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Sample records for sequential proteolytic cleavage

  1. In-capillary self-assembly and proteolytic cleavage of polyhistidine peptide capped quantum dots

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    Wang, Jianhao; Li, Jingyan; Li, Jinchen; Liu, Feifei [School of Pharmaceutical Engineering and Life Science, Changzhou University, Changzhou, Jiangsu, 213164 (China); Zhou, Xiang; Yao, Yi [Changzhou Qianhong Bio-pharma Co. Ltd, Changzhou 213164, Jiangsu (China); Wang, Cheli [School of Pharmaceutical Engineering and Life Science, Changzhou University, Changzhou, Jiangsu, 213164 (China); Qiu, Lin, E-mail: linqiupjj@gmail.com [School of Pharmaceutical Engineering and Life Science, Changzhou University, Changzhou, Jiangsu, 213164 (China); Jiang, Pengju, E-mail: pengju.jiang@gmail.com [School of Pharmaceutical Engineering and Life Science, Changzhou University, Changzhou, Jiangsu, 213164 (China); State Key Laboratory of Pharmaceutical Biotechnology, Nanjing, Jiangsu (China)

    2015-10-01

    A new method using fluorescence coupled capillary electrophoresis (CE-FL) for monitoring self-assembly and proteolytic cleavage of hexahistidine peptide capped quantum dots (QDs) inside a capillary has been developed in this report. QDs and the ATTO 590-labeled hexahistidine peptide (H6-ATTO) were injected into a capillary, sequentially. Their self-assembly inside the capillary was driven by a metal-affinity force which yielded a new fluorescence signal due to Förster resonance energy transfer (FRET). The highly efficient separation of fluorescent complexes and the FRET process were analyzed using CE-FL. The self-assembly of QDs and biomolecules was found to effectively take place inside the capillary. The kinetics of the assembly was monitored by CE-FL, and the approach was extended to the study of proteolytic cleavage of surface conjugated peptides. Being the first in-depth analysis of in-capillary nanoparticle–biomolecule assembly, the novel approach reported here provides inspiration to the development of QD-based FRET probes for biomedical applications. - Highlights: • We examined the self-assembly QDs with H6-ATTO inside a capillary. • We prove CE-FL to be a powerful method to resolve QDs-H6-ATTO complex. • We achieve chromatographic separation of QDs-H6-ATTO complex. • We discovered a novel strategy for the online detection of thrombin. • This technique integrated “injection, mixing, reaction, separation and detection”.

  2. The p2 domain of human immunodeficiency virus type 1 Gag regulates sequential proteolytic processing and is required to produce fully infectious virions.

    Science.gov (United States)

    Pettit, S C; Moody, M D; Wehbie, R S; Kaplan, A H; Nantermet, P V; Klein, C A; Swanstrom, R

    1994-12-01

    The proteolytic processing sites of the human immunodeficiency virus type 1 (HIV-1) Gag precursor are cleaved in a sequential manner by the viral protease. We investigated the factors that regulate sequential processing. When full-length Gag protein was digested with recombinant HIV-1 protease in vitro, four of the five major processing sites in Gag were cleaved at rates that differ by as much as 400-fold. Three of these four processing sites were cleaved independently of the others. The CA/p2 site, however, was cleaved approximately 20-fold faster when the adjacent downstream p2/NC site was blocked from cleavage or when the p2 domain of Gag was deleted. These results suggest that the presence of a C-terminal p2 tail on processing intermediates slows cleavage at the upstream CA/p2 site. We also found that lower pH selectively accelerated cleavage of the CA/p2 processing site in the full-length precursor and as a peptide primarily by a sequence-based mechanism rather than by a change in protein conformation. Deletion of the p2 domain of Gag results in released virions that are less infectious despite the presence of the processed final products of Gag. These findings suggest that the p2 domain of HIV-1 Gag regulates the rate of cleavage at the CA/p2 processing site during sequential processing in vitro and in infected cells and that p2 may function in the proper assembly of virions.

  3. Proteolytic Cleavage of ProBDNF into Mature BDNF in the Basolateral Amygdala Is Necessary for Defeat-Induced Social Avoidance

    Science.gov (United States)

    Dulka, Brooke N.; Ford, Ellen C.; Lee, Melissa A.; Donnell, Nathaniel J.; Goode, Travis D.; Prosser, Rebecca; Cooper, Matthew A.

    2016-01-01

    Brain-derived neurotrophic factor (BDNF) is essential for memory processes. The present study tested whether proteolytic cleavage of proBDNF into mature BDNF (mBDNF) within the basolateral amygdala (BLA) regulates the consolidation of defeat-related memories. We found that acute social defeat increases the expression of mBDNF, but not proBDNF, in…

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

    Directory of Open Access Journals (Sweden)

    Lin PY

    2015-10-01

    Full Text Available Pao-Yen Lin1,2 1Department of Psychiatry, Kaohsiung Chang Gung Memorial Hospital, Chang Gung University College of Medicine, 2Center for Translational Research in Biomedical Sciences, Kaohsiung Chang Gung Memorial Hospital, Kaohsiung, Taiwan Abstract: Evidence has supported the role of brain-derived neurotrophic factor (BDNF in antidepressant effect. The precursor of BDNF (proBDNF often exerts opposing biological effects on mature BDNF (mBDNF. Hence, the balance between proBDNF and mBDNF might be critical in total neurotrophic effects, leading to susceptibility to or recovery from depression. In the current study, we measured the protein expression levels of proBDNF, and its proteolytic products, truncated BDNF, and mBDNF, in human SH-SY5Y cells treated with different antidepressants. We found that the treatment significantly increased the production of mBDNF, but decreased the production of truncated BDNF and proBDNF. These results support that antidepressants can promote proBDNF cleavage. Further studies are needed to clarify whether proBDNF cleavage plays a role in antidepressant mechanisms. Keywords: antidepressant, mature BDNF, neurotrophic effect, proBDNF cleavage 

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

    International Nuclear Information System (INIS)

    Craft, Willie Warren; Dutch, Rebecca Ellis

    2005-01-01

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

  6. The epithelial sodium channel γ-subunit is processed proteolytically in human kidney

    DEFF Research Database (Denmark)

    Langkilde, Rikke Zachar; Skjødt, Karsten; Marcussen, Niels

    2015-01-01

    The epithelial sodium channel (ENaC) of the kidney is necessary for extracellular volume homeostasis and normal arterial BP. Activity of ENaC is enhanced by proteolytic cleavage of the gamma-subunit and putative release of a 43-amino acid inhibitory tract from the gamma-subunit ectodomain. We......ENaC was detected consistently only in tissue from patients with proteinuria and observed in collecting ducts. In conclusion, human kidney gammaENaC is subject to proteolytic cleavage, yielding fragments compatible with furin cleavage, and proteinuria is associated with cleavage at the putative prostasin...

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

    Science.gov (United States)

    Mazor, Rafi; Schmid-Schönbein, Geert W

    2015-01-01

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

  8. Proteolytic cleavage and PKA phosphorylation of α1C subunit are not required for adrenergic regulation of CaV1.2 in the heart.

    Science.gov (United States)

    Katchman, Alexander; Yang, Lin; Zakharov, Sergey I; Kushner, Jared; Abrams, Jeffrey; Chen, Bi-Xing; Liu, Guoxia; Pitt, Geoffrey S; Colecraft, Henry M; Marx, Steven O

    2017-08-22

    Calcium influx through the voltage-dependent L-type calcium channel (Ca V 1.2) rapidly increases in the heart during "fight or flight" through activation of the β-adrenergic and protein kinase A (PKA) signaling pathway. The precise molecular mechanisms of β-adrenergic activation of cardiac Ca V 1.2, however, are incompletely known, but are presumed to require phosphorylation of residues in α 1C and C-terminal proteolytic cleavage of the α 1C subunit. We generated transgenic mice expressing an α 1C with alanine substitutions of all conserved serine or threonine, which is predicted to be a potential PKA phosphorylation site by at least one prediction tool, while sparing the residues previously shown to be phosphorylated but shown individually not to be required for β-adrenergic regulation of Ca V 1.2 current (17-mutant). A second line included these 17 putative sites plus the five previously identified phosphoregulatory sites (22-mutant), thus allowing us to query whether regulation requires their contribution in combination. We determined that acute β-adrenergic regulation does not require any combination of potential PKA phosphorylation sites conserved in human, guinea pig, rabbit, rat, and mouse α 1C subunits. We separately generated transgenic mice with inducible expression of proteolytic-resistant α 1C Prevention of C-terminal cleavage did not alter β-adrenergic stimulation of Ca V 1.2 in the heart. These studies definitively rule out a role for all conserved consensus PKA phosphorylation sites in α 1C in β-adrenergic stimulation of Ca V 1.2, and show that phosphoregulatory sites on α 1C are not redundant and do not each fractionally contribute to the net stimulatory effect of β-adrenergic stimulation. Further, proteolytic cleavage of α 1C is not required for β-adrenergic stimulation of Ca V 1.2.

  9. Transcript levels, alternative splicing and proteolytic cleavage of TFIIIA control 5S rRNA accumulation during Arabidopsis thaliana development.

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    Layat, Elodie; Cotterell, Sylviane; Vaillant, Isabelle; Yukawa, Yasushi; Tutois, Sylvie; Tourmente, Sylvette

    2012-07-01

    Ribosome biogenesis is critical for eukaryotic cells and requires coordinated synthesis of the protein and rRNA moieties of the ribosome, which are therefore highly regulated. 5S ribosomal RNA, an essential component of the large ribosomal subunit, is transcribed by RNA polymerase III and specifically requires transcription factor IIIA (TFIIIA). To obtain insight into the regulation of 5S rRNA transcription, we have investigated the expression of 5S rRNA and the exon-skipped (ES) and exon-including (EI) TFIIIA transcripts, two transcript isoforms that result from alternative splicing of the TFIIIA gene, and TFIIIA protein amounts with respect to requirements for 5S rRNA during development. We show that 5S rRNA quantities are regulated through distinct but complementary mechanisms operating through transcriptional and post-transcriptional control of TFIIIA transcripts as well as at the post-translational level through proteolytic cleavage of the TFIIIA protein. During the reproductive phase, high expression of the TFIIIA gene together with low proteolytic cleavage contributes to accumulation of functional, full-length TFIIIA protein, and results in 5S rRNA accumulation in the seed. In contrast, just after germination, the levels of TFIIIA-encoding transcripts are low and stable. Full-length TFIIIA protein is undetectable, and the level of 5S rRNA stored in the embryo progressively decreases. After day 4, in correlation with the reorganization of 5S rDNA chromatin to a mature state, full-length TFIIIA protein with transcriptional activity accumulates and permits de novo transcription of 5S rRNA. © 2012 The Authors. The Plant Journal © 2012 Blackwell Publishing Ltd.

  10. CleavPredict: A Platform for Reasoning about Matrix Metalloproteinases Proteolytic Events.

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    Sonu Kumar

    Full Text Available CleavPredict (http://cleavpredict.sanfordburnham.org is a Web server for substrate cleavage prediction for matrix metalloproteinases (MMPs. It is intended as a computational platform aiding the scientific community in reasoning about proteolytic events. CleavPredict offers in silico prediction of cleavage sites specific for 11 human MMPs. The prediction method employs the MMP specific position weight matrices (PWMs derived from statistical analysis of high-throughput phage display experimental results. To augment the substrate cleavage prediction process, CleavPredict provides information about the structural features of potential cleavage sites that influence proteolysis. These include: secondary structure, disordered regions, transmembrane domains, and solvent accessibility. The server also provides information about subcellular location, co-localization, and co-expression of proteinase and potential substrates, along with experimentally determined positions of single nucleotide polymorphism (SNP, and posttranslational modification (PTM sites in substrates. All this information will provide the user with perspectives in reasoning about proteolytic events. CleavPredict is freely accessible, and there is no login required.

  11. Deletion of the thrombin cleavage domain of osteopontin mediates breast cancer cell adhesion, proteolytic activity, tumorgenicity, and metastasis

    International Nuclear Information System (INIS)

    Beausoleil, Michel S; Schulze, Erika B; Goodale, David; Postenka, Carl O; Allan, Alison L

    2011-01-01

    Osteopontin (OPN) is a secreted phosphoprotein often overexpressed at high levels in the blood and primary tumors of breast cancer patients. OPN contains two integrin-binding sites and a thrombin cleavage domain located in close proximity to each other. To study the role of the thrombin cleavage site of OPN, MDA-MB-468 human breast cancer cells were stably transfected with either wildtype OPN (468-OPN), mutant OPN lacking the thrombin cleavage domain (468-ΔTC) or an empty vector (468-CON) and assessed for in vitro and in vivo functional differences in malignant/metastatic behavior. All three cell lines were found to equivalently express thrombin, tissue factor, CD44, αvβ5 integrin and β1 integrin. Relative to 468-OPN and 468-CON cells, 468-ΔTC cells expressing OPN with a deleted thrombin cleavage domain demonstrated decreased cell adhesion (p < 0.001), decreased mRNA expression of MCAM, maspin and TRAIL (p < 0.01), and increased uPA expression and activity (p < 0.01) in vitro. Furthermore, injection of 468-ΔTC cells into the mammary fat pad of nude mice resulted in decreased primary tumor latency time (p < 0.01) and increased primary tumor growth and lymph node metastatic burden (p < 0.001) compared to 468-OPN and 468-CON cells. The results presented here suggest that expression of thrombin-uncleavable OPN imparts an early tumor formation advantage as well as a metastatic advantage for breast cancer cells, possibly due to increased proteolytic activity and decreased adhesion and apoptosis. Clarification of the mechanisms responsible for these observations and the translation of this knowledge into the clinic could ultimately provide new therapeutic opportunities for combating breast cancer

  12. The p2 domain of human immunodeficiency virus type 1 Gag regulates sequential proteolytic processing and is required to produce fully infectious virions.

    OpenAIRE

    Pettit, S C; Moody, M D; Wehbie, R S; Kaplan, A H; Nantermet, P V; Klein, C A; Swanstrom, R

    1994-01-01

    The proteolytic processing sites of the human immunodeficiency virus type 1 (HIV-1) Gag precursor are cleaved in a sequential manner by the viral protease. We investigated the factors that regulate sequential processing. When full-length Gag protein was digested with recombinant HIV-1 protease in vitro, four of the five major processing sites in Gag were cleaved at rates that differ by as much as 400-fold. Three of these four processing sites were cleaved independently of the others. The CA/p...

  13. Site-specific proteolytic degradation of IgG monoclonal antibodies expressed in tobacco plants.

    Science.gov (United States)

    Hehle, Verena K; Lombardi, Raffaele; van Dolleweerd, Craig J; Paul, Mathew J; Di Micco, Patrizio; Morea, Veronica; Benvenuto, Eugenio; Donini, Marcello; Ma, Julian K-C

    2015-02-01

    Plants are promising hosts for the production of monoclonal antibodies (mAbs). However, proteolytic degradation of antibodies produced both in stable transgenic plants and using transient expression systems is still a major issue for efficient high-yield recombinant protein accumulation. In this work, we have performed a detailed study of the degradation profiles of two human IgG1 mAbs produced in plants: an anti-HIV mAb 2G12 and a tumour-targeting mAb H10. Even though they use different light chains (κ and λ, respectively), the fragmentation pattern of both antibodies was similar. The majority of Ig fragments result from proteolytic degradation, but there are only a limited number of plant proteolytic cleavage events in the immunoglobulin light and heavy chains. All of the cleavage sites identified were in the proximity of interdomain regions and occurred at each interdomain site, with the exception of the VL /CL interface in mAb H10 λ light chain. Cleavage site sequences were analysed, and residue patterns characteristic of proteolytic enzymes substrates were identified. The results of this work help to define common degradation events in plant-produced mAbs and raise the possibility of predicting antibody degradation patterns 'a priori' and designing novel stabilization strategies by site-specific mutagenesis. © 2014 Society for Experimental Biology, Association of Applied Biologists and John Wiley & Sons Ltd.

  14. Bicarbonate-dependent secretion and proteolytic processing of recombinant myocilin.

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    José-Daniel Aroca-Aguilar

    Full Text Available Myocilin is an extracellular glycoprotein of poorly understood function. Mutations of this protein are involved in glaucoma, an optic neuropathy characterized by a progressive and irreversible visual loss and frequently associated with elevated intraocular pressure. We previously showed that recombinant myocilin undergoes an intracellular proteolytic processing by calpain II which cleaves the central region of the protein, releasing one N- and one C-terminal fragment. Myocilin cleavage is reduced by glaucoma mutations and it has been proposed to participate in intraocular pressure modulation. To identify possible factors regulating the proteolytic processing of recombinant myocilin, we used a cellular model in which we analyzed how different culture medium parameters (i.e., culture time, cell density, pH, bicarbonate concentration, etc. affect the presence of the extracellular C-terminal fragment. Extracellular bicarbonate depletion associated with culture medium acidification produced a reversible intracellular accumulation of full-length recombinant myocilin and incremented its intracellular proteolytic processing, raising the extracellular C-terminal fragment percentage. It was also determined that myocilin intracellular accumulation depends on its N-terminal region. These data suggest that aqueous humor bicarbonate variations could also modulate the secretion and cleavage of myocilin present in ocular tissues.

  15. Determination of proteolytic activity using L-[4,5-3H]leucine-labelled globin as a substrate

    International Nuclear Information System (INIS)

    Maliopoulou, T.B.; Dionyssiou-Asteriou, A.; Loucopoulos, D.

    1980-01-01

    A simple and sensitive method for the assay of proteolytic enzyme activity is described. This is based on the digestion of L-[4,5- 3 H]leucine globin by proteolytic enzymes and radioactivity measurement of the trichloroacetic acid soluble cleavage products. (Auth.)

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

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    Joseph Chavarría-Smith

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

  17. Proteolytic processing of poliovirus polypeptides: antibodies to polypeptide P3-7c inhibit cleavage at glutamine-glycine pairs

    International Nuclear Information System (INIS)

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

    1982-01-01

    Proteolytic processing of poliovirus polypeptides was examined by the addition of antibodies directed against the viral proteins P3-7c and P2-X to a cell-free translation extract prepared from infected HeLa cells. Antisera to P3-7c specifically inhibited in vitro processing at Gln-Gly pairs. Partial amino acid sequence analysis revealed a second Tyr-Gly pair that is utilized in protein processing. Neither Tyr-Gly cleavage is affected by antibody to P3-7C. Anti-P3-7c antibodies react not only with P3-7c but also with P3-6a and P3-2, two viral polypeptides NH 2 -coterminal with P3-7c. Preimmune and anti-P2-X antibodies had no effect on the processing of poliovirus proteins in vitro. The authors conclude that the activity responsible for processing poliovirus polypeptides at Gln-Gly pairs resides in the primary structure of P3-7c and not in P2-X

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

    International Nuclear Information System (INIS)

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

    2016-01-01

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

  19. Tyrosine phosphorylation and proteolytic cleavage of Notch are required for non-canonical Notch/Abl signaling in Drosophila axon guidance.

    Science.gov (United States)

    Kannan, Ramakrishnan; Cox, Eric; Wang, Lei; Kuzina, Irina; Gu, Qun; Giniger, Edward

    2018-01-17

    Notch signaling is required for the development and physiology of nearly every tissue in metazoans. Much of Notch signaling is mediated by transcriptional regulation of downstream target genes, but Notch controls axon patterning in Drosophila by local modulation of Abl tyrosine kinase signaling, via direct interactions with the Abl co-factors Disabled and Trio. Here, we show that Notch-Abl axonal signaling requires both of the proteolytic cleavage events that initiate canonical Notch signaling. We further show that some Notch protein is tyrosine phosphorylated in Drosophila , that this form of the protein is selectively associated with Disabled and Trio, and that relevant tyrosines are essential for Notch-dependent axon patterning but not for canonical Notch-dependent regulation of cell fate. Based on these data, we propose a model for the molecular mechanism by which Notch controls Abl signaling in Drosophila axons. © 2018. Published by The Company of Biologists Ltd.

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

    Science.gov (United States)

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

    2015-03-01

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

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

    Energy Technology Data Exchange (ETDEWEB)

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

    2015-12-25

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

  2. Dopaminergic neurotoxicant 6-OHDA induces oxidative damage through proteolytic activation of PKCδ in cell culture and animal models of Parkinson's disease

    International Nuclear Information System (INIS)

    Latchoumycandane, Calivarathan; Anantharam, Vellareddy; Jin, Huajun; Kanthasamy, Anumantha; Kanthasamy, Arthi

    2011-01-01

    The neurotoxicant 6-hydroxydopamine (6-OHDA) is used to investigate the cellular and molecular mechanisms underlying selective degeneration of dopaminergic neurons in Parkinson's disease (PD). Oxidative stress and caspase activation contribute to the 6-OHDA-induced apoptotic cell death of dopaminergic neurons. In the present study, we sought to systematically characterize the key downstream signaling molecule involved in 6-OHDA-induced dopaminergic degeneration in cell culture and animal models of PD. Treatment of mesencephalic dopaminergic neuronal N27 cells with 6-OHDA (100 μM) for 24 h significantly reduced mitochondrial activity and increased cytosolic cytochrome c, followed by sequential activation of caspase-9 and caspase-3. Co-treatment with the free radical scavenger MnTBAP (10 μM) significantly attenuated 6-OHDA-induced caspase activities. Interestingly, 6-OHDA induced proteolytic cleavage and activation of protein kinase C delta (PKCδ) was completely suppressed by treatment with a caspase-3-specific inhibitor, Z-DEVD-FMK (50 μM). Furthermore, expression of caspase-3 cleavage site-resistant mutant PKCδ D327A and kinase dead PKCδ K376R or siRNA-mediated knockdown of PKCδ protected against 6-OHDA-induced neuronal cell death, suggesting that caspase-3-dependent PKCδ promotes oxidative stress-induced dopaminergic degeneration. Suppression of PKCδ expression by siRNA also effectively protected N27 cells from 6-OHDA-induced apoptotic cell death. PKCδ cleavage was also observed in the substantia nigra of 6-OHDA-injected C57 black mice but not in control animals. Viral-mediated delivery of PKCδ D327A protein protected against 6-OHDA-induced PKCδ activation in mouse substantia nigra. Collectively, these results strongly suggest that proteolytic activation of PKCδ is a key downstream event in dopaminergic degeneration, and these results may have important translational value for development of novel treatment strategies for PD.

  3. Intrinsic Tau Acetylation Is Coupled to Auto-Proteolytic Tau Fragmentation.

    Directory of Open Access Journals (Sweden)

    Todd J Cohen

    Full Text Available Tau proteins are abnormally aggregated in a range of neurodegenerative tauopathies including Alzheimer's disease (AD. Recently, tau has emerged as an extensively post-translationally modified protein, among which lysine acetylation is critical for normal tau function and its pathological aggregation. Here, we demonstrate that tau isoforms have different propensities to undergo lysine acetylation, with auto-acetylation occurring more prominently within the lysine-rich microtubule-binding repeats. Unexpectedly, we identified a unique intrinsic property of tau in which auto-acetylation induces proteolytic tau cleavage, thereby generating distinct N- and C-terminal tau fragments. Supporting a catalytic reaction-based mechanism, mapping and mutagenesis studies showed that tau cysteines, which are required for acetyl group transfer, are also essential for auto-proteolytic tau processing. Further mass spectrometry analysis identified the C-terminal 2nd and 4th microtubule binding repeats as potential sites of auto-cleavage. The identification of acetylation-mediated auto-proteolysis provides a new biochemical mechanism for tau self-regulation and warrants further investigation into whether auto-catalytic functions of tau are implicated in AD and other tauopathies.

  4. Blocking of proteolytic processing and deletion of glycosaminoglycan side chain of mouse DMP1 by substituting critical amino acid residues.

    Science.gov (United States)

    Peng, Tao; Huang, Bingzhen; Sun, Yao; Lu, Yongbo; Bonewald, Lynda; Chen, Shuo; Butler, William T; Feng, Jerry Q; D'Souza, Rena N; Qin, Chunlin

    2009-01-01

    Dentin matrix protein 1 (DMP1) is present in the extracellular matrix (ECM) of dentin and bone as processed NH(2)- and COOH-terminal fragments, resulting from proteolytic cleavage at the NH(2) termini of 4 aspartic acid residues during rat DMP1 processing. One cleavage site residue, Asp(181) (corresponding to Asp(197) of mouse DMP1), and its flanking region are highly conserved across species. We speculate that cleavage at the NH(2) terminus of Asp(197) of mouse DMP1 represents an initial, first-step scission in the whole cascade of proteolytic processing. To test if Asp(197) is critical for initiating the proteolytic processing of mouse DMP1, we substituted Asp(197) with Ala(197) by mutating the corresponding nucleotides of mouse cDNA that encode this amino acid residue. This mutant DMP1 cDNA was cloned into a pcDNA3.1 vector. Data from transfection experiments indicated that this single substitution blocked the proteolytic processing of mouse DMP1 in HEK-293 cells, indicating that cleavage at the NH(2) terminus of Asp(197) is essential for exposing other cleavage sites for the conversion of DMP1 to its fragments. The NH(2)-terminal fragment of DMP1 occurs as a proteoglycan form (DMP1-PG) that contains a glycosaminoglycan (GAG) chain. Previously, we showed that a GAG chain is linked to Ser(74) in rat DMP1 (Ser(89) in mouse DMP1). To confirm that mouse DMP1-PG possesses a single GAG chain attached to Ser(89), we substituted Ser(89) by Gly(89). Data from transfection analysis indicated that this substitution completely prevented formation of the GAG-containing form, confirming that DMP1-PG contains a single GAG chain attached to Ser(89) in mouse DMP1. Copyright 2008 S. Karger AG, Basel.

  5. Combined Quantification of the Global Proteome, Phosphoproteome, and Proteolytic Cleavage to Characterize Altered Platelet Functions in the Human Scott Syndrome.

    Science.gov (United States)

    Solari, Fiorella A; Mattheij, Nadine J A; Burkhart, Julia M; Swieringa, Frauke; Collins, Peter W; Cosemans, Judith M E M; Sickmann, Albert; Heemskerk, Johan W M; Zahedi, René P

    2016-10-01

    The Scott syndrome is a very rare and likely underdiagnosed bleeding disorder associated with mutations in the gene encoding anoctamin-6. Platelets from Scott patients are impaired in various Ca 2+ -dependent responses, including phosphatidylserine exposure, integrin closure, intracellular protein cleavage, and cytoskeleton-dependent morphological changes. Given the central role of anoctamin-6 in the platelet procoagulant response, we used quantitative proteomics to understand the underlying molecular mechanisms and the complex phenotypic changes in Scott platelets compared with control platelets. Therefore, we applied an iTRAQ-based multi-pronged strategy to quantify changes in (1) the global proteome, (2) the phosphoproteome, and (3) proteolytic events between resting and stimulated Scott and control platelets. Our data indicate a limited number of proteins with decreased (70) or increased (64) expression in Scott platelets, among those we confirmed the absence of anoctamin-6 and the strong up-regulation of aquaporin-1 by parallel reaction monitoring. The quantification of 1566 phosphopeptides revealed major differences between Scott and control platelets after stimulation with thrombin/convulxin or ionomycin. In Scott platelets, phosphorylation levels of proteins regulating cytoskeletal or signaling events were increased. Finally, we quantified 1596 N-terminal peptides in activated Scott and control platelets, 180 of which we identified as calpain-regulated, whereas a distinct set of 23 neo-N termini was caspase-regulated. In Scott platelets, calpain-induced cleavage of cytoskeleton-linked and signaling proteins was downregulated, in accordance with an increased phosphorylation state. Thus, multipronged proteomic profiling of Scott platelets provides detailed insight into their protection against detrimental Ca 2+ -dependent changes that are normally associated with phosphatidylserine exposure. © 2016 by The American Society for Biochemistry and Molecular

  6. Developing a capillary electrophoresis based method for dynamically monitoring enzyme cleavage activity using quantum dots-peptide assembly.

    Science.gov (United States)

    Wang, Jianhao; Fan, Jie; Liu, Li; Ding, Shumin; Liu, Xiaoqian; Wang, Jianpeng; Gao, Liqian; Chattopadhaya, Souvik; Miao, Peng; Xia, Jiang; Qiu, Lin; Jiang, Pengju

    2017-10-01

    Herein, a novel assay has been developed for monitoring PreScission protease (His-PSP) mediated enzyme cleavage of ATTO 590 labeled peptide substrate (ATTO-LEV). This novel method is based on combining the use of capillary electrophoresis and fluorescence detection (CE-FL) to dynamically monitor the enzyme cleavage activity. A multivalent peptide substrate was first constructed by immobilizing His-tagged ATTO 590 labeled peptide substrate (ATTO-LEVH6) onto the surface of CdSe/ZnS quantum dots (QDs). Once successfully immobilized, the novel multivalent peptide substrate resulted in the Förster resonance energy transfer (FRET) from QDs to ATTO 590. The ATTO-LEVH6-QD assembly was then incubated with His-PSP to study the proteolytic cleavage of surface bound ATTO-LEVH6 by CE-FL. Our data suggests that PreScission-mediated proteolytic cleavage is enzyme concentration- and incubation time-dependent. By combining capillary electrophoresis, QDs and FRET, our study herein not only provides a new method for the detection and dynamically monitoring of PSP enzyme cleavage activity, but also can be extended to the detection of many other enzymes and proteases. © 2017 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  7. Elastinolytic and proteolytic enzymes.

    Science.gov (United States)

    Kessler, Efrat; Safrin, Mary

    2014-01-01

    Pseudomonas aeruginosa secretes into its environment at least seven extracellular proteases: pseudolysin (LasB protease; elastase), aeruginolysin (alkaline proteinase), staphylolysin (staphylolytic endopeptidase; LasA protease), lysyl endopeptidase (protease IV; PrpL), PASP (P. aeruginosa small protease), LepA (Large ExoProtease A), and an aminopeptidase. Their action on host proteins, both individually and synergistically, plays important roles in pathogenesis of P. aeruginosa infections. Methods to measure/detect their activities are fundamental for understanding their physiological functions, roles in pathogenesis, mechanisms of action, regulation, and secretion. Most assays for determination/detection of proteolytic activity employ modified/non-modified casein or gelatin as substrates. In the quantitative assay, fragments generated from azocasein are separated from undigested substrate by trichloroacetic acid precipitation and their absorbance is measured. In non-quantitative assays, proteolytic activity is detected as clearing zones around bacterial growth or samples of culture supernatants on casein containing solid media formed due to local casein degradation. In zymography, individual proteases are detected as clear bands in gelatin/casein containing gels after SDS-PAGE separation, renaturation and protein staining. The elastinolytic capacity of P. aeruginosa is reflected by clearing zones on nutrient agar plates containing insoluble elastin instead of casein. Mueller-Hinton agar plates on which S. aureus cells are grown as a lawn are used to assess the susceptibility of S. aureus isolates to staphylolysin. A clear zone around a staphylolysin-containing sample indicates inhibition of S. aureus growth. Methods for measuring the activity of individual proteases are based on their cleavage specificity. These include assays of elastinolytic activity of pseudolysin and/or staphylolysin using elastin-Congo red as a substrate, a method for determination of

  8. Coronavirus 3CL(pro) proteinase cleavage sites: Possible relevance to SARS virus pathology

    DEFF Research Database (Denmark)

    Kiemer, Lars; Lund, Ole; Brunak, Søren

    2004-01-01

    the picornaviruses it is known that pathology is related to proteolytic cleavage of host proteins by viral proteinases. Furthermore, several studies indicate that virus proliferation can be arrested using specific proteinase inhibitors supporting the belief that proteinases are indeed important during infection...

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

    Science.gov (United States)

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

    2017-07-14

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

  10. Dopaminergic neurotoxicant 6-OHDA induces oxidative damage through proteolytic activation of PKC{delta} in cell culture and animal models of Parkinson's disease

    Energy Technology Data Exchange (ETDEWEB)

    Latchoumycandane, Calivarathan; Anantharam, Vellareddy; Jin, Huajun; Kanthasamy, Anumantha; Kanthasamy, Arthi, E-mail: arthik@iastate.edu

    2011-11-15

    The neurotoxicant 6-hydroxydopamine (6-OHDA) is used to investigate the cellular and molecular mechanisms underlying selective degeneration of dopaminergic neurons in Parkinson's disease (PD). Oxidative stress and caspase activation contribute to the 6-OHDA-induced apoptotic cell death of dopaminergic neurons. In the present study, we sought to systematically characterize the key downstream signaling molecule involved in 6-OHDA-induced dopaminergic degeneration in cell culture and animal models of PD. Treatment of mesencephalic dopaminergic neuronal N27 cells with 6-OHDA (100 {mu}M) for 24 h significantly reduced mitochondrial activity and increased cytosolic cytochrome c, followed by sequential activation of caspase-9 and caspase-3. Co-treatment with the free radical scavenger MnTBAP (10 {mu}M) significantly attenuated 6-OHDA-induced caspase activities. Interestingly, 6-OHDA induced proteolytic cleavage and activation of protein kinase C delta (PKC{delta}) was completely suppressed by treatment with a caspase-3-specific inhibitor, Z-DEVD-FMK (50 {mu}M). Furthermore, expression of caspase-3 cleavage site-resistant mutant PKC{delta}{sup D327A} and kinase dead PKC{delta}{sup K376R} or siRNA-mediated knockdown of PKC{delta} protected against 6-OHDA-induced neuronal cell death, suggesting that caspase-3-dependent PKC{delta} promotes oxidative stress-induced dopaminergic degeneration. Suppression of PKC{delta} expression by siRNA also effectively protected N27 cells from 6-OHDA-induced apoptotic cell death. PKC{delta} cleavage was also observed in the substantia nigra of 6-OHDA-injected C57 black mice but not in control animals. Viral-mediated delivery of PKC{delta}{sup D327A} protein protected against 6-OHDA-induced PKC{delta} activation in mouse substantia nigra. Collectively, these results strongly suggest that proteolytic activation of PKC{delta} is a key downstream event in dopaminergic degeneration, and these results may have important translational value for

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

    Science.gov (United States)

    Lu, Rui-Nan; Yang, Shangbin; Wu, Haifeng M.; Zheng, X. Long

    2015-01-01

    Summary Background Bilirubin is a yellow breakdown product of heme catabolism. Increased serum levels of unconjugated bilirubin are conditions commonly seen in premature neonates and adults with acute hemolysis including thrombotic microangiopathy. Previous studies have shown that unconjugated bilirubin lowers plasma ADAMTS13 activity, but the mechanism is not fully understood. Objectives The study is to determine whether unconjugated bilirubin directly inhibits the cleavage of von Willebrand factor (VWF) and its analogs by ADAMTS13. Methods Fluorogenic, SELDI-TOF mass spectrometric assay, and Western blotting analyses were employed to address this question. Results Unconjugated bilirubin inhibits the cleavage of F485-rVWF73-H, D633-rVWF73-H, and GST-rVWF71-11K by ADAMTS13 in a concentration-dependent manner with a half-maximal inhibitory concentration (IC50) of ~13 μM, ~70 μM, and ~17 μM, respectively. Unconjugated bilirubin also dose-dependently inhibits the cleavage of multimeric VWF by ADAMTS13 under denaturing conditions. The inhibitory activity of bilirubin on the cleavage of D633-rVWF73-H and multimeric VWF, but not F485-rVWF73-H, was eliminated after incubation with bilirubin oxidase that converts bilirubin to biliverdin. Furthermore, plasma ADAMTS13 activity in patients with hyperbilirubinemia is lower prior to than after treatment with bilirubin oxidase. Conclusions unconjugated bilirubin directly inhibits ADAMTS13’s ability to cleave both peptidyl and native VWF substrates in addition to its interference with certain fluorogenic assays. Our findings may help proper interpretation of ADAMTS13 results under pathological conditions. Whether elevated serum unconjugated bilirubin has an adverse effect in vivo remains to be determined in our future study. PMID:25782102

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

    Science.gov (United States)

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

    2014-12-01

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

  13. Proteolytic activation transforms heparin cofactor II into a host defense molecule.

    Science.gov (United States)

    Kalle, Martina; Papareddy, Praveen; Kasetty, Gopinath; Tollefsen, Douglas M; Malmsten, Martin; Mörgelin, Matthias; Schmidtchen, Artur

    2013-06-15

    The abundant serine proteinase inhibitor heparin cofactor II (HCII) has been proposed to inhibit extravascular thrombin. However, the exact physiological role of this plasma protein remains enigmatic. In this study, we demonstrate a previously unknown role for HCII in host defense. Proteolytic cleavage of the molecule induced a conformational change, thereby inducing endotoxin-binding and antimicrobial properties. Analyses employing representative peptide epitopes mapped these effects to helices A and D. Mice deficient in HCII showed increased susceptibility to invasive infection by Pseudomonas aeruginosa, along with a significantly increased cytokine response. Correspondingly, decreased levels of HCII were observed in wild-type animals challenged with bacteria or endotoxin. In humans, proteolytically cleaved HCII forms were detected during wounding and in association with bacteria. Thus, the protease-induced uncovering of cryptic epitopes in HCII, which transforms the molecule into a host defense factor, represents a previously unknown regulatory mechanism in HCII biology and innate immunity.

