd.

  12. Multiple functions of the von Willebrand Factor A domain in matrilins: secretion, assembly, and proteolysis

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    Kanbe Katsuaki

    2008-06-01

    Full Text Available Abstract The von Willebrand Factor A (vWF A domain is one of the most widely distributed structural modules in cell-matrix adhesive molecules such as intergrins and extracellular matrix proteins. Mutations in the vWF A domain of matrilin-3 cause multiple epiphyseal dysplasia (MED, however the pathological mechanism remains to be determined. Previously we showed that the vWF A domain in matrilin-1 mediates formation of a filamentous matrix network through metal-ion dependent adhesion sites in the domain. Here we show two new functions of the vWF A domain in cartilage-specific matrilins (1 and 3. First, vWF A domain regulates oligomerization of matrilins. Insertion of a vWF A domain into matrilin-3 converts the formation of a mixture of matrilin-3 tetramer, trimer, and dimer into a tetramer only, while deletion of a vWF A domain from matrilin-1 converts the formation of the native matrilin-1 trimer into a mixture of trimer and dimer. Second, the vWF A domain protects matrilin-1 from proteolysis. We identified a latent proteolytic site next to the vWF A2 domain in matrilin-1, which is sensitive to the inhibitors of matrix proteases. Deletion of the abutting vWF A domain results in degradation of matrilin-1, presumably by exposing the adjacent proteolytic site. In addition, we also confirmed the vWF A domain is vital for the secretion of matrilin-3. Secretion of the mutant matrilin-3 harbouring a point mutation within the vWF A domain, as occurred in MED patients, is markedly reduced and delayed, resulting from intracellular retention of the mutant matrilin-3. Taken together, our data suggest that different mutations/deletions of the vWF A domain in matrilins may lead to distinct pathological mechanisms due to the multiple functions of the vWF A domain.

  13. Structural inferences for the native skeletal muscle sodium channel as derived from patterns of endogenous proteolysis

    International Nuclear Information System (INIS)

    Kraner, S.; Yang, J.; Barchi, R.

    1989-01-01

    The alpha subunit (Mr approximately 260,000) of the rat skeletal muscle sodium channel is sensitive to cleavage by endogenous proteases during the isolation of muscle surface membrane. Antisera against synthetic oligopeptides were used to map the resultant fragments in order to identify protease-sensitive regions of the channel's structure in its native membrane environment. Antibodies to the amino terminus labeled major fragments of Mr approximately 130,000 and 90,000 and lesser amounts of other peptides as small as Mr approximately 12,000. Antisera to epitopes within the carboxyl-terminal half of the primary sequence recognized two fragments of Mr approximately 110,000 and 78,000. Individual antisera also selectively labeled smaller polypeptides in the most extensively cleaved preparations. The immunoreactivity patterns of monoclonal antibodies previously raised against the purified channel were then surveyed. The binding sites for one group of monoclonals, including several that recognize subtype-specific epitopes in the channel structure, were localized within a 12-kDa fragment near the amino terminus. The distribution of carbohydrate along the primary structure of the channel was also assessed by quantitating 125 I-wheat germ agglutinin and 125I-concanavalin A binding to the proteolytic peptides. Most of the carbohydrate detected by these lectins was located between 22 and 90 kDa from the amino terminus of the protein. No lectin binding was detected to fragments arising from carboxyl-terminal half of the protein. These results were analyzed in terms of current models of sodium channel tertiary structure. In its normal membrane environment, the skeletal muscle sodium channel appears sensitive to cleavage by endogenous proteases in regions predicted to link the four repeat domains on the cytoplasmic side of the membrane while the repeat domains themselves are resistant to proteolysis

  14. LSD1 demethylase and the methyl-binding protein PHF20L1 prevent SET7 methyltransferase-dependent proteolysis of the stem-cell protein SOX2.

    Science.gov (United States)

    Zhang, Chunxiao; Hoang, Nam; Leng, Feng; Saxena, Lovely; Lee, Logan; Alejo, Salvador; Qi, Dandan; Khal, Anthony; Sun, Hong; Lu, Fei; Zhang, Hui

    2018-03-09

    The pluripotency-controlling stem-cell protein SRY-box 2 (SOX2) plays a pivotal role in maintaining the self-renewal and pluripotency of embryonic stem cells and also of teratocarcinoma or embryonic carcinoma cells. SOX2 is monomethylated at lysine 119 (Lys-119) in mouse embryonic stem cells by the SET7 methyltransferase, and this methylation triggers ubiquitin-dependent SOX2 proteolysis. However, the molecular regulators and mechanisms controlling SET7-induced SOX2 proteolysis are unknown. Here, we report that in human ovarian teratocarcinoma PA-1 cells, methylation-dependent SOX2 proteolysis is dynamically regulated by the LSD1 lysine demethylase and a methyl-binding protein, PHD finger protein 20-like 1 (PHF20L1). We found that LSD1 not only removes the methyl group from monomethylated Lys-117 (equivalent to Lys-119 in mouse SOX2), but it also demethylates monomethylated Lys-42 in SOX2, a reaction that SET7 also regulated and that also triggered SOX2 proteolysis. Our studies further revealed that PHF20L1 binds both monomethylated Lys-42 and Lys-117 in SOX2 and thereby prevents SOX2 proteolysis. Down-regulation of either LSD1 or PHF20L1 promoted SOX2 proteolysis, which was prevented by SET7 inactivation in both PA-1 and mouse embryonic stem cells. Our studies also disclosed that LSD1 and PHF20L1 normally regulate the growth of pluripotent mouse embryonic stem cells and PA-1 cells by preventing methylation-dependent SOX2 proteolysis. In conclusion, our findings reveal an important mechanism by which the stability of the pluripotency-controlling stem-cell protein SOX2 is dynamically regulated by the activities of SET7, LSD1, and PHF20L1 in pluripotent stem cells. © 2018 by The American Society for Biochemistry and Molecular Biology, Inc.

  15. Proteolysis of MDA5 and IPS-1 is not required for inhibition of the type I IFN response by poliovirus.

    Science.gov (United States)

    Kotla, Swathi; Gustin, Kurt E

    2015-10-06

    The type I interferon (IFN) response is a critical component of the innate immune response to infection by RNA viruses and is initiated via recognition of viral nucleic acids by RIG-like receptors (RLR). Engagement of these receptors in the cytoplasm initiates a signal transduction pathway leading to activation of the transcription factors NF-κB, ATF-2 and IRF-3 that coordinately upregulate transcription of type I IFN genes, such as that encoding IFN-β. In this study the impact of poliovirus infection on the type I interferon response has been examined. The type I IFN response was assessed by measuring IFN-β mRNA levels using qRT-PCR and normalizing to levels of β-actin mRNA. The status of host factors involved in activation of the type I IFN response was examined by immunoblot, immunofluorescence microcopy and qRT-PCR. The results show that poliovirus infection results in induction of very low levels of IFN-β mRNA despite clear activation of NF-κB and ATF-2. In contrast, analysis of IRF-3 revealed no transcriptional induction of an IRF-3-responsive promoter or homodimerization of IRF-3 indicating it is not activated in poliovirus-infected cells. Exposure of poliovirus-infected cells to poly(I:C) results in lower levels of IFN-β mRNA synthesis and IRF-3 activation compared to mock-infected cells. Analysis of MDA-5 and IPS-1 revealed that these components of the RLR pathway were largely intact at times when the type I IFN response was suppressed. Collectively, these results demonstrate that poliovirus infection actively suppresses the host type I interferon response by blocking activation of IRF-3 and suggests that this is not mediated by cleavage of MDA-5 or IPS-1.

  16. Regulation of progranulin expression in human microglia and proteolysis of progranulin by matrix metalloproteinase-12 (MMP-12.

    Directory of Open Access Journals (Sweden)

    Hyeon-Sook Suh

    Full Text Available The essential role of progranulin (PGRN as a neurotrophic factor has been demonstrated by the discovery that haploinsufficiency due to GRN gene mutations causes frontotemporal lobar dementia. In addition to neurons, microglia in vivo express PGRN, but little is known about the regulation of PGRN expression by microglia.In the current study, we examined the regulation of expression and function of PGRN, its proteolytic enzyme macrophage elastase (MMP-12, as well as the inhibitor of PGRN proteolysis, secretory leukocyte protease inhibitor (SLPI, in human CNS cells.Cultures of primary human microglia and astrocytes were stimulated with the TLR ligands (LPS or poly IC, Th1 cytokines (IL-1/IFNγ, or Th2 cytokines (IL-4, IL-13. Results were analyzed by Q-PCR, immunoblotting or ELISA. The roles of MMP-12 and SLPI in PGRN cleavage were also examined.Unstimulated microglia produced nanogram levels of PGRN, and PGRN release from microglia was suppressed by the TLR ligands or IL-1/IFNγ, but increased by IL-4 or IL-13. Unexpectedly, while astrocytes stimulated with proinflammatory factors released large amounts of SLPI, none were detected in microglial cultures. We also identified MMP-12 as a PGRN proteolytic enzyme, and SLPI as an inhibitor of MMP-12-induced PGRN proteolysis. Experiments employing PGRN siRNA demonstrated that microglial PGRN was involved in the cytokine and chemokine production following TLR3/4 activation, with its effect on TNFα being the most conspicuous.Our study is the first detailed examination of PGRN in human microglia. Our results establish microglia as a significant source of PGRN, and MMP-12 and SLPI as modulators of PGRN proteolysis. Negative and positive regulation of microglial PGRN release by the proinflammatory/Th1 and the Th2 stimuli, respectively, suggests a fundamentally different aspect of PGRN regulation compared to other known microglial activation products. Microglial PGRN appears to function as an endogenous

  17. Proteolysis of virulence regulator ToxR is associated with entry of Vibrio cholerae into a dormant state.

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    Salvador Almagro-Moreno

    2015-04-01

    Full Text Available Vibrio cholerae O1 is a natural inhabitant of aquatic environments and causes the diarrheal disease, cholera. Two of its primary virulence regulators, TcpP and ToxR, are localized in the inner membrane. TcpP is encoded on the Vibrio Pathogenicity Island (VPI, a horizontally acquired mobile genetic element, and functions primarily in virulence gene regulation. TcpP has been shown to undergo regulated intramembrane proteolysis (RIP in response to environmental conditions that are unfavorable for virulence gene expression. ToxR is encoded in the ancestral genome and is present in non-pathogenic strains of V. cholerae, indicating it has roles outside of the human host. In this study, we show that ToxR undergoes RIP in V. cholerae in response to nutrient limitation at alkaline pH, a condition that occurs during the stationary phase of growth. This process involves the site-2 protease RseP (YaeL, and is dependent upon the RpoE-mediated periplasmic stress response, as deletion mutants for the genes encoding these two proteins cannot proteolyze ToxR under nutrient limitation at alkaline pH. We determined that the loss of ToxR, genetically or by proteolysis, is associated with entry of V. cholerae into a dormant state in which the bacterium is normally found in the aquatic environment called viable but nonculturable (VBNC. Strains that can proteolyze ToxR, or do not encode it, lose culturability, experience a change in morphology associated with cells in VBNC, yet remain viable under nutrient limitation at alkaline pH. On the other hand, mutant strains that cannot proteolyze ToxR remain culturable and maintain the morphology of cells in an active state of growth. Overall, our findings provide a link between the proteolysis of a virulence regulator and the entry of a pathogen into an environmentally persistent state.

  18. Time-Course of Muscle Mass Loss, Damage, and Proteolysis in Gastrocnemius following Unloading and Reloading: Implications in Chronic Diseases

    Science.gov (United States)

    Chacon-Cabrera, Alba; Lund-Palau, Helena; Gea, Joaquim; Barreiro, Esther

    2016-01-01

    Background Disuse muscle atrophy is a major comorbidity in patients with chronic diseases including cancer. We sought to explore the kinetics of molecular mechanisms shown to be involved in muscle mass loss throughout time in a mouse model of disuse muscle atrophy and recovery following immobilization. Methods Body and muscle weights, grip strength, muscle phenotype (fiber type composition and morphometry and muscle structural alterations), proteolysis, contractile proteins, systemic troponin I, and mitochondrial content were assessed in gastrocnemius of mice exposed to periods (1, 2, 3, 7, 15 and 30 days) of non-invasive hindlimb immobilization (plastic splint, I cohorts) and in those exposed to reloading for different time-points (1, 3, 7, 15, and 30 days, R cohorts) following a seven-day period of immobilization. Groups of control animals were also used. Results Compared to non-exposed controls, muscle weight, limb strength, slow- and fast-twitch cross-sectional areas, mtDNA/nDNA, and myosin content were decreased in mice of I cohorts, whereas tyrosine release, ubiquitin-proteasome activity, muscle injury and systemic troponin I levels were increased. Gastrocnemius reloading following splint removal improved muscle mass loss, strength, fiber atrophy, injury, myosin content, and mtDNA/nDNA, while reducing ubiquitin-proteasome activity and proteolysis. Conclusions A consistent program of molecular and cellular events leading to reduced gastrocnemius muscle mass and mitochondrial content and reduced strength, enhanced proteolysis, and injury, was seen in this non-invasive mouse model of disuse muscle atrophy. Unloading of the muscle following removal of the splint significantly improved the alterations seen during unloading, characterized by a specific kinetic profile of molecular events involved in muscle regeneration. These findings have implications in patients with chronic diseases including cancer in whom physical activity may be severely compromised. PMID

  19. Role of calpain in eccentric contraction-induced proteolysis of Ca2+-regulatory proteins and force depression in rat fast-twitch skeletal muscle.

    Science.gov (United States)

    Kanzaki, Keita; Watanabe, Daiki; Kuratani, Mai; Yamada, Takashi; Matsunaga, Satoshi; Wada, Masanobu

    2017-02-01

    The aim of this study was to examine the in vivo effects of eccentric contraction (ECC) on calpain-dependent proteolysis of Ca 2+ -regulatory proteins and force production in fast-twitch skeletal muscles. Rat extensor digitorum longus muscles were exposed to 200 repeated ECC in situ and excised immediately [recovery 0 (REC0)] or 3 days [recovery 3 (REC3)] after cessation of ECC. Calpain inhibitor (CI)-treated rats were intraperitoneally injected with MDL-28170 before ECC and during REC3. Tetanic force was markedly reduced at REC0 and remained reduced at REC3. CI treatment ameliorated the ECC-induced force decline but only at REC3. No evidence was found for proteolysis of dihydropyridine receptor (DHPR), junctophilin (JP)1, JP2, ryanodine receptor (RyR), sarcoplasmic reticulum Ca 2+ -ATPase (SERCA)1a, or junctional face protein-45 at REC0. At REC3, ECC resulted in decreases in DHPR, JP1, JP2, RyR, and SERCA1a. CI treatment prevented the decreases in DHPR, JP1, and JP2, whereas it had little effect on RyR and SERCA1a. These findings suggest that DHPR, JP1, and JP2, but not RyR and SERCA1a, undergo calpain-dependent proteolysis in in vivo muscles subjected to ECC and that impaired function of DHPR and/or JP might cause prolonged force deficits with ECC. NEW & NOTEWORTHY Calpain-dependent proteolysis is one of the contributing factors to muscle damage that occurs with eccentric contraction (ECC). It is unclear, however, whether calpains account for proteolysis of Ca 2+ -regulatory proteins in in vivo muscles subjected to ECC. Here, we provide evidence that dihydropyridine receptor and junctophilin, but not ryanodine receptor and sarcoplasmic reticulum Ca 2+ -ATPase, undergo calpain-dependent proteolysis. Copyright © 2017 the American Physiological Society.

  20. Effect of monoclonal antibodies on limited proteolysis of native glycoprotein gD of herpes simplex virus type 1

    International Nuclear Information System (INIS)

    Eisenberg, R.J.; Long, D.; Pereira, L.; Hampar, B.; Zweig, M.; Cohen, G.H.

    1982-01-01

    We examined the properties of 17 monoclonal antibodies to glycoprotein gD of herpes simplex type 1 (HSV-1) (gD-1) and HSV-2 (gD-2). The antibodies recognized eight separate determinants of gD, based on differences in radioimmuno-precipitation and neutralization assays. The determinants were distributed as follows: three were gD-1 specific, one was gD-2 specific, and four were type common. Several type-specific and type-common determinants appeared to be involved in neutralization. We developed a procedure for examining the effect that binding of monoclonal antibody has on proteolysis of native gD-1 by Staphylococcus aureus protease V8. We showed that several different patterns of protease V8 cleavage were obtained, depending on the monoclonal antibody used. The proteolysis patterns were generally consistent with the immunological groupings. With four groups of antibodies, we found that fragments of gD-1 remained bound to antibody after V8 treatment. A 38,000-dalton fragment remained bound to antibodies in three different groups of monoclonal antibodies. This fragment appeared to contain one type-common and two type-specific determinants. A 12,000-dalton fragment remained bound to antibodies belonging to one type-common group of monoclonal antibodies. Tryptic peptide analysis revealed that the 12,000-dalton fragment represented a portion of the 38,000-dalton fragment and was enriched in a type-common arginine tryptic peptide

  1. Effect of pulsed electric field on the proteolysis of cold boned beef M. Longissimus lumborum and M. Semimembranosus.

    Science.gov (United States)

    Suwandy, Via; Carne, Alan; van de Ven, Remy; Bekhit, Alaa El-Din A; Hopkins, David L

    2015-02-01

    The effects of pulsed electric field (PEF) and ageing (3, 7, 14 and 21 days) on the shear force, protein profile, and post-mortem proteolysis of beef loins (M. Longissimus lumborum, LL) and topsides (M. Semimembranosus, SM) were investigated using a range of pulsed electric field treatments [voltages (5 and 10 kV) and frequencies (20, 50, and 90 Hz)]. PEF treatment decreased the shear force of beef LL and SM muscles by up to 19%. The reduction in the shear force in the LL was not affected by the treatment intensity whereas the reduction in the SM was dependent on PEF frequency. PEF treated beef loins showed increased proteolysis, both early post-mortem and during subsequent post-mortem storage reflected by increased degradation of troponin-T and desmin. The most prominent troponin-T degradation was found in samples treated with 5 kV-90 Hz, 10 kV-20 Hz at day 3 and day 7 post-treatment in addition to 10 kV-50 Hz in subsequent post-treatment times. The degradation of desmin in PEF treated beef loins increased with ageing time.

  2. The Vip3Ag4 Insecticidal Protoxin from Bacillus thuringiensis Adopts A Tetrameric Configuration That Is Maintained on Proteolysis

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    Leopoldo Palma

    2017-05-01

    Full Text Available The Vip3 proteins produced during vegetative growth by strains of the bacterium Bacillus thuringiensis show insecticidal activity against lepidopteran insects with a mechanism of action that may involve pore formation and apoptosis. These proteins are promising supplements to our arsenal of insecticidal proteins, but the molecular details of their activity are not understood. As a first step in the structural characterisation of these proteins, we have analysed their secondary structure and resolved the surface topology of a tetrameric complex of the Vip3Ag4 protein by transmission electron microscopy. Sites sensitive to proteolysis by trypsin are identified and the trypsin-cleaved protein appears to retain a similar structure as an octomeric complex comprising four copies each of the ~65 kDa and ~21 kDa products of proteolysis. This processed form of the toxin may represent the active toxin. The quality and monodispersity of the protein produced in this study make Vip3Ag4 a candidate for more detailed structural analysis using cryo-electron microscopy.

  3. Characterisation of cysteine proteinases responsible for digestive proteolysis in guts of larval Western corn rootworm (Diabrotica virgifera) by expression in the yeast Pichia pastoris

    NARCIS (Netherlands)

    Bown, D.P.; Wilkinson, H.S.; Jongsma, M.A.; Gatehouse, J.A.

    2004-01-01

    Cysteine proteinases are the major class of enzymes responsible for digestive proteolysis in western corn rootworm (Diabrotica virgifera), a serious pest of maize. A larval gut extract hydrolysed typical cathepsin substrates, such as Z-phe-arg-AMC and Z-arg-arg-AMC, and hydrolysis was inhibited by

  4. PROTEOLYSIS DURING MANUFACTURE AND RIPENING/STORING OF “OLOMOUCKÉ TVARŮŽKY” CHEESE (PGI

    Directory of Open Access Journals (Sweden)

    Vendula Pachlová

    2015-02-01

    Full Text Available Twenty-two free amino acid (FAA concentrations were observed during manufacture (1st, 3rd and 7th days of production and ripening period (42 days storing at 8°C of “Olomoucké tvarůžky” (PGI, smear acid cheese. Sensory attributes were also analysed during ripening period. The free amino acids were determined by means of ion-exchange chromatography. The development of the individual FAA content positively correlated with the ripening period (r = 0.7734–0.9229; P < 0.01.The results gave information about the development precursors (FAA of typically sensory active compound in “Olomoucké tvarůžky” (PGI during its production and especially ripening. In conclusion, we found that free amino acid concentration as finally products of proteolysis are positive with improved flavour.

  5. Cloning and expression of the receptor for human urokinase plasminogen activator, a central molecule in cell surface, plasmin dependent proteolysis

    DEFF Research Database (Denmark)

    Roldan, A.L.; Cubellis, M.V.; Masucci, M.T.

    1990-01-01

    , and therefore the capacity of cells to migrate and invade neighboring tissues. We have isolated a 1.4 kb cDNA clone coding for the entire human uPAR. An oligonucleotide synthesized on the basis of the N-terminal sequence of the purified protein was used to screen a cDNA library made from SV40 transformed human......, a size very close to that of the cloned cDNA. Expression of the uPAR cDNA in mouse cells confirms that the clone is complete and expresses a functional uPA binding protein, located on the cell surface and with properties similar to the human uPAR. Caseinolytic plaque assay, immunofluorescence analysis......The surface receptor for urokinase plasminogen activator (uPAR) has been recognized in recent years as a key molecule in regulating plasminogen mediated extracellular proteolysis. Surface plasminogen activation controls the connections between cells, basement membrane and extracellular matrix...

  6. Initiation of proteolysis of yeast fructose-1,6-bisphosphatase by pH-control of adenylate cyclase

    International Nuclear Information System (INIS)

    Holzer, H.; Purwin, C.; Pohlig, G.; Scheffers, W.A.; Nicolay, K.

    1986-01-01

    Addition of fermentable sugars or uncouplers such as CCCP to resting yeast cells grown on glucose initiates phosphorylation of fructose-1,6-bisphosphatase (FBPase). There is good evidence that phosphorylation marks FBPase for proteolytic degradation. 31 P-NMR measurements of the cytosolic pH of yeast cells demonstrated a decrease of the cytosolic pH from 7.0 to 6.5 after addition of glucose or CCCP to starved yeast. Activity of adenylate cyclase in permeabilized yeast cells increases 2-3-fold when the pH is lowered from 7.0 to 6.5. It is concluded that pH controlled activation of adenylate cyclase causes the previously described increase in cyclic AMP which leads to phosphorylation of FBPase and finally to proteolysis of FBPase

  7. Proteolysis of His-Phe-Arg-Trp-Pro-Gly-Pro in the blood and brain of rats in vivo.

    Science.gov (United States)

    Shevchenko, K V; Nagaev, I Yu; Babakov, V N; Andreeva, L A; Shevchenko, V P; Radilov, A S; Myasoedov, N F

    2015-01-01

    The kinetics of the content of His-Phe-Arg-Trp-Pro-Gly-Pro (ACTH (6-9)PGP) and its hydrolysis products in the blood and brain of rats in the case of intranasal administration and intravenous injection of tritiated ACTH(6-9)PGP was studied. The parameters of bioavailability of ACTH(6-9)PGP administered intranasally were higher, indicating certain prospects in the intranasal application in clinical practice. We also found that the factor that determines ACTH(6-9)PGP proteolysis in experiments both in vivo and in vitro is aminopeptidases. The main products of ACTH(6-9)PGP during its metabolism in rats are short peptides and amino acids.

  8. Impact of pasteurization of human milk on preterm newborn in vitro digestion: Gastrointestinal disintegration, lipolysis and proteolysis.

    Science.gov (United States)

    de Oliveira, Samira C; Bourlieu, Claire; Ménard, Olivia; Bellanger, Amandine; Henry, Gwénaële; Rousseau, Florence; Dirson, Emelyne; Carrière, Frédéric; Dupont, Didier; Deglaire, Amélie

    2016-11-15

    Human milk feeding is an important recommendation for preterm newborns considering their vulnerability and digestive immaturity. Holder pasteurization (62.5°C, 30min) applied in milk banks modifies its biological quality and its microstructure. We investigated the impact of pasteurization of preterm human milk on its gastrointestinal kinetics of lipolysis, proteolysis and structural disintegration. An in vitro dynamic system was set up to simulate the gastrointestinal digestion of preterm newborns. A pool of preterm human milk was digested as raw or after Holder pasteurization. Pasteurization impacted the microstructure of undigested human milk, its gastrointestinal disintegration and tended to limit the intestinal lipolysis. Furthermore, the gastrointestinal bioaccessibility of some fatty acids was decreased by pasteurization, while the intestinal bioaccessibility of some amino acids was selectively modulated. The impact of pasteurization on the digestion of human milk may have nutritional relevance in vivo and potentially modulates preterm development and growth. Copyright © 2016 Elsevier Ltd. All rights reserved.

  9. Ascorbate attenuates pulmonary emphysema by inhibiting tobacco smoke and Rtp801-triggered lung protein modification and proteolysis.

    Science.gov (United States)

    Gupta, Indranil; Ganguly, Souradipta; Rozanas, Christine R; Stuehr, Dennis J; Panda, Koustubh

    2016-07-19

    Cigarette smoking causes emphysema, a fatal disease involving extensive structural and functional damage of the lung. Using a guinea pig model and human lung cells, we show that oxidant(s) present in tobacco smoke not only cause direct oxidative damage of lung proteins, contributing to the major share of lung injury, but also activate Rtp801, a key proinflammatory cellular factor involved in tobacco smoke-induced lung damage. Rtp801 triggers nuclear factor κB and consequent inducible NOS (iNOS)-mediated overproduction of NO, which in combination with excess superoxide produced during Rtp801 activation, contribute to increased oxido-nitrosative stress and lung protein nitration. However, lung-specific inhibition of iNOS with a iNOS-specific inhibitor, N6-(1-iminoethyl)-L-lysine, dihydrochloride (L-NIL) solely restricts lung protein nitration but fails to prevent or reverse the major tobacco smoke-induced oxidative lung injury. In comparison, the dietary antioxidant, ascorbate or vitamin C, can substantially prevent such damage by inhibiting both tobacco smoke-induced lung protein oxidation as well as activation of pulmonary Rtp801 and consequent iNOS/NO-induced nitration of lung proteins, that otherwise lead to increased proteolysis of such oxidized or nitrated proteins by endogenous lung proteases, resulting in emphysematous lung damage. Vitamin C also restricts the up-regulation of matrix-metalloproteinase-9, the major lung protease involved in the proteolysis of such modified lung proteins during tobacco smoke-induced emphysema. Overall, our findings implicate tobacco-smoke oxidant(s) as the primary etiopathogenic factor behind both the noncellular and cellular damage mechanisms governing emphysematous lung injury and demonstrate the potential of vitamin C to accomplish holistic prevention of such damage.

  10. MMP-13 regulates growth of wound granulation tissue and modulates gene expression signatures involved in inflammation, proteolysis, and cell viability.

    Directory of Open Access Journals (Sweden)

    Mervi Toriseva

    Full Text Available Proteinases play a pivotal role in wound healing by regulating cell-matrix interactions and availability of bioactive molecules. The role of matrix metalloproteinase-13 (MMP-13 in granulation tissue growth was studied in subcutaneously implanted viscose cellulose sponge in MMP-13 knockout (Mmp13(-/- and wild type (WT mice. The tissue samples were harvested at time points day 7, 14 and 21 and subjected to histological analysis and gene expression profiling. Granulation tissue growth was significantly reduced (42% at day 21 in Mmp13(-/- mice. Granulation tissue in Mmp13(-/- mice showed delayed organization of myofibroblasts, increased microvascular density at day 14, and virtual absence of large vessels at day 21. Gene expression profiling identified differentially expressed genes in Mmp13(-/- mouse granulation tissue involved in biological functions including inflammatory response, angiogenesis, cellular movement, cellular growth and proliferation and proteolysis. Among genes linked to angiogenesis, Adamts4 and Npy were significantly upregulated in early granulation tissue in Mmp13(-/- mice, and a set of genes involved in leukocyte motility including Il6 were systematically downregulated at day 14. The expression of Pdgfd was downregulated in Mmp13(-/- granulation tissue in all time points. The expression of matrix metalloproteinases Mmp2, Mmp3, Mmp9 was also significantly downregulated in granulation tissue of Mmp13(-/- mice compared to WT mice. Mmp13(-/- mouse skin fibroblasts displayed altered cell morphology and impaired ability to contract collagen gel and decreased production of MMP-2. These results provide evidence for an important role for MMP-13 in wound healing by coordinating cellular activities important in the growth and maturation of granulation tissue, including myofibroblast function, inflammation, angiogenesis, and proteolysis.

  11. Recombinant PrPSc shares structural features with brain-derived PrPSc: Insights from limited proteolysis.

    Science.gov (United States)

    Sevillano, Alejandro M; Fernández-Borges, Natalia; Younas, Neelam; Wang, Fei; R Elezgarai, Saioa; Bravo, Susana; Vázquez-Fernández, Ester; Rosa, Isaac; Eraña, Hasier; Gil, David; Veiga, Sonia; Vidal, Enric; Erickson-Beltran, Melissa L; Guitián, Esteban; Silva, Christopher J; Nonno, Romolo; Ma, Jiyan; Castilla, Joaquín; R Requena, Jesús

    2018-01-01

    Very solid evidence suggests that the core of full length PrPSc is a 4-rung β-solenoid, and that individual PrPSc subunits stack to form amyloid fibers. We recently used limited proteolysis to map the β-strands and connecting loops that make up the PrPSc solenoid. Using high resolution SDS-PAGE followed by epitope analysis, and mass spectrometry, we identified positions ~116/118, 133-134, 141, 152-153, 162, 169 and 179 (murine numbering) as Proteinase K (PK) cleavage sites in PrPSc. Such sites likely define loops and/or borders of β-strands, helping us to predict the threading of the β-solenoid. We have now extended this approach to recombinant PrPSc (recPrPSc). The term recPrPSc refers to bona fide recombinant prions prepared by PMCA, exhibiting infectivity with attack rates of ~100%. Limited proteolysis of mouse and bank vole recPrPSc species yielded N-terminally truncated PK-resistant fragments similar to those seen in brain-derived PrPSc, albeit with varying relative yields. Along with these fragments, doubly N- and C-terminally truncated fragments, in particular ~89/97-152, were detected in some recPrPSc preparations; similar fragments are characteristic of atypical strains of brain-derived PrPSc. Our results suggest a shared architecture of recPrPSc and brain PrPSc prions. The observed differences, in particular the distinct yields of specific PK-resistant fragments, are likely due to differences in threading which result in the specific biochemical characteristics of recPrPSc. Furthermore, recombinant PrPSc offers exciting opportunities for structural studies unachievable with brain-derived PrPSc.

  12. Andrographolide alleviates imiquimod-induced psoriasis in mice via inducing autophagic proteolysis of MyD88.

    Science.gov (United States)

    Shao, Fenli; Tan, Tao; Tan, Yang; Sun, Yang; Wu, Xingxin; Xu, Qiang

    2016-09-01

    Psoriasis is a chronic inflammatory skin disease with excessive activation of toll-like receptors (TLRs), which play important roles in developing psoriasis. Targeting TLR signaling remains a challenge for treating psoriasis. Here, we found that andrographolide (Andro), a small-molecule natural product, alleviated imiquimod- but not interleukin 23 (IL-23)-induced psoriasis in mice with reducing expressions of IL-23 and IL-1β in the skin. The improvement in imiquimod-induced psoriasis by Andro was not observed in microtubule-associated protein 1 light chain 3 beta (MAP1LC3B) knockout mice. Furthermore, Andro inhibited mRNA expressions of IL-23, IL-6 and IL-1β but not CD80 and CD86 in bone-marrow derived dendritic cells (BMDCs) treated with lipopolysaccharide (LPS) in a MAP1LC3B-dependent manner. In addition, Andro inhibited imiquimod-induced mRNA expressions of IL-23, IL-6, IL-1β, CD80 and CD86 in BMDCs from mice. Interestingly, Andro induced a degradation of myeloid differentiation factor 88 (MyD88) and blocked the recruitment of TNF receptor-associated factor 6 (TRAF6) to MyD88 upon LPS stimulation in BMDCs from mice. Blockade of autophagic proteolysis using NH4Cl or MAP1LC3B(-/-) BMDCs abolished the Andro-induced MyD88 degradation. In conclusion, Andro controls activation of MyD88-dependent cytokines and alleviates psoriasis in mice via inducing autophagic proteolysis of MyD88, which could be a novel strategy to treat psoriasis. Copyright © 2016 Elsevier Inc. All rights reserved.

  13. In vivo effect of an antilipolytic drug (3,5'-dimethylpyrazole) on autophagic proteolysis and autophagy-related gene expression in rat liver

    International Nuclear Information System (INIS)

    Donati, Alessio; Ventruti, Annamaria; Cavallini, Gabriella; Masini, Matilde; Vittorini, Simona; Chantret, Isabelle; Codogno, Patrice; Bergamini, Ettore

    2008-01-01

    Autophagy is an intracellular pathway induced by starvation, inhibited by nutrients, that is responsible for degradation of long-lived proteins and altered cell organelles. This process is involved in cell maintenance could be induced by antilipolytic drugs and may have anti-aging effects [A. Donati, The involvement of macroautophagy in aging and anti-aging interventions, Mol. Aspects Med. 27 (2006) 455-470]. We analyzed the effect of an intraperitoneal injection of an antilipolytic agent (3,5'-dimethylpyrazole, DMP, 12 mg/kg b.w.), that mimics nutrient shortage on autophagy and expression of autophagic genes in the liver of male 3-month-old Sprague-Dawley albino rats. Autophagy was evaluated by observing electron micrographs of the liver autophagosomal compartment and by monitoring protein degradation assessed by the release of valine into the bloodstream. LC3 gene expression, whose product is one of the best known markers of autophagy, was also monitored. As expected, DMP decreased the plasma levels of free fatty acids, glucose, and insulin and increased autophagic vacuoles and proteolysis. DMP treatment caused an increase in the expression of the LC3 gene although this occurred later than the induction of authophagic proteolysis caused by DMP. Glucose treatment rescued the effects caused by DMP on glucose and insulin plasma levels and negatively affected the rate of autophagic proteolysis, but did not suppress the positive regulatory effect on LC3 mRNA levels. In conclusion, antilipolytic drugs may induce both autophagic proteolysis and higher expression of an autophagy-related gene and the effect on autophagy gene expression might not be secondary to the stimulation of autophagic proteolysis

  14. Myocardial overexpression of TIMP3 after myocardial infarction exerts beneficial effects by promoting angiogenesis and suppressing early proteolysis.

    Science.gov (United States)

    Takawale, Abhijit; Zhang, Pu; Azad, Abul; Wang, Wang; Wang, Xiuhua; Murray, Allan G; Kassiri, Zamaneh

    2017-08-01

    Myocardial infarction (MI) results in loss of cardiomyocytes, adverse extracellular matrix (ECM) and structural remodeling, and left ventricular (LV) dilation and dysfunction. Tissue inhibitors of metalloproteinase (TIMPs) inhibit matrix metalloproteinases (MMPs), the main regulators of ECM turnover. TIMPs also have MMP-independent functions. TIMP3 levels are reduced in the heart within 24 h of MI in mice. We investigated if overexpression of TIMP3 post-MI limits adverse remodeling and LV dilation and dysfunction. MI was induced by left anterior descending coronary artery ligation in 10- to 12-wk-old male C57BL/6J mice, and adenoviral constructs expressing human (h)TIMP3 (Ad-hTIMP3) or no TIMP (Ad-Null) were injected in the peri-infarct zone (5.4 × 10 7 plaque-forming units/heart, 5 injections/heart). Cardiac function assessed by echocardiography showed improved LV physiology and reduced LV dilation after TIMP3 overexpression compared with the Ad-Null-MI group. Post-MI adverse remodeling was attenuated in the Ad-hTIMP3-MI group, as assessed by greater cardiomyocyte density, less infarct expansion, and ECM disruption. TIMP3 overexpression blunted the early rise in proteolytic activities post-MI. A higher density of coronary arteries and a greater number of proliferating endothelial cells were detected in the infarct and peri-infarct regions in the Ad-hTIMP3-MI group compared with the Ad-Null-MI group. In vitro three-dimensional angiogenesis assay confirmed that recombinant TIMP3 promotes angiogenesis in human endothelial cells, although biphasically and in a dose-dependent manner. Intriguingly, overexpression of Ad-hTIMP3 at 10-fold higher concentration had no beneficial effects, consistent with antiangiogenic effects of TIMP3 at higher doses. In conclusion, optimal overexpression of TIMP3 can be a promising therapeutic approach to limit adverse post-MI remodeling by dually inhibiting early proteolysis and promoting angiogenesis. NEW & NOTEWORTHY Here, we report

  15. [Influence of delta-sleep inducing peptide on the state of lysosomal membranes and intensity of lysosomal proteolysis in different rat tissues during physiological aging of the organism].

    Science.gov (United States)

    Kutilin, D S; Bondarenko, T I; Mikhaleva, I I

    2014-01-01

    It is shown that subcutaneous injection of exogenous delta-sleep inducing peptide (DSIP) to rats aged 2-24 months in a dose of 100 μg/kg animal body weight by courses of 5 consecutive days per month has a stabilizing effect on the state of lysosomal membranes in rat tissues (brain, heart muscle and liver) at different ontogenetic stages, and this effect is accompanied by increasing intensity of lysosomal proteolysis in these tissues.

  16. Effect of protein degradation on spot Mr distribution in 2-D gels – a case study of proteolysis during development of Streptomyces coelicolor cultures

    Czech Academy of Sciences Publication Activity Database

    Vohradský, Jiří; Branny, Pavel; Li, X.-M.; Thompson, CH. J.

    2008-01-01

    Roč. 8, č. 12 (2008), s. 2371-2375 ISSN 1615-9853 R&D Projects: GA ČR GA310/07/1009 Grant - others:AT(AT) EC Integrated Project ActinoGEN, LSHM-CT-2004-005224 Institutional research plan: CEZ:AV0Z50200510 Keywords : 2-de * proteolysis * streptomyces Subject RIV: EE - Microbiology, Virology Impact factor: 4.586, year: 2008

  17. Activated thrombin-activatable fibrinolysis inhibitor (TAFIa) attenuates breast cancer cell metastatic behaviors through inhibition of plasminogen activation and extracellular proteolysis

    International Nuclear Information System (INIS)

    Bazzi, Zainab A.; Lanoue, Danielle; El-Youssef, Mouhanned; Romagnuolo, Rocco; Tubman, Janice; Cavallo-Medved, Dora; Porter, Lisa A.; Boffa, Michael B.

    2016-01-01

    Thrombin activatable fibrinolysis inhibitor (TAFI) is a plasma zymogen, which can be converted to activated TAFI (TAFIa) through proteolytic cleavage by thrombin, plasmin, and most effectively thrombin in complex with the endothelial cofactor thrombomodulin (TM). TAFIa is a carboxypeptidase that cleaves carboxyl terminal lysine and arginine residues from protein and peptide substrates, including plasminogen-binding sites on cell surface receptors. Carboxyl terminal lysine residues play a pivotal role in enhancing cell surface plasminogen activation to plasmin. Plasmin has many critical functions including cleaving components of the extracellular matrix (ECM), which enhances invasion and migration of cancer cells. We therefore hypothesized that TAFIa could act to attenuate metastasis. To assess the role of TAFIa in breast cancer metastasis, in vitro migration and invasion assays, live cell proteolysis and cell proliferation using MDA-MB-231 and SUM149 cells were carried out in the presence of a TAFIa inhibitor, recombinant TAFI variants, or soluble TM. Inhibition of TAFIa with potato tuber carboxypeptidase inhibitor increased cell invasion, migration and proteolysis of both cell lines, whereas addition of TM resulted in a decrease in all these parameters. A stable variant of TAFIa, TAFIa-CIIYQ, showed enhanced inhibitory effects on cell invasion, migration and proteolysis. Furthermore, pericellular plasminogen activation was significantly decreased on the surface of MDA-MB-231 and SUM149 cells following treatment with various concentrations of TAFIa. Taken together, these results indicate a vital role for TAFIa in regulating pericellular plasminogen activation and ultimately ECM proteolysis in the breast cancer microenvironment. Enhancement of TAFI activation in this microenvironment may be a therapeutic strategy to inhibit invasion and prevent metastasis of breast cancer cells

  18. Continuous fast Fourier transforms cyclic voltammetry as a new approach for investigation of skim milk k-casein proteolysis, a comparative study.

    Science.gov (United States)

    Shayeh, Javad Shabani; Sefidbakht, Yahya; Siadat, Seyed Omid Ranaei; Niknam, Kaveh

    2017-10-01

    Cheese production is relied upon the action of Rennet on the casein micelles of milk. Chymosin assay methods are usually time consuming and offline. Herein, we report a new electrochemical technique for studying the proteolysis of K-casein. The interaction of rennet and its substrate was studied by fast Fourier transform continuous cyclic voltammetry (FFTCCV) based on a determination of k-casein in aqueous solution. FFTCCV technique is a very useful method for studying the enzymatic procedures. Fast response, no need of modified electrodes or complex equipment is some of FFTCCV advantages. Various concentrations of enzyme and substrate were selected and the increase in the appearance of charged species in solution as a result of the addition of rennet was studied. Data obtained using FFTCCV technique were also confirmed by turbidity analysis. The results show that rennet proteolysis activity occurs in much shorter time scales compare with its aggregation. Hence, following the appearance of charged segments as a result of proteolysis could be under consideration as a rapid and online method. Copyright © 2017 Elsevier B.V. All rights reserved.

  19. Effect of Aloe vera (Aloe barbadensis Miller) on survivability, extent of proteolysis and ACE inhibition of potential probiotic cultures in fermented milk.

    Science.gov (United States)

    Basannavar, Santosh; Pothuraju, Ramesh; Sharma, Raj Kumar

    2014-10-01

    In the present investigation, the effect of Aloe vera gel powder on angiotensin-converting enzyme (ACE) inhibitory activity, extent of proteolysis during fermentation and survival of Lactobacillus casei NCDC19 during storage of fermented milk was studied. Among the different cultures screened for ACE inhibitory activity, Lactobacillus casei NCDC 19 exhibited the highest ACE inhibition (approx. 40%) as well as extent of proteolysis (0.37, Abs₃₄₀). In the presence of Aloe vera (0.5% and 1% w/v) an increase in extent of proteolysis (0.460 ± 0.047 and 0.480 ± 0.027) and percent ACE inhibitory activity (44.32 ± 2.83 and 47.52 ± 1.83) was observed in comparison to control. Aloe vera powder addition also led to an increase in viable counts (>11 log cfu mL⁻¹) of L. casei NCDC 19 in fermented milk during storage for 7 days and the counts were maintained in sufficiently higher numbers. The study suggests Aloe vera to be a good functional ingredient which can be further explored for different health attributes. © 2014 Society of Chemical Industry.

  20. Regulated proteolysis of Trop2 drives epithelial hyperplasia and stem cell self-renewal via β-catenin signaling.

    Science.gov (United States)

    Stoyanova, Tanya; Goldstein, Andrew S; Cai, Houjian; Drake, Justin M; Huang, Jiaoti; Witte, Owen N

    2012-10-15

    The cell surface protein Trop2 is expressed on immature stem/progenitor-like cells and is overexpressed in many epithelial cancers. However the biological function of Trop2 in tissue maintenance and tumorigenesis remains unclear. In this study, we demonstrate that Trop2 is a regulator of self-renewal, proliferation, and transformation. Trop2 controls these processes through a mechanism of regulated intramembrane proteolysis that leads to cleavage of Trop2, creating two products: the extracellular domain and the intracellular domain. The intracellular domain of Trop2 is released from the membrane and accumulates in the nucleus. Heightened expression of the Trop2 intracellular domain promotes stem/progenitor self-renewal through signaling via β-catenin and is sufficient to initiate precursor lesions to prostate cancer in vivo. Importantly, we demonstrate that loss of β-catenin or Trop2 loss-of-function cleavage mutants abrogates Trop2-driven self-renewal and hyperplasia in the prostate. These findings suggest that heightened expression of Trop2 is selected for in epithelial cancers to enhance the stem-like properties of self-renewal and proliferation. Defining the mechanism of Trop2 function in self-renewal and transformation is essential to identify new therapeutic strategies to block Trop2 activation in cancer.