  14. Polycystin-1 Cleavage and the Regulation of Transcriptional Pathways

    Science.gov (United States)

    Merrick, David; Bertuccio, Claudia A.; Chapin, Hannah C.; Lal, Mark; Chauvet, Veronique; Caplan, Michael J.

    2013-01-01

    Autosomal dominant polycystic kidney disease (ADPKD) is the most common genetic cause of end stage renal disease, affecting ~1 in 1,000 people. The disease is characterized by the development of numerous large fluid filled renal cysts over the course of decades. These cysts compress the surrounding renal parenchyma and impair its function. Mutations in two genes are responsible for ADPKD. The protein products of both of these genes, polycystin-1 and polycystin-2, localize to the primary cilium and participate in a wide variety of signaling pathways. Polycystin-1 undergoes several proteolytic cleavages that produce fragments that manifest biological activities. Recent results suggest that the production of polycystin-1 cleavage fragments is necessary and sufficient to account for at least some, although certainly not all, of the physiological functions of the parent protein. PMID:23824180

  15. Differential proteolytic activation of factor VIII-von Willebrand factor complex by thrombin

    International Nuclear Information System (INIS)

    Hill-Eubanks, D.C.; Parker, C.G.; Lollar, P.

    1989-01-01

    Blood coagulation factor VIII (fVIII) is a plasma protein that is decreased or absent in hemophilia A. It is isolated as a mixture of heterodimers that contain a variably sized heavy chain and a common light chain. Thrombin catalyzes the activation of fVIII in a reaction that is associated with cleavages in both types of chain. The authors isolated a serine protease from Bothrops jararacussu snake venom that catalyzes thrombin-like heavy-chain cleavage but not light-chain cleavage in porcine fVIII as judged by NaDodSO 4 /PAGE and N-terminal sequence analysis. Using a plasma-free assay of the ability of activated 125 I-fVIII to function as a cofactor in the activation of factor X by factor IXa, they found that fVIII is activated by the venom enzyme. The venom enzyme-activated fVIII was isolated in stable form by cation-exchange HPLC. von Willebrand factor inhibited venom enzyme-activated fVIII but not thrombin-activated fVIII. These results suggest that the binding of fVIII to von Willebrand factor depends on the presence of an intact light chain and that activated fVIII must dissociate from von Willebrand factor to exert its cofactor effect. Thus, proteolytic activation of fVIII-von Willebrand factor complex appears to be differentially regulated by light-chain cleavage to dissociate the complex and heavy-chain cleavage to activate the cofactor function

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

    Directory of Open Access Journals (Sweden)

    Nicole Hofmann

    Full Text Available ABCA3 is a lipid transporter in the limiting membrane of lamellar bodies in alveolar type II cells. Mutations in the ABCA3 gene cause respiratory distress syndrome in new-borns and childhood interstitial lung disease. ABCA3 is N-terminally cleaved by an as yet unknown protease, a process believed to regulate ABCA3 activity.The exact site where ABCA3 is cleaved was localized using mass spectrometry (MS. Proteases involved in ABCA3 processing were identified using small molecule inhibitors and siRNA mediated gene knockdown. Results were verified by in vitro digestion of a synthetic peptide substrate mimicking ABCA3's cleavage region, followed by MS analysis.We found that cleavage of ABCA3 occurs after Lys174 which is located in the proteins' first luminal loop. Inhibition of cathepsin L and, to a lesser extent, cathepsin B resulted in attenuation of ABCA3 cleavage. Both enzymes showed activity against the ABCA3 peptide in vitro with cathepsin L being more active.We show here that, like some other proteins of the lysosomal membrane, ABCA3 is a substrate of cathepsin L. Therefore, cathepsin L may represent a potential target to therapeutically influence ABCA3 activity in ABCA3-associated lung disease.

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

    OpenAIRE

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

    1990-01-01

    Tomato black ring virus RNA-1 was translated in a rabbit reticulocyte lysate. The primary translation product of Mr 250K, which corresponds to its whole coding capacity, was synthesized within 45 min and, during further incubation in the translation medium, was proteolytically processed. Essentially, four cleavage products (P190, P120, P60 and P50) were detected and located within P250 by pulse-chase and immunoprecipitation experiments. P190 is an intermediate cleavage product which is furthe...

  18. A mature and fusogenic form of the Nipah virus fusion protein requires proteolytic processing by cathepsin L

    International Nuclear Information System (INIS)

    Pager, Cara Theresia; Craft, Willie Warren; Patch, Jared; Dutch, Rebecca Ellis

    2006-01-01

    The Nipah virus fusion (F) protein is proteolytically processed to F 1 + F 2 subunits. We demonstrate here that cathepsin L is involved in this important maturation event. Cathepsin inhibitors ablated cleavage of Nipah F. Proteolytic processing of Nipah F and fusion activity was dramatically reduced in cathepsin L shRNA-expressing Vero cells. Additionally, Nipah virus F-mediated fusion was inhibited in cathepsin L-deficient cells, but coexpression of cathepsin L restored fusion activity. Both purified cathepsin L and B could cleave immunopurified Nipah F protein, but only cathepsin L produced products of the correct size. Our results suggest that endosomal cathepsins can cleave Nipah F, but that cathepsin L specifically converts Nipah F to a mature and fusogenic form

  19. Proteolytic processing of the vitellogenin precursor in the boll weevil, Anthonomus grandis.

    Science.gov (United States)

    Heilmann, L J; Trewitt, P M; Kumaran, A K

    1993-01-01

    The soluble proteins of the eggs of the coleopteran insect Anthonomus grandis Boheman, the cotton boll weevil, consist almost entirely of two vitellin types with M(r)s of 160,000 and 47,000. We sequenced their N-terminal ends and one internal cyanogen bromide fragment of the large vitellin and compared these sequences with the deduced amino acid sequence from the vitellogenin gene. The results suggest that both the boll weevil vitellin proteins are products of the proteolytic cleavage of a single precursor protein. The smaller 47,000 M(r) vitellin protein is derived from the N-terminal portion of the precursor adjacent to an 18 amino acid signal peptide. The cleavage site between the large and small vitellins at amino acid 362 is adjacent to a pentapeptide sequence containing two pairs of arginine residues. Comparison of the boll weevil sequences with limited known sequences from the single 180,000 M(r) honey bee protein show that the honey bee vitellin N-terminal exhibits sequence homology to the N-terminal of the 47,000 M(r) boll weevil vitellin. Treatment of the vitellins with an N-glycosidase results in a decrease in molecular weight of both proteins, from 47,000 to 39,000 and from 160,000 to 145,000, indicating that about 10-15% of the molecular weight of each vitellin consists of N-linked carbohydrate. The molecular weight of the deglycosylated large vitellin is smaller than that predicted from the gene sequence, indicating possible further proteolytic processing at the C-terminal of that protein.

  20. Calsyntenin-1 shelters APP from proteolytic processing during anterograde axonal transport

    Directory of Open Access Journals (Sweden)

    Martin Steuble

    2012-06-01

    Endocytosis of amyloid-β precursor protein (APP is thought to represent the major source of substrate for the production of the amyloidogenic Aβ peptide by the β-secretase BACE1. The irreversible nature of proteolytic cleavage implies the existence of an efficient replenishment route for APP from its sites of synthesis to the cell surface. We recently found that APP exits the trans-Golgi network in intimate association with calsyntenin-1, a transmembrane cargo-docking protein for Kinesin-1-mediated vesicular transport. Here we characterized the function of calsyntenin-1 in neuronal APP transport using selective immunoisolation of intracellular trafficking organelles, immunocytochemistry, live-imaging, and RNAi. We found that APP is co-transported with calsyntenin-1 along axons to early endosomes in the central region of growth cones in carriers that exclude the α-secretase ADAM10. Intriguingly, calsyntenin-1/APP organelles contained BACE1, suggesting premature cleavage of APP along its anterograde path. However, we found that APP contained in calsyntenin-1/APP organelles was stable. We further analyzed vesicular trafficking of APP in cultured hippocampal neurons, in which calsyntenin-1 was reduced by RNAi. We found a markedly increased co-localization of APP and ADAM10 in axons and growth cones, along with increased proteolytic processing of APP and Aβ secretion in these neurons. This suggested that the reduced capacity for calsyntenin-1-dependent APP transport resulted in mis-sorting of APP into additional axonal carriers and, therefore, the premature encounter of unprotected APP with its ectodomain proteases. In combination, our results characterize calsyntenin-1/APP organelles as carriers for sheltered anterograde axonal transport of APP.

  1. PROTEOLYTIC PROCESSING OF VON WILLEBRAND FACTOR BY ADAMTS13 AND LEUKOCYTE PROTEASES

    Directory of Open Access Journals (Sweden)

    Stefano Lancellotti

    2013-09-01

    Full Text Available ADAMTS13 is a 190 kDa zinc protease encoded by a gene located on chromosome 9q34.   This protease specifically hydrolyzes von Willebrand factor (VWF multimers, thus causing VWF size reduction. ADAMTS13 belongs to the A Disintegrin And Metalloprotease with ThromboSpondin type 1 repeats (ADAMTS family, involved in proteolytic processing of many matrix proteins. ADAMTS13 consists of numerous domains including a metalloprotease domain, a disintegrin domain, several thrombospondin type 1 (TSP1 repeats, a cysteine-rich domain, a spacer domain and 2 CUB (Complement c1r/c1s, sea Urchin epidermal growth factor, and Bone morphogenetic protein domains. ADAMTS13 cleaves a single peptide bond (Tyr1605-Met1606 in the central A2 domain of the VWF molecule. This proteolytic cleavage is essential to reduce the size of ultra-large VWF polymers, which, when exposed to high shear stress in the microcirculation, are prone to form with platelets clumps, which cause severe syndromes called thrombotic microangiopathies (TMAs. In this review, we a discuss the current knowledge of structure-function aspects of ADAMTS13 and its involvement in the pathogenesis of TMAs, b address the recent findings concerning proteolytic processing of VWF multimers by different proteases, such as the leukocyte-derived serine and metallo-proteases and c indicate the direction of future investigations

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

    Science.gov (United States)

    Lewis, John G; Elder, Peter A

    2017-07-01

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

  3. Low Proteolytic Clipping of Histone H3 in Cervical Cancer

    Science.gov (United States)

    Sandoval-Basilio, Jorge; Serafín-Higuera, Nicolás; Reyes-Hernandez, Octavio D.; Serafín-Higuera, Idanya; Leija-Montoya, Gabriela; Blanco-Morales, Magali; Sierra-Martínez, Monica; Ramos-Mondragon, Roberto; García, Silvia; López-Hernández, Luz Berenice; Yocupicio-Monroy, Martha; Alcaraz-Estrada, Sofia L.

    2016-01-01

    Chromatin in cervical cancer (CC) undergoes chemical and structural changes that alter the expression pattern of genes. Recently, a potential mechanism, which regulates gene expression at transcriptional levels is the proteolytic clipping of histone H3. However, until now this process in CC has not been reported. Using HeLa cells as a model of CC and human samples from patients with CC, we identify that the H3 cleavage was lower in CC compared with control tissue. Additionally, the histone H3 clipping was performed by serine and aspartyl proteases in HeLa cells. These results suggest that histone H3 clipping operates as part of post-translational modification system in CC. PMID:27698925

  4. Proteolytic activation of proapoptotic kinase protein kinase Cδ by tumor necrosis factor α death receptor signaling in dopaminergic neurons during neuroinflammation

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    Gordon Richard

    2012-04-01

    Full Text Available Abstract Background The mechanisms of progressive dopaminergic neuronal loss in Parkinson’s disease (PD remain poorly understood, largely due to the complex etiology and multifactorial nature of disease pathogenesis. Several lines of evidence from human studies and experimental models over the last decade have identified neuroinflammation as a potential pathophysiological mechanism contributing to disease progression. Tumor necrosis factor α (TNF has recently emerged as the primary neuroinflammatory mediator that can elicit dopaminergic cell death in PD. However, the signaling pathways by which TNF mediates dopaminergic cell death have not been completely elucidated. Methods In this study we used a dopaminergic neuronal cell model and recombinant TNF to characterize intracellular signaling pathways activated during TNF-induced dopaminergic neurotoxicity. Etanercept and neutralizing antibodies to tumor necrosis factor receptor 1 (TNFR1 were used to block TNF signaling. We confirmed the results from our mechanistic studies in primary embryonic mesencephalic cultures and in vivo using the stereotaxic lipopolysaccharide (LPS model of nigral dopaminergic degeneration. Results TNF signaling in dopaminergic neuronal cells triggered the activation of protein kinase Cδ (PKCδ, an isoform of the novel PKC family, by caspase-3 and caspase-8 dependent proteolytic cleavage. Both TNFR1 neutralizing antibodies and the soluble TNF receptor Etanercept blocked TNF-induced PKCδ proteolytic activation. Proteolytic activation of PKCδ was accompanied by translocation of the kinase to the nucleus. Notably, inhibition of PKCδ signaling by small interfering (siRNA or overexpression of a PKCδ cleavage-resistant mutant protected against TNF-induced dopaminergic neuronal cell death. Further, primary dopaminergic neurons obtained from PKCδ knockout (−/− mice were resistant to TNF toxicity. The proteolytic activation of PKCδ in the mouse substantia nigra in the

  5. Kallistatin Ameliorates Influenza Virus Pathogenesis by Inhibition of Kallikrein-Related Peptidase 1-Mediated Cleavage of Viral Hemagglutinin

    Science.gov (United States)

    Leu, Chia-Hsing; Yang, Mei-Lin; Chung, Nai-Hui; Huang, Yen-Jang; Su, Yu-Chu; Chen, Yi-Cheng; Lin, Chia-Cheng; Shieh, Gia-Shing; Chang, Meng-Ya; Wang, Shainn-Wei; Chang, Yao; Chao, Julie; Chao, Lee

    2015-01-01

    Proteolytic cleavage of the hemagglutinin (HA) of influenza virus by host trypsin-like proteases is required for viral infectivity. Some serine proteases are capable of cleaving influenza virus HA, whereas some serine protease inhibitors (serpins) inhibit the HA cleavage in various cell types. Kallikrein-related peptidase 1 (KLK1, also known as tissue kallikrein) is a widely distributed serine protease. Kallistatin, a serpin synthesized mainly in the liver and rapidly secreted into the circulation, forms complexes with KLK1 and inhibits its activity. Here, we investigated the roles of KLK1 and kallistatin in influenza virus infection. We show that the levels of KLK1 increased, whereas those of kallistatin decreased, in the lungs of mice during influenza virus infection. KLK1 cleaved H1, H2, and H3 HA molecules and consequently enhanced viral production. In contrast, kallistatin inhibited KLK1-mediated HA cleavage and reduced viral production. Cells transduced with the kallistatin gene secreted kallistatin extracellularly, which rendered them more resistant to influenza virus infection. Furthermore, lentivirus-mediated kallistatin gene delivery protected mice against lethal influenza virus challenge by reducing the viral load, inflammation, and injury in the lung. Taking the data together, we determined that KLK1 and kallistatin contribute to the pathogenesis of influenza virus by affecting the cleavage of the HA peptide and inflammatory responses. This study provides a proof of principle for the potential therapeutic application of kallistatin or other KLK1 inhibitors for influenza. Since proteolytic activation also enhances the infectivity of some other viruses, kallistatin and other kallikrein inhibitors may be explored as antiviral agents against these viruses. PMID:26149981

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

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    Blom Nikolaj

    2004-06-01

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

  7. Measurement of separase proteolytic activity in single living cells by a fluorogenic flow cytometry assay.

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    Wiltrud Haaß

    Full Text Available ESPL1/Separase, an endopeptidase, is required for centrosome duplication and separation of sister-chromatides in anaphase of mitosis. Overexpression and deregulated proteolytic activity of Separase as frequently observed in human cancers is associated with the occurrence of supernumerary centrosomes, chromosomal missegregation and aneuploidy. Recently, we have hypothesized that increased Separase proteolytic activity in a small subpopulation of tumor cells may serve as driver of tumor heterogeneity and clonal evolution in chronic myeloid leukemia (CML. Currently, there is no quantitative assay to measure Separase activity levels in single cells. Therefore, we have designed a flow cytometry-based assay that utilizes a Cy5- and rhodamine 110 (Rh110-biconjugated Rad21 cleavage site peptide ([Cy5-D-R-E-I-M-R]2-Rh110 as smart probe and intracellular substrate for detection of Separase enzyme activity in living cells. As measured by Cy5 fluorescence the cellular uptake of the fluorogenic peptide was fast and reached saturation after 210 min of incubation in human histiocytic lymphoma U937 cells. Separase activity was recorded as the intensity of Rh110 fluorescence released after intracellular peptide cleavage providing a linear signal gain within a 90-180 min time slot. Compared to conventional cell extract-based methods the flow cytometric assay delivers equivalent results but is more reliable, bypasses the problem of vague loading controls and unspecific proteolysis associated with whole cell extracts. Especially suited for the investigaton of blood- and bone marrow-derived hematopoietic cells the flow cytometric Separase assay allows generation of Separase activity profiles that tell about the number of Separase positive cells within a sample i.e. cells that currently progress through mitosis and about the range of intercellular variation in Separase activity levels within a cell population. The assay was used to quantify Separase proteolytic

  8. Soybean P34 Probable Thiol Protease Probably Has Proteolytic Activity on Oleosins.

    Science.gov (United States)

    Zhao, Luping; Kong, Xiangzhen; Zhang, Caimeng; Hua, Yufei; Chen, Yeming

    2017-07-19

    P34 probable thiol protease (P34) and Gly m Bd 30K (30K) show high relationship with the protease of 24 kDa oleosin of soybean oil bodies. In this study, 9 day germinated soybean was used to separate bioprocessed P34 (P32) from bioprocessed 30K (28K). Interestingly, P32 existed as dimer, whereas 28K existed as monomer; a P32-rich sample had proteolytic activity and high cleavage site specificity (Lys-Thr of 24 kDa oleosin), whereas a 28K-rich sample showed low proteolytic activity; the P32-rich sample contained one thiol protease. After mixing with purified oil bodies, all P32 dimers were dissociated and bound to 24 kDa oleosins to form P32-24 kDa oleosin complexes. By incubation, 24 kDa oleosin was preferentially hydrolyzed, and two hydrolyzed products (HPs; 17 and 7 kDa) were confirmed. After most of 24 kDa oleosin was hydrolyzed, some P32 existed as dimer, and the other as P32-17 kDa HP. It was suggested that P32 was the protease.

  9. Proteolytic Digestion and TiO2 Phosphopeptide Enrichment Microreactor for Fast MS Identification of Proteins

    Science.gov (United States)

    Deng, Jingren; Lazar, Iulia M.

    2016-04-01

    The characterization of phosphorylation state(s) of a protein is best accomplished by using isolated or enriched phosphoprotein samples or their corresponding phosphopeptides. The process is typically time-consuming as, often, a combination of analytical approaches must be used. To facilitate throughput in the study of phosphoproteins, a microreactor that enables a novel strategy for performing fast proteolytic digestion and selective phosphopeptide enrichment was developed. The microreactor was fabricated using 100 μm i.d. fused-silica capillaries packed with 1-2 mm beds of C18 and/or TiO2 particles. Proteolytic digestion-only, phosphopeptide enrichment-only, and sequential proteolytic digestion/phosphopeptide enrichment microreactors were developed and tested with standard protein mixtures. The protein samples were adsorbed on the C18 particles, quickly digested with a proteolytic enzyme infused over the adsorbed proteins, and further eluted onto the TiO2 microreactor for enrichment in phosphopeptides. A number of parameters were optimized to speed up the digestion and enrichments processes, including microreactor dimensions, sample concentrations, digestion time, flow rates, buffer compositions, and pH. The effective time for the steps of proteolytic digestion and enrichment was less than 5 min. For simple samples, such as standard protein mixtures, this approach provided equivalent or better results than conventional bench-top methods, in terms of both enzymatic digestion and selectivity. Analysis times and reagent costs were reduced ~10- to 15-fold. Preliminary analysis of cell extracts and recombinant proteins indicated the feasibility of integration of these microreactors in more advanced workflows amenable for handling real-world biological samples.

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

    International Nuclear Information System (INIS)

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

    2007-01-01

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

  11. Transthyretin protects against A-beta peptide toxicity by proteolytic cleavage of the peptide: a mechanism sensitive to the Kunitz protease inhibitor.

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    Rita Costa

    Full Text Available Alzheimer's disease (AD is a neurodegenerative disorder characterized by the deposition of amyloid beta-peptide (A-Beta in the brain. Transthyretin (TTR is a tetrameric protein of about 55 kDa mainly produced in the liver and choroid plexus of the brain. The known physiological functions of TTR are the transport of thyroid hormone T(4 and retinol, through binding to the retinol binding protein. TTR has also been established as a cryptic protease able to cleave ApoA-I in vitro. It has been described that TTR is involved in preventing A-Beta fibrilization, both by inhibiting and disrupting A-Beta fibrils, with consequent abrogation of toxicity. We further characterized the nature of the TTR/A-Beta interaction and found that TTR, both recombinant or isolated from human sera, was able to proteolytically process A-Beta, cleaving the peptide after aminoacid residues 1, 2, 3, 10, 13, 14,16, 19 and 27, as determined by mass spectrometry, and reversed phase chromatography followed by N-terminal sequencing. A-Beta peptides (1-14 and (15-42 showed lower amyloidogenic potential than the full length counterpart, as assessed by thioflavin binding assay and ultrastructural analysis by transmission electron microscopy. A-Beta cleavage by TTR was inhibited in the presence of an alphaAPP peptide containing the Kunitz Protease Inhibitor (KPI domain but not in the presence of the secreted alphaAPP derived from the APP isoform 695 without the KPI domain. TTR was also able to degrade aggregated forms of A-Beta peptide. Our results confirmed TTR as a protective molecule in AD, and prompted A-Beta proteolysis by TTR as a protective mechanism in this disease. TTR may prove to be a useful therapeutic agent for preventing or retarding the cerebral amyloid plaque formation implicated in AD pathology.

  12. Multiplex profiling of tumor-associated proteolytic activity in serum of colorectal cancer patients.

    Science.gov (United States)

    Yepes, Diego; Costina, Victor; Pilz, Lothar R; Hofheinz, Ralf; Neumaier, Michael; Findeisen, Peter

    2014-06-01

    The monitoring of tumor-associated protease activity in blood specimens has recently been proposed as new diagnostic tool in cancer research. In this paper, we describe the screening of a peptide library for identification of reporter peptides (RPs) that are selectively cleaved in serum specimens from colorectal cancer patients and investigate the benefits of RP multiplexing. A library of 144 RPs was constructed that contained amino acid sequences of abundant plasma proteins. Proteolytic cleavage of RPs was monitored with MS. Five RPs that were selectively cleaved in serum specimens from tumor patients were selected for further validation in serum specimens of colorectal tumor patients (n = 30) and nonmalignant controls (n = 60). RP spiking and subsequent quantification of proteolytic fragments with LC-MS showed good reproducibility with CVs always below 26%. The linear discriminant analysis and PCA revealed that a combination of RPs for diagnostic classification is superior to single markers. Classification accuracy reached 88% (79/90) when all five markers were combined. Functional protease profiling with RPs might improve the laboratory-based diagnosis, monitoring and prognosis of malignant disease, and has to be evaluated thoroughly in future studies. © 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

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

    International Nuclear Information System (INIS)

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

    1981-01-01

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

  14. Angiogenic activity of bFGF and VEGF suppressed by proteolytic cleavage by neutrophil elastase

    International Nuclear Information System (INIS)

    Ai, Shingo; Cheng Xianwu; Inoue, Aiko; Nakamura, Kae; Okumura, Kenji; Iguchi, Akihisa; Murohara, Toyoaki; Kuzuya, Masafumi

    2007-01-01

    Neutrophil elastase (NE), a serine protease released from the azurophil granules of activated neutrophil, proteolytically cleaves multiple cytokines, and cell surface proteins. In the present study, we examined whether NE affects the biological abilities of angiogenic growth factors such as basic-fibroblast growth factor (bFGF) and vascular endothelial growth factor (VEGF). NE degraded bFGF and VEGF in a time- and concentration-dependent manner, and these degradations were suppressed by sivelestat, a synthetic inhibitor of NE. The bFGF- or VEGF-mediated proliferative activity of human umbilical vein endothelial cells was inhibited by NE, and the activity was recovered by sivelestat. Furthermore, NE reduced the bFGF- or VEGF-induced tubulogenic response of the mice aortas, ex vivo angiogenesis assay, and these effects were also recovered by sivelestat. Neutrophil-derived NE degraded potent angiogenic factors, resulting in loss of their angiogenic activity. These findings provide additional insight into the role played by neutrophils in the angiogenesis process at sites of inflammation

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

    Science.gov (United States)

    Demangeat, G; Greif, C; Hemmer, O; Fritsch, C

    1990-08-01

    Tomato black ring virus RNA-1 was translated in a rabbit reticulocyte lysate. The primary translation product of Mr 250K, which corresponds to its whole coding capacity, was synthesized within 45 min and, during further incubation in the translation medium, was proteolytically processed. Essentially, four cleavage products (P190, P120, P60 and P50) were detected and located within P250 by pulse-chase and immunoprecipitation experiments. P190 is an intermediate cleavage product which is further cleaved to form P60 and P120. P120, which contains the region that has been assigned to the virus protease and the virus polymerase, was not further cleaved in vitro.

  16. Analysis of proteolytic processes and enzymatic activities in the generation of huntingtin n-terminal fragments in an HEK293 cell model.

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    Andrew T N Tebbenkamp

    Full Text Available N-terminal fragments of mutant huntingtin (htt that terminate between residues 90-115, termed cleavage product A or 1 (cp-A/1, form intracellular and intranuclear inclusion bodies in the brains of patients with Huntington's disease (HD. These fragments appear to be proteolytic products of the full-length protein. Here, we use an HEK293 cell culture model to investigate huntingtin proteolytic processing; previous studies of these cells have demonstrated cleavage of htt to cp-A/1 like htt fragments.Recombinant N-terminal htt fragments, terminating at residue 171 (also referred to as cp-B/2 like, were efficiently cleaved to produce cp-A/1 whereas fragments representing endogenous caspase, calpain, and metalloproteinase cleavage products, terminating between residues 400-600, were inefficiently cleaved. Using cysteine-labeling techniques and antibody binding mapping, we localized the C-terminus of the cp-A/1 fragments produced by HEK293 cells to sequences minimally limited by cysteine 105 and an antibody epitope composed of residues 115-124. A combination of genetic and pharmacologic approaches to inhibit potential proteases, including γ-secretase and calpain, proved ineffective in preventing production of cp-A/1.Our findings indicate that HEK293 cells express a protease that is capable of efficiently cleaving cp-B/2 like fragments of htt with normal or expanded glutamine repeats. For reasons that remain unclear, this protease cleaves longer htt fragments, with normal or expanded glutamine expansions, much less efficiently. The protease in HEK293 cells that is capable of generating a cp-A/1 like htt fragment may be a novel protease with a high preference for a cp-B/2-like htt fragment as substrate.

  17. Mechanisms for lymphocytic choriomeningitis virus glycoprotein cleavage, transport, and incorporation into virions

    International Nuclear Information System (INIS)

    Kunz, Stefan; Edelmann, Kurt H.; Torre, Juan-Carlos de la; Gorney, Robert; Oldstone, Michael B.A.

    2003-01-01

    The glycoprotein (GP) of lymphocytic choriomeningitis virus (LCMV) serves as virus attachment protein to its receptor on host cells and is a key determinant for cell tropism, pathogenesis, and epidemiology of the virus. The GP of LCMV is posttranslationally cleaved by the subtilase SKI-1/S1P into two subunits, the peripheral GP1, which is implicated in receptor binding, and the transmembrane GP2 that is structurally similar to the fusion active membrane proximal portions of the glycoproteins of other enveloped viruses. The present study shows that cleavage by SKI-1/S1P is not required for cell surface expression of LCMVGP on infected cells but is essential for its incorporation into virions and for the production of infectious virus particles. In absence of SKI-1/S1P cleavage, cell-to-cell propagation of the virus was markedly reduced. Further, proteolytic processing of LCMVGP depends on the presence of a cluster of basic amino acids at the C-terminus of the cytoplasmic domain of GP2, a structural motif that is conserved in Old World arenaviruses. The effect of the truncation of the cytoplasmic tail on cleavage suggests a structural interdependence between the cytoplasmic domain and the ectodomains of LCMVGP

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

    Science.gov (United States)

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

    2013-08-16

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

  19. Activation of the Arabidopsis membrane-bound transcription factor bZIP28 is mediated by site-2 protease, but not site-1 protease.

    Science.gov (United States)

    Iwata, Yuji; Ashida, Makoto; Hasegawa, Chisa; Tabara, Kazuki; Mishiba, Kei-Ichiro; Koizumi, Nozomu

    2017-08-01

    The unfolded protein response (UPR) is a homeostatic cellular response conserved in eukaryotic cells to alleviate the accumulation of unfolded proteins in the endoplasmic reticulum (ER). Arabidopsis bZIP28 is a membrane-bound transcription factor activated by proteolytic cleavage in response to ER stress, thereby releasing its cytosolic portion containing the bZIP domain from the membrane to translocate into the nucleus where it induces the transcription of genes encoding ER-resident molecular chaperones and folding enzymes. It has been widely recognized that the proteolytic activation of bZIP28 is mediated by the sequential cleavage of site-1 protease (S1P) and site-2 protease (S2P). In the present study we provide evidence that bZIP28 protein is cleaved by S2P, but not by S1P. We demonstrated that wild-type and s1p mutant plants produce the active, nuclear form of bZIP28 in response to the ER stress inducer tunicamycin. In contrast, tunicamycin-treated s2p mutants do not accumulate the active, nuclear form of bZIP28. Consistent with these observations, s2p mutants, but not s1p mutants, exhibited a defective transcriptional response of ER stress-responsive genes and significantly higher sensitivity to tunicamycin. Interestingly, s2p mutants accumulate two membrane-bound bZIP28 fragments with a shorter ER lumen-facing C-terminal domain. Importantly, the predicted cleavage sites are located far from the canonical S1P recognition motif previously described. We propose that ER stress-induced proteolytic activation of bZIP28 is mediated by the sequential actions of as-yet-unidentified protease(s) and S2P, and does not require S1P. © 2017 The Authors The Plant Journal © 2017 John Wiley & Sons Ltd.