  1. Bacillus licheniformis BlaR1 L3 Loop Is a Zinc Metalloprotease Activated by Self-Proteolysis

    Science.gov (United States)

    Sépulchre, Jérémy; Amoroso, Ana; Joris, Bernard

    2012-01-01

    In Bacillus licheniformis 749/I, BlaP β-lactamase is induced by the presence of a β-lactam antibiotic outside the cell. The first step in the induction mechanism is the detection of the antibiotic by the membrane-bound penicillin receptor BlaR1 that is composed of two functional domains: a carboxy-terminal domain exposed outside the cell, which acts as a penicillin sensor, and an amino-terminal domain anchored to the cytoplasmic membrane, which works as a transducer-transmitter. The acylation of BlaR1 sensor domain by the antibiotic generates an intramolecular signal that leads to the activation of the L3 cytoplasmic loop of the transmitter by a single-point cleavage. The exact mechanism of L3 activation and the nature of the secondary cytoplasmic signal launched by the activated transmitter remain unknown. However, these two events seem to be linked to the presence of a HEXXH zinc binding motif of neutral zinc metallopeptidases. By different experimental approaches, we demonstrated that the L3 loop binds zinc ion, belongs to Gluzincin metallopeptidase superfamily and is activated by self-proteolysis. PMID:22623956

  2. Therapeutic effects of remediating autophagy failure in a mouse model of Alzheimer disease by enhancing lysosomal proteolysis.

    Science.gov (United States)

    Yang, Dun-Sheng; Stavrides, Philip; Mohan, Panaiyur S; Kaushik, Susmita; Kumar, Asok; Ohno, Masuo; Schmidt, Stephen D; Wesson, Daniel W; Bandyopadhyay, Urmi; Jiang, Ying; Pawlik, Monika; Peterhoff, Corrinne M; Yang, Austin J; Wilson, Donald A; St George-Hyslop, Peter; Westaway, David; Mathews, Paul M; Levy, Efrat; Cuervo, Ana M; Nixon, Ralph A

    2011-07-01

    The extensive autophagic-lysosomal pathology in Alzheimer disease (AD) brain has revealed a major defect: in the proteolytic clearance of autophagy substrates. Autophagy failure contributes on several levels to AD pathogenesis and has become an important therapeutic target for AD and other neurodegenerative diseases. We recently observed broad therapeutic effects of stimulating autophagic-lysosomal proteolysis in the TgCRND8 mouse model of AD that exhibits defective proteolytic clearance of autophagic substrates, robust intralysosomal amyloid-β peptide (Aβ) accumulation, extracellular β-amyloid deposition and cognitive deficits. By genetically deleting the lysosomal cysteine protease inhibitor, cystatin B (CstB), to selectively restore depressed cathepsin activities, we substantially cleared Aβ, ubiquitinated proteins and other autophagic substrates from autolysosomes/lysosomes and rescued autophagic-lysosomal pathology, as well as reduced total Aβ40/42 levels and extracellular amyloid deposition, highlighting the underappreciated importance of the lysosomal system for Aβ clearance. Most importantly, lysosomal remediation prevented the marked learning and memory deficits in TgCRND8 mice. Our findings underscore the pathogenic significance of autophagic-lysosomal dysfunction in AD and demonstrate the value of reversing this dysfunction as an innovative therapeautic strategy for AD.

  3. Pronounced energy restriction with elevated protein intake results in no change in proteolysis and reductions in skeletal muscle protein synthesis that are mitigated by resistance exercise.

    Science.gov (United States)

    Hector, Amy J; McGlory, Chris; Damas, Felipe; Mazara, Nicole; Baker, Steven K; Phillips, Stuart M

    2018-01-01

    Preservation of lean body mass (LBM) may be important during dietary energy restriction (ER) and requires equal rates of muscle protein synthesis (MPS) and muscle protein breakdown (MPB). Currently, the relative contribution of MPS and MPB to the loss of LBM during ER in humans is unknown. We aimed to determine the impact of dietary protein intake and resistance exercise on MPS and MPB during a controlled short-term energy deficit. Adult men (body mass index, 28.6 ± 0.6 kg/m 2 ; age 22 ± 1 yr) underwent 10 d of 40%-reduced energy intake while performing unilateral resistance exercise and consuming lower protein (1.2 g/kg/d, n = 12) or higher protein (2.4 g/kg/d, n = 12). Pre- and postintervention testing included dual-energy X-ray absorptiometry, primed constant infusion of ring -[ 13 C 6 ]phenylalanine, and 15 [N]phenylalanine to measure acute postabsorptive MPS and MPB; D 2 O to measure integrated MPS; and gene and protein expression. There was a decrease in acute MPS after ER (higher protein, 0.059 ± 0.006 to 0.051 ± 0.009%/h; lower protein, 0.061 ± 0.005 to 0.045 ± 0.006%/h; P resistance exercise (higher protein, 0.067 ± 0.01%/h; lower protein, 0.061 ± 0.006%/h), and integrated MPS followed a similar pattern. There was no change in MPB (energy balance, 0.080 ± 0.01%/hr; ER rested legs, 0.078 ± 0.008%/hr; ER exercised legs, 0.079 ± 0.006%/hr). We conclude that a reduction in MPS is the main mechanism that underpins LBM loss early in ER in adult men.-Hector, A. J., McGlory, C., Damas, F., Mazara, N., Baker, S. K., Phillips, S. M. Pronounced energy restriction with elevated protein intake results in no change in proteolysis and reductions in skeletal muscle protein synthesis that are mitigated by resistance exercise. © FASEB.

  4. Coronavirus cell entry occurs through the endo-/lysosomal pathway in a proteolysis-dependent manner.

    Directory of Open Access Journals (Sweden)

    Christine Burkard

    2014-11-01

    Full Text Available Enveloped viruses need to fuse with a host cell membrane in order to deliver their genome into the host cell. While some viruses fuse with the plasma membrane, many viruses are endocytosed prior to fusion. Specific cues in the endosomal microenvironment induce conformational changes in the viral fusion proteins leading to viral and host membrane fusion. In the present study we investigated the entry of coronaviruses (CoVs. Using siRNA gene silencing, we found that proteins known to be important for late endosomal maturation and endosome-lysosome fusion profoundly promote infection of cells with mouse hepatitis coronavirus (MHV. Using recombinant MHVs expressing reporter genes as well as a novel, replication-independent fusion assay we confirmed the importance of clathrin-mediated endocytosis and demonstrated that trafficking of MHV to lysosomes is required for fusion and productive entry to occur. Nevertheless, MHV was shown to be less sensitive to perturbation of endosomal pH than vesicular stomatitis virus and influenza A virus, which fuse in early and late endosomes, respectively. Our results indicate that entry of MHV depends on proteolytic processing of its fusion protein S by lysosomal proteases. Fusion of MHV was severely inhibited by a pan-lysosomal protease inhibitor, while trafficking of MHV to lysosomes and processing by lysosomal proteases was no longer required when a furin cleavage site was introduced in the S protein immediately upstream of the fusion peptide. Also entry of feline CoV was shown to depend on trafficking to lysosomes and processing by lysosomal proteases. In contrast, MERS-CoV, which contains a minimal furin cleavage site just upstream of the fusion peptide, was negatively affected by inhibition of furin, but not of lysosomal proteases. We conclude that a proteolytic cleavage site in the CoV S protein directly upstream of the fusion peptide is an essential determinant of the intracellular site of fusion.

  5. Lipolysis and proteolysis profiles of fresh artisanal goat cheese made with raw milk with 3 different fat contents.

    Science.gov (United States)

    Sánchez-Macías, D; Morales-Delanuez, A; Moreno-Indias, I; Hernández-Castellano, L E; Mendoza-Grimón, V; Castro, N; Argüello, A

    2011-12-01

    The objective of this study was to describe the proteolysis and lipolysis profiles in goat cheese made in the Canary Islands (Spain) using raw milk with 3 different fat contents (0.5, 1.5, and 5%) and ripened for 1, 7, 14, and 28 d. β-Casein was the most abundant protein in all cheeses and at all ripening times. Quantitative analysis showed a general decrease in caseins as ripening progressed, and degradation rates were higher for α(S1)-casein than for β-casein and α(S2)-casein. Furthermore, the degradation rate during the experimental time decreased with lower fat contents. The α(S2)-casein and α(S1)-casein levels that remained in full-fat and reduced-fat cheeses were less than those in low-fat cheese. In contrast, β-casein also showed degradation along with ripening, but differences in degradation among the 3 cheese types were not significant at 28 d. The degradation products increased with the ripening time in all cheeses, but they were higher in full-fat cheese than in reduced-fat and low-fat cheeses. The free fatty acid concentration per 100g of cheese was higher in full-fat cheese than in reduced- and low-fat cheese; however, when the results were expressed as milligrams of free fatty acids per gram of fat in cheese, then lipolysis occurred more rapidly in low-fat cheese than in reduced- and full-fat cheeses. These results may explain the atypical texture and off-flavors found in low-fat goat cheeses, likely the main causes of non-acceptance. Copyright © 2011 American Dairy Science Association. Published by Elsevier Inc. All rights reserved.

  6. Rapid micro-scale proteolysis of proteins for MALDI-MS peptide mapping using immobilized trypsin

    Science.gov (United States)

    Gobom, Johan; Nordhoff, Eckhard; Ekman, Rolf; Roepstorff, Peter

    1997-12-01

    In this study we present a rapid method for tryptic digestion of proteins using micro-columns with enzyme immobilized on perfusion chromatography media. The performance of the method is exemplified with acyl-CoA-binding protein and reduced carbamidomethylated bovine serum albumin. The method proved to be significantly faster and yielded a better sequence coverage and an improved signal-to-noise ratio for the MALDI-MS peptide maps, compared to in-solution- and on-target digestion. Only a single sample transfer step is required, and therefore sample loss due to adsorption to surfaces is reduced, which is a critical issue when handling low picomole to femtomole amounts of proteins. An example is shown with on-column proteolytic digestion and subsequent elution of the digest into a reversed-phase micro-column. This is useful if the sample contains large amounts of salt or is too diluted for MALDI-MS analysis. Furthermore, by step-wise elution from the reversedphase column, a complex digest can be fractionated, which reduces signal suppression and facilitates data interpretation in the subsequent MS-analysis. The method also proved useful for consecutive digestions with enzymes of different cleavage specificity. This is exemplified with on-column tryptic digestion, followed by reversed-phase step-wise elution, and subsequent on-target V8 protease digestion.

  7. Proteases, proteolysis and inflammatory molecules in the tears of people with keratoconus.

    Science.gov (United States)

    Balasubramanian, Sivaraman Arumugam; Mohan, Sujatha; Pye, David Cecil; Willcox, Mark Duncan Perry

    2012-06-01

    To investigate the expression of proteases, proteolytic activity and cytokines in the tear film of people with keratoconus. Basal tears from people with keratoconus, from individuals who had undergone corneal collagen cross-linking for the treatment of keratoconus, and from normal controls were collected using a capillary tube. Corneal curvature of each subject was mapped. The total protein in tears was estimated. Levels and activity of proteases in the tears were analysed using specific antibody arrays and activity assays. The total tear protein level was significantly reduced in keratoconus (4.1 ± 0.9 mg/ml) compared with normals (6.7 ± 1.4 mg/ml) (p tear expression of matrix metalloproteinases (MMP) -1, -3, -7, -13, interleukins (IL) -4, -5, -6, -8 and tumour necrosis factor (TNF) -α, -β were evident in keratoconus. Tear IL-6 was the only cytokine significantly (p tears of keratoconus subjects compared with the collagen cross-linked group. No significant difference in tear proteases were observed between the normal and the cross-linked groups, although the expression of TNF-α was significantly (p tears from keratoconus compared with normal subjects. The activity of tear gelatinases (69.6 ± 22.2 FIU) and collagenases (5.7 ± 3.3 FIU) in the collagen cross-linked group was not significantly different compared with either keratoconus or normals. Tears of people with keratoconus had 1.9 times higher levels of proteolytic activity and over expression of several MMPs and cytokines compared with tears from controls. Further investigations are required to study the possible implications of these changes and whether they can be used to monitor disease progression or determine the success of corneal collagen cross-linking. © 2012 The Authors. Acta Ophthalmologica © 2012 Acta Ophthalmologica Scandinavica Foundation.

  8. Molecular mechanism of APC/C activation by mitotic phosphorylation.

    Science.gov (United States)

    Zhang, Suyang; Chang, Leifu; Alfieri, Claudio; Zhang, Ziguo; Yang, Jing; Maslen, Sarah; Skehel, Mark; Barford, David

    2016-05-12

    In eukaryotes, the anaphase-promoting complex (APC/C, also known as the cyclosome) regulates the ubiquitin-dependent proteolysis of specific cell-cycle proteins to coordinate chromosome segregation in mitosis and entry into the G1 phase. The catalytic activity of the APC/C and its ability to specify the destruction of particular proteins at different phases of the cell cycle are controlled by its interaction with two structurally related coactivator subunits, Cdc20 and Cdh1. Coactivators recognize substrate degrons, and enhance the affinity of the APC/C for its cognate E2 (refs 4-6). During mitosis, cyclin-dependent kinase (Cdk) and polo-like kinase (Plk) control Cdc20- and Cdh1-mediated activation of the APC/C. Hyperphosphorylation of APC/C subunits, notably Apc1 and Apc3, is required for Cdc20 to activate the APC/C, whereas phosphorylation of Cdh1 prevents its association with the APC/C. Since both coactivators associate with the APC/C through their common C-box and Ile-Arg tail motifs, the mechanism underlying this differential regulation is unclear, as is the role of specific APC/C phosphorylation sites. Here, using cryo-electron microscopy and biochemical analysis, we define the molecular basis of how phosphorylation of human APC/C allows for its control by Cdc20. An auto-inhibitory segment of Apc1 acts as a molecular switch that in apo unphosphorylated APC/C interacts with the C-box binding site and obstructs engagement of Cdc20. Phosphorylation of the auto-inhibitory segment displaces it from the C-box-binding site. Efficient phosphorylation of the auto-inhibitory segment, and thus relief of auto-inhibition, requires the recruitment of Cdk-cyclin in complex with a Cdk regulatory subunit (Cks) to a hyperphosphorylated loop of Apc3. We also find that the small-molecule inhibitor, tosyl-l-arginine methyl ester, preferentially suppresses APC/C(Cdc20) rather than APC/C(Cdh1), and interacts with the binding sites of both the C-box and Ile-Arg tail motifs. Our

  9. Proteolysis of plasminogen activator inhibitor-1 by Yersinia pestis remodulates the host environment to promote virulence.

    Science.gov (United States)

    Eddy, J L; Schroeder, J A; Zimbler, D L; Caulfield, A J; Lathem, W W

    2016-09-01

    Essentials Effect of plasminogen activator inhibitor (PAI)-1 on plague and its Y. pestis cleavage is unknown. An intranasal mouse model of infection was used to determine the role of PAI-1 in pneumonic plague. PAI-1 is cleaved and inactivated by the Pla protease of Y. pestis in the lung airspace. PAI-1 impacts both bacterial outgrowth and the immune response to respiratory Y. pestis infection. Click to hear Dr Bock discuss pathogen activators of plasminogen. Background The hemostatic regulator plasminogen activator inhibitor-1 (PAI-1) inactivates endogenous plasminogen activators and aids in the immune response to bacterial infection. Yersinia pestis, the causative agent of plague, produces the Pla protease, a virulence factor that is required during plague. However, the specific hemostatic proteins cleaved by Pla in vivo that contribute to pathogenesis have not yet been fully elucidated. Objectives To determine whether PAI-1 is cleaved by the Pla protease during pneumonic plague, and to define the impact of PAI-1 on Y. pestis respiratory infection in the presence or absence of Pla. Methods An intranasal mouse model of pneumonic plague was used to assess the levels of total and active PAI-1 in the lung airspace, and the impact of PAI-1 deficiency on bacterial pathogenesis, the host immune response and plasmin generation following infection with wild-type or ∆pla Y. pestis. Results We found that Y. pestis cleaves and inactivates PAI-1 in the lungs in a Pla-dependent manner. The loss of PAI-1 enhances Y. pestis outgrowth in the absence of Pla, and is associated with increased conversion of plasminogen to plasmin. Furthermore, we found that PAI-1 regulates immune cell recruitment, cytokine production and tissue permeability during pneumonic plague. Conclusions Our data demonstrate that PAI-1 is an in vivo target of the Pla protease in the lungs, and that PAI-1 is a key regulator of the pulmonary innate immune response. We conclude that the inactivation of PAI-1 by Y

  10. Toxoplasma gondii infection shifts dendritic cells into an amoeboid rapid migration mode encompassing podosome dissolution, secretion of TIMP-1, and reduced proteolysis of extracellular matrix.

    Science.gov (United States)

    Ólafsson, Einar B; Varas-Godoy, Manuel; Barragan, Antonio

    2018-03-01

    Dendritic cells (DCs) infected by Toxoplasma gondii rapidly acquire a hypermigratory phenotype that promotes systemic parasite dissemination by a "Trojan horse" mechanism in mice. Recent paradigms of leukocyte migration have identified the amoeboid migration mode of DCs as particularly suited for rapid locomotion in extracellular matrix and tissues. Here, we have developed a microscopy-based high-throughput approach to assess motility and matrix degradation by Toxoplasma-challenged murine and human DCs. DCs challenged with T. gondii exhibited dependency on metalloproteinase activity for hypermotility and transmigration but, strikingly, also dramatically reduced pericellular proteolysis. Toxoplasma-challenged DCs up-regulated expression and secretion of tissue inhibitor of metalloproteinases-1 (TIMP-1) and their supernatants impaired matrix degradation by naïve DCs and by-stander DCs dose dependently. Gene silencing of TIMP-1 by short hairpin RNA restored matrix degradation activity in Toxoplasma-infected DCs. Additionally, dissolution of podosome structures in parasitised DCs coincided with abrogated matrix degradation. Toxoplasma lysates inhibited pericellular proteolysis in a MyD88-dependent fashion whereas abrogated proteolysis persevered in Toxoplasma-infected MyD88-deficient DCs. This indicated that both TLR/MyD88-dependent and TLR/MyD88-independent signalling pathways mediated podosome dissolution and the abrogated matrix degradation. We report that increased TIMP-1 secretion and cytoskeletal rearrangements encompassing podosome dissolution are features of Toxoplasma-induced hypermigration of DCs with an impact on matrix degradation. Jointly, the data highlight how an obligate intracellular parasite orchestrates key regulatory cellular processes consistent with non-proteolytic amoeboid migration of the vehicle cells that facilitate its dissemination. © 2017 John Wiley & Sons Ltd.

  11. Probing the 3-D Structure, Dynamics, and Stability of Bacterial Collagenase Collagen Binding Domain (apo- versus holo-) by Limited Proteolysis MALDI-TOF MS

    Science.gov (United States)

    Sides, Cynthia R.; Liyanage, Rohana; Lay, Jackson O.; Philominathan, Sagaya Theresa Leena; Matsushita, Osamu; Sakon, Joshua

    2012-03-01

    Pairing limited proteolysis and matrix-assisted laser desorption/ionization-time of flight mass spectrometry (MALDI-TOF MS) to probe clostridial collagenase collagen binding domain (CBD) reveals the solution dynamics and stability of the protein, as these factors are crucial to CBD effectiveness as a drug-delivery vehicle. MS analysis of proteolytic digests indicates initial cleavage sites, thereby specifying the less stable and highly accessible regions of CBD. Modulation of protein structure and stability upon metal binding is shown through MS analysis of calcium-bound and cobalt-bound CBD proteolytic digests. Previously determined X-ray crystal structures illustrate that calcium binding induces secondary structure transformation in the highly mobile N-terminal arm and increases protein stability. MS-based detection of exposed residues confirms protein flexibility, accentuates N-terminal dynamics, and demonstrates increased global protein stability exported by calcium binding. Additionally, apo- and calcium-bound CBD proteolysis sites correlate well with crystallographic B-factors, accessibility, and enzyme specificity. MS-observed cleavage sites with no clear correlations are explained either by crystal contacts of the X-ray crystal structures or by observed differences between Molecules A and B in the X-ray crystal structures. The study newly reveals the absence of the βA strand and thus the very dynamic N-terminal linker, as corroborated by the solution X-ray scattering results. Cobalt binding has a regional effect on the solution phase stability of CBD, as limited proteolysis data implies the capture of an intermediate-CBD solution structure when cobalt is bound.

  12. Age-related differences in lean mass, protein synthesis and skeletal muscle markers of proteolysis after bed rest and exercise rehabilitation

    DEFF Research Database (Denmark)

    Tanner, Ruth E; Brunker, Lucille B; Agergaard, Jakob

    2015-01-01

    during a constant stable isotope infusion in the postabsorptive state and after essential amino acid (EAA) ingestion on three occasions: before (PRE), after bed rest and after rehabilitation. Samples were assessed for protein synthesis, mTORC1 signalling, REDD1/2 expression and molecular markers related...... to muscle proteolysis (MURF1, MAFBX, AMPKα, LC3II/I, Beclin1). We found that leg lean mass and strength decreased in older but not younger adults after bedrest (P protein synthesis increased before bed rest in both age groups...... (P protein synthesis rates and increased MAFBX mRNA, p-AMPKα and the LC3II/I ratio (P

  13. Surface expression and limited proteolysis of ADAM10 are increased by a dominant negative inhibitor of dynamin

    Directory of Open Access Journals (Sweden)

    Slack Barbara E

    2011-05-01

    Full Text Available Abstract Background The amyloid precursor protein (APP is cleaved by β- and γ-secretases to generate toxic amyloid β (Aβ peptides. Alternatively, α-secretases cleave APP within the Aβ domain, precluding Aβ formation and releasing the soluble ectodomain, sAPPα. We previously showed that inhibition of the GTPase dynamin reduced APP internalization and increased release of sAPPα, apparently by prolonging the interaction between APP and α-secretases at the plasma membrane. This was accompanied by a reduction in Aβ generation. In the present study, we investigated whether surface expression of the α-secretase ADAM (a disintegrin and metalloprotease10 is also regulated by dynamin-dependent endocytosis. Results Transfection of human embryonic kidney (HEK cells stably expressing M3 muscarinic receptors with a dominant negative dynamin I mutant (dyn I K44A, increased surface expression of both immature, and mature, catalytically active forms of co-expressed ADAM10. Surface levels of ADAM10 were unaffected by activation of protein kinase C (PKC or M3 receptors, indicating that receptor-coupled shedding of the ADAM substrate APP is unlikely to be mediated by inhibition of ADAM10 endocytosis in this cell line. Dyn I K44A strongly increased the formation of a C-terminal fragment of ADAM10, consistent with earlier reports that the ADAM10 ectodomain is itself a target for sheddases. The abundance of this fragment was increased in the presence of a γ-secretase inhibitor, but was not affected by M3 receptor activation. The dynamin mutant did not affect the distribution of ADAM10 and its C-terminal fragment between raft and non-raft membrane compartments. Conclusions Surface expression and limited proteolysis of ADAM10 are regulated by dynamin-dependent endocytosis, but are unaffected by activation of signaling pathways that upregulate shedding of ADAM substrates such as APP. Modulation of ADAM10 internalization could affect cellular behavior in two

  14. Determination of protein-carbonyls and ubiquitin-mediated proteolysis as biomarkers of oxidative-stress in bivalvia and anthozoa

    International Nuclear Information System (INIS)

    Walker, Stephen Thomas

    2002-01-01

    blotting and immunohistochemistry in host and zooxanthellae tissues of A. agaricites, depth-dependant differential expression is suggested. These data suggest that differential capacities to mitigate oxidative-stress play a role in setting species distribution limits and that selective proteolysis can be considered an important defence against free-radical mediated protein oxidation. (author)

  15. Determination of protein-carbonyls and ubiquitin-mediated proteolysis as biomarkers of oxidative-stress in bivalvia and anthozoa

    Energy Technology Data Exchange (ETDEWEB)

    Walker, Stephen Thomas

    2002-07-01

    between species. PC=Os can be detected by DNPH-reactivity/Western blotting assay in host A. agaricites. UPCs can be assayed via Western blotting and immunohistochemistry in host and zooxanthellae tissues of A. agaricites, depth-dependant differential expression is suggested. These data suggest that differential capacities to mitigate oxidative-stress play a role in setting species distribution limits and that selective proteolysis can be considered an important defence against free-radical mediated protein oxidation. (author)

  16. Concomitant external pneumatic compression treatment with consecutive days of high intensity interval training reduces markers of proteolysis.

    Science.gov (United States)

    Haun, Cody T; Roberts, Michael D; Romero, Matthew A; Osburn, Shelby C; Healy, James C; Moore, Angelique N; Mobley, Christopher B; Roberson, Paul A; Kephart, Wesley C; Mumford, Petey W; Goodlett, Michael D; Pascoe, David D; Martin, Jeffrey S

    2017-12-01

    To compare the effects of external pneumatic compression (EPC) and sham when used concurrently with high intensity interval training (HIIT) on performance-related outcomes and recovery-related molecular measures. Eighteen recreationally endurance-trained male participants (age: 21.6 ± 2.4 years, BMI: 25.7 ± 0.5 kg/m 2 , VO 2peak : 51.3 ± 0.9 mL/kg/min) were randomized to balanced sham and EPC treatment groups. Three consecutive days of HIIT followed by EPC/sham treatment (Days 2-4) and 3 consecutive days of recovery (Days 5-7) with EPC/sham only on Days 5-6 were employed. Venipuncture, flexibility and pressure-to-pain threshold (PPT) measurements were made throughout. Vastus lateralis muscle was biopsied at PRE (i.e., Day 1), 1-h post-EPC/sham treatment on Day 2 (POST1), and 24-h post-EPC/sham treatment on Day 7 (POST2). 6-km run time trial performance was tested at PRE and POST2. No group × time interaction was observed for flexibility, PPT, or serum measures of creatine kinase (CK), hsCRP, and 8-isoprostane. However, there was a main effect of time for serum CK (p = 0.005). Change from PRE in 6-km run times at POST2 were not significantly different between groups. Significant between-groups differences existed for change from PRE in atrogin-1 mRNA (p = 0.018) at the POST1 time point (EPC: - 19.7 ± 8.1%, sham: + 7.7 ± 5.9%) and atrogin-1 protein concentration (p = 0.013) at the POST2 time point (EPC: - 31.8 ± 7.5%, sham: + 96.0 ± 34.7%). In addition, change from PRE in poly-Ub proteins was significantly different between groups at both the POST1 (EPC: - 26.0 ± 10.3%, sham: + 34.8 ± 28.5%; p = 0.046) and POST2 (EPC: - 33.7 ± 17.2%, sham: + 21.4 ± 14.9%; p = 0.037) time points. EPC when used concurrently with HIIT and in subsequent recovery days reduces skeletal muscle markers of proteolysis.

  17. Decreased rate of protein synthesis, caspase-3 activity, and ubiquitin-proteasome proteolysis in soleus muscles from growing rats fed a low-protein, high-carbohydrate diet.

    Science.gov (United States)

    Batistela, Emanuele; Pereira, Mayara Peron; Siqueira, Juliany Torres; Paula-Gomes, Silvia; Zanon, Neusa Maria; Oliveira, Eduardo Brandt; Navegantes, Luiz Carlos Carvalho; Kettelhut, Isis C; Andrade, Claudia Marlise Balbinotti; Kawashita, Nair Honda; Baviera, Amanda Martins

    2014-06-01

    The aim of this study was to investigate the changes in the rates of both protein synthesis and breakdown, and the activation of intracellular effectors that control these processes in soleus muscles from growing rats fed a low-protein, high-carbohydrate (LPHC) diet for 15 days. The mass and the protein content, as well as the rate of protein synthesis, were decreased in the soleus from LPHC-fed rats. The availability of amino acids was diminished, since the levels of various essential amino acids were decreased in the plasma of LPHC-fed rats. Overall rate of proteolysis was also decreased, explained by reductions in the mRNA levels of atrogin-1 and MuRF-1, ubiquitin conjugates, proteasome activity, and in the activity of caspase-3. Soleus muscles from LPHC-fed rats showed increased insulin sensitivity, with increased levels of insulin receptor and phosphorylation levels of AKT, which probably explains the inhibition of both the caspase-3 activity and the ubiquitin-proteasome system. The fall of muscle proteolysis seems to represent an adaptive response that contributes to spare proteins in a condition of diminished availability of dietary amino acids. Furthermore, the decreased rate of protein synthesis may be the driving factor to the lower muscle mass gain in growing rats fed the LPHC diet.

  18. JNK signaling pathway regulates sorbitol-induced Tau proteolysis and apoptosis in SH-SY5Y cells by targeting caspase-3.

    Science.gov (United States)

    Olivera Santa-Catalina, Marta; Caballero Bermejo, Montaña; Argent, Ricardo; Alonso, Juan C; Centeno, Francisco; Lorenzo, María J

    2017-12-15

    Growing evidence suggests that Diabetes Mellitus increases the risk of developing Alzheimer's disease. It is well known that hyperglycemia, a key feature of Diabetes Mellitus, may induce plasma osmolarity disturbances. Both hyperglycemia and hyperosmolarity promote the altered post-translational regulation of microtubule-associated protein Tau. Interestingly, abnormal hyperphosphorylation and cleavage of Tau have been proven to lead to the genesis of filamentous structures referred to as neurofibrillary tangles, the main pathological hallmark of Alzheimer's disease. We have previously described that hyperosmotic stress induced by sorbitol promotes Tau proteolysis and apoptosis in SH-SY5Y cells via caspase-3 activation. In order to gain insights into the regulatory mechanisms of such processes, in this work we explored the intracellular signaling pathways that regulate these events. We found that sorbitol treatment significantly enhanced the activation of conventional families of MAPK in SH-SY5Y cells. Tau proteolysis was completely prevented by JNK inhibition but not affected by either ERK1/2 or p38 MAPK blockade. Moreover, inhibition of JNK, but not ERK1/2 or p38 MAPK, efficiently prevented sorbitol-induced apoptosis and caspase-3 activation. In summary, we provide evidence that JNK signaling pathway is an upstream regulator of hyperosmotic stress-induced Tau cleavage and apoptosis in SH-SY5Y through the control of caspase-3 activation. Copyright © 2017 Elsevier Inc. All rights reserved.

  19. Cooperation of two ADAMTS metalloproteases in closure of the mouse palate identifies a requirement for versican proteolysis in regulating palatal mesenchyme proliferation

    OpenAIRE

    Enomoto, Hiroyuki; Nelson, Courtney M.; Somerville, Robert P. T.; Mielke, Katrina; Dixon, Laura J.; Powell, Kimerly; Apte, Suneel S.

    2010-01-01

    We have identified a role for two evolutionarily related, secreted metalloproteases of the ADAMTS family, ADAMTS20 and ADAMTS9, in palatogenesis. Adamts20 mutations cause the mouse white-spotting mutant belted (bt), whereas Adamts9 is essential for survival beyond 7.5 days gestation (E7.5). Functional overlap of Adamts9 with Adamts20 was identified using Adamts9+/–;bt/bt mice, which have a fully penetrant cleft palate. Palate closure was delayed, although eventually completed, in both Adamts9...

  20. Inhibition of post-traumatic septic proteolysis and ureagenesis and stimulation of hepatic acute-phase protein production by branched-chain amino acid TPN.

    Science.gov (United States)

    Chiarla, C; Siegel, J H; Kidd, S; Coleman, B; Mora, R; Tacchino, R; Placko, R; Gum, M; Wiles, C E; Belzberg, H

    1988-08-01

    Previous studies have shown that severe sepsis after major trauma results in the reprioritization of release of hepatic acute-phase proteins (APP). They suggest competition for leucine for nutritional utilization may be responsible. To test this hypothesis, a branched-chain enriched (46.6%) amino acid mixture (BCAA) was administered on a prospective randomized basis with standard TPN therapy to 16 septic post-trauma patients. After sepsis was diagnosed, a randomized therapy (control-TPN or BCAA-TPN) was given for 12 days, or until death occurred. Total calories and amino acid nitrogen (N) administered were not different in the two groups (t-test) and q 8 h (347 study periods) amino acid clearances, urinary urea nitrogen excretion, muscle proteolysis from 3-methyl-histidine (3-MH) excretion, and standard indices of sepsis severity and hepatic function were measured, as well as platelets (PLAT), leucocytes (WBC), albumin (ALB), and six acute-phase proteins: C-reactive protein (CRP), alpha-1-antitrypsin (A1TRIP), fibrinogen (FIBRIN), alpha-2-macroglobulin (AMACRO), ceruloplasmin (CERUL), and transferrin (TRANS). Using Scheffé analysis of all contrasts the data showed: BCAA resulted in a fall in 24-hour urea N excretion (24.0 to 20.0 gm/24 hr) and in proteolysis (138 to 126 gm/24 hr) (p less than 0.0001). Prestudy CRP levels were all elevated, but compared to control where APP reprioritization occurred, over the initial 10 days of therapy BCAA patients had a more rapid fall in CRP with a more rapid rise in FIBRIN, TRANS, CERUL, ALBUMIN, AMACRO, and A1TRIP (all p less than 0.0001) relative to CRP. Also, the sepsis-reduced clearances of glutamine and glutamate, alanine, and proline were increased (p less than 0.0001) during BCAA even though urea nitrogen production was reduced (p less than 0.0001). The increase in leucine clearance with BCAA-enriched TPN was positively correlated (r2 = 0.601; p less than 0.0001) with the increase in the sum of all APP and ALB and was

  1. Defining the molecular basis of BubR1 kinetochore interactions and APC/C-CDC20 inhibition.

    Science.gov (United States)

    D'Arcy, Sheena; Davies, Owen R; Blundell, Tom L; Bolanos-Garcia, Victor M

    2010-05-07

    BubR1 is essential for the mitotic checkpoint that prevents aneuploidy in cellular progeny by triggering anaphase delay in response to kinetochores incorrectly/not attached to the mitotic spindle. Here, we define the molecular architecture of the functionally significant N-terminal region of human BubR1 and present the 1.8 A crystal structure of its tetratricopeptide repeat (TPR) domain. The structure reveals divergence from the classical TPR fold and is highly similar to the TPR domain of budding yeast Bub1. Shared distinctive features include a disordered loop insertion, a 3(10)-helix, a tight turn involving glycine positive Phi angles, and noncanonical packing of and between the TPR motifs. We also define the molecular determinants of the interaction between BubR1 and kinetochore protein Blinkin. We identify a shallow groove on the concave surface of the BubR1 TPR domain that forms multiple discrete and potentially cooperative interactions with Blinkin. Finally, we present evidence for a direct interaction between BubR1 and Bub1 mediated by regions C-terminal to their TPR domains. This interaction provides a mechanism for Bub1-dependent kinetochore recruitment of BubR1. We thus present novel molecular insights into the structure of BubR1 and its interactions at the kinetochore-microtubule interface. Our studies pave the way for future structure-directed engineering aimed at dissecting the roles of kinetochore-bound and other pools of BubR1 in vivo.

  2. Effect of processing on proteolysis and biogenic amines formation in a Portuguese traditional dry-fermented ripened sausage "Chouriço Grosso de Estremoz e Borba PGI".

    Science.gov (United States)

    Roseiro, L C; Gomes, A; Gonçalves, H; Sol, M; Cercas, R; Santos, C

    2010-01-01

    The influence of alternative drying environmental conditions on the proteolysis of a traditional Portuguese fermented sausage was evaluated, in relation to different ripening periods. Traditional sausages (batch T) had lower pH than counterparts (batch M), with differences (Palanine>taurine>serine>valine and taurine>alanine>leucine>serine>valine sequences in the former product from batches T and M, respectively, and, coincidently, the same sequence for both batches in the later (serine>leucine>alanine>proline>valine). Such effect on FAA concentrations led to a distinct (Psweet, bitter, acidic and aged sensorial attributes between batches, in S8 and S9 products. BA typical quantitative sequences varied between batches according to the ripening stage, with differences in S6 and S7 end products also reflecting the distinct microbial development rates and profiles observed. Overall, the total BA mean concentration was higher (P<0.05) in products from batch T.

  3. Expression, limited proteolysis and preliminary crystallographic analysis of IpaD, a component of the Shigella flexneri type III secretion system

    Energy Technology Data Exchange (ETDEWEB)

    Johnson, Steven [Laboratory of Molecular Biophysics, Department of Biochemistry, University of Oxford (United Kingdom); Sir William Dunn School of Pathology, University of Oxford (United Kingdom); Roversi, Pietro [Laboratory of Molecular Biophysics, Department of Biochemistry, University of Oxford (United Kingdom); Espina, Marianela [Department of Molecular Biosciences, University of Kansas (United States); Deane, Janet E. [Laboratory of Molecular Biophysics, Department of Biochemistry, University of Oxford (United Kingdom); Birket, Susan; Picking, William D. [Department of Molecular Biosciences, University of Kansas (United States); Blocker, Ariel [Sir William Dunn School of Pathology, University of Oxford (United Kingdom); Picking, Wendy L. [Department of Molecular Biosciences, University of Kansas (United States); Lea, Susan M., E-mail: susan.lea@path.ox.ac.uk [Laboratory of Molecular Biophysics, Department of Biochemistry, University of Oxford (United Kingdom); Sir William Dunn School of Pathology, University of Oxford (United Kingdom)

    2006-09-01

    IpaD, the putative needle-tip protein of the S. flexneri type III secretion system, has been crystallized in a variety of crystal forms using in-drop proteolysis. Native and selenomethionine-labelled data collection and preliminary analyses are reported. IpaD, the putative needle-tip protein of the Shigella flexneri type III secretion system, has been overexpressed and purified. Crystals were grown of the native protein in space group P2{sub 1}2{sub 1}2{sub 1}, with unit-cell parameters a = 55.9, b = 100.7, c = 112.0 Å, and data were collected to 2.9 Å resolution. Analysis of the native Patterson map revealed a peak at 50% of the origin on the Harker section v = 0.5, suggesting twofold non-crystallographic symmetry parallel to the b crystallographic axis. As attempts to derivatize or grow selenomethionine-labelled protein crystals failed, in-drop proteolysis was used to produce new crystal forms. A trace amount of subtilisin Carlsberg was added to IpaD before sparse-matrix screening, resulting in the production of several new crystal forms. This approach produced SeMet-labelled crystals and diffraction data were collected to 3.2 Å resolution. The SeMet crystals belong to space group C2, with unit-cell parameters a = 139.4, b = 45.0, c = 99.5 Å, β = 107.9°. An anomalous difference Patterson map revealed peaks on the Harker section v = 0, while the self-rotation function indicates the presence of a twofold noncrystallographic symmetry axis, which is consistent with two molecules per asymmetric unit.

  4. Expression, limited proteolysis and preliminary crystallographic analysis of IpaD, a component of the Shigella flexneri type III secretion system

    International Nuclear Information System (INIS)

    Johnson, Steven; Roversi, Pietro; Espina, Marianela; Deane, Janet E.; Birket, Susan; Picking, William D.; Blocker, Ariel; Picking, Wendy L.; Lea, Susan M.

    2006-01-01

    IpaD, the putative needle-tip protein of the S. flexneri type III secretion system, has been crystallized in a variety of crystal forms using in-drop proteolysis. Native and selenomethionine-labelled data collection and preliminary analyses are reported. IpaD, the putative needle-tip protein of the Shigella flexneri type III secretion system, has been overexpressed and purified. Crystals were grown of the native protein in space group P2 1 2 1 2 1 , with unit-cell parameters a = 55.9, b = 100.7, c = 112.0 Å, and data were collected to 2.9 Å resolution. Analysis of the native Patterson map revealed a peak at 50% of the origin on the Harker section v = 0.5, suggesting twofold non-crystallographic symmetry parallel to the b crystallographic axis. As attempts to derivatize or grow selenomethionine-labelled protein crystals failed, in-drop proteolysis was used to produce new crystal forms. A trace amount of subtilisin Carlsberg was added to IpaD before sparse-matrix screening, resulting in the production of several new crystal forms. This approach produced SeMet-labelled crystals and diffraction data were collected to 3.2 Å resolution. The SeMet crystals belong to space group C2, with unit-cell parameters a = 139.4, b = 45.0, c = 99.5 Å, β = 107.9°. An anomalous difference Patterson map revealed peaks on the Harker section v = 0, while the self-rotation function indicates the presence of a twofold noncrystallographic symmetry axis, which is consistent with two molecules per asymmetric unit

  5. Arsenite reduces insulin secretion in rat pancreatic β-cells by decreasing the calcium-dependent calpain-10 proteolysis of SNAP-25

    International Nuclear Information System (INIS)

    Diaz-Villasenor, Andrea; Burns, Anna L.; Salazar, Ana Maria; Sordo, Monserrat; Hiriart, Marcia; Cebrian, Mariano E.; Ostrosky-Wegman, Patricia

    2008-01-01

    An increase in the prevalence of type 2 diabetes has been consistently observed among residents of high arsenic exposure areas. We have previously shown that in rat pancreatic β-cells, low arsenite doses impair the secretion of insulin without altering its synthesis. To further study the mechanism by which arsenite reduces insulin secretion, we evaluated the effects of arsenite on the calcium-calpain pathway that triggers insulin exocytosis in RINm5F cells. Cell cycle and proliferation analysis were also performed to complement the characterization. Free [Ca 2+ ]i oscillations needed for glucose-stimulated insulin secretion were abated in the presence of subchronic low arsenite doses (0.5-2 μM). The global activity of calpains increased with 2 μM arsenite. However, during the secretion of insulin stimulated with glucose (15.6 mM), 1 μM arsenite decreased the activity of calpain-10, measured as SNAP-25 proteolysis. Both proteins are needed to fuse insulin granules with the membrane to produce insulin exocytosis. Arsenite also induced a slowdown in the β cell line proliferation in a dose-dependent manner, reflected by a reduction of dividing cells and in their arrest in G2/M. Data obtained showed that one of the mechanisms by which arsenite impairs insulin secretion is by decreasing the oscillations of free [Ca 2+ ]i, thus reducing calcium-dependent calpain-10 partial proteolysis of SNAP-25. The effects in cell division and proliferation observed with arsenite exposure can be an indirect consequence of the decrease in insulin secretion

  6. In-capillary enrichment, proteolysis and separation using capillary electrophoresis with discontinuous buffers: application on proteins with moderately acidic and basic isoelectric points.

    Science.gov (United States)

    Nesbitt, Chandra A; Yeung, Ken K-C

    2009-01-01

    Advances in mass spectrometry and capillary-format separation continue to improve the sensitivity of protein analysis. Of equal importance is the miniaturization of sample pretreatment such as enrichment and proteolysis. In a previous report (Nesbitt et al., Electrophoresis, 2008, 29, 466-474), nanoliter-volume protein enrichment, tryptic digestion, and partial separation was demonstrated in capillary electrophoresis followed by MALDI mass spectral analysis. A discontinuous buffer system, consisting of ammonium (pH 10) and acetate (pH 4), was used to create a pH junction inside the capillary, trapping a protein with a neutral isoelectric point, myoglobin (pI 7.2). Moreover, co-enrichment of myoglobin with trypsin led to an in-capillary digestion. In this paper, the ability of this discontinuous buffer system to perform similar in-capillary sample pretreatment on proteins with moderately acidic and basic pI was studied and reported. Lentil lectin (pI 8.6) and a multi-phosphorylated protein, beta-casein (pI 5.1), were selected as model proteins. In addition to the previously shown tryptic digestion, proteolysis with endoproteinase Asp-N was also performed. Digestion of these acidic and basic pI proteins produced a few peptides with extreme pI values lying outside the trapping range of the discontinuous buffer. An alteration in the peptide trapping procedure was made to accommodate these analytes. Offline MALDI mass spectral analysis confirmed the presence of the expected peptides. The presented miniaturized sample pretreatment methodology was proven to be applicable on proteins with a moderately wide range of pI. Flexibility in the choice of protease was also evident.

  7. Insulin does not stimulate muscle protein synthesis during increased plasma branched-chain amino acids alone but still decreases whole body proteolysis in humans.

    Science.gov (United States)

    Everman, Sarah; Meyer, Christian; Tran, Lee; Hoffman, Nyssa; Carroll, Chad C; Dedmon, William L; Katsanos, Christos S

    2016-10-01

    Insulin stimulates muscle protein synthesis when the levels of total amino acids, or at least the essential amino acids, are at or above their postabsorptive concentrations. Among the essential amino acids, branched-chain amino acids (BCAA) have the primary role in stimulating muscle protein synthesis and are commonly sought alone to stimulate muscle protein synthesis in humans. Fourteen healthy young subjects were studied before and after insulin infusion to examine whether insulin stimulates muscle protein synthesis in relation to the availability of BCAA alone. One half of the subjects were studied in the presence of postabsorptive BCAA concentrations (control) and the other half in the presence of increased plasma BCAA (BCAA). Compared with that prior to the initiation of the insulin infusion, fractional synthesis rate of muscle protein (%/h) did not change (P > 0.05) during insulin in either the control (0.04 ± 0.01 vs 0.05 ± 0.01) or the BCAA (0.05 ± 0.02 vs. 0.05 ± 0.01) experiments. Insulin decreased (P BCAA (0.89 ± 0.07 vs 0.61 ± 0.03) experiments, but the change was not different between the two experiments (P > 0.05). In conclusion, insulin does not stimulate muscle protein synthesis in the presence of increased circulating levels of plasma BCAA alone. Insulin's suppressive effect on proteolysis is observed independently of the levels of circulating plasma BCAA. Copyright © 2016 the American Physiological Society.