  20. Computational analysis and modeling of cleavage by the immunoproteasome and the constitutive proteasome

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    Lafuente Esther M

    2010-09-01

    Full Text Available Abstract Background Proteasomes play a central role in the major histocompatibility class I (MHCI antigen processing pathway. They conduct the proteolytic degradation of proteins in the cytosol, generating the C-terminus of CD8 T cell epitopes and MHCI-peptide ligands (P1 residue of cleavage site. There are two types of proteasomes, the constitutive form, expressed in most cell types, and the immunoproteasome, which is constitutively expressed in mature dendritic cells. Protective CD8 T cell epitopes are likely generated by the immunoproteasome and the constitutive proteasome, and here we have modeled and analyzed the cleavage by these two proteases. Results We have modeled the immunoproteasome and proteasome cleavage sites upon two non-overlapping sets of peptides consisting of 553 CD8 T cell epitopes, naturally processed and restricted by human MHCI molecules, and 382 peptides eluted from human MHCI molecules, respectively, using N-grams. Cleavage models were generated considering different epitope and MHCI-eluted fragment lengths and the same number of C-terminal flanking residues. Models were evaluated in 5-fold cross-validation. Judging by the Mathew's Correlation Coefficient (MCC, optimal cleavage models for the proteasome (MCC = 0.43 ± 0.07 and the immunoproteasome (MCC = 0.36 ± 0.06 were obtained from 12-residue peptide fragments. Using an independent dataset consisting of 137 HIV1-specific CD8 T cell epitopes, the immunoproteasome and proteasome cleavage models achieved MCC values of 0.30 and 0.18, respectively, comparatively better than those achieved by related methods. Using ROC analyses, we have also shown that, combined with MHCI-peptide binding predictions, cleavage predictions by the immunoproteasome and proteasome models significantly increase the discovery rate of CD8 T cell epitopes restricted by different MHCI molecules, including A*0201, A*0301, A*2402, B*0702, B*2705. Conclusions We have developed models that are specific

  1. Proteolytic enzymes of lactic acid bacteria

    NARCIS (Netherlands)

    Law, J; Haandrikman, A

    The proteolytic system of lactic acid bacteria is essential for their growth in milk and contributes significantly to flavour development in fermented milk products where these microorganisms are used as starter cultures. The proteolytic system is composed of proteinases which initially cleave the

  2. Coupling fibroblast growth factor 23 production and cleavage: iron deficiency, rickets, and kidney disease.

    Science.gov (United States)

    Wolf, Myles; White, Kenneth E

    2014-07-01

    High levels of fibroblast growth factor 23 (FGF23) cause the rare disorders of hypophosphatemic rickets and are a risk factor for cardiovascular disease and death in patients with chronic kidney disease (CKD). Despite major advances in understanding FGF23 biology, fundamental aspects of FGF23 regulation in health and in CKD remain mostly unknown. Autosomal dominant hypophosphatemic rickets (ADHR) is caused by gain-of-function mutations in FGF23 that prevent its proteolytic cleavage, but affected individuals experience a waxing and waning course of phosphate wasting. This led to the discovery that iron deficiency is an environmental trigger that stimulates FGF23 expression and hypophosphatemia in ADHR. Unlike osteocytes in ADHR, normal osteocytes couple increased FGF23 production with commensurately increased FGF23 cleavage to ensure that normal phosphate homeostasis is maintained in the event of iron deficiency. Simultaneous measurement of FGF23 by intact and C-terminal assays supported these breakthroughs by providing minimally invasive insight into FGF23 production and cleavage in bone. These findings also suggest a novel mechanism of FGF23 elevation in patients with CKD, who are often iron deficient and demonstrate increased FGF23 production and decreased FGF23 cleavage, consistent with an acquired state that mimics the molecular pathophysiology of ADHR. Iron deficiency stimulates FGF23 production, but normal osteocytes couple increased FGF23 production with increased cleavage to maintain normal circulating levels of biologically active hormone. These findings uncover a second level of FGF23 regulation within osteocytes, failure of which culminates in elevated levels of biologically active FGF23 in ADHR and perhaps CKD.

  3. Proteolytic Activity of Prostate-Specific Antigen (PSA) towards Protein Substrates and Effect of Peptides Stimulating PSA Activity

    Science.gov (United States)

    Mattsson, Johanna M.; Ravela, Suvi; Hekim, Can; Jonsson, Magnus; Malm, Johan; Närvänen, Ale; Stenman, Ulf-Håkan; Koistinen, Hannu

    2014-01-01

    Prostate-specific antigen (PSA or kallikrein-related peptidase-3, KLK3) exerts chymotrypsin-like proteolytic activity. The main biological function of PSA is the liquefaction of the clot formed after ejaculation by cleavage of semenogelins I and II in seminal fluid. PSA also cleaves several other substrates, which may explain its putative functions in prostate cancer and its antiangiogenic activity. We compared the proteolytic efficiency of PSA towards several protein and peptide substrates and studied the effect of peptides stimulating the activity of PSA with these substrates. An endothelial cell tube formation model was used to analyze the effect of PSA-degraded protein fragments on angiogenesis. We showed that PSA degrades semenogelins I and II much more efficiently than other previously identified protein substrates, e.g., fibronectin, galectin-3 and IGFBP-3. We identified nidogen-1 as a new substrate for PSA. Peptides B2 and C4 that stimulate the activity of PSA towards small peptide substrates also enhanced the proteolytic activity of PSA towards protein substrates. Nidogen-1, galectin-3 or their fragments produced by PSA did not have any effect on endothelial cell tube formation. Although PSA cleaves several other protein substrates, in addition to semenogelins, the physiological importance of this activity remains speculative. The PSA levels in prostate are very high, but several other highly active proteases, such as hK2 and trypsin, are also expressed in the prostate and may cleave protein substrates that are weakly cleaved by PSA. PMID:25237904

  4. Affect of Presenilin Mutations on APP Cleavage; Insights into the Pathogenesis of FAD

    Directory of Open Access Journals (Sweden)

    Nuomin eLi

    2016-03-01

    Full Text Available Alzheimer disease (AD is characterized by progressive memory loss, reduction in cognitive functions, and damage to the brain. The β-amyloid precursor protein can be sequentially cleaved by β- secretase and γ-secretase. Mutations in the presenilin1(PS1) are the most common cause of Familial Alzheimer’s disease ( FAD. PS1 mutations can alter the activity of γ-secretase on the cleavage of the β-amyloid precursor protein, causing increased Aβ production. Previous studies show that the βAPP-C-terminal fragment is first cleaved by β-scretase, primarily generating long fragments of Aβ48 and Aβ49, followed by the stepwise cleavage of every three amino acid residues at the C terminus, resulting in Aβ48-, 45-, 42 line and Aβ49-, 46-, 43-, 40 line. Here, we used LC-MS/MS to analyze unique peptides IAT, VVIA, ITL,TVI,IVI through sequential cleavage, combined with ELISA to test the level of Aβ42 and Aβ40 for validation. The results show that most FAD mutant PS1 can alter the level of Aβ42 and Aβ40 monitored by the Aβ42/Aβ40 ratio. Among them, 6 mutants (I143T, H163P, S170F, Q223R, M233V and G384A affect the Aβ42/40 ratio through both Aβ49-40 and Aβ48-38 lines; L166P through decreasing the Aβ49-40 line, 6 mutants (I143V, M146V, G217A, E280A, L381V and L392V through increasing the Aβ48-42 line. More importantly, we found some mutations can affect the γ-secretase cleavage preference of α-CTF and β-CTF. In conclusion, we found that the FAD PS1 mutations mainly increase the generation of Aβ42 by decreasing the cleavage of Aβ42-Aβ38 and Aβ43-Aβ40.

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

    International Nuclear Information System (INIS)

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

    1988-01-01

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

  6. The proteolytic system of Lactococcus lactis

    NARCIS (Netherlands)

    Kunji, Edmundus Richardus Stephanus

    1997-01-01

    The bacterium Lactococcus lactis usues an extencive proteolytic system to utilize milk proteins (caseins) in orde to meet its need for amino acids. The genetic and biochemical properties of the putative components of the proteolytic pathway are well-described. However, little is known about the role

  7. Sequencing Lys-N Proteolytic Peptides by ESI and MALDI Tandem Mass Spectrometry

    Science.gov (United States)

    Dupré, Mathieu; Cantel, Sonia; Verdié, Pascal; Martinez, Jean; Enjalbal, Christine

    2011-02-01

    In this study, we explored the MS/MS behavior of various synthetic peptides that possess a lysine residue at the N-terminal position. These peptides were designed to mimic peptides produced upon proteolysis by the Lys-N enzyme, a metalloendopeptidase issued from a Japanese fungus Grifola frondosa that was recently investigated in proteomic studies as an alternative to trypsin digestion, as a specific cleavage at the amide X-Lys chain is obtained that provides N-terminal lysine peptide fragments. In contrast to tryptic peptides exhibiting a lysine or arginine residue solely at the C-terminal position, and are thus devoid of such basic amino acids within the sequence, these Lys-N proteolytic peptides can contain the highly basic arginine residue anywhere within the peptide chain. The fragmentation patterns of such sequences with the ESI-QqTOF and MALDI-TOF/TOF mass spectrometers commonly used in proteomic bottom-up experiments were investigated.

  8. Atypical cleavage of protonated N-fatty acyl amino acids derived from aspartic acid evidenced by sequential MS3 experiments.

    Science.gov (United States)

    Boukerche, Toufik Taalibi; Alves, Sandra; Le Faouder, Pauline; Warnet, Anna; Bertrand-Michel, Justine; Bouchekara, Mohamed; Belbachir, Mohammed; Tabet, Jean-Claude

    2016-12-01

    Lipidomics calls for information on detected lipids and conjugates whose structural elucidation by mass spectrometry requires to rationalization of their gas phase dissociations toward collision-induced dissociation (CID) processes. This study focused on activated dissociations of two lipoamino acid (LAA) systems composed of N-palmitoyl acyl coupled with aspartic and glutamic acid mono ethyl esters (as LAA (*D) and LAA (*E) ). Although in MS/MS, their CID spectra show similar trends, e.g., release of water and ethanol, the [(LAA (*D/*E) +H)-C 2 H 5 OH] + product ions dissociate via distinct pathways in sequential MS 3 experiments. The formation of all the product ions is rationalized by charge-promoted cleavages often involving stepwise processes with ion isomerization into ion-dipole prior to dissociation. The latter explains the maleic anhydride or ketene neutral losses from N-palmitoyl acyl aspartate and glutamate anhydride fragment ions, respectively. Consequently, protonated palmitoyl acid amide is generated from LAA (*D), whereas LAA (*E) leads to the [*E+H-H 2 O] + anhydride. The former releases ammonia to provide acylium, which gives the C n H (2n-1) and C n H (2n-3) carbenium series. This should offer structural information, e.g., to locate either unsaturation(s) or alkyl group branching present on the various fatty acyl moieties of lipo-aspartic acid in further studies based on MS n experiments.

  9. Proteolytic activity of prostate-specific antigen (PSA towards protein substrates and effect of peptides stimulating PSA activity.

    Directory of Open Access Journals (Sweden)

    Johanna M Mattsson

    Full Text Available Prostate-specific antigen (PSA or kallikrein-related peptidase-3, KLK3 exerts chymotrypsin-like proteolytic activity. The main biological function of PSA is the liquefaction of the clot formed after ejaculation by cleavage of semenogelins I and II in seminal fluid. PSA also cleaves several other substrates, which may explain its putative functions in prostate cancer and its antiangiogenic activity. We compared the proteolytic efficiency of PSA towards several protein and peptide substrates and studied the effect of peptides stimulating the activity of PSA with these substrates. An endothelial cell tube formation model was used to analyze the effect of PSA-degraded protein fragments on angiogenesis. We showed that PSA degrades semenogelins I and II much more efficiently than other previously identified protein substrates, e.g., fibronectin, galectin-3 and IGFBP-3. We identified nidogen-1 as a new substrate for PSA. Peptides B2 and C4 that stimulate the activity of PSA towards small peptide substrates also enhanced the proteolytic activity of PSA towards protein substrates. Nidogen-1, galectin-3 or their fragments produced by PSA did not have any effect on endothelial cell tube formation. Although PSA cleaves several other protein substrates, in addition to semenogelins, the physiological importance of this activity remains speculative. The PSA levels in prostate are very high, but several other highly active proteases, such as hK2 and trypsin, are also expressed in the prostate and may cleave protein substrates that are weakly cleaved by PSA.

  10. Sequential activation of proteases in radiation induced apoptosis

    International Nuclear Information System (INIS)

    Watters, D.; Waterhouse, N.

    1997-01-01

    Full text: Significant advances have been made in recent years in unraveling the molecular mechanisms of apoptosis particularly in relation to Fas- and TNF-mediated cell death, however there are considerable gaps in our knowledge of the processes involved in apoptosis induced by ionizing radiation. We have used the degradation of specific proteolytic targets in a pair of isogenic Burkitt's Iymphoma cells lines (BL30A, sensitive and BL30K resistant) to study the sequence of events in the execution of radiation-induced apoptosis. Fodrin can be cleaved to fragments of 150 kDa and 120 kDa. In the case of Fas-mediated apoptosis both cleavages are inhibited by the caspase inhibitor zVAD-fmk at 10 μM, a concentration which inhibits all the hallmarks of apoptosis. However in radiation-induced apoptosis, inhibition of the clevage of fodrin to the 150 kDa fragment requires 100 μM zVAD-fink while apoptosis itself is inhibited at 10 μM. This suggests that different enzymes are responsible for the generation of the 150 kDa fragment in the two models of apoptosis. Fodrin has been reported to be cleaved by μ-calpain to a 150 kDa fragment however, the involvement of μ-calpain in apoptosis has not yet been established. In murine fodrin there is a caspase cleavage site within 1 kDa of the calpain cleavage site. In vitro studies using purified enzymes showed that only caspase-3 and μ-calpain could cleave fodrin in untreated cell extracts to the same sized fragments as seen during apoptosis in vivo. We provide evidence for the early activation of μ-calpain after ionizing radiation in the sensitive BL30A cell line, and show that the time course of μ-calpain activation parallels that of the appearance of the 150 kDa fragment. Caspase-3 is activated much later and is likely to be responsible for the generation of the 120 kDa fragment. μ-Calpain was not activated in the resistant cell line. Based on these results we propose a model for the proteolytic cascade in radiation

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

    Directory of Open Access Journals (Sweden)

    Alexander Jonathan S

    2010-12-01

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

  12. TAILS N-Terminomics and Proteomics Show Protein Degradation Dominates over Proteolytic Processing by Cathepsins in Pancreatic Tumors

    Directory of Open Access Journals (Sweden)

    Anna Prudova

    2016-08-01

    Full Text Available Deregulated cathepsin proteolysis occurs across numerous cancers, but in vivo substrates mediating tumorigenesis remain ill-defined. Applying 8-plex iTRAQ terminal amine isotopic labeling of substrates (TAILS, a systems-level N-terminome degradomics approach, we identified cathepsin B, H, L, S, and Z in vivo substrates and cleavage sites with the use of six different cathepsin knockout genotypes in the Rip1-Tag2 mouse model of pancreatic neuroendocrine tumorigenesis. Among 1,935 proteins and 1,114 N termini identified by TAILS, stable proteolytic products were identified in wild-type tumors compared with one or more different cathepsin knockouts (17%–44% of 139 cleavages. This suggests a lack of compensation at the substrate level by other cathepsins. The majority of neo-N termini (56%–83% for all cathepsins was consistent with protein degradation. We validated substrates, including the glycolytic enzyme pyruvate kinase M2 associated with the Warburg effect, the ER chaperone GRP78, and the oncoprotein prothymosin-alpha. Thus, the identification of cathepsin substrates in tumorigenesis improves the understanding of cathepsin functions in normal physiology and cancer.

  13. Intracellular Cleavage of the Cx43 C-Terminal Domain by Matrix-Metalloproteases: A Novel Contributor to Inflammation?

    Directory of Open Access Journals (Sweden)

    Marijke De Bock

    2015-01-01

    Full Text Available The coordination of tissue function is mediated by gap junctions (GJs that enable direct cell-cell transfer of metabolic and electric signals. GJs are formed by connexin (Cx proteins of which Cx43 is most widespread in the human body. Beyond its role in direct intercellular communication, Cx43 also forms nonjunctional hemichannels (HCs in the plasma membrane that mediate the release of paracrine signaling molecules in the extracellular environment. Both HC and GJ channel function are regulated by protein-protein interactions and posttranslational modifications that predominantly take place in the C-terminal domain of Cx43. Matrix metalloproteases (MMPs are a major group of zinc-dependent proteases, known to regulate not only extracellular matrix remodeling, but also processing of intracellular proteins. Together with Cx43 channels, both GJs and HCs, MMPs contribute to acute inflammation and a small number of studies reports on an MMP-Cx43 link. Here, we build further on these reports and present a novel hypothesis that describes proteolytic cleavage of the Cx43 C-terminal domain by MMPs and explores possibilities of how such cleavage events may affect Cx43 channel function. Finally, we set out how aberrant channel function resulting from cleavage can contribute to the acute inflammatory response during tissue injury.

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

    Science.gov (United States)

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

    1995-01-10

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

  15. Serum disposition of bovine lactoferrin after oral and anal administration and its proteolytic cleavage by gastric transit in rainbow trout (Oncorhynchus mykiss W.).

    Science.gov (United States)

    Cecchini, Stefano; Caputo, Anna R

    2009-01-01

    Several studies have shown an immunomodulatory effect of orally administered bovine lactoferrin (LF) in fish, but the process of digestion was not characterized. In the present study, we investigated the fate of bovine LF after oral and anal administration, and studied the appearance of intact LF in the bloodstream and its proteolytic attack during the gastric transit in rainbow trout (Oncorhynchus mykiss) held at 9 degrees C and 18 degrees C. Data obtained showed the presence of intact bovine LF in the bloodstream only after anal administration in fish held at 18 degrees C and the presence of several peptides derived from bovine LF in the gastric content. Immunoblotting analysis showed that only a part of bovine LF-derived peptides reacted with the applied anti-bovine LF antibody. The concentration of intact bovine LF, after 30 min of administration, in the gastric content of fish reared at 18 degrees C, being extremely low, if any, led us to suspect that the immunoregulatory effect of dietary bovine LF shown in fish by several authors is not due to the intact form but to bioactive fragments, originated by the proteolytic attack during the gastric transit, as demonstrated in higher vertebrates.

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

    International Nuclear Information System (INIS)

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

    1988-01-01

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

  17. Native Frames: Disentangling Sequential from Concerted Three-Body Fragmentation

    Science.gov (United States)

    Rajput, Jyoti; Severt, T.; Berry, Ben; Jochim, Bethany; Feizollah, Peyman; Kaderiya, Balram; Zohrabi, M.; Ablikim, U.; Ziaee, Farzaneh; Raju P., Kanaka; Rolles, D.; Rudenko, A.; Carnes, K. D.; Esry, B. D.; Ben-Itzhak, I.

    2018-03-01

    A key question concerning the three-body fragmentation of polyatomic molecules is the distinction of sequential and concerted mechanisms, i.e., the stepwise or simultaneous cleavage of bonds. Using laser-driven fragmentation of OCS into O++C++S+ and employing coincidence momentum imaging, we demonstrate a novel method that enables the clear separation of sequential and concerted breakup. The separation is accomplished by analyzing the three-body fragmentation in the native frame associated with each step and taking advantage of the rotation of the intermediate molecular fragment, CO2 + or CS2 + , before its unimolecular dissociation. This native-frame method works for any projectile (electrons, ions, or photons), provides details on each step of the sequential breakup, and enables the retrieval of the relevant spectra for sequential and concerted breakup separately. Specifically, this allows the determination of the branching ratio of all these processes in OCS3 + breakup. Moreover, we find that the first step of sequential breakup is tightly aligned along the laser polarization and identify the likely electronic states of the intermediate dication that undergo unimolecular dissociation in the second step. Finally, the separated concerted breakup spectra show clearly that the central carbon atom is preferentially ejected perpendicular to the laser field.

  18. Research Applications of Proteolytic Enzymes in Molecular Biology

    Directory of Open Access Journals (Sweden)

    József Tőzsér

    2013-11-01

    Full Text Available Proteolytic enzymes (also termed peptidases, proteases and proteinases are capable of hydrolyzing peptide bonds in proteins. They can be found in all living organisms, from viruses to animals and humans. Proteolytic enzymes have great medical and pharmaceutical importance due to their key role in biological processes and in the life-cycle of many pathogens. Proteases are extensively applied enzymes in several sectors of industry and biotechnology, furthermore, numerous research applications require their use, including production of Klenow fragments, peptide synthesis, digestion of unwanted proteins during nucleic acid purification, cell culturing and tissue dissociation, preparation of recombinant antibody fragments for research, diagnostics and therapy, exploration of the structure-function relationships by structural studies, removal of affinity tags from fusion proteins in recombinant protein techniques, peptide sequencing and proteolytic digestion of proteins in proteomics. The aim of this paper is to review the molecular biological aspects of proteolytic enzymes and summarize their applications in the life sciences.

  19. The N-terminal cleavage of chondromodulin-I in growth-plate cartilage at the hypertrophic and calcified zones during bone development.

    Directory of Open Access Journals (Sweden)

    Shigenori Miura

    Full Text Available Chondromodulin-I (ChM-I is a 20-25 kDa anti-angiogenic glycoprotein in cartilage matrix. In the present study, we identified a novel 14-kDa species of ChM-I by immunoblotting, and purified it by immunoprecipitation with a newly raised monoclonal antibody against ChM-I. The N-terminal amino acid sequencing indicated that it was an N-terminal truncated form of ChM-I generated by the proteolytic cleavage at Asp37-Asp38. This 14-kDa ChM-I was shown by the modified Boyden chamber assay to have very little inhibitory activity on the VEGF-A-induced migration of vascular endothelial cells in contrast to the intact 20-25 kDa form of ChM-I (ID50 = 8 nM. Immunohistochemistry suggested that 20-25 kDa ChM-I was exclusively localized in the avascular zones, i.e. the resting, proliferating, and prehypertrophic zones, of the cartilaginous molds of developing long bone, whereas the 14-kDa form of ChM-I was found in hypertrophic and calcified zones. Immunoblotting demonstrated that mature growth-plate chondrocytes isolated from rat costal cartilage actively secrete ChM-I almost exclusively as the intact 20-25 kDa form into the medium in primary culture. Taken together, our results suggest that intact 20-25 kDa ChM-I is stored as a component of extracellular matrix in the avascular cartilage zones, but it is inactivated by a single N-terminal proteolytic cleavage in the hypertrophic zone of growth-plate cartilage.

  20. Effect of Presenilin Mutations on APP Cleavage; Insights into the Pathogenesis of FAD

    OpenAIRE

    Li, Nuomin; Liu, Kefu; Qiu, Yunjie; Ren, Zehui; Dai, Rongji; Deng, Yulin; Qing, Hong

    2016-01-01

    Alzheimer disease (AD) is characterized by progressive memory loss, reduction in cognitive functions, and damage to the brain. The β-amyloid precursor protein can be sequentially cleaved by β- secretase and γ-secretase. Mutations in the presenilin1(PS1) are the most common cause of Familial Alzheimer’s disease (FAD). PS1 mutations can alter the activity of γ-secretase on the cleavage of the β-amyloid precursor protein, causing increased Aβ production. Previous studies show that the βAPP-C-ter...

  1. The multifaceted nature of amyloid precursor protein and its proteolytic fragments: friends and foes.

    Science.gov (United States)

    Nhan, Hoang S; Chiang, Karen; Koo, Edward H

    2015-01-01

    The amyloid precursor protein (APP) has occupied a central position in Alzheimer's disease (AD) pathophysiology, in large part due to the seminal role of amyloid-β peptide (Aβ), a proteolytic fragment derived from APP. Although the contribution of Aβ to AD pathogenesis is accepted by many in the research community, recent studies have unveiled a more complicated picture of APP's involvement in neurodegeneration in that other APP-derived fragments have been shown to exert pathological influences on neuronal function. However, not all APP-derived peptides are neurotoxic, and some even harbor neuroprotective effects. In this review, we will explore this complex picture by first discussing the pleiotropic effects of the major APP-derived peptides cleaved by multiple proteases, including soluble APP peptides (sAPPα, sAPPβ), various C- and N-terminal fragments, p3, and APP intracellular domain fragments. In addition, we will highlight two interesting sequences within APP that likely contribute to this duality in APP function. First, it has been found that caspase-mediated cleavage of APP in the cytosolic region may release a cytotoxic peptide, C31, which plays a role in synapse loss and neuronal death. Second, recent studies have implicated the -YENPTY- motif in the cytoplasmic region as a domain that modulates several APP activities through phosphorylation and dephosphorylation of the first tyrosine residue. Thus, this review summarizes the current understanding of various APP proteolytic products and the interplay among them to gain deeper insights into the possible mechanisms underlying neurodegeneration and AD pathophysiology.

  2. Dominant negative umuD mutations decreasing RecA-mediated cleavage suggest roles for intact UmuD in modulation of SOS mutagenesis

    International Nuclear Information System (INIS)

    Battista, J.R.; Ohta, Toshihiro; Nohmi, Takehiko; Sun, W.; Walker, G.C.

    1990-01-01

    The products of the SOS-regulated umuDC operon are required for most UV and chemical mutagenesis in Escherichia coli. The UmuD protein shares homology with a family of proteins that includes LexA and several bacteriophage repressors. UmuD is posttranslationally activated for its role n mutagenesis by a RecA-mediated proteolytic cleavage that yields UmuD'. A set of missense mutants of umuD was isolated and shown to encode mutant UmuD proteins that are deficient in RecA-mediated cleavage in vivo. Most of these mutations are dominant to umuD + with respect to UV mutagenesis yet do not interfere with SOS induction. Although both UmuD and UmuD' form homodimers, the authors provide evidence that they preferentially form heterodimers. The relationship of UmuD to LexA, λ repressor, and other members of the family of proteins is discussed and possible roles intact UmuD in modulating SOS mutagenesis are discussed

  3. Characterization of proteolytic and anti-proteolytic activity involvement in sterlet spermatozoon maturation

    Czech Academy of Sciences Publication Activity Database

    Dzyuba, V.; Słowińska, M.; Cosson, J.; Ciereszko, A.; Boryshpolets, S.; Štěrba, Ján; Rodina, M.; Linhart, O.; Dzyuba, B.

    2016-01-01

    Roč. 42, č. 6 (2016), s. 1755-1766 ISSN 0920-1742 R&D Projects: GA MŠk(CZ) EE2.3.30.0032 Institutional support: RVO:60077344 Keywords : amidase activity * anti-proteolytic activity * casein and gelatin zymography * siberian sturgeon sperm * spermatozoon maturation * sterlet sperm Subject RIV: ED - Physiology Impact factor: 1.647, year: 2016

  4. Atorvastatin prevents Aβ oligomer-induced neurotoxicity in cultured rat hippocampal neurons by inhibiting Tau cleavage

    Science.gov (United States)

    Sui, Hai-juan; Zhang, Ling-ling; Liu, Zhou; Jin, Ying

    2015-01-01

    Aim: The proteolytic cleavage of Tau is involved in Aβ-induced neuronal dysfunction and cell death. In this study, we investigated whether atorvastatin could prevent Tau cleavage and hence prevent Aβ1–42 oligomer (AβO)-induced neurotoxicity in cultured cortical neurons. Methods: Cultured rat hippocampal neurons were incubated in the presence of AβOs (1.25 μmol/L) with or without atorvastatin pretreatment. ATP content and LDH in the culture medium were measured to assess the neuronal viability. Caspase-3/7 and calpain protease activities were detected. The levels of phospho-Akt, phospho-Erk1/2, phospho-GSK3β, p35 and Tau proteins were measured using Western blotting. Results: Treatment of the neurons with AβO significantly decreased the neuronal viability, induced rapid activation of calpain and caspase-3/7 proteases, accompanied by Tau degradation and relatively stable fragments generated in the neurons. AβO also suppressed Akt and Erk1/2 kinase activity, while increased GSK3β and Cdk5 activity in the neurons. Pretreatment with atorvastatin (0.5, 1, 2.5 μmol/L) dose-dependently inhibited AβO-induced activation of calpain and caspase-3/7 proteases, and effectively diminished the generation of Tau fragments, attenuated synaptic damage and increased neuronal survival. Atorvastatin pretreatment also prevented AβO-induced decreases in Akt and Erk1/2 kinase activity and the increases in GSK3β and Cdk5 kinase activity. Conclusion: Atorvastatin prevents AβO-induced neurotoxicity in cultured rat hippocampal neurons by inhibiting calpain- and caspase-mediated Tau cleavage. PMID:25891085

  5. Host cell killing by the West Nile Virus NS2B-NS3 proteolytic complex: NS3 alone is sufficient to recruit caspase-8-based apoptotic pathway

    International Nuclear Information System (INIS)

    Ramanathan, Mathura P.; Chambers, Jerome A.; Pankhong, Panyupa; Chattergoon, Michael; Attatippaholkun, Watcharee; Dang, Kesen; Shah, Neelima; Weiner, David B.

    2006-01-01

    The West Nile Virus (WNV) non-structural proteins 2B and 3 (NS2B-NS3) constitute the proteolytic complex that mediates the cleavage and processing of the viral polyprotein. NS3 recruits NS2B and NS5 proteins to direct protease and replication activities. In an effort to investigate the biology of the viral protease, we cloned cDNA encoding the NS2B-NS3 proteolytic complex from brain tissue of a WNV-infected dead crow, collected from the Lower Merion area (Merion strain). Expression of the NS2B-NS3 gene cassette induced apoptosis within 48 h of transfection. Electron microscopic analysis of NS2B-NS3-transfected cells revealed ultra-structural changes that are typical of apoptotic cells including membrane blebbing, nuclear disintegration and cytoplasmic vacuolations. The role of NS3 or NS2B in contributing to host cell apoptosis was examined. NS3 alone triggers the apoptotic pathways involving caspases-8 and -3. Experimental results from the use of caspase-specific inhibitors and caspase-8 siRNA demonstrated that the activation of caspase-8 was essential to initiate apoptotic signaling in NS3-expressing cells. Downstream of caspase-3 activation, we observed nuclear membrane ruptures and cleavage of the DNA-repair enzyme, PARP in NS3-expressing cells. Nuclear herniations due to NS3 expression were absent in the cells treated with a caspase-3 inhibitor. Expression of protease and helicase domains themselves was sufficient to trigger apoptosis generating insight into the apoptotic pathways triggered by NS3 from WNV

  6. Analysis by the reductive-cleavage method of linkage positions in a polysaccharide containing 4-linked D-glucopyranosyluronic residues.

    Science.gov (United States)

    Vodonik, S A; Gray, G R

    1988-04-01

    The fate of 4-linked D-glucopyranosyluronic residues under reductive-cleavage conditions was investigated by using the Klebsiella aerogenes type 54 strain A3 capsular polysaccharide. Treatment of the fully methylated polysaccharide with triethylsilane and trimethylsilyl trifluoromethanesulfonate in dichloromethane, followed by in situ acetylation, yielded 1,5-anhydro-2,3,4,6-tetra-O-methyl-D-glucitol, 3,4-di-O-acetyl-1,5-anhydro-2,6-di-O-methyl-D-glucitol, and 3-O-acetyl-1,5-anhydro-2,4-di-O-methyl-L-fucitol, as expected, but the expected product of reductive cleavage of the 4-linked D-glucopyranosyluronic residue, namely, methyl 3-O-acetyl-2,6-anhydro-4,5-di-O-methyl-L-gulonate, was not observed. Instead, methyl 2-O-acetyl-3,6-anhydro-4,5-di-O-methyl-L-gulonate (6) was identified as the sole product of reductive cleavage of the 4-linked D-glucopyranosyluronic residue. That compound 6 arose as a result of rearrangement during reductive cleavage rather than by reductive cleavage of a 5-linked D-glucofuranosyluronic residue, was established by reductive cleavage of the fully methylated polysaccharide following reduction of its ester groups with either lithium aluminum hydride or lithium aluminum deuteride. The products of the latter reductive cleavage were the same as before, except for the absence of 6 and the presence of 4,6-di-O-acetyl-1,5-anhydro-2,3-di-O-methyl-D-glucitol, or its 6,6-dideuterio isomer. Although the reductive-cleavage technique is suitable for the direct analysis of polysaccharides containing 4-linked D-glucopyranosyluronic residues, it does not establish whether the uronic residue is a 4-linked pyranoside or a 5-linked furanoside. The expected product is, however, derived from the 4-linked D-glucopyranosyluronic residue after sequential methylation, reduction of its ester group and reductive cleavage.

  7. Comparison of human embryomorphokinetic parameters in sequential or global culture media.

    Science.gov (United States)

    Kazdar, Nadia; Brugnon, Florence; Bouche, Cyril; Jouve, Guilhem; Veau, Ségolène; Drapier, Hortense; Rousseau, Chloé; Pimentel, Céline; Viard, Patricia; Belaud-Rotureau, Marc-Antoine; Ravel, Célia

    2017-08-01

    A prospective study on randomized patients was conducted to determine how morphokinetic parameters are altered in embryos grown in sequential versus global culture media. Eleven morphokinetic parameters of 160 single embryos transferred were analyzed by time lapse imaging involving two University-affiliated in vitro fertilization (IVF) centers. We found that the fading of the two pronuclei occurred earlier in global (22.56±2.15 hpi) versus sequential media (23.63±2.71 hpi; p=0.0297). Likewise, the first cleavage started earlier at 24.52±2.33 hpi vs 25.76±2.95 hpi (p=0.0158). Also, the first cytokinesis was shorter in global medium, lasting 18±10.2 minutes in global versus 36±37.8 minutes in sequential culture medium (p culture medium. Our study highlights the need to adapt morphokinetic analysis accordingly to the type of media used to best support human early embryo development.

  8. MHC molecules protect T cell epitopes against proteolytic destruction

    DEFF Research Database (Denmark)

    Mouritsen, S; Meldal, M; Werdelin, O

    1992-01-01

    There is a subtle duality in the role of proteolytic enzymes in Ag processing. They are required to fragment protein Ag ingested by APC. However, prolonged exposure to proteolytic enzymes may lead to a complete degradation of the Ag, leaving nothing for the T cell system to recognize. What ensures...

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

    Science.gov (United States)

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

    2016-01-01

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

  10. Evidence for proteolytic cleavage of brevican by the ADAMTSs in the dentate gyrus after excitotoxic lesion of the mouse entorhinal cortex

    Directory of Open Access Journals (Sweden)

    Gottschall Paul E

    2005-08-01

    Full Text Available Abstract Background Brevican is a member of the lectican family of aggregating extracellular matrix (ECM proteoglycans that bear chondroitin sulfate (CS chains. It is highly expressed in the central nervous system (CNS and is thought to stabilize synapses and inhibit neural plasticity and as such, neuritic or synaptic remodeling would be less likely to occur in regions with intact and abundant, lectican-containing, ECM complexes. Neural plasticity may occur more readily when these ECM complexes are broken down by endogenous proteases, the ADAMTSs (adisintegrin and metalloproteinase with thrombospondin motifs, that selectively cleave the lecticans. The purpose of these experiments was to determine whether the production of brevican or the ADAMTS-cleaved fragments of brevican were altered after deafferentation and reinnervation of the dentate gyrus via entorhinal cortex lesion (ECL. Results In the C57Bl6J mouse, synaptic density in the molecular layer of the dentate gyrus, as measured by synaptophysin levels in ELISA, was significantly attenuated 2 days (nearly 50% of contralateral and 7 days after lesion and returned to levels not different from the contralateral region at 30 days. Immunoreactive brevican in immunoblot was elevated 2 days after lesion, whereas there was a significant increase in the proteolytic product at 7, but not 30 days post-lesion. ADAMTS activity, estimated using the ratio of the specific ADAMTS-derived brevican fragment and intact brevican levels was increased at 7 days, but was not different from the contralateral side at 2 or 30 days after deafferentation. Conclusion These findings indicate that ADAMTS activity in the dentate outer molecular layer (OML is elevated during the initial synaptic reinnervation period (7 days after lesion. Therefore, proteolytic processing of brevican appears to be a significant extracellular event in the remodeling of the dentate after EC lesion, and may modulate the process of sprouting and

  11. Structural and functional characterization of cleavage and inactivation of human serine protease inhibitors by the bacterial SPATE protease EspPα from enterohemorrhagic E. coli.