  8. Age-related differences in lean mass, protein synthesis and skeletal muscle markers of proteolysis after bed rest and exercise rehabilitation.

    Science.gov (United States)

    Tanner, Ruth E; Brunker, Lucille B; Agergaard, Jakob; Barrows, Katherine M; Briggs, Robert A; Kwon, Oh Sung; Young, Laura M; Hopkins, Paul N; Volpi, Elena; Marcus, Robin L; LaStayo, Paul C; Drummond, Micah J

    2015-09-15

    Bed rest-induced muscle loss and impaired muscle recovery may contribute to age-related sarcopenia. It is unknown if there are age-related differences in muscle mass and muscle anabolic and catabolic responses to bed rest. A secondary objective was to determine if rehabilitation could reverse bed rest responses. Nine older and fourteen young adults participated in a 5-day bed rest challenge (BED REST). This was followed by 8 weeks of high intensity resistance exercise (REHAB). Leg lean mass (via dual-energy X-ray absorptiometry; DXA) and strength were determined. Muscle biopsies were collected during a constant stable isotope infusion in the postabsorptive state and after essential amino acid (EAA) ingestion on three occasions: before (PRE), after bed rest and after rehabilitation. Samples were assessed for protein synthesis, mTORC1 signalling, REDD1/2 expression and molecular markers related to muscle proteolysis (MURF1, MAFBX, AMPKα, LC3II/I, Beclin1). We found that leg lean mass and strength decreased in older but not younger adults after bedrest (P protein synthesis increased before bed rest in both age groups (P protein synthesis rates and increased MAFBX mRNA, p-AMPKα and the LC3II/I ratio (P protein synthesis and a marginal increase in proteolytic markers. Finally, rehabilitation restored bed rest-induced deficits in lean mass and strength in older adults. © 2015 The Authors. The Journal of Physiology © 2015 The Physiological Society.

  9. Limited proteolysis combined with isotope labeling and quantitative LC-MALDI MS for monitoring protein conformational changes: a study on calcium-binding sites of cardiac Troponin C

    International Nuclear Information System (INIS)

    McDonald, Chris; Li Liang

    2005-01-01

    Studies of protein-protein and protein-ligand interactions are important for understanding biological functions of proteins. A new technique based on the partial proteolysis of proteins combined with quantitative mass spectrometry is developed as a means of tracking structural changes after the formation of a protein-ligand complex. In this technique, a protein of interest with and without the binding of a ligand is digested with an enzyme to generate a set of peptides, followed by separation of the peptides by liquid chromatography. Matrix-assisted laser desorption ionization (MALDI) mass spectrometry (MS) is used to identify chromatographically separated peptides, and locate their sequence alignments in the parent protein. Using an isotopically labeled protein as a sample against an unlabeled protein standard, quantitative information can be gathered. This overcomes the inherent lack of quantitative capability of MALDI MS. The utility of the technique to investigate protein-ligand interactions is demonstrated in a model system involving calcium binding to cardiac Troponin C (cTnC). Using this technique, the general location of the three calcium-binding sites of cTnC can be determined by using several different enzymes to generate overlapping peptide maps of cTnC

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

    Directory of Open Access Journals (Sweden)

    Marie Trudel

    2016-01-01

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

  11. Proteolysis of complement factors iC3b and C5 by the serine protease prostate-specific antigen in prostatic fluid and seminal plasma.

    Science.gov (United States)

    Manning, Michael L; Williams, Simon A; Jelinek, Christine A; Kostova, Maya B; Denmeade, Samuel R

    2013-03-15

    Prostate-specific Ag (PSA) is a serine protease that is expressed exclusively by normal and malignant prostate epithelial cells. The continued high-level expression of PSA by the majority of men with both high- and low-grade prostate cancer throughout the course of disease progression, even in the androgen-ablated state, suggests that PSA has a role in the pathogenesis of disease. Current experimental and clinical evidence suggests that chronic inflammation, regardless of the cause, may predispose men to prostate cancer. The responsibility of the immune system in immune surveillance and eventually tumor progression is well appreciated but not completely understood. In this study, we used a mass spectrometry-based evaluation of prostatic fluid obtained from diseased prostates after removal by radical prostatectomy to identify potential immunoregulatory proteins. This analysis revealed the presence of Igs and the complement system proteins C3, factor B, and clusterin. Verification of these findings by Western blot confirmed the high-level expression of C3 in the prostatic fluid and the presence of a previously uncharacterized C-terminal C3 cleavage product. Biochemical analysis of this C3 cleavage fragment revealed a putative PSA cleavage site after tyrosine-1348. Purified PSA was able to cleave iC3b and the related complement protein C5. These results suggest a previously uncharacterized function of PSA as an immunoregulatory protease that could help to create an environment hospitable to malignancy through proteolysis of the complement system.

  12. Enhancement of 9α-Hydroxy-4-androstene-3,17-dione Production from Soybean Phytosterols by Deficiency of a Regulated Intramembrane Proteolysis Metalloprotease in Mycobacterium neoaurum.

    Science.gov (United States)

    Xiong, Liang-Bin; Sun, Wan-Ju; Liu, Yong-Jun; Wang, Feng-Qing; Wei, Dong-Zhi

    2017-12-06

    Modification of the sterol catabolism pathway in mycobacteria may result in the accumulation of some valuable steroid pharmaceutical intermediates, such as 9α-hydroxy-4-androstene-3,17-dione (9-OHAD). In previous work, sigma factor D (SigD) was identified as a negative factor of the 9-OHAD production in Mycobacterium neoaurum. Here, the deficiency of rip1 putatively coding for a regulated intramembrane proteolysis metalloprotease (Rip1), which could cleave the negative regulator of SigD (anti-SigD), enhanced the transcription of some key genes (choM1, kshA, and hsd4A) in the sterol catabolic pathway. Furthermore, the deletion of rip1 increased the consumption of phytosterols by 37.8% after 96 h of growth in M. neoaurum. The production of 9-OHAD in the engineered M. neoaurumΔkstD1ΔkstD2ΔkstD3Δrip1 (MnΔk123Δrip1) strain was ultimately increased by 27.3% compared to that in its parental strain M. neoaurumΔkstD1ΔkstD2ΔkstD3 (MnΔk123). This study further confirms the important role of SigD-related factors in the catabolism of sterols.

  13. Methylated DNMT1 and E2F1 are targeted for proteolysis by L3MBTL3 and CRL4DCAF5 ubiquitin ligase.

    Science.gov (United States)

    Leng, Feng; Yu, Jiekai; Zhang, Chunxiao; Alejo, Salvador; Hoang, Nam; Sun, Hong; Lu, Fei; Zhang, Hui

    2018-04-24

    Many non-histone proteins are lysine methylated and a novel function of this modification is to trigger the proteolysis of methylated proteins. Here, we report that the methylated lysine 142 of DNMT1, a major DNA methyltransferase that preserves epigenetic inheritance of DNA methylation patterns during DNA replication, is demethylated by LSD1. A novel methyl-binding protein, L3MBTL3, binds the K142-methylated DNMT1 and recruits a novel CRL4 DCAF5 ubiquitin ligase to degrade DNMT1. Both LSD1 and PHF20L1 act primarily in S phase to prevent DNMT1 degradation by L3MBTL3-CRL4 DCAF5 . Mouse L3MBTL3/MBT-1 deletion causes accumulation of DNMT1 protein, increased genomic DNA methylation, and late embryonic lethality. DNMT1 contains a consensus methylation motif shared by many non-histone proteins including E2F1, a key transcription factor for S phase. We show that the methylation-dependent E2F1 degradation is also controlled by L3MBTL3-CRL4 DCAF5 . Our studies elucidate for the first time a novel mechanism by which the stability of many methylated non-histone proteins are regulated.

  14. In Situ Proteolysis for Crystallization of Membrane Bound Cytochrome P450 17A1 and 17A2 Proteins from Zebrafish.

    Science.gov (United States)

    Lei, Li; Egli, Martin

    2016-04-01

    Fish and human cytochrome P450 (P450) 17A1 catalyze both steroid 17α-hydroxylation and 17α,20-lyase reactions. Fish P450 17A2 catalyzes only 17α-hydroxylation. Both enzymes are microsomal-type P450s, integral membrane proteins that bind to the membrane through their N-terminal hydrophobic segment, the signal anchor sequence. The presence of this N-terminal region renders expression of full-length proteins challenging or impossible. For some proteins, variable truncation of the signal anchor sequence precludes expression or results in poor expression levels. To crystallize P450 17A1 and 17A2 in order to gain insight into their different activities, we used an alternative N-terminal sequence to boost expression together with in situ proteolysis. Key features of our approach to identify crystallizable P450 fragments were the use of an N-terminal leader sequence, a screen composed of 12 proteases to establish optimal cleavage, variations of protease concentration in combination with an SDS-PAGE assay, and analysis of the resulting fragments using Edman sequencing. Described in this unit are protocols for vector preparation, expression, purification, and in situ proteolytic crystallization of two membrane-bound P450 proteins. Copyright © 2016 John Wiley & Sons, Inc.

  15. (67/68)Ga-labeling agent that liberates (67/68)Ga-NOTA-methionine by lysosomal proteolysis of parental low molecular weight polypeptides to reduce renal radioactivity levels.

    Science.gov (United States)

    Uehara, Tomoya; Rokugawa, Takemi; Kinoshita, Mai; Nemoto, Souki; Fransisco Lazaro, Guerra Gomez; Hanaoka, Hirofumi; Arano, Yasushi

    2014-11-19

    The renal localization of gallium-67 or gallium-68 ((67/68)Ga)-labeled low molecular weight (LMW) probes such as peptides and antibody fragments constitutes a problem in targeted imaging. Wu et al. previously showed that (67)Ga-labeled S-2-(4-isothiocyanatobenzyl)-1,4,7-triazacyclononane-1,4,7-triacetic acid (SCN-Bz-NOTA)-conjugated methionine ((67)Ga-NOTA-Met) was rapidly excreted from the kidney in urine following lysosomal proteolysis of the parental (67)Ga-NOTA-Bz-SCN-disulfide-stabilized Fv fragment (Bioconjugate Chem., (1997) 8, 365-369). In the present study, a new (67/68)Ga-labeling reagent for LMW probes that liberates (67/68)Ga-NOTA-Met was designed, synthesized, and evaluated using longer-lived (67)Ga in order to reduce renal radioactivity levels. We employed a methionine-isoleucine (MI) dipeptide bond as the cleavable linkage. The amine residue of MI was coupled with SCN-Bz-NOTA for (67)Ga-labeling, while the carboxylic acid residue of MI was derivatized to maleimide for antibody conjugation in order to synthesize NOTA-MI-Mal. A Fab fragment of the anti-Her2 antibody was thiolated with iminothiolane, and NOTA-MI-Mal was conjugated with the antibody fragment by maleimide-thiol chemistry. The Fab fragment was also conjugated with SCN-Bz-NOTA (NOTA-Fab) for comparison. (67)Ga-NOTA-MI-Fab was obtained at radiochemical yields of over 95% and was stable in murine serum for 24 h. In the biodistribution study using normal mice, (67)Ga-NOTA-MI-Fab registered significantly lower renal radioactivity levels from 1 to 6 h postinjection than those of (67)Ga-NOTA-Fab. An analysis of urine samples obtained 6 h after the injection of (67)Ga-NOTA-MI-Fab showed that the majority of radioactivity was excreted as (67)Ga-NOTA-Met. In the biodistribution study using tumor-bearing mice, the tumor to kidney ratios of (67)Ga-NOTA-MI-Fab were 4 times higher (6 h postinjection) than those of (67)Ga-NOTA-Fab. Although further studies including the structure of radiometabolites and

  16. Cellular Models of Aggregation-dependent Template-directed Proteolysis to Characterize Tau Aggregation Inhibitors for Treatment of Alzheimer Disease.

    Science.gov (United States)

    Harrington, Charles R; Storey, John M D; Clunas, Scott; Harrington, Kathleen A; Horsley, David; Ishaq, Ahtsham; Kemp, Steven J; Larch, Christopher P; Marshall, Colin; Nicoll, Sarah L; Rickard, Janet E; Simpson, Michael; Sinclair, James P; Storey, Lynda J; Wischik, Claude M

    2015-04-24

    Alzheimer disease (AD) is a degenerative tauopathy characterized by aggregation of Tau protein through the repeat domain to form intraneuronal paired helical filaments (PHFs). We report two cell models in which we control the inherent toxicity of the core Tau fragment. These models demonstrate the properties of prion-like recruitment of full-length Tau into an aggregation pathway in which template-directed, endogenous truncation propagates aggregation through the core Tau binding domain. We use these in combination with dissolution of native PHFs to quantify the activity of Tau aggregation inhibitors (TAIs). We report the synthesis of novel stable crystalline leucomethylthioninium salts (LMTX®), which overcome the pharmacokinetic limitations of methylthioninium chloride. LMTX®, as either a dihydromesylate or a dihydrobromide salt, retains TAI activity in vitro and disrupts PHFs isolated from AD brain tissues at 0.16 μM. The Ki value for intracellular TAI activity, which we have been able to determine for the first time, is 0.12 μM. These values are close to the steady state trough brain concentration of methylthioninium ion (0.18 μM) that is required to arrest progression of AD on clinical and imaging end points and the minimum brain concentration (0.13 μM) required to reverse behavioral deficits and pathology in Tau transgenic mice. © 2015 by The American Society for Biochemistry and Molecular Biology, Inc.

  17. Effect of germination temperatures on proteolysis of the gluten-free grains sorghum and millet during malting and mashing.

    Science.gov (United States)

    Chiba, Y; Bryce, J H; Goodfellow, V; MacKinlay, J; Agu, R C; Brosnan, J M; Bringhurst, T A; Harrison, B

    2012-04-11

    Our study showed that sorghum and millet followed a similar pattern of changes when they were malted under similar conditions. When the malt from these cereals was mashed, both cereal types produced wide spectra of substrates (sugars and amino acids) that are required for yeast fermentation when malted at either lower or higher temperatures. At the germination temperatures of 20, 25, and 30 °C used in malting both cereal types, production of reducing sugars and that of free amino nitrogen (FAN) were similar. This is an important quality attribute for both cereals because it implies that variation in temperature during the malting of sorghum and millet, especially when malting temperature is difficult to control, and also reflecting temperature variations, experienced in different countries, will not have an adverse effect on the production and release of amino acids and sugars required by yeast during fermentation. Such consistency in the availability of yeast food (substrates) for metabolism during fermentation when sorghum and millet are malted at various temperatures is likely to reduce processing issues when their malts are used for brewing. Although sorghum has gained wide application in the brewing industry, and has been used extensively in brewing gluten-free beer on industrial scale, this is not the case with millet. The work described here provides novel information regarding the potential of millet for brewing. When both cereals were malted, the results obtained for millet in this study followed patterns similar to those of sorghum. This suggests that millet, in terms of sugars and amino acids, can play a role similar to that of sorghum in the brewing industry. This further suggests that millet, like sorghum, would be a good raw material for brewing gluten-free beer. Inclusion of millet as a brewing raw material will increase the availability of suitable materials (raw material sustainability) for use in the production of gluten-free beer, beverages, and

  18. Oxidative and proteolysis-related parameters of skeletal muscle from hamsters with experimental pulmonary emphysema: a comparison between papain and elastase induction.

    Science.gov (United States)

    Brunnquell, Cláudia R; Vieira, Nichelle A; Sábio, Laís R; Sczepanski, Felipe; Cecchini, Alessandra L; Cecchini, Rubens; Guarnier, Flávia A

    2015-06-01

    The objective of this study was to investigate whether emphysema induced by elastase or papain triggers the same effects on skeletal muscle, related to oxidative stress and proteolysis, in hamsters. For this purpose, we evaluated pulmonary lesions, body weight, muscle loss, oxidative stress (thiobarbituric acid-reactive substances, total and oxidized glutathiones, chemiluminescence stimulated by tert-butyl hydroperoxide and carbonyl proteins), chymotrypsin-like and calpain-like proteolytic activities and muscle fibre cross-sectional area in the gastrocnemius muscles of emphysemic hamsters. Two groups of animals received different intratracheal inductions of experimental emphysema: by 40 mg/ml papain (EP) or 5.2 IU/100 g animal (EE) elastase (n = 10 animals/group). The control group received intratracheal instillation of 300 μl sterile NaCl 0.9%. Compared with the control group, the EP group had reduced muscle weight (18.34%) and the EE group had increased muscle weight (8.37%). Additionally, tert-butyl hydroperoxide-initiated chemiluminescence, carbonylated proteins and chymotrypsin-like proteolytic activity were all elevated in the EP group compared to the CS group, while total glutathione was decreased compared to the EE group. The EE group showed more fibres with increased cross-sectional areas and increased calpain-like activity. Together, these data show that elastase and papain, when used to induce experimental models of emphysema, lead to different speeds and types of adaptation. These findings provide more information on choosing a suitable experimental model for studying skeletal muscle adaptations in emphysema. © 2015 The Authors. International Journal of Experimental Pathology © 2015 International Journal of Experimental Pathology.

  19. Effects of FlAsH/Tetracysteine (TC) tag on PrP proteolysis and PrPres formation by TC-scanning

    Science.gov (United States)

    Taguchi, Yuzuru; Hohsfield, Lindsay A.; Hollister, Jason R.

    2014-01-01

    The FlAsH/tetracysteine (FlAsH/TC) tag is a powerful tool for fluorescent labeling of proteins. However, even small tags such as FlAsH/TC could alter the behavior of the tagged proteins, especially if the insertion occurs at internal sites. Defining the influence of FlAsH/TC on nearby protein-protein interactions might aid in selecting appropriate positions for internal TC insertions and allow the exploitation of serial FlAsH/TC insertions (TC-scanning) as a probe to characterize sites of protein-protein interaction. To explore this application in the context of substrate-protease interactions, we analyzed the effect of FlAsH/TC insertions on proteolysis of cellular prion protein (PrPsen) in in vitro reactions and generation of the C1 metabolic fragment of PrPsen in live neuroblastoma cells. The influence of FlAsH/TC insertion was evaluated by TC-scanning across the cleavage sites of each protease. The results showed that FlAsH/TC inhibited protease cleavage only within limited ranges of the cleavage sites that varied from about 1 to 6 residues-wide depending on the protease, providing an estimate of the PrP residues interacting with each protease. TC-scanning was also used to probe a different type of protein-protein interaction, the conformational conversion of FlAsH-PrPsen to the prion disease-associated isoform, PrPres. PrP constructs with FlAsH/TC insertions at residues 90–96 but not 97–101 were converted to FlAsH-PrPres, identifying a boundary separating loosely versus compactly folded regions of PrPres. Our observations demonstrate that TC-scanning with the FlAsH/TC tag can be a versatile method for probing protein-protein interactions and folding processes. PMID:23943295

  20. Novel BET protein proteolysis-targeting chimera exerts superior lethal activity than bromodomain inhibitor (BETi) against post-myeloproliferative neoplasm secondary (s) AML cells.

    Science.gov (United States)

    Saenz, D T; Fiskus, W; Qian, Y; Manshouri, T; Rajapakshe, K; Raina, K; Coleman, K G; Crew, A P; Shen, A; Mill, C P; Sun, B; Qiu, P; Kadia, T M; Pemmaraju, N; DiNardo, C; Kim, M-S; Nowak, A J; Coarfa, C; Crews, C M; Verstovsek, S; Bhalla, K N

    2017-09-01

    The PROTAC (proteolysis-targeting chimera) ARV-825 recruits bromodomain and extraterminal (BET) proteins to the E3 ubiquitin ligase cereblon, leading to degradation of BET proteins, including BRD4. Although the BET-protein inhibitor (BETi) OTX015 caused accumulation of BRD4, treatment with equimolar concentrations of ARV-825 caused sustained and profound depletion (>90%) of BRD4 and induced significantly more apoptosis in cultured and patient-derived (PD) CD34+ post-MPN sAML cells, while relatively sparing the CD34+ normal hematopoietic progenitor cells. RNA-Seq, Reverse Phase Protein Array and mass cytometry 'CyTOF' analyses demonstrated that ARV-825 caused greater perturbations in messenger RNA (mRNA) and protein expressions than OTX015 in sAML cells. Specifically, compared with OTX015, ARV-825 treatment caused more robust and sustained depletion of c-Myc, CDK4/6, JAK2, p-STAT3/5, PIM1 and Bcl-xL, while increasing the levels of p21 and p27. Compared with OTX015, PROTAC ARV-771 treatment caused greater reduction in leukemia burden and further improved survival of NSG mice engrafted with luciferase-expressing HEL92.1.7 cells. Co-treatment with ARV-825 and JAK inhibitor ruxolitinib was synergistically lethal against established and PD CD34+ sAML cells. Notably, ARV-825 induced high levels of apoptosis in the in vitro generated ruxolitinib-persister or ruxolitinib-resistant sAML cells. These findings strongly support the in vivo testing of the BRD4-PROTAC based combinations against post-MPN sAML.

  1. Calpain-mediated proteolysis of polycystin-1 C-terminus induces JAK2 and ERK signal alterations

    Energy Technology Data Exchange (ETDEWEB)

    Kim, Hyunho [Transplantation Research Institute, Seoul National University Medical Research Center, Seoul (Korea, Republic of); Department of Medicine, University of Maryland, Baltimore, MD (United States); Kang, Ah-Young [Transplantation Research Institute, Seoul National University Medical Research Center, Seoul (Korea, Republic of); Department of Medicine, Program of Immunology, Graduate School, Seoul National University, Seoul (Korea, Republic of); Ko, Ah-ra [Clinical Research Center, Samsung Biomedical Research Institute, Seoul (Korea, Republic of); Park, Hayne Cho [Transplantation Research Institute, Seoul National University Medical Research Center, Seoul (Korea, Republic of); Research Coordination Center for Rare Diseases, Seoul National University Hospital, Seoul (Korea, Republic of); So, Insuk [Department of Physiology, Seoul National University College of Medicine, Seoul (Korea, Republic of); Park, Jong Hoon [Department of Biological Science, Sookmyung Women’s University, Seoul (Korea, Republic of); Cheong, Hae Il [Research Coordination Center for Rare Diseases, Seoul National University Hospital, Seoul (Korea, Republic of); Department of Pediatrics, Seoul National University Children’s Hospital, Seoul (Korea, Republic of); Kidney Research Institute, Medical Research Center, Seoul National University College of Medicine, Seoul (Korea, Republic of); Hwang, Young-Hwan [Research Coordination Center for Rare Diseases, Seoul National University Hospital, Seoul (Korea, Republic of); Department of Internal Medicine, Eulji General Hospital, Eulji University College of Medicine, Seoul (Korea, Republic of); and others

    2014-01-01

    Autosomal dominant polycystic kidney disease (ADPKD), a hereditary renal disease caused by mutations in PKD1 (85%) or PKD2 (15%), is characterized by the development of gradually enlarging multiple renal cysts and progressive renal failure. Polycystin-1 (PC1), PKD1 gene product, is an integral membrane glycoprotein which regulates a number of different biological processes including cell proliferation, apoptosis, cell polarity, and tubulogenesis. PC1 is a target of various proteolytic cleavages and proteosomal degradations, but its role in intracellular signaling pathways remains poorly understood. Herein, we demonstrated that PC1 is a novel substrate for μ- and m-calpains, which are calcium-dependent cysteine proteases. Overexpression of PC1 altered both Janus-activated kinase 2 (JAK2) and extracellular signal-regulated kinase (ERK) signals, which were independently regulated by calpain-mediated PC1 degradation. They suggest that the PC1 function on JAK2 and ERK signaling pathways might be regulated by calpains in response to the changes in intracellular calcium concentration. - Highlights: • Polycystin-1 is a target of ubiquitin-independent degradation by calpains. • The PEST domain is required for calpain-mediated degradation of polycystin-1. • Polycystin-1 may independently regulate JAK2 and ERK signaling pathways.

  2. CYLD Proteolysis Protects Macrophages from TNF-Mediated Auto-necroptosis Induced by LPS and Licensed by Type I IFN

    Directory of Open Access Journals (Sweden)

    Diana Legarda

    2016-06-01

    Full Text Available Tumor necrosis factor (TNF induces necroptosis, a RIPK3/MLKL-dependent form of inflammatory cell death. In response to infection by Gram-negative bacteria, multiple receptors on macrophages, including TLR4, TNF, and type I IFN receptors, are concurrently activated, but it is unclear how they crosstalk to regulate necroptosis. We report that TLR4 activates CASPASE-8 to cleave and remove the deubiquitinase cylindromatosis (CYLD in a TRIF- and RIPK1-dependent manner to disable necroptosis in macrophages. Inhibiting CASPASE-8 leads to CYLD-dependent necroptosis caused by the TNF produced in response to TLR4 ligation. While lipopolysaccharides (LPS-induced necroptosis was abrogated in Tnf−/− macrophages, a soluble TNF antagonist was not able to do so in Tnf+/+ macrophages, indicating that necroptosis occurs in a cell-autonomous manner. Surprisingly, TNF-mediated auto-necroptosis of macrophages requires type I IFN, which primes the expression of key necroptosis-signaling molecules, including TNFR2 and MLKL. Thus, the TNF necroptosis pathway is regulated by both negative and positive crosstalk.

  3. L-lysine escinat, thiotriazolin, gordox and mydocalm influence on oxygen tension in the intestinal wall and acid-base balance and limited proteolysis in intestinal venous blood in terms of intraabdominal hypertension modeling

    Directory of Open Access Journals (Sweden)

    Sapegin V.I.

    2014-11-01

    Full Text Available In acute experiments on rabbits there were studied changes in oxygen tension in the intestinal wall tissues, acid-base balance and limited proteolysis and its inhibitors in intestinal venous blood, protective action of L-lysine escinat (0,15 mg/kg / single dose, thiotriazolin (25 mg/kg / single dose, aprotinin (gordox (10,000 units/kg / single dose in sequential modeling of standard levels increasing of intra-abdominal hypertension (IAH — 50, 100, 150, 200, 250, 300, 350 m H2O, and also of tolperison (mydocalm (5 mg/kg / single dose on modeling of stable 3-hour IAH 200 m H2O. The IAH modeling was performed by means of stand of our construction. Under the influence of IAH the compensated metabolic acidosis in intestinal venous blood with a compensative hyperpnoe develops, decline of oxygen tension in tissues and activating of a limited proteolysis as well as decline of its inhibitors activity in intestinal venous blood occur. By the degree of metabolic acidosis prevention investigational preparations were distributed as follows gordox > thiotriazolin = L-lysine escinat = mydocalm, and by prevention of decline of oxygen tension in tissues — thiotriazolin > L-lysine escinat > mydocalm > gordox, it is is connected with different rate of methabolic products excretion into the blood, due to the influence on blood circulation and transcapilary exchange. By the degree of prevention of proteolytic activity and inhibitory potential changes, investigational preparations were distributed as follows: gordox > mydocalm > thiotriazolin > L-lysine escinat, this is connected with inhibition of proteolysis in gordox, and in other ones – with reduction of ischemic damage of tissues. Owing to different mechanism of action thiotriazolin, L-lysine escinat and mydocalm may be simultaneously recommended for a conservative treatment of patients with intraabdominal hypertension syndrome.

  4. Differences in heavy-ion-induced DNA double-strand breaks in a mouse DNA repair-deficient mutant cell line (SL3-147) before and after chromatin proteolysis

    International Nuclear Information System (INIS)

    Murakami, Masahiro; Eguchi-Kasai, Kiyomi; Sato, Koki; Minohara, Shinichi; Kanai, Tatsuaki; Yatagai, Fumio.

    1995-01-01

    DNA double-strand breaks induced by X- or neon beam-irradiation in a DNA double-strand break-repair-deficient mutant cell line (SL3-147) were examined. The increase in the number of DNA double-strand breaks was dose-depend after irradiation with X-rays and neon beams and was enhanced by chromatin-proteolysis treatment before irradiation. These results suggest that the induction of DNA double-strand breaks by ionizing radiation, including heavy-ions, is influenced by the chromatin structure. (author)

  5. Proteolysis breaks tolerance toward intact α345(IV) collagen, eliciting novel anti-GBM autoantibodies specific for α345NC1 hexamers

    Science.gov (United States)

    Olaru, Florina; Wang, Xu-Ping; Luo, Wentian; Ge, Linna; Miner, Jeffrey H; Kleinau, Sandra; Geiger, Xochiquetzal J.; Wasiluk, Andrew; Heidet, Laurence; Kitching, A. Richard; Borza, Dorin-Bogdan

    2012-01-01

    Goodpasture disease is an autoimmune kidney disease mediated by autoAbs against NC1 monomers of α3(IV) collagen that bind to the glomerular basement membrane (GBM), usually causing rapidly progressive glomerulonephritis. We identified a novel type of human IgG4-restricted anti-GBM autoAbs associated with mild non-progressive glomerulonephritis, which specifically targeted α345NC1 hexamers but not α3NC1 monomers. The mechanisms eliciting these anti-GBM autoAbs were investigated in mouse models recapitulating this phenotype. Wild type and FcγRIIB−/− mice immunized with autologous murine GBM NC1 hexamers produced mouse IgG1-restricted autoAbs specific for α345NC1 hexamers, which bound to the GBM in vivo but did not cause glomerulonephritis. In these mice, intact collagen IV from murine GBM was not immunogenic. However, in Col4a3−/− Alport mice, both intact collagen IV and NC1 hexamers from murine GBM elicited IgG antibodies specific for α3α4α5NC1 hexamers, which were not subclass restricted. As heterologous antigen in COL4A3-humanized mice, murine GBM NC1 hexamers elicited mouse IgG1, IgG2a and IgG2b autoAbs specific for α345NC1 hexamers and induced anti-GBM Ab glomerulonephritis. These findings indicate that tolerance toward autologous intact α3α4α5(IV) collagen is established in hosts expressing this antigen, even though autoreactive B cells specific for α345NC1 hexamers are not purged from their repertoire. Proteolysis selectively breaches this tolerance by generating autoimmunogenic α3α4α5NC1 hexamers. This provides a mechanism eliciting autoAbs specific for α345NC1 hexamers, which are restricted to non-inflammatory IgG subclasses and non-nephritogenic. In Alport syndrome, lack of tolerance toward α3α4α5(IV) collagen promotes production of alloantibodies to α345NC1 hexamers, including pro-inflammatory IgG subclasses which mediate post-transplant anti-GBM nephritis. PMID:23303673

  6. Where do the immunostimulatory effects of oral proteolytic enzymes ('systemic enzyme therapy') come from? Microbial proteolysis as a possible starting point.

    Science.gov (United States)

    Biziulevicius, Gediminas A

    2006-01-01

    Enteric-coated proteolytic enzyme preparations like Wobenzym and Phlogenzym are widely used for the so-called 'systemic enzyme therapy' both in humans and animals. Numerous publications reveal that oral proteolytic enzymes are able to stimulate directly the activity of immune competent cells as well as to increase efficiency of some of their products. But origins of the immunostimulatory effects of oral proteolytic enzymes are still unclear. The hypothesis described here suggests that it may be proteolysis of intestinal microorganisms that makes the immune competent cells to work in the immunostimulatory manner. The hypothesis was largely formed by several scientific observations: First, microbial lysis products (lipopolysaccharides, muropeptides and other peptidoglycan fragments, beta-glucans, etc.) are well known for their immunostimulatory action. Second, a normal human being hosts a mass of intestinal microorganisms equivalent to about 1 kg. The biomass (mainly due to naturally occurring autolysis) continuously supplies the host's organism with immunostimulatory microbial cell components. Third, the immunostimulatory effects resulting from the oral application of exogenously acting antimicrobial (lytic) enzyme preparations, such as lysozyme and lysosubtilin, are likely to be a result of the action of microbial lysis products. Fourth, cell walls of most microorganisms contain a considerable amount of proteins/peptides, a possible target for exogenous proteolytic enzymes. In fact, several authors have already shown that a number of proteases possess an ability to lyse the microbial cells in vitro. Fifth, the pretreatment of microbial cells (at least of some species) in vitro with proteolytic enzymes makes them more sensitive to the lytic action of lysozyme and, otherwise, pretreatment with lysozyme makes them more susceptible to proteolytic degradation. Sixth, exogenous proteases, when in the intestines, may participate in final steps of food-protein digestion

  7. Software requirements

    CERN Document Server

    Wiegers, Karl E

    2003-01-01

    Without formal, verifiable software requirements-and an effective system for managing them-the programs that developers think they've agreed to build often will not be the same products their customers are expecting. In SOFTWARE REQUIREMENTS, Second Edition, requirements engineering authority Karl Wiegers amplifies the best practices presented in his original award-winning text?now a mainstay for anyone participating in the software development process. In this book, you'll discover effective techniques for managing the requirements engineering process all the way through the development cy

  8. Assessment of proteolysis and sensory characteristics of prato cheese with adjunct cultureAvaliação da proteólise e das características sensoriais de queijo prato com cultura adjunta

    Directory of Open Access Journals (Sweden)

    Priscila Cristina Bizam Vianna

    2012-02-01

    Full Text Available Influence of adjunct cultures on the chemical and sensory characteristics, and proteolysis of Prato cheese was investigated. Cheeses were manufactured using a commercial starter culture and Lactobacillus strains (Lactobacillus plantarum or Lactobacillus helveticus as adjunct cultures. Control cheeses lacked the adjunct culture. The chemical composition was analyzed at day 5 after manufacture and the proteolysis at days 5, 25, 45 and 65 of ripening. The sensory acceptance was assessed at 60 days. A split-plot design was used and the complete experiment was carried out in triplicate. The results were evaluated by ANOVA and Tukey’s test test at 5% significance level. There were no significant differences in chemical composition among the cheeses. A significant increase in proteolysis occurred during ripening period for the cheeses with adjunct culture when compared to cheeses without adjunct culture. Cheese with Lactobacillus helveticus showed higher scores for flavor, texture and purchase intent compared with the others treatments. Use of adjunct Lactobacillus suggests that the proteolysis of Prato cheese should be accelerated in order to reduce ripening period. A influência de culturas adjuntas sobre as características químicas e sensoriais, e sobre a proteólise do queijo Prato foi avaliada. Os queijos foram fabricados com cultura starter comercial e cepas de Lactobacillus (Lactobacillus plantarum ou Lactobacillus helveticus como culturas adjuntas. Os queijos controle não foram adicionados de cultura adjunta. A composição química foi analisada no dia 5 após a fabricação e a proteólise nos dias 5, 25, 45 e 65 de maturação. A aceitação sensorial foi avaliada em 60 dias. Um delineamento de parcelas subdivididas foi utilizado e o experimento completo foi realizado em triplicata. Os resultados foram avaliados pela análise de variância e teste de Tukey no nível de 5% de probabilidade. Os queijos não apresentaram diferen

  9. Closure requirements

    International Nuclear Information System (INIS)

    Hutchinson, I.P.G.; Ellison, R.D.

    1992-01-01

    Closure of a waste management unit can be either permanent or temporary. Permanent closure may be due to: economic factors which make it uneconomical to mine the remaining minerals; depletion of mineral resources; physical site constraints that preclude further mining and beneficiation; environmental, regulatory or other requirements that make it uneconomical to continue to develop the resources. Temporary closure can occur for a period of several months to several years, and may be caused by factors such as: periods of high rainfall or snowfall which prevent mining and waste disposal; economic circumstances which temporarily make it uneconomical to mine the target mineral; labor problems requiring a cessation of operations for a period of time; construction activities that are required to upgrade project components such as the process facilities and waste management units; and mine or process plant failures that require extensive repairs. Permanent closure of a mine waste management unit involves the provision of durable surface containment features to protect the waters of the State in the long-term. Temporary closure may involve activities that range from ongoing maintenance of the existing facilities to the installation of several permanent closure features in order to reduce ongoing maintenance. This paper deals with the permanent closure features

  10. Protein Requirements and Recommendations for Older People: A Review

    Directory of Open Access Journals (Sweden)

    Caryl Nowson

    2015-08-01

    Full Text Available Declines in skeletal muscle mass and strength are major contributors to increased mortality, morbidity and reduced quality of life in older people. Recommended Dietary Allowances/Intakes have failed to adequately consider the protein requirements of the elderly with respect to function. The aim of this paper was to review definitions of optimal protein status and the evidence base for optimal dietary protein. Current recommended protein intakes for older people do not account for the compensatory loss of muscle mass that occurs on lower protein intakes. Older people have lower rates of protein synthesis and whole-body proteolysis in response to an anabolic stimulus (food or resistance exercise. Recommendations for the level of adequate dietary intake of protein for older people should be informed by evidence derived from functional outcomes. Randomized controlled trials report a clear benefit of increased dietary protein on lean mass gain and leg strength, particularly when combined with resistance exercise. There is good consistent evidence (level III-2 to IV that consumption of 1.0 to 1.3 g/kg/day dietary protein combined with twice-weekly progressive resistance exercise reduces age-related muscle mass loss. Older people appear to require 1.0 to 1.3 g/kg/day dietary protein to optimize physical function, particularly whilst undertaking resistance exercise recommendations.

  11. Defective lysosomal proteolysis and axonal transport are early pathogenic events that worsen with age leading to increased APP metabolism and synaptic Abeta in transgenic APP/PS1 hippocampus.

    Science.gov (United States)

    Torres, Manuel; Jimenez, Sebastian; Sanchez-Varo, Raquel; Navarro, Victoria; Trujillo-Estrada, Laura; Sanchez-Mejias, Elisabeth; Carmona, Irene; Davila, Jose Carlos; Vizuete, Marisa; Gutierrez, Antonia; Vitorica, Javier

    2012-11-22

    Axonal pathology might constitute one of the earliest manifestations of Alzheimer disease. Axonal dystrophies were observed in Alzheimer's patients and transgenic models at early ages. These axonal dystrophies could reflect the disruption of axonal transport and the accumulation of multiple vesicles at local points. It has been also proposed that dystrophies might interfere with normal intracellular proteolysis. In this work, we have investigated the progression of the hippocampal pathology and the possible implication in Abeta production in young (6 months) and aged (18 months) PS1(M146L)/APP(751sl) transgenic mice. Our data demonstrated the existence of a progressive, age-dependent, formation of axonal dystrophies, mainly located in contact with congophilic Abeta deposition, which exhibited tau and neurofilament hyperphosphorylation. This progressive pathology was paralleled with decreased expression of the motor proteins kinesin and dynein. Furthermore, we also observed an early decrease in the activity of cathepsins B and D, progressing to a deep inhibition of these lysosomal proteases at late ages. This lysosomal impairment could be responsible for the accumulation of LC3-II and ubiquitinated proteins within axonal dystrophies. We have also investigated the repercussion of these deficiencies on the APP metabolism. Our data demonstrated the existence of an increase in the amyloidogenic pathway, which was reflected by the accumulation of hAPPfl, C99 fragment, intracellular Abeta in parallel with an increase in BACE and gamma-secretase activities. In vitro experiments, using APPswe transfected N2a cells, demonstrated that any imbalance on the proteolytic systems reproduced the in vivo alterations in APP metabolism. Finally, our data also demonstrated that Abeta peptides were preferentially accumulated in isolated synaptosomes. A progressive age-dependent cytoskeletal pathology along with a reduction of lysosomal and, in minor extent, proteasomal activity could be

  12. Feed tank transfer requirements

    International Nuclear Information System (INIS)

    Freeman-Pollard, J.R.

    1998-01-01

    This document presents a definition of tank turnover; DOE responsibilities; TWRS DST permitting requirements; TWRS Authorization Basis (AB) requirements; TWRS AP Tank Farm operational requirements; unreviewed safety question (USQ) requirements; records and reporting requirements, and documentation which will require revision in support of transferring a DST in AP Tank Farm to a privatization contractor for use during Phase 1B

  13. Regulated Proteolysis of Arabidopsis Argonaute1

    DEFF Research Database (Denmark)

    Kausika, Swathi Pranavi

    . These are large multi domain proteins found in both eukaryotes and prokaryotes. They consist of four domains namely N, PAZ, MID and PIWI domain. The PAZ and MID domains bind small RNA 3´ and 5´ends while the PIWI domain harbors the endonucleolytic activity responsible for target RNA cleavage. This study focuses...

  14. Feed tank transfer requirements

    International Nuclear Information System (INIS)

    Freeman-Pollard, J.R.

    1998-01-01

    This document presents a definition of tank turnover. Also, DOE and PC responsibilities; TWRS DST permitting requirements; TWRS Authorization Basis (AB) requirements; TWRS AP Tank Farm operational requirements; unreviewed safety question (USQ) requirements are presented for two cases (i.e., tank modifications occurring before tank turnover and tank modification occurring after tank turnover). Finally, records and reporting requirements, and documentation which will require revision in support of transferring a DST in AP Tank Farm to a privatization contractor are presented

  15. Feed tank transfer requirements

    Energy Technology Data Exchange (ETDEWEB)

    Freeman-Pollard, J.R.

    1998-09-16

    This document presents a definition of tank turnover. Also, DOE and PC responsibilities; TWRS DST permitting requirements; TWRS Authorization Basis (AB) requirements; TWRS AP Tank Farm operational requirements; unreviewed safety question (USQ) requirements are presented for two cases (i.e., tank modifications occurring before tank turnover and tank modification occurring after tank turnover). Finally, records and reporting requirements, and documentation which will require revision in support of transferring a DST in AP Tank Farm to a privatization contractor are presented.

  16. N-Glycosylation of Lipocalin 2 Is Not Required for Secretion or Exosome Targeting

    Directory of Open Access Journals (Sweden)

    Erawan Borkham-Kamphorst

    2018-04-01

    Full Text Available Lipocalin 2 (LCN2 is a highly conserved secreted adipokine acting as a serum transport protein for small hydrophobic molecules such as fatty acids and steroids. In addition, LCN2 limits bacterial growth by sequestering iron-containing siderophores and further protects against intestinal inflammation and tumorigenesis associated with alterations in the microbiota. Human LCN2 contains one N-glycosylation site conserved in other species. It was postulated that this post-translational modification could facilitate protein folding, protects from proteolysis, is required for proper trafficking from the Golgi apparatus to the cell surface, and might be relevant for effective secretion. We here show that the homologous nucleoside antibiotic tunicamycin blocks N-linked glycosylation but not secretion of LCN2 in primary murine hepatocytes, derivatives thereof, human lung carcinoma cell line A549, and human prostate cancer cell line PC-3. Moreover, both the glycosylated and the non-glycosylated LCN2 variants are equally targeted to exosomes, demonstrating that this post-translational modification is not necessary for proper trafficking of LCN2 into these membranous extracellular vesicles. Furthermore, a hydrophobic cluster analysis revealed that the N-glycosylation site is embedded in a highly hydrophobic evolutionarily conserved surrounding. In sum, our data indicate that the N-glycosylation of LCN2 is not required for proper secretion and exosome cargo recruitment in different cell types, but might be relevant to increase overall solubility.