    Directory of Open Access Journals (Sweden)

    André Weiss

    Full Text Available EspPα and EspI are serine protease autotransporters found in enterohemorrhagic Escherichia coli. They both belong to the SPATE autotransporter family and are believed to contribute to pathogenicity via proteolytic cleavage and inactivation of different key host proteins during infection. Here, we describe the specific cleavage and functional inactivation of serine protease inhibitors (serpins by EspPα and compare this activity with the related SPATE EspI. Serpins are structurally related proteins that regulate vital protease cascades, such as blood coagulation and inflammatory host response. For the rapid determination of serpin cleavage sites, we applied direct MALDI-TOF-MS or ESI-FTMS analysis of coincubations of serpins and SPATE proteases and confirmed observed cleavage positions using in-gel-digest of SDS-PAGE-separated degradation products. Activities of both serpin and SPATE protease were assessed in a newly developed photometrical assay using chromogenic peptide substrates. EspPα cleaved the serpins α1-protease inhibitor (α1-PI, α1-antichymotrypsin, angiotensinogen, and α2-antiplasmin. Serpin cleavage led to loss of inhibitory function as demonstrated for α1-PI while EspPα activity was not affected. Notably, EspPα showed pronounced specificity and cleaved procoagulatory serpins such as α2-antiplasmin while the anticoagulatory antithrombin III was not affected. Together with recently published research, this underlines the interference of EspPα with hemostasis or inflammatory responses during infection, while the observed interaction of EspI with serpins is likely to be not physiologically relevant. EspPα-mediated serpin cleavage occurred always in flexible loops, indicating that this structural motif might be required for substrate recognition.

  12. Research Applications of Proteolytic Enzymes in Molecular Biology

    OpenAIRE

    Mótyán, János András; Tóth, Ferenc; Tőzsér, József

    2013-01-01

    Proteolytic enzymes (also termed peptidases, proteases and proteinases) are capable of hydrolyzing peptide bonds in proteins. They can be found in all living organisms, from viruses to animals and humans. Proteolytic enzymes have great medical and pharmaceutical importance due to their key role in biological processes and in the life-cycle of many pathogens. Proteases are extensively applied enzymes in several sectors of industry and biotechnology, furthermore, numerous research applications ...

  13. Functional role of proteolytic cleavage at arginine-275 of human tissue plasminogen activator as assessed by site-directed mutagenesis

    International Nuclear Information System (INIS)

    Tate, K.M.; Higgins, D.L.; Holmes, W.E.; Winkler, M.E.; Heyneker, H.L.; Vehar, G.A.

    1987-01-01

    Activation of the zymogen form of a serine protease is associated with a conformational change that follows proteolysis at a specific site. Tissue-type plasminogen activator (t-PA) is homologous to mammalian serine proteases and contains an apparent activation cleavage site at arginine-275. To clarify the functional consequences of cleavage at arginine-275 of t-PA, site-specific mutagenesis was performed to convert arginine-275 to a glutamic acid. The mutant enzyme (designated Arg-275 → Glu t-PA) could be converted to the two-chain form by Staphylococcus aureus V8 protease but not by plasmin. The one-chain form was 8 times less active against the tripeptide substrate H-D-isoleucyl-L-prolyl-L-arginine-rho-nitroanilide (S-2288), and the ability of the enzyme to activate plasminogen in the absence of fibrinogen was reduced 20-50 times compared to the two-chain form. In contrast, one-chain Arg-275 → Glu t-PA has equal activity to the two-chain form when assayed in the presence of physiological levels of fibrinogen and plasminogen. Fibrin bound significantly more of the one-chain form of t-PA than the two-chain form for both the wild-type and mutated enzymes. One- and two-chain forms of the wild-type and mutated plasminogen activators slowly formed complexes with plasma protease inhibitors, although the one-chain forms showed decreased complex formation with → 2 -macroglobulin. The one-chain form of t-PA therefore is fully functional under physiologic conditions and has a increased fibrin binding compared to the two-chain form

  14. Biosynthesis of intestinal microvillar proteins. Intracellular processing of lactase-phlorizin hydrolase

    DEFF Research Database (Denmark)

    Danielsen, E M; Skovbjerg, H; Norén, Ove

    1984-01-01

    of the Mr 160 000 form but not that of the Mr 245 000 polypeptide, suggesting that the proteolytic cleavage takes place after trimming and complex glycosylation. The proteolytic cleavage was not essential for the transport since the precursor was expressed in the microvillar membrane in the presence...

  15. Nuclear import of influenza B virus nucleoprotein: Involvement of an N-terminal nuclear localization signal and a cleavage-protection motif

    International Nuclear Information System (INIS)

    Wanitchang, Asawin; Narkpuk, Jaraspim; Jongkaewwattana, Anan

    2013-01-01

    The nucleoprotein of influenza B virus (BNP) shares several characteristics with its influenza A virus counterpart (ANP), including localization in the host's nucleus. However, while the nuclear localization signal(s) (NLS) of ANP are well characterized, little is known about those of BNP. In this study, we showed that the fusion protein bearing the BNP N-terminus fused with GFP (N70–GFP) is exclusively nuclear, and identified a highly conserved KRXR motif spanning residues 44–47 as a putative NLS. In addition, we demonstrated that residues 3–15 of BNP, though not an NLS, are also crucial for nuclear import. Results from mutational analyses of N70–GFP and the full-length BNP suggest that this region may be required for protection of the N-terminus from proteolytic cleavage. Altogether, we propose that the N-terminal region of BNP contains the NLS and cleavage-protection motif, which together drive its nuclear localization. - Highlights: • The N-terminal region of BNP is required for nuclear accumulation. • The conserved motif at position 44–47 is a putative nuclear localization signal. • The first 15 amino acids of BNP may function as a cleavage-protection motif. • BNP may get access to the nucleus via a mechanism distinct from ANP

  16. Possible cleavage sites of glutelin partial degradation confirmed by immunological analysis in globulin-less mutants of rice (Oryza sativa L.).

    Science.gov (United States)

    Khan, Nadar; Yamaguchi, Satoru; Katsube-Tanaka, Tomoyuki

    2017-10-01

    Proteolytic cleavage or partial degradation of proteins is one of the important post-translational modifications for various biological processes, but it is difficult to analyze. Previously, we demonstrated that some subunits of the major rice (Oryza sativa L.) seed storage protein glutelin are partially degraded to produce newly identified polypeptides X1-X5 in mutants in which another major seed storage protein globulin is absent. In this study, the new polypeptides X3 and X4/X5 were immunologically confirmed to be derived from GluA3 and GluA1/GluA2 subunits, respectively. Additionally, the new polypeptides X1 and X2 were at least in part the α polypeptides of the GluB4 subunit partially degraded at the C-terminus. Simulated 2D-PAGE migration patterns of intact and partially degraded α polypeptides based on the calculation of their MWs and pIs enabled us to narrow or predict the possible locations of cleavage sites. The predicted cleavage sites were also verified by the comparison of 2D-PAGE patterns between seed-extracted and E. coli-expressed proteins of the intact and truncated α polypeptides. The results and methodologies demonstrated here would be useful for analyses of partial degradation of proteins and the structure-function relationships of rice seed protein bodies. © 2017 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  17. Nuclear import of influenza B virus nucleoprotein: Involvement of an N-terminal nuclear localization signal and a cleavage-protection motif

    Energy Technology Data Exchange (ETDEWEB)

    Wanitchang, Asawin; Narkpuk, Jaraspim; Jongkaewwattana, Anan, E-mail: anan.jon@biotec.or.th

    2013-08-15

    The nucleoprotein of influenza B virus (BNP) shares several characteristics with its influenza A virus counterpart (ANP), including localization in the host's nucleus. However, while the nuclear localization signal(s) (NLS) of ANP are well characterized, little is known about those of BNP. In this study, we showed that the fusion protein bearing the BNP N-terminus fused with GFP (N70–GFP) is exclusively nuclear, and identified a highly conserved KRXR motif spanning residues 44–47 as a putative NLS. In addition, we demonstrated that residues 3–15 of BNP, though not an NLS, are also crucial for nuclear import. Results from mutational analyses of N70–GFP and the full-length BNP suggest that this region may be required for protection of the N-terminus from proteolytic cleavage. Altogether, we propose that the N-terminal region of BNP contains the NLS and cleavage-protection motif, which together drive its nuclear localization. - Highlights: • The N-terminal region of BNP is required for nuclear accumulation. • The conserved motif at position 44–47 is a putative nuclear localization signal. • The first 15 amino acids of BNP may function as a cleavage-protection motif. • BNP may get access to the nucleus via a mechanism distinct from ANP.

  18. The influence of radiation on bacterial cells and their proteolytic properties

    International Nuclear Information System (INIS)

    Szulc, M.; Stefaniakowa, A.; Stanczak, B.; Peconek, J.

    1980-01-01

    The suspension of bacterial cells and their spores were exposed to X rays in the environment with and without protein. The doses of radiation ranged from 1 to 100 Gy and in case of spores of B. subtilis from 50 to 1000 Gy. It was found that irradiation to Proteus vulgaris, Pseudomonas fluorescens and Ps. aeruginosa caused an inconsiderable decrease of proteolytic properties of the generation originated from irradiated bacteria. Irradiation of B. subtilis spores did not influence the proteolytic activity of bacterial cells derived from the exposed spores. The degree of wasting away of bacteria exposed to the same radiation was higher than the rate of proteolytic properties decrease. The presence of protein in the surroundings had no influence on proteolytic characteristics of new generations. (author)

  19. Picking up the pieces: a generic porous Si biosensor for probing the proteolytic products of enzymes.

    Science.gov (United States)

    Shtenberg, Giorgi; Massad-Ivanir, Naama; Moscovitz, Oren; Engin, Sinem; Sharon, Michal; Fruk, Ljiljana; Segal, Ester

    2013-02-05

    A multifunctional porous Si biosensor that can both monitor the enzymatic activity of minute samples and allow subsequent retrieval of the entrapped proteolytic products for mass spectrometry analysis is described. The biosensor is constructed by DNA-directed/reversible immobilization of enzymes onto a Fabry-Pérot thin film. We demonstrate high enzymatic activity levels of the immobilized enzymes (more than 80%), while maintaining their specificity. Mild dehybridization conditions allow enzyme recycling and facile surface regeneration for consecutive biosensing analysis. The catalytic activity of the immobilized enzymes is monitored in real time by reflective interferometric Fourier transform spectroscopy. The real-time analysis of minute quantities of enzymes (concentrations at least 1 order of magnitude lower, 0.1 mg mL(-1), in comparison to previous reports, 1 mg mL(-1)), in particular proteases, paves the way for substrate profiling and the identification of cleavage sites. The biosensor configuration is compatible with common proteomic methods and allows for a successful downstream mass spectrometry analysis of the reaction products.

  20. Proteolytic crosstalk in multi-protease networks

    Science.gov (United States)

    Ogle, Curtis T.; Mather, William H.

    2016-04-01

    Processive proteases, such as ClpXP in E. coli, are conserved enzyme assemblies that can recognize and rapidly degrade proteins. These proteases are used for a number of purposes, including degrading mistranslated proteins and controlling cellular stress response. However, proteolytic machinery within the cell is limited in capacity and can lead to a bottleneck in protein degradation, whereby many proteins compete (‘queue’) for proteolytic resources. Previous work has demonstrated that such queueing can lead to pronounced statistical relationships between different protein counts when proteins compete for a single common protease. However, real cells contain many different proteases, e.g. ClpXP, ClpAP, and Lon in E. coli, and it is not clear how competition between proteins for multiple classes of protease would influence the dynamics of cellular networks. In the present work, we theoretically demonstrate that a multi-protease proteolytic bottleneck can substantially couple the dynamics for both simple and complex (oscillatory) networks, even between substrates with substantially different affinities for protease. For these networks, queueing often leads to strong positive correlations between protein counts, and these correlations are strongest near the queueing theoretic point of balance. Furthermore, we find that the qualitative behavior of these networks depends on the relative size of the absolute affinity of substrate to protease compared to the cross affinity of substrate to protease, leading in certain regimes to priority queue statistics.

  1. Top-down proteomics for the analysis of proteolytic events - Methods, applications and perspectives.

    Science.gov (United States)

    Tholey, Andreas; Becker, Alexander

    2017-11-01

    Mass spectrometry based proteomics is an indispensable tool for almost all research areas relevant for the understanding of proteolytic processing, ranging from the identification of substrates, products and cleavage sites up to the analysis of structural features influencing protease activity. The majority of methods for these studies are based on bottom-up proteomics performing analysis at peptide level. As this approach is characterized by a number of pitfalls, e.g. loss of molecular information, there is an ongoing effort to establish top-down proteomics, performing separation and MS analysis both at intact protein level. We briefly introduce major approaches of bottom-up proteomics used in the field of protease research and highlight the shortcomings of these methods. We then discuss the present state-of-the-art of top-down proteomics. Together with the discussion of known challenges we show the potential of this approach and present a number of successful applications of top-down proteomics in protease research. This article is part of a Special Issue entitled: Proteolysis as a Regulatory Event in Pathophysiology edited by Stefan Rose-John. Copyright © 2017 Elsevier B.V. All rights reserved.

  2. Sirtuins and Proteolytic Systems: Implications for Pathogenesis of Synucleinopathies

    Directory of Open Access Journals (Sweden)

    Belém Sampaio-Marques

    2015-05-01

    Full Text Available Insoluble and fibrillar forms of α-synuclein are the major components of Lewy bodies, a hallmark of several sporadic and inherited neurodegenerative diseases known as synucleinopathies. α-Synuclein is a natural unfolded and aggregation-prone protein that can be degraded by the ubiquitin-proteasomal system and the lysosomal degradation pathways. α-Synuclein is a target of the main cellular proteolytic systems, but it is also able to alter their function further, contributing to the progression of neurodegeneration. Aging, a major risk for synucleinopathies, is associated with a decrease activity of the proteolytic systems, further aggravating this toxic looping cycle. Here, the current literature on the basic aspects of the routes for α-synuclein clearance, as well as the consequences of the proteolytic systems collapse, will be discussed. Finally, particular focus will be given to the sirtuins’s role on proteostasis regulation, since their modulation emerged as a promising therapeutic strategy to rescue cells from α-synuclein toxicity. The controversial reports on the potential role of sirtuins in the degradation of α-synuclein will be discussed. Connection between sirtuins and proteolytic systems is definitely worth of further studies to increase the knowledge that will allow its proper exploration as new avenue to fight synucleinopathies.

  3. A novel immune evasion strategy of candida albicans: proteolytic cleavage of a salivary antimicrobial peptide.

    Directory of Open Access Journals (Sweden)

    Timothy F Meiller

    Full Text Available Oropharyngeal candidiasis is an opportunistic infection considered to be a harbinger of AIDS. The etiologic agent Candida albicans is a fungal species commonly colonizing human mucosal surfaces. However, under conditions of immune dysfunction, colonizing C. albicans can become an opportunistic pathogen causing superficial or even life-threatening infections. The reasons behind this transition, however, are not clear. In the oral cavity, salivary antimicrobial peptides are considered to be an important part of the host innate defense system in the prevention of microbial colonization. Histatin-5 specifically has exhibited potent activity against C. albicans. Our previous studies have shown histatin-5 levels to be significantly reduced in the saliva of HIV+ individuals, indicating an important role for histatin-5 in keeping C. albicans in its commensal stage. The versatility in the pathogenic potential of C. albicans is the result of its ability to adapt through the regulation of virulence determinants, most notably of which are proteolytic enzymes (Saps, involved in tissue degradation. In this study, we show that C. albicans cells efficiently and rapidly degrade histatin-5, resulting in loss of its anti-candidal potency. In addition, we demonstrate that this cellular activity is due to proteolysis by a member of the secreted aspartic proteases (Sap family involved in C. albicans pathogenesis. Specifically, the proteolysis was attributed to Sap9, in turn identifying histatin-5 as the first host-specific substrate for that isoenzyme. These findings demonstrate for the first time the ability of a specific C. albicans enzyme to degrade and deactivate a host antimicrobial peptide involved in the protection of the oral mucosa against C. albicans, thereby providing new insights into the factors directing the transition of C. albicans from commensal to pathogen, with important clinical implications for alternative therapy. This report characterizes the

  4. Perturbation with intrabodies reveals that calpain cleavage is required for degradation of huntingtin exon 1.

    Directory of Open Access Journals (Sweden)

    Amber L Southwell

    2011-01-01

    Full Text Available Proteolytic processing of mutant huntingtin (mHtt, the protein that causes Huntington's disease (HD, is critical for mHtt toxicity and disease progression. mHtt contains several caspase and calpain cleavage sites that generate N-terminal fragments that are more toxic than full-length mHtt. Further processing is then required for the degradation of these fragments, which in turn, reduces toxicity. This unknown, secondary degradative process represents a promising therapeutic target for HD.We have used intrabodies, intracellularly expressed antibody fragments, to gain insight into the mechanism of mutant huntingtin exon 1 (mHDx-1 clearance. Happ1, an intrabody recognizing the proline-rich region of mHDx-1, reduces the level of soluble mHDx-1 by increasing clearance. While proteasome and macroautophagy inhibitors reduce turnover of mHDx-1, Happ1 is still able to reduce mHDx-1 under these conditions, indicating Happ1-accelerated mHDx-1 clearance does not rely on these processes. In contrast, a calpain inhibitor or an inhibitor of lysosomal pH block Happ1-mediated acceleration of mHDx-1 clearance. These results suggest that mHDx-1 is cleaved by calpain, likely followed by lysosomal degradation and this process regulates the turnover rate of mHDx-1. Sequence analysis identifies amino acid (AA 15 as a potential calpain cleavage site. Calpain cleavage of recombinant mHDx-1 in vitro yields fragments of sizes corresponding to this prediction. Moreover, when the site is blocked by binding of another intrabody, V(L12.3, turnover of soluble mHDx-1 in living cells is blocked.These results indicate that calpain-mediated removal of the 15 N-terminal AAs is required for the degradation of mHDx-1, a finding that may have therapeutic implications.

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

    Science.gov (United States)

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

    2008-02-01

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

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

    Directory of Open Access Journals (Sweden)

    Joshua T Schiffer

    Full Text Available Most chronic viral infections are managed with small molecule therapies that inhibit replication but are not curative because non-replicating viral forms can persist despite decades of suppressive treatment. There are therefore numerous strategies in development to eradicate all non-replicating viruses from the body. We are currently engineering DNA cleavage enzymes that specifically target hepatitis B virus covalently closed circular DNA (HBV cccDNA, the episomal form of the virus that persists despite potent antiviral therapies. DNA cleavage enzymes, including homing endonucleases or meganucleases, zinc-finger nucleases (ZFNs, TAL effector nucleases (TALENs, and CRISPR-associated system 9 (Cas9 proteins, can disrupt specific regions of viral DNA. Because DNA repair is error prone, the virus can be neutralized after repeated cleavage events when a target sequence becomes mutated. DNA cleavage enzymes will be delivered as genes within viral vectors that enter hepatocytes. Here we develop mathematical models that describe the delivery and intracellular activity of DNA cleavage enzymes. Model simulations predict that high vector to target cell ratio, limited removal of delivery vectors by humoral immunity, and avid binding between enzyme and its DNA target will promote the highest level of cccDNA disruption. Development of de novo resistance to cleavage enzymes may occur if DNA cleavage and error prone repair does not render the viral episome replication incompetent: our model predicts that concurrent delivery of multiple enzymes which target different vital cccDNA regions, or sequential delivery of different enzymes, are both potentially useful strategies for avoiding multi-enzyme resistance. The underlying dynamics of cccDNA persistence are unlikely to impact the probability of cure provided that antiviral therapy is given concurrently during eradication trials. We conclude by describing experiments that can be used to validate the model, which

  7. Inhibition of Lassa virus glycoprotein cleavage and multicycle replication by site 1 protease-adapted alpha(1-antitrypsin variants.

    Directory of Open Access Journals (Sweden)

    Anna Maisa

    2009-06-01

    Full Text Available Proteolytic processing of the Lassa virus envelope glycoprotein precursor GP-C by the host proprotein convertase site 1 protease (S1P is a prerequisite for the incorporation of the subunits GP-1 and GP-2 into viral particles and, hence, essential for infectivity and virus spread. Therefore, we tested in this study the concept of using S1P as a target to block efficient virus replication.We demonstrate that stable cell lines inducibly expressing S1P-adapted alpha(1-antitrypsin variants inhibit the proteolytic maturation of GP-C. Introduction of the S1P recognition motifs RRIL and RRLL into the reactive center loop of alpha(1-antitrypsin resulted in abrogation of GP-C processing by endogenous S1P to a similar level observed in S1P-deficient cells. Moreover, S1P-specific alpha(1-antitrypsins significantly inhibited replication and spread of a replication-competent recombinant vesicular stomatitis virus expressing the Lassa virus glycoprotein GP as well as authentic Lassa virus. Inhibition of viral replication correlated with the ability of the different alpha(1-antitrypsin variants to inhibit the processing of the Lassa virus glycoprotein precursor.Our data suggest that glycoprotein cleavage by S1P is a promising target for the development of novel anti-arenaviral strategies.

  8. Characterization of a bioactive 15 kDa fragment produced by proteolytic cleavage of chicken growth hormone.

    Science.gov (United States)

    Arámburo, C; Carranza, M; Reyes, M; Luna, M; Martinez-Coria, H; Berúmen, L; Scanes, C G

    2001-07-01

    There is evidence for a cleaved form of GH in the chicken pituitary gland. A 25 kDa band of immunoreactive-(ir-)GH, as well as the 22 kDa monomeric form and some oligomeric forms were observed when purified GH or fresh pituitary extract were subjected to SDS-PAGE under nonreducing conditions. Under reducing conditions, the 25 kDa ir-GH was no longer observed, being replaced by a 15 kDa band, consistent with reduction of the disulfide bridges of the cleaved form. The type of protease involved was investigated using exogenous proteases and monomeric cGH. Cleaved forms of chicken GH were generated by thrombin or collagenase. The site of cleavage was found in position Arg133-Gly134 as revealed by sequencing the fragments produced. The NH2-terminal sequence of 40 amino acid residues in the 15 kDa form was identical to that of the rcGH and analysis of the remaining 7 kDa fragment showed an exact identity with positions 134-140 of cGH structure. The thrombin cleaved GH and the 15 kDa form showed reduced activity (0.8% and 0.5% of GH, respectively) in a radioreceptor assay employing a chicken liver membrane preparation. However, this fragment had a clear bioactivity in an angiogenic bioassay and was capable to inhibit the activity of deiodinase type III in the chicken liver.

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

    Energy Technology Data Exchange (ETDEWEB)

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

    2014-10-24

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

  10. Vertebrate Embryonic Cleavage Pattern Determination.

    Science.gov (United States)

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

    2017-01-01

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

  11. Rapid paracellular transmigration of Campylobacter jejuni across polarized epithelial cells without affecting TER: role of proteolytic-active HtrA cleaving E-cadherin but not fibronectin

    LENUS (Irish Health Repository)

    Boehm, Manja

    2012-04-25

    AbstractBackgroundCampylobacter jejuni is one of the most important bacterial pathogens causing food-borne illness worldwide. Crossing the intestinal epithelial barrier and host cell entry by C. jejuni is considered the primary reason of damage to the intestinal tissue, but the molecular mechanisms as well as major bacterial and host cell factors involved in this process are still widely unclear.ResultsIn the present study, we characterized the serine protease HtrA (high-temperature requirement A) of C. jejuni as a secreted virulence factor with important proteolytic functions. Infection studies and in vitro cleavage assays showed that C. jejuni’s HtrA triggers shedding of the extracellular E-cadherin NTF domain (90 kDa) of non-polarised INT-407 and polarized MKN-28 epithelial cells, but fibronectin was not cleaved as seen for H. pylori’s HtrA. Deletion of the htrA gene in C. jejuni or expression of a protease-deficient S197A point mutant did not lead to loss of flagella or reduced bacterial motility, but led to severe defects in E-cadherin cleavage and transmigration of the bacteria across polarized MKN-28 cell layers. Unlike other highly invasive pathogens, transmigration across polarized cells by wild-type C. jejuni is highly efficient and is achieved within a few minutes of infection. Interestingly, E-cadherin cleavage by C. jejuni occurs in a limited fashion and transmigration required the intact flagella as well as HtrA protease activity, but does not reduce transepithelial electrical resistance (TER) as seen with Salmonella, Shigella, Listeria or Neisseria.ConclusionThese results suggest that HtrA-mediated E-cadherin cleavage is involved in rapid crossing of the epithelial barrier by C. jejuni via a very specific mechanism using the paracellular route to reach basolateral surfaces, but does not cleave the fibronectin receptor which is necessary for cell entry.

  12. Rapid paracellular transmigration of Campylobacter jejuni across polarized epithelial cells without affecting TER: role of proteolytic-active HtrA cleaving E-cadherin but not fibronectin.

    Science.gov (United States)

    Boehm, Manja; Hoy, Benjamin; Rohde, Manfred; Tegtmeyer, Nicole; Bæk, Kristoffer T; Oyarzabal, Omar A; Brøndsted, Lone; Wessler, Silja; Backert, Steffen

    2012-04-25

    Campylobacter jejuni is one of the most important bacterial pathogens causing food-borne illness worldwide. Crossing the intestinal epithelial barrier and host cell entry by C. jejuni is considered the primary reason of damage to the intestinal tissue, but the molecular mechanisms as well as major bacterial and host cell factors involved in this process are still widely unclear. In the present study, we characterized the serine protease HtrA (high-temperature requirement A) of C. jejuni as a secreted virulence factor with important proteolytic functions. Infection studies and in vitro cleavage assays showed that C. jejuni's HtrA triggers shedding of the extracellular E-cadherin NTF domain (90 kDa) of non-polarised INT-407 and polarized MKN-28 epithelial cells, but fibronectin was not cleaved as seen for H. pylori's HtrA. Deletion of the htrA gene in C. jejuni or expression of a protease-deficient S197A point mutant did not lead to loss of flagella or reduced bacterial motility, but led to severe defects in E-cadherin cleavage and transmigration of the bacteria across polarized MKN-28 cell layers. Unlike other highly invasive pathogens, transmigration across polarized cells by wild-type C. jejuni is highly efficient and is achieved within a few minutes of infection. Interestingly, E-cadherin cleavage by C. jejuni occurs in a limited fashion and transmigration required the intact flagella as well as HtrA protease activity, but does not reduce transepithelial electrical resistance (TER) as seen with Salmonella, Shigella, Listeria or Neisseria. These results suggest that HtrA-mediated E-cadherin cleavage is involved in rapid crossing of the epithelial barrier by C. jejuni via a very specific mechanism using the paracellular route to reach basolateral surfaces, but does not cleave the fibronectin receptor which is necessary for cell entry.

  13. Use of Proteolytic Enzymes in the Treatment of Proteinaceous Esophageal Food Impaction.

    Science.gov (United States)

    Morse, Christopher R; Wang, Howard; Donahue, Dean M; Garrity, Julie M; Allan, James S

    2016-01-01

    Proteinaceous esophageal food impaction typically requires endoscopic intervention. An alternative approach is the use of proteolytic enzymes. Concerns regarding the use of proteolytic enzymes include the risk of perforation and aspiration pneumonitis. We retrospectively reviewed our series of 69 patients treated with papain to determine the safety and efficacy of proteolytic enzymes. Patients were retrospectively reviewed if treated for an esophageal food impaction from 1999 through 2008. Median age was 56 years (range 19-91 years), with 46 male and 23 female patients. In 27 patients (39%) this was their first presentation, in 14 (20%) it was the second, and 28 (41%) had multiple previous episodes. Meat was the cause in 49 (71%), chicken in 6 (9%), fish in 3 (4%), and unspecified in 11 (16%). All patients presented with dysphagia for solids, 56 (81%) could not tolerate liquids. Papain solution, 1 tsp in 8 oz of water, was given to patients in an unlimited quantity. Papain was successful in relieving the obstruction in 60 patients (87%). The remaining 9 patients (13%) underwent endoscopy with successful retrieval. No patient suffered a perforation, either with papain ingestion or endoscopy. There were no episodes of pneumonitis or pneumonia. We have used proteolytic enzymes with a high success rate and with minimal complication. Further, if proteolytic enzymes fail, endoscopy can be performed safely and effectively. We recommend the use of proteolytic enzymes as the initial management in all patients with proteinaceous food impaction of the esophagus. Published by Elsevier Inc.

  14. Microbiological Monitoring and Proteolytic Study of Clinical Samples From Burned and Burned Wounded Patients

    International Nuclear Information System (INIS)

    Toema, M.A.; El-Bazza, Z.E.; El-Hifnawi, H.N.; Abd-El-Hakim, E.E.

    2013-01-01

    In this study, clinical samples were collected from 100 patients admitted to Burn and Plastic Surgery Department, Faculty of Medicine, Ain Shams University, Egypt, over a period of 12 months. The proteolytic activity of 110 clinical samples taken from surfaces swabs which taken from burned and burned wounded patients with different ages and gender was examined. Screening for the proteolytic activity produced by pathogenic bacteria isolated from burned and burned wounded patients was evaluated as gram positive Bacilli and gram negative bacilli showed high proteolytic activity (46.4%) while 17.9% showed no activity. The isolated bacteria proved to have proteolytic activity were classified into high, moderate and weak. The pathogenic bacteria isolated from burned and burned wounded patients and showing proteolytic activity were identified as Pseudomonas aeruginosa, Proteus mirabilis, Proteus vulgaris, Bacillus megaterium, Bacillus cereus, Staphylococcus aureus, Escherichia coli, Klebsiella ozaeanae, Klebsiella oxytoca, Klebsiella pneumoniae and Pseudomonas fluoresces.

  15. Amyloid-β production via cleavage of amyloid-β protein precursor is modulated by cell density.

    Science.gov (United States)

    Zhang, Can; Browne, Andrew; Divito, Jason R; Stevenson, Jesse A; Romano, Donna; Dong, Yuanlin; Xie, Zhongcong; Tanzi, Rudolph E

    2010-01-01

    Mounting evidence suggests that Alzheimer's disease (AD) is caused by the accumulation of the small peptide, amyloid-β (Aβ), a proteolytic cleavage product of amyloid-β protein precursor (AβPP). Aβ is generated through a serial cleavage of AβPP by β- and γ-secretase. Aβ40 and Aβ42 are the two main components of amyloid plaques in AD brains, with Aβ42 being more prone to aggregation. AβPP can also be processed by α-secretase, which cleaves AβPP within the Aβ sequence, thereby preventing the generation of Aβ. Little is currently known regarding the effects of cell density on AβPP processing and Aβ generation. Here we assessed the effects of cell density on AβPP processing in neuronal and non-neuronal cell lines, as well as mouse primary cortical neurons. We found that decreased cell density significantly increases levels of Aβ40, Aβ42, total Aβ, and the ratio of Aβ42: Aβ40. These results also indicate that cell density is a significant modulator of AβPP processing. Overall, these findings carry profound implications for both previous and forthcoming studies aiming to assess the effects of various conditions and genetic/chemical factors, e.g., novel drugs on AβPP processing and Aβ generation in cell-based systems. Moreover, it is interesting to speculate whether cell density changes in vivo may also affect AβPP processing and Aβ levels in the AD brain.

  16. Proteolytic activity regarding Sarconesiopsis magellanica (Diptera: Calliphoridae) larval excretions and secretions.

    Science.gov (United States)

    Pinilla, Yudi T; Moreno-Pérez, Darwin A; Patarroyo, Manuel A; Bello, Felio J

    2013-12-01

    Sarconesiopsis magellanica (Diptera: Calliphoridae) is a medically important necrophagous fly which is used for establishing the post-mortem interval. Diptera maggots release proteolytic enzymes contained in larval excretion and secretion (ES) products playing a key role in digestion. Special interest in proteolytic enzymes has also been aroused regarding understanding their role in wound healing since they degrade necrotic tissue during larval therapy. This study was thus aimed at identifying and characterising S. magellanica proteolytic enzyme ES products for the first time. These products were obtained from first-, second- and third-instar larvae taken from a previously-established colony. ES proteins were separated by SDS-PAGE and their proteolytic activity was characterised by zymograms and inhibition assays involving BAPNA (Nα-benzoyl-dl-Arg-p-nitroanilide) and SAPNA substrates, using synthetic inhibitors. The protein profile ranged from ∼69kDa to ∼23kDa; several of them coincided with the Lucilia sericata ES protein profile. Serine-protease hydrolysis activity (measured by zymogram) was confirmed when a ∼25kDa band disappeared upon ES incubation with PMSF inhibitor at pH 7.8. Analysis of larval ES proteolytic activity on BAPNA and SAPNA substrates (determined by using TLCK and TPCK specific inhibitors) suggested a greater amount of trypsin-like protease. These results support the need for further experiments aimed at validating S. magellanica use in larval therapy. Copyright © 2013 Elsevier B.V. All rights reserved.

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

    Directory of Open Access Journals (Sweden)

    Lin Qu

    2011-09-01

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

  18. Isolation and removal of proteolytic enzymes with magnetic cross-linked erythrocytes

    International Nuclear Information System (INIS)

    Safarik, I. Ivo; Safarikova, Mirka

    2001-01-01

    New magnetic adsorbents for batch isolation and removal of various proteolytic enzymes were prepared by glutaraldehyde cross-linking of bovine, porcine and human erythrocytes in the presence of fine magnetic particles. Trypsin, chymotrypsin, alkaline bacterial protease and proteases present in various commercial enzyme preparations were efficiently adsorbed on these adsorbents; on the contrary, proteins without proteolytic activity were not adsorbed

  19. Role of Proteolytic Enzymes in the Interaction of Phytopathogenic Microorganisms with Plants.

    Science.gov (United States)

    Valueva, T A; Zaichik, B Ts; Kudryavtseva, N N

    2016-12-01

    Various forms of participation of proteolytic enzymes in pathogenesis and defense in plants are reviewed. Along with extracellular proteinases, phytopathogenic microorganisms produce specific effectors having proteolytic activity and capable of acting on proteins inside plant cells. In turn, for defense against pathogens, plants use both extracellular and intracellular proteinases.