  17. Efeito do uso de cultura adjunta (Lactobacillus helveticus na proteólise, propriedades viscoelásticas e aceitação sensorial de queijo prato light Effect of adjunct culture (Lactobacillus helveticus on proteolysis, viscoelastic properties and sensory acceptance of reduced fat prato cheese

    Directory of Open Access Journals (Sweden)

    Christiane Maciel V. Barros

    2006-03-01

    Full Text Available A proteólise, as propriedades viscoelásticas e a aceitação sensorial de queijo prato light fabricado com e sem adição de cultura adjunta (CAD foram avaliadas. Os queijos foram fabricados a partir de leite microfiltrado. Dois tratamentos foram testados em duplicata: o queijo controle foi fabricado apenas com cultura mesófila tradicional (acidificante e aromatizante, e o outro foi fabricado com adição de CAD (Lactobacillus helveticus, além da cultura tradicional. A composição dos queijos foi determinada no quinto dia após a fabricação. A proteólise e as propriedades reológicas foram avaliadas nos dias 5, 25 e 45 após a fabricação. Os parâmetros viscoelásticos foram obtidos a partir de testes de relaxação. As amostras foram avaliadas sensorialmente por meio de testes de aceitação. Não houve diferença significativa (p>0,05 na composição dos queijos. Os índices de profundidade de proteólise foram significativamente (p0,05. Nos testes de aceitação sensorial, o queijo produzido com CAD obteve notas significativamente (pProteolysis, viscoelastic properties and sensory acceptance of reduced fat Prato cheeses made with and without adjunt culture (AC were evaluated. The cheeses were made from microfiltered milk. Two different treatments were replicated twice: control cheese was made only with traditional starter, while the other was made with the addition of both AC (Lactobacillus helveticus and traditional starter. Cheese composition was determined after 5 days of manufacture. Proteolysis and rheological properties were evaluated after 5, 25 and 45 days. Viscoelastic parameters were obtained using relaxation tests. Cheese sensory properties were evaluated using acceptability tests. There was no statistical difference (p>0,05 in cheese composition. The proteolysis depth indexes were significantly higher (p0.05 in viscoelastic parameters for cheeses made with and without AC. Sensory acceptability tests indicated significant

  18. Composição, proteólise, capacidade de derretimento e formação de "blisters" do queijo mussarela obtido pelos métodos tradicional e de ultrafiltração: composition, proteolysis, melting capacity and blisters formation Mozzarella by ultrafiltration

    Directory of Open Access Journals (Sweden)

    Patrícia D. Pizaia

    2003-12-01

    Full Text Available O objetivo deste trabalho foi comparar a composição, a proteólise, a capacidade de derretimento e a formação de "blisters" (bolhas em queijos tipo Mussarela fabricados com retentado de leite (MR de fator de concentração volumétrica (FCV de 2,34:1, com um queijo Mussarela padrão (MP fabricado com leite não ultrafiltrado. Foi realizado um ensaio de produção com 3 lotes de MR e um lote de MP. Determinou-se a composição do leite, retentado, soro, água de filagem e queijos e a proteólise, a capacidade de derretimento e a formação de "blisters" nos queijos com 7, 15, 30 e 60 dias de armazenamento refrigerado. MRs apresentaram maiores valores de pH e de porcentagem de cinzas e de proteína total e menores porcentagens de acidez titulável, gordura, gordura no extrato seco e sal quando comparadas a MP. Durante o tempo de estocagem, as MRs apresentaram menor proteólise e capacidade de derretimento, em todas as datas analisadas. A porcentagem de área coberta por 'blisters" na pizza e o diâmetro médio dos mesmos foram maiores na MP durante o primeiro mês de estocagem e depois ambos os tipos de queijos apresentaram comportamentos similares para estes 2 parâmetros.The objective of this research was to compare the composition, proteolysis, melting capacity and blisters formation in Mozzarella cheese manufactured with milk retentate (MR of a volumetric concentration factor (FCV of 2.34:1, with a standard Mozzarella cheese (MP manufactured with non ultrafiltrated milk. It was realized one production assay with 3 batches of MRs and one of MP. It was evaluated the milk, retentate, whey, stretching water and cheeses composition and the proteolysis, melting capacity and the blisters formation on cheeses with 7, 15, 30 and 60 days of refrigerates storage. MRs presented larger pH, ash and total protein contents and lower titratable acidity and fat, fat on dry matter and salt contents when compared to MP. Along the storage time the MRs

  19. Assessing Requirements Quality through Requirements Coverage

    Science.gov (United States)

    Rajan, Ajitha; Heimdahl, Mats; Woodham, Kurt

    2008-01-01

    In model-based development, the development effort is centered around a formal description of the proposed software system the model. This model is derived from some high-level requirements describing the expected behavior of the software. For validation and verification purposes, this model can then be subjected to various types of analysis, for example, completeness and consistency analysis [6], model checking [3], theorem proving [1], and test-case generation [4, 7]. This development paradigm is making rapid inroads in certain industries, e.g., automotive, avionics, space applications, and medical technology. This shift towards model-based development naturally leads to changes in the verification and validation (V&V) process. The model validation problem determining that the model accurately captures the customer's high-level requirements has received little attention and the sufficiency of the validation activities has been largely determined through ad-hoc methods. Since the model serves as the central artifact, its correctness with respect to the users needs is absolutely crucial. In our investigation, we attempt to answer the following two questions with respect to validation (1) Are the requirements sufficiently defined for the system? and (2) How well does the model implement the behaviors specified by the requirements? The second question can be addressed using formal verification. Nevertheless, the size and complexity of many industrial systems make formal verification infeasible even if we have a formal model and formalized requirements. Thus, presently, there is no objective way of answering these two questions. To this end, we propose an approach based on testing that, when given a set of formal requirements, explores the relationship between requirements-based structural test-adequacy coverage and model-based structural test-adequacy coverage. The proposed technique uses requirements coverage metrics defined in [9] on formal high-level software

  20. Postmarket Requirements and Commitments

    Data.gov (United States)

    U.S. Department of Health & Human Services — Provides information to the public on postmarket requirements and commitments. The phrase postmarket requirements and commitments refers to studies and clinical...

  1. Requirements for existing buildings

    DEFF Research Database (Denmark)

    Thomsen, Kirsten Engelund; Wittchen, Kim Bjarne

    This report collects energy performance requirements for existing buildings in European member states by June 2012.......This report collects energy performance requirements for existing buildings in European member states by June 2012....

  2. Replacing reserve requirements

    OpenAIRE

    Edward J. Stevens

    1993-01-01

    An examination of the fading significance of the Federal Reserve System's reserve requirements and the recent flowering of required clearing balances, a rapidly growing feature of Reserve Bank operations.

  3. PIT Coating Requirements Analysis

    International Nuclear Information System (INIS)

    MINTEER, D.J.

    2000-01-01

    This study identifies the applicable requirements for procurement and installation of a coating intended for tank farm valve and pump pit interior surfaces. These requirements are intended to be incorporated into project specification documents and design media. This study also evaluates previously recommended coatings and identifies requirement-compliant coating products

  4. PIT Coating Requirements Analysis

    Energy Technology Data Exchange (ETDEWEB)

    MINTEER, D.J.

    2000-10-20

    This study identifies the applicable requirements for procurement and installation of a coating intended for tank farm valve and pump pit interior surfaces. These requirements are intended to be incorporated into project specification documents and design media. This study also evaluates previously recommended coatings and identifies requirement-compliant coating products.

  5. Future Home Network Requirements

    DEFF Research Database (Denmark)

    Charbonnier, Benoit; Wessing, Henrik; Lannoo, Bart

    This paper presents the requirements for future Home Area Networks (HAN). Firstly, we discuss the applications and services as well as their requirements. Then, usage scenarios are devised to establish a first specification for the HAN. The main requirements are an increased bandwidth (towards 1...

  6. User Requirements for Wireless

    DEFF Research Database (Denmark)

    in the elicitation process. Cases and user requirement elements discussed in the book include: User requirements elicitation processes for children, construction workers, and farmers User requirements for personalized services of a broadcast company Variations in user involvement Practical elements of user...

  7. TRANSPORTATION SYSTEM REQUIREMENTS DOCUMENT

    International Nuclear Information System (INIS)

    2004-01-01

    This document establishes the Transportation system requirements for the U.S. Department of Energy's (DOE's) Civilian Radioactive Waste Management System (CRWMS). These requirements are derived from the Civilian Radioactive Waste Management System Requirements Document (CRD). The Transportation System Requirements Document (TSRD) was developed in accordance with LP-3.1Q-OCRWM, Preparation, Review, and Approval of Office of National Transportation Level-2 Baseline Requirements. As illustrated in Figure 1, the TSRD forms a part of the DOE Office of Civilian Radioactive Waste Management (OCRWM) Technical Baseline

  8. Transportation System Requirements Document

    International Nuclear Information System (INIS)

    1993-09-01

    This Transportation System Requirements Document (Trans-SRD) describes the functions to be performed by and the technical requirements for the Transportation System to transport spent nuclear fuel (SNF) and high-level radioactive waste (HLW) from Purchaser and Producer sites to a Civilian Radioactive Waste Management System (CRWMS) site, and between CRWMS sites. The purpose of this document is to define the system-level requirements for Transportation consistent with the CRWMS Requirement Document (CRD). These requirements include design and operations requirements to the extent they impact on the development of the physical segments of Transportation. The document also presents an overall description of Transportation, its functions, its segments, and the requirements allocated to the segments and the system-level interfaces with Transportation. The interface identification and description are published in the CRWMS Interface Specification

  9. Environmental Requirements Management

    Energy Technology Data Exchange (ETDEWEB)

    Cusack, Laura J.; Bramson, Jeffrey E.; Archuleta, Jose A.; Frey, Jeffrey A.

    2015-01-08

    CH2M HILL Plateau Remediation Company (CH2M HILL) is the U.S. Department of Energy (DOE) prime contractor responsible for the environmental cleanup of the Hanford Site Central Plateau. As part of this responsibility, the CH2M HILL is faced with the task of complying with thousands of environmental requirements which originate from over 200 federal, state, and local laws and regulations, DOE Orders, waste management and effluent discharge permits, Comprehensive Environmental Response, Compensation, and Liability Act (CERCLA) response and Resource Conservation and Recovery Act (RCRA) corrective action documents, and official regulatory agency correspondence. The challenge is to manage this vast number of requirements to ensure they are appropriately and effectively integrated into CH2M HILL operations. Ensuring compliance with a large number of environmental requirements relies on an organization’s ability to identify, evaluate, communicate, and verify those requirements. To ensure that compliance is maintained, all changes need to be tracked. The CH2M HILL identified that the existing system used to manage environmental requirements was difficult to maintain and that improvements should be made to increase functionality. CH2M HILL established an environmental requirements management procedure and tools to assure that all environmental requirements are effectively and efficiently managed. Having a complete and accurate set of environmental requirements applicable to CH2M HILL operations will promote a more efficient approach to: • Communicating requirements • Planning work • Maintaining work controls • Maintaining compliance

  10. Requirements for Ion Sources

    International Nuclear Information System (INIS)

    Scrivens, R

    2013-01-01

    Ion sources produce beams for a large variety of different physical experiments, industrial processes and medical applications. In order to characterize the beam delivered by them, a list of requirements is necessary. In this chapter the list of principal requirements is specified and definitions for them are given. (author)

  11. Entrepreneurial learning requires action

    DEFF Research Database (Denmark)

    Brink, Tove; Madsen, Svend Ole

    2014-01-01

    that is enhanced by essential large-scale industry players and other SME managers are required to create action and value in learning. An open-mindedness to new learning approaches by SME managers and an open-mindedness to multi- and cross-disciplinary collaboration with SME managers by facilitators is required....

  12. Writing testable software requirements

    Energy Technology Data Exchange (ETDEWEB)

    Knirk, D. [Sandia National Labs., Albuquerque, NM (United States)

    1997-11-01

    This tutorial identifies common problems in analyzing requirements in the problem and constructing a written specification of what the software is to do. It deals with two main problem areas: identifying and describing problem requirements, and analyzing and describing behavior specifications.

  13. Engineering Requirements for crowds

    Directory of Open Access Journals (Sweden)

    Rogeiro Silva

    2015-12-01

    Full Text Available In the software project the interested parts are highly distributed and form numerous and heterogeneous groups, online or face, constituting what could be called crowds. The development of social applications and cloud computing and mobile has generated a marked increase in environments based requirements in crowds. Technical Requirements Engineering (RE traditional face these scalability issues, and require the co-presence of interested and engineers in joint meetings that can not be made in common physical environments. While different approaches have been introduced to partially automate RE in these contexts, still is required a multi-method approach to semi-automate all activities related to work with crowds. In this paper is propose an approach that integrates existing elicitation techniques and requirements analysis and is complemented by introducing new concepts. The information is collected through direct interaction and social collaboration, and through data mining techniques.

  14. Timely activation of budding yeast APCCdh1 involves degradation of its inhibitor, Acm1, by an unconventional proteolytic mechanism.

    Directory of Open Access Journals (Sweden)

    Michael Melesse

    Full Text Available Regulated proteolysis mediated by the ubiquitin proteasome system is a fundamental and essential feature of the eukaryotic cell division cycle. Most proteins with cell cycle-regulated stability are targeted for degradation by one of two related ubiquitin ligases, the Skp1-cullin-F box protein (SCF complex or the anaphase-promoting complex (APC. Here we describe an unconventional cell cycle-regulated proteolytic mechanism that acts on the Acm1 protein, an inhibitor of the APC activator Cdh1 in budding yeast. Although Acm1 can be recognized as a substrate by the Cdc20-activated APC (APCCdc20 in anaphase, APCCdc20 is neither necessary nor sufficient for complete Acm1 degradation at the end of mitosis. An APC-independent, but 26S proteasome-dependent, mechanism is sufficient for complete Acm1 clearance from late mitotic and G1 cells. Surprisingly, this mechanism appears distinct from the canonical ubiquitin targeting pathway, exhibiting several features of ubiquitin-independent proteasomal degradation. For example, Acm1 degradation in G1 requires neither lysine residues in Acm1 nor assembly of polyubiquitin chains. Acm1 was stabilized though by conditional inactivation of the ubiquitin activating enzyme Uba1, implying some requirement for the ubiquitin pathway, either direct or indirect. We identified an amino terminal predicted disordered region in Acm1 that contributes to its proteolysis in G1. Although ubiquitin-independent proteasome substrates have been described, Acm1 appears unique in that its sensitivity to this mechanism is strictly cell cycle-regulated via cyclin-dependent kinase (Cdk phosphorylation. As a result, Acm1 expression is limited to the cell cycle window in which Cdk is active. We provide evidence that failure to eliminate Acm1 impairs activation of APCCdh1 at mitotic exit, justifying its strict regulation by cell cycle-dependent transcription and proteolytic mechanisms. Importantly, our results reveal that strict cell

  15. Timely Activation of Budding Yeast APCCdh1 Involves Degradation of Its Inhibitor, Acm1, by an Unconventional Proteolytic Mechanism

    Science.gov (United States)

    Melesse, Michael; Choi, Eunyoung; Hall, Hana; Walsh, Michael J.; Geer, M. Ariel; Hall, Mark C.

    2014-01-01

    Regulated proteolysis mediated by the ubiquitin proteasome system is a fundamental and essential feature of the eukaryotic cell division cycle. Most proteins with cell cycle-regulated stability are targeted for degradation by one of two related ubiquitin ligases, the Skp1-cullin-F box protein (SCF) complex or the anaphase-promoting complex (APC). Here we describe an unconventional cell cycle-regulated proteolytic mechanism that acts on the Acm1 protein, an inhibitor of the APC activator Cdh1 in budding yeast. Although Acm1 can be recognized as a substrate by the Cdc20-activated APC (APCCdc20) in anaphase, APCCdc20 is neither necessary nor sufficient for complete Acm1 degradation at the end of mitosis. An APC-independent, but 26S proteasome-dependent, mechanism is sufficient for complete Acm1 clearance from late mitotic and G1 cells. Surprisingly, this mechanism appears distinct from the canonical ubiquitin targeting pathway, exhibiting several features of ubiquitin-independent proteasomal degradation. For example, Acm1 degradation in G1 requires neither lysine residues in Acm1 nor assembly of polyubiquitin chains. Acm1 was stabilized though by conditional inactivation of the ubiquitin activating enzyme Uba1, implying some requirement for the ubiquitin pathway, either direct or indirect. We identified an amino terminal predicted disordered region in Acm1 that contributes to its proteolysis in G1. Although ubiquitin-independent proteasome substrates have been described, Acm1 appears unique in that its sensitivity to this mechanism is strictly cell cycle-regulated via cyclin-dependent kinase (Cdk) phosphorylation. As a result, Acm1 expression is limited to the cell cycle window in which Cdk is active. We provide evidence that failure to eliminate Acm1 impairs activation of APCCdh1 at mitotic exit, justifying its strict regulation by cell cycle-dependent transcription and proteolytic mechanisms. Importantly, our results reveal that strict cell-cycle expression profiles

  16. Requirements in engineering projects

    CERN Document Server

    Fernandes, João M

    2016-01-01

    This book focuses on various topics related to engineering and management of requirements, in particular elicitation, negotiation, prioritisation, and documentation (whether with natural languages or with graphical models). The book provides methods and techniques that help to characterise, in a systematic manner, the requirements of the intended engineering system.  It was written with the goal of being adopted as the main text for courses on requirements engineering, or as a strong reference to the topics of requirements in courses with a broader scope. It can also be used in vocational courses, for professionals interested in the software and information systems domain.   Readers who have finished this book will be able to: - establish and plan a requirements engineering process within the development of complex engineering systems; - define and identify the types of relevant requirements in engineering projects; - choose and apply the most appropriate techniques to elicit the requirements of a giv...

  17. Creativity in Requirement Identification

    DEFF Research Database (Denmark)

    Sørensen, Lene Tolstrup; Olesen, Henning

    Traditional requirements engineering focuses mainly on analysis and elicitation. However, current trends in new system, device and software are towards involving all stakeholders in the early stages of the engineering process to define the user requirements. Creativity is here seen as a major...... keystone in this process in order to open up stakeholder's mind to new technologies, which do not yet exist. This paper dis-cusses the application of creativity in the requirements process and illustrate through cases from the MAGNET and MAGNET Beyond projects....

  18. FAA Financial Requirements

    Science.gov (United States)

    1997-06-04

    In June 1995, the FAA developed a "total requirements" estimate for the period : FY 97-FY 02 to help explain the difficulty of supporting a dynamic, growing : aviation industry under a federal budget picture which projected flat or reduced : funding ...

  19. TWRSview system requirements specification

    International Nuclear Information System (INIS)

    Caldwell, J.A.; Lee, A.K.

    1995-12-01

    This document provides the system requirements specification for the TWRSview software system. The TWRSview software system is being developed to integrate electronic data supporting the development of the TWRS technical baseline

  20. Estimating ISABELLE shielding requirements

    International Nuclear Information System (INIS)

    Stevens, A.J.; Thorndike, A.M.

    1976-01-01

    Estimates were made of the shielding thicknesses required at various points around the ISABELLE ring. Both hadron and muon requirements are considered. Radiation levels at the outside of the shield and at the BNL site boundary are kept at or below 1000 mrem per year and 5 mrem/year respectively. Muon requirements are based on the Wang formula for pion spectra, and the hadron requirements on the hadron cascade program CYLKAZ of Ranft. A muon shield thickness of 77 meters of sand is indicated outside the ring in one area, and hadron shields equivalent to from 2.7 to 5.6 meters in thickness of sand above the ring. The suggested safety allowance would increase these values to 86 meters and 4.0 to 7.2 meters respectively. There are many uncertainties in such estimates, but these last figures are considered to be rather conservative

  1. Utility requirements for fusion

    International Nuclear Information System (INIS)

    Vondrasek, R.J.

    1982-02-01

    This report describes work done and results obtained during performance of Task 1 of a study of Utility Requirements and Criteria for Fusion Options. The work consisted of developing a list of utility requirements for fusion optics containing definition of the requirements and showing their relative importance to the utility industry. The project team members developed a preliminary list which was refined by discussions and literature searches. The refined list was recast as a questionnaire which was sent to a substantial portion of the utility industry in this country. Forty-three questionnaire recipients responded including thirty-two utilities. A workshop was held to develop a revised requirements list using the survey responses as a major input. The list prepared by the workshop was further refined by a panel consisting of vice presidents of the three project team firms. The results of the study indicate that in addition to considering the cost of energy for a power plant, utilities consider twenty-three other requirements. Four of the requirements were judged to be vital to plant acceptability: Plant Capital Cost, Financial Liability, Plant Safety and Licensability

  2. Range Flight Safety Requirements

    Science.gov (United States)

    Loftin, Charles E.; Hudson, Sandra M.

    2018-01-01

    The purpose of this NASA Technical Standard is to provide the technical requirements for the NPR 8715.5, Range Flight Safety Program, in regards to protection of the public, the NASA workforce, and property as it pertains to risk analysis, Flight Safety Systems (FSS), and range flight operations. This standard is approved for use by NASA Headquarters and NASA Centers, including Component Facilities and Technical and Service Support Centers, and may be cited in contract, program, and other Agency documents as a technical requirement. This standard may also apply to the Jet Propulsion Laboratory or to other contractors, grant recipients, or parties to agreements to the extent specified or referenced in their contracts, grants, or agreements, when these organizations conduct or participate in missions that involve range flight operations as defined by NPR 8715.5.1.2.2 In this standard, all mandatory actions (i.e., requirements) are denoted by statements containing the term “shall.”1.3 TailoringTailoring of this standard for application to a specific program or project shall be formally documented as part of program or project requirements and approved by the responsible Technical Authority in accordance with NPR 8715.3, NASA General Safety Program Requirements.

  3. NP Science Network Requirements

    Energy Technology Data Exchange (ETDEWEB)

    Dart, Eli [Lawrence Berkeley National Laboratory (LBNL), Berkeley, CA (United States); Rotman, Lauren [Lawrence Berkeley National Laboratory (LBNL), Berkeley, CA (United States); Tierney, Brian [Lawrence Berkeley National Laboratory (LBNL), Berkeley, CA (United States)

    2011-08-26

    The Energy Sciences Network (ESnet) is the primary provider of network connectivity for the U.S. Department of Energy (DOE) Office of Science (SC), the single largest supporter of basic research in the physical sciences in the United States. To support SC programs, ESnet regularly updates and refreshes its understanding of the networking requirements of the instruments, facilities, scientists, and science programs it serves. This focus has helped ESnet to be a highly successful enabler of scientific discovery for over 20 years. In August 2011, ESnet and the Office of Nuclear Physics (NP), of the DOE SC, organized a workshop to characterize the networking requirements of the programs funded by NP. The requirements identified at the workshop are summarized in the Findings section, and are described in more detail in the body of the report.

  4. Quantifying requirements volatility effects

    NARCIS (Netherlands)

    Kulk, G.P.; Verhoef, C.

    2008-01-01

    In an organization operating in the bancassurance sector we identified a low-risk IT subportfolio of 84 IT projects comprising together 16,500 function points, each project varying in size and duration, for which we were able to quantify its requirements volatility. This representative portfolio

  5. Requirements for Xenon International

    Energy Technology Data Exchange (ETDEWEB)

    Hayes, James C.; Ely, James H.

    2013-09-26

    This document defines the requirements for the new Xenon International radioxenon system. The output of this project will be a Pacific Northwest National Laboratory (PNNL) developed prototype and a manufacturer-developed production prototype. The two prototypes are intended to be as close to matching as possible; this will be facilitated by overlapping development cycles and open communication between PNNL and the manufacturer.

  6. Requirements for Xenon International

    Energy Technology Data Exchange (ETDEWEB)

    Hayes, James C. [Pacific Northwest National Lab. (PNNL), Richland, WA (United States); Ely, James H. [Pacific Northwest National Lab. (PNNL), Richland, WA (United States); Haas, Derek A. [Pacific Northwest National Lab. (PNNL), Richland, WA (United States); Harper, Warren W. [Pacific Northwest National Lab. (PNNL), Richland, WA (United States); Heimbigner, Tom R. [Pacific Northwest National Lab. (PNNL), Richland, WA (United States); Hubbard, Charles W. [Pacific Northwest National Lab. (PNNL), Richland, WA (United States); Humble, Paul H. [Pacific Northwest National Lab. (PNNL), Richland, WA (United States); Madison, Jill C. [Pacific Northwest National Lab. (PNNL), Richland, WA (United States); Morris, Scott J. [Pacific Northwest National Lab. (PNNL), Richland, WA (United States); Panisko, Mark E. [Pacific Northwest National Lab. (PNNL), Richland, WA (United States); Ripplinger, Mike D. [Pacific Northwest National Lab. (PNNL), Richland, WA (United States); Stewart, Timothy L. [Pacific Northwest National Lab. (PNNL), Richland, WA (United States)

    2015-12-30

    This document defines the requirements for the new Xenon International radioxenon system. The output of this project will be a Pacific Northwest National Laboratory (PNNL) developed prototype and a manufacturer-developed production prototype. The two prototypes are intended to be as close to matching as possible; this will be facilitated by overlapping development cycles and open communication between PNNL and the manufacturer.

  7. Requirements for enrichment tools

    NARCIS (Netherlands)

    Boer, A.; Winkels, R.; Trompper, M.

    2016-01-01

    This report gives a high level overview of requirements for Enrichment tools in the Openlaws.eu project. Openlaws.eu aims to initiate a platform and develop a vision for Big Open Legal Data (BOLD): an open framework for legislation, case law, and legal literature from across Europe.

  8. Data Crosscutting Requirements Review

    Energy Technology Data Exchange (ETDEWEB)

    Kleese van Dam, Kerstin [Pacific Northwest National Lab. (PNNL), Richland, WA (United States); Shoshani, Arie [Pacific Northwest National Lab. (PNNL), Richland, WA (United States); Plata, Charity [Pacific Northwest National Lab. (PNNL), Richland, WA (United States)

    2013-04-01

    In April 2013, a diverse group of researchers from the U.S. Department of Energy (DOE) scientific community assembled to assess data requirements associated with DOE-sponsored scientific facilities and large-scale experiments. Participants in the review included facilities staff, program managers, and scientific experts from the offices of Basic Energy Sciences, Biological and Environmental Research, High Energy Physics, and Advanced Scientific Computing Research. As part of the meeting, review participants discussed key issues associated with three distinct aspects of the data challenge: 1) processing, 2) management, and 3) analysis. These discussions identified commonalities and differences among the needs of varied scientific communities. They also helped to articulate gaps between current approaches and future needs, as well as the research advances that will be required to close these gaps. Moreover, the review provided a rare opportunity for experts from across the Office of Science to learn about their collective expertise, challenges, and opportunities. The "Data Crosscutting Requirements Review" generated specific findings and recommendations for addressing large-scale data crosscutting requirements.

  9. Ongoing experiments: diagnostics requirements

    International Nuclear Information System (INIS)

    Dickerman, C.E.

    1976-01-01

    The paper reviews the fuel motion diagnostics needs for ongoing LMFBR safety experiments over approximately the next five years, with the discussion centered on TREAT. Brief comments on the direction in which clad motion diagnostics requirements are expected to develop are also presented

  10. Human Systems Integration Requirements

    Science.gov (United States)

    2009-09-01

    52 Stratofortress, C/ KC -135 Stratotanker, E-3 Sentry and contractor logistics support aircraft; as well as a substantial jet engine inventory...www.hqda.army.mil/ teo /Sutability%20Terms%20MOA%20Oct%2005.pdf q. ISO 9241-11, Ergonomic requirements for office work with visual display terminals (VDTs

  11. The molecular chaperone Hsp90 is required for cell cycle exit in Drosophila melanogaster.

    Directory of Open Access Journals (Sweden)

    Jennifer L Bandura

    Full Text Available The coordination of cell proliferation and differentiation is crucial for proper development. In particular, robust mechanisms exist to ensure that cells permanently exit the cell cycle upon terminal differentiation, and these include restraining the activities of both the E2F/DP transcription factor and Cyclin/Cdk kinases. However, the full complement of mechanisms necessary to restrain E2F/DP and Cyclin/Cdk activities in differentiating cells are not known. Here, we have performed a genetic screen in Drosophila melanogaster, designed to identify genes required for cell cycle exit. This screen utilized a PCNA-miniwhite(+ reporter that is highly E2F-responsive and results in a darker red eye color when crossed into genetic backgrounds that delay cell cycle exit. Mutation of Hsp83, the Drosophila homolog of mammalian Hsp90, results in increased E2F-dependent transcription and ectopic cell proliferation in pupal tissues at a time when neighboring wild-type cells are postmitotic. Further, these Hsp83 mutant cells have increased Cyclin/Cdk activity and accumulate proteins normally targeted for proteolysis by the anaphase-promoting complex/cyclosome (APC/C, suggesting that APC/C function is inhibited. Indeed, reducing the gene dosage of an inhibitor of Cdh1/Fzr, an activating subunit of the APC/C that is required for timely cell cycle exit, can genetically suppress the Hsp83 cell cycle exit phenotype. Based on these data, we propose that Cdh1/Fzr is a client protein of Hsp83. Our results reveal that Hsp83 plays a heretofore unappreciated role in promoting APC/C function during cell cycle exit and suggest a mechanism by which Hsp90 inhibition could promote genomic instability and carcinogenesis.

  12. The molecular chaperone Hsp90 is required for cell cycle exit in Drosophila melanogaster.

    Science.gov (United States)

    Bandura, Jennifer L; Jiang, Huaqi; Nickerson, Derek W; Edgar, Bruce A

    2013-01-01

    The coordination of cell proliferation and differentiation is crucial for proper development. In particular, robust mechanisms exist to ensure that cells permanently exit the cell cycle upon terminal differentiation, and these include restraining the activities of both the E2F/DP transcription factor and Cyclin/Cdk kinases. However, the full complement of mechanisms necessary to restrain E2F/DP and Cyclin/Cdk activities in differentiating cells are not known. Here, we have performed a genetic screen in Drosophila melanogaster, designed to identify genes required for cell cycle exit. This screen utilized a PCNA-miniwhite(+) reporter that is highly E2F-responsive and results in a darker red eye color when crossed into genetic backgrounds that delay cell cycle exit. Mutation of Hsp83, the Drosophila homolog of mammalian Hsp90, results in increased E2F-dependent transcription and ectopic cell proliferation in pupal tissues at a time when neighboring wild-type cells are postmitotic. Further, these Hsp83 mutant cells have increased Cyclin/Cdk activity and accumulate proteins normally targeted for proteolysis by the anaphase-promoting complex/cyclosome (APC/C), suggesting that APC/C function is inhibited. Indeed, reducing the gene dosage of an inhibitor of Cdh1/Fzr, an activating subunit of the APC/C that is required for timely cell cycle exit, can genetically suppress the Hsp83 cell cycle exit phenotype. Based on these data, we propose that Cdh1/Fzr is a client protein of Hsp83. Our results reveal that Hsp83 plays a heretofore unappreciated role in promoting APC/C function during cell cycle exit and suggest a mechanism by which Hsp90 inhibition could promote genomic instability and carcinogenesis.

  13. Spent fuel storage requirements

    International Nuclear Information System (INIS)

    Fletcher, J.

    1982-06-01

    Spent fuel storage requirements, as projected through the year 2000 for U.S. LWRs, were calculated using information supplied by the utilities reflecting plant status as of December 31, 1981. Projections through the year 2000 combined fuel discharge projections of the utilities with the assumed discharges of typical reactors required to meet the nuclear capacity of 165 GWe projected by the Energy Information Administration (EIA) for the year 2000. Three cases were developed and are summarized. A reference case, or maximum at-reactor (AR) capacity case, assumes that all reactor storage pools are increased to their maximum capacities as estimated by the utilities for spent fuel storage utilizing currently licensed technologies. The reference case assumes no transshipments between pools except as currently licensed by the Nuclear Regulatory Commission (NRC). This case identifies an initial requirement for 13 MTU of additional storage in 1984, and a cumulative requirement for 14,490 MTU additional storage in the year 2000. The reference case is bounded by two alternative cases. One, a current capacity case, assumes that only those pool storage capacity increases currently planned by the operating utilities will occur. The second, or maximum capacity with transshipment case, assumes maximum development of pool storage capacity as described above and also assumes no constraints on transshipment of spent fuel among pools of reactors of like type (BWR, PWR) within a given utility. In all cases, a full core discharge capability (full core reserve or FCR) is assumed to be maintained for each reactor, except that only one FCR is maintained when two reactors share a common pool. For the current AR capacity case the indicated storage requirements in the year 2000 are indicated to be 18,190 MTU; for the maximum capacity with transshipment case they are 11,320 MTU

  14. Next Generation Microbiology Requirements

    Science.gov (United States)

    Ott, C. M.; Oubre, C. M.; Elliott, T. F.; Castro, V. A.; Pierson, D. L.

    2012-01-01

    As humans continue to explore deep into space, microorganisms will travel with them. The primary means to mitigate the risk of infectious disease are a combination of prudent spacecraft design and rigorous operational controls. The effectiveness of these methods are evaluated by microbiological monitoring of spacecraft, food, water, and the crew that is performed preflight, in-flight, and post-flight. Current NASA requirements associated with microbiological monitoring are based on culture-based methodology where microorganisms are grown on a semi-solid growth medium and enumerated. Subsequent identification of the organisms requires specialized labor and large equipment, which historically has been performed on Earth. Requirements that rely strictly on culture-based units limit the use of non-culture based monitoring technology. Specifically, the culture-based "measurement criteria" are Colony Forming Units (CFU, representing the growth of one microorganism at a single location on the agar medium) per a given volume, area, or sample size. As the CFU unit by definition is culture-based, these requirements limit alternative technologies for spaceflight applications. As spaceflight missions such as those to Mars extend further into space, culture-based technology will become difficult to implement due to the (a) limited shelf life of the culture media, (b) mass/volume necessary to carry these consumables, and (c) problems associated with the production of biohazardous material in the habitable volume of the spacecraft. In addition, an extensive amount of new knowledge has been obtained during the Space Shuttle, NASA-Mir, and International Space Station Programs, which gave direction for new or modified microbial control requirements for vehicle design and mission operations. The goal of this task is to develop and recommend a new set of requirements for vehicle design and mission operations, including microbiological monitoring, based upon "lessons learned" and new

  15. Optical CDMA components requirements

    Science.gov (United States)

    Chan, James K.

    1998-08-01

    Optical CDMA is a complementary multiple access technology to WDMA. Optical CDMA potentially provides a large number of virtual optical channels for IXC, LEC and CLEC or supports a large number of high-speed users in LAN. In a network, it provides asynchronous, multi-rate, multi-user communication with network scalability, re-configurability (bandwidth on demand), and network security (provided by inherent CDMA coding). However, optical CDMA technology is less mature in comparison to WDMA. The components requirements are also different from WDMA. We have demonstrated a video transport/switching system over a distance of 40 Km using discrete optical components in our laboratory. We are currently pursuing PIC implementation. In this paper, we will describe the optical CDMA concept/features, the demonstration system, and the requirements of some critical optical components such as broadband optical source, broadband optical amplifier, spectral spreading/de- spreading, and fixed/programmable mask.

  16. Preanalytical requirements of urinalysis

    Science.gov (United States)

    Delanghe, Joris; Speeckaert, Marijn

    2014-01-01

    Urine may be a waste product, but it contains an enormous amount of information. Well-standardized procedures for collection, transport, sample preparation and analysis should become the basis of an effective diagnostic strategy for urinalysis. As reproducibility of urinalysis has been greatly improved due to recent technological progress, preanalytical requirements of urinalysis have gained importance and have become stricter. Since the patients themselves often sample urine specimens, urinalysis is very susceptible to preanalytical issues. Various sampling methods and inappropriate specimen transport can cause important preanalytical errors. The use of preservatives may be helpful for particular analytes. Unfortunately, a universal preservative that allows a complete urinalysis does not (yet) exist. The preanalytical aspects are also of major importance for newer applications (e.g. metabolomics). The present review deals with the current preanalytical problems and requirements for the most common urinary analytes. PMID:24627718

  17. Users' requirements for IFMIF

    International Nuclear Information System (INIS)

    Noda, K.; Jitsukawa, S.; Ehrlich, K.; Moeslang, A.

    1998-01-01

    The International Fusion Materials Irradiation Facility (IFMIF) is a high energy neutron irradiation facility which generates an intense neutron flux with D-Li stripping reactions for fusion materials testing. The role of IFMIF is (1) development of various fusion reactor materials, (2) determination of design-relevant engineering databases for the DEMO fusion reactor, (3) calibration and validation of data generated from fission reactor irradiations and the other simulation experiments, etc. The conceptual design activity (CDA) of IFMIF was initiated in February 1995 as an IEA collaborative activity to complete a reference conceptual design of IFMIF in December 1996. Users' requirements for the conceptual design of IFMIF were developed for materials to be tested, types of experiments, small specimen test technology and irradiation conditions. Furthermore, the neutron irradiation field characteristics (spectrum, flux/volume, etc.) of IFMIF were evaluated for the conceptual design parameters and were shown to meet the essential requirements of the users. (orig.)

  18. LHCb Online Networking Requirements

    CERN Document Server

    Jost, B

    2003-01-01

    This document describes the networking requirements of the LHCb online installation. It lists both quantitative aspects such as the number of required switch ports, as well as some qualitative features of the equipment, such as minimum buffer sizes in switches. The document comprises both the data acquisition network and the controls/general-purpose network. While the numbers represent our best current knowledge and are intended to give (in particular) network equipment manufacturers an overview of our needs, this document should not be confused with a market survey questionnaire or a formal tendering document. However the information contained in this document will be the input of any such document. A preliminary schedule for procurement and installation is also given.

  19. Remoção de células somáticas pela microfiltração não afeta a composição e a proteólise do leite Somatic cell removal by microfiltration does not affect composition and proteolysis of milk

    Directory of Open Access Journals (Sweden)

    Marcos Veiga dos Santos

    2006-10-01

    Full Text Available O objetivo do presente estudo foi avaliar os efeitos da retirada mecânica das células somáticas do leite cru sobre a composição e a proteólise durante o armazenamento refrigerado do leite pasteurizado. O delineamento experimental utilizado foi o de blocos generalizados ao acaso, no qual foram considerados como blocos as repetições (n=3 e o nível de gordura do leite (desnatado e integral. Utilizou-se um arranjo fatorial de tratamento do tipo 2 x 2, constituído por: dois níveis de contagem de células somáticas - CCS (baixa e alta CCS e pela aplicação ou não da microfiltração ao leite. Foram realizadas, ainda, medidas repetidas no tempo, as quais corresponderam aos dias de coleta do leite pasteurizado durante o período de armazenamento (1, 7, 14 e 21 dias. Os lotes de leite cru de alta (1.000.000cél. mL-1 e baixa (100.000cél. mL-1 CCS foram submetidos ao desnate centrífugo, à microfiltração em sistema a vácuo e, em seguida, os lotes de todos os tratamentos foram pasteurizados e armazenados por 21 dias sob refrigeração a 6°C. Não foi identificado efeito da microfiltração sobre a proteólise do leite, indicando que este tratamento não reduziu a taxa de proteólise do leite de alta CCS durante o período de armazenamento. Foi observado efeito significativo do tempo de armazenamento sobre a proteólise, indicando a manutenção de atividade proteolítica mesmo após a pasteurização do leite. Pode-se concluir que o leite com alta contagem de células somáticas apresenta maior taxa de proteólise durante o período de armazenamento que o leite de baixa contagem de células somáticas. A microfiltração como processo de retirada mecânica das células somáticas do leite não reduz a proteólise do leite durante o armazenamento.This study was aimed at evaluating the effects of raw milk somatic cell removal by microfiltration on the composition and proteolysis during refrigerated storage of pasturized milk. A completely

  20. TANK FARM ENVIRONMENTAL REQUIREMENTS

    International Nuclear Information System (INIS)

    TIFFT, S.R.

    2003-01-01

    Through regulations, permitting or binding negotiations, Regulators establish requirements, limits, permit conditions and Notice of Construction (NOC) conditions with which the Office of River Protection (ORP) and the Tank Farm Contractor (TFC) must comply. Operating Specifications are technical limits which are set on a process to prevent injury to personnel, or damage to the facility or environment, The main purpose of this document is to provide specification limits and recovery actions for the TFC Environmental Surveillance Program at the Hanford Site. Specification limits are given for monitoring frequencies and permissible variation of readings from an established baseline or previous reading. The requirements in this document are driven by environmental considerations and data analysis issues, rather than facility design or personnel safety issues. This document is applicable to all single-shell tank (SST) and double-shell tank (DST) waste tanks, and the associated catch tanks and receiver tanks, and transfer systems. This Tank Farm Environmental Specifications Document (ESD) implements environmental-regulatory limits on the configuration and operation of the Hanford Tank Farms facility that have been established by Regulators. This ESD contains specific field operational limits and recovery actions for compliance with airborne effluent regulations and agreements, liquid effluents regulations and agreements, and environmental tank system requirements. The scope of this ESD is limited to conditions that have direct impact on Operations/Projects or that Operations Projects have direct impact upon. This document does not supercede or replace any Department of Energy (DOE) Orders, regulatory permits, notices of construction, or Regulatory agency agreements binding on the ORP or the TFC. Refer to the appropriate regulation, permit, or Notice of Construction for an inclusive listing of requirements

  1. Utility requirements for HTGRs

    International Nuclear Information System (INIS)

    Nicholls, D.R.

    1997-01-01

    Eskom, the state utility of South Africa, is currently evaluating the technical and economic feasibility of the helium cooled Pebble Bed Modular Reactor with a closed cycle gas turbine power conversion system for future power generating additions to its electric system. This paper provides an overview of the Eskom system including the needs of the utility for future generation capacity and the key performance requirements necessary for incorporation of this gas cooled reactor plant. (author)

  2. ENVIRONMENTAL SPECIFICATION REQUIREMENTS

    International Nuclear Information System (INIS)

    TIFFT, S.R.

    2003-01-01

    Through regulations, permitting or binding negotiations, Regulators establish requirements, limits, permit conditions and Notice of Construction (NOC) conditions with which the Office of River Protection (ORP) and the Tank Farm Contractor (TFC) must comply. Operating Specifications are technical limits which are set on a process to prevent injury to personnel, or damage to the facility or environment. The main purpose of this document is to provide specification limits and recovery actions for the TFC Environmental Surveillance Program at the Hanford Site. Specification limits are given for monitoring frequencies and permissible variation of readings from an established baseline or previous reading. The requirements in this document are driven by environmental considerations and data analysis issues, rather than facility design or personnel safety issues. This document is applicable to all single-shell tank (SST) and double-shell tank (DST) waste tanks, and the associated catch tanks and receiver tanks, and transfer systems. This Tank Farm Environmental Specifications Document (ESD) implements environmental-regulatory limits on the configuration and operation of the Hanford Tank Farms facility that have been established by Regulators. This ESD contains specific field operational limits and recovery actions for compliance with airborne effluent regulations and agreements, liquid effluents regulations and agreements, and environmental tank system requirements. The scope of this ESD is limited to conditions that have direct impact on Operations Projects or that Operations/Projects have direct impact upon. This document does not supercede or replace any DOE Orders, regulatory permits, notices of construction, or Regulatory agency agreements binding on the ORP or the TFC. Refer to the appropriate regulation, permit, or NOC for an inclusive listing of requirements

  3. ENVIRONMENTAL SPECIFICATION REQUIREMENTS

    International Nuclear Information System (INIS)

    TIFFT, S.R.

    2003-01-01

    Through regulations, permitting or binding negotiations, Regulators establish requirements, limits, permit conditions and Notice of Construction (NOC) conditions with which the Office of River Protection (ORP) and the Tank Farm Contractor (TFC) must comply. Operating Specifications are technical limits which are set on a process to prevent injury to personnel, or damage to the facility or environment. The main purpose of this document is to provide specification limits and recovery actions for the TFC Environmental Surveillance Program at the Hanford Site. Specification limits are given for monitoring frequencies and permissible variation of readings from an established baseline or previous reading. The requirements in this document are driven by environmental considerations and data analysis issues, rather than facility design or personnel safety issues. This document is applicable to all SST and DST waste tanks, and the associated catch tanks and receiver tanks, and transfer systems. This Tank Farm ESD implements environmental-regulatory limits on the configuration and operation of the Hanford Tank Farms facility that have been established by Regulators. This ESD contains specific field operational limits and recovery actions for compliance with airborne effluent regulations and agreements, liquid effluents regulations and agreements, and environmental tank system requirements. The scope of this ESD is limited to conditions that have direct impact on Operations/Projects or that Operations/Projects have direct impact upon. This document does not supercede or replace any DOE Orders, regulatory permits, notices of construction, or Regulatory agency agreements binding on the ORP or the TFC. Refer to the appropriate regulation, permit, or NOC for an inclusive listing of requirements

  4. BER Science Network Requirements

    Energy Technology Data Exchange (ETDEWEB)

    Alapaty, Kiran; Allen, Ben; Bell, Greg; Benton, David; Brettin, Tom; Canon, Shane; Dart, Eli; Cotter, Steve; Crivelli, Silvia; Carlson, Rich; Dattoria, Vince; Desai, Narayan; Egan, Richard; Tierney, Brian; Goodwin, Ken; Gregurick, Susan; Hicks, Susan; Johnston, Bill; de Jong, Bert; Kleese van Dam, Kerstin; Livny, Miron; Markowitz, Victor; McGraw, Jim; McCord, Raymond; Oehmen, Chris; Regimbal, Kevin; Shipman, Galen; Strand, Gary; Flick, Jeff; Turnbull, Susan; Williams, Dean; Zurawski, Jason

    2010-11-01

    The Energy Sciences Network (ESnet) is the primary provider of network connectivity for the US Department of Energy Office of Science, the single largest supporter of basic research in the physical sciences in the United States. In support of the Office of Science programs, ESnet regularly updates and refreshes its understanding of the networking requirements of the instruments, facilities, scientists, and science programs that it serves. This focus has helped ESnet to be a highly successful enabler of scientific discovery for over 20 years. In April 2010 ESnet and the Office of Biological and Environmental Research, of the DOE Office of Science, organized a workshop to characterize the networking requirements of the science programs funded by BER. The requirements identified at the workshop are summarized and described in more detail in the case studies and the Findings section. A number of common themes emerged from the case studies and workshop discussions. One is that BER science, like many other disciplines, is becoming more and more distributed and collaborative in nature. Another common theme is that data set sizes are exploding. Climate Science in particular is on the verge of needing to manage exabytes of data, and Genomics is on the verge of a huge paradigm shift in the number of sites with sequencers and the amount of sequencer data being generated.

  5. LEGACY MANAGEMENT REQUIRES INFORMATION

    International Nuclear Information System (INIS)

    CONNELL, C.W.; HILDEBRAND, R.D.

    2006-01-01

    ''Legacy Management Requires Information'' describes the goal(s) of the US Department of Energy's Office of Legacy Management (LM) relative to maintaining critical records and the way those goals are being addressed at Hanford. The paper discusses the current practices for document control, as well as the use of modern databases for both storing and accessing the data to support cleanup decisions. In addition to the information goals of LM, the Hanford Federal Facility Agreement and Consent Order, known as the ''Tri-Party Agreement'' (TPA) is one of the main drivers in documentation and data management. The TPA, which specifies discrete milestones for cleaning up the Hanford Site, is a legally binding agreement among the US Department of Energy (DOE), the Washington State Department of Ecology (Ecology), and the US Environmental Protection Agency (EPA). The TPA requires that DOE provide the lead regulatory agency with the results of analytical laboratory and non-laboratory tests/readings to help guide them in making decisions. The Agreement also calls for each signatory to preserve--for at least ten years after the Agreement has ended--all of the records in its or its contractors, possession related to sampling, analysis, investigations, and monitoring conducted. The tools used at Hanford to meet TPA requirements are also the tools that can satisfy the needs of LM

  6. BES Science Network Requirements

    Energy Technology Data Exchange (ETDEWEB)

    Biocca, Alan; Carlson, Rich; Chen, Jackie; Cotter, Steve; Tierney, Brian; Dattoria, Vince; Davenport, Jim; Gaenko, Alexander; Kent, Paul; Lamm, Monica; Miller, Stephen; Mundy, Chris; Ndousse, Thomas; Pederson, Mark; Perazzo, Amedeo; Popescu, Razvan; Rouson, Damian; Sekine, Yukiko; Sumpter, Bobby; Dart, Eli; Wang, Cai-Zhuang -Z; Whitelam, Steve; Zurawski, Jason

    2011-02-01

    The Energy Sciences Network (ESnet) is the primary provider of network connectivityfor the US Department of Energy Office of Science (SC), the single largest supporter of basic research in the physical sciences in the United States. In support of the Office ofScience programs, ESnet regularly updates and refreshes its understanding of the networking requirements of the instruments, facilities, scientists, and science programs that it serves. This focus has helped ESnet to be a highly successful enabler of scientific discovery for over 20 years.

  7. BES Science Network Requirements

    International Nuclear Information System (INIS)

    Dart, Eli; Tierney, Brian; Biocca, A.; Carlson, R.; Chen, J.; Cotter, S.; Dattoria, V.; Davenport, J.; Gaenko, A.; Kent, P.; Lamm, M.; Miller, S.; Mundy, C.; Ndousse, T.; Pederson, M.; Perazzo, A.; Popescu, R.; Rouson, D.; Sekine, Y.; Sumpter, B.; Wang, C.-Z.; Whitelam, S.; Zurawski, J.

    2011-01-01

    The Energy Sciences Network (ESnet) is the primary provider of network connectivity for the US Department of Energy Office of Science (SC), the single largest supporter of basic research in the physical sciences in the United States. In support of the Office of Science programs, ESnet regularly updates and refreshes its understanding of the networking requirements of the instruments, facilities, scientists, and science programs that it serves. This focus has helped ESnet to be a highly successful enabler of scientific discovery for over 20 years.

  8. Section 4: Requirements Intertwining

    Science.gov (United States)

    Loucopoulos, Pericles

    Business analysts are being asked to develop increasingly complex and varied business systems that need to cater to the changing and dynamic market conditions of the new economy. This is particularly acute in today’s turbulent business environment where powerful forces such as deregulation, globalisation, mergers, advances in information and telecommunications technologies, and increasing education of people provide opportunities for organising work in ways that have never before been possible. Enterprises attempt to create wealth either by getting better at improving their products and services or by harnessing creativity and human-centred management to create innovative solutions. In these business settings, requirements become critical in bridging system solutions to organisational and societal problems. They intertwine organisational, social, cognitive, and implementation considerations and they can provide unique insights to change in systems and their business context. Such design situations often involve multiple stakeholders from different participating organisations, subcontractors, divisions, etc., who may have a diversity of expertise, come from different organisational cultures and often have competing goals. The success or failure of many projects depends, to a large extent, on understanding the contextual setting of requirements and their interaction amongst a diverse population of stakeholders.

  9. Beauty Requires Thought.

    Science.gov (United States)

    Brielmann, Aenne A; Pelli, Denis G

    2017-05-22

    The experience of beauty is a pleasure, but common sense and philosophy suggest that feeling beauty differs from sensuous pleasures such as eating or sex. Immanuel Kant [1, 2] claimed that experiencing beauty requires thought but that sensuous pleasure can be enjoyed without thought and cannot be beautiful. These venerable hypotheses persist in models of aesthetic processing [3-7] but have never been tested. Here, participants continuously rated the pleasure felt from a nominally beautiful or non-beautiful stimulus and then judged whether they had experienced beauty. The stimuli, which engage various senses, included seeing images, tasting candy, and touching a teddy bear. The observer reported the feelings that the stimulus evoked. The time course of pleasure, across stimuli, is well-fit by a model with one free parameter: pleasure amplitude. Pleasure amplitude increases linearly with the feeling of beauty. To test Kant's claim of a need for thought, we reduce cognitive capacity by adding a "two-back" task to distract the observer's thoughts. The distraction greatly reduces the beauty and pleasure experienced from stimuli that otherwise produce strong pleasure and spares that of less-pleasant stimuli. We also find that strong pleasure is always beautiful, whether produced reliably by beautiful stimuli or just occasionally by sensuous stimuli. In sum, we confirm Kant's claim that only the pleasure associated with feeling beauty requires thought and disprove his claim that sensuous pleasures cannot be beautiful. Copyright © 2017 The Authors. Published by Elsevier Ltd.. All rights reserved.

  10. Display Parameters and Requirements

    Science.gov (United States)

    Bahadur, Birendra

    The following sections are included: * INTRODUCTION * HUMAN FACTORS * Anthropometry * Sensory * Cognitive * Discussions * THE HUMAN VISUAL SYSTEM - CAPABILITIES AND LIMITATIONS * Cornea * Pupil and Iris * Lens * Vitreous Humor * Retina * RODS - NIGHT VISION * CONES - DAY VISION * RODS AND CONES - TWILIGHT VISION * VISUAL PIGMENTS * MACULA * BLOOD * CHOROID COAT * Visual Signal Processing * Pathways to the Brain * Spatial Vision * Temporal Vision * Colour Vision * Colour Blindness * DICHROMATISM * Protanopia * Deuteranopia * Tritanopia * ANOMALOUS TRICHROMATISM * Protanomaly * Deuteranomaly * Tritanomaly * CONE MONOCHROMATISM * ROD MONOCHROMATISM * Using Colour Effectively * COLOUR MIXTURES AND THE CHROMATICITY DIAGRAM * Colour Matching Functions and Chromaticity Co-ordinates * CIE 1931 Colour Space * CIE PRIMARIES * CIE COLOUR MATCHING FUNCTIONS AND CHROMATICITY CO-ORDINATES * METHODS FOR DETERMINING TRISTIMULUS VALUES AND COLOUR CO-ORDINATES * Spectral Power Distribution Method * Filter Method * CIE 1931 CHROMATICITY DIAGRAM * ADDITIVE COLOUR MIXTURE * CIE 1976 Chromaticity Diagram * CIE Uniform Colour Spaces and Colour Difference Formulae * CIELUV OR L*u*v* * CIELAB OR L*a*b* * CIE COLOUR DIFFERENCE FORMULAE * Colour Temperature and CIE Standard Illuminants and source * RADIOMETRIC AND PHOTOMETRIC QUANTITIES * Photopic (Vλ and Scotopic (Vλ') Luminous Efficiency Function * Photometric and Radiometric Flux * Luminous and Radiant Intensities * Incidence: Illuminance and Irradiance * Exitance or Emittance (M) * Luminance and Radiance * ERGONOMIC REQUIREMENTS OF DISPLAYS * ELECTRO-OPTICAL PARAMETERS AND REQUIREMENTS * Contrast and Contrast Ratio * Luminance and Brightness * Colour Contrast and Chromaticity * Glare * Other Aspects of Legibility * SHAPE AND SIZE OF CHARACTERS * DEFECTS AND BLEMISHES * FLICKER AND DISTORTION * ANGLE OF VIEW * Switching Speed * Threshold and Threshold Characteristic * Measurement Techniques For Electro-optical Parameters * RADIOMETRIC

  11. N-linked glycans are required on epithelial Na+ channel subunits for maturation and surface expression.

    Science.gov (United States)

    Kashlan, Ossama B; Kinlough, Carol L; Myerburg, Michael M; Shi, Shujie; Chen, Jingxin; Blobner, Brandon M; Buck, Teresa M; Brodsky, Jeffrey L; Hughey, Rebecca P; Kleyman, Thomas R

    2018-03-01

    Epithelial Na + channel (ENaC) subunits undergo N-linked glycosylation in the endoplasmic reticulum where they assemble into an αβγ complex. Six, 13, and 5 consensus sites (Asn-X-Ser/Thr) for N-glycosylation reside in the extracellular domains of the mouse α-, β-, and γ-subunits, respectively. Because the importance of ENaC N-linked glycans has not been fully addressed, we examined the effect of preventing N-glycosylation of specific subunits on channel function, expression, maturation, and folding. Heterologous expression in Xenopus oocytes or Fischer rat thyroid cells with αβγ-ENaC lacking N-linked glycans on a single subunit reduced ENaC activity as well as the inhibitory response to extracellular Na + . The lack of N-linked glycans on the β-subunit also precluded channel activation by trypsin. However, channel activation by shear stress was N-linked glycan independent, regardless of which subunit was modified. We also discovered that the lack of N-linked glycans on any one subunit reduced the total and surface levels of cognate subunits. The lack of N-linked glycans on the β-subunit had the largest effect on total levels, with the lack of N-linked glycans on the γ- and α-subunits having intermediate and modest effects, respectively. Finally, channels with wild-type β-subunits were more sensitive to limited trypsin proteolysis than channels lacking N-linked glycans on the β-subunit. Our results indicate that N-linked glycans on each subunit are required for proper folding, maturation, surface expression, and function of the channel.

  12. Knowing requires data

    Science.gov (United States)

    Naranjo, Ramon C.

    2017-01-01

    Groundwater-flow models are often calibrated using a limited number of observations relative to the unknown inputs required for the model. This is especially true for models that simulate groundwater surface-water interactions. In this case, subsurface temperature sensors can be an efficient means for collecting long-term data that capture the transient nature of physical processes such as seepage losses. Continuous and spatially dense network of diverse observation data can be used to improve knowledge of important physical drivers, conceptualize and calibrate variably saturated groundwater flow models. An example is presented for which the results of such analysis were used to help guide irrigation districts and water management decisions on costly upgrades to conveyance systems to improve water usage, farm productivity and restoration efforts to improve downstream water quality and ecosystems.

  13. SOFG: Standards requirements

    International Nuclear Information System (INIS)

    Gerganov, T.; Grigorov, S.; Kozhukharov, V.; Brashkova, N.

    2005-01-01

    It is well-known that Solid Oxide Fuel Cells will have industrial application in the nearest future. In this context, the problem of SOFC materials and SOFC systems standardization is of high level of priority. In the present study the attention is focused on the methods for physical and chemical characterization of the materials for SOFC components fabrication and about requirements on single SOFC cells tests. The status of the CEN, ISO, ASTM (ANSI, ASSN) and JIS class of standards has been verified. Standards regarding the test methods for physical-chemical characterization of vitreous materials (as sealing SOFC component), ceramic materials (as electrodes and electrolyte components, including alternative materials used) and metallic materials (interconnect components) are subject of overview. It is established that electrical, mechanical, surface and interfacial phenomena, chemical durability and thermal corrosion behaviour are the key areas for standardization of the materials for SOFC components

  14. Equipment Operational Requirements

    Energy Technology Data Exchange (ETDEWEB)

    Greenwalt, B; Henderer, B; Hibbard, W; Mercer, M

    2009-06-11

    The Iraq Department of Border Enforcement is rich in personnel, but poor in equipment. An effective border control system must include detection, discrimination, decision, tracking and interdiction, capture, identification, and disposition. An equipment solution that addresses only a part of this will not succeed, likewise equipment by itself is not the answer without considering the personnel and how they would employ the equipment. The solution should take advantage of the existing in-place system and address all of the critical functions. The solutions are envisioned as being implemented in a phased manner, where Solution 1 is followed by Solution 2 and eventually by Solution 3. This allows adequate time for training and gaining operational experience for successively more complex equipment. Detailed descriptions of the components follow the solution descriptions. Solution 1 - This solution is based on changes to CONOPs, and does not have a technology component. It consists of observers at the forts and annexes, forward patrols along the swamp edge, in depth patrols approximately 10 kilometers inland from the swamp, and checkpoints on major roads. Solution 2 - This solution adds a ground sensor array to the Solution 1 system. Solution 3 - This solution is based around installing a radar/video camera system on each fort. It employs the CONOPS from Solution 1, but uses minimal ground sensors deployed only in areas with poor radar/video camera coverage (such as canals and streams shielded by vegetation), or by roads covered by radar but outside the range of the radar associated cameras. This document provides broad operational requirements for major equipment components along with sufficient operational details to allow the technical community to identify potential hardware candidates. Continuing analysis will develop quantities required and more detailed tactics, techniques, and procedures.

  15. ASCR Science Network Requirements

    Energy Technology Data Exchange (ETDEWEB)

    Dart, Eli; Tierney, Brian

    2009-08-24

    The Energy Sciences Network (ESnet) is the primary provider of network connectivity for the US Department of Energy Office of Science, the single largest supporter of basic research in the physical sciences in the United States. In support of the Office of Science programs, ESnet regularly updates and refreshes its understanding of the networking requirements of the instruments, facilities, scientists, and science programs that it serves. This focus has helped ESnet to be a highly successful enabler of scientific discovery for over 20 years. In April 2009 ESnet and the Office of Advanced Scientific Computing Research (ASCR), of the DOE Office of Science, organized a workshop to characterize the networking requirements of the programs funded by ASCR. The ASCR facilities anticipate significant increases in wide area bandwidth utilization, driven largely by the increased capabilities of computational resources and the wide scope of collaboration that is a hallmark of modern science. Many scientists move data sets between facilities for analysis, and in some cases (for example the Earth System Grid and the Open Science Grid), data distribution is an essential component of the use of ASCR facilities by scientists. Due to the projected growth in wide area data transfer needs, the ASCR supercomputer centers all expect to deploy and use 100 Gigabit per second networking technology for wide area connectivity as soon as that deployment is financially feasible. In addition to the network connectivity that ESnet provides, the ESnet Collaboration Services (ECS) are critical to several science communities. ESnet identity and trust services, such as the DOEGrids certificate authority, are widely used both by the supercomputer centers and by collaborations such as Open Science Grid (OSG) and the Earth System Grid (ESG). Ease of use is a key determinant of the scientific utility of network-based services. Therefore, a key enabling aspect for scientists beneficial use of high

  16. Requirements of quality standards

    International Nuclear Information System (INIS)

    Mueller, J.

    1977-01-01

    The lecture traces the development of nuclear standards, codes, and Federal regulations on quality assurance (QA) for nuclear power plants and associated facilities. The technical evolution of the last twelve years, especially in the area of nuclear technology, led to different activities and regulatory initiatives, and the present result is: several nations have their own homemade standards. The lecture discusses the former and especially current activities in standard development, and gives a description of the requirements of QA-standards used in USA and Europe, especially Western Germany. Furthermore the lecture attempts to give a comparison and an evaluation of the international quality standards from the author's viewpoint. Finally the lecture presents an outlook for the future international implications of QA-standards. There is an urgent need within the nuclear industry for simplification and standardization of QA-standards. The relationship between the various standards, and the applicability of the standards need clarification and a better transparancy. To point out these problems is the purpose of the lecture. (orig.) [de

  17. mTORC1 Activator SLC38A9 Is Required to Efflux Essential Amino Acids from Lysosomes and Use Protein as a Nutrient.

    Science.gov (United States)

    Wyant, Gregory A; Abu-Remaileh, Monther; Wolfson, Rachel L; Chen, Walter W; Freinkman, Elizaveta; Danai, Laura V; Vander Heiden, Matthew G; Sabatini, David M

    2017-10-19

    The mTORC1 kinase is a master growth regulator that senses many environmental cues, including amino acids. Activation of mTORC1 by arginine requires SLC38A9, a poorly understood lysosomal membrane protein with homology to amino acid transporters. Here, we validate that SLC38A9 is an arginine sensor for the mTORC1 pathway, and we uncover an unexpectedly central role for SLC38A9 in amino acid homeostasis. SLC38A9 mediates the transport, in an arginine-regulated fashion, of many essential amino acids out of lysosomes, including leucine, which mTORC1 senses through the cytosolic Sestrin proteins. SLC38A9 is necessary for leucine generated via lysosomal proteolysis to exit lysosomes and activate mTORC1. Pancreatic cancer cells, which use macropinocytosed protein as a nutrient source, require SLC38A9 to form tumors. Thus, through SLC38A9, arginine serves as a lysosomal messenger that couples mTORC1 activation to the release from lysosomes of the essential amino acids needed to drive cell growth. Copyright © 2017 Elsevier Inc. All rights reserved.

  18. Delivery of the autofluorescent protein R-phycoerythrin by calcium phosphate nanoparticles into four different eukaryotic cell lines (HeLa, HEK293T, MG-63, MC3T3): Highly efficient, but leading to endolysosomal proteolysis in HeLa and MC3T3 cells.

    Science.gov (United States)

    Kopp, Mathis; Rotan, Olga; Papadopoulos, Chrisovalantis; Schulze, Nina; Meyer, Hemmo; Epple, Matthias

    2017-01-01

    Nanoparticles can be used as carriers to transport biomolecules like proteins and synthetic molecules across the cell membrane because many molecules are not able to cross the cell membrane on their own. The uptake of nanoparticles together with their cargo typically occurs via endocytosis, raising concerns about the possible degradation of the cargo in the endolysosomal system. As the tracking of a dye-labelled protein during cellular uptake and processing is not indicative of the presence of the protein itself but only for the fluorescent label, a label-free tracking was performed with the red-fluorescing model protein R-phycoerythrin (R-PE). Four different eukaryotic cell lines were investigated: HeLa, HEK293T, MG-63, and MC3T3. Alone, the protein was not taken up by any cell line; only with the help of calcium phosphate nanoparticles, an efficient uptake occurred. After the uptake into HeLa cells, the protein was found in early endosomes (shown by the marker EEA1) and lysosomes (shown by the marker Lamp1). There, it was still intact and functional (i.e. properly folded) as its red fluorescence was detected. However, a few hours after the uptake, proteolysis started as indicated by the decreasing red fluorescence intensity in the case of HeLa and MC3T3 cells. 12 h after the uptake, the protein was almost completely degraded in HeLa cells and MC3T3 cells. In HEK293T cells and MG-63 cells, no degradation of the protein was observed. In the presence of Bafilomycin A1, an inhibitor of acidification and protein degradation in lysosomes, the fluorescence of R-PE remained intact over the whole observation period in the four cell lines. These results indicate that despite an efficient nanoparticle-mediated uptake of proteins by cells, a rapid endolysosomal degradation may prevent the desired (e.g. therapeutic) effect of a protein inside a cell.

  19. Functional requirements of road lighting.

    NARCIS (Netherlands)

    Schreuder, D.A.

    1975-01-01

    The functional, technical and visual requirements for public lighting are discussed. The improvement of the presentation of information to the road user is the main functional requirement. The visual requirements can be deduced from the functional requirement of enabling drivers to follow the

  20. Neutrosophy for software requirement prioritization

    Directory of Open Access Journals (Sweden)

    Ronald Barriga Dias

    2017-09-01

    Full Text Available Software engineers are involved in complex decisions that require multiples viewpoints. A specific case is the requirement prioritization process. This process is used to decide which software requirement to develop in certain release from a group of candidate requirements. Criteria involved in this process can involve indeterminacy. In this paper a software requirement prioritization model is develop based SVN numbers. Finally, an illustrative example is presented in order to show the proposed model.

  1. THE EQUALITY PRINCIPLE REQUIREMENTS

    Directory of Open Access Journals (Sweden)

    CLAUDIA ANDRIŢOI

    2013-05-01

    Full Text Available The problem premises and the objectives followed: the idea of inserting the equality principle between the freedom and the justice principles is manifested in positive law in two stages, as a general idea of all judicial norms and as requirement of the owner of a subjective right of the applicants of an objective law. Equality in face of the law and of public authorities can not involve the idea of standardization, of uniformity, of enlisting of all citizens under the mark of the same judicial regime, regardless of their natural or socio-professional situation. Through the Beijing Platform and the position documents of the European Commission we have defined the integrative approach of equality as representing an active and visible integration of the gender perspective in all sectors and at all levels. The research methods used are: the conceptualist method, the logical method and the intuitive method necessary as means of reasoning in order to argue our demonstration. We have to underline the fact that the system analysis of the research methods of the judicial phenomenon doesn’t agree with “value ranking”, because one value cannot be generalized in rapport to another. At the same time, we must fight against a methodological extremism. The final purpose of this study is represented by the reaching of the perfecting/excellence stage by all individuals through the promotion of equality and freedom. This supposes the fact that the existence of a non-discrimination favourable frame (fairness represents a means and a condition of self-determination, and the state of perfection/excellency is a result of this self-determination, the condition necessary for the obtaining of this nondiscrimination frame for all of us and in conditions of freedom for all individuals, represents the same condition that promotes the state of perfection/excellency. In conclusion we may state the fact that the equality principle represents a true catalyst of the

  2. Waste management system requirements document

    International Nuclear Information System (INIS)

    1991-02-01

    This volume defines the top level requirements for the Mined Geologic Disposal System (MGDS). It is designed to be used in conjunction with Volume 1 of the WMSR, General System Requirements. It provides a functional description expanding the requirements allocated to the MGDS in Volume 1 and elaborates on each requirement by providing associated performance criteria as appropriate. Volumes 1 and 4 of the WMSR provide a minimum set of requirements that must be satisfied by the final MGDS design. This document sets forth specific requirements that must be fulfilled. It is not the intent or purpose of this top level document to describe how each requirement is to be satisfied in the final MGDS design. Each subsequent level of the technical document hierarchy must provide further guidance and definition as to how each of these requirements is to be implemented in the design. It is expected that each subsequent level of requirements will be significantly more detailed. Section 2 of this volume provides a functional description of the MGDS. Each function is addressed in terms of requirements, and performance criteria. Section 3 provides a list of controlling documents. Each document cited in a requirement of Chapter 2 is included in this list and is incorporated into this document as a requirement on the final system. The WMSR addresses only federal requirements (i.e., laws, regulations and DOE orders). State and local requirements are not addressed. However, it will be specifically noted at the potentially affected WMSR requirements that there could be additional or more stringent regulations imposed by a state or local requirements or administering agency over the cited federal requirements

  3. IRET: requirements for service platforms

    OpenAIRE

    Baresi, Luciano; Ripa, Gianluca; Pasquale, Liliana

    2013-01-01

    peer-reviewed This paper describes IRENE (Indenica Requirements ElicitatioN mEthod), a methodology to elicit and model the requirements of service platforms, and IRET (IREne Tool), the Eclipse-based modeling framework we developed for IRENE

  4. Physician Requirements-1990. For Cardiology.

    Science.gov (United States)

    Tracy, Octavious; Birchette-Pierce, Cheryl

    Professional requirements for physicians specializing in cardiology were estimated to assist policymakers in developing guidelines for graduate medical education. The determination of physician requirements was based on an adjusted needs rather than a demand or utilization model. For each illness, manpower requirements were modified by the…

  5. Waste Management System Requirement document

    International Nuclear Information System (INIS)

    1990-04-01

    This volume defines the top level technical requirements for the Monitored Retrievable Storage (MRS) facility. It is designed to be used in conjunction with Volume 1, General System Requirements. Volume 3 provides a functional description expanding the requirements allocated to the MRS facility in Volume 1 and, when appropriate, elaborates on requirements by providing associated performance criteria. Volumes 1 and 3 together convey a minimum set of requirements that must be satisfied by the final MRS facility design without unduly constraining individual design efforts. The requirements are derived from the Nuclear Waste Policy Act of 1982 (NWPA), the Nuclear Waste Policy Amendments Act of 1987 (NWPAA), the Environmental Protection Agency's (EPA) Environmental Standards for the Management and Disposal of Spent Nuclear Fuel (40 CFR 191), NRC Licensing Requirements for the Independent Storage of Spent Nuclear and High-Level Radioactive Waste (10 CFR 72), and other federal statutory and regulatory requirements, and major program policy decisions. This document sets forth specific requirements that will be fulfilled. Each subsequent level of the technical document hierarchy will be significantly more detailed and provide further guidance and definition as to how each of these requirements will be implemented in the design. Requirements appearing in Volume 3 are traceable into the MRS Design Requirements Document. Section 2 of this volume provides a functional breakdown for the MRS facility. 1 tab

  6. Capital Requirements and Banks' Leniency

    DEFF Research Database (Denmark)

    Dietrich, J. Kimball; Wihlborg, Clas

    2003-01-01

    We investigate the effect of changes in capital regulation on the strictness(leniency) of loan terms using a simple model of bank capital requirements andasset quality examinations. Banks offer different levels of `leniency' in the senseof willingness to offer automatic extensions of loans...... rates. As capital requirements increase thedifference between initial capital levels and between interest rates of strict andlenient banks decrease. Thus, higher capital requirements in recessions tend toreduce the interest rate premium paid for leniency. If a recession is interpreted asan increase...... in the required return, the interest rate premium paid for leniency isincreased in recession at a given level of required capital....

  7. Mcl-1 dynamics influence mitotic slippage and death in mitosis.

    Science.gov (United States)

    Sloss, Olivia; Topham, Caroline; Diez, Maria; Taylor, Stephen

    2016-02-02

    Microtubule-binding drugs such as taxol are frontline treatments for a variety of cancers but exactly how they yield patient benefit is unclear. In cell culture, inhibiting microtubule dynamics prevents spindle assembly, leading to mitotic arrest followed by either apoptosis in mitosis or slippage, whereby a cell returns to interphase without dividing. Myeloid cell leukaemia-1 (Mcl-1), a pro-survival member of the Bcl-2 family central to the intrinsic apoptosis pathway, is degraded during a prolonged mitotic arrest and may therefore act as a mitotic death timer. Consistently, we show that blocking proteasome-mediated degradation inhibits taxol-induced mitotic apoptosis in a Mcl-1-dependent manner. However, this degradation does not require the activity of either APC/C-Cdc20, FBW7 or MULE, three separate E3 ubiquitin ligases implicated in targeting Mcl-1 for degradation. This therefore challenges the notion that Mcl-1 undergoes regulated degradation during mitosis. We also show that Mcl-1 is continuously synthesized during mitosis and that blocking protein synthesis accelerates taxol induced death-in-mitosis. Modulating Mcl-1 levels also influences slippage; overexpressing Mcl-1 extends the time from mitotic entry to mitotic exit in the presence of taxol, while inhibiting Mcl-1 accelerates it. We suggest that Mcl-1 competes with Cyclin B1 for binding to components of the proteolysis machinery, thereby slowing down the slow degradation of Cyclin B1 responsible for slippage. Thus, modulating Mcl-1 dynamics influences both death-in-mitosis and slippage. However, because mitotic degradation of Mcl-1 appears not to be under the control of an E3 ligase, we suggest that the notion of network crosstalk is used with caution.

  8. The protease degrading sperm histones post-fertilization in sea urchin eggs is a nuclear cathepsin L that is further required for embryo development.

    Directory of Open Access Journals (Sweden)

    Violeta Morin

    Full Text Available Proteolysis of sperm histones in the sea urchin male pronucleus is the consequence of the activation at fertilization of a maternal cysteine protease. We previously showed that this protein is required for male chromatin remodelling and for cell-cycle progression in the newly formed embryos. This enzyme is present in the nucleus of unfertilized eggs and is rapidly recruited to the male pronucleus after insemination. Interestingly, this cysteine-protease remains co-localized with chromatin during S phase of the first cell cycle, migrates to the mitotic spindle in M-phase and is re-located to the nuclei of daughter cells after cytokinesis. Here we identified the protease encoding cDNA and found a high sequence identity to cathepsin proteases of various organisms. A phylogenetical analysis clearly demonstrates that this sperm histone protease (SpHp belongs to the cathepsin L sub-type. After an initial phase of ubiquitous expression throughout cleavage stages, SpHp gene transcripts become restricted to endomesodermic territories during the blastula stage. The transcripts are localized in the invaginating endoderm during gastrulation and a gut specific pattern continues through the prism and early pluteus stages. In addition, a concomitant expression of SpHp transcripts is detected in cells of the skeletogenic lineage and in accordance a pharmacological disruption of SpHp activity prevents growth of skeletal rods. These results further document the role of this nuclear cathepsin L during development.

  9. Characterization of DNA polymerase X from Thermus thermophilus HB8 reveals the POLXc and PHP domains are both required for 3'-5' exonuclease activity.

    Science.gov (United States)

    Nakane, Shuhei; Nakagawa, Noriko; Kuramitsu, Seiki; Masui, Ryoji

    2009-04-01

    The X-family DNA polymerases (PolXs) comprise a highly conserved DNA polymerase family found in all kingdoms. Mammalian PolXs are known to be involved in several DNA-processing pathways including repair, but the cellular functions of bacterial PolXs are less known. Many bacterial PolXs have a polymerase and histidinol phosphatase (PHP) domain at their C-termini in addition to a PolX core (POLXc) domain, and possess 3'-5' exonuclease activity. Although both domains are highly conserved in bacteria, their molecular functions, especially for a PHP domain, are unknown. We found Thermus thermophilus HB8 PolX (ttPolX) has Mg(2+)/Mn(2+)-dependent DNA/RNA polymerase, Mn(2+)-dependent 3'-5' exonuclease and DNA-binding activities. We identified the domains of ttPolX by limited proteolysis and characterized their biochemical activities. The POLXc domain was responsible for the polymerase and DNA-binding activities but exonuclease activity was not detected for either domain. However, the POLXc and PHP domains interacted with each other and a mixture of the two domains had Mn(2+)-dependent 3'-5' exonuclease activity. Moreover, site-directed mutagenesis revealed catalytically important residues in the PHP domain for the 3'-5' exonuclease activity. Our findings provide a molecular insight into the functional domain organization of bacterial PolXs, especially the requirement of the PHP domain for 3'-5' exonuclease activity.

  10. p75 neurotrophin receptor cleavage by α- and γ-secretases is required for neurotrophin-mediated proliferation of brain tumor-initiating cells.

    Science.gov (United States)

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

    2014-03-21

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

  11. Anticipating requirements changes-using futurology in requirements elicitation

    OpenAIRE

    Pimentel, João Henrique; Santos, Emanuel; Castro, Jaelson; Franch Gutiérrez, Javier

    2012-01-01

    It is well known that requirements changes in a later phase of software developments is a major source of software defects and costs. Thus, the need of techniques to control or reduce the amount of changes during software development projects. The authors advocate the use of foresight methods as a valuable input to requirements elicitation, with the potential to decrease the number of changes that would be required after deployment, by anticipating them. In this paper, the authors define a pr...

  12. Tool-based requirement traceability between requirement and design artifacts

    CERN Document Server

    Turban, Bernhard

    2013-01-01

    Processes for developing safety-critical systems impose special demands on ensuring requirements traceability. Achieving valuable traceability information, however, is especially difficult concerning the transition from requirements to design. Bernhard Turban analyzes systems and software engineering theories cross-cutting the issue (embedded systems development, systems engineering, software engineering, requirements engineering and management, design theory and processes for safety-critical systems). As a solution, the author proposes a new tool approach to support designers in their thinkin

  13. Delivery of the autofluorescent protein R-phycoerythrin by calcium phosphate nanoparticles into four different eukaryotic cell lines (HeLa, HEK293T, MG-63, MC3T3: Highly efficient, but leading to endolysosomal proteolysis in HeLa and MC3T3 cells.

    Directory of Open Access Journals (Sweden)

    Mathis Kopp

    Full Text Available Nanoparticles can be used as carriers to transport biomolecules like proteins and synthetic molecules across the cell membrane because many molecules are not able to cross the cell membrane on their own. The uptake of nanoparticles together with their cargo typically occurs via endocytosis, raising concerns about the possible degradation of the cargo in the endolysosomal system. As the tracking of a dye-labelled protein during cellular uptake and processing is not indicative of the presence of the protein itself but only for the fluorescent label, a label-free tracking was performed with the red-fluorescing model protein R-phycoerythrin (R-PE. Four different eukaryotic cell lines were investigated: HeLa, HEK293T, MG-63, and MC3T3. Alone, the protein was not taken up by any cell line; only with the help of calcium phosphate nanoparticles, an efficient uptake occurred. After the uptake into HeLa cells, the protein was found in early endosomes (shown by the marker EEA1 and lysosomes (shown by the marker Lamp1. There, it was still intact and functional (i.e. properly folded as its red fluorescence was detected. However, a few hours after the uptake, proteolysis started as indicated by the decreasing red fluorescence intensity in the case of HeLa and MC3T3 cells. 12 h after the uptake, the protein was almost completely degraded in HeLa cells and MC3T3 cells. In HEK293T cells and MG-63 cells, no degradation of the protein was observed. In the presence of Bafilomycin A1, an inhibitor of acidification and protein degradation in lysosomes, the fluorescence of R-PE remained intact over the whole observation period in the four cell lines. These results indicate that despite an efficient nanoparticle-mediated uptake of proteins by cells, a rapid endolysosomal degradation may prevent the desired (e.g. therapeutic effect of a protein inside a cell.

  14. National Ignition Facility site requirements

    International Nuclear Information System (INIS)

    1996-07-01

    The Site Requirements (SR) provide bases for identification of candidate host sites for the National Ignition Facility (NIF) and for the generation of data regarding potential actual locations for the facilities. The SR supplements the NIF Functional Requirements (FR) with information needed for preparation of responses to queries for input to HQ DOE site evaluation. The queries are to include both documents and explicit requirements for the potential host site responses. The Sr includes information extracted from the NIF FR (for convenience), data based on design approaches, and needs for physical and organization infrastructure for a fully operational NIF. The FR and SR describe requirements that may require new construction or may be met by use or modification of existing facilities. The SR do not establish requirements for NIF design or construction project planning. The SR document does not constitute an element of the NIF technical baseline

  15. UTM TCL2 Software Requirements

    Science.gov (United States)

    Smith, Irene S.; Rios, Joseph L.; McGuirk, Patrick O.; Mulfinger, Daniel G.; Venkatesan, Priya; Smith, David R.; Baskaran, Vijayakumar; Wang, Leo

    2017-01-01

    The Unmanned Aircraft Systems (UAS) Traffic Management (UTM) Technical Capability Level (TCL) 2 software implements the UTM TCL 2 software requirements described herein. These software requirements are linked to the higher level UTM TCL 2 System Requirements. Each successive TCL implements additional UTM functionality, enabling additional use cases. TCL 2 demonstrated how to enable expanded multiple operations by implementing automation for beyond visual line-of-sight, tracking operations, and operations flying over sparsely populated areas.

  16. Capital Requirements and Credit Rationing

    OpenAIRE

    Itai Agur

    2010-01-01

    This paper analyzes the trade-off between financial stability and credit rationing that arises when increasing capital requirements. It extends the Stiglitz-Weiss model of credit rationing to allow for bank default. Bank capital structure then matters for lending incentives. With default and rationing endogenous, optimal capital requirements can be analyzed. Introducing bank financiers, the paper also shows that uninsured funding raises the sensitivity of rationing to capital requirements. In...

  17. Information requirements for enterprise systems

    OpenAIRE

    Sommerville, Ian; Lock, Russell; Storer, Tim

    2012-01-01

    In this paper, we discuss an approach to system requirements engineering, which is based on using models of the responsibilities assigned to agents in a multi-agency system of systems. The responsibility models serve as a basis for identifying the stakeholders that should be considered in establishing the requirements and provide a basis for a structured approach, described here, for information requirements elicitation. We illustrate this approach using a case study drawn from civil emergenc...

  18. World enrichment requirements to 2005

    International Nuclear Information System (INIS)

    Anon.

    1991-01-01

    The primary enrichment suppliers-Eurodif, Techsnabexport, Urenco, and the US DOE - are positioning themselves to take advantage of the post - 1995 market. Overall, unfilled requirements represent about 40 percent of world requirements in the year 2000. The USA will be the primary market, as US utilities' unfilled enrichment requirements account for over 60 percent of the world's total unfilled requirements. The enrichment market is moving toward more global competition, as each supplier tries to maintain its current regional market base and then to capture additional market share in other regions

  19. Addressing the Resource Requirements Mismatch

    National Research Council Canada - National Science Library

    Braun, William

    2003-01-01

    ... on the other, appear to be developing a requirements-resource mismatch. The goals and objectives of the transformation rhetoric intuitively resonate with the military's increasingly technologic culture...

  20. Federal Environmental Requirements for Construction

    Data.gov (United States)

    Department of Veterans Affairs — This guide provides information on federal environmental requirements for construction projects. It is written primarily for owners of construction projects and for...

  1. Autolysis of Lactococcus lactis is influenced by proteolysis

    NARCIS (Netherlands)

    Buist, G; Venema, G; Kok, J.

    1998-01-01

    The autolysin AcmA of Lactococcus lactis was shown to be degraded by the extracellular Lactococcal proteinase PrtP. Autolysis, as evidenced by reduction in optical density of a stationary-phase culture and concomitant release of intracellular proteins, was greatly reduced when L. lactis MG1363 cells

  2. The effect of different packaging materials on proteolysis, sensory ...

    African Journals Online (AJOL)

    Administrator

    2011-05-23

    May 23, 2011 ... In this study, tulum cheese was manufactured using raw ewe's milk and was ripened in goat's skin and plastic bags. The effect of ... terms of gross composition due to the porous structure of skin bag, which causes moisture loses during ripening. ... pasteurized milk and starter culture for tulum cheese.

  3. Proteolysis of platelet receptors in humans and other species.

    Science.gov (United States)

    Qiao, Jian L; Shen, Yang; Gardiner, Elizabeth E; Andrews, Robert K

    2010-08-01

    In the past 5 years, metalloproteinase-mediated ectodomain shedding of platelet receptors has emerged as a new mechanism for modulating platelet function. By regulating surface expression of the platelet-specific receptors, glycoprotein (GP)VI that binds collagen, and GPIbalpha (the major ligand-binding subunit of the GPIb-IX-V complex) that binds von Willebrand factor (VWF) and other procoagulant and proinflammatory ligands, shedding not only irreversibly downregulates GPVI/GPIbalpha function, but generates proteolytic fragments that might be unique biomarkers or modulators in plasma. This is potentially significant because GPVI and GPIbalpha are involved in initiating thrombotic diseases such as heart attack and stroke, as well as autoimmune diseases where anti-platelet antibodies result in thrombocytopenia. Altered expression levels of GPIbalpha/GPVI are associated with both thrombotic propensity and platelet aging, suggesting an additional role in platelet clearance. Although emerging data are elucidating molecular mechanisms underlying GPIbalpha/GPVI shedding, evidence for the functional consequences of shedding in vivo, either clinically or in animal models, is far more limited. Here we consider recent published evidence for GPVI or GPIbalpha shedding in humans, nonhuman primates and mice, and whether conservation of sheddase cleavage sites across species points to a functional role for metalloproteolytic shedding in vivo.

  4. Calpain-Dependent Proteolysis of the Androgen Receptor

    Science.gov (United States)

    2009-11-01

    Thompson, P.J. Goodman, C.M. Tangen, M.S. Lucia , G.J. Miller, L.G. Ford, M.M. Lieber, R.D. Cespedes, J.N. Atkins, S.M. Lippman, S.M. Carlin, A. Ryan...for the treatment of cancer. Oncogene 2007;26(22):3291-310. 30. Price DT, Della Rocca G, Guo C, Ballo MS, Schwinn DA, Luttrell LM. Activation of

  5. Degradation signals for ubiquitin system proteolysis in Saccharomyces cerevisiae.

    Science.gov (United States)

    Gilon, T; Chomsky, O; Kulka, R G

    1998-01-01

    Combinations of different ubiquitin-conjugating (Ubc) enzymes and other factors constitute subsidiary pathways of the ubiquitin system, each of which ubiquitinates a specific subset of proteins. There is evidence that certain sequence elements or structural motifs of target proteins are degradation signals which mark them for ubiquitination by a particular branch of the ubiquitin system and for subsequent degradation. Our aim was to devise a way of searching systematically for degradation signals and to determine to which ubiquitin system subpathways they direct the proteins. We have constructed two reporter gene libraries based on the lacZ or URA3 genes which, in Saccharomyces cerevisiae, express fusion proteins with a wide variety of C-terminal extensions. From these, we have isolated clones producing unstable fusion proteins which are stabilized in various ubc mutants. Among these are 10 clones whose products are stabilized in ubc6, ubc7 or ubc6ubc7 double mutants. The C-terminal extensions of these clones, which vary in length from 16 to 50 amino acid residues, are presumed to contain degradation signals channeling proteins for degradation via the UBC6 and/or UBC7 subpathways of the ubiquitin system. Some of these C-terminal tails share similar sequence motifs, and a feature common to almost all of these sequences is a highly hydrophobic region such as is usually located inside globular proteins or inserted into membranes. PMID:9582269

  6. Structural and mechanistic principles of intramembrane proteolysis - lessons from rhomboids

    Czech Academy of Sciences Publication Activity Database

    Stříšovský, Kvido; Vinothkumar, K. R.; Sharpe, H.; Adrain, C.; Stevens, E.; Freeman, M.

    2013-01-01

    Roč. 20, č. 1 (2013), s. 18-18 ISSN 1211-5894. [Discussions in Structural Molecular Biology. Annual Meeting of the Czech Society for Structural Biology /11./. 14.03.2013-16.03.2013, Nové Hrady] Institutional support: RVO:61388963 Keywords : intramembrane protease * rhomboid * crystal structure Subject RIV: CE - Biochemistry

  7. Physiological hypercortisolemia increases proteolysis, glutamine, and alanine production

    International Nuclear Information System (INIS)

    Darmaun, D.; Matthews, D.E.; Bier, D.M.

    1988-01-01

    Physiological elevations of plasma cortisol levels, as are encountered in stress and severe trauma, were produced in six normal subjects by infusing them with hydrocortisone for 64 h. Amino acid kinetics were measured in the postabsorptive state using three 4-h infusions of L-[1- 13 C]leucine, L-phenyl[ 2 H 5 ]phenylalanine, L-[2- 15 N]glutamine, and L-[1- 13 C]alanine tracers (1) before, (2) at 12 h, and (3) at 60 h of cortisol infusion. Before and throughout the study, the subjects ate a normal diet of adequate protein and energy intake. The cortisol infusion raised plasma cortisol levels significantly from 10 ± 1 to 32 ± 4 μg/dl, leucine flux from 83 ± 3 to 97 ± 3 μmol·kg -1 ·h -1 , and phenylalanine flux from 34 ± 1 to 39 ± 1 (SE) μmol·kg -1 ·h -1 after 12 h of cortisol infusion. These increases were maintained until the cortisol infusion was terminated. These nearly identical 15% increases in two different essential amino acid appearance rates are reflective of increased whole body protein breakdown. Glutamine flux rose by 12 h of cortisol infusion and remained elevated at the same level at 64 h. The increase in flux was primarily due to a 55% increase in glutamine de novo synthesis. Alanine flux increased with acute hypercortisolemia and increased further at 60 h of cortisol infusion, a result primarily of increased alanine de novo synthesis. Insulin, alanine, and lactate plasma levels responded similarly with significant rises between the acute and chronic periods of cortisol infusion. Thus hypercortisolemia increases both protein breakdown and the turnover of important nonessential amino acids for periods of up to 64 h

  8. Cathepsin E deficiency impairs autophagic proteolysis in macrophages.

    Directory of Open Access Journals (Sweden)

    Takayuki Tsukuba

    Full Text Available Cathepsin E is an endosomal aspartic proteinase that is predominantly expressed in immune-related cells. Recently, we showed that macrophages derived from cathepsin E-deficient (CatE(-/- mice display accumulation of lysosomal membrane proteins and abnormal membrane trafficking. In this study, we demonstrated that CatE(-/- macrophages exhibit abnormalities in autophagy, a bulk degradation system for aggregated proteins and damaged organelles. CatE(-/- macrophages showed increased accumulation of autophagy marker proteins such as LC3 and p62, and polyubiquitinated proteins. Cathepsin E deficiency also altered autophagy-related signaling pathways such as those mediated by the mammalian target of rapamycin (mTOR, Akt, and extracellular signal-related kinase (ERK. Furthermore, immunofluorescence microscopy analyses showed that LC3-positive vesicles were merged with acidic compartments in wild-type macrophages, but not in CatE(-/- macrophages, indicating inhibition of fusion of autophagosome with lysosomes in CatE(-/- cells. Delayed degradation of LC3 protein was also observed under starvation-induced conditions. Since the autophagy system is involved in the degradation of damaged mitochondria, we examined the accumulation of damaged mitochondria in CatE(-/- macrophages. Several mitochondrial abnormalities such as decreased intracellular ATP levels, depolarized mitochondrial membrane potential, and decreased mitochondrial oxygen consumption were observed. Such mitochondrial dysfunction likely led to the accompanying oxidative stress. In fact, CatE(-/- macrophages showed increased reactive oxygen species (ROS production and up-regulation of oxidized peroxiredoxin-6, but decreased antioxidant glutathione. These results indicate that cathepsin E deficiency causes autophagy impairment concomitantly with increased aberrant mitochondria as well as increased oxidative stress.