  20. Molecular mechanism of the intramembrane cleavage of the β-carboxyl terminal fragment of amyloid precursor protein by γ-secretase

    Directory of Open Access Journals (Sweden)

    Maho eMorishima-Kawashima

    2014-11-01

    Full Text Available Amyloid β-protein (Aβ plays a central role in the pathogenesis of Alzheimer’s disease, the most common age-associated neurodegenerative disorder. Aβ is generated through intramembrane proteolysis of the β-carboxyl terminal fragment (βCTF of β-amyloid precursor protein (APP by γ-secretase. The initial cleavage by γ-secretase occurs in the membrane/cytoplasm boundary of the βCTF, liberating the APP intracellular domain (AICD. The remaining βCTFs, which are truncated at the C-terminus (longer Aβs, are then cropped sequentially in a stepwise manner, predominantly at three residue intervals, to generate Aβ. There are two major Aβ product lines which generate Aβ40 and Aβ42 with concomitant release of three and two tripeptides, respectively. Additionally, many alternative cleavages occur, releasing peptides with three to six residues. These modulate the Aβ product lines and define the species and quantity of Aβ generated. Here, we review our current understanding of the intramembrane cleavage of the βCTF by γ-secretase, which may contribute to the future goal of developing an efficient therapeutic strategy for Alzheimer’s disease.

  1. Detection of proteolytic signatures for Parkinson's disease

    DEFF Research Database (Denmark)

    Jordal, Peter Lüttge; Dyrlund, Thomas F.; Winge, Kristian

    2016-01-01

    Aim: To investigate if idiopathic Parkinson's disease (IPD) is associated with distinct proteolytic signatures relative to non-neurodegenerative controls (NND) and patients with multiple system atrophy (MSA). Materials & methods: A subtiligase-based N-terminomics screening method was exploited...

  2. Proteolytic enzymes involved in MHC class I antigen processing: A guerrilla army that partners with the proteasome.

    Science.gov (United States)

    Lázaro, Silvia; Gamarra, David; Del Val, Margarita

    2015-12-01

    Major histocompatibility complex class I proteins (MHC-I) load short peptides derived from proteolytic cleavage of endogenous proteins in any cell of the body, in a process termed antigen processing and presentation. When the source proteins are altered self or encoded by a pathogen, recognition of peptide/MHC-I complexes at the plasma membrane leads to CD8(+) T-lymphocyte responses that clear infections and probably underlie tumor immune surveillance. On the other hand, presentation of self peptides may cause some types of autoimmunity. The peptides that are presented determine the specificity and efficiency of pathogen clearance or, conversely, of immunopathology. In this review we highlight the growing number of peptidases which, as a by-product of their regular activity, can generate peptide epitopes for immune surveillance. These ∼20 peptidases collectively behave as a guerrilla army partnering with the regular proteasome army in generating a variety of peptides for presentation by MHC-I and thus optimally signaling infection. Copyright © 2015 The Authors. Published by Elsevier Ltd.. All rights reserved.

  3. Biological and Proteolytic Variation in the Venom of Crotalus scutulatus scutulatus from Mexico

    Directory of Open Access Journals (Sweden)

    Miguel Borja

    2018-01-01

    Full Text Available Rattlesnake venoms may be classified according to the presence/absence and relative abundance of the neurotoxic phospholipases A 2 s (PLA 2 s, such as Mojave toxin, and snake venom metalloproteinases (SVMPs. In Mexico, studies to determine venom variation in Mojave Rattlesnakes (Crotalus scutulatus scutulatus are limited and little is known about the biological and proteolytic activities in this species. Tissue (34 and venom (29 samples were obtained from C. s. scutulatus from different locations within their distribution in Mexico. Mojave toxin detection was carried out at the genomic (by PCR and protein (by ELISA levels for all tissue and venom samples. Biological activity was tested on representative venoms by measuring LD 50 and hemorrhagic activity. To determine the approximate amount of SVMPs, 15 venoms were separated by RP-HPLC and variation in protein profile and proteolytic activity was evaluated by SDS-PAGE (n = 28 and Hide Powder Azure proteolytic analysis (n = 27. Three types of venom were identified in Mexico which is comparable to the intraspecific venom diversity observed in the Sonoran Desert of Arizona, USA: Venom Type A (∼Type II, with Mojave toxin, highly toxic, lacking hemorrhagic activity, and with scarce proteolytic activity; Type B (∼Type I, without Mojave toxin, less toxic than Type A, highly hemorrhagic and proteolytic; and Type A + B, containing Mojave toxin, as toxic as venom Type A, variable in hemorrhagic activity and with intermediate proteolytic activity. We also detected a positive correlation between SVMP abundance and hemorrhagic and proteolytic activities. Although more sampling is necessary, our results suggest that venoms containing Mojave toxin and venom lacking this toxin are distributed in the northwest and southeast portions of the distribution in Mexico, respectively, while an intergradation in the middle of both zones is present.

  4. PhnY and PhnZ comprise a new oxidative pathway for enzymatic cleavage of a carbon-phosphorus bond

    DEFF Research Database (Denmark)

    McSorley, Fern R.; Wyatt, Peter W.; Martinez, Ascuncion

    2012-01-01

    The sequential activities of PhnY, an α-ketoglutarate/Fe(II)-dependent dioxygenase, and PhnZ, a Fe(II)-dependent enzyme of the histidine-aspartate motif hydrolase family, cleave the carbon-phosphorus bond of the organophosphonate natural product 2-aminoethylphosphonic acid. PhnY adds a hydroxyl...... group to the α-carbon, yielding 2-amino-1-hydroxyethylphosphonic acid, which is oxidatively converted by PhnZ to inorganic phosphate and glycine. The PhnZ reaction represents a new enzyme mechanism for metabolic cleavage of a carbon-phosphorus bond....

  5. Proteolytic fragmentation of inositol 1,4,5-trisphosphate receptors: a novel mechanism regulating channel activity?

    Science.gov (United States)

    Wang, Liwei; Alzayady, Kamil J; Yule, David I

    2016-06-01

    Inositol 1,4,5-trisphosphate receptors (IP3 Rs) are a family of ubiquitously expressed intracellular Ca(2+) release channels. Regulation of channel activity by Ca(2+) , nucleotides, phosphorylation, protein binding partners and other cellular factors is thought to play a major role in defining the specific spatiotemporal characteristics of intracellular Ca(2+) signals. These properties are, in turn, believed pivotal for the selective and specific physiological activation of Ca(2+) -dependent effectors. IP3 Rs are also substrates for the intracellular cysteine proteases, calpain and caspase. Cleavage of the IP3 R has been proposed to play a role in apoptotic cell death by uncoupling regions important for IP3 binding from the channel domain, leaving an unregulated leaky Ca(2+) pore. Contrary to this hypothesis, we demonstrate following proteolysis that N- and C-termini of IP3 R1 remain associated, presumably through non-covalent interactions. Further, we show that complementary fragments of IP3 R1 assemble into tetrameric structures and retain their ability to be regulated robustly by IP3 . While peptide continuity is clearly not necessary for IP3 -gating of the channel, we propose that cleavage of the IP3 R peptide chain may alter other important regulatory events to modulate channel activity. In this scenario, stimulation of the cleaved IP3 R may support distinct spatiotemporal Ca(2+) signals and activation of specific effectors. Notably, in many adaptive physiological events, the non-apoptotic activities of caspase and calpain are demonstrated to be important, but the substrates of the proteases are poorly defined. We speculate that proteolytic fragmentation may represent a novel form of IP3 R regulation, which plays a role in varied adaptive physiological processes. © 2015 The Authors. The Journal of Physiology © 2015 The Physiological Society.

  6. Cleavage and creep fracture of rock salt

    International Nuclear Information System (INIS)

    Chan, K.S.; Munson, D.E.; Bodner, S.R.

    1996-01-01

    The dominant failure mechanism in rock salt at ambient temperature is either cleavage or creep fracture. Since the transition of creep fracture to cleavage in a compressive stress field is not well understood, failure of rock salt by cleavage and creep fracture is analyzed in this paper to elucidate the effect of stress state on the competition between these two fracture mechanisms. For cleavage fracture, a shear crack is assumed to cause the formation and growth of a symmetric pair of wing cracks in a predominantly compressive stress field. The conditions for wing-crack instability are derived and presented as the cleavage fracture boundary in the fracture mechanism map. Using an existing creep fracture model, stress conditions for the onset of creep fracture and isochronous failure curves of specified times-to-rupture are calculated and incorporated into the fracture mechanism map. The regimes of dominance by cleavage and creep fracture are established and compared with experimental data. The result indicates that unstable propagation of cleavage cracks occurs only in the presence of tensile stress. The onset of creep fracture is promoted by a tensile stress, but can be totally suppressed by a high confining pressure. Transition of creep fracture to cleavage occurs when critical conditions of stress difference and tensile stress for crack instability are exceeded

  7. Biochemical analyses indicate that binding and cleavage specificities define the ordered processing of human Okazaki fragments by Dna2 and FEN1.

    Science.gov (United States)

    Gloor, Jason W; Balakrishnan, Lata; Campbell, Judith L; Bambara, Robert A

    2012-08-01

    In eukaryotic Okazaki fragment processing, the RNA primer is displaced into a single-stranded flap prior to removal. Evidence suggests that some flaps become long before they are cleaved, and that this cleavage involves the sequential action of two nucleases. Strand displacement characteristics of the polymerase show that a short gap precedes the flap during synthesis. Using biochemical techniques, binding and cleavage assays presented here indicate that when the flap is ∼ 30 nt long the nuclease Dna2 can bind with high affinity to the flap and downstream double strand and begin cleavage. When the polymerase idles or dissociates the Dna2 can reorient for additional contacts with the upstream primer region, allowing the nuclease to remain stably bound as the flap is further shortened. The DNA can then equilibrate to a double flap that can bind Dna2 and flap endonuclease (FEN1) simultaneously. When Dna2 shortens the flap even more, FEN1 can displace the Dna2 and cleave at the flap base to make a nick for ligation.

  8. Translation of Polioviral mRNA Is Inhibited by Cleavage of Polypyrimidine Tract-Binding Proteins Executed by Polioviral 3Cpro

    Science.gov (United States)

    Back, Sung Hoon; Kim, Yoon Ki; Kim, Woo Jae; Cho, Sungchan; Oh, Hoe Rang; Kim, Jung-Eun; Jang, Sung Key

    2002-01-01

    The translation of polioviral mRNA occurs through an internal ribosomal entry site (IRES). Several RNA-binding proteins, such as polypyrimidine tract-binding protein (PTB) and poly(rC)-binding protein (PCBP), are required for the poliovirus IRES-dependent translation. Here we report that a poliovirus protein, 3Cpro (and/or 3CDpro), cleaves PTB isoforms (PTB1, PTB2, and PTB4). Three 3Cpro target sites (one major target site and two minor target sites) exist in PTBs. PTB fragments generated by poliovirus infection are redistributed to the cytoplasm from the nucleus, where most of the intact PTBs are localized. Moreover, these PTB fragments inhibit polioviral IRES-dependent translation in a cell-based assay system. We speculate that the proteolytic cleavage of PTBs may contribute to the molecular switching from translation to replication of polioviral RNA. PMID:11836431

  9. The proteolytic systems of lactic acid bacteria

    NARCIS (Netherlands)

    Kunji, Edmund R.S.; Mierau, Igor; Hagting, Anja; Poolman, Bert; Konings, Wil N.

    1996-01-01

    Proteolysis in dairy lactic acid bacteria has been studied in great detail by genetic, biochemical and ultrastructural methods. From these studies the picture emerges that the proteolytic systems of lactococci and lactobacilli are remarkably similar in their components and mode of action. The

  10. Proteolytic activity of alkaliphilic, salt-tolerant actinomycetes from ...

    African Journals Online (AJOL)

    tolerant alkaliphilic. All the isolates need to be further studied for the ability of their potential protease enzyme production. Key words: Alkaliphilic actinomycetes, salt tolerant actinomycetes, desert soil, isolation, proteolytic activity.

  11. [Proteolytic activity of IgG-antibodies of mice, immunized by calf thymus histones].

    Science.gov (United States)

    Kit, Iu Ia; Korniĭ, N; Kril', I Ĭ; Mahorivs'ka, I B; Tkachenko, V; Bilyĭ, R O; Stoĭka, R S

    2014-01-01

    The main goal of the study was to determine the ability of histones to induce production of the proteolytically active IgG-antibodies in BALB/c mice. In order to perform this study 8 mice were immunized with the fraction of total calf thymus histones. IgGs were isolated from the serum of the immunized and not immunized animals by means of precipitation with 33% ammonium sulfate, followed by affinity chromatography on protein G-Sepharose column. Histones, myelin basic protein (MBP), lysozyme, BSA, ovalbumin, macroglobulin, casein and cytochrome c served as substrates for determining the proteolytic activity. It was found that IgGs from the blood serum of immunized mice are capable of hydrolyzing histone H1, core histone and MBP. On the contrary, the proteolytic activity of IgGs from the blood serum of not immunized mice was not detected. The absence of proteolytical enzymes in the fraction of IgGs was proven by HPLC chromatography. High levels of proteolytic activity toward histones have been also detected in affinity purified IgGs from blood serum of patients with rheumatoid arthritis, but not in healthy donors. These data indicate that eukaryotic histones may induce production of protabzymes in mammals. The possible origin of these protabzymes and their potential biological role in mammalians is discussed.

  12. New radionuclide method for determination of gastric content proteolytic activity. (Experiments)

    International Nuclear Information System (INIS)

    Sergienko, V.B.; Popova, L.V.; Khodarev, N.N.; Astashenkova, K.Yu.

    1988-01-01

    A possibility of the use of a radionuclide tubeless rapid method for measuring gastric content proteolytic activity (GCPA) with the help of a protein (gelatin) RP containing capsule was demonstrated in experiments in vitro. There was correlation between the time of dissolution of RP containing capsules and GCPA determined after Mett's method. Reference time intervals were established for normal, raised and lowered proteolytic activity. The method was shown to be physiological, simple and timesaving

  13. The proteolytic profile of human cancer procoagulant suggests that it promotes cancer metastasis at the level of activation rather than degradation.

    Science.gov (United States)

    Kee, Nalise Low Ah; Krause, Jason; Blatch, Gregory L; Muramoto, Koji; Sakka, Kazuo; Sakka, Makiko; Naudé, Ryno J; Wagner, Leona; Wolf, Raik; Rahfeld, Jens-Ulrich; Demuth, Hans-Ulrich; Mielicki, Wojciech P; Frost, Carminita L

    2015-10-01

    Proteases are essential for tumour progression and many are over-expressed during this time. The main focus of research was the role of these proteases in degradation of the basement membrane and extracellular matrix (ECM), thereby enabling metastasis to occur. Cancer procoagulant (CP), a protease present in malignant tumours, but not normal tissue, is a known activator of coagulation factor X (FX). The present study investigated the function of CP in cancer progression by focussing on its enzymatic specificity. FX cleavage was confirmed using SDS-PAGE and MALDI-TOF MS and compared to the proteolytic action of CP on ECM proteins, including collagen type IV, laminin and fibronectin. Contrary to previous reports, CP cleaved FX at the conventional activation site (between Arg-52 and Ile-53). Additionally, degradation of FX by CP occurred at a much slower rate than degradation by conventional activators. Complete degradation of the heavy chain of FX was only visible after 24 h, while degradation by RVV was complete after 30 min, supporting postulations that the procoagulant function of CP may be of secondary importance to its role in cancer progression. Of the ECM proteins tested, only fibronectin was cleaved. The substrate specificity of CP was further investigated by screening synthetic peptide substrates using a novel direct CP assay. The results indicate that CP is not essential for either cancer-associated blood coagulation or the degradation of ECM proteins. Rather, they suggest that this protease may be required for the proteolytic activation of membrane receptors.

  14. Novel carotenoid cleavage dioxygenase catalyzes the first dedicated step in saffron crocin biosynthesis

    KAUST Repository

    Frusciante, Sarah; Diretto, Gianfranco; Bruno, Mark; Ferrante, Paola; Pietrella, Marco; Prado-Cabrero, Alfonso; Rubio-Moraga, Á ngela L.; Beyer, Peter D.; Gó mez-Gó mez, Lourdes; Al-Babili, Salim; Giuliano, Giovanni

    2014-01-01

    Crocus sativus stigmas are the source of the saffron spice and accumulate the apocarotenoids crocetin, crocins, picrocrocin, and safranal, responsible for its color, taste, and aroma. Through deep transcriptome sequencing, we identified a novel dioxygenase, carotenoid cleavage dioxygenase 2 (CCD2), expressed early during stigma development and closely related to, but distinct from, the CCD1 dioxygenase family. CCD2 is the only identified member of a novel CCD clade, presents the structural features of a bona fide CCD, and is able to cleave zeaxanthin, the presumed precursor of saffron apocarotenoids, both in Escherichia coli and in maize endosperm. The cleavage products, identified through high-resolution mass spectrometry and comigration with authentic standards, are crocetin dialdehyde and crocetin, respectively. In vitro assays show that CCD2 cleaves sequentially the 7,8 and 7′,8′ double bonds adjacent to a 3-OH-β-ionone ring and that the conversion of zeaxanthin to crocetin dialdehyde proceeds via the C30 intermediate 3-OH-β-apo-8′-carotenal. In contrast, zeaxanthin cleavage dioxygenase (ZCD), an enzyme previously claimed to mediate crocetin formation, did not cleave zeaxanthin or 3-OH-β-apo-8′-carotenal in the test systems used. Sequence comparison and structure prediction suggest that ZCD is an N-truncated CCD4 form, lacking one blade of the β-propeller structure conserved in all CCDs. These results constitute strong evidence that CCD2 catalyzes the first dedicated step in crocin biosynthesis. Similar to CCD1, CCD2 has a cytoplasmic localization, suggesting that it may cleave carotenoids localized in the chromoplast outer envelope.

  15. Novel carotenoid cleavage dioxygenase catalyzes the first dedicated step in saffron crocin biosynthesis

    KAUST Repository

    Frusciante, Sarah

    2014-08-05

    Crocus sativus stigmas are the source of the saffron spice and accumulate the apocarotenoids crocetin, crocins, picrocrocin, and safranal, responsible for its color, taste, and aroma. Through deep transcriptome sequencing, we identified a novel dioxygenase, carotenoid cleavage dioxygenase 2 (CCD2), expressed early during stigma development and closely related to, but distinct from, the CCD1 dioxygenase family. CCD2 is the only identified member of a novel CCD clade, presents the structural features of a bona fide CCD, and is able to cleave zeaxanthin, the presumed precursor of saffron apocarotenoids, both in Escherichia coli and in maize endosperm. The cleavage products, identified through high-resolution mass spectrometry and comigration with authentic standards, are crocetin dialdehyde and crocetin, respectively. In vitro assays show that CCD2 cleaves sequentially the 7,8 and 7′,8′ double bonds adjacent to a 3-OH-β-ionone ring and that the conversion of zeaxanthin to crocetin dialdehyde proceeds via the C30 intermediate 3-OH-β-apo-8′-carotenal. In contrast, zeaxanthin cleavage dioxygenase (ZCD), an enzyme previously claimed to mediate crocetin formation, did not cleave zeaxanthin or 3-OH-β-apo-8′-carotenal in the test systems used. Sequence comparison and structure prediction suggest that ZCD is an N-truncated CCD4 form, lacking one blade of the β-propeller structure conserved in all CCDs. These results constitute strong evidence that CCD2 catalyzes the first dedicated step in crocin biosynthesis. Similar to CCD1, CCD2 has a cytoplasmic localization, suggesting that it may cleave carotenoids localized in the chromoplast outer envelope.

  16. EFFECT OF MARINATION WITH PROTEOLYTIC ENZYMES ON QUALITY OF BEEF MUSCLE

    Directory of Open Access Journals (Sweden)

    Daniela Istrati

    2012-03-01

    Full Text Available During storage and thermal treatment meat suffers a number of biochemical and physical-chemical changes in the substrate protein, changes that take place with varying intensity depending on the method of preservation utilized and temperature of thermal treatment applied. Application of different treatments aimed to influence the proteolytic activity as is the case of enzymatic tenderization of beef.Improving the meat tenderness with proteolytic enzymes is promising, but current legislation restricting the use of proteolytic enzymes from bacterial origin and recommended tenderizers salts containing papain, ficin and bromelain. Recent research revealed that meat marinating before grilling results in a reduction of heterocyclic amine content after thermal treatment. Also, the addition of fruit pulp, garlic or other spices contributes to decreased production of heterocyclic amines because of their antioxidant activity. In the present study was aimed influence of exogenous proteolytic enzymes on adult beef tenderness. To increase the tenderness of adult beef were used exogenous enzymes preparations (papain and bromelain and natural sources of enzymes using pineapple and papaya fruit. It was intended to establish the correlation between enzymatic activity of enzymes used in the study, the processing technology and changes in the physical-chemical and biochemical characteristics that occur during storage in refrigerated conditions (evolution of the rigidity index and water holding capacity, cooking losses and cooking yield of the samples injected/marinated with enzymes.

  17. Seasonal variation in the temperature sensitivity of proteolytic enzyme activity in temperate forest soils

    Science.gov (United States)

    Brzostek, Edward R.; Finzi, Adrien C.

    2012-03-01

    Increasing soil temperature has the potential to alter the activity of the extracellular enzymes that mobilize nitrogen (N) from soil organic matter (SOM) and ultimately the availability of N for primary production. Proteolytic enzymes depolymerize N from proteinaceous components of SOM into amino acids, and their activity is a principal driver of the within-system cycle of soil N. The objectives of this study were to investigate whether the soils of temperate forest tree species differ in the temperature sensitivity of proteolytic enzyme activity over the growing season and the role of substrate limitation in regulating temperature sensitivity. Across species and sampling dates, proteolytic enzyme activity had relatively low sensitivity to temperature with a mean activation energy (Ea) of 33.5 kJ mol-1. Ea declined in white ash, American beech, and eastern hemlock soils across the growing season as soils warmed. By contrast, Eain sugar maple soil increased across the growing season. We used these data to develop a species-specific empirical model of proteolytic enzyme activity for the 2009 calendar year and studied the interactive effects of soil temperature (ambient or +5°C) and substrate limitation (ambient or elevated protein) on enzyme activity. Declines in substrate limitation had a larger single-factor effect on proteolytic enzyme activity than temperature, particularly in the spring. There was, however, a large synergistic effect of increasing temperature and substrate supply on proteolytic enzyme activity. Our results suggest limited increases in N availability with climate warming unless there is a parallel increase in the availability of protein substrates.

  18. Sequential reduction–oxidation for photocatalytic degradation of tetrabromobisphenol A: Kinetics and intermediates

    International Nuclear Information System (INIS)

    Guo, Yaoguang; Lou, Xiaoyi; Xiao, Dongxue; Xu, Lei; Wang, Zhaohui; Liu, Jianshe

    2012-01-01

    Highlights: ► Sequential photocatalytic reduction–oxidation degradation of TBBPA was firstly examined. ► Different atmospheres were found to have significant effect on debromination reaction. ► A possible sequential photocatalytic reduction–oxidation pathway was proposed. - Abstract: C-Br bond cleavage is considered as a key step to reduce their toxicities and increase degradation rates for most brominated organic pollutants. Here a sequential reduction/oxidation strategy (i.e. debromination followed by photocatalytic oxidation) for photocatalytic degradation of tetrabromobisphenol A (TBBPA), one of the most frequently used brominated flame retardants, was proposed on the basis of kinetic analysis and intermediates identification. The results demonstrated that the rates of debromination and even photodegradation of TBBPA strongly depended on the atmospheres, initial TBBPA concentrations, pH of the reaction solution, hydrogen donors, and electron acceptors. These kinetic data and byproducts identification obtained by GC–MS measurement indicated that reductive debromination reaction by photo-induced electrons dominated under N 2 -saturated condition, while oxidation reaction by photoexcited holes or hydroxyl radicals played a leading role when air was saturated. It also suggested that the reaction might be further optimized for pretreatment of TBBPA-contaminated wastewater by a two-stage reductive debromination/subsequent oxidative decomposition process in the UV-TiO 2 system by changing the reaction atmospheres.

  19. Microstructure and cleavage in lath martensitic steels

    International Nuclear Information System (INIS)

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

    2013-01-01

    In this paper we discuss the microstructure of lath martensitic steels and the mechanisms by which it controls cleavage fracture. The specific experimental example is a 9Ni (9 wt% Ni) steel annealed to have a large prior austenite grain size, then examined and tested in the as-quenched condition to produce a relatively coarse lath martensite. The microstructure is shown to approximate the recently identified ‘classic’ lath martensite structure: prior austenite grains are divided into packets, packets are subdivided into blocks, and blocks contain interleaved laths whose variants are the two Kurjumov–Sachs relations that share the same Bain axis of the transformation. When the steel is fractured in brittle cleavage, the laths in the block share {100} cleavage planes and cleave as a unit. However, cleavage cracks deflect or blunt at the boundaries between blocks with different Bain axes. It follows that, as predicted, the block size governs the effective grain size for cleavage. (paper)

  20. Proteolytic and antimicrobial activity of lactic acid bacteria grown in goat milk.

    Science.gov (United States)

    Atanasova, Jivka; Moncheva, Penka; Ivanova, Iskra

    2014-11-02

    We examined 62 strains and 21 trade starter cultures from the collection of LB Bulgaricum PLC for proteolytic and antimicrobial activity of lactic acid bacteria (LAB) grown in goat milk. The aim of this study was to investigate the fermentation of caseins, α-lactalbumin and β-lactoglobulin by LAB, using the o -phthaldialdehyde (OPA) spectrophotometric assay and sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE). The proteolysis targeted mainly caseins, especially β-casein. Whey proteins were proteolyzed, essentially β-lactoglobulin. The proteolytic activity of Lactococcus lactis l598, Streptococcus thermophilus t3D1, Dt1, Lactobacillus lactis 1043 and L. delbrueckii subsp. bulgaricus b38, b122 and b24 was notably high. The proteolysis process gave rise to medium-sized peptide populations. Most of the examined strains showed antimicrobial activity against some food pathogens, such as Escherichia coli , Staphylococcus aureus , Salmonella cholere enteridis , Listeria monocytogenes , Listeria innocua and Enterobacter aerogenes . The most active producers of antimicrobial-active peptides were strains of L. delbrueckii subsp. bulgaricus and S. thermophilus , which are of practical importance. The starter cultures containing the examined species showed high proteolytic and antimicrobial activity in skimmed goat milk. The greatest antimicrobial activity of the cultures was detected against E. aerogenes . The obtained results demonstrated the significant proteolytic potential of the examined strains in goat milk and their potential for application in the production of dairy products from goat's milk. The present results could be considered as the first data on the proteolytic capacity of strains and starter cultures in goat milk for the purposes of trade interest of LB Bulgaricum PLC.

  1. Proteolytic Enzymes Clustered in Specialized Plasma-Membrane Domains Drive Endothelial Cells' Migration.

    Directory of Open Access Journals (Sweden)

    Monica Salamone

    Full Text Available In vitro cultured endothelial cells forming a continuous monolayer establish stable cell-cell contacts and acquire a "resting" phenotype; on the other hand, when growing in sparse conditions these cells acquire a migratory phenotype and invade the empty area of the culture. Culturing cells in different conditions, we compared expression and clustering of proteolytic enzymes in cells having migratory versus stationary behavior. In order to observe resting and migrating cells in the same microscopic field, a continuous cell monolayer was wounded. Increased expression of proteolytic enzymes was evident in cell membranes of migrating cells especially at sprouting sites and in shed membrane vesicles. Gelatin zymography and western blotting analyses confirmed that in migrating cells, expression of membrane-bound and of vesicle-associated proteolytic enzymes are increased. The enzymes concerned include MMP-2, MMP-9, MT1-MMP, seprase, DPP4 (DiPeptidyl Peptidase 4 and uPA. Shed membrane vesicles were shown to exert degradative activity on ECM components and produce substrates facilitating cell migration. Vesicles shed by migrating cells degraded ECM components at an increased rate; as a result their effect on cell migration was amplified. Inhibiting either Matrix Metallo Proteases (MMPs or Serine Integral Membrane Peptidases (SIMPs caused a decrease in the stimulatory effect of vesicles, inhibiting the spontaneous migratory activity of cells; a similar result was also obtained when a monoclonal antibody acting on DPP4 was tested. We conclude that proteolytic enzymes have a synergistic stimulatory effect on cell migration and that their clustering probably facilitates the proteolytic activation cascades needed to produce maximal degradative activity on cell substrates during the angiogenic process.

  2. Proteolytic Enzymes Clustered in Specialized Plasma-Membrane Domains Drive Endothelial Cells' Migration.

    Science.gov (United States)

    Salamone, Monica; Carfì Pavia, Francesco; Ghersi, Giulio

    2016-01-01

    In vitro cultured endothelial cells forming a continuous monolayer establish stable cell-cell contacts and acquire a "resting" phenotype; on the other hand, when growing in sparse conditions these cells acquire a migratory phenotype and invade the empty area of the culture. Culturing cells in different conditions, we compared expression and clustering of proteolytic enzymes in cells having migratory versus stationary behavior. In order to observe resting and migrating cells in the same microscopic field, a continuous cell monolayer was wounded. Increased expression of proteolytic enzymes was evident in cell membranes of migrating cells especially at sprouting sites and in shed membrane vesicles. Gelatin zymography and western blotting analyses confirmed that in migrating cells, expression of membrane-bound and of vesicle-associated proteolytic enzymes are increased. The enzymes concerned include MMP-2, MMP-9, MT1-MMP, seprase, DPP4 (DiPeptidyl Peptidase 4) and uPA. Shed membrane vesicles were shown to exert degradative activity on ECM components and produce substrates facilitating cell migration. Vesicles shed by migrating cells degraded ECM components at an increased rate; as a result their effect on cell migration was amplified. Inhibiting either Matrix Metallo Proteases (MMPs) or Serine Integral Membrane Peptidases (SIMPs) caused a decrease in the stimulatory effect of vesicles, inhibiting the spontaneous migratory activity of cells; a similar result was also obtained when a monoclonal antibody acting on DPP4 was tested. We conclude that proteolytic enzymes have a synergistic stimulatory effect on cell migration and that their clustering probably facilitates the proteolytic activation cascades needed to produce maximal degradative activity on cell substrates during the angiogenic process.

  3. Proteolytic Enzymes Clustered in Specialized Plasma-Membrane Domains Drive Endothelial Cells’ Migration

    Science.gov (United States)

    Salamone, Monica; Carfì Pavia, Francesco

    2016-01-01

    In vitro cultured endothelial cells forming a continuous monolayer establish stable cell-cell contacts and acquire a “resting” phenotype; on the other hand, when growing in sparse conditions these cells acquire a migratory phenotype and invade the empty area of the culture. Culturing cells in different conditions, we compared expression and clustering of proteolytic enzymes in cells having migratory versus stationary behavior. In order to observe resting and migrating cells in the same microscopic field, a continuous cell monolayer was wounded. Increased expression of proteolytic enzymes was evident in cell membranes of migrating cells especially at sprouting sites and in shed membrane vesicles. Gelatin zymography and western blotting analyses confirmed that in migrating cells, expression of membrane-bound and of vesicle-associated proteolytic enzymes are increased. The enzymes concerned include MMP-2, MMP-9, MT1-MMP, seprase, DPP4 (DiPeptidyl Peptidase 4) and uPA. Shed membrane vesicles were shown to exert degradative activity on ECM components and produce substrates facilitating cell migration. Vesicles shed by migrating cells degraded ECM components at an increased rate; as a result their effect on cell migration was amplified. Inhibiting either Matrix Metallo Proteases (MMPs) or Serine Integral Membrane Peptidases (SIMPs) caused a decrease in the stimulatory effect of vesicles, inhibiting the spontaneous migratory activity of cells; a similar result was also obtained when a monoclonal antibody acting on DPP4 was tested. We conclude that proteolytic enzymes have a synergistic stimulatory effect on cell migration and that their clustering probably facilitates the proteolytic activation cascades needed to produce maximal degradative activity on cell substrates during the angiogenic process. PMID:27152413

  4. Modeling and inferring cleavage patterns in proliferating epithelia.

    Directory of Open Access Journals (Sweden)

    Ankit B Patel

    2009-06-01

    Full Text Available The regulation of cleavage plane orientation is one of the key mechanisms driving epithelial morphogenesis. Still, many aspects of the relationship between local cleavage patterns and tissue-level properties remain poorly understood. Here we develop a topological model that simulates the dynamics of a 2D proliferating epithelium from generation to generation, enabling the exploration of a wide variety of biologically plausible cleavage patterns. We investigate a spectrum of models that incorporate the spatial impact of neighboring cells and the temporal influence of parent cells on the choice of cleavage plane. Our findings show that cleavage patterns generate "signature" equilibrium distributions of polygonal cell shapes. These signatures enable the inference of local cleavage parameters such as neighbor impact, maternal influence, and division symmetry from global observations of the distribution of cell shape. Applying these insights to the proliferating epithelia of five diverse organisms, we find that strong division symmetry and moderate neighbor/maternal influence are required to reproduce the predominance of hexagonal cells and low variability in cell shape seen empirically. Furthermore, we present two distinct cleavage pattern models, one stochastic and one deterministic, that can reproduce the empirical distribution of cell shapes. Although the proliferating epithelia of the five diverse organisms show a highly conserved cell shape distribution, there are multiple plausible cleavage patterns that can generate this distribution, and experimental evidence suggests that indeed plants and fruitflies use distinct division mechanisms.

  5. Beta-scission of alkoxyl radicals on peptides and proteins can give rise to backbone cleavage and loss of side-chains

    International Nuclear Information System (INIS)

    Headlam, H.A.; Davies, M.J.; Mortimer, A.; Easton, C.J.

    2000-01-01

    Full text: Exposure of proteins to radicals in the presence of O 2 brings about multiple changes including side-chain oxidation, backbone fragmentation, cross-linking, unfolding, changes in hydrophobicity and conformation, altered susceptibility to proteolytic enzymes and formation of new reactive groups (e.g. hydroperoxides and 3,4-dihydroxyphenylalanine). All of these processes can result in loss of structural or enzymatic activity. The mechanisms that give rise to backbone cleavage are only partly understood. Whilst it is known that direct hydrogen atom abstraction at a-carbon sites gives backbone cleavages it has also been proposed that initial attack at side-chain sites might also give rise to backbone cleavage. In this study we have examined whether initial attack at the β- (C-3) position can give rise to α-carbon radicals (and hence backbone cleavage) via the formation, and subsequent β- scission, of C-3 alkoxyl radicals. This process has been observed previously with protected amino acids in organic solvents (J. Chem. Soc. Perkin Trans. 2, 1997, 503-507) but the occurrence of such reactions with proteins in aqueous solution has not been explored. Alkoxyl radicals were generated at the C-3 position of a variety of protected amino acids and small peptides by two methods: metal-ion catalysed decomposition of hydroperoxides formed as a result of γ-radiolysis in the presence of O 2 , and UV photolysis of nitrate esters. In most cases radicals have been detected by EPR spectroscopy using nitroso and nitrone spin traps, which can be assigned by comparison with literature data to α-carbon radicals; in some case assignments were confirmed by the generation of the putative species by other routes. With Ala peptide hydroperoxides and nitrate esters, and MNP as the spin trap, the major radical detected in each case has been assigned to the adduct of an α-carbon radical with partial structure - NH- . CH-C(O) - consistent with the rapid occurrence of the above

  6. Activation of human monocytes by proteolytic fragments of gliadin

    Czech Academy of Sciences Publication Activity Database

    Jelínková, Lenka; Tučková, Ludmila; Cinová, Jana; Cimburek, Zdeněk; Tlaskalová, Helena

    2003-01-01

    Roč. 87, č. 1 (2003), s. 08 ISSN 0165-2478 Institutional research plan: CEZ:AV0Z5020903 Keywords : proteolytic * fragments * gliadin Subject RIV: EE - Microbiology, Virology Impact factor: 1.710, year: 2003

  7. Planar integrated optical waveguide used as a transducer to yield chemical information: detection of the activity of proteolytic enzymes e.g. serine-proteases

    Science.gov (United States)

    Zhylyak, Gleb; Ramoz-Perez, Victor; Linnhoff, Michael; Hug, Thomas; Citterio, Daniel; Spichiger-Keller, Ursula E.

    2005-03-01

    The paper shows the very first results of a feasibility study where the activity of proteolytic enzymes towards dye-labelled artificial substrates immobilized on the surface of planar optical Ta2O5 waveguide was investigated. Within this project, a chromophore label was developed, synthesized and attached to the carboxy-terminus of specific tripeptides. The goal was to develop a highly sensitive optical assay in order to monitor the activity of serine-proteases by cleavage of the amide bond between peptide and chromophore. On the one hand, a strategy was developed to immobilize the labeled tripeptide unto integrated planar waveguides. On the other hand, an instrument, the so-called "chip-reader" was developed to detect the biological process on the surface of the integrated planar optical waveguide. Surface characteristics were analyzed by XPS, TOF-SIMS and contact angle measurements. A comparison between the effectivity of ATR-photometry on chip using TE0 mode and photometry in transmission mode is discussed.

  8. Intermolecular cleavage by UmuD-like mutagenesis proteins

    Science.gov (United States)

    McDonald, John P.; Frank, Ekaterina G.; Levine, Arthur S.; Woodgate, Roger

    1998-01-01

    The activity of a number of proteins is regulated by self-processing reactions. Elegant examples are the cleavage of the prokaryotic LexA and λCI transcriptional repressors and the UmuD-like mutagenesis proteins. Various studies support the hypothesis that LexA and λCI cleavage reactions are predominantly intramolecular in nature. The recently described crystal structure of the Escherichia coli UmuD′ protein (the posttranslational cleavage product of the UmuD protein) suggests, however, that the region of the protein corresponding to the cleavage site is at least 50 Å away from the catalytic active site. We considered the possibility, therefore, that the UmuD-like proteins might undergo self-processing that, in contrast to LexA and λCI, occurs via an intermolecular rather than intramolecular reaction. To test this hypothesis, we introduced into E. coli compatible plasmids with mutations at either the cleavage or the catalytic site of three UmuD-like proteins. Cleavage of these proteins only occurs in the presence of both plasmids, indicating that the reaction is indeed intermolecular in nature. Furthermore, this intermolecular reaction is completely dependent upon the multifunctional RecA protein and leads to the restoration of cellular mutagenesis in nonmutable E. coli strains. Intermolecular cleavage of a biotinylated UmuD active site mutant was also observed in vitro in the presence of the wild-type UmuD′ protein, indicating that in addition to the intact UmuD protein, the normal cleavage product (UmuD′) can also act as a classical enzyme. PMID:9465040

  9. Regioselectivity in the Reductive Bond Cleavage of Diarylalkylsulfonium Salts

    DEFF Research Database (Denmark)

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

    2009-01-01

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

  10. Secondary isotope effects on alpha-cleavage reactions

    International Nuclear Information System (INIS)

    Ingemann, S.; Hammerum, S.

    1980-01-01

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

  11. Optical Imaging of Matrix Metalloproteinase-7 Activity in Vivo Using a Proteolytic Nanobeacon

    Directory of Open Access Journals (Sweden)

    Randy L. Scherer

    2008-05-01

    Full Text Available Matrix metalloproteinases (MMPs are extracellular proteolytic enzymes involved in tumor progression. We present the in vivo detection and quantitation of MMP7 activity using a specific near-infrared polymer-based proteolytic beacon, PB-M7NIR. PB-M7NIR is a pegylated polyamidoamine PAMAM-Generation 4 dendrimer core covalently coupled to a Cy5.5-labeled peptide representing a selective substrate that monitors MMP7 activity (sensor and AF750 as an internal reference to monitor relative substrate concentration (reference. In vivo imaging of tumors expressing MMP7 had a median sensor to reference ratio 2.2-fold higher than a that of a bilateral control tumor. Ex vivo imaging of intestines of multiple intestinal neoplasia (APCMin mice injected systemically with PB-M7NIR revealed a sixfold increase in the sensor to reference ratio in the adenomas of APCMin mice compared with control intestinal tissue or adenomas from MMP7-null Min mice. PB-M7NIR detected tumor sizes as small as 0.01 cm2, and the sensor to reference ratio was independent of tumor size. Histologic sectioning of xenograft tumors localized the proteolytic signal to the extracellular matrix; MMP7-overexpressing tumors displayed an approximately 300-fold enhancement in the sensor to reference ratio compared with nonexpressing tumor cells. In APCMin adenomas, the proteolytic signal colocalized with the endogenously expressed MMP7 protein, with sensor to reference ratios approximately sixfold greater than that of normal intestinal epithelium. PB-M7NIR provides a useful reagent for the in vivo and ex vivo quantitation and localization of MMP-selective proteolytic activity.

  12. Characterization of gel-separated glycoproteins using two-step proteolytic digestion combined with sequential microcolumns and mass spectrometry

    DEFF Research Database (Denmark)

    Larsen, Martin Røssel; Højrup, Peter; Roepstorff, Peter

    2005-01-01

    present a new technique that allows characterization of low amounts of glycoproteins separated by gel electrophoresis. The method takes advantage of sequential specific and nonspecific enzymatic treatment followed by selective purification and characterization of the glycopeptides using graphite powder......Protein glycosylation can be vital for changing the function or physiochemical properties of a protein. Abnormal glycosylation can lead to protein malfunction, resulting in severe diseases. Therefore, it is important to develop techniques for characterization of such modifications in proteins...... at a sensitivity level comparable with state-of-the-art proteomics. Whereas techniques exist for characterization of high abundance glycoproteins, no single method is presently capable of providing information on both site occupancy and glycan structure on a single band excised from an electrophoretic gel. We...

  13. Macrophage-mediated proteolytic remodeling of the extracellular matrix in atherosclerosis results in neoepitopes

    DEFF Research Database (Denmark)

    Skjøt-Arkil, Helene; Barascuk, Natasha; Register, Thomas

    2010-01-01

    in almost all stages of atherosclerosis, by both initiating atherosclerotic plaques and degrading them through the secretion of proteolytic enzymes leading to rupture. This review summarizes the literature on the role of macrophages and their proteolytic activity on proteins in the extracellular matrix (ECM......) of the atherosclerotic plaque with a view to suggest a novel approach for identification of vulnerable plaques and turnover by the use of a new type of biomarker. The PubMed database was searched using the terms macrophages, foam cells, atherosclerosis, CVD, ECM remodeling, biomarker, neoepitope, matrix...... of the constituents of the ECM of the atherosclerotic plaque. At present it is not clear which proteases play pivotal roles at distinct stages of pathogenesis, rather that the combined proteolytic potential with some proteases at early stages and other at later stages may result in plaque rupture. This macrophage...

  14. Dual regulatory switch confers tighter control on HtrA2 proteolytic activity.

    Science.gov (United States)

    Singh, Nitu; D'Souza, Areetha; Cholleti, Anuradha; Sastry, G Madhavi; Bose, Kakoli

    2014-05-01

    High-temperature requirement protease A2 (HtrA2), a multitasking serine protease that is involved in critical biological functions and pathogenicity, such as apoptosis and cancer, is a potent therapeutic target. It is established that the C-terminal post-synaptic density protein, Drosophila disc large tumor suppressor, zonula occludens-1 protein (PDZ) domain of HtrA2 plays pivotal role in allosteric modulation, substrate binding and activation, as commonly reported in other members of this family. Interestingly, HtrA2 exhibits an additional level of functional modulation through its unique N-terminus, as is evident from 'inhibitor of apoptosis proteins' binding and cleavage. This phenomenon emphasizes multiple activation mechanisms, which so far remain elusive. Using conformational dynamics, binding kinetics and enzymology studies, we addressed this complex behavior with respect to defining its global mode of regulation and activity. Our findings distinctly demonstrate a novel N-terminal ligand-mediated triggering of an allosteric switch essential for transforming HtrA2 to a proteolytically competent state in a PDZ-independent yet synergistic activation process. Dynamic analyses suggested that it occurs through a series of coordinated structural reorganizations at distal regulatory loops (L3, LD, L1), leading to a population shift towards the relaxed conformer. This precise synergistic coordination among different domains might be physiologically relevant to enable tighter control upon HtrA2 activation for fostering its diverse cellular functions. Understanding this complex rheostatic dual switch mechanism offers an opportunity for targeting various disease conditions with tailored site-specific effector molecules. © 2014 FEBS.

  15. Urinary Proteolytic Activation of Renal Epithelial Na+ Channels in Chronic Heart Failure.

    Science.gov (United States)

    Zheng, Hong; Liu, Xuefei; Sharma, Neeru M; Li, Yulong; Pliquett, Rainer U; Patel, Kaushik P

    2016-01-01

    One of the key mechanisms involved in renal Na(+) retention in chronic heart failure (CHF) is activation of epithelial Na(+) channels (ENaC) in collecting tubules. Proteolytic cleavage has an important role in activating ENaC. We hypothesized that enhanced levels of proteases in renal tubular fluid activate ENaC, resulting in renal Na(+) retention in rats with CHF. CHF was produced by left coronary artery ligation in rats. By immunoblotting, we found that several urinary serine proteases were significantly increased in CHF rats compared with sham rats (fold increases: furin 6.7, prostasin 23.6, plasminogen 2.06, and plasmin 3.57 versus sham). Similar increases were observed in urinary samples from patients with CHF. Whole-cell patch clamp was conducted in cultured renal collecting duct M-1 cells to record Na(+) currents. Protease-rich urine (from rats and patients with CHF) significantly increased the Na(+) inward current in M-1 cells. Two weeks of protease inhibitor treatment significantly abrogated the enhanced diuretic and natriuretic responses to ENaC inhibitor benzamil in rats with CHF. Increased podocyte lesions were observed in the kidneys of rats with CHF by transmission electron microscopy. Consistent with these results, podocyte damage markers desmin and podocin expressions were also increased in rats with CHF (increased ≈2-folds). These findings suggest that podocyte damage may lead to increased proteases in the tubular fluid, which in turn contributes to the enhanced renal ENaC activity, providing a novel mechanistic insight for Na(+) retention commonly observed in CHF. © 2015 American Heart Association, Inc.

  16. Urinary proteolytic activation of renal epithelial Na+ channels in chronic heart failure

    Science.gov (United States)

    Zheng, Hong; Liu, Xuefei; Sharma, Neeru M.; Li, Yulong; Pliquett, Rainer U; Patel, Kaushik P.

    2015-01-01

    One of the key mechanisms involved in renal Na+ retention in chronic heart failure (CHF) is activation of epithelial Na+ channels (ENaC) in collecting tubules. Proteolytic cleavage has an important role in activating ENaC. We hypothesized that enhanced levels of proteases in renal tubular fluid activate ENaC resulting in renal Na+ retention in rats with CHF. CHF was produced by left coronary artery ligation in rats. By immunoblotting, we found that several urinary serine proteases were significantly increased in CHF rats compared to sham rats (fold increases: furin 6.7, prostasin 23.6, plasminogen 2.06 and plasmin 3.57 vs. sham). Similar increases were observed in urinary samples from patients with CHF. Whole-cell patch-clamp was conducted in cultured renal collecting duct M-1 cells to record Na+ currents. Protease-rich urine (from rats and patients with CHF) significantly increased the Na+ inward current in M-1 cells. Two weeks of protease inhibitor treatment significantly abrogated the enhanced diuretic and natriuretic responses to ENaC inhibitor benzamil in rats with CHF. Increased podocyte lesions were observed in the kidneys of rats with CHF by transmission electron microscopy. Consistent with these results, podocyte damage markers desmin and podocin expressions were also increased in rats with CHF (increased ~2 folds). These findings suggest that podocyte damage may lead to increased proteases in the tubular fluid which in turn contributes to the enhanced renal ENaC activity, providing a novel mechanistic insight for Na+ retention commonly observed in CHF. PMID:26628676

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

    Science.gov (United States)

    Meluzzi, Dario; Arya, Gaurav

    2016-01-08

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

  18. Factors affecting proteolytic action of Lactococcus lactis in cheese

    NARCIS (Netherlands)

    Youssef, Y.B.

    1992-01-01

    Model cheeses were developed to study the behaviour of proteolytic agents involved in cheese maturation under conditions that closely resemble those in normal cheese. The models were applied to study protein breakdown by Lactococcus lactis ssp. cremoris HP , as a

  19. Dataset of cocoa aspartic protease cleavage sites

    Directory of Open Access Journals (Sweden)

    Katharina Janek

    2016-09-01

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

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

    International Nuclear Information System (INIS)

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

    2012-01-01

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

  1. Glucocorticoids activate the ATP-ubiquitin-dependent proteolytic system in skeletal muscle during fasting

    Science.gov (United States)

    Wing, S. S.; Goldberg, A. L.; Goldberger, A. L. (Principal Investigator)

    1993-01-01

    Glucocorticoids are essential for the increase in protein breakdown in skeletal muscle normally seen during fasting. To determine which proteolytic pathway(s) are activated upon fasting, leg muscles from fed and fasted normal rats were incubated under conditions that block or activate different proteolytic systems. After food deprivation (1 day), the nonlysosomal ATP-dependent process increased by 250%, as shown in experiments involving depletion of muscle ATP. Also, the maximal capacity of the lysosomal process increased 60-100%, but no changes occurred in the Ca(2+)-dependent or the residual energy-independent proteolytic processes. In muscles from fasted normal and adrenalectomized (ADX) rats, the protein breakdown sensitive to inhibitors of the lysosomal or Ca(2+)-dependent pathways did not differ. However, the ATP-dependent process was 30% slower in muscles from fasted ADX rats. Administering dexamethasone to these animals or incubating their muscles with dexamethasone reversed this defect. During fasting, when the ATP-dependent process rises, muscles show a two- to threefold increase in levels of ubiquitin (Ub) mRNA. However, muscles of ADX animals failed to show this response. Injecting dexamethasone into the fasted ADX animals increased muscle Ub mRNA within 6 h. Thus glucocorticoids activate the ATP-Ub-dependent proteolytic pathway in fasting apparently by enhancing the expression of components of this system such as Ub.

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

    Directory of Open Access Journals (Sweden)

    Hyung Chul Lee

    Full Text Available This study was undertaken to elucidate detailed event of early embryogenesis in chicken embryos using a noninvasive egg retrieval technique before oviposition. White Leghorn intrauterine eggs were retrieved from 95 cyclic hens aged up to 54-56 weeks and morphogenetic observation was made under both bright field and fluorescent image in a time course manner. Differing from mammals, asymmetric cleavage to yield preblastodermal cells was observed throughout early embryogenesis. The first two divisions occurred synchronously and four polarized preblastodermal cells resulted after cruciform cleavage. Then, asynchronous cleavage continued in a radial manner and overall cell size in the initial cleavage region was smaller than that in the distal area. Numerous sperms were visible, regardless of zygotic nuclei formation. Condensed sperm heads were present mainly in the perivitelline space and cytoplasm, and rarely in the yolk region, while decondensed sperm heads were only visible in the yolk. In conclusion, apparent differences in sperm dynamics and early cleavage events compared with mammalian embryos were detected in chick embryo development, which demonstrated polarized cleavage with penetrating supernumerary sperm into multiple regions.

  3. Inhibition of proteolytic ferments by taurin in radiation sickness

    International Nuclear Information System (INIS)

    Dokshina, G.A.; Klimova, A.D.; Yartsev, E.I.; Yakovlev, V.G.

    1975-01-01

    The application of taurin potassium phosphate (TKPh) as radioprotective remedy resulted in a considerable influence on the activity of the proteolytic ferments in the irradiated organism. White male rats with a weight of 160 to 180 g being kept in cages without any food for 12 hours before this experiment were irradiated with a gamma unit GUM-Co-60 with a dose of 700 rad (LDsub(70/30)). A 4% solution of the preparation was injected in an amount of 40 mg per rat 1, 3, 5, 7 days after irradiation. Under the effect of ionizing radiation there was a progredient increase of proteolytic ferment activity of liver and spleen which was detected already 30 min after irradiation with maximum rate of proteolysis on the 21st day. After injection of the preparation a two-phase reaction developed: on the 7th to 12th day an increased activity of cathepsins in the tissue and in the following time up to the 30th day of observation an inhibition of ferment activity was demonstrated. Simultaneously it was found that the radiation-induced corticosteroid level was prevented by the preparation. A similar effect was also shown by TKPh inhibiting proteolytic ferment activity in experiments with rats with preceding application of hydrocortisone in high doses. The obtained results permit the assumption that the radioprotective activity of taurin comes about by its effect in the direction of structural integrity of the cell membranes leading to a normalization of the hormonal and fermentative events

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

    Science.gov (United States)

    Heo, Jinsol; Kim, Se Hyeuk

    2013-01-01

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

  5. An association between RBMX, a heterogeneous nuclear ribonucleoprotein, and ARTS-1 regulates extracellular TNFR1 release

    International Nuclear Information System (INIS)

    Adamik, Barbara; Islam, Aminul; Rouhani, Farshid N.; Hawari, Feras I.; Zhang Jing; Levine, Stewart J.

    2008-01-01

    The type I, 55-kDa tumor necrosis factor receptor (TNFR1) is released to the extracellular space by two mechanisms, the constitutive release of TNFR1 exosome-like vesicles and the inducible proteolytic cleavage of TNFR1 ectodomains. Both pathways appear to be regulated by an interaction between TNFR1 and ARTS-1 (aminopeptidase regulator of TNFR1 shedding). Here, we sought to identify ARTS-1-interacting proteins that modulate TNFR1 release. Co-immunoprecipitation identified an association between ARTS-1 and RBMX (RNA-binding motif gene, X chromosome), a 43-kDa heterogeneous nuclear ribonucleoprotein. RNA interference attenuated RBMX expression, which reduced both the constitutive release of TNFR1 exosome-like vesicles and the IL-1β-mediated inducible proteolytic cleavage of soluble TNFR1 ectodomains. Reciprocally, over-expression of RBMX increased TNFR1 exosome-like vesicle release and the IL-1β-mediated inducible shedding of TNFR1 ectodomains. This identifies RBMX as an ARTS-1-associated protein that regulates both the constitutive release of TNFR1 exosome-like vesicles and the inducible proteolytic cleavage of TNFR1 ectodomains

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

    Directory of Open Access Journals (Sweden)

    Robert J Lake

    2009-08-01

    Full Text Available A ligand-independent cleavage (S1 in the extracellular domain of the mammalian Notch receptor results in what is considered to be the canonical heterodimeric form of Notch on the cell surface. The in vivo consequences and significance of this cleavage on Drosophila Notch signaling remain unclear and contradictory. We determined the cleavage site in Drosophila and examined its in vivo function by a transgenic analysis of receptors that cannot be cleaved. Our results demonstrate a correlation between loss of cleavage and loss of in vivo function of the Notch receptor, supporting the notion that S1 cleavage is an in vivo mechanism of Notch signal control.

  7. Genetics of the proteolytic system of lactic acid bacteria

    NARCIS (Netherlands)

    Kok, Jan

    1990-01-01

    The proteolytic system of lactic acid bacteria is of eminent importance for the rapid growth of these organisms in protein-rich media. The combined action of proteinases and peptidases provides the cell with small peptides and essential amino acids. The amino acids and peptides thus liberated have

  8. In vitro measurement of beta-carotene cleavage activity : methodological considerations and the effect of other carotenoids on beta-carotene cleavage

    NARCIS (Netherlands)

    Vliet, T. van; Schaik, F. van; Schreurs, W.H.P.; Berg, H. van den

    1996-01-01

    In view of controversies about assessment of the β-carotene cleavage activity, methodological aspects and problems of the dioxygenase assay are described. Using rat and hamster intestinal preparations the method was optimized on retinal formation, the only cleavage product we could demonstrate. It

  9. FAP finds FGF21 easy to digest

    DEFF Research Database (Denmark)

    Gillum, Matthew P; Potthoff, Matthew J

    2016-01-01

    Fibroblast growth factor 21 (FGF21) is an endocrine hormone that regulates carbohydrate and lipid metabolism. In humans, circulating FGF21 is inactivated by proteolytic cleavage of its C-terminus, thereby preventing signalling through a receptor complex. The mechanism for this cleavage event and ...

  10. Neutrophil elastase processing of Gelatinase A is mediated by extracellular matrix

    Energy Technology Data Exchange (ETDEWEB)

    Rice, A.; Banda, M.J. [Univ. of California, San Franciso, CA (United States)

    1995-07-18

    Gelatinase A (72-kDa type IV collagenase) is a metalloproteinase that is expressed by many cells in culture and is overexpressed by some tumor cells. It has been suggested that the serine proteinase neutrophil elastase might play a role iii the posttranslational processing of gelatinase A and that noncatalytic interactions between gelatinase A and components of the extracellular matrix might alter potential processing pathways. These questions were addressed with the use of gelatin substrate zymography, gelatinolytic activity assays, and amino acid sequence analysis. We found that neutrophil elastase does proteolytically modify gelatinase A by cleaving at a number of sites within gelatinase A. Sequential treatment of gelatinase A with 4-aminophenylmercuric acetate (APMA) and neutrophil elastase yielded an active gelatinase with a 4-fold increase in gelatinolytic activity. The increased gelatinolytic activity correlated with that of a 40-kDa fragment of gelatinase A. Matrix components altered the proteolytic modifications in gelatinase A that were mediated by neutrophil elastase. In the absence of gelatin, neutrophil elastase destructively degraded gelatinase A by hydrolyzing at least two bonds within the fibronectin-like gelatin-binding domain of gelatinase A. In the presence of gelatin, these two inactivating cleavage sites were protected, and cleavage at a site within the hemopexin-like carboxyl-terminal domain resulted in a truncated yet active gelatinase. The results suggest a regulatory role for extracellular matrix molecules in stabilizing gelatinase A fragments and in altering the availability of sites susceptible to destructive proteolysis by neutrophil elastase. 32 refs., 10 figs.

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

    Directory of Open Access Journals (Sweden)

    Salmi Jussi

    2010-06-01

    Full Text Available Abstract Background Caspases are a family of proteases that have central functions in programmed cell death (apoptosis and inflammation. Caspases mediate their effects through aspartate-specific cleavage of their target proteins, and at present almost 400 caspase substrates are known. There are several methods developed to predict caspase cleavage sites from individual proteins, but currently none of them can be used to predict caspase cleavage sites from multiple proteins or entire proteomes, or to use several classifiers in combination. The possibility to create a database from predicted caspase cleavage products for the whole genome could significantly aid in identifying novel caspase targets from tandem mass spectrometry based proteomic experiments. Results Three different pattern recognition classifiers were developed for predicting caspase cleavage sites from protein sequences. Evaluation of the classifiers with quality measures indicated that all of the three classifiers performed well in predicting caspase cleavage sites, and when combining different classifiers the accuracy increased further. A new tool, Pripper, was developed to utilize the classifiers and predict the caspase cut sites from an arbitrary number of input sequences. A database was constructed with the developed tool, and it was used to identify caspase target proteins from tandem mass spectrometry data from two different proteomic experiments. Both known caspase cleavage products as well as novel cleavage products were identified using the database demonstrating the usefulness of the tool. Pripper is not restricted to predicting only caspase cut sites, but it gives the possibility to scan protein sequences for any given motif(s and predict cut sites once a suitable cut site prediction model for any other protease has been developed. Pripper is freely available and can be downloaded from http://users.utu.fi/mijopi/Pripper. Conclusions We have developed Pripper, a tool for

  12. [Inhibitors of proteolytic enzymes under abiotic stresses in plants (review)].

    Science.gov (United States)

    Mosolov, V V; Valueva, T A

    2011-01-01

    Data on the role of proteolytic enzyme inhibitors in plant adaptation to various unfavorable environmental abiotic factors--water deficiency, salinization of soil, extreme temperatures, etc.--and also probable functions of proteinases inhibitors in natural plant senescense are considered.

  13. Selective condensation drives partitioning and sequential secretion of cyst wall proteins in differentiating Giardia lamblia.

    Directory of Open Access Journals (Sweden)

    Christian Konrad

    2010-04-01

    Full Text Available Controlled secretion of a protective extracellular matrix is required for transmission of the infective stage of a large number of protozoan and metazoan parasites. Differentiating trophozoites of the highly minimized protozoan parasite Giardia lamblia secrete the proteinaceous portion of the cyst wall material (CWM consisting of three paralogous cyst wall proteins (CWP1-3 via organelles termed encystation-specific vesicles (ESVs. Phylogenetic and molecular data indicate that Diplomonads have lost a classical Golgi during reductive evolution. However, neogenesis of ESVs in encysting Giardia trophozoites transiently provides basic Golgi functions by accumulating presorted CWM exported from the ER for maturation. Based on this "minimal Golgi" hypothesis we predicted maturation of ESVs to a trans Golgi-like stage, which would manifest as a sorting event before regulated secretion of the CWM. Here we show that proteolytic processing of pro-CWP2 in maturing ESVs coincides with partitioning of CWM into two fractions, which are sorted and secreted sequentially with different kinetics. This novel sorting function leads to rapid assembly of a structurally defined outer cyst wall, followed by slow secretion of the remaining components. Using live cell microscopy we find direct evidence for condensed core formation in maturing ESVs. Core formation suggests that a mechanism controlled by phase transitions of the CWM from fluid to condensed and back likely drives CWM partitioning and makes sorting and sequential secretion possible. Blocking of CWP2 processing by a protease inhibitor leads to mis-sorting of a CWP2 reporter. Nevertheless, partitioning and sequential secretion of two portions of the CWM are unaffected in these cells. Although these cysts have a normal appearance they are not water resistant and therefore not infective. Our findings suggest that sequential assembly is a basic architectural principle of protective wall formation and requires

  14. Quantitative characterization of cleavage and hydrogen-assisted quasi-cleavage fracture surfaces with the use of confocal laser scanning microscopy

    International Nuclear Information System (INIS)

    Merson, E.; Kudrya, A.V.; Trachenko, V.A.; Merson, D.; Danilov, V.; Vinogradov, A.

    2016-01-01

    “True” cleavage (TC) and quasi-cleavage (QC) fracture surfaces of low-carbon steel specimens tested in liquid nitrogen and after hydrogen charging respectively were investigated by quantitative confocal laser scanning microscopy (CLSM) and conventional scanning electron microscopy (SEM) with electron-backscattered diffraction (EBSD). Topological and crystallographic features of the TC fracture surface are found in good agreement with the generally accepted cleavage mechanism: TC facets diameters correspond to those of grains; the crack path strictly follows the crystallographic orientation of grains and the most of the cleavage cracks are parallel to {100} planes. On the 2D SEM images, the QC facets appeared resembling the TC ones in terms of river line patterns, shapes and sizes. However, the substantial differences between the topography of these two kinds of fracture surfaces were revealed by 3D CLSM: the average misorientation angle between QC facets and the roughness of the QC fracture surface were much lower than those measured for TC. It is demonstrated that all these features are attributed to the specific fracture mechanism operating during hydrogen-assisted cracking.

  15. Quantitative characterization of cleavage and hydrogen-assisted quasi-cleavage fracture surfaces with the use of confocal laser scanning microscopy

    Energy Technology Data Exchange (ETDEWEB)

    Merson, E. [Institute of Advanced Technologies, Togliatti State University, 445667 (Russian Federation); Kudrya, A.V.; Trachenko, V.A. [Department of Physical Metallurgy and the Physics of Strength, NUST MISiS, Moscow 119490 (Russian Federation); Merson, D. [Institute of Advanced Technologies, Togliatti State University, 445667 (Russian Federation); Laboratory for Advanced Materials, Kazan Federal University, Naberezhnye Chelny 423812, Republic of Tatarstan (Russian Federation); Danilov, V. [Institute of Advanced Technologies, Togliatti State University, 445667 (Russian Federation); Vinogradov, A. [Institute of Advanced Technologies, Togliatti State University, 445667 (Russian Federation); Department of Engineering Design and Materials, Norwegian University of Science and Technology – NTNU, N-7491 Trondheim (Norway)

    2016-05-17

    “True” cleavage (TC) and quasi-cleavage (QC) fracture surfaces of low-carbon steel specimens tested in liquid nitrogen and after hydrogen charging respectively were investigated by quantitative confocal laser scanning microscopy (CLSM) and conventional scanning electron microscopy (SEM) with electron-backscattered diffraction (EBSD). Topological and crystallographic features of the TC fracture surface are found in good agreement with the generally accepted cleavage mechanism: TC facets diameters correspond to those of grains; the crack path strictly follows the crystallographic orientation of grains and the most of the cleavage cracks are parallel to {100} planes. On the 2D SEM images, the QC facets appeared resembling the TC ones in terms of river line patterns, shapes and sizes. However, the substantial differences between the topography of these two kinds of fracture surfaces were revealed by 3D CLSM: the average misorientation angle between QC facets and the roughness of the QC fracture surface were much lower than those measured for TC. It is demonstrated that all these features are attributed to the specific fracture mechanism operating during hydrogen-assisted cracking.

  16. Effects of Sunphenon and Polyphenon 60 on proteolytic pathways ...

    Indian Academy of Sciences (India)

    2016-08-26

    Aug 26, 2016 ... The effect of Sunphenon and Polyphenon 60 in oxidative stress response, myogenic regulatory factors, inflammatory cytokines, apoptotic and proteolytic pathways on H2O2-induced myotube atrophy was addressed. Cellular responses of H2O2-induced C2C12cells were examined, including mRNA ...

  17. 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.

  18. Functional analysis of coordinated cleavage in V(D)J recombination.

    Science.gov (United States)

    Kim, D R; Oettinger, M A

    1998-08-01

    V(D)J recombination in vivo requires a pair of signals with distinct spacer elements of 12 and 23 bp that separate conserved heptamer and nonamer motifs. Cleavage in vitro by the RAG1 and RAG2 proteins can occur at individual signals when the reaction buffer contains Mn2+, but cleavage is restricted to substrates containing two signals when Mg2+ is the divalent cation. By using a novel V(D)J cleavage substrate, we show that while the RAG proteins alone establish a moderate preference for a 12/23 pair versus a 12/12 pair, a much stricter dependence of cleavage on the 12/23 signal pair is produced by the inclusion of HMG1 and competitor double-stranded DNA. The competitor DNA serves to inhibit the cleavage of substrates carrying a 12/12 or 23/23 pair, as well as the cutting at individual signals in 12/23 substrates. We show that a 23/33 pair is more efficiently recombined than a 12/33 pair, suggesting that the 12/23 rule can be generalized to a requirement for spacers that differ from each other by a single helical turn. Furthermore, we suggest that a fixed spatial orientation of signals is required for cleavage. In general, the same signal variants that can be cleaved singly can function under conditions in which a signal pair is required. However, a chemically modified substrate with one noncleavable signal enables us to show that formation of a functional cleavage complex is mechanistically separable from the cleavage reaction itself and that although cleavage requires a pair of signals, cutting does not have to occur simultaneously at both. The implications of these results are discussed with respect to the mechanism of V(D)J recombination and the generation of chromosomal translocations.

  19. Independent evolution of neurotoxin and flagellar genetic loci in proteolytic Clostridium botulinum.

    Science.gov (United States)

    Carter, Andrew T; Paul, Catherine J; Mason, David R; Twine, Susan M; Alston, Mark J; Logan, Susan M; Austin, John W; Peck, Michael W

    2009-03-19

    Proteolytic Clostridium botulinum is the causative agent of botulism, a severe neuroparalytic illness. Given the severity of botulism, surprisingly little is known of the population structure, biology, phylogeny or evolution of C. botulinum. The recent determination of the genome sequence of C. botulinum has allowed comparative genomic indexing using a DNA microarray. Whole genome microarray analysis revealed that 63% of the coding sequences (CDSs) present in reference strain ATCC 3502 were common to all 61 widely-representative strains of proteolytic C. botulinum and the closely related C. sporogenes tested. This indicates a relatively stable genome. There was, however, evidence for recombination and genetic exchange, in particular within the neurotoxin gene and cluster (including transfer of neurotoxin genes to C. sporogenes), and the flagellar glycosylation island (FGI). These two loci appear to have evolved independently from each other, and from the remainder of the genetic complement. A number of strains were atypical; for example, while 10 out of 14 strains that formed type A1 toxin gave almost identical profiles in whole genome, neurotoxin cluster and FGI analyses, the other four strains showed divergent properties. Furthermore, a new neurotoxin sub-type (A5) has been discovered in strains from heroin-associated wound botulism cases. For the first time, differences in glycosylation profiles of the flagella could be linked to differences in the gene content of the FGI. Proteolytic C. botulinum has a stable genome backbone containing specific regions of genetic heterogeneity. These include the neurotoxin gene cluster and the FGI, each having evolved independently of each other and the remainder of the genetic complement. Analysis of these genetic components provides a high degree of discrimination of strains of proteolytic C. botulinum, and is suitable for clinical and forensic investigations of botulism outbreaks.