  9. Direct imaging of APP proteolysis in living cells

    Directory of Open Access Journals (Sweden)

    Niccoló Parenti

    2017-04-01

    Full Text Available Alzheimer’s disease is a multifactorial disorder caused by the interaction of genetic, epigenetic and environmental factors. The formation of cytotoxic oligomers consisting of Aβ peptide is widely accepted as being one of the main key events triggering the development of Alzheimer’s disease. Aβ peptide production results from the specific proteolytic processing of the amyloid precursor protein (APP. Deciphering the factors governing the activity of the secretases responsible for the cleavage of APP is still a critical issue. Kits available commercially measure the enzymatic activity of the secretases from cells lysates, in vitro. By contrast, we have developed a prototypal rapid bioassay that provides visible information on the proteolytic processing of APP directly in living cells. APP was fused to a monomeric variant of the green fluorescent protein and a monomeric variant of the red fluorescent protein at the C-terminal and N-terminal (mChAPPmGFP, respectively. Changes in the proteolytic processing rate in transfected human neuroblastoma and rat neuronal cells were imaged with confocal microscopy as changes in the red/green fluorescence intensity ratio. The significant decrease in the mean red/green ratio observed in cells over-expressing the β-secretase BACE1, or the α-secretase ADAM10, fused to a monomeric blue fluorescent protein confirms that the proteolytic site is still accessible. Specific siRNA was used to evaluate the contribution of endogenous BACE1. Interestingly, we found that the degree of proteolytic processing of APP is not completely homogeneous within the same single cell, and that there is a high degree of variability between cells of the same type. We were also able to follow with a fluorescence spectrometer the changes in the red emission intensity of the extracellular medium when BACE1 was overexpressed. This represents a complementary approach to fluorescence microscopy for rapidly detecting changes in the proteolytic processing of APP in real time. In order to allow the discrimination between the α- and the β-secretase activity, we have created a variant of mChAPPmGFP with a mutation that inhibits the α-secretase cleavage without perturbing the β-secretase processing. Moreover, we obtained a quantitatively robust estimate of the changes in the red/green ratio for the above conditions by using a flow cytometer able to simultaneously excite and measure the red and green fluorescence. Our novel approach lay the foundation for a bioassay suitable to study the effect of drugs or particular conditions, to investigate in an unbiased way the the proteolytic processing of APP in single living cells in order, and to elucidate the causes of the variability and the factors driving the processing of APP.

  10. Direct imaging of APP proteolysis in living cells.

    Science.gov (United States)

    Parenti, Niccoló; Del Grosso, Ambra; Antoni, Claudia; Cecchini, Marco; Corradetti, Renato; Pavone, Francesco S; Calamai, Martino

    2017-01-01

    Alzheimer's disease is a multifactorial disorder caused by the interaction of genetic, epigenetic and environmental factors. The formation of cytotoxic oligomers consisting of A β peptide is widely accepted as being one of the main key events triggering the development of Alzheimer's disease. A β peptide production results from the specific proteolytic processing of the amyloid precursor protein (APP). Deciphering the factors governing the activity of the secretases responsible for the cleavage of APP is still a critical issue. Kits available commercially measure the enzymatic activity of the secretases from cells lysates, in vitro . By contrast, we have developed a prototypal rapid bioassay that provides visible information on the proteolytic processing of APP directly in living cells. APP was fused to a monomeric variant of the green fluorescent protein and a monomeric variant of the red fluorescent protein at the C-terminal and N-terminal (mChAPPmGFP), respectively. Changes in the proteolytic processing rate in transfected human neuroblastoma and rat neuronal cells were imaged with confocal microscopy as changes in the red/green fluorescence intensity ratio. The significant decrease in the mean red/green ratio observed in cells over-expressing the β -secretase BACE1, or the α -secretase ADAM10, fused to a monomeric blue fluorescent protein confirms that the proteolytic site is still accessible. Specific siRNA was used to evaluate the contribution of endogenous BACE1. Interestingly, we found that the degree of proteolytic processing of APP is not completely homogeneous within the same single cell, and that there is a high degree of variability between cells of the same type. We were also able to follow with a fluorescence spectrometer the changes in the red emission intensity of the extracellular medium when BACE1 was overexpressed. This represents a complementary approach to fluorescence microscopy for rapidly detecting changes in the proteolytic processing of APP in real time. In order to allow the discrimination between the α - and the β -secretase activity, we have created a variant of mChAPPmGFP with a mutation that inhibits the α -secretase cleavage without perturbing the β -secretase processing. Moreover, we obtained a quantitatively robust estimate of the changes in the red/green ratio for the above conditions by using a flow cytometer able to simultaneously excite and measure the red and green fluorescence. Our novel approach lay the foundation for a bioassay suitable to study the effect of drugs or particular conditions, to investigate in an unbiased way the the proteolytic processing of APP in single living cells in order, and to elucidate the causes of the variability and the factors driving the processing of APP.

  11. Nuclear energy and investment requirements

    International Nuclear Information System (INIS)

    Voeltzel, D.

    1978-01-01

    The author assesses the investment requirements of the French nuclear programme within the framework of the national economy. He then evokes the means of financing these requirements as well as drawing attention to certain constraints which must be taken into account [fr

  12. Utility requirements for fusion power

    International Nuclear Information System (INIS)

    DeBellis, R.J.

    1977-03-01

    A four-man month study was undertaken to identify utility requirements of fusion power and define a role for the utilities in the fusion development process during the 1980s. This report, preliminary in nature, serves mainly as a planning document for future requirements analyses. A requirements organization was defined to consist of three major chronological phases: research and development, plant installation, and plant operation. Thirty-seven requirements were identified, covering all categories. In addition, training, environment, safety, licensing, and utility model were identified as five matrix-type requirements. As the requirement definition process continued during the study period, comments received from utility representatives revealed a consistency of key issues in the fusion development process. These issues form the basis for the eventual establishment of definitive roles for the utilities during the 1980s. The issues are not meant to reflect a negative view of fusion, but are items that must be solved before fusion can be introduced commercially as an electrical power source. As a result of this requirements study, preliminary candidate roles for the utilities in the fusion development process during the 1980s were identified as public education, commercialization studies, industry investment analyses, training plan implementation, alternate reactor concept development, ERDA concept design review, and requirements refinement

  13. Requirements Engineering for Pervasive Services

    NARCIS (Netherlands)

    Kolos, L.; Poulisse, Gert-Jan; van Eck, Pascal; Videira lopes, C.; Schaefer, S.; Clarke, S.; Elrad, T.; Jahnke, J.

    2005-01-01

    Developing pervasive mobile services for a mass market of end customers entails large up-front investments and therefore a good understanding of customer requirements is of paramount importance. This paper presents an approach for developing requirements engineering method that takes distinguishing

  14. Consensus standard requirements and guidance

    International Nuclear Information System (INIS)

    Putman, V.L.

    1995-01-01

    This report presents information from the ANS Criticality Alarm System Workshop relating to the consensus standard requirements and guidance. Topics presented include: definition; nomenclature; requirements and recommendations; purpose of criticality alarms; design criteria; signal characteristics; reliability, dependability and durability; tests; and emergency preparedness and planning

  15. Humidity requirements in WSCF Laboratories

    International Nuclear Information System (INIS)

    Evans, R.A.

    1994-01-01

    The purpose of this paper is to develop and document a position on Relative Humidity (RH) requirements in the WSCF Laboratories. A current survey of equipment vendors for Organic, Inorganic and Radiochemical laboratories indicate that 25% - 80% relative humidity may meet the environmental requirements for safe operation and protection of all the laboratory equipment

  16. Utility requirements for fusion power

    International Nuclear Information System (INIS)

    DeBellis, R.J.

    1977-03-01

    A four-man-month study, jointly funded by EPRI and McDonnell Douglas Astronautics Company-EAST, was undertaken to identify the utility requirements of fusion power and define a role for the utilities in the fusion development process during the 1980's. This report, preliminary in nature, serves mainly as a planning document for future requirements analyses. A requirements organization was defined to consist of three major chronological phases: research and development, plant installation, and plant operation. Thirty-seven requirements were identified, covering all categories. In addition, training, environment, safety, licensing, and utility model were identified as five matrix-type requirements. As the requirement definition process continued during the study period, comments received from utility representatives revealed a consistency of key issues in the fusion development process. These issues form the basis for the eventual establishment of definitive roles for the utilities during the 1980's. The issues are not meant to reflect a negative view of fusion, but are items which must be solved before fusion can be introduced commercially as an electrical power source. As a result of this requirements study, preliminary candidate roles for the utilities in the fusion development process during the 1980's were identified as public education, commercialization studies, industry investment analyses, training plan implementation, alternate reactor concept development, ERDA concept design review, and requirements refinement

  17. Security and trust requirements engineering

    NARCIS (Netherlands)

    Giorgini, P.; Massacci, F.; Zannone, N.; Aldini, A.; Gorrieri, R.; Martinelli, F.

    2005-01-01

    Integrating security concerns throughout the whole software development process is one of today’s challenges in software and requirements engineering research. A challenge that so far has proved difficult to meet. The major difficulty is that providing security does not only require to solve

  18. Subsurface Contamination Focus Area technical requirements. Volume 1: Requirements summary

    International Nuclear Information System (INIS)

    Nickelson, D.; Nonte, J.; Richardson, J.

    1996-10-01

    This document summarizes functions and requirements for remediation of source term and plume sites identified by the Subsurface Contamination Focus Area. Included are detailed requirements and supporting information for source term and plume containment, stabilization, retrieval, and selective retrieval remedial activities. This information will be useful both to the decision-makers within the Subsurface Contamination Focus Area (SCFA) and to the technology providers who are developing and demonstrating technologies and systems. Requirements are often expressed as graphs or charts, which reflect the site-specific nature of the functions that must be performed. Many of the tradeoff studies associated with cost savings are identified in the text

  19. The NLC Software Requirements Methodology

    Energy Technology Data Exchange (ETDEWEB)

    Shoaee, Hamid

    2002-08-20

    We describe the software requirements and development methodology developed for the NLC control system. Given the longevity of that project, and the likely geographical distribution of the collaborating engineers, the planned requirements management process is somewhat more formal than the norm in high energy physics projects. The short term goals of the requirements process are to accurately estimate costs, to decompose the problem, and to determine likely technologies. The long term goal is to enable a smooth transition from high level functional requirements to specific subsystem and component requirements for individual programmers, and to support distributed development. The methodology covers both ends of that life cycle. It covers both the analytical and documentary tools for software engineering, and project management support. This paper introduces the methodology, which is fully described in [1].

  20. Autonomous Real Time Requirements Tracing

    Science.gov (United States)

    Plattsmier, George; Stetson, Howard

    2014-01-01

    One of the more challenging aspects of software development is the ability to verify and validate the functional software requirements dictated by the Software Requirements Specification (SRS) and the Software Detail Design (SDD). Insuring the software has achieved the intended requirements is the responsibility of the Software Quality team and the Software Test team. The utilization of Timeliner-TLX(sup TM) Auto- Procedures for relocating ground operations positions to ISS automated on-board operations has begun the transition that would be required for manned deep space missions with minimal crew requirements. This transition also moves the auto-procedures from the procedure realm into the flight software arena and as such the operational requirements and testing will be more structured and rigorous. The autoprocedures would be required to meet NASA software standards as specified in the Software Safety Standard (NASASTD- 8719), the Software Engineering Requirements (NPR 7150), the Software Assurance Standard (NASA-STD-8739) and also the Human Rating Requirements (NPR-8705). The Autonomous Fluid Transfer System (AFTS) test-bed utilizes the Timeliner-TLX(sup TM) Language for development of autonomous command and control software. The Timeliner-TLX(sup TM) system has the unique feature of providing the current line of the statement in execution during real-time execution of the software. The feature of execution line number internal reporting unlocks the capability of monitoring the execution autonomously by use of a companion Timeliner-TLX(sup TM) sequence as the line number reporting is embedded inside the Timeliner-TLX(sup TM) execution engine. This negates I/O processing of this type data as the line number status of executing sequences is built-in as a function reference. This paper will outline the design and capabilities of the AFTS Autonomous Requirements Tracker, which traces and logs SRS requirements as they are being met during real-time execution of the

  1. Development of transportation operations requirements

    International Nuclear Information System (INIS)

    Grady, S.T.; Best, R.E.; Danese, F.L.; Peterson, R.W.; Pope, R.B.

    1990-01-01

    Transport conditions at various utility sties vary dramatically in terms of characteristics at and near the site, requirements, administrative procedures, and other factors. Continuation of design efforts for the OCRWM transportation operations system requires that the operating requirements for the transportation system -- quantity of fuel per unit time per site -- be identified so that the effect the variations have on the system can be accommodated. The approach outlined in this paper provides for an identification of specific sites, evaluation of shipment capabilities at each site, and integration of the sites into multi-site shipping campaigns to scope the logistics management problem for the transportation operations system. 1 fig., 1 tab

  2. Signage Required Term and Condition

    Science.gov (United States)

    The recipient is required to place a sign at construction sites supported under this award displaying the EPA logo in a manner that informs the public that the project is funded in part or wholly by the EPA.

  3. LH2 airport requirements study

    Science.gov (United States)

    Brewer, G. D. (Editor)

    1976-01-01

    A preliminary assessment of the facilities and equipment which will be required at a representative airport is provided so liquid hydrogen LH2 can be used as fuel in long range transport aircraft in 1995-2000. A complete facility was conceptually designed, sized to meet the projected air traffic requirement. The facility includes the liquefaction plant, LH2, storage capability, and LH2 fuel handling system. The requirements for ground support and maintenance for the LH2 fueled aircraft were analyzed. An estimate was made of capital and operating costs which might be expected for the facility. Recommendations were made for design modifications to the reference aircraft, reflecting results of the analysis of airport fuel handling requirements, and for a program of additional technology development for air terminal related items.

  4. Deaf mobile application accessibility requirements

    Science.gov (United States)

    Nathan, Shelena Soosay; Hussain, Azham; Hashim, Nor Laily

    2016-08-01

    Requirement for deaf mobile applications need to be analysed to ensure the disabilities need are instilled into the mobile applications developed for them. Universal design is understandable to comply every user needs, however specific disability is argued by the authors to have different need and requirements. These differences are among the reasons for these applications being developed to target for a specific group of people, however they are less usable and later abandoned. This study focuses on deriving requirements that are needed by the deaf in their mobile applications that are meant specifically for them. Studies on previous literature was conducted it can be concluded that graphic, text, multimedia and sign language interpreter are among mostly required features to be included in their mobile application to ensure the applications are usable for this community.

  5. Guest Comment: Universal Language Requirement.

    Science.gov (United States)

    Sherwood, Bruce Arne

    1979-01-01

    Explains that reading English among Scientists is almost universal, however, there are enormous problems with spoken English. Advocates the use of Esperanto as a viable alternative, and as a language requirement for graduate work. (GA)

  6. Fusion technology status and requirements

    International Nuclear Information System (INIS)

    Thomassen, K.I.

    1982-01-01

    This paper summarizes the status of fusion technology and discusses the requirements to be met in order to build a demonstration fusion plant. Strategies and programmatic considerations in pursuing engineering feasibility are also outlined

  7. Grant Closeout Requirements and Reports

    Science.gov (United States)

    Requirements and reports to comply with grant closeout, including Final Federal Financial Report (FFR, SF425); Final Research Performance Progress Report (FRPPR); Interim Research Performance Progress Report (IRPPR); Final Invention Statement (FIS, HHS

  8. Electrocardiogram Scanner-System Requirements

    Science.gov (United States)

    1973-03-01

    An experimental and analytical study has been conducted to establish the feasibility for scanning and digitizing electrocardiogram records. The technical requirements and relative costs for two systems are discussed herein. One is designed to automat...

  9. PFP requirements development planning guide

    International Nuclear Information System (INIS)

    SINCLAIR, J.C.

    1999-01-01

    The PFP Requirements Development Planning Guide presents the strategy and process used for the identification, allocation, and maintenance of requirements within the Plutonium Finishing Plant (PFP) integrated project baseline. Future revisions to this document will be included as attachments (e.g., results of the PFP Requirements Analysis attributable to this approach). This document is intended be a Project-owned management tool. As such, this document will periodically require revisions resulting from improvements of the information, processes, and techniques as now described. Future updates may be made to this document by PFP management and final approval of the content will be accomplished in a Baseline Change Request as it impacts the Multi-Year Work Plan, or baseline information managed in the Hanford Site Systems Engineering Baseline

  10. Requirements for Space Settlement Design

    Science.gov (United States)

    Gale, Anita E.; Edwards, Richard P.

    2004-02-01

    When large space settlements are finally built, inevitably the customers who pay for them will start the process by specifying requirements with a Request for Proposal (RFP). Although we are decades away from seeing the first of these documents, some of their contents can be anticipated now, and provide insight into the variety of elements that must be researched and developed before space settlements can happen. Space Settlement Design Competitions for High School students present design challenges in the form of RFPs, which predict basic requirements for space settlement attributes in the future, including structural features, infrastructure, living conveniences, computers, business areas, and safety. These requirements are generically summarized, and unique requirements are noted for specific space settlement locations and applications.

  11. Capturing Requirements for Autonomous Spacecraft with Autonomy Requirements Engineering

    Science.gov (United States)

    Vassev, Emil; Hinchey, Mike

    2014-08-01

    The Autonomy Requirements Engineering (ARE) approach has been developed by Lero - the Irish Software Engineering Research Center within the mandate of a joint project with ESA, the European Space Agency. The approach is intended to help engineers develop missions for unmanned exploration, often with limited or no human control. Such robotics space missions rely on the most recent advances in automation and robotic technologies where autonomy and autonomic computing principles drive the design and implementation of unmanned spacecraft [1]. To tackle the integration and promotion of autonomy in software-intensive systems, ARE combines generic autonomy requirements (GAR) with goal-oriented requirements engineering (GORE). Using this approach, software engineers can determine what autonomic features to develop for a particular system (e.g., a space mission) as well as what artifacts that process might generate (e.g., goals models, requirements specification, etc.). The inputs required by this approach are the mission goals and the domain-specific GAR reflecting specifics of the mission class (e.g., interplanetary missions).

  12. Puberty menorrhagia Requiring Inpatient Admission

    Directory of Open Access Journals (Sweden)

    AH Khosla

    2010-06-01

    Full Text Available INTRODUCTION: Puberty menorrhagia is a significant health problem in adolescent age group and severe cases may require admission and blood transfusion. Aim of this study was to evaluate the causes, associated complications and management of puberty menorrhagia. METHODS: Hospital records of all patients of puberty menorrhagia requiring admission were analyzed for etiology, duration since menarche, duration of bleeding, investigation profile and management. RESULTS: There were 18 patients of puberty menorrhagia requiring hospital admission. Etiology was anovulatory bleeding in 11 patients, bleeding disorders in five which included idiopathic thrombocytopenia purpura in three and one each with Von-Willebrand disease and leukemia. Two patients had hypothyroidism as the cause. Fourteen patients presented with severe anaemia and required blood transfusion. All except one responded to oral hormonal therapy. CONCLUSIONS: Puberty menorrhagia can be associated with severe complications and requiring blood transfusion. Although most common cause is anovulation but bleeding disorder, other medical condition and other organic causes must be ruled out in any patient of Puberty menorrhagia. KEYWORDS: anovulation, bleeding disorder, puberty, menorrhagia, anaemia.

  13. The JPL functional requirements tool

    Science.gov (United States)

    Giffin, Geoff; Skinner, Judith; Stoller, Richard

    1987-01-01

    Planetary spacecraft are complex vehicles which are built according to many thousands of requirements. Problems encountered in documenting and maintaining these requirements led to the current attempt to reduce or eliminate these problems by a computer automated data base Functional Requirements Tool. The tool developed at JPL and in use on several JPL Projects is described. The organization and functionality of the Tool, together with an explanation of the data base inputs, their relationships, and use are presented. Methods of interfacing with external documents, representation of tables and figures, and methods of approval and change processing are discussed. The options available for disseminating information from the Tool are identified. The implementation of the Requirements Tool is outlined, and the operation is summarized. The conclusions drawn from this work is that the Requirements Tool represents a useful addition to the System Engineer's Tool kit, it is not currently available elsewhere, and a clear development path exists to expand the capabilities of the Tool to serve larger and more complex projects.

  14. Quality Assurance Requirements and Description

    International Nuclear Information System (INIS)

    Ram Murthy

    2002-01-01

    The Quality Assurance Requirements and Description (QARD) is the principal Quality Assurance (QA) document for the Civilian Radioactive Waste Management Program (Program). It establishes the minimum requirements for the QA program [INTRODUCTION :1p2s (NOT A REQUIREMENT)]. The QARD contains regulatory requirements and program commitments necessary for the development of an effective QA program [INTRODUCTION :1p3s (NOT A REQUIREMENT)]. Implementing documents must be based on, and be consistent with the QARD. The QARD applies to the following: (1) Acceptance of spent nuclear fuel and high-level waste. (2) Transport of spent nuclear fuel and high-level waste. (3) Storage of spent nuclear fuel through receipt of storage cask certification or a facility operating license. (4) Monitored Geologic Repository, including the site characterization activities [Exploratory Studies Facility (ESF) and surface based testing], through receipt of an operating license. (5) High-level waste form development through qualification, production, and acceptance. (6) Characterization of DOE spent nuclear fuel, and conditioning through acceptance of DOE spent nuclear fuel. Section 2.0, Quality Assurance Program, defines in greater detail criteria for determining work subject to the QARD

  15. Authorization basis requirements comparison report

    Energy Technology Data Exchange (ETDEWEB)

    Brantley, W.M.

    1997-08-18

    The TWRS Authorization Basis (AB) consists of a set of documents identified by TWRS management with the concurrence of DOE-RL. Upon implementation of the TWRS Basis for Interim Operation (BIO) and Technical Safety Requirements (TSRs), the AB list will be revised to include the BIO and TSRs. Some documents that currently form part of the AB will be removed from the list. This SD identifies each - requirement from those documents, and recommends a disposition for each to ensure that necessary requirements are retained when the AB is revised to incorporate the BIO and TSRs. This SD also identifies documents that will remain part of the AB after the BIO and TSRs are implemented. This document does not change the AB, but provides guidance for the preparation of change documentation.

  16. Fusion Energy Sciences Network Requirements

    Energy Technology Data Exchange (ETDEWEB)

    Dart, Eli [ESNet, Berkeley, CA (United States); Tierney, Brian [ESNet, Berkeley, CA (United States)

    2012-09-26

    The Energy Sciences Network (ESnet) is the primary provider of network connectivity for the U.S. Department of Energy Office of Science, the single largest supporter of basic research in the physical sciences in the United States. In support of the Office of Science programs, ESnet regularly updates and refreshes its understanding of the networking requirements of the instruments, facilities, scientists, and science programs that it serves. This focus has helped ESnet to be a highly successful enabler of scientific discovery for over 25 years. In December 2011, ESnet and the Office of Fusion Energy Sciences (FES), of the DOE Office of Science (SC), organized a workshop to characterize the networking requirements of the programs funded by FES. The requirements identified at the workshop are summarized in the Findings section, and are described in more detail in the body of the report.

  17. Non-functional Avionics Requirements

    Science.gov (United States)

    Paulitsch, Michael; Ruess, Harald; Sorea, Maria

    Embedded systems in aerospace become more and more integrated in order to reduce weight, volume/size, and power of hardware for more fuel-effi ciency. Such integration tendencies change architectural approaches of system ar chi tec tures, which subsequently change non-functional requirements for plat forms. This paper provides some insight into state-of-the-practice of non-func tional requirements for developing ultra-critical embedded systems in the aero space industry, including recent changes and trends. In particular, formal requi re ment capture and formal analysis of non-functional requirements of avionic systems - including hard-real time, fault-tolerance, reliability, and per for mance - are exemplified by means of recent developments in SAL and HiLiTE.

  18. Authorization basis requirements comparison report

    International Nuclear Information System (INIS)

    Brantley, W.M.

    1997-01-01

    The TWRS Authorization Basis (AB) consists of a set of documents identified by TWRS management with the concurrence of DOE-RL. Upon implementation of the TWRS Basis for Interim Operation (BIO) and Technical Safety Requirements (TSRs), the AB list will be revised to include the BIO and TSRs. Some documents that currently form part of the AB will be removed from the list. This SD identifies each - requirement from those documents, and recommends a disposition for each to ensure that necessary requirements are retained when the AB is revised to incorporate the BIO and TSRs. This SD also identifies documents that will remain part of the AB after the BIO and TSRs are implemented. This document does not change the AB, but provides guidance for the preparation of change documentation

  19. Overview of SSC accelerator requirements

    International Nuclear Information System (INIS)

    Dugan, G.

    1992-03-01

    This paper will present a general overview of the requirements of the Superconducting Super Collider (SSC) accelerators. Each accelerator in the injector chain will be discussed separately, followed by a discussion of the collider itself. In conclusion, the top level requirements of the overall accelerator system will be presented. For each accelerator, the primary operating parameters will be presented in tabular form. A brief narrative discussion of the principal technical features of each machine will be given. Finally, the principal technical design challenges for the machine will be noted, together with the currently planned solution to these challenges

  20. Containment leakage rate testing requirements

    International Nuclear Information System (INIS)

    Arndt, E.G.

    1992-01-01

    This report presents the status of several documents under revision or development that provide requirements and guidance for testing nuclear power plant containment systems for leakage rates. These documents include the general revision to 10 CFR Part 50, Appendix J; the regulatory guide affiliated with the revision to Appendix J; the national standard that the regulatory guide endorses, ANSI/ANS-56.8, 'Containment System Leakage Rate Testing Requirements'; and the draft industry Licensing Topical Report, 'Standardized Program for Primary Containment Integrity Testing'. The actual or potential relationships between these documents are also explored

  1. Requirements Modeling with Agent Programming

    Science.gov (United States)

    Dasgupta, Aniruddha; Krishna, Aneesh; Ghose, Aditya K.

    Agent-oriented conceptual modeling notations are highly effective in representing requirements from an intentional stance and answering questions such as what goals exist, how key actors depend on each other, and what alternatives must be considered. In this chapter, we review an approach to executing i* models by translating these into set of interacting agents implemented in the CASO language and suggest how we can perform reasoning with requirements modeled (both functional and non-functional) using i* models. In this chapter we particularly incorporate deliberation into the agent design. This allows us to benefit from the complementary representational capabilities of the two frameworks.

  2. Buddy Tag CONOPS and Requirements.

    Energy Technology Data Exchange (ETDEWEB)

    Brotz, Jay Kristoffer [Sandia National Lab. (SNL-NM), Albuquerque, NM (United States); Deland, Sharon M. [Sandia National Lab. (SNL-NM), Albuquerque, NM (United States)

    2015-12-01

    This document defines the concept of operations (CONOPS) and the requirements for the Buddy Tag, which is conceived and designed in collaboration between Sandia National Laboratories and Princeton University under the Department of State Key VerificationAssets Fund. The CONOPS describe how the tags are used to support verification of treaty limitations and is only defined to the extent necessary to support a tag design. The requirements define the necessary functions and desired non-functional features of the Buddy Tag at a high level

  3. Requirements Engineering: Solutions and Trends

    NARCIS (Netherlands)

    Ebert, C.; Wieringa, Roelf J.; Aurum, A.; Wohlin, C.

    2005-01-01

    This last chapter of the book describes solutions and trends in the discipline of RE. Starting from a wrap-up of what was presented throughout this book, it suggests a framework of requirements engineering and indicates what current solutions are available in this framework. Beyond providing a short

  4. Utilizing inheritance in requirements engineering

    Science.gov (United States)

    Kaindl, Hermann

    1994-01-01

    The scope of this paper is the utilization of inheritance for requirements specification, i.e., the tasks of analyzing and modeling the domain, as well as forming and defining requirements. Our approach and the tool supporting it are named RETH (Requirements Engineering Through Hypertext). Actually, RETH uses a combination of various technologies, including object-oriented approaches and artificial intelligence (in particular frames). We do not attempt to exclude or replace formal representations, but try to complement and provide means for gradually developing them. Among others, RETH has been applied in the CERN (Conseil Europeen pour la Rechereche Nucleaire) Cortex project. While it would be impossible to explain this project in detail here, it should be sufficient to know that it deals with a generic distributed control system. Since this project is not finished yet, it is difficult to state its size precisely. In order to give an idea, its final goal is to substitute the many existing similar control systems at CERN by this generic approach. Currently, RETH is also tested using real-world requirements for the Pastel Mission Planning System at ESOC in Darmstadt. First, we outline how hypertext is integrated into a frame system in our approach. Moreover, the usefulness of inheritance is demonstrated as performed by the tool RETH. We then summarize our experiences of utilizing inheritance in the Cortex project. Lastly, RETH will be related to existing work.

  5. An Introduction to Requirements Traceability

    NARCIS (Netherlands)

    Wieringa, Roelf J.

    This report surveys the requirements traceability literature and gives some recommendations for further research and for an approach to consultancy concerning traceability in the 2RARE project. The problem of maintaining traceability in a development project is viewed as the problem of maintaining

  6. 76 FR 39259 - Manual Requirements

    Science.gov (United States)

    2011-07-06

    ... established new requirements for the certification, operation, and maintenance of light-sport aircraft under...-6474; e-mail [email protected] . SUPPLEMENTARY INFORMATION: The FAA published a final rule entitled ``Certification of Aircraft and Airmen for the Operation of Light-Sport Aircraft,'' in the Federal...

  7. Requirements in Functional IT Management

    NARCIS (Netherlands)

    Blaauboer, F.A.; Blaauboer, F.A.

    Requirements engineering and functional IT management have never been researched as to containing similar activities. This paper describes and compares both disciplines, where the BiSL-framework is used for functional IT management. The similarities and differences between the two disciplines are

  8. Requirements management: A CSR's perspective

    Science.gov (United States)

    Thompson, Joanie

    1991-01-01

    The following subject areas are covered: customer service overview of network service request processing; Customer Service Representative (CSR) responsibility matrix; extract from a sample Memorandum of Understanding; Network Service Request Form and its instructions sample notification of receipt; and requirements management in the NASA Science Internet.

  9. Requirements for flexible learner monitoring

    NARCIS (Netherlands)

    Glahn, Christian; Specht, Marcus; Koper, Rob

    2007-01-01

    Glahn, C., Specht, M., & Koper, R. (2007). Requirements for flexible learner monitoring. In T. Navarette, J. Blat & R. Koper (Eds.). Proceedings of the 3rd TENCompetence Open Workshop 'Current Research on IMS Learning Design and Lifelong Competence Development Infrastructures' (pp. 89-96). June,

  10. SRS control system upgrade requirements

    International Nuclear Information System (INIS)

    Hill, L.F.

    1998-01-01

    This document defines requirements for an upgrade of the Sodium Removal System (SRS) control system. The upgrade is being performed to solve a number of maintainability and operability issues. The upgraded system will provide the same functions, controls and interlocks as the present system, and in addition provide enhanced functionality in areas discussed in this document

  11. Coke quality requirements in POSCO

    Energy Technology Data Exchange (ETDEWEB)

    Song, J.; Yi, J.; Wang, H. [POSCO (Republic of Korea). Cokemaking Dept.

    2001-07-01

    The 26 overheads describes coke quality requirements in POSCO, Republic of Korea. It is concluded that it is necessary to develop new coal and to prompt technical development in order to produce high quality coke. To improve coke quality. Posco had applied DMAIC (define, measurement, analysis, improvement, control) technique which is part of Six-Sigma activity.

  12. Conversion of dependability deterministic requirements into probabilistic requirements

    International Nuclear Information System (INIS)

    Bourgade, E.; Le, P.

    1993-02-01

    This report concerns the on-going survey conducted jointly by the DAM/CCE and NRE/SR branches on the inclusion of dependability requirements in control and instrumentation projects. Its purpose is to enable a customer (the prime contractor) to convert into probabilistic terms dependability deterministic requirements expressed in the form ''a maximum permissible number of failures, of maximum duration d in a period t''. The customer shall select a confidence level for each previously defined undesirable event, by assigning a maximum probability of occurrence. Using the formulae we propose for two repair policies - constant rate or constant time - these probabilized requirements can then be transformed into equivalent failure rates. It is shown that the same formula can be used for both policies, providing certain realistic assumptions are confirmed, and that for a constant time repair policy, the correct result can always be obtained. The equivalent failure rates thus determined can be included in the specifications supplied to the contractors, who will then be able to proceed to their previsional justification. (author), 8 refs., 3 annexes

  13. Physical requirements in Olympic sailing

    DEFF Research Database (Denmark)

    Bojsen-Møller, J; Larsson, B; Aagaard, Per

    2015-01-01

    Abstract Physical fitness and muscular strength are important performance parameters in Olympic sailing although their relative importance changes between classes. The Olympic format consists of eight yacht types combined into 10 so-called events with total 15 sailors (male and female) in a compl...... to yacht types, and reviews the existing knowledge on physical requirements in modern Olympic sailing. Finally, recommendations for future research in sailing are given.......Abstract Physical fitness and muscular strength are important performance parameters in Olympic sailing although their relative importance changes between classes. The Olympic format consists of eight yacht types combined into 10 so-called events with total 15 sailors (male and female......) in a complete national Olympic delegation. The yachts have different requirements with respect to handling, and moreover, each sailor plays a specific role when sailing. Therefore physical demands remain heterogeneous for Olympic sailors. Previous studies have mainly examined sailors where 'hiking' (the task...

  14. ROS signalling - specificity is required

    DEFF Research Database (Denmark)

    Møller, Ian M; Sweetlove, Lee J

    2010-01-01

    Reactive oxygen species (ROS) production increases in plants under stress. ROS can damage cellular components, but they can also act in signal transduction to help the cell counteract the oxidative damage in the stressed compartment. H2O2 might induce a general stress response, but it does not have...... the required specificity to selectively regulate nuclear genes required for dealing with localized stress, e.g. in chloroplasts or mitochondria. Here we argue that peptides deriving from proteolytic breakdown of oxidatively damaged proteins have the requisite specificity to act as secondary ROS messengers...... and regulate source-specific genes and in this way contribute to retrograde ROS signalling during oxidative stress. Likewise, unmodified peptides deriving from the breakdown of redundant proteins could help coordinate organellar and nuclear gene expression...

  15. The European Utility Requirement Document

    International Nuclear Information System (INIS)

    Roche, I.I.

    1999-01-01

    The major European electricity producers work on a common requirement document for future LWR plants since 1992. They aim at requirements acceptable together by the owners, the public and the authorities. Thus the designers can develop standard LWR designs acceptable everywhere in Europe and the utilities can open their consultations to vendors on common bases. Such a standardisation promotes an improvement of generation costs and of safety : public and authorities acceptance should be improved as well ; significant savings are expected in development and construction costs. Since the early stages of the project, the EUR group has grown significantly. It now includes utilities from nine European countries. Utilities from two other European countries are joining the group. Specific cooperation agreements are also in progress with a few extra-European partners

  16. Grading of quality assurance requirements

    International Nuclear Information System (INIS)

    1991-01-01

    The present Manual provides guidance and illustrative examples for applying a method by which graded quality assurance requirements may be determined and adapted to the items and services of a nuclear power plant in conformance with the requirements of the IAEA Nuclear Safety Standards (NUSS) Code and Safety Guides on quality assurance. The Manual replaces the previous publication IAEA-TECDOC-303 on the same subject. Various methods of grading quality assurance are available in a number of Member States. During the development of the present Manual it was not considered practical to attempt to resolve the differences between those methods and it was preferred to identify and benefit from the good practices available in all the methods. The method presented in this Manual deals with the aspects of management, documentation, control, verification and administration which affect quality. 1 fig., 4 tabs

  17. Documentation requirements for radiation sterilization

    DEFF Research Database (Denmark)

    Miller, A.

    1995-01-01

    Several standards are recently approved or are under development by the standard organizations ISO and CEN in the field of radiation sterilization. Particularly in Europe these standards define new requirements on some issues and on other issues they emphasize the necessary documentation for appr......Several standards are recently approved or are under development by the standard organizations ISO and CEN in the field of radiation sterilization. Particularly in Europe these standards define new requirements on some issues and on other issues they emphasize the necessary documentation...... for approval of radiation sterilized products. The impact of these standards on the radiation sterilization is discussed, with special attention given to a few special issues, mainly traceability and uncertainty of measurement results....

  18. Regulatory requirements for radiation protection

    International Nuclear Information System (INIS)

    Mason, E.A.; Cunningham, R.E.; Hard, J.E.; Mattson, R.J.; Smith, R.D.; Peterson, H.T. Jr.

    1977-01-01

    Regulatory requirements for radiation protection have evolved and matured over several decades. Due to the wide adoption of recommendations of the International Commission on Radiation Protection (ICRP), there exists international agreement on the principles to be followed for radiation protection. This foundation will be increasingly important due to the growing need for international agreements and standards for radiation protection and radioactive materials management. During the infancy of the commercial nuclear industry, primary reliance was placed on the protection of the individual, both in the work force and as a member of the public. With the growth of nuclear power in the 1960's and 1970's, environmental impact assessments and expert reviews of bio-effects data have focused attention on statistical risks to large population groups and the use of the collective dose commitment concept to estimate potential effects. The potential release of long-lived radionuclides from the nuclear fuel cycle requires further consideration of radionuclide accumulation in the biosphere and calls for controls conceived and implemented at the international level. The initial development efforts for addressing these concerns already have been instituted by the ICRP and the IAEA. However, formal international agreements and a unified set of international standards may be required to implement the recommendations of these groups. Further international efforts in the field of radiation protection are also called for in developing waste management practices and radioactive effluent control technology, in site selection for fuel reprocessing plants and waste dispersal facilities, and for ensuring safe transport of high-level wastes in various forms. Since the regulation of very low dose rates and doses will be involved, it will be useful to reexamine dose-effect relationships and societal goals for health protection. Improved criteria and methodologies for ''as low as readily

  19. Water requirements of selected industries

    Science.gov (United States)

    ,; Mussey, Orville D.; Conklin, Howard L.; Durfor, Charles N.; Otts, Louis Ethelbert; Walling, Faulkner B.

    1955-01-01

    The early industries in America generally were established when and where demands for the products of industry arose. Most of the early industries were so located that their increasing requirements for transportation, raw materials, market, labor, and water supply could be satisfied economically. Many of these original plant locations have continued as modern industrial centers and their output has increased manyfold in meeting the demands of our growing Nation. The recent and current industrial expansion and the trend toward the growth of chemical industries, many Of which are heavy users of water, has resulted in a tremendous increase in the total withdrawal of water for industrial use as well as a large increase in the per capita use of water. This increase in industrial water requirement has strained the capacity of the developed water supplies in many areas, and in some instances the adequacy of the potential water supplies is questionable. The Geological Survey is engaged in preparing and publishing a series of reports describing the developed and undeveloped water resources of many important industrial areas. This work was started initially at the request of the National Securities Resources Board as a means to insure that water supplies are adequate for our rapidly expanding industrial development. Although many factors contribute to establishing the feasibility or even the limits of future industrial development, the one relating to available water supply is extremely important. A knowledge of the water requirements of various industries is valuable therefore in planning the logical development in any area where water supply is a critical factor. Thus far very little suitable information on the water requirements of our major industries is available for general planning. An inventory of unit water-use values in industry therefore would be generally helpful and also might tend to stimulate water-conservation methods. To obtain such information

  20. Accelerator requirments for strategic defense

    International Nuclear Information System (INIS)

    Gullickson, R.L.

    1987-01-01

    The authors discuss how directed energy applications require accelerators with high brightness and large gradients to minimize size and weight for space systems. Several major directed energy applications are based upon accelerator technology. The radio-frequency linear accelerator is the basis for both space-based neutral particle beam (NPB) and free electron laser (FEL) devices. The high peak current of the induction linac has made it a leading candidate for ground based free electron laser applications

  1. Physical system requirements - Accept waste

    International Nuclear Information System (INIS)

    1992-08-01

    The Nuclear Waste Policy Act (NWPA) assigned to the Department of Energy (DOE) the responsibility for managing the disposal of spent nuclear fuel and high-level radioactive waste and established the Office of Civilian Radioactive Waste Management (OCRWM) for that purpose. The Secretary of Energy, in his November 1989 report to Congress (DOE/RW-0247), announced new initiatives for the conduct of the Civilian Radioactive Waste Management (CRWM) program. One of these initiatives was to establish improved management structure and procedures. In response, OCRWM performed a management study and the OCRWM Director subsequently issued the Management Systems improvement Strategy (MSIS) on August 10, 1990, calling for a rigorous implementation of systems engineering principles with a special emphasis on functional analysis. The functional analysis approach establishes a framework for integrating the program management efforts with the technical requirements analysis into a single, unified, and consistent program. This approach recognizes that just as the facilities and equipment comprising the physical waste management system must perform certain functions, so must certain programmatic and management functions be performed within the program in order to successfully bring the physical system into being. Thus, a comprehensive functional analysis effort has been undertaken which is intended to: Identify the functions that must be performed to fulfill the waste disposal mission; Identify the corresponding requirements imposed on each of the functions; and Identify the conceptual architecture that will be used to satisfy the requirements. The principal purpose of this requirements document is to present the results that were obtained from the conduct of a functional analysis effort for the Accept Waste mission

  2. Requirements for light water reactors

    International Nuclear Information System (INIS)

    Hedin, F.

    2009-01-01

    The EUR (European Utilities Requirements) is an organization founded in 1991 whose aim was to write down the European specifications and requirements for the future reactors of third generation. EUR gathers most of the nuclear power producers of Europe. The EUR document has been built on the large and varied experience of EUR members and can be used to elaborate invitations to tender for nuclear projects. 4000 requirements only for the nuclear part of the plant are listed, among which we have: -) the probability of core meltdown for a reactor must be less than 10 -6 per year, -) the service life of every component that is not replaceable must be 60 years, -) the capacity of the spent fuel pool must be sufficient to store 10-15 years of production without clearing out. The EUR document is both open and complete: every topic has been considered, it does not favor any type of reactor but can ban any technology that is too risky or has an unfavourable feedback experience. The assessment of the conformity with the EUR document of 7 reactor projects (BWR 90/, EPR, EP1000, SWR1000, ABWR, AP1000 and VVER-AES-92) has already be made. (A.C.)

  3. 2008 wind farm submission requirements

    Energy Technology Data Exchange (ETDEWEB)

    NONE

    2008-05-01

    Wind turbines used to generate electricity can have important visual, environmental and amenity impacts depending on their location. Planning can contribute to the provision of renewable energy by enabling wind energy development in appropriate locations in a manner that appropriately balances their environmental, social and economic benefits with any demonstrated visual, environmental and amenity impacts. The County of Bruce, Ontario and member municipalities are attempting to balance these matters in favour of an overall net community benefit. Planning applications need to include sufficient information and explanation to allow the approval authorities to come to a sound and timely decision, in order to facilitate a viable wind energy industry. This document outlined the County of Bruce application requirements in order to assist in the design and siting of proposed wind energy facilities and in preparing planning applications. The County of Bruce official plan and policies for large wind energy conversion systems (LWECS) were presented. Submissions requirements include an environmental screening report; a federal environmental assessment clearance; a prime agricultural land justification report; a general project description; turbine specifications; noise evaluation; shadow flicker calculations; visual effect modelling; NAV Canada/Transport Canada clearance; grid connections and routing; project staging; overshadowing study; electromagnetic interference study; turbine foundation drawings; and an environmental management plan. Other requirements include lot level maps; project area maps; sensitive receptor table; shadow flicker; stage 2 archaeological study; type certification; statement of planning policy conformity; and a submission checklist. 6 refs., 1 tab., 3 appendices.

  4. Misplaced Cervical Screws Requiring Reoperation.

    Science.gov (United States)

    Peterson, Jeremy C; Arnold, Paul M; Smith, Zachary A; Hsu, Wellington K; Fehlings, Michael G; Hart, Robert A; Hilibrand, Alan S; Nassr, Ahmad; Rahman, Ra'Kerry K; Tannoury, Chadi A; Tannoury, Tony; Mroz, Thomas E; Currier, Bradford L; De Giacomo, Anthony F; Fogelson, Jeremy L; Jobse, Bruce C; Massicotte, Eric M; Riew, K Daniel

    2017-04-01

    A multicenter, retrospective case series. In the past several years, screw fixation of the cervical spine has become commonplace. For the most part, this is a safe, low-risk procedure. While rare, screw backout or misplaced screws can lead to morbidity and increased costs. We report our experiences with this uncommon complication. A multicenter, retrospective case series was undertaken at 23 institutions in the United States. Patients were included who underwent cervical spine surgery from January 1, 2005, to December 31, 2011, and had misplacement of screws requiring reoperation. Institutional review board approval was obtained at all participating institutions, and detailed records were sent to a central data center. A total of 12 903 patients met the inclusion criteria and were analyzed. There were 11 instances of screw backout requiring reoperation, for an incidence of 0.085%. There were 7 posterior procedures. Importantly, there were no changes in the health-related quality-of-life metrics due to this complication. There were no new neurologic deficits; a patient most often presented with pain, and misplacement was diagnosed on plain X-ray or computed tomography scan. The most common location for screw backout was C6 (36%). This study represents the largest series to tabulate the incidence of misplacement of screws following cervical spine surgery, which led to revision procedures. The data suggest this is a rare event, despite the widespread use of cervical fixation. Patients suffering this complication can require revision, but do not usually suffer neurologic sequelae. These patients have increased cost of care. Meticulous technique and thorough knowledge of the relevant anatomy are the best means of preventing this complication.

  5. Requirements on high resolution detectors

    Energy Technology Data Exchange (ETDEWEB)

    Koch, A. [European Synchrotron Radiation Facility, Grenoble (France)

    1997-02-01

    For a number of microtomography applications X-ray detectors with a spatial resolution of 1 {mu}m are required. This high spatial resolution will influence and degrade other parameters of secondary importance like detective quantum efficiency (DQE), dynamic range, linearity and frame rate. This note summarizes the most important arguments, for and against those detector systems which could be considered. This article discusses the mutual dependencies between the various figures which characterize a detector, and tries to give some ideas on how to proceed in order to improve present technology.

  6. Testing requirements for SCWR radiolysis

    International Nuclear Information System (INIS)

    Guzonas, D.; Stuart, C.; Jay-Gerin, J.-P.; Meesungnoen, J.

    2010-01-01

    The Generation IV supercritical water-cooled reactor (SCWR) designs under consideration would operate at ∼430-625 o C and 25 MPa, i.e., well beyond the thermodynamic critical point of water. Supercritical (light/heavy) water (SCW)-cooled reactors would enable thermodynamic cycle efficiencies as high as ∼44% (versus ∼33% for existing water reactors), thus generating lower-cost electricity. The purpose of this report is to survey the literature on SCW radiolysis, describe the recent modeling performed at the Universite de Sherbrooke, and to broadly outline the testing required to develop a benchmarked SCW radiolysis model. (author)

  7. The development of safety requirements

    International Nuclear Information System (INIS)

    Jorel, M.

    2009-01-01

    This document describes the safety approach followed in France for the design of nuclear reactors. This safety approach is based on safety principles from which stem safety requirements that set limiting values for specific parameters. The improvements in computerized simulation, the use of more adequate new materials, a better knowledge of the concerned physical processes, the changes in the reactor operations (higher discharge burnups for instance) have to be taken into account for the definition of safety criteria and the setting of limiting values. The developments of the safety criteria linked to the risks of cladding failure and loss of primary coolant are presented. (A.C.)

  8. Energy requirements of tire pulling.

    Science.gov (United States)

    Fredriksen, Per M; Mamen, Asgeir

    2017-10-01

    We have investigated the effect using walking poles and pulling tires at 4 and 6 km·h-1 (1.11 and 1.67 m·s-1) speeds on oxygen uptake (V̇O2) and heart rate. Eleven subjects, 6 males, with a mean (SD) age of 25.2 (6.9) years participated in field tests involving walking without poles, walking with poles and tire pulling with poles. Increasing the load caused the largest increases in energy demand, more than 4 MET. Speed increase also caused substantial energy increase, approximately 4 MET. Increasing the inclination only modestly increased the oxygen uptake, approximately 2 MET. In both level walking and uphill walking, using poles marginally increased oxygen uptake compared to working without poles. Pulling one tire (12.5 kg) required an oxygen uptake of 27 (4) mL·kg-1·min-1 at 4 km·h-1 and 0% inclination. Adding one more tire (6 kg) drove the oxygen uptake further up to 39 (4) mL·kg-1·min-1. This is close to the requirement of level running at 10.5 km·h-1. Pulling both tires at 6 km·h-1 and 5% inclination required a V̇O2 of 54 (6) mL·kg-1·min-1, equal to running uphill at 5% inclination and 12.5 km·h-1 speed. Heart rate rose comparably with oxygen uptake. At 4 km·h-1 and 0% inclination the increase was 29 bpm, from 134 (21) to 163 (22) bpm when going from pulling one tire to two tires. In the hardest exercise, 6 km·h-1 and 5% inclination, heart rate reached 174 (14) bpm. The study showed that tire pulling even at slow speeds has an energy requirement that is so large that the activity may be feasible as endurance training.

  9. Technology Requirements for Information Management

    Science.gov (United States)

    Graves, Sara; Knoblock, Craig A.; Lannom, Larry

    2002-01-01

    This report provides the results of a panel study conducted into the technology requirements for information management in support of application domains of particular government interest, including digital libraries, mission operations, and scientific research. The panel concluded that it was desirable to have a coordinated program of R&D that pursues a science of information management focused on an environment typified by applications of government interest - highly distributed with very large amounts of data and a high degree of heterogeneity of sources, data, and users.

  10. Quality requirements for EHR archetypes.

    Science.gov (United States)

    Kalra, Dipak; Tapuria, Archana; Austin, Tony; De Moor, Georges

    2012-01-01

    The realisation of semantic interoperability, in which any EHR data may be communicated between heterogeneous systems and fully understood by computers as well as people on receipt, is a challenging goal. Despite the use of standardised generic models for the EHR and standard terminology systems, too much optionality and variability exists in how particular clinical entries may be represented. Clinical archetypes provide a means of defining how generic models should be shaped and bound to terminology for specific kinds of clinical data. However, these will only contribute to semantic interoperability if libraries of archetypes can be built up consistently. This requires the establishment of design principles, editorial and governance policies, and further research to develop ways for archetype authors to structure clinical data and to use terminology consistently. Drawing on several years of work within communities of practice developing archetypes and implementing systems from them, this paper presents quality requirements for the development of archetypes. Clinical engagement on a wide scale is also needed to help grow libraries of good quality archetypes that can be certified. Vendor and eHealth programme engagement is needed to validate such archetypes and achieve safe, meaningful exchange of EHR data between systems.

  11. Tokamak and RFP ignition requirements

    International Nuclear Information System (INIS)

    Werley, K.A.

    1991-01-01

    A plasma model is applied to calculate numerically transport- confinement (nτ E ) requirements and steady-state operation tokamak. The CIT tokamak and RFP ignition conditions are examined. Physics differences between RFP and tokamaks, and their consequences for a DT ignition machine, are discussed. The ignition RFP, compared to a tokamak, has many physics advantages, including ohmic heating to ignition (no need for auxiliary heating systems), higher beta, low ignition current, less sensitivity of ignition requirements to impurity effects, no hard disruptions (associated with beta or density limits), and successful operation with high radiation fractions (f RAD ∼ 0.95). These physics advantages, coupled with important engineering advantages associated with lower external magnetic fields, larger aspect ratios, and smaller plasma cross sections translate into significant cost reductions for both ignition and power reactor. The primary drawback of the RFP is the uncertainty that the present confinement scaling will extrapolate to reactor regimes. The 4-MA ZTH was expected to extend the nτ E transport scaling data three order of magnitude above ZT-40M results, and if the present scaling held, to achieve a DT-equivalent scientific energy breakeven, Q=1. A basecase RFP ignition point is identified with a plasma current of 8.1 MA and no auxiliary heating. 16 refs., 4 figs., 1 tab

  12. Nuclear Energy, Long Term Requirements

    International Nuclear Information System (INIS)

    Knapp, V.

    2006-01-01

    There are serious warnings about depletion of oil and gas and even more serious warnings about dangers of climate change caused by emission of carbon dioxide. Should developed countries be called to replace CO2 emitting energy sources as soon as possible, and the time available may not be longer then few decades, can nuclear energy answer the call and what are the requirements? Assuming optimistic contribution of renewable energy sources, can nuclear energy expand to several times present level in order to replace large part of fossil fuels use? Paper considers intermediate and long-term requirements. Future of nuclear power depends on satisfactory answers on several questions. First group of questions are those important for near and intermediate future. They deal with economics and safety of nuclear power stations in the first place. On the same time scale a generally accepted concept for radioactive waste disposal is also required. All these issues are in the focus of present research and development. Safer and more economical reactors are targets of international efforts in Generation IV and INPRO projects, but aiming further ahead these innovative projects are also addressing issues such as waste reduction and proliferation resistance. However, even assuming successful technical development of these projects, and there is no reason to doubt it, long term and large-scale nuclear power use is thereby not yet secured. If nuclear power is to play an essential role in the long-term future energy production and in reduction of CO2 emission, than several additional questions must be replied. These questions will deal with long-term nuclear fuel sufficiency, with necessary contribution of nuclear power in sectors of transport and industrial processes and with nuclear proliferation safety. This last issue is more political then technical, thus sometimes neglected by nuclear engineers, yet it will have essential role for the long-term prospects of nuclear power. The

  13. Requirements and Markets for Nanoelectronics

    Science.gov (United States)

    Hoefflinger, Bernd

    The semiconductor market grew 2010 by 70Bio. against 2009, more than in the previous 9 years taken together, and the semiconductor industry launched the biggest investment program in its history with 100Bio. over a 2-year period. This was the overture to a decade with great potential and great challenges. We look at the market segments and the required electronic functions, and we highlight four product and service areas: Approaching 6 Billion mobile-phone subscribers Access to education for any child One Carebot (personal robot) per family Efficient and safe personal mobility. At the level of over four billion active mobile phones 2010, it is clear that mobile electronic companions have become the drivers of nanoelectronic innovations with growth only limited by the creation and support of new, attractive features and services. Energy, bandwidth, size and weight requirements of these consumer products provide the largest pressure for System-on-Chip (SoC) architectures. Other exemplary new products are selected for their significance, some for their lengthy path into the market. Health care is such an example: The non-invasive glucose sensor and the portable ECG recorder" with automatic, neuroprocessor-driven event detection in the size of a quarter would serve hundreds of millions of people. Nanoelectronics for self-guided health is an area of public policy in view of the cost of "a posteriori" medical care. Access to information and education for any child/student will be provided by 1 tablets where service contracts and the spin-offs from surfing and cloud-computing will generate the revenue. Personal robots, coined by the ageing Japanese nation as the key product after the PC and ridiculed by others, will arrive as carebots for education, entertainment, rehabilitation, and home-service, accepted as a large-scale need by 2020 in most developed countries including China. Accident prevention systems on rail and road already would make millions of units per year

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

    Science.gov (United States)

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

    2014-01-01

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

  15. Requirements engineering for digital health

    CERN Document Server

    Thümmler, Christoph; Gavras, Anastasius

    2015-01-01

    Healthcare and well-being have captured the attention of established software companies, start-ups, and investors. Software is starting to play a central role for addressing the problems of the aging society and the escalating cost of healthcare services. Enablers of such digital health are a growing number of sensors for sensing the human body and communication infrastructure for remote meetings, data sharing, and messaging. The challenge that lies in front of us is how to effectively make use of these capabilities, for example to empower patients and to free the scarce resources of medical personnel. Requirements engineering is the process by which the capabilities of a software product are aligned with stakeholder needs and a shared understanding between the stakeholders and development team established. This book provides guide for what to look for and do when inquiring and specifying software that targets healthcare and well-being, helping readers avoid the pitfalls of the highly regulated and sensible h...

  16. Moral enhancement requires multiple virtues.

    Science.gov (United States)

    Hughes, James J

    2015-01-01

    Some of the debates around the concept of moral enhancement have focused on whether the improvement of a single trait, such as empathy or intelligence, would be a good in general, or in all circumstances. All virtue theories, however, both secular and religious, have articulated multiple virtues that temper and inform one another in the development of a mature moral character. The project of moral enhancement requires a reengagement with virtue ethics and contemporary moral psychology to develop an empirically grounded model of the virtues and a fuller model of character development. Each of these virtues may be manipulable with electronic, psychopharmaceutical, and genetic interventions. A set of interdependent virtues is proposed, along with some of the research pointing to ways such virtues could be enhanced.

  17. Aligning seminars with Bologna requirements

    DEFF Research Database (Denmark)

    Lueg, Rainer; Lueg, Klarissa; Lauridsen, Ole

    2016-01-01

    Changes in public policy, such as the Bologna Process, require students to be equipped with multifunctional competencies to master relevant tasks in unfamiliar situations. Achieving this goal might imply a change in many curricula toward deeper learning. As a didactical means to achieve deep...... learning results, the authors suggest reciprocal peer tutoring (RPT); as a conceptual framework the authors suggest the SOLO (Structure of Observed Learning Outcomes) taxonomy and constructive alignment as suggested by Biggs and Tang. Our study presents results from the introduction of RPT in a large...... and students, suggest that RPT fosters deeper learning than does teacher-led instruction. Based on these findings, the authors also offer guidelines regarding how to implement RPT and how to overcome barriers....

  18. Reversed field pinch ignition requirements

    International Nuclear Information System (INIS)

    Werley, K.A.

    1991-01-01

    Plasma models are described and used to calculated numerically the transport confinement (nτ E ) requirements and steady state operation points for both the reversed field pinch (RFP) and the tokamak. The models are used to examine the CIT tokamak ignition conditions and the RFP experimental and ignition conditions. Physics differences between RFPs and tokamaks and their consequences for a D-T ignition machine are discussed. Compared with a tokamak, the ignition RFP has many physics advantages, including Ohmic heating to ignition (no need for auxiliary heating systems), higher beta, lower ignition current, less sensitivity of ignition requirements to impurity effects, no hard disruptions (associated with beta or density limits) and successful operation with high radiation fractions (f RAD ∼ 0.95). These physics advantages, coupled with important engineering advantages associated with lower external magnetic field, larger aspect ratios and smaller plasma cross-sections, translate to significant cost reductions for both ignition and reactor applications. The primary drawback of the RFP is the uncertainty that the present scaling will extrapolate to reactor regimes. Devices that are under construction should go a long way toward resolving this scaling uncertainty. The 4 MA ZTH is expected to extend the nτ E transport scaling data by three orders of magnitude above the results of ZT-40M, and, if the present scaling holds, ZTH is expected to achieve a D-T equivalent scientific energy breakeven, Q = 1. A base case RFP ignition point is identified with a plasma current of 8.1 MA and no auxiliary heating. (author). 19 refs, 11 figs, 3 tabs

  19. Requirements for an energy policy

    International Nuclear Information System (INIS)

    Conant, M.A.

    1987-01-01

    The central issue facing the US today lies in the rise of oil imports. No supergiant (5 billion barrels) oil discoveries have been made in the US. Production from existing fields is declining. The 1985-86 oil price collapse from $26 to less than $15 per barrel had a disastrous effect on the budgets of smaller oil companies which do most of the exploring, and on the service industry. Budgets for overseas exploration has been generally sustained. Oil prices are not expected to sustain domestic exploration. Gulf oil sources will, in the next five years, supply some 75 percent of all oil in international trade. Without an energy policy, involvement in Middle East oil will grow exponentially, as will the needs of others for Gulf oil. The natural gas situation is different, with a spare producing capacity of one trillion cubic feet this year, which could double next year. Natural gas deregulation has created an unbelievable mess in the requirements of producers/suppliers and purchasers to have dependable business arrangements. Coal is plentiful and will be until the end of time. Public opposition to emission problems and the greenhouse effect are an obstacle to greater use of coal. The nuclear option may be dead, with no new orders since 1978. Statistics are provided on proven world reserves of conventional crude oil, recoverable heavy oils, tar sands, and shale oil; which indicates for the long term an ability to transform the Geopolitics of oil away from the middle east. Energy options require energy R ampersand D, use of Alaskan gas, conservation and efficiency in energy use, strategic reserves, close energy relations with allies, and a government-industry link which insures meeting the US oil needs from the Western Hemisphere

  20. Physical System Requirements: Transport Waste

    International Nuclear Information System (INIS)

    1992-04-01

    The Nuclear Waste Policy Act (NWPA) of 1982 assigned to the Department of Energy (DOE) the responsibility for managing the disposal of spent nuclear fuel and high-level radioactive waste and established the Office of Civilian Radioactive Waste Management (OCRWM) for that purpose. The Secretary of Energy, in his November 1989 report to Congress (DOE/RW-0247), announced three new initiatives for the conduct of the Civilian Radioactive Waste Management (CRWM) program. One of these initiatives was to establish improved management structure and procedures. In response, OCRWM performed a management study and the Director subsequently issued the Management Systems Improvement Strategy (MSIS) on August 10, 1990, calling for a rigorous implementation of systems engineering principles with a special emphasis on functional analysis. The functional analysis approach establishes a framework for integrating the program management efforts with the technical requirements analysis into a single, unified, and consistent program. This approach recognizes that just as the facilities and equipment comprising the physical waste management system must perform certain functions, so must certain programmatic and management functions be performed within the program in order to successfully bring the physical system into being. The objective of this document is to establish the essential functions, requirements, interfaces, and system architecture for the Transport Waste mission. Based upon the Nuclear Waste Policy Act, the mission of the Waste Transportation System is to transport SNF and/or HLW from the purchaser's/producer's facilities to, and between, NWMS facilities in a manner that protects the health and safety of the public and of workers and the quality of the environment makes effective use of financial and other resources, and to the fullest extent possible uses the private sector

  1. 7 CFR 4284.18 - Audit requirements.

    Science.gov (United States)

    2010-01-01

    ... Grant Programs § 4284.18 Audit requirements. Grantees must comply with the audit requirements of 7 CFR part 3052. The audit requirements apply to the years in which grant funds are received and years in...

  2. Design requirement on HYPER blanket fuel assembly

    International Nuclear Information System (INIS)

    Hwang, Woan; Lee, B. O.; Nam, C.; Ryu, W. S.; Lee, B. S.; Park, W. S.

    2000-07-01

    This document describes design requirements which are needed for designing the blanket assembly of the HYPER as design guidance. The blanket assembly of the HYPER consists of blanket fuel rods, mounting rail, spacer, upper nozzle with handling socket, bottom nozzle with mounting rail and skeleton structure. The blanket fuel rod consists of top end plug, bottom end plug with key way, blanket fuel slug, and cladding. In the assembly, the rods are in a triangular pitch array. This report contains functional requirements, performance and operational requirements, interfacing systems requirements, core restraint and interface requirements, design limits and strength requirements, system configuration and essential feature requirements, seismic requirements, structural requirements, environmental requirements, reliability and safety requirements, standard and codes, QA programs, and other requirements for the blanket fuel assembly of the HYPER

  3. Rapid quality assurance with requirements smells

    OpenAIRE

    Femmer, Henning; Méndez Fernández, Daniel; Wagner, Stefan; Eder, Sebastian

    2016-01-01

    Context: Bad requirements quality can cause expensive consequences during the software development lifecycle, especially if iterations are long and feedback comes late. Objectives: We aim at a light-weight static requirements analysis approach that allows for rapid checks immediately when requirements are written down. Method: We transfer the concept of code smells to Requirements Engineering as Requirements Smells. To evaluate the benefits and limitations, we define Requirements Smells, real...

  4. Specifying Functional Requirements Dependency in the REWiki

    OpenAIRE

    ZHANG, ZHANG

    2013-01-01

    Most of the individual requirements cannot be treated in isolation. Requirements may affect each other in various ways. The dependency between requirements impacts a number of software development aspects and activities. How to classify and specify requirements dependency remains a classic research topic. This research aims at providing an approach of specifying functional requirements dependency. In this thesis we generalize a classification of functional requirements dependency. We also pro...

  5. Cold vacuum drying facility design requirements

    International Nuclear Information System (INIS)

    IRWIN, J.J.

    1999-01-01

    This document provides the detailed design requirements for the Spent Nuclear Fuel Project Cold Vacuum Drying Facility. Process, safety, and quality assurance requirements and interfaces are specified

  6. Cold vacuum drying facility design requirements

    Energy Technology Data Exchange (ETDEWEB)

    IRWIN, J.J.

    1999-07-01

    This document provides the detailed design requirements for the Spent Nuclear Fuel Project Cold Vacuum Drying Facility. Process, safety, and quality assurance requirements and interfaces are specified.

  7. VO₂ requirements of boxing exercises.

    Science.gov (United States)

    Arseneau, Eric; Mekary, Saïd; Léger, Luc A

    2011-02-01

    The purpose of this study was to quantify the physiological requirements of various boxing exercises such as sparring, pad work, and punching bag. Because it was not possible to measure the oxygen uptake (VO₂) of "true" sparring with a collecting gas valve in the face, we developed and validated a method to measure VO₂ of "true" sparring based on "postexercise" measurements. Nine experienced male amateur boxers (Mean ± SD: age = 22.0 ± 3.5 years, height = 176.0 ± 8.0 cm, weight = 71.4 ± 10.9 kg, number of fights = 13.0 ± 9.5) of regional and provincial level volunteered to participate in 3 testing sessions: (a) maximal treadmill test in the LAB, (b) standardized boxing training in the GYM, and (c) standardized boxing exercises in the LAB. Measures of VO₂, heart rate (HR), blood lactate concentration [LA], rated perceived exertion level, and punching frequencies were collected. VO₂ values of 43.4 ± 5.9, 41.1 ± 5.1, 24.7 ± 6.1, 30.4 ± 5.8, and 38.3 ± 6.5 ml·kg⁻¹·min⁻¹ were obtained, which represent 69.7 ± 8.0, 66.1 ± 8.0, 39.8 ± 10.4, 48.8 ± 8.5, and 61.7 ± 10.3%VO₂peak for sparring, pad work, and punching bag at 60, 120, and 180 b·min⁻¹, respectively. Except for lower VO₂ values for punching the bag at 60 and 120 b·min⁻¹ (p < 0.05), there was no VO₂ difference between exercises. Similar pattern was obtained for %HRmax with respective values of 85.5 ± 5.9, 83.6 ± 6.3, 67.5 ± 3.5, 74.8 ± 5.9, and 83.0 ± 6.0. Finally, sparring %HRmax and [LA] were slightly higher in the GYM (91.7 ± 4.3 and 9.4 ± 2.2 mmol·L⁻¹) vs. LAB (85.5 ± 5.9 and 6.1 ± 2.3 mmol·L⁻¹). Thus, in this study simulated LAB sparring and pad work required similar VO₂ (43-41 ml·kg⁻¹·min⁻¹, respectively), which corresponds to ~70%VO₂peak. These results underline the importance of a minimum of aerobic fitness for boxers and draw some guidelines for the intensity of training.

  8. Global Land Transport Infrastructure Requirements

    Energy Technology Data Exchange (ETDEWEB)

    NONE

    2013-06-01

    Over the next four decades, global passenger and freight travel is expected to double over 2010 levels. In order to accommodate this growth, it is expected that the world will need to add nearly 25 million paved road lane-kilometres and 335 000 rail track kilometres. In addition, it is expected that between 45 000 square kilometres and 77 000 square kilometres of new parking spaces will be added to accommodate vehicle stock growth. These land transport infrastructure additions, when combined with operations, maintenance and repairs, are expected to cost as much as USD 45 trillion by 2050. This publication reports on the International Energy Agency’s (IEA) analysis of infrastructure requirements to support projected road and rail travel through 2050, using the IEA Mobility Model. It considers land transport infrastructure additions to support travel growth to 2050. It also considers potential savings if countries pursue “avoid and shift” policies: in this scenario, cumulative global land transport infrastructure spending could decrease as much as USD 20 trillion by 2050 over baseline projections.

  9. Physical system requirements: Overall system

    International Nuclear Information System (INIS)

    1992-01-01

    The Nuclear Waste Policy Act (NWPA) of 1982 assigned to the Department of Energy (DOE) the responsibility for managing the disposal of spent nuclear fuel and high-level radioactive waste and established the Office of Civilian Radioactive Waste Management (OCRWM) for that purpose. The Secretary of Energy, in his November 1989 report to Congress (DOE/RW-0247), announced three new initiatives for conduct of the Civilian Radioactive Waste Management (CRWM) program. One of these initiatives was to establish improved management structure and procedures. In response, OCRWM performed a management study and the Direct subsequently issued the Management Systems Improvement Strategy (MSIS) on August 10, 1990, calling for a rigorous implementation of systems engineering principles with a special emphasis on functional analysis. This approach establishes a framework for integrating the program management efforts with the technical requirements analysis into a single, unified, and consistent program. The functional analysis approach recognizes that just the facilities and equipment comprising the physical waste management system must perform certain functions, so must certain programmatic and management functions be performed within the program in order to successfully bring the physical system into being

  10. Ageing management requirements in Sweden

    International Nuclear Information System (INIS)

    Gott, K.

    2007-01-01

    Since the adoption of the 1997 act of parliament concerning the abolishment of nuclear power in Sweden plant lives are no longer limited until 2010 as they were following the referendum in 1980. The Swedish Nuclear Power Inspectorate (SKI) has therefore in its most recent general regulations from 2004 introduced requirements for plants to develop ageing management programmes which should have been in place by the end of 2005. An ageing management programme for a nuclear power plant can be viewed as a high level coordinated programme consisting of several other programmes including maintenance and inspection programmes, and as such it should be an integrated part of the quality assurance system of the plant. The goals of the programme are to ensure that there is long term management of ageing and avoidance of the unexpected. For the Swedish regulator the safety aspects and not the economic aspects of ageing management are of primary importance. But there are clear advantages to utilities if large surprises which result in unplanned outages can be avoided. (author)

  11. General lighting requirements for photosynthesis

    Energy Technology Data Exchange (ETDEWEB)

    Geiger, D.R. [Univ. of Dayton, OH (United States)

    1994-12-31

    A review of the general lighting requirements for photosynthesis reveals that four aspects of light are important: irradiance, quality, timing and duration. These properties of light affect photosynthesis by providing the energy that drives carbon assimilation as well as by exerting control over physiology, structure and morphology of plants. Irradiance, expressed as energy flux, W m{sup -2}, or photon irradiance, {mu}mol m{sup -2} s{sup -1}, determines the rate at which energy is being delivered to the photosynthetic reaction centers. Spectral quality, the wavelength composition of light, is important because photons differ in their probability of being absorbed by the light harvesting complex and hence their ability to drive carbon assimilation. Also the various light receptors for light-mediated regulation of plant form and physiology have characteristic absorption spectra and hence photons differ in their effectiveness for eliciting responses. Duration is important because both carbon assimilation and regulation are affected by the total energy or integrated irradiance delivered during a given period. Many processes associated with photosynthesis are time-dependent, increasing or decreasing with duration. Timing is important because the effectiveness of light in the regulation of plant processes varies with the phase of the diumal cycle as determined by the plant`s time-measuring mechanisms.

  12. The penalty: function and requirements

    Directory of Open Access Journals (Sweden)

    Iván Meini

    2013-12-01

    Full Text Available Legitimacy of criminal sanction is originated on its own purposes pursued in a state governed by the Rule of Law. That legitimacy should include the penalty as well as security measures, bearing in mind that both are imposed to someone breaking a rule of conduct, and therefore, someone capable to do it. Reviewing penal capacity or criminal liability concepts is required because if penal capacity means the capacity to understand the reality and adjust the behavior to it, and if every legitimate criminal sanction have to be imposed to someone who have the capacity of break it, then security measures also have to be imposed only to people responsible, capable to understand rules and act in accordance. With regard to people not subject to criminal liability they are standing outside Criminal Law and punish them would be illegitimate. In this line, criminal liability should be seen not only as a crime assumption but also as a basic statement for any dialogue the state shall have with the citizens: at the level of crime itself, proceedings and sentence execution .

  13. Space station propulsion requirements study

    Science.gov (United States)

    Wilkinson, C. L.; Brennan, S. M.

    1985-01-01

    Propulsion system requirements to support Low Earth Orbit (LEO) manned space station development and evolution over a wide range of potential capabilities and for a variety of STS servicing and space station operating strategies are described. The term space station and the overall space station configuration refers, for the purpose of this report, to a group of potential LEO spacecraft that support the overall space station mission. The group consisted of the central space station at 28.5 deg or 90 deg inclinations, unmanned free-flying spacecraft that are both tethered and untethered, a short-range servicing vehicle, and a longer range servicing vehicle capable of GEO payload transfer. The time phasing for preferred propulsion technology approaches is also investigated, as well as the high-leverage, state-of-the-art advancements needed, and the qualitative and quantitative benefits of these advancements on STS/space station operations. The time frame of propulsion technologies applicable to this study is the early 1990's to approximately the year 2000.

  14. The Anti-sigma Factor RsiV Is a Bacterial Receptor for Lysozyme: Co-crystal Structure Determination and Demonstration That Binding of Lysozyme to RsiV Is Required for σV Activation.

    Directory of Open Access Journals (Sweden)

    Jessica L Hastie

    2016-09-01

    Full Text Available σ factors provide RNA polymerase with promoter specificity in bacteria. Some σ factors require activation in order to interact with RNA polymerase and transcribe target genes. The Extra-Cytoplasmic Function (ECF σ factor, σV, is encoded by several Gram-positive bacteria and is specifically activated by lysozyme. This activation requires the proteolytic destruction of the anti-σ factor RsiV via a process of regulated intramembrane proteolysis (RIP. In many cases proteases that cleave at site-1 are thought to directly sense a signal and initiate the RIP process. We previously suggested binding of lysozyme to RsiV initiated the proteolytic destruction of RsiV and activation of σV. Here we determined the X-ray crystal structure of the RsiV-lysozyme complex at 2.3 Å which revealed that RsiV and lysozyme make extensive contacts. We constructed RsiV mutants with altered abilities to bind lysozyme. We find that mutants that are unable to bind lysozyme block site-1 cleavage of RsiV and σV activation in response to lysozyme. Taken together these data demonstrate that RsiV is a receptor for lysozyme and binding of RsiV to lysozyme is required for σV activation. In addition, the co-structure revealed that RsiV binds to the lysozyme active site pocket. We provide evidence that in addition to acting as a sensor for the presence of lysozyme, RsiV also inhibits lysozyme activity. Thus we have demonstrated that RsiV is a protein with multiple functions. RsiV inhibits σV activity in the absence of lysozyme, RsiV binds lysozyme triggering σV activation and RsiV inhibits the enzymatic activity of lysozyme.

  15. Refinement of boards' role required.

    Science.gov (United States)

    Umbdenstock, R J

    1987-01-01

    The governing board's role in health care is not changing, but new competitive forces necessitate a refinement of the board's approach to fulfilling its role. In a free-standing, community, not-for-profit hospital, the board functions as though it were the "owner." Although it does not truly own the facility in the legal sense, the board does have legal, fiduciary, and financial responsibilities conferred on it by the state. In a religious-sponsored facility, the board fulfills these same obligations on behalf of the sponsoring institute, subject to the institute's reserved powers. In multi-institutional systems, the hospital board's power and authority depend on the role granted it by the system. Boards in all types of facilities are currently faced with the following challenges: Fulfilling their basic responsibilities, such as legal requirements, financial duties, and obligations for the quality of care. Encouraging management and the board itself to "think strategically" in attacking new competitive market forces while protecting the organization's traditional mission and values. Assessing recommended strategies in light of consequences if constituencies think the organization is abandoning its commitments. Boards can take several steps to match their mode of operation with the challenges of the new environment. Boards must rededicate themselves to the hospital's mission. Trustees must expand their understanding of health care trends and issues and their effect on the organization. Boards must evaluate and help strengthen management's performance, rather than acting as a "watchdog" in an adversarial position. Boards must think strategically, rather than focusing solely on operational details. Boards must evaluate the methods they use for conducting business.

  16. ILK statement about ATWS requirements

    International Nuclear Information System (INIS)

    2005-01-01

    A controversial debate is going on in Germany about the management of operating transients in case of the failure, additionally assumed, of the scram system (ATWS=Anticipated Transients without Scram). It was triggered by a recommendation by the German Advisory Committee on Reactor Safeguards (RSK) in a statement of May 3, 2001 according to which the demonstration that ATWS events were under control was to deviate from requirements in the RSK Guidelines for pressurized water reactors of 1981 (last amended in 1996) and not to take credit of the effects of one-off measures initiated actively, especially shutdown of the main coolant pumps. ILK therefore expresses its opinion in this Statement about the criteria to be met in demonstrations that ATWS is under control in pressurized water reactors. Also in boiling water reactors, studies of ATWS transients are part of the licensing procedure. However, the assumptions to be made there in demonstrating effective pressure limitation have been unchanged and uncontested long since. ILK included in its considerations especially also practices in the United States, France and Finland. In doing so, the Committee found the basic approach in dealing with ATWS to be the same in Germany, the United States and in France, namely to show that the consequences remain tolerable without the application of aggravating postulates. ILK feels that the approach so far employed in demonstrating safety in ATWS events results in balanced risk mitigation. The initiating event already has a very low probability of occurrence. Reliable measures are in place to manage it. (orig.)

  17. 40 CFR 141.70 - General requirements.

    Science.gov (United States)

    2010-07-01

    ...) NATIONAL PRIMARY DRINKING WATER REGULATIONS Filtration and Disinfection § 141.70 General requirements. (a... regulations establish criteria under which filtration is required as a treatment technique for public water... filtration requirements in § 141.73 and the disinfection requirements in § 141.72(b). (c) Each public water...

  18. 46 CFR 11.707 - Examination requirements.

    Science.gov (United States)

    2010-10-01

    ... 46 Shipping 1 2010-10-01 2010-10-01 false Examination requirements. 11.707 Section 11.707 Shipping... OFFICER ENDORSEMENTS Professional Requirements for Pilots § 11.707 Examination requirements. (a) An... required to pass the examination described in subpart I of this part. (b) An applicant for an extension of...

  19. 21 CFR 1304.11 - Inventory requirements.

    Science.gov (United States)

    2010-04-01

    ... the inventory of the registered location to which they are subject to control or to which the person... 21 Food and Drugs 9 2010-04-01 2010-04-01 false Inventory requirements. 1304.11 Section 1304.11... REGISTRANTS Inventory Requirements § 1304.11 Inventory requirements. (a) General requirements. Each inventory...

  20. Meeting Quay 2k30's requirements

    NARCIS (Netherlands)

    Wijnants, G.H.; Toorn, A. van der; Schuylenburg, M.; Heijnen, H.P.J.; Gijt, J.G. de; Molenaar, W.F.; Ligteringen, H.; Krom, A.H.M.

    2005-01-01

    The requirements that a quay design should meet in order to yield a viable port infrastructure, vary widely from flexibility due to future customers requirements to durability due to owners requirements. In a Port of Rotterdam backed project, current and future requirements have been aggregated by

  1. 20 CFR 655.152 - Advertising requirements.

    Science.gov (United States)

    2010-04-01

    ... 20 Employees' Benefits 3 2010-04-01 2010-04-01 false Advertising requirements. 655.152 Section 655... Employment in the United States (H-2A Workers) Post-Acceptance Requirements § 655.152 Advertising requirements. All advertising conducted to satisfy the required recruitment activities under § 655.151 must...

  2. Specifying semantic information on functional requirements

    OpenAIRE

    YAO, WUPING

    2012-01-01

    Requirements engineering is a challenging process in software development projects. Requirements, in general, are documented in natural language. They often have issues related to ambiguity, completeness and consistency. How to improve the quality of requirements documentation remains a classic research topic. This research aims at improving the way of editing and documenting functional requirements. We propose a meta-model to specify the semantic information of functional requirements, and d...

  3. 24 CFR 266.105 - Application requirements.

    Science.gov (United States)

    2010-04-01

    ... HOUSING FINANCE AGENCY RISK-SHARING PROGRAM FOR INSURED AFFORDABLE MULTIFAMILY PROJECT LOANS Housing Finance Agency Requirements § 266.105 Application requirements. (a) Applications for approval as a HUD...

  4. 24 CFR 266.110 - Reserve requirements.

    Science.gov (United States)

    2010-04-01

    ... HOUSING FINANCE AGENCY RISK-SHARING PROGRAM FOR INSURED AFFORDABLE MULTIFAMILY PROJECT LOANS Housing Finance Agency Requirements § 266.110 Reserve requirements. (a) HFAs with top-tier designation or overall...

  5. Ecological flow requirements for South African rivers

    CSIR Research Space (South Africa)

    Ferrar, AA

    1989-01-01

    Full Text Available This document contains the proceedings of a workshop which was convened to debate the ecological flow requirements of South African rivers. Topics which are discussed include the influence of weirs and impoundments, the quantity requirements...

  6. 38 CFR 39.10 - Application requirements.

    Science.gov (United States)

    2010-07-01

    ...-10) Grant Requirements and Procedures § 39.10 Application requirements. (a) For a project to be..., etc., (10) Economic activities, (11) Cultural resources, (12) Aesthetics, (13) Residential population...

  7. I-15 integrated corridor management : system requirements.

    Science.gov (United States)

    2011-07-01

    This document is intended as a listing and discussion of the Requirements for the I-15 Integrated Corridor Management System : (ICMS) Demonstration Project in San Diego. This document describes what the system is to do (the functional requirements), ...

  8. Requirements Elicitation Problems: A Literature Analysis

    Directory of Open Access Journals (Sweden)

    Bill Davey

    2015-06-01

    Full Text Available Requirements elicitation is the process through which analysts determine the software requirements of stakeholders. Requirements elicitation is seldom well done, and an inaccurate or incomplete understanding of user requirements has led to the downfall of many software projects. This paper proposes a classification of problem types that occur in requirements elicitation. The classification has been derived from a literature analysis. Papers reporting on techniques for improving requirements elicitation practice were examined for the problem the technique was designed to address. In each classification the most recent or prominent techniques for ameliorating the problems are presented. The classification allows the requirements engineer to be sensitive to problems as they arise and the educator to structure delivery of requirements elicitation training.

  9. Insulin requirements in type 1 diabetic pregnancy

    DEFF Research Database (Denmark)

    Callesen, Nicoline; Ringholm, Lene; Stage, Edna

    2012-01-01

    To evaluate the insulin requirements in women with type 1 diabetes during twin pregnancy compared with singleton pregnancy.......To evaluate the insulin requirements in women with type 1 diabetes during twin pregnancy compared with singleton pregnancy....

  10. Requirements Engineering for Software Integrity and Safety

    Science.gov (United States)

    Leveson, Nancy G.

    2002-01-01

    Requirements flaws are the most common cause of errors and software-related accidents in operational software. Most aerospace firms list requirements as one of their most important outstanding software development problems and all of the recent, NASA spacecraft losses related to software (including the highly publicized Mars Program failures) can be traced to requirements flaws. In light of these facts, it is surprising that relatively little research is devoted to requirements in contrast with other software engineering topics. The research proposed built on our previous work. including both criteria for determining whether a requirements specification is acceptably complete and a new approach to structuring system specifications called Intent Specifications. This grant was to fund basic research on how these ideas could be extended to leverage innovative approaches to the problems of (1) reducing the impact of changing requirements, (2) finding requirements specification flaws early through formal and informal analysis, and (3) avoiding common flaws entirely through appropriate requirements specification language design.

  11. 8 CFR 1216.2 - Notification requirements.

    Science.gov (United States)

    2010-01-01

    ... second time of the requirement that the alien and the petitioning spouse or alien entrepreneur must file... does not relieve the alien and the petitioning spouse, or alien entrepreneur of the requirement to file...

  12. Rapid quality assurance with Requirements Smells

    OpenAIRE

    Femmer, H.; Fernández, D. Méndez; Wagner, S.; Eder, S.

    2016-01-01

    Bad requirements quality can cause expensive consequences during the software development lifecycle, especially if iterations are long and feedback comes late. %-- the faster a problem is found, the cheaper it is to fix. This makes explicit the need of a lightweight detection mechanism of requirements quality violations. We aim at a light-weight static requirements analysis approach that allows for rapid checks immediately when requirements are written down. We transfer the concept of code sm...

  13. Airspace Operations Demo Functional Requirements Matrix

    Science.gov (United States)

    2005-01-01

    The Flight IPT assessed the reasonableness of demonstrating each of the Access 5 Step 1 functional requirements. The functional requirements listed in this matrix are from the September 2005 release of the Access 5 Functional Requirements Document. The demonstration mission considered was a notional Western US mission (WUS). The conclusion of the assessment is that 90% of the Access 5 Step 1 functional requirements can be demonstrated using the notional Western US mission.

  14. A Design Theory for Requirements Mining Systems

    OpenAIRE

    Meth, Hendrik

    2013-01-01

    Software requirements are often communicated in unstructured text documents, which need to be analyzed in order to identify and classify individual needs. This process is referred to as requirements mining in the context of this thesis. It is known to be time-consuming and error-prone when performed manually by a requirements engineer. Thus, there is a demand to support requirements mining through information technology. However, little research has been conducted to conceptualize theoretical...

  15. Dosimeter characteristics and service performance requirements

    International Nuclear Information System (INIS)

    Ambrosi, P.; Bartlett, D.T.

    1999-01-01

    The requirements for personal dosimeters and dosimetry services given by ICRP 26, ICRP 35, ICRP 60 and ICRP 75 are summarised and compared with the requirements given in relevant international standards. Most standards could be made more relevant to actual workplace conditions. In some standards, the required tests of energy and angular dependence of the response are not sufficient, or requirements on overall uncertainty are lacking. (author)

  16. Reserve requirement systems in OECD countries

    OpenAIRE

    Yueh-Yun C. O’Brien

    2007-01-01

    This paper compares the reserve requirements of OECD countries. Reserve requirements are the minimum percentages or amounts of liabilities that depository institutions are required to keep in cash or as deposits with their central banks. To facilitate monetary policy implementation, twenty-four of the thirty OECD countries impose reserve requirements to influence their banking systems’ demand for liquidity. These include twelve OECD countries that are also members of the European Economic and...

  17. 28 CFR 80.12 - Accounting requirements.

    Science.gov (United States)

    2010-07-01

    ... 28 Judicial Administration 2 2010-07-01 2010-07-01 false Accounting requirements. 80.12 Section 80... PROCEDURE § 80.12 Accounting requirements. Neither the submission of a request for an FCPA Opinion, its... comply with the accounting requirements of 15 U.S.C. 78m(b)(2) and (3). ...

  18. Capturing security requirements for software systems.

    Science.gov (United States)

    El-Hadary, Hassan; El-Kassas, Sherif

    2014-07-01

    Security is often an afterthought during software development. Realizing security early, especially in the requirement phase, is important so that security problems can be tackled early enough before going further in the process and avoid rework. A more effective approach for security requirement engineering is needed to provide a more systematic way for eliciting adequate security requirements. This paper proposes a methodology for security requirement elicitation based on problem frames. The methodology aims at early integration of security with software development. The main goal of the methodology is to assist developers elicit adequate security requirements in a more systematic way during the requirement engineering process. A security catalog, based on the problem frames, is constructed in order to help identifying security requirements with the aid of previous security knowledge. Abuse frames are used to model threats while security problem frames are used to model security requirements. We have made use of evaluation criteria to evaluate the resulting security requirements concentrating on conflicts identification among requirements. We have shown that more complete security requirements can be elicited by such methodology in addition to the assistance offered to developers to elicit security requirements in a more systematic way.

  19. 27 CFR 7.28 - General requirements.

    Science.gov (United States)

    2010-04-01

    ... statements of alcoholic content, all mandatory information required on labels by this part shall be in script.... Except for statements of alcoholic content, all mandatory information required on labels by this part... OF THE TREASURY LIQUORS LABELING AND ADVERTISING OF MALT BEVERAGES Labeling Requirements for Malt...

  20. 29 CFR 96.12 - Audit requirements.

    Science.gov (United States)

    2010-07-01

    .... (b) The audit requirements contained in 29 CFR part 99 shall be followed for audits of all fiscal... 29 Labor 1 2010-07-01 2010-07-01 true Audit requirements. 96.12 Section 96.12 Labor Office of the Secretary of Labor AUDIT REQUIREMENTS FOR GRANTS, CONTRACTS, AND OTHER AGREEMENTS Audits of States, Local...

  1. 7 CFR 1948.96 - Audit requirements.

    Science.gov (United States)

    2010-01-01

    ... Program § 1948.96 Audit requirements. (a) Audit requirements for Site Development and Acquisition Grants will be made in accordance with FmHA Instruction 1942-G. (b) Audits for planning grants made in... 7 Agriculture 13 2010-01-01 2009-01-01 true Audit requirements. 1948.96 Section 1948.96...

  2. Organizing Performance Requirements For Dynamical Systems

    Science.gov (United States)

    Malchow, Harvey L.; Croopnick, Steven R.

    1990-01-01

    Paper describes methodology for establishing performance requirements for complicated dynamical systems. Uses top-down approach. In series of steps, makes connections between high-level mission requirements and lower-level functional performance requirements. Provides systematic delineation of elements accommodating design compromises.