  20. Independent evolution of neurotoxin and flagellar genetic loci in proteolytic Clostridium botulinum

    Directory of Open Access Journals (Sweden)

    Twine Susan M

    2009-03-01

    Full Text Available Abstract Background Proteolytic Clostridium botulinum is the causative agent of botulism, a severe neuroparalytic illness. Given the severity of botulism, surprisingly little is known of the population structure, biology, phylogeny or evolution of C. botulinum. The recent determination of the genome sequence of C. botulinum has allowed comparative genomic indexing using a DNA microarray. Results Whole genome microarray analysis revealed that 63% of the coding sequences (CDSs present in reference strain ATCC 3502 were common to all 61 widely-representative strains of proteolytic C. botulinum and the closely related C. sporogenes tested. This indicates a relatively stable genome. There was, however, evidence for recombination and genetic exchange, in particular within the neurotoxin gene and cluster (including transfer of neurotoxin genes to C. sporogenes, and the flagellar glycosylation island (FGI. These two loci appear to have evolved independently from each other, and from the remainder of the genetic complement. A number of strains were atypical; for example, while 10 out of 14 strains that formed type A1 toxin gave almost identical profiles in whole genome, neurotoxin cluster and FGI analyses, the other four strains showed divergent properties. Furthermore, a new neurotoxin sub-type (A5 has been discovered in strains from heroin-associated wound botulism cases. For the first time, differences in glycosylation profiles of the flagella could be linked to differences in the gene content of the FGI. Conclusion Proteolytic C. botulinum has a stable genome backbone containing specific regions of genetic heterogeneity. These include the neurotoxin gene cluster and the FGI, each having evolved independently of each other and the remainder of the genetic complement. Analysis of these genetic components provides a high degree of discrimination of strains of proteolytic C. botulinum, and is suitable for clinical and forensic investigations of botulism

  1. Proteolytic and lipolytic microbiota of refrigerated raw milk from northeast and southern regions of Brazil

    Directory of Open Access Journals (Sweden)

    Jose Carlos Ribeiro Junior

    2015-12-01

    Full Text Available The shelf life of milk and milk derivatives is directly related to the microbiological quality of refrigerated raw milk. Spoilage microorganisms with proteolytic and/or lipolytic properties are primarily responsible for the decrease in the quality of milk, which is reflected in the shelf life of pasteurized milk and all derivatives. The aim of this study was to determine the spoilage microbial load of refrigerated raw milk from the northeast and southern regions of Brazil, which have different climatic and technological conditions of production. We evaluated 46 samples of milk from the state of Paraná in the southern region, and 10 samples of milk from the state of Maranhão in the northeast region, totaling 56 samples collected from November 2013 to November 2014. The producers of Paraná were divided into large (20 or small (26 according to the average daily production. All producers of Maranhão were considered small (<500L/day. The proteolytic and lipolytic microorganism counts were conducted in milk agar and tributyrin agar, respectively. Milk from the large producers of Paraná had average counts of 1.4 × 104 CFU/mL for proteolytic microorganisms and 1.2 × 103 CFU/mL for lipolytics microorganisms, significantly (p <0.05 lower than the small producers in the same state, and the producers of Maranhão. Producers of Maranhao had counts of 1.1 × 105 CFU/mL for proteolytic microorganisms and 2 × 105 CFU/mL for lipolytic microorganisms, with the proteolytic count significantly lower than that of small Paraná producers. The amount of proteolytic and lipolytic spoilage microorganisms in milk is influenced by the adaptation of the microorganisms to cold, promoted by the cooling of milk, which is practiced less frequently in the country’s northeastern region. The amount of spoilage microorganisms is also affected by the implementation of milking hygiene practices, which reduce contamination. Such practices are more frequently and efficiently

  2. Persistence of bacterial proteolytic enzymes in lake ecosystems.

    Science.gov (United States)

    Kiersztyn, Bartosz; Siuda, Waldemar; Chróst, Ryszard J

    2012-04-01

    This study analyzes proteolytic enzyme persistence and the role of dead (or metabolically inactive) aquatic bacteria in organic matter cycling. Samples from four lakes of different trophic status were used. Irrespective of the trophic status of the examined lakes, bacterial aminopeptidases remained active even 72 h after the death of the bacteria that produced them. The total pool of proteolytic enzymes in natural lake water samples was also stable. We found that the rates of amino acid enzymatic release from proteinaceous matter added to preserved lake water sample were constant for at least 96 h (r(2)  = 0.99, n = 17, P ≤ 0.0001, V(max)  = 84.6 nM h(-1) ). We also observed that proteases built into bacterial cell debris fragments remained active for a long time, even after the total destruction of cells. Moreover, during 24 h of incubation time, about 20% of these enzymatically active fragments adsorbed onto natural seston particles, becoming a part of the 'attached enzymes system' that is regarded as the 'hot-spot' of protein degradation in aquatic ecosystems. © 2011 Federation of European Microbiological Societies. Published by Blackwell Publishing Ltd. All rights reserved.

  3. Cleavage sites within the poliovirus capsid protein precursors

    International Nuclear Information System (INIS)

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

    1982-01-01

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

  4. A new cultural cleavage in post-modern society

    Directory of Open Access Journals (Sweden)

    Jan-Erik Lane

    2007-09-01

    Full Text Available The attitudes towards gender and homosexuality tend to be linked at the micro level (individuals, which explains the political saliency of this newly emerging cleavage. At the macro level (country, the main finding is that the value orientations towards gender and homosexuality are strongly embedded in the basic cultural or civilisation differences among countries. As developing countries modernise and enter post-modernity, they will also experience the gender cleavage, especially when they adhere to an individualistic culture. Cultural cleavages in the post-modern society, whether in rich or developing countries, can only be properly researched by the survey method. It opens up a large area for both micro and macro analyses in the social sciences.

  5. Furin proteolytically processes the heparin-binding region of extracellular superoxide dismutase

    DEFF Research Database (Denmark)

    Bowler, Russell P; Nicks, Mike; Olsen, Dorte Aa

    2002-01-01

    Extracellular superoxide dismutase (EC-SOD) is an antioxidant enzyme that attenuates brain and lung injury from oxidative stress. A polybasic region in the carboxyl terminus distinguishes EC-SOD from other superoxide dismutases and determines EC-SOD's tissue half-life and affinity for heparin....... There are two types of EC-SOD that differ based on the presence or absence of this heparin-binding region. It has recently been shown that proteolytic removal of the heparin-binding region is an intracellular event (Enghild, J. J., Thogersen, I. B., Oury, T. D., Valnickova, Z., Hojrup, P., and Crapo, J. D...... of intracellular proteases implicate furin as a processing protease. In vitro experiments using furin and purified EC-SOD suggest that furin proteolytically cleaves EC-SOD in the middle of the polybasic region and then requires an additional carboxypeptidase to remove the remaining lysines and arginines...

  6. Unexpected tolerance of alpha-cleavage of the prion protein to sequence variations.

    Directory of Open Access Journals (Sweden)

    José B Oliveira-Martins

    Full Text Available The cellular form of the prion protein, PrP(C, undergoes extensive proteolysis at the alpha site (109K [see text]H110. Expression of non-cleavable PrP(C mutants in transgenic mice correlates with neurotoxicity, suggesting that alpha-cleavage is important for PrP(C physiology. To gain insights into the mechanisms of alpha-cleavage, we generated a library of PrP(C mutants with mutations in the region neighbouring the alpha-cleavage site. The prevalence of C1, the carboxy adduct of alpha-cleavage, was determined for each mutant. In cell lines of disparate origin, C1 prevalence was unaffected by variations in charge and hydrophobicity of the region neighbouring the alpha-cleavage site, and by substitutions of the residues in the palindrome that flanks this site. Instead, alpha-cleavage was size-dependently impaired by deletions within the domain 106-119. Almost no cleavage was observed upon full deletion of this domain. These results suggest that alpha-cleavage is executed by an alpha-PrPase whose activity, despite surprisingly limited sequence specificity, is dependent on the size of the central region of PrP(C.

  7. Proteolytic activity of IgGs from blood serum of wistar rats at experimental rheumatoid arthritis

    Directory of Open Access Journals (Sweden)

    Yu. Ya. Kit

    2014-10-01

    Full Text Available The aim of this work was to study the proteolytic activity of IgGs purified from blood serum of Wistar rats at experimental rheumatoid arthritis (ERA induced by an injection of bovine collagen of type II. Twenty rats were immunized with a preparation of bovine collagen II (Sigma-Aldrich, USA in the presence of complete Freund’s adjuvant. ERA development was determined by inflammation in limbs of treated animals. IgG preparations were isolated from blood serum of immunized and non-immunized animals by precipitation of antibodies with 33% ammonium sulfate followed by chromatography on the Protein G-Sepharose column. Human histone H1, bovine collagen II, calf thymus histones, myelin basic protein (MBP, bovine serum albumin (BSA, and bovine casein were used as substrates of the proteolytic activity of IgGs. It was found that IgG preparations from blood serum of rats with ERA were capable of cleaving histone H1 and MBP, however, they were catalytically inactive towards collagen II, casein, BSA, and core histones. IgGs from blood serum of non-immunized rats were proteolytically inactive towards all used protein substrates. Thus, we demonstrated that immunization of rats with bovine collagen II induced IgG-antibodies possessing the proteolytic activity towards histone H1 and MBP. This activity might be associated with the development of inflammatory processes in the immunized rats.

  8. Influence of autoclaved saprotrophic fungal mycelia on proteolytic activity in ectomycorrhizal fungi.

    Science.gov (United States)

    Mucha, Joanna; Dahm, Hanna; Werner, Antoni

    2007-07-01

    The production of proteolytic enzymes by several strains of ectomycorrhizal fungi i.e., Amanita muscaria (16-3), Laccaria laccata (9-12), L. laccata (9-1), Suillus bovinus (15-4), Suillus bovinus (15-3), Suillus luteus (14-7) on mycelia of Trichoderma harzianum, Trichoderma virens and Mucor hiemalis and sodium caseinate, yeast extract was evaluated. The strains of A. muscaria (16-3) and L. laccata (9-12) were characterized by the highest activity of the acidic and neutral proteases. Taking the mycelia of saprotrophic fungi into consideration, the mycelium of M. hiemalis was the best inductor for proteolytic activity. The examined ectomycorrhizal fungi exhibited higher activity of acidic proteases than neutral ones on the mycelia of saprotrophic fungi, which may imply the participation of acidic proteases in nutrition.

  9. Comparative and phylogenetic perspectives of the cleavage process in tailed amphibians.

    Science.gov (United States)

    Desnitskiy, Alexey G; Litvinchuk, Spartak N

    2015-10-01

    The order Caudata includes about 660 species and displays a variety of important developmental traits such as cleavage pattern and egg size. However, the cleavage process of tailed amphibians has never been analyzed within a phylogenetic framework. We use published data on the embryos of 36 species concerning the character of the third cleavage furrow (latitudinal, longitudinal or variable) and the magnitude of synchronous cleavage period (up to 3-4 synchronous cell divisions in the animal hemisphere or a considerably longer series of synchronous divisions followed by midblastula transition). Several species from basal caudate families Cryptobranchidae (Andrias davidianus and Cryptobranchus alleganiensis) and Hynobiidae (Onychodactylus japonicus) as well as several representatives from derived families Plethodontidae (Desmognathus fuscus and Ensatina eschscholtzii) and Proteidae (Necturus maculosus) are characterized by longitudinal furrows of the third cleavage and the loss of synchrony as early as the 8-cell stage. By contrast, many representatives of derived families Ambystomatidae and Salamandridae have latitudinal furrows of the third cleavage and extensive period of synchronous divisions. Our analysis of these ontogenetic characters mapped onto a phylogenetic tree shows that the cleavage pattern of large, yolky eggs with short series of synchronous divisions is an ancestral trait for the tailed amphibians, while the data on the orientation of third cleavage furrows seem to be ambiguous with respect to phylogeny. Nevertheless, the midblastula transition, which is characteristic of the model species Ambystoma mexicanum (Caudata) and Xenopus laevis (Anura), might have evolved convergently in these two amphibian orders.

  10. A Comparative Study of New Aspergillus Strains for Proteolytic Enzymes Production by Solid State Fermentation

    Directory of Open Access Journals (Sweden)

    Gastón Ezequiel Ortiz

    2016-01-01

    Full Text Available A comparative study of the proteolytic enzymes production using twelve Aspergillus strains previously unused for this purpose was performed by solid state fermentation. A semiquantitative and quantitative evaluation of proteolytic activity were carried out using crude enzymatic extracts obtained from the fermentation cultures, finding seven strains with high and intermediate level of protease activity. Biochemical, thermodynamics, and kinetics features such as optimum pH and temperature values, thermal stability, activation energy (Ea, quotient energy (Q10, Km, and Vmax were studied in four enzymatic extracts from the selected strains that showed the highest productivity. Additionally, these strains were evaluated by zymogram analysis obtaining protease profiles with a wide range of molecular weight for each sample. From these four strains with the highest productivity, the proteolytic extract of A. sojae ATCC 20235 was shown to be an appropriate biocatalyst for hydrolysis of casein and gelatin substrates, increasing its antioxidant activities in 35% and 125%, respectively.

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

    Directory of Open Access Journals (Sweden)

    Rémi Baroso

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

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

    Science.gov (United States)

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

    2010-04-02

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

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

    Science.gov (United States)

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

    2010-01-01

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

  14. Detection of nucleic acid sequences by invader-directed cleavage

    Science.gov (United States)

    Brow, Mary Ann D.; Hall, Jeff Steven Grotelueschen; Lyamichev, Victor; Olive, David Michael; Prudent, James Robert

    1999-01-01

    The present invention relates to means for the detection and characterization of nucleic acid sequences, as well as variations in nucleic acid sequences. The present invention also relates to methods for forming a nucleic acid cleavage structure on a target sequence and cleaving the nucleic acid cleavage structure in a site-specific manner. The 5' nuclease activity of a variety of enzymes is used to cleave the target-dependent cleavage structure, thereby indicating the presence of specific nucleic acid sequences or specific variations thereof. The present invention further relates to methods and devices for the separation of nucleic acid molecules based by charge.

  15. Characterisation of a novel proteolytic enzyme localised to goblet cells in rat and man

    DEFF Research Database (Denmark)

    Poulsen, Steen Seier

    1984-01-01

    A proteolytic enzyme, ingobsin , purified from rat duodenal extracts is shown to be localised to intestinal goblet cells of both man and rat. Enzyme positive cells decrease in number from duodenum to colon. The enzyme is a 33 000 Mr protein with an isoelectric point of 5.1. The pH optimum...... for enzymatic activity is 7.4-8.0. Based on substrate specificity for arg-x, lys-x and to a lesser degree tyr-x, on the effect of diisopropylphosphorofluoride , Trasylol and phenylmethylsulfonylfluoride and on proteolytic activity towards intact proteins, ingobsin is classified as a serine proteinase...

  16. Proteolytic activity of recombinant DegP from Chromohalobacter salexigens BKL5

    Directory of Open Access Journals (Sweden)

    Dewi Fitriani

    2017-09-01

    Conclusions: Recombinant DegP from C. salexigens BKL5 showed proteolytic activity when β-casein was used as a substrate. In silico analysis indicated that recombinant DegP had characteristics similar to those of halophilic proteins depending on its amino acid composition.

  17. Proteolytic activity and cooperative hemolytic effect of dermatophytes with different species of bacteria

    Science.gov (United States)

    Pakshir, K; Mohamadi, T; Khodadadi, H; Motamedifar, M; Zomorodian, K; Alipour, S; Motamedi, M

    2016-01-01

    Background and Purpose: Globally, dermatophytes are the most common filamentous group of fungi causing cutaneous mycoses. Dermatophytes were shown to secrete a multitude of enzymes that play a role in their pathogenesis. There is limited data on co-hemolytic (CAMP-like) effect of different bacterial species on dermatophyte species. In this study, we sought to the evaluate exoenzyme activity and co-hemolytic effect of four bacteria on clinical dermatophytes isolated from patients in Shiraz, Iran. Materials and Methods: A total of 84 clinical dermatophyte species were isolated from patients suffering dermatophytosis and identified by conventional methods. Hemolytic activity was evaluated with Columbia 5% sheep blood agar. Proteolytic activity was determined by plate clearance assay method, using gelatin 8% agar. CAMP-like factor was evaluated with four bacteria, namely, S. areus, S. saprophyticus, S. pyogenes, and S. agalactiae. Fisher's exact test was run for statistical analysis. Results: T. mentagrophytes was the most predominant agent (27 [32.1%]) followed by T. verrucosum(20 [23.8%]), T. tonsurans (10 [11.9%]), Microsporum canis (7 [8.3%]), T. rubrum (6 [7.1%]), E. floccosum (6 [7.1%]), M. gypseum (5 [6%]), and T. violaceum (3[3.6%]). The most common clinical area of dermatophytosis was the skin. All the isolates expressed the zone of incomplete alpha hemolysis. All the isolates had CAMP- positive reaction with S. aureus and the other bacteria were CAMP-negative. All the isolates expressed proteolytic activity and no significant differences were noted among diverse genera of dermatophytes and severities of proteolytic activity. Conclusion: This study indicated that hemolysin and proteolytic enzymes potentially play a role in dermatophyte pathogenesis and S. aureus could be considered as a main bacterium for creation of co-hemolytic effect in association with dermatophyte species. PMID:28959790

  18. Physical-chemical and biocatalytic properties of a proteolytic complex of the preparation "Protepsin"

    Directory of Open Access Journals (Sweden)

    L. V. Antipova

    2016-01-01

    Full Text Available Enzymatic technologies were included strongly into practical activities of the person, the volume of the world market constantly grows and is updated. However the domestic production of enzymatic preparations very lags behind world level that is in many respects connected with insufficient scientific and technical base for a wide circulation of technologies in large-scale production. At the same time there were Russian producers of enzymatic preparations from animal fabrics and bodies for processing of raw materials of an animal origin, according to forecasts, of interest in rational use of resources of an animal origin. In article data on research of properties of the enzymatic preparation "Protepsin" and an assessment of prospects of application are provided in processing of raw materials of an animal origin. The enzymatic preparation "Protepsin" made in the conditions of JSC Plant of Endocrine Enzymes (Rzhavki, Moscow region activity at action on proteins of meat shows, including the strengthened structure, has milk-clotting effect, is active in the field of pH 4,0-6,0 and temperature 20-45zs. The proteinaceous complex includes 4 fractions, 2 from which possess the general proteolytic activity. One of them shows the general proteolytic and milk-clotting activity. Enzymes differ in an amino-acid set and molecular weight. The method of a disk electrophoresis determined molecular-mass structure of "Protepsin". The preparation inactivation conditions guaranteeing its safety in the production technology of foodstuff as active proteolytic enzymes in the course of digestion can cause violations of integrity of fabrics and corresponding diseases are shown. Thus, conditions of use of a perspective proteolytic preparation in technology of a wide range of food of an animal origin are in a complex proved and picked up.

  19. Controllable laser thermal cleavage of sapphire wafers

    Science.gov (United States)

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

    2018-03-01

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

  20. The effects of Capn1 gene inactivation on skeletal muscle growth, development, and atrophy, and the compensatory role of other proteolytic systems.

    Science.gov (United States)

    Kemp, C M; Oliver, W T; Wheeler, T L; Chishti, A H; Koohmaraie, M

    2013-07-01

    Myofibrillar protein turnover is a key component of muscle growth and degeneration, requiring proteolytic enzymes to degrade the skeletal muscle proteins. The objective of this study was to investigate the role of the calpain proteolytic system in muscle growth development using μ-calpain knockout (KO) mice in comparison with control wild-type (WT) mice, and evaluate the subsequent effects of silencing this gene on other proteolytic systems. No differences in muscle development between genotypes were observed during the early stages of growth due to the up regulation of other proteolytic systems. The KO mice showed significantly greater m-calpain protein abundance (P proteolytic systems to ensure muscle protein homeostasis in vivo. Furthermore, these data contribute to the existing evidence of the importance of the calpain system's involvement in muscle growth, development, and atrophy. Collectively, these data suggest that there are opportunities to target the calpain system to promote the growth and/or restoration of skeletal muscle mass.

  1. The Impact of the ‘Austrian’ Mutation of the Amyloid Precursor Protein Transmembrane Helix is Communicated to the Hinge Region

    DEFF Research Database (Denmark)

    Stelzer, Walter; Scharnagl, Christina; Leurs, Ulrike

    2016-01-01

    The transmembrane helix of the amyloid precursor protein is subject to proteolytic cleavages by γ-secretase at different sites resulting in Aβ peptides of different length and toxicity. A number of point mutations within this transmembrane helix alter the cleavage pattern thus enhancing production...... destabilizes amide hydrogen bonds in the hinge which connects dimerization and cleavage regions. Weaker intrahelical hydrogen bonds at the hinge may enhance helix bending and thereby affect recognition of the transmembrane substrate by the enzyme and/or presentation of its cleavage sites to the catalytic cleft....

  2. Production of extracellular proteolytic enzymes by Beauveria bassiana

    Directory of Open Access Journals (Sweden)

    Józefa Chrzanowska

    2014-08-01

    Full Text Available The production of proteolytic enzymes by two strains of Beauveria bassiana 278, B. bassiana 446 and one strain of Ascosphera apis 496 was analysed. It was demonstrated that the strain of B. bassiana 278 proved to be the best producer of basic and acid proteases. The influence of different environmental factors such as nitrogen and carbon sources on the production of extracellular hydrolytic enzymes was assessed. In addition the acid protease from B. bassiana was partially characterized.

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

    African Journals Online (AJOL)

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

  4. Cleavage preference distinguishes the two-component NS2B-NS3 serine proteinases of Dengue and West Nile viruses.

    Science.gov (United States)

    Shiryaev, Sergey A; Kozlov, Igor A; Ratnikov, Boris I; Smith, Jeffrey W; Lebl, Michal; Strongin, Alex Y

    2007-02-01

    Regulated proteolysis of the polyprotein precursor by the NS2B-NS3 protease is required for the propagation of infectious virions. Unless the structural and functional parameters of NS2B-NS3 are precisely determined, an understanding of its functional role and the design of flaviviral inhibitors will be exceedingly difficult. Our objectives were to define the substrate recognition pattern of the NS2B-NS3 protease of West Nile and Dengue virises (WNV and DV respectively). To accomplish our goals, we used an efficient, 96-well plate format, method for the synthesis of 9-mer peptide substrates with the general P4-P3-P2-P1-P1'-P2'-P3'-P4'-Gly structure. The N-terminus and the constant C-terminal Gly of the peptides were tagged with a fluorescent tag and with a biotin tag respectively. The synthesis was followed by the proteolytic cleavage of the synthesized, tagged peptides. Because of the strict requirement for the presence of basic amino acid residues at the P1 and the P2 substrate positions, the analysis of approx. 300 peptide sequences was sufficient for an adequate representation of the cleavage preferences of the WNV and DV proteinases. Our results disclosed the strict substrate specificity of the WNV protease for which the (K/R)(K/R)R/GG amino acid motifs was optimal. The DV protease was less selective and it tolerated well the presence of a number of amino acid residue types at either the P1' or the P2' site, as long as the other position was occupied by a glycine residue. We believe that our data represent a valuable biochemical resource and a solid foundation to support the design of selective substrates and synthetic inhibitors of flaviviral proteinases.

  5. Proteolytically modified human beta 2-microglobulin augments the specific cytotoxic activity in murine mixed lymphocyte culture

    DEFF Research Database (Denmark)

    Nissen, Mogens Holst; Claësson, M H

    1987-01-01

    the endogenous production of interleukin 2 in the MLC culture; monoclonal antibody which reacts with both the native beta 2-m and M-beta 2-m molecule blocks the augmentation of cytotoxic T lymphocyte production induced by M-beta 2-m; murine as well as human MLC responder cells can proteolytically modify native......A proteolytically modified form of beta 2-microglobulin (beta 2-m) present in the serum of patients suffering from autoimmune, immunodeficient diseases and cancer has been reported in the literature. In the present study we show that human beta 2-m as well as the proteolytically modified human form...... (M-beta 2-m) bind to murine lymphocytes expressing H-2 class I antigens; M-beta 2-m, when added at day 0 and 1 of culture in nanomolar concentrations to a one-way murine allogeneic mixed lymphocyte culture (MLC) augments the generation of specific cytotoxic T lymphocytes; M-beta 2-m increases...

  6. Altered cleavage patterns in human tripronuclear embryos and their association to fertilization method

    DEFF Research Database (Denmark)

    Joergensen, Mette Warming; Agerholm, Inge; Hindkjaer, Johnny

    2014-01-01

    PURPOSE: To analyze the cleavage patterns in dipronuclear (2PN) and tripronuclear (3PN) embryos in relation to fertilization method. METHOD: Time-lapse analysis. RESULTS: Compared to 2PN, more 3PN IVF embryos displayed early cleavage into 3 cells (p ... stage (p embryos, the 2nd and 3rd cleavage cycles were completed within the expected time frame. However, timing of the cell divisions within the cleavage cycles differed between the two groups. In contrast......, the completion of the 1st, 2nd, and 3rd cleavage cycle was delayed, but with a similar division pattern for 3PN ICSI compared with the 2PN ICSI embryos. 3PN, more often than 2PN ICSI embryos, displayed early cleavage into 3 cells (p = 0.03) and arrested development from the compaction stage and onwards (p = 0...

  7. Proteolytic Activity in Reduced-Fat Cheddar Cheese Made with Lactic Acid Bacteria and Camel Chymosin

    DEFF Research Database (Denmark)

    Børsting, Mette Winther

    be the need of an extended ripening period to reach a similar cheese structure as in cheeses produced with BC. The aim of this project was to compensate for the lower proteolytic activity in cheese produced with CC compared to BC. Selection of dairy lactic acid bacteria (LAB) for cheese production with high....... lactis subsp lactis, 10 thermophilic Lactobacillus strains and 15 frozen direct vat set strains of thermopholic Lactobacillus) to hydrolyse αS1-CN, candidates were selected for cheese-making experiments. None of the selected proteolytic strains contributed significantly to softening the cheese structure...

  8. RecA-mediated cleavage reaction of Lambda repressor and DNA ...

    African Journals Online (AJOL)

    PRECIOUS

    2010-01-11

    Jan 11, 2010 ... hydrolyze ATP at all, but fulfills RecA functions such as cleavage of Lambda repressor and strand .... DNA binding properties of RecA and may result in an in- .... AMP-PNP there is no cleavage of Lambda repressor (Figure.

  9. Sequential Power-Dependence Theory

    NARCIS (Netherlands)

    Buskens, Vincent; Rijt, Arnout van de

    2008-01-01

    Existing methods for predicting resource divisions in laboratory exchange networks do not take into account the sequential nature of the experimental setting. We extend network exchange theory by considering sequential exchange. We prove that Sequential Power-Dependence Theory—unlike

  10. Acquisition of a novel eleven amino acid insertion directly N-terminal to a tetrabasic cleavage site confers intracellular cleavage of an H7N7 influenza virus hemagglutinin

    International Nuclear Information System (INIS)

    Hamilton, Brian S.; Sun, Xiangjie; Chung, Changik; Whittaker, Gary R.

    2012-01-01

    A critical feature of highly pathogenic avian influenza viruses (H5N1 and H7N7) is the efficient intracellular cleavage of the hemagglutinin (HA) protein. H7N7 viruses also exist in equine species, and a unique feature of the equine H7N7 HA is the presence of an eleven amino acid insertion directly N-terminal to a tetrabasic cleavage site. Here, we show that three histidine residues within the unique insertion of the equine H7N7 HA are essential for intracellular cleavage. An asparagine residue within the insertion-derived glycosylation site was also found to be essential for intracellular cleavage. The presence of the histidine residues also appear to be involved in triggering fusion, since mutation of the histidine residues resulted in a destabilizing effect. Importantly, the addition of a tetrabasic site and the eleven amino acid insertion conferred efficient intracellular cleavage to the HA of an H7N3 low pathogenicity avian influenza virus. Our studies show that acquisition of the eleven amino acid insertion offers an alternative mechanism for intracellular cleavage of influenza HA.

  11. Acquisition of a novel eleven amino acid insertion directly N-terminal to a tetrabasic cleavage site confers intracellular cleavage of an H7N7 influenza virus hemagglutinin

    Energy Technology Data Exchange (ETDEWEB)

    Hamilton, Brian S.; Sun, Xiangjie; Chung, Changik [Department of Microbiology and Immunology, College of Veterinary Medicine, Cornell University, Ithaca NY 14853 (United States); New York Center of Excellence for Influenza Research and Surveillance, University of Rochester Medical Center, Rochester NY 14627 (United States); Whittaker, Gary R., E-mail: grw7@cornell.edu [Department of Microbiology and Immunology, College of Veterinary Medicine, Cornell University, Ithaca NY 14853 (United States); New York Center of Excellence for Influenza Research and Surveillance, University of Rochester Medical Center, Rochester NY 14627 (United States)

    2012-12-05

    A critical feature of highly pathogenic avian influenza viruses (H5N1 and H7N7) is the efficient intracellular cleavage of the hemagglutinin (HA) protein. H7N7 viruses also exist in equine species, and a unique feature of the equine H7N7 HA is the presence of an eleven amino acid insertion directly N-terminal to a tetrabasic cleavage site. Here, we show that three histidine residues within the unique insertion of the equine H7N7 HA are essential for intracellular cleavage. An asparagine residue within the insertion-derived glycosylation site was also found to be essential for intracellular cleavage. The presence of the histidine residues also appear to be involved in triggering fusion, since mutation of the histidine residues resulted in a destabilizing effect. Importantly, the addition of a tetrabasic site and the eleven amino acid insertion conferred efficient intracellular cleavage to the HA of an H7N3 low pathogenicity avian influenza virus. Our studies show that acquisition of the eleven amino acid insertion offers an alternative mechanism for intracellular cleavage of influenza HA.

  12. Short RNA guides cleavage by eukaryotic RNase III.

    Directory of Open Access Journals (Sweden)

    Bruno Lamontagne

    Full Text Available In eukaryotes, short RNAs guide a variety of enzymatic activities that range from RNA editing to translation repression. It is hypothesized that pre-existing proteins evolved to bind and use guide RNA during evolution. However, the capacity of modern proteins to adopt new RNA guides has never been demonstrated. Here we show that Rnt1p, the yeast orthologue of the bacterial dsRNA-specific RNase III, can bind short RNA transcripts and use them as guides for sequence-specific cleavage. Target cleavage occurred at a constant distance from the Rnt1p binding site, leaving the guide RNA intact for subsequent cleavage. Our results indicate that RNase III may trigger sequence-specific RNA degradation independent of the RNAi machinery, and they open the road for a new generation of precise RNA silencing tools that do not trigger a dsRNA-mediated immune response.

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

    Directory of Open Access Journals (Sweden)

    Kurth Reinhard

    2011-05-01

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

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

    Science.gov (United States)

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

    2011-05-09

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

  15. Proteolytic signatures define unique thrombin-derived peptides present in human wound fluid in vivo.

    Science.gov (United States)

    Saravanan, Rathi; Adav, Sunil S; Choong, Yeu Khai; van der Plas, Mariena J A; Petrlova, Jitka; Kjellström, Sven; Sze, Siu Kwan; Schmidtchen, Artur

    2017-10-13

    The disease burden of failing skin repair and non-healing ulcers is extensive. There is an unmet need for new diagnostic approaches to better predict healing activity and wound infection. Uncontrolled and excessive protease activity, of endogenous or bacterial origin, has been described as a major contributor to wound healing impairments. Proteolytic peptide patterns could therefore correlate and "report" healing activity and infection. This work describes a proof of principle delineating a strategy by which peptides from a selected protein, human thrombin, are detected and attributed to proteolytic actions. With a particular focus on thrombin-derived C-terminal peptides (TCP), we show that distinct peptide patterns are generated in vitro by the human S1 peptidases human neutrophil elastase and cathepsin G, and the bacterial M4 peptidases Pseudomonas aeruginosa elastase and Staphylococcus aureus aureolysin, respectively. Corresponding peptide sequences were identified in wound fluids from acute and non-healing ulcers, and notably, one peptide, FYT21 (FYTHVFRLKKWIQKVIDQFGE), was only present in wound fluid from non-healing ulcers colonized by P. aeruginosa and S. aureus. Our result is a proof of principle pointing at the possibility of defining peptide biomarkers reporting distinct proteolytic activities, of potential implication for improved diagnosis of wound healing and infection.

  16. Interactions between Lactobacillus sakei and CNC (Staphylococcus xylosus and Kocuria varians) and their influence on proteolytic activity.

    Science.gov (United States)

    Tremonte, P; Reale, A; Di Renzo, T; Tipaldi, L; Di Luccia, A; Coppola, R; Sorrentino, E; Succi, M

    2010-11-01

    To evaluate interactions between Lactobacillus sakei and coagulase negative cocci (CNC) (Staphylococcus xylosus and Kocuria varians) and to investigate the influence of these interactions on their own proteolytic activity. Interactions occurring between strains of Lact. sakei and CNC were assessed by spectrophotometric analysis. The growth of 35 strains of Lact. sakei, used as indicators, was compared to that obtained combining the same strains with growing cells or cell-free supernatants of 20 CNC (18 Staph. xylosus and 2 K. varians). The proteolytic activity expressed by single strains or by their combinations was assessed on sarcoplasmic protein extracts by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The results evidenced that interactions are able to affect not only the growth but also the in vitro proteolytic activity of Lact. sakei and CNC used in combination. A relationship between the presence of interactions among useful strains and the strength of technological characteristics, such as proteolysis, was defined. The study highlighted that CNC are able to stimulate the growth of some Lact. sakei strains. At the same time, this interaction positively influences the proteolytic activity of strains used in combination. Given the importance of proteolysis during the ripening of fermented meats, this phenomenon should be taken into account to select meat starter cultures. © 2010 The Authors. © 2010 The Society for Applied Microbiology.

  17. Proteolytic system of blood-feeding ticks: An update on protein structures

    Czech Academy of Sciences Publication Activity Database

    Kovářová, Zuzana; Hobizalová, Radka; Žebrakovská, Iva; Brynda, Jiří; Řezáčová, Pavlína; Horn, Martin; Mareš, Michael

    2015-01-01

    Roč. 22, č. 1 (2015), s. 43-44 ISSN 1211-5894. [Discussions in Structural Molecular Biology. Annual Meeting of the Czech Society for Structural Biology /13./. 19.03.2015-21.03.2015, Nové Hrady] Institutional support: RVO:61388963 ; RVO:68378050 Keywords : blood-feeding ticks * protein structure * proteolytic system Subject RIV: CE - Biochemistry

  18. Investigation of plant latices of Asteraceae and Campanulaceae regarding proteolytic activity.

    Science.gov (United States)

    Sytwala, Sonja; Domsalla, André; Melzig, Matthias F

    2015-12-01

    Occurrence of plant latices is widespread, there are more than 40 families of plants characterized to establish lactiferous structures. The appearance of hydrolytic active proteins, incorporated in latices is already characterized, and hydrolytic active proteins are considerable, and for several plant families, the occurrence of hydrolytic active proteins is already specified e.g. Apocynaceae Juss., Caricaceae Dumort, Euphorbiaceae Juss., Moraceae Gaudich and Papaveraceae Juss. In our investigation, focused on latex bearing plants of order Asterales, Asteraceae and Campanulaceae in particular. The present outcomes represent a comprehensive study, relating to the occurrence of proteolytic active enzymes of order Asterales for the first time. 131 different species of Asteraceae and Campanulaceae were tested, and the appearance of plant latex proteases were determined in different quantities. Proteolytic activity was investigated by inhibitory studies and determination of residual activity in the following, enable us to characterize the proteases. Most of the considered species exhibit a serine protease activity and a multiplicity of species exhibited two or more subclasses of proteases. Copyright © 2015 Elsevier Masson SAS. All rights reserved.

  19. Proteolytic activity and cooperative hemolytic effect of dermatophytes with different species of bacteria

    Directory of Open Access Journals (Sweden)

    Keyvan Pakshir

    2016-12-01

    Conclusion: This study indicated that hemolysin and proteolytic enzymes potentially play a role in dermatophyte pathogenesis and S. aureus could be considered as a main bacterium for creation of co-hemolytic effect in association with dermatophyte species.

  20. [Treatment of surface burns with proteolytic enzymes: mathematic description of lysis kinetics].

    Science.gov (United States)

    Domogatskaia, A S; Domogatskiĭ, S P; Ruuge, E K

    2003-01-01

    The lysis of necrotic tissue by a proteolytic enzyme applied to the surface of a burn wound was studied. A mathematical model was proposed, which describes changes in the thickness of necrotic tissue as a function of the proteolytic activity of the enzyme. The model takes into account the inward-directed diffusion of the enzyme, the counterflow of interstitial fluid (exudates) containing specific inhibitors, and the extracellular matrix proteolysis. It was shown in terms of the quasi-stationary approach that the thickness of the necrotic tissue layer decreases exponentially with time; i.e., the lysis slows down as the thickness of the necrotic tissue layer decreases. The dependence of the characteristic time of this decrease on enzyme concentration was obtained. It was shown that, at high enzyme concentrations (more than 5 mg/ml), the entire time of lysis (after the establishment of quasi-stationary equilibrium) is inversely proportional to the concentration of the enzyme.

  1. PROTEOLYTIC AND FIBRINOLYTIC ACTIVITIES OF HALOPHILIC LACTIC ACID BACTERIA FROM TWO INDONESIAN FERMENTED FOODS

    Directory of Open Access Journals (Sweden)

    Asep A. Prihanto

    2013-04-01

    Full Text Available Exploration of fermented foods as sources of fibrinolytic enzymes is increased in the last decades. Terasi and Jambal roti is Indonesian traditional fermented fish products, which were famous in Java Island. Both are important products in Indonesian dishes, especially in Java. Investigation on halophilic lactic acid bacteria using MRS and M-17 agar obtained seventy four isolated strains. Their proteolytic and fibrinolytic activities were determined using skim milk agar and plasminogen-free fibrin plate. Twenty five isolates showed protease activities, while only four of them secreted fibrinolitic enzyme. The highest proteolytic and fibrinolytic activity was shown by TB1 strain, which is identified as Bacillus coagulans. The 16s rDNA is still in investigating to confirm the TB1 strain identity.

  2. Effect of microstructure on the cleavage fracture strength of low carbon Mn-Ni-Mo bainitic steels

    International Nuclear Information System (INIS)

    Im, Young-Roc; Lee, Byeong-Joo; Oh, Yong Jun; Hong, Jun Hwa; Lee, Hu-Chul

    2004-01-01

    The effects of the microstructure on the cleavage fracture strength of low carbon Mn-Ni-Mo bainitic steels were examined. A four-point bend test and double-notched bend specimens were used to measure the cleavage fracture strength of the alloys and identify the cleavage initiating micro-cracks, respectively. The cleavage fracture strength and DBTT of Mn-Ni-Mo bainitic steels were strongly affected by the alloy carbon content. The decrease in the alloy carbon content resulted in a decrease in the inter-lath cementite-crowded layers and higher cleavage fracture strength. Micro-cracks that formed across the inter-lath cementite-crowded layers were observed to initiate cleavage fracture. The width of these inter-lath cementite-crowded layers was accepted as a cleavage initiating micro-crack size in the micro-mechanical modeling of the cleavage fracture, and the measured cleavage strength values of the bainitic Mn-Ni-Mo steels were well represented by the modified Griffith relationship

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

    Directory of Open Access Journals (Sweden)

    Jack Xie

    2010-08-01

    Full Text Available The study evaluated substrate cleavage product(s generated by three botulinum neurotoxin serotype A (BoNT/A medicinal drug products utilizing a novel and highly specific, light-chain activity, high-performance liquid chromatography (LCA-HPLC method. Samples were reacted with a commercially available BoNT/A fluorescent substrate derived from the SNAP-25 sequence. Reaction products were separated by reversed-phase HPLC. The method detected an atypical cleavage pattern by one of the formulated drug products. IncobotulinumtoxinA produced two cleavage fragments rather than the single fragment typically generated by BoNT/A. Identification confirmed the secondary cleavage at a position corresponding to SNAP-25 Arg198–Ala199 (normal BoNT/A cleavage is Gln197–Arg198. Arg198–Ala199 is also the cleavage site for trypsin and serotype C toxin. Normal cleavage was observed for all other BoNT/A drug product samples, as well as 900-kD and 150-kD bulk toxin BoNT/A. The reason for this unexpected secondary cleavage pattern by one formulated BoNT/A drug product is unknown. Possible explanations include a contaminating protease and/or damage to the 150-kD type-A toxin causing nonspecific substrate recognition and subsequent cleavage uncharacteristic of type-A toxin. The BoNT/A drug products were also analyzed via the LCA-HPLC assay using a commercial BoNT/C fluorescent substrate derived from the syntaxin sequence. Cleavage of the serotype C substrate by incobotulinumtoxinA was also confirmed whilst neither of the other drug products cleaved the syntaxin substrate.

  4. Post-transcription cleavage generates the 3' end of F17R transcripts in vaccinia virus

    International Nuclear Information System (INIS)

    D'Costa, Susan M.; Antczak, James B.; Pickup, David J.; Condit, Richard C.

    2004-01-01

    Most vaccinia virus intermediate and late mRNAs possess 3' ends that are extremely heterogeneous in sequence. However, late mRNAs encoding the cowpox A-type inclusion protein (ATI), the second largest subunit of the RNA polymerase, and the late telomeric transcripts possess homogeneous 3' ends. In the case of the ATI mRNA, it has been shown that the homogeneous 3' end is generated by a post-transcriptional endoribonucleolytic cleavage event. We have determined that the F17R gene also produces homogeneous transcripts generated by a post-transcriptional cleavage event. Mapping of in vivo mRNA shows that the major 3' end of the F17R transcript maps 1262 nt downstream of the F17R translational start site. In vitro transcripts spanning the in vivo 3' end are cleaved in an in vitro reaction using extracts from virus infected cells, and the site of cleavage is the same both in vivo and in vitro. Cleavage is not observed using extract from cells infected in the presence of hydroxyurea; therefore, the cleavage factor is either virus-coded or virus-induced during the post-replicative phase of virus replication. The cis-acting sequence responsible for cleavage is orientation specific and the factor responsible for cleavage activity has biochemical properties similar to the factor required for cleavage of ATI transcripts. Partially purified cleavage factor generates cleavage products of expected size when either the ATI or F17R substrates are used in vitro, strongly suggesting that cleavage of both transcripts is mediated by the same factor

  5. Oxidative cleavage of the octyl side chain of 1-(3,4-dichlorobenzyl)-5-octylbiguanide (OPB-2045) in rat and dog liver preparations.

    Science.gov (United States)

    Umehara, K; Kudo, S; Hirao, Y; Morita, S; Uchida, M; Odomi, M; Miyamoto, G

    2000-08-01

    The metabolism of 1-(3,4-dichlorobenzyl)-5-octylbiguanide (OPB-2045), a new potent biguanide antiseptic, was investigated using rat and dog liver preparations to elucidate the mechanism of OPB-2045 metabolite formation, in which the octyl side chain is reduced to four, five, or six carbon atoms. Chemical structures of metabolites were characterized by 1H NMR, fast atom bombardment/mass spectrometry, and liquid chromatography/electrospray ionization-tandem mass spectrometry. Three main metabolites were observed during incubation of OPB-2045 with rat liver S9: 2-octanol (M-1), 3-octanol (M-2), and 4-octanol (M-3). In the incubation of OPB-2045 with dog liver S9, eight metabolites were observed, seven of which being M-1, M-2, M-3, 2-octanone (M-4), threo-2,3-octandiol (M-5), erythro-2,3-octandiol (M-6), and 1,2-octandiol (M-7). M-5 and M-6 were further biotransformed to a ketol derivative and C-C bond cleavage metabolite (hexanoic acid derivative), an in vivo end product, in the incubation with dog liver microsomes. The reactions required NADPH as a cofactor and were significantly inhibited by the various inhibitors of cytochrome P450 (i.e., CO, n-octylamine, SKF 525-A, metyrapone, and alpha-naphthoflavone). The results indicate that the degraded products of OPB-2045 are produced by C-C bond cleavage after monohydroxylation, dihydroxylation, and ketol formation at the site of the octyl side chain with possible involvement of cytochrome P450 systems. This aliphatic C-C bond cleavage by sequential oxidative reactions may play an important role in the metabolism of other drugs or endogenous compounds that possess aliphatic chains.

  6. Smart biomaterials: Surfaces functionalized with proteolytically stable osteoblast-adhesive peptides

    Directory of Open Access Journals (Sweden)

    Annj Zamuner

    2017-09-01

    Full Text Available Engineered scaffolds for bone tissue regeneration are designed to promote cell adhesion, growth, proliferation and differentiation. Recently, covalent and selective functionalization of glass and titanium surfaces with an adhesive peptide (HVP mapped on [351–359] sequence of human Vitronectin allowed to selectively increase osteoblast attachment and adhesion strength in in vitro assays, and to promote osseointegration in in vivo studies. For the first time to our knowledge, in this study we investigated the resistance of adhesion sequences to proteolytic digestion: HVP was completely cleaved after 5 h. In order to overcome the enzymatic degradation of the native peptide under physiological conditions we synthetized three analogues of HVP sequence. A retro-inverted peptide D-2HVP, composed of D amino acids, was completely stable in serum-containing medium. In addition, glass surfaces functionalized with D-2HVP increased human osteoblast adhesion as compared to the native peptide and maintained deposition of calcium. Interestingly, D-2HVP increased expression of IBSP, VTN and SPP1 genes as compared to HVP functionalized surfaces. Total internal reflection fluorescence microscope analysis showed cells with numerous filopodia spread on D-2HVP-functionalized surfaces. Therefore, the D-2HVP sequence is proposed as new osteoblast adhesive peptide with increased bioactivity and high proteolytic resistance.

  7. Smart biomaterials: Surfaces functionalized with proteolytically stable osteoblast-adhesive peptides.

    Science.gov (United States)

    Zamuner, Annj; Brun, Paola; Scorzeto, Michele; Sica, Giuseppe; Castagliuolo, Ignazio; Dettin, Monica

    2017-09-01

    Engineered scaffolds for bone tissue regeneration are designed to promote cell adhesion, growth, proliferation and differentiation. Recently, covalent and selective functionalization of glass and titanium surfaces with an adhesive peptide (HVP) mapped on [351-359] sequence of human Vitronectin allowed to selectively increase osteoblast attachment and adhesion strength in in vitro assays, and to promote osseointegration in in vivo studies. For the first time to our knowledge, in this study we investigated the resistance of adhesion sequences to proteolytic digestion: HVP was completely cleaved after 5 h. In order to overcome the enzymatic degradation of the native peptide under physiological conditions we synthetized three analogues of HVP sequence. A retro-inverted peptide D-2HVP, composed of D amino acids, was completely stable in serum-containing medium. In addition, glass surfaces functionalized with D-2HVP increased human osteoblast adhesion as compared to the native peptide and maintained deposition of calcium. Interestingly, D-2HVP increased expression of IBSP, VTN and SPP1 genes as compared to HVP functionalized surfaces. Total internal reflection fluorescence microscope analysis showed cells with numerous filopodia spread on D-2HVP-functionalized surfaces. Therefore, the D-2HVP sequence is proposed as new osteoblast adhesive peptide with increased bioactivity and high proteolytic resistance.

  8. Comparing Proteolytic Fingerprints of Antigen-Presenting Cells during Allergen Processing.

    Science.gov (United States)

    Hofer, Heidi; Weidinger, Tamara; Briza, Peter; Asam, Claudia; Wolf, Martin; Twaroch, Teresa E; Stolz, Frank; Neubauer, Angela; Dall, Elfriede; Hammerl, Peter; Jacquet, Alain; Wallner, Michael

    2017-06-08

    Endolysosomal processing has a critical influence on immunogenicity as well as immune polarization of protein antigens. In industrialized countries, allergies affect around 25% of the population. For the rational design of protein-based allergy therapeutics for immunotherapy, a good knowledge of T cell-reactive regions on allergens is required. Thus, we sought to analyze endolysosomal degradation patterns of inhalant allergens. Four major allergens from ragweed, birch, as well as house dust mites were produced as recombinant proteins. Endolysosomal proteases were purified by differential centrifugation from dendritic cells, macrophages, and B cells, and combined with allergens for proteolytic processing. Thereafter, endolysosomal proteolysis was monitored by protein gel electrophoresis and mass spectrometry. We found that the overall proteolytic activity of specific endolysosomal fractions differed substantially, whereas the degradation patterns of the four model allergens obtained with the different proteases were extremely similar. Moreover, previously identified T cell epitopes were assigned to endolysosomal peptides and indeed showed a good overlap with known T cell epitopes for all four candidate allergens. Thus, we propose that the degradome assay can be used as a predictor to determine antigenic peptides as potential T cell epitopes, which will help in the rational design of protein-based allergy vaccine candidates.

  9. Bacterial and Fungal Proteolytic Enzymes: Production, Catalysis and Potential Applications.

    Science.gov (United States)

    da Silva, Ronivaldo Rodrigues

    2017-09-01

    Submerged and solid-state bioprocesses have been extensively explored worldwide and employed in a number of important studies dealing with microbial cultivation for the production of enzymes. The development of these production technologies has facilitated the generation of new enzyme-based products with applications in pharmaceuticals, food, bioactive peptides, and basic research studies, among others. The applicability of microorganisms in biotechnology is potentiated because of their various advantages, including large-scale production, short time of cultivation, and ease of handling. Currently, several studies are being conducted to search for new microbial peptidases with peculiar biochemical properties for industrial applications. Bioprospecting, being an important prerequisite for research and biotechnological development, is based on exploring the microbial diversity for enzyme production. Limited information is available on the production of specific proteolytic enzymes from bacterial and fungal species, especially on the subgroups threonine and glutamic peptidases, and the seventh catalytic type, nonhydrolytic asparagine peptide lyase. This gap in information motivated the present study about these unique biocatalysts. In this study, the biochemical and biotechnological aspects of the seven catalytic types of proteolytic enzymes, namely aspartyl, cysteine, serine, metallo, glutamic, and threonine peptidase, and asparagine peptide lyase, are summarized, with an emphasis on new studies, production, catalysis, and application of these enzymes.

  10. Proteolytic stability in colloidal systems : interaction of proteins with the solid-water interface

    NARCIS (Netherlands)

    Maste, M.C.L.

    1996-01-01


    Proteolytic enzymes in liquid detergents suffer from lack of stability in the sense that activity diminishes with time. Although the phenomenon could be attributed to several factors, the influence of colloidal surfaces on the enzymatic stability was investigated. Besides the types of

  11. AKTIVITAS PROTEOLITIK BAKTERI ASAM LAKTAT DALAM FERMENTASI SUSU KEDELAI [Proteolytic Activities of Lactic Acid Bacteria in Fermentation of Soymilk

    Directory of Open Access Journals (Sweden)

    Yusmarini1,2*

    2010-12-01

    Full Text Available Some lactic acid bacteria (LAB strains had been isolated from spontaneously fermented soymilk which have proteolytic system. The purpose of this research was to study ability of isolates in fermentation of soymilk. The changes in bacterial growth, pH, titrable acidity, and proteolytic activities during fermentation were examined. Isolates of Lactobacillus plantarum 1 R.1.3.2; L. plantarum 1 R.11.1.2 and L. acidophilus FNCC 0051 (as a control were capable growing in soymilk. The results indicated that initial pH of soymilk was 6,6 and decreased to 4,6 after fermentation and titrable acidity of 0.11 increased to 0.34 after fermentation. The proteolytic activities were 0.352 U/ml – 0.468 U/ml. The electrophoretic pattern of the proteins showed changes during fermentation of soymilk.

  12. Mixed-substrate (glycerol tributyrate and fibrin) zymography for simultaneous detection of lipolytic and proteolytic enzymes on a single gel.

    Science.gov (United States)

    Choi, Nack-Shick; Choi, Jong Hyun; Kim, Bo-Hye; Han, Yun-Jon; Kim, Joong Su; Lee, Seung-Goo; Song, Jae Jun

    2009-06-01

    A new zymography method for simultaneous detection of two different enzymatic activities (lipolytic and proteolytic) using a single SDS-containing or native-conformation gel and a mixed-substrate (glycerol tributyrate and fibrin) (MS)(1) gel was developed. After routine electrophoresis, SDS in the gel was removed by treatment with Triton X-100. Gel proteins were electrotransferred to the MS gel. To visualize lipolytic activity, the MS gel was incubated at 37 degrees C (for 6 or 24 h) until clear bands against an opaque background were observed. To detect proteolytic activity, the same MS gel was stained with Coomassie brilliant blue. Using this method, we show that six lipolytic enzymes from Staphylococcus pasteuri NJ-1 and four proteolytic enzymes from two Bacillus strains, B. licheniformis DJ-2 and B. licheniformis NJ-5, isolated from soil, can be simultaneously detected.

  13. Prediction of proteasome cleavage motifs by neural networks

    DEFF Research Database (Denmark)

    Kesimir, C.; Nussbaum, A.K.; Schild, H.

    2002-01-01

    physiological conditions. Our algorithm has been trained not only on in vitro data, but also on MHC Class I ligand data, which reflect a combination of immunoproteasome and constitutive proteasome specificity. This feature, together with the use of neural networks, a non-linear classification technique, make...... the prediction of MHC Class I ligand boundaries more accurate: 65% of the cleavage sites and 85% of the non-cleavage sites are correctly determined. Moreover, we show that the neural networks trained on the constitutive proteasome data learns a specificity that differs from that of the networks trained on MHC...

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

    International Nuclear Information System (INIS)

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

    2011-01-01

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

  15. A novel processing system of sterol regulatory element-binding protein-1c regulated by polyunsaturated fatty acid.

    Science.gov (United States)

    Nakakuki, Masanori; Kawano, Hiroyuki; Notsu, Tatsuto; Imada, Kazunori; Mizuguchi, Kiyoshi; Shimano, Hitoshi

    2014-05-01

    The proteolytic cascade is the key step in transactivation of sterol regulatory element-binding proteins (SREBPs), a transcriptional factor of lipid synthesis. Proteolysis of SREBP-2 is strictly regulated by sterols, but that of SREBP-1c was not strongly sterol-regulated, but inhibited by polyunsaturated fatty acids (PUFAs). In this study, the proteolytic processing of SREBP-1 and -2 was examined by transfection studies of cDNA-encoding mutants in which all the known cleavage sites were disrupted. In cultured cells, sterol-regulated SREBP-2 processing was completely eliminated by mutation of cleavage sites. In contrast, the corresponding SREBP-1c mutants as well as wild type exhibited large amounts of cleaved products in the nuclear extracts from culture cells and murine liver in vivo. The nuclear form of the mutant SREBP-1c was induced by delipidated condition and suppressed by eicosapentaenoic acid, an n-3 PUFA, but not by sterols. This novel processing mechanism was affected by neither SREBP cleavage-activating protein (SCAP) nor insulin-induced gene (Insig)-1, unlike SREBP-2, but abolished by a serine protease inhibitor. Through analysis of deletion mutant, a site-2 protease recognition sequence (DRSR) was identified to be involved in this novel processing. These findings suggest that SREBP-1c cleavage could be subjected to a novel PUFA-regulated cleavage system in addition to the sterol-regulatory SCAP/Insig system.

  16. Genetics of Proteolytic Enzymes of Lactococci and Their Role in Cheese Flavor Development

    NARCIS (Netherlands)

    Kok, Jan

    In recent years, knowledge of the genetics and biochemistry of the enzymes that constitute the proteolytic system of starter lactococci has increased tremendously. This paper summarizes the data obtained largely in the last 5 yr of intensive research by various research groups operative in this

  17. Pregnancy-associated plasma protein-A (PAPP-A) modulates early developmental rate in zebrafish independent of its proteolytic activity

    DEFF Research Database (Denmark)

    Kjær-Sørensen, Kasper; Engholm, Ditte Høyer; Kamei, Hiroyasu

    2013-01-01

    the developmental rate beginning during gastrulation without affecting the normal patterning of the embryo. This phenotype is different from those resulting from deficiency of Igf receptor or ligand in zebrafish, suggesting a function of Papp-a outside the Igf system. Biochemical analysis of recombinant zebrafish...... Papp-a demonstrates conservation of proteolytic activity, specificity, and intrinsic regulatory mechanism. However, in vitro transcribed mRNA, which encodes a proteolytically inactive Papp-a mutant, recues the papp-a knockdown phenotype as efficient as wild-type Papp-a. Thus, the developmental...

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

    Directory of Open Access Journals (Sweden)

    Ranganathan Shoba

    2008-12-01

    Full Text Available Abstract Background Signal peptides (SPs mediate the targeting of secretory precursor proteins to the correct subcellular compartments in prokaryotes and eukaryotes. Identifying these transient peptides is crucial to the medical, food and beverage and biotechnology industries yet our understanding of these peptides remains limited. This paper examines the most common type of signal peptides cleavable by the endoprotease signal peptidase I (SPase I, and the residues flanking the cleavage sites of three groups of signal peptide sequences, namely (i eukaryotes (Euk (ii Gram-positive (Gram+ bacteria, and (iii Gram-negative (Gram- bacteria. Results In this study, 2352 secretory peptide sequences from a variety of organisms with amino-terminal SPs are extracted from the manually curated SPdb database for analysis based on physicochemical properties such as pI, aliphatic index, GRAVY score, hydrophobicity, net charge and position-specific residue preferences. Our findings show that the three groups share several similarities in general, but they display distinctive features upon examination in terms of their amino acid compositions and frequencies, and various physico-chemical properties. Thus, analysis or prediction of their sequences should be separated and treated as distinct groups. Conclusion We conclude that the peptide segment recognized by SPase I extends to the start of the mature protein to a limited extent, upon our survey of the amino acid residues surrounding the cleavage processing site. These flanking residues possibly influence the cleavage processing and contribute to non-canonical cleavage sites. Our findings are applicable in defining more accurate prediction tools for recognition and identification of cleavage site of SPs.

  19. Staggering in the cleavage pattern of E. coli ABC-excinuclease

    International Nuclear Information System (INIS)

    Myles, G.M.; Van Houten, B.; Sancar, A.

    1986-01-01

    E. coli ABC excinuclease is a complex of three proteins encoded by the uvrA, uvrB, and uvrC genes. The enzyme repairs DNA mono and diadducts by the single strand cleavage of DNA eight phosphodiester bond 5' and four or five phosphodiester bonds 3' to a DNA lesion and facilitates the removal of the resulting twelve or thirteen nucleotide fragment. In this study, the authors have investigated the excision pattern for ultraviolet (UV) induced diadducts, i.e. cyclobutane pyrimidine dimers and pyrimidine-pyrimidone (6-4) photoproducts. Terminally (5' or 3') labeled DNA was irradiated with 254nm UV and treated with ABC excinuclease before and after photoreactivation of cyclobutane dimers by E. coli DNA photolyase. In this way, the authors were able to differentiate between the cleavage pattern of pyrimidine dimers and of (6-4) photoproducts. Their results show that certain TT cyclobutane dimers and rare TT (6-4) photoproducts are excised by cleavage seven and, less frequently, six phosphodiester bonds to the 5' side of the DNA lesion in addition to the primary cutting site at the eight 5' phosphodiester bond. The 3' cleavage sites are maintained at the fourth and fifth phosphodiester bonds for the these UV induced lesions. These data indicate that the cleavage pattern of the ABC excinuclease may be dependent upon both the type of DNA lesion as well as it surrounding nucleotide sequence. In addition, the authors analysis shows that (6-4) photoproducts are much better substrates for ABC excinuclease than are pyrimidine dimers

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

    Science.gov (United States)

    Marincola, Gabriella; Wolz, Christiane

    2017-06-02

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

  1. Blastocyst Development in a Single Medium Compared to Sequential Media: A Prospective Study With Sibling Oocytes.

    Science.gov (United States)

    Sfontouris, Ioannis A; Kolibianakis, Efstratios M; Lainas, George T; Petsas, George K; Tarlatzis, Basil C; Lainas, Trifon G

    2017-09-01

    The aim of the present study was to compare blastocyst formation rates after embryo culture in a single medium (Global) as compared to sequential media (ISM1/BlastAssist). In this prospective trial with sibling oocytes, 542 metaphase II (ΜΙΙ) oocytes from 31 women were randomly and equally divided to be fertilized and cultured to the blastocyst stage in either sequential media (ISM1/BlastAssist; n = 271 MII oocytes) or a single medium (Global; n = 271 MII oocytes). In both groups, embryos were cultured in an interrupted fashion with media changes on day 3. Embryo transfer was performed on day 5. Blastocyst formation rates on day 5 (61.7% ± 19.9% vs 37.0% ± 25.5%, P ISM1/BlastAssist, respectively. Fertilization rates, cleavage rates, and percentage of good quality embryos on day 3 were similar between Global and ISM1/BlastAssist, respectively. The percentages of good quality blastocysts (63.0% ± 24.8% vs 32.1% ± 37.2%, P ISM1/BlastAssist, respectively. In conclusion, culture in Global was associated with higher blastocyst formation rates compared to ISM1/BlastAssist, suggesting that the single medium may provide better support to the developing embryo.

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

    Directory of Open Access Journals (Sweden)

    Poetsch Ansgar

    2006-03-01

    Full Text Available Abstract Background Membrane proteins still remain elusive in proteomic studies. This is in part due to the distribution of the amino acids lysine and arginine, which are less frequent in integral membrane proteins and almost absent in transmembrane helices. As these amino acids are cleavage targets for the commonly used protease trypsin, alternative cleavage conditions, which should improve membrane protein analysis, were tested by in silico digestion for the three organisms Saccharomyces cerevisiae, Halobacterium sp. NRC-1, and Corynebacterium glutamicum as hallmarks for eukaryotes, archea and eubacteria. Results For the membrane proteomes from all three analyzed organisms, we identified cleavage conditions that achieve better sequence and proteome coverage than trypsin. Greater improvement was obtained for bacteria than for yeast, which was attributed to differences in protein size and GRAVY. It was demonstrated for bacteriorhodopsin that the in silico predictions agree well with the experimental observations. Conclusion For all three examined organisms, it was found that a combination of chymotrypsin and staphylococcal peptidase I gave significantly better results than trypsin. As some of the improved cleavage conditions are not more elaborate than trypsin digestion and have been proven useful in practice, we suppose that the cleavage at both hydrophilic and hydrophobic amino acids should facilitate in general the analysis of membrane proteins for all organisms.

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

    Science.gov (United States)

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

    2015-01-01

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

  4. LINGO-1 promotes lysosomal degradation of amyloid-β protein precursor

    Directory of Open Access Journals (Sweden)

    Rian de Laat

    2015-03-01

    Full Text Available Sequential proteolytic cleavages of amyloid-β protein precursor (AβPP by β-secretase and γ-secretase generate amyloid β (Aβ peptides, which are thought to contribute to Alzheimer's disease (AD. Much of this processing occurs in endosomes following endocytosis of AβPP from the plasma membrane. However, this pathogenic mode of processing AβPP may occur in competition with lysosomal degradation of AβPP, a common fate of membrane proteins trafficking through the endosomal system. Following up on published reports that LINGO-1 binds and promotes the amyloidogenic processing of AβPP we have examined the consequences of LINGO-1/AβPP interactions. We report that LINGO-1 and its paralogs, LINGO-2 and LINGO-3, decrease processing of AβPP in the amyloidogenic pathway by promoting lysosomal degradation of AβPP. We also report that LINGO-1 levels are reduced in AD brain, representing a possible pathogenic mechanism stimulating the generation of Aβ peptides in AD.

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

    Directory of Open Access Journals (Sweden)

    Ting Wei

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

  6. Modelling sequentially scored item responses

    NARCIS (Netherlands)

    Akkermans, W.

    2000-01-01

    The sequential model can be used to describe the variable resulting from a sequential scoring process. In this paper two more item response models are investigated with respect to their suitability for sequential scoring: the partial credit model and the graded response model. The investigation is

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

    Science.gov (United States)

    Kuyumcu-Martinez, Muge; Belliot, Gaël; Sosnovtsev, Stanislav V; Chang, Kyeong-Ok; Green, Kim Y; Lloyd, Richard E

    2004-08-01

    Caliciviruses are single-stranded RNA viruses that cause a wide range of diseases in both humans and animals, but little is known about the regulation of cellular translation during infection. We used two distinct calicivirus strains, MD145-12 (genus Norovirus) and feline calicivirus (FCV) (genus Vesivirus), to investigate potential strategies used by the caliciviruses to inhibit cellular translation. Recombinant 3C-like proteinases (r3CL(pro)) from norovirus and FCV were found to cleave poly(A)-binding protein (PABP) in the absence of other viral proteins. The norovirus r3CL(pro) PABP cleavage products were indistinguishable from those generated by poliovirus (PV) 3C(pro) cleavage, while the FCV r3CL(pro) products differed due to cleavage at an alternate cleavage site 24 amino acids downstream of one of the PV 3C(pro) cleavage sites. All cleavages by calicivirus or PV proteases separated the C-terminal domain of PABP that binds translation factors eIF4B and eRF3 from the N-terminal RNA-binding domain of PABP. The effect of PABP cleavage by the norovirus r3CL(pro) was analyzed in HeLa cell translation extracts, and the presence of r3CL(pro) inhibited translation of both endogenous and exogenous mRNAs. Translation inhibition was poly(A) dependent, and replenishment of the extracts with PABP restored translation. Analysis of FCV-infected feline kidney cells showed that the levels of de novo cellular protein synthesis decreased over time as virus-specific proteins accumulated, and cleavage of PABP occurred in virus-infected cells. Our data indicate that the calicivirus 3CL(pro), like PV 3C(pro), mediates the cleavage of PABP as part of its strategy to inhibit cellular translation. PABP cleavage may be a common mechanism among certain virus families to manipulate cellular translation.

  8. The potato carotenoid cleavage dioxygenase 4 catalyzes a single cleavage of β-ionone ring-containing carotenes and non-epoxidated xanthophylls

    KAUST Repository

    Bruno, Mark

    2015-04-01

    Down-regulation of the potato carotenoid cleavage dioxygenase 4 (StCCD4) transcript level led to tubers with altered morphology and sprouting activity, which also accumulated higher levels of violaxanthin and lutein leading to elevated carotenoid amounts. This phenotype indicates a role of this enzyme in tuber development, which may be exerted by a cleavage product. In this work, we investigated the enzymatic activity of StCCD4, by expressing the corresponding cDNA in carotenoid accumulating Escherichia coli strains and by performing in vitro assays with heterologously expressed enzyme. StCCD4 catalyzed the cleavage of all-. trans-β-carotene at the C9\\'-C10\\' double bond, leading to β-ionone and all-. trans-β-apo-10\\'-carotenal, both in vivo and in vitro. The enzyme also cleaved β,β-cryptoxanthin, zeaxanthin and lutein either at the C9\\'-C10\\' or the C9-C10 double bond in vitro. In contrast, we did not observe any conversion of violaxanthin and only traces of activity with 9-. cis-β-carotene, which led to 9-. cis-β-apo-10\\'-carotenal. Our data indicate that all-. trans-β-carotene is the likely substrate of StCCD4 in planta, and that this carotene may be precursor of an unknown compound involved in tuber development.

  9. Improved proteolytic stability and potent activity against Leishmania infantum trypanothione reductase of α/β-peptide foldamers conjugated to cell-penetrating peptides.

    Science.gov (United States)

    de Lucio, Héctor; Gamo, Ana María; Ruiz-Santaquiteria, Marta; de Castro, Sonia; Sánchez-Murcia, Pedro A; Toro, Miguel A; Gutiérrez, Kilian Jesús; Gago, Federico; Jiménez-Ruiz, Antonio; Camarasa, María-José; Velázquez, Sonsoles

    2017-11-10

    The objective of the current study was to enhance the proteolytic