  3. Capturing security requirements for software systems

    Directory of Open Access Journals (Sweden)

    Hassan El-Hadary

    2014-07-01

    Full Text Available Security is often an afterthought during software development. Realizing security early, especially in the requirement phase, is important so that security problems can be tackled early enough before going further in the process and avoid rework. A more effective approach for security requirement engineering is needed to provide a more systematic way for eliciting adequate security requirements. This paper proposes a methodology for security requirement elicitation based on problem frames. The methodology aims at early integration of security with software development. The main goal of the methodology is to assist developers elicit adequate security requirements in a more systematic way during the requirement engineering process. A security catalog, based on the problem frames, is constructed in order to help identifying security requirements with the aid of previous security knowledge. Abuse frames are used to model threats while security problem frames are used to model security requirements. We have made use of evaluation criteria to evaluate the resulting security requirements concentrating on conflicts identification among requirements. We have shown that more complete security requirements can be elicited by such methodology in addition to the assistance offered to developers to elicit security requirements in a more systematic way.

  4. 49 CFR 7.4 - Publication required.

    Science.gov (United States)

    2010-10-01

    ... 49 Transportation 1 2010-10-01 2010-10-01 false Publication required. 7.4 Section 7.4 Transportation Office of the Secretary of Transportation PUBLIC AVAILABILITY OF INFORMATION Information Required To Be Made Public by DOT § 7.4 Publication required. (a) General. The material described in § 7.3...

  5. 20 CFR 302.6 - Publication requirements.

    Science.gov (United States)

    2010-04-01

    ... 20 Employees' Benefits 1 2010-04-01 2010-04-01 false Publication requirements. 302.6 Section 302.6 Employees' Benefits RAILROAD RETIREMENT BOARD REGULATIONS UNDER THE RAILROAD UNEMPLOYMENT INSURANCE ACT QUALIFIED EMPLOYEE § 302.6 Publication requirements. (a) Publication of base year compensation requirement...

  6. 29 CFR 1620.32 - Recordkeeping requirements.

    Science.gov (United States)

    2010-07-01

    ... Regulations Relating to Labor (Continued) EQUAL EMPLOYMENT OPPORTUNITY COMMISSION THE EQUAL PAY ACT § 1620.32 Recordkeeping requirements. (a) Employers having employees subject to the Act are required to keep records in... subject to the equal pay provisions of the Act shall maintain and preserve all records required by the...

  7. Gamified Requirements Engineering: Model and Experimentation

    NARCIS (Netherlands)

    Lombriser, Philipp; Dalpiaz, Fabiano; Lucassen, Garm; Brinkkemper, Sjaak

    2016-01-01

    [Context & Motivation] Engaging stakeholders in requirements engineering (RE) influences the quality of the requirements and ultimately of the system to-be. Unfortunately, stakeholder engagement is often insufficient, leading to too few, low-quality requirements. [Question/problem] We aim to

  8. Understand the Design Requirement in Companies

    DEFF Research Database (Denmark)

    Li, Xuemeng; Ahmed-Kristensen, Saeema

    2015-01-01

    requirements can lead to inappropriate products (Hall, et al., 2002). Understanding the nature of design requirements and the sources, from where they can or should be generated, is critical to before developing methods and processes to support this process. Requirement Engineering research, originated from...

  9. A Metamodeling Approach for Reasoning about Requirements

    NARCIS (Netherlands)

    Göknil, Arda; Ivanov, Ivan; van den Berg, Klaas; Schieferdecker, I.; Hartman, A.

    In requirements engineering, there are several approaches for requirements modeling such as goal-oriented, aspect-driven, and system requirements modeling. In practice, companies often customize a given approach to their specific needs. Thus, we seek a solution that allows customization in a

  10. 48 CFR 1430.202 - Disclosure requirements.

    Science.gov (United States)

    2010-10-01

    ... 48 Federal Acquisition Regulations System 5 2010-10-01 2010-10-01 false Disclosure requirements. 1430.202 Section 1430.202 Federal Acquisition Regulations System DEPARTMENT OF THE INTERIOR GENERAL CONTRACTING REQUIREMENTS COST ACCOUNTING STANDARDS ADMINISTRATION CAS Program Requirements 1430.202 Disclosure...

  11. 48 CFR 430.202 - Disclosure requirements.

    Science.gov (United States)

    2010-10-01

    ... 48 Federal Acquisition Regulations System 4 2010-10-01 2010-10-01 false Disclosure requirements. 430.202 Section 430.202 Federal Acquisition Regulations System DEPARTMENT OF AGRICULTURE GENERAL CONTRACTING REQUIREMENTS COST ACCOUNTING STANDARDS ADMINISTRATION CAS Program Requirements 430.202 Disclosure...

  12. 48 CFR 30.202 - Disclosure requirements.

    Science.gov (United States)

    2010-10-01

    ... 48 Federal Acquisition Regulations System 1 2010-10-01 2010-10-01 false Disclosure requirements. 30.202 Section 30.202 Federal Acquisition Regulations System FEDERAL ACQUISITION REGULATION GENERAL CONTRACTING REQUIREMENTS COST ACCOUNTING STANDARDS ADMINISTRATION CAS Program Requirements 30.202 Disclosure...

  13. 48 CFR 9903.202 - Disclosure requirements.

    Science.gov (United States)

    2010-10-01

    ... ACCOUNTING STANDARDS CONTRACT COVERAGE CAS Program Requirements 9903.202 Disclosure requirements. ... 48 Federal Acquisition Regulations System 7 2010-10-01 2010-10-01 false Disclosure requirements. 9903.202 Section 9903.202 Federal Acquisition Regulations System COST ACCOUNTING STANDARDS BOARD...

  14. 48 CFR 1330.202 - Disclosure requirements.

    Science.gov (United States)

    2010-10-01

    ... 48 Federal Acquisition Regulations System 5 2010-10-01 2010-10-01 false Disclosure requirements. 1330.202 Section 1330.202 Federal Acquisition Regulations System DEPARTMENT OF COMMERCE GENERAL CONTRACTING REQUIREMENTS COST ACCOUNTING STANDARDS ADMINISTRATION CAS Program Requirements 1330.202 Disclosure...

  15. 40 CFR 141.80 - General requirements.

    Science.gov (United States)

    2010-07-01

    ... service line replacement, and public education. These requirements are triggered, in some cases, by lead... requirements. (1) All water systems shall install and operate optimal corrosion control treatment as defined in... specified by the State under § 141.83. (f) Lead service line replacement requirements. Any system exceeding...

  16. 7 CFR 718.9 - Signature requirements.

    Science.gov (United States)

    2010-01-01

    ... 7 Agriculture 7 2010-01-01 2010-01-01 false Signature requirements. 718.9 Section 718.9... MULTIPLE PROGRAMS General Provisions § 718.9 Signature requirements. (a) When a program authorized by this chapter or Chapter XIV of this title requires the signature of a producer; landowner; landlord; or tenant...

  17. 42 CFR 424.36 - Signature requirements.

    Science.gov (United States)

    2010-10-01

    ... 42 Public Health 3 2010-10-01 2010-10-01 false Signature requirements. 424.36 Section 424.36... (CONTINUED) MEDICARE PROGRAM CONDITIONS FOR MEDICARE PAYMENT Claims for Payment § 424.36 Signature requirements. (a) General rule. The beneficiary's own signature is required on the claim unless the beneficiary...

  18. 40 CFR 63.347 - Reporting requirements.

    Science.gov (United States)

    2010-07-01

    ... for Chromium Emissions From Hard and Decorative Chromium Electroplating and Chromium Anodizing Tanks... required each time that an affected source becomes subject to the requirements of this subpart. (2) If the... time a notification of compliance status is required under this part, the owner or operator of an...

  19. 47 CFR 80.1081 - Functional requirements.

    Science.gov (United States)

    2010-10-01

    ... 47 Telecommunication 5 2010-10-01 2010-10-01 false Functional requirements. 80.1081 Section 80... STATIONS IN THE MARITIME SERVICES Global Maritime Distress and Safety System (GMDSS) Equipment Requirements for Ship Stations § 80.1081 Functional requirements. Ships, while at sea, must be capable: (a) Except...

  20. 22 CFR 126.13 - Required information.

    Science.gov (United States)

    2010-04-01

    ... 22 Foreign Relations 1 2010-04-01 2010-04-01 false Required information. 126.13 Section 126.13... PROVISIONS § 126.13 Required information. (a) All applications for licenses (DSP-5, DSP-61, DSP-73, and DSP... are multiple consignors, consignees or freight forwarders, and all the required information cannot be...

  1. 76 FR 50881 - Required Scale Tests

    Science.gov (United States)

    2011-08-17

    ... RIN 0580-AB10 Required Scale Tests AGENCY: Grain Inspection, Packers and Stockyards Administration... required scale tests. Those documents defined ``limited seasonal basis'' incorrectly. This document... 20, 2011 (76 FR 3485) and on April 4, 2011 (76 FR 18348), concerning required scale tests. Those...

  2. School Library Media Certification Requirements: 1990 Update.

    Science.gov (United States)

    Perritt, Patsy H.

    1990-01-01

    Presents a compilation of school library media certification requirements taken from responses to a national survey. For each state, existing certificates or endorsements are listed, along with credit hours and/or experience required, whether it is an accredited or approved program, and required subject areas or competencies. A directory of…

  3. Reserve Requirements and Monetary Management; An Introduction

    OpenAIRE

    International Monetary Fund

    1993-01-01

    Reserve requirements are widely used by central banks as a means to improve monetary control, an instrument for policy implementation, a source of revenue, and a safeguard of bank liquidity. The effectiveness of reserve requirements in fulfilling these functions is reviewed, and the detailed modalities of their use are examined. Reserve requirements in a sample of developing countries are described.

  4. 42 CFR 417.934 - Reserve requirement.

    Science.gov (United States)

    2010-10-01

    ... 42 Public Health 3 2010-10-01 2010-10-01 false Reserve requirement. 417.934 Section 417.934 Public... PLANS Administration of Outstanding Loans and Loan Guarantees § 417.934 Reserve requirement. (a) Timing... section 1305 of the PHS Act was required to establish a restricted reserve account on the earlier of the...

  5. 7 CFR 1942.117 - General requirements.

    Science.gov (United States)

    2010-01-01

    ... § 1942.117 General requirements. (a) Reserve requirements. Loans under this subpart are subject to the... 7 Agriculture 13 2010-01-01 2009-01-01 true General requirements. 1942.117 Section 1942.117 Agriculture Regulations of the Department of Agriculture (Continued) RURAL HOUSING SERVICE, RURAL BUSINESS...

  6. Capturing security requirements for software systems

    Science.gov (United States)

    El-Hadary, Hassan; El-Kassas, Sherif

    2014-01-01

    Security is often an afterthought during software development. Realizing security early, especially in the requirement phase, is important so that security problems can be tackled early enough before going further in the process and avoid rework. A more effective approach for security requirement engineering is needed to provide a more systematic way for eliciting adequate security requirements. This paper proposes a methodology for security requirement elicitation based on problem frames. The methodology aims at early integration of security with software development. The main goal of the methodology is to assist developers elicit adequate security requirements in a more systematic way during the requirement engineering process. A security catalog, based on the problem frames, is constructed in order to help identifying security requirements with the aid of previous security knowledge. Abuse frames are used to model threats while security problem frames are used to model security requirements. We have made use of evaluation criteria to evaluate the resulting security requirements concentrating on conflicts identification among requirements. We have shown that more complete security requirements can be elicited by such methodology in addition to the assistance offered to developers to elicit security requirements in a more systematic way. PMID:25685514

  7. 49 CFR 383.111 - Required knowledge.

    Science.gov (United States)

    2010-10-01

    ... importance of proper visual search, and proper visual search methods. (6) Communication. The principles and procedures for proper communications and the hazards of failure to signal properly. (7) Speed management. The... STANDARDS; REQUIREMENTS AND PENALTIES Required Knowledge and Skills § 383.111 Required knowledge. All...

  8. Nicotinamide impairs entry into and exit from meiosis I in mouse oocytes.

    Science.gov (United States)

    Riepsamen, Angelique; Wu, Lindsay; Lau, Laurin; Listijono, Dave; Ledger, William; Sinclair, David; Homer, Hayden

    2015-01-01

    Following exit from meiosis I, mammalian oocytes immediately enter meiosis II without an intervening interphase, accompanied by rapid reassembly of a bipolar spindle that maintains condensed chromosomes in a metaphase configuration (metaphase II arrest). Here we study the effect of nicotinamide (NAM), a non-competitive pan-sirtuin inhibitor, during meiotic maturation in mouse oocytes. Sirtuins are a family of seven NAD+-dependent deacetylases (Sirt1-7), which are involved in multiple cellular processes and are emerging as important regulators in oocytes and embryos. We found that NAM significantly delayed entry into meiosis I associated with delayed accumulation of the Cdk1 co-activator, cyclin B1. GVBD was also inhibited by the Sirt2-specific inhibitor, AGK2, and in a very similar pattern to NAM, supporting the notion that as in somatic cells, NAM inhibits sirtuins in oocytes. NAM did not affect subsequent spindle assembly, chromosome alignment or the timing of first polar body extrusion (PBE). Unexpectedly, however, in the majority of oocytes with a polar body, chromatin was decondensed and a nuclear structure was present. An identical phenotype was observed when flavopiridol was used to induce Cdk1 inactivation during late meiosis I prior to PBE, but not if Cdk1 was inactivated after PBE when metaphase II arrest was already established, altogether indicating that NAM impaired establishment rather than maintenance of metaphase II arrest. During meiosis I exit in NAM-treated medium, we found that cyclin B1 levels were lower and inhibitory Cdk1 phosphorylation was increased compared with controls. Although activation of the anaphase-promoting complex-Cdc20 (APC-Cdc20) occurred on-time in NAM-treated oocytes, Cdc20 levels were higher in very late meiosis I, pointing to exaggerated APC-Cdc20-mediated proteolysis as a reason for lower cyclin B1 levels. Collectively, therefore, our data indicate that by disrupting Cdk1 regulation, NAM impairs entry into meiosis I and

  9. From document to database: modernizing requirements management

    International Nuclear Information System (INIS)

    Giajnorio, J.; Hamilton, S.

    2007-01-01

    The creation, communication, and management of design requirements are central to the successful completion of any large engineering project, both technically and commercially. Design requirements in the Canadian nuclear industry are typically numbered lists in multiple documents created using word processing software. As an alternative, GE Nuclear Products implemented a central requirements management database for a major project at Bruce Power. The database configured the off-the-shelf software product, Telelogic Doors, to GE's requirements structure. This paper describes the advantages realized by this scheme. Examples include traceability from customer requirements through to test procedures, concurrent engineering, and automated change history. (author)

  10. Fiscal year 1999 waste information requirements document

    International Nuclear Information System (INIS)

    Adams, M.R.

    1998-01-01

    The Waste Information Requirements Document (WIRD) has the following purposes: To describe the overall drivers that require characterization information and to document their source; To define how characterization is going to satisfy the drivers, close issues, and measure and report progress; and To describe deliverables and acceptance criteria for characterization. Characterization information is required to maintain regulatory compliance, perform operations and maintenance, resolve safety issues, and prepare for disposal of waste. Commitments addressing these requirements are derived from the Hanford Federal Facility Agreement and Consent Order, also known as the Tri-Party Agreement; the Recommendation 93-5 Implementation Plan (DOE-RL 1996a) to the Defense Nuclear Facilities Safety Board (DNFSB); and other requirement sources listed in Section 2.0. The Waste Information Requirements Document replaces the tank waste analysis plans and the tank characterization plan previously required by the Tri-Party Agreement, Milestone M-44-01 and M-44-02 series

  11. Requirements management at Westinghouse Electric Company

    International Nuclear Information System (INIS)

    Gustavsson, Henrik

    2014-01-01

    Field studies and surveys made in various industry branches support the Westinghouse opinion that qualitative systems engineering and requirements management have a high value in the development of complex systems and products. Two key issues causing overspending and schedule delays in projects are underestimation of complexity and misunderstandings between the different sub-project teams. These issues often arise when a project jumps too early into detail design. Good requirements management practice before detail design helps the project teams avoid such issues. Westinghouse therefore puts great effort into requirements management. The requirements management methodology at Westinghouse rests primarily on four key cornerstones: 1 - Iterative team work when developing requirements specifications, 2 - Id number tags on requirements, 3 - Robust change routine, and 4 - Requirements Traceability Matrix. (authors)

  12. TRACER - TRACING AND CONTROL OF ENGINEERING REQUIREMENTS

    Science.gov (United States)

    Turner, P. R.

    1994-01-01

    TRACER (Tracing and Control of Engineering Requirements) is a database/word processing system created to document and maintain the order of both requirements and descriptive material associated with an engineering project. A set of hierarchical documents are normally generated for a project whereby the requirements of the higher level documents levy requirements on the same level or lower level documents. Traditionally, the requirements are handled almost entirely by manual paper methods. The problem with a typical paper system, however, is that requirements written and changed continuously in different areas lead to misunderstandings and noncompliance. The purpose of TRACER is to automate the capture, tracing, reviewing, and managing of requirements for an engineering project. The engineering project still requires communications, negotiations, interactions, and iterations among people and organizations, but TRACER promotes succinct and precise identification and treatment of real requirements separate from the descriptive prose in a document. TRACER permits the documentation of an engineering project's requirements and progress in a logical, controllable, traceable manner. TRACER's attributes include the presentation of current requirements and status from any linked computer terminal and the ability to differentiate headers and descriptive material from the requirements. Related requirements can be linked and traced. The program also enables portions of documents to be printed, individual approval and release of requirements, and the tracing of requirements down into the equipment specification. Requirement "links" can be made "pending" and invisible to others until the pending link is made "binding". Individuals affected by linked requirements can be notified of significant changes with acknowledgement of the changes required. An unlimited number of documents can be created for a project and an ASCII import feature permits existing documents to be incorporated

  13. EPR compared to international requirements (Mainly EUR)

    International Nuclear Information System (INIS)

    Broecker, B.

    1996-01-01

    A number of European Utilities have entered an agreement to write common requirements dedicated to future light water nuclear power plants to be built in Europe. The activities are known under the sign EUR (European Utilities Requirements). EPR, the future European Pressurized water Reactor, is the first installation of this type which will be operational from the year 2000 onwards, must fulfill the European requirements. EPR will serve as a test whether these requirements are realistic and well balanced. At the basic design stage of EPR, this paper concentrates on four main topics: the requirements which are new compared with existing reactors and which put a major challenge to the designer; the requirements today still open and the way they can be met by the EPR or not; the points for which already today the EPR special requirements exceed the EUR; the examples where the design of the EPR has given feedback which has led to a change of the EUR. EPR and EUR are different approaches to the reactor of the future. EUR is a set of requirements which leaves a flexibility to the designer while EPR is a real project which defines the technical solutions. EPR will fulfill the EUR and will at the same time serve as a test whether these requirements are realistic. EPR will also fulfill international requirements with minor changes. (J.S.). 7 figs

  14. 77 FR 6704 - Exemptions From Entry Requirements and Report of Arrival Requirements for Certain Department of...

    Science.gov (United States)

    2012-02-09

    ... requirements and thus also subject to advanced electronic presentation of cargo information requirements. Under..., Aircraft, Airports, Alcohol and alcoholic beverages, Cigars and cigarettes, Cuba, Customs duties and...

  15. Management system requirements for small reactors

    Energy Technology Data Exchange (ETDEWEB)

    Jones, K.A., E-mail: kenneth.jones@cnsc-ccsn.gc.ca [Canadian Nuclear Safety Commission, Ottawa, Ontario (Canada)

    2013-07-01

    This abstract identifies the management system requirements for the life cycle of small reactors from initial conception through completion of decommissioning. For small reactors, the requirements for management systems remain the same as those for 'large' reactors regardless of the licensee' business model and objectives. The CSA N-Series of standards provides an interlinked set of requirements for the management of nuclear facilities. CSA N286 provides overall direction to management to develop and implement sound management practices and controls, while other CSA nuclear standards provide technical requirements and guidance that support the management system. CSA N286 is based on a set of principles. The principles are then supported by generic requirements that are applicable to the life cycle of nuclear facilities. CNSC regulatory documents provide further technical requirements and guidance. (author)

  16. Requirements Analysis in the Value Methodology

    Energy Technology Data Exchange (ETDEWEB)

    Conner, Alison Marie

    2001-05-01

    The Value Methodology (VM) study brings together a multidisciplinary team of people who own the problem and have the expertise to identify and solve it. With the varied backgrounds and experiences the team brings to the study, come different perspectives on the problem and the requirements of the project. A requirements analysis step can be added to the Information and Function Analysis Phases of a VM study to validate whether the functions being performed are required, either regulatory or customer prescribed. This paper will provide insight to the level of rigor applied to a requirements analysis step and give some examples of tools and techniques utilized to ease the management of the requirements and functions those requirements support for highly complex problems.

  17. Disposal of Radioactive Waste. Specific Safety Requirements

    International Nuclear Information System (INIS)

    2011-01-01

    This publication establishes requirements applicable to all types of radioactive waste disposal facility. It is linked to the fundamental safety principles for each disposal option and establishes a set of strategic requirements that must be in place before facilities are developed. Consideration is also given to the safety of existing facilities developed prior to the establishment of present day standards. The requirements will be complemented by Safety Guides that will provide guidance on good practice for meeting the requirements for different types of waste disposal facility. Contents: 1. Introduction; 2. Protection of people and the environment; 3. Safety requirements for planning for the disposal of radioactive waste; 4. Requirements for the development, operation and closure of a disposal facility; 5. Assurance of safety; 6. Existing disposal facilities; Appendices.

  18. Waste Acceptance System Requirements document (WASRD)

    International Nuclear Information System (INIS)

    1993-01-01

    This Waste Acceptance System Requirements document (WA-SRD) describes the functions to be performed and the technical requirements for a Waste Acceptance System for accepting spent nuclear fuel (SNF) and high-level radioactive waste (HLW) into the Civilian Radioactive Waste Management System (CRWMS). This revision of the WA-SRD addresses the requirements for the acceptance of HLW. This revision has been developed as a top priority document to permit DOE's Office of Environmental Restoration and Waste Management (EM) to commence waste qualification runs at the Savannah River Site's (SRS) Defense Waste Processing Facility (DWPF) in a timely manner. Additionally, this revision of the WA-SRD includes the requirements from the Physical System Requirements -- Accept Waste document for the acceptance of SNF. A subsequent revision will fully address requirements relative to the acceptance of SNF

  19. Mined Geologic Disposal System Requirements Document

    International Nuclear Information System (INIS)

    1993-01-01

    This Mined Geologic Disposal System Requirements document (MGDS-RD) describes the functions to be performed by, and the requirements for, a Mined Geologic Disposal System (MGDS) for the permanent disposal of spent nuclear fuel (SNF) and commercial and defense high level radioactive waste (HLW) in support of the Civilian Radioactive Waste Management System (CRWMS). The development and control of the MGDS-RD is quality-affecting work and is subject to the Department of Energy (DOE) Office of Civilian Radioactive Waste Management (OCRWM) Quality Assurance Requirements Document (QARD). As part of the technical requirements baseline, it is also subject to Baseline Management Plan controls. The MGDS-RD and the other program-level requirements documents have been prepared and managed in accordance with the Technical Document Preparation Plan (TDPP) for the Preparation of System Requirements Documents

  20. Legal requirements governing proxy voting in Denmark

    DEFF Research Database (Denmark)

    Werlauff, Erik

    2008-01-01

    The requirements in Danish company law concerning proxy voting in companies whose shares have been accepted for listing on a regulated market have been successively tightened in recent years, and corporate governance principles have also led to the introduction of several requirements concerning...... proxy holders. A thorough knowledge of these requirements is important not only for the listed companies but also for their advisers and investors in Denmark and abroad. This article considers these requirements as well as the additional requirements which will derive from Directive 2007....../36 on the exercise of shareholders' rights in listed companies, which must be implemented by 3 August 2009. It is pointed out that companies may provide with advantage in their articles of association for both the existing and the forthcoming requirements at this early stage....

  1. A Requirement Specification Language for AADL

    Science.gov (United States)

    2016-06-01

    models. The objective of ReqSpec is to support the elicitation , definition, and modeling of requirements for real-time embedded systems in an iterative...A Requirement Specification Language for AADL Peter H. Feiler Julien Delange Lutz Wrage June 2016 TECHNICAL REPORT CMU/SEI-2016-TR-008...is required for any other external and/or commercial use. Requests for permission should be directed to the Software En- gineering Institute at

  2. Civilian Radioactive Waste Management System Requirements Document

    International Nuclear Information System (INIS)

    1992-12-01

    This document specifies the top-level requirements for the Civilian Radioactive Waste Management System (CRWMS). The document is referred to herein as the CRD, for CRWMS Requirements document. The OCRWM System Engineering Management Plan (SEMP) establishes the technical document hierarchy (hierarchy of technical requirements and configuration baseline documents) for the CRWMS program. The CRD is the top-level document in this hierarchy. The immediate subordinate documents are the System Requirements Documents (SRDS) for the four elements of the CRWMS and the Interface Specification (IFS). The four elements of the CRWMS are the Waste Acceptance System, the Transportation System, the Monitored Retrievable Storage (MRS) System and the Mined Geologic Disposal System (MGDS). The Interface Specification describes the six inter-element interfaces between the four elements. This hierarchy establishes the requirements to be addressed by the design of the system elements. Many of the technical requirements for the CRWMS are documented in a variety of Federal regulations, DOE directives and other Government documentation. It is the purpose of the CRD to establish the technical requirements for the entire program. In doing so, the CRD summarizes source documentation for requirements that must be addressed by the program, specifies particular requirements, and documents derived requirements that are not covered in regulatory and other Government documentation, but are necessary to accomplish the mission of the CRWMS. The CRD defines the CRWMS by identifying the top-level functions the elements must perform (These top-level functions were derived using functional analysis initially documented in the Physical System Requirements (PSR) documents). The CRD also defines the top-level physical architecture of the system and allocates the functions and requirements to the architectural elements of the system

  3. Training Requirements and Information Management System

    Energy Technology Data Exchange (ETDEWEB)

    Cillan, T.F.; Hodgson, M.A.

    1992-05-01

    This is the software user's guide for the Training Requirements and Information Management System. This guide defines and describes the software operating procedures as they apply to the end user of the software program. This guide is intended as a reference tool for the user who already has an indepth knowledge of the Training Requirements and Information Management System functions and data reporting requirement.

  4. Required reserves as a credit policy tool

    OpenAIRE

    Mimir, Yasin; Sunel, Enes; Taskin, Temel

    2012-01-01

    This paper conducts a quantitative investigation of the role of reserve requirements as a macroprudential policy tool. We build a monetary DSGE model with a banking sector in which (i) an agency problem between households and banks leads to endogenous capital constraints for banks in obtaining funds from households, (ii) banks are subject to time-varying reserve requirements that countercyclically respond to expected credit growth, (iii) households face cash-in-advance constraints, requiring ...

  5. Safety of magnetic fusion facilities: Requirements

    International Nuclear Information System (INIS)

    1996-05-01

    This Standard identifies safety requirements for magnetic fusion facilities. Safety functions are used to define outcomes that must be achieved to ensure that exposures to radiation, hazardous materials, or other hazards are maintained within acceptable limits. Requirements applicable to magnetic fusion facilities have been derived from Federal law, policy, and other documents. In addition to specific safety requirements, broad direction is given in the form of safety principles that are to be implemented and within which safety can be achieved

  6. Energy requirements for racing endurance sled dogs*

    OpenAIRE

    Loftus, John P.; Yazwinski, Molly; Milizio, Justin G.; Wakshlag, Joseph J.

    2014-01-01

    Endurance sled dogs have unique dietary energy requirements. At present, there is disparity in the literature regarding energy expenditure and thus energy requirements of these dogs. We sought to further elucidate energy requirements for endurance sled dogs under field conditions. Three sled dog teams completing the 2011 Yukon Quest volunteered to provide diet history. Nutritional content was evaluated and a mock meal was analysed for each team. Race data were obtained from www.yukonquest.com...

  7. Identify and Manage the Software Requirements Volatility

    OpenAIRE

    Khloud Abd Elwahab; Mahmoud Abd EL Latif; Sherif Kholeif

    2016-01-01

    Management of software requirements volatility through development of life cycle is a very important stage. It helps the team to control significant impact all over the project (cost, time and effort), and also it keeps the project on track, to finally satisfy the user which is the main success criteria for the software project. In this research paper, we have analysed the root causes of requirements volatility through a proposed framework presenting the requirements volatility causes and how...

  8. Design requirements for the new reactor

    International Nuclear Information System (INIS)

    Koski, S.

    2005-01-01

    This presentation deals with the safety related design requirements specified for the new nuclear power plant to be built in Finland (FINS). The legislation, codes and standards, on which the design requirements are based, can be arranged into a hierarchical pyramid as follows: The safety related design criteria are based on the three uppermost hierarchical levels: Finnish legislation (e.g. decisions of the State Council) Basic Regulations (75-INSAG-3, USNRC General Design Criteria) Process oriented nuclear documents (YVL- guides or corresponding US/German rules). The European Utility Requirements (EUR) document was used as the starting point for the writing of the design requirements document. The structure and headlines of EUR could be kept, but in many cases the contents had to be deleted and rewritten to correspond to the requirement level of the above codes and standards. This was the case, for example, with the requirements concerning safety classification or application of failure criteria. In the presentation, the most important safety related design criteria are reviewed, with an emphasis on those requirements which exceed the requirement level applied on the existing plant units. Some hints are also given on the main differences between Finnish and international safety requirements. (orig.)

  9. Responsibilities in the Usability Requirements Elicitation Process

    Directory of Open Access Journals (Sweden)

    Marianella Aveledo

    2008-12-01

    Full Text Available Like any other software system quality attribute, usability places requirements on software components. In particular, it has been demonstrated that certain usability features have a direct impact throughout the software process. This paper details an approach that looks at how to deal with certain usability features in the early software development stages. In particular, we consider usability features as functional usability requirements using patterns that have been termed usability patterns to elicit requirements. Additionally, we clearly establish the responsibilities of all the players at the usability requirements elicitation stage.

  10. Daily Water Requirements when Wearing Body Armor

    National Research Council Canada - National Science Library

    Montain, Scott

    2000-01-01

    .... This report presents the results of model simulations predicting the individual daily water requirements under a broad range of energy expenditures and weather conditions when wearing battle dress...

  11. Safety of Research Reactors. Safety Requirements

    International Nuclear Information System (INIS)

    2010-01-01

    The main objective of this Safety Requirements publication is to provide a basis for safety and a basis for safety assessment for all stages in the lifetime of a research reactor. Another objective is to establish requirements on aspects relating to regulatory control, the management of safety, site evaluation, design, operation and decommissioning. Technical and administrative requirements for the safety of research reactors are established in accordance with these objectives. This Safety Requirements publication is intended for use by organizations engaged in the site evaluation, design, manufacturing, construction, operation and decommissioning of research reactors as well as by regulatory bodies

  12. A Requirements Analysis Model Based on QFD

    Institute of Scientific and Technical Information of China (English)

    TANG Zhi-wei; Nelson K.H.Tang

    2004-01-01

    The enterprise resource planning (ERP) system has emerged to offer an integrated IT solution and more and more enterprises are increasing by adopting this system and regarding it as an important innovation. However, there is already evidence of high failure risks in ERP project implementation, one major reason is poor analysis of the requirements for system implementation. In this paper, the importance of requirements analysis for ERP project implementation is highlighted, and a requirements analysis model by applying quality function deployment (QFD) is presented, which will support to conduct requirements analysis for ERP project.

  13. Hydrogen tomorrow: Demands and technology requirements

    Science.gov (United States)

    1975-01-01

    National needs for hydrogen are projected and the technologies of production, handling, and utilization are evaluated. Research and technology activities required to meet the projected needs are determined.

  14. 12 CFR 564.3 - Appraisals required; transactions requiring a State certified or licensed appraiser.

    Science.gov (United States)

    2010-01-01

    ... 12 Banks and Banking 5 2010-01-01 2010-01-01 false Appraisals required; transactions requiring a State certified or licensed appraiser. 564.3 Section 564.3 Banks and Banking OFFICE OF THRIFT SUPERVISION, DEPARTMENT OF THE TREASURY APPRAISALS § 564.3 Appraisals required; transactions requiring a State...

  15. CH2M Hill Hanford Group, Inc., Standards and Requirements Identification Document (SRID) Requirements Management System and Requirements Specification

    International Nuclear Information System (INIS)

    JOHNSON, A.L.

    2000-01-01

    The current Tank Farm Contractor (TFC) for the U. S. Department of Energy, Office of River Protection (ORP), River Protection Project (RPP), CH2M Hill Hanford Group, Inc. (CHG), will use a computer based requirements management system. The system will serve as a tool to assist in identifying, capturing, and maintaining the Standards/Requirements Identification Document (S/RID) requirements and links to implementing procedures and other documents. By managing requirements as one integrated set, CHG will be able to carry out its mission more efficiently and effectively. CHG has chosen the Dynamic Object Oriented Requirements System (DOORS(trademark)) as the preferred computer based requirements management system. Accordingly, the S/RID program will use DOORS(trademark). DOORS(trademark) will replace the Environmental Requirements Management Interface (ERMI) system as the tool for S/RID data management. The DOORS(trademark) S/RID test project currently resides on the DOORSTM test server. The S/RID project will be migrated to the DOORS(trademark) production server. After the migration the S/RID project will be considered a production project and will no longer reside on the test server

  16. 40 CFR 61.186 - Reporting requirements.

    Science.gov (United States)

    2010-07-01

    ...) NATIONAL EMISSION STANDARDS FOR HAZARDOUS AIR POLLUTANTS National Emission Standard for Inorganic Arsenic Emissions From Arsenic Trioxide and Metallic Arsenic Production Facilities § 61.186 Reporting requirements... at least 30 days prior notice of each reference opacity level determination required in § 61.183(a...

  17. 7 CFR 926.17 - Reporting requirements.

    Science.gov (United States)

    2010-01-01

    ... Federal cranberry marketing order (7 CFR Part 926) shall be required to submit four times annually, for... Agriculture Regulations of the Department of Agriculture (Continued) AGRICULTURAL MARKETING SERVICE (Marketing... RECORDKEEPING REQUIREMENTS APPLICABLE TO CRANBERRIES NOT SUBJECT TO THE CRANBERRY MARKETING ORDER § 926.17...

  18. 13 CFR 120.860 - Required objectives.

    Science.gov (United States)

    2010-01-01

    ... 13 Business Credit and Assistance 1 2010-01-01 2010-01-01 false Required objectives. 120.860 Section 120.860 Business Credit and Assistance SMALL BUSINESS ADMINISTRATION BUSINESS LOANS Development Company Loan Program (504) Project Economic Development Goals § 120.860 Required objectives. A Project...

  19. 49 CFR 229.206 - Design requirements.

    Science.gov (United States)

    2010-10-01

    ...-climber, emergency egress, emergency interior lighting, and interior configuration design requirements set... 49 Transportation 4 2010-10-01 2010-10-01 false Design requirements. 229.206 Section 229.206..., DEPARTMENT OF TRANSPORTATION RAILROAD LOCOMOTIVE SAFETY STANDARDS Locomotive Crashworthiness Design...

  20. 32 CFR 298.5 - Information requirements.

    Science.gov (United States)

    2010-07-01

    ... 32 National Defense 2 2010-07-01 2010-07-01 false Information requirements. 298.5 Section 298.5... OF INFORMATION ACT PROGRAM DEFENSE INVESTIGATIVE SERVICE (DIS) FREEDOM OF INFORMATION ACT PROGRAM § 298.5 Information requirements. The DIS Office of Information and Public Affairs is responsible for...

  1. Nuclear plant requirements during power system restoration

    International Nuclear Information System (INIS)

    Adamski, G.; Jenkins, R.; Gill, P.

    1995-01-01

    This paper is one of a series presented on behalf of the System Operation Subcommittee with the intent of focusing industry attention on power system restoration issues. This paper discusses a number of nuclear power plant requirements that require special attention during power system restoration

  2. 27 CFR 4.38 - General requirements.

    Science.gov (United States)

    2010-04-01

    ... mandatory information required on labels by this part, except the alcoholic content statement, shall be in... OF THE TREASURY LIQUORS LABELING AND ADVERTISING OF WINE Labeling Requirements for Wine § 4.38... descriptive or explanatory information, the script, type, or printing of the mandatory information shall be of...

  3. 8 CFR 312.1 - Literacy requirements.

    Science.gov (United States)

    2010-01-01

    ... 8 Aliens and Nationality 1 2010-01-01 2010-01-01 false Literacy requirements. 312.1 Section 312.1... FOR NATURALIZATION § 312.1 Literacy requirements. (a) General. Except as otherwise provided in... determining English proficiency, as outlined in paragraph(c) of this section. (c) Literacy examination—(1...

  4. 7 CFR 1770.13 - Accounting requirements.

    Science.gov (United States)

    2010-01-01

    ... accrual basis of accounting. All transactions shall be recorded in the period in which they occur and... 7 Agriculture 12 2010-01-01 2010-01-01 false Accounting requirements. 1770.13 Section 1770.13... AGRICULTURE (CONTINUED) ACCOUNTING REQUIREMENTS FOR RUS TELECOMMUNICATIONS BORROWERS Uniform System of...

  5. Linoleic acid intake and vitamin E requirement

    NARCIS (Netherlands)

    Jager, F.C.

    1973-01-01

    In experiments with rats and Peking ducklings it has been investigated to what extent the linoleic acid content of the diet is of influence on the requirement of vitamin E. This requirement was determined by adding D-α-tocopheryl acetate in increasing doses to vitamin E-free diets and to determine

  6. 40 CFR 63.707 - Reporting requirements.

    Science.gov (United States)

    2010-07-01

    ... proposed limit; and (iv) A statement signed by a responsible official of the company that the control... monitoring system performance report and summary reports shall be submitted as required by § 63.10(e). (1... the performance test required by § 63.7: (i) The performance test or CEM data collected to establish...

  7. Grid Code Requirements for Wind Power Integration

    DEFF Research Database (Denmark)

    Wu, Qiuwei

    2018-01-01

    This chapter reviews the grid code requirements for integration of wind power plants (WPPs). The grid codes reviewed are from the UK, Ireland, Germany, Denmark, Spain, Sweden, the USA, and Canada. Transmission system operators (TSOs) around the world have specified requirements for WPPs under...

  8. 17 CFR 41.2 - Required records.

    Science.gov (United States)

    2010-04-01

    ... 17 Commodity and Securities Exchanges 1 2010-04-01 2010-04-01 false Required records. 41.2 Section 41.2 Commodity and Securities Exchanges COMMODITY FUTURES TRADING COMMISSION SECURITY FUTURES PRODUCTS General Provisions § 41.2 Required records. A designated contract market or registered derivatives...

  9. 16 CFR 307.2 - Required warnings.

    Science.gov (United States)

    2010-01-01

    ... 16 Commercial Practices 1 2010-01-01 2010-01-01 false Required warnings. 307.2 Section 307.2 Commercial Practices FEDERAL TRADE COMMISSION REGULATIONS UNDER SPECIFIC ACTS OF CONGRESS REGULATIONS UNDER... Comprehensive Smokeless Tobacco Health Education Act of 1986 is the law that requires the enactment of these...

  10. 15 CFR 970.517 - Diligence requirements.

    Science.gov (United States)

    2010-01-01

    ... 15 Commerce and Foreign Trade 3 2010-01-01 2010-01-01 false Diligence requirements. 970.517 Section 970.517 Commerce and Foreign Trade Regulations Relating to Commerce and Foreign Trade (Continued..., Conditions and Restrictions Terms, Conditions, and Restrictions § 970.517 Diligence requirements. The terms...

  11. 15 CFR 971.418 - Diligence requirements.

    Science.gov (United States)

    2010-01-01

    ... 15 Commerce and Foreign Trade 3 2010-01-01 2010-01-01 false Diligence requirements. 971.418 Section 971.418 Commerce and Foreign Trade Regulations Relating to Commerce and Foreign Trade (Continued...: Terms, Conditions and Restrictions Terms, Conditions and Restrictions § 971.418 Diligence requirements...

  12. ISS Crew Transportation and Services Requirements Document

    Science.gov (United States)

    Bayt, Robert L. (Compiler); Lueders, Kathryn L. (Compiler)

    2016-01-01

    The ISS Crew Transportation and Services Requirements Document (CCT-REQ-1130) contains all technical, safety, and crew health medical requirements that are mandatory for achieving a Crew Transportation System Certification that will allow for International Space Station delivery and return of NASA crew and limited cargo. Previously approved on TN23183.

  13. 30 CFR 250.1000 - General requirements.

    Science.gov (United States)

    2010-07-01

    ... 30 Mineral Resources 2 2010-07-01 2010-07-01 false General requirements. 250.1000 Section 250.1000... OPERATIONS IN THE OUTER CONTINENTAL SHELF Pipelines and Pipeline Rights-of-Way § 250.1000 General....1001, must meet the requirements in §§ 250.1000 through 250.1008. (2) A pipeline right-of-way grant...

  14. Core Requirements for the Economics Major

    Science.gov (United States)

    Petkus, Marie; Perry, John J.; Johnson, Bruce K.

    2014-01-01

    In this article, the authors are the first to describe the core economics curriculum requirements for economics majors at all American colleges and universities, as opposed to a sample of institutions. Not surprisingly, principles of economics is nearly universally required and implemented as a two-semester course in 85 percent of economics major…

  15. An Online Graduate Requirements Engineering Course

    Science.gov (United States)

    Kilicay-Ergin, N.; Laplante, P. A.

    2013-01-01

    Requirements engineering is one of the fundamental knowledge areas in software and systems engineering graduate curricula. Recent changes in educational delivery and student demographics have created new challenges for requirements engineering education. In particular, there is an increasing demand for online education for working professionals.…

  16. 9 CFR 93.106 - Quarantine requirements.

    Science.gov (United States)

    2010-01-01

    ... Section 93.106 Animals and Animal Products ANIMAL AND PLANT HEALTH INSPECTION SERVICE, DEPARTMENT OF... other than ratites and hatching eggs of ratites. Each lot of pet birds, except as provided for in § 93.... (2) Physical plant requirements. The facility shall comply with the following requirements: (i...

  17. 40 CFR 63.1018 - Reporting requirements.

    Science.gov (United States)

    2010-07-01

    ...) National Emission Standards for Equipment Leaks-Control Level 1 § 63.1018 Reporting requirements. (a... § 63.1007(e)(2) and those pumps complying with the closed vent system provisions of § 63.1007(e)(3). (iv) Number of compressors subject to the requirements of § 63.1012, excluding those compressors...

  18. 12 CFR 563g.9 - Escrow requirement.

    Science.gov (United States)

    2010-01-01

    ... 12 Banks and Banking 5 2010-01-01 2010-01-01 false Escrow requirement. 563g.9 Section 563g.9 Banks and Banking OFFICE OF THRIFT SUPERVISION, DEPARTMENT OF THE TREASURY SECURITIES OFFERINGS § 563g.9 Escrow requirement. (a) Any funds received in an offering which is offered and sold on a best efforts all...

  19. Critical Review of NOAA's Observation Requirements Process

    Science.gov (United States)

    LaJoie, M.; Yapur, M.; Vo, T.; Templeton, A.; Bludis, D.

    2017-12-01

    NOAA's Observing Systems Council (NOSC) maintains a comprehensive database of user observation requirements. The requirements collection process engages NOAA subject matter experts to document and effectively communicate the specific environmental observation measurements (parameters and attributes) needed to produce operational products and pursue research objectives. User observation requirements documented using a structured and standardized manner and framework enables NOAA to assess its needs across organizational lines in an impartial, objective, and transparent manner. This structure provides the foundation for: selecting, designing, developing, acquiring observing technologies, systems and architectures; budget and contract formulation and decision-making; and assessing in a repeatable fashion the productivity, efficiency and optimization of NOAA's observing system enterprise. User observation requirements are captured independently from observing technologies. Therefore, they can be addressed by a variety of current or expected observing capabilities and allow flexibility to be remapped to new and evolving technologies. NOAA's current inventory of user observation requirements were collected over a ten-year period, and there have been many changes in policies, mission priorities, and funding levels during this time. In light of these changes, the NOSC initiated a critical, in-depth review to examine all aspects of user observation requirements and associated processes during 2017. This presentation provides background on the NOAA requirements process, major milestones and outcomes of the critical review, and plans for evolving and connecting observing requirements processes in the next year.

  20. Requirements elicitation for geo-information solutions

    NARCIS (Netherlands)

    Robbi Sluter, Claudia; van Elzakker, Corné P.J.M.; Ivanova, Ivana

    2017-01-01

    Geo-information solutions can achieve a higher level of quality if they are developed in accordance with a user-centred design that requires definition of the user requirements in the first step of solution construction. We treat a geo-information solution as a system designed to support human-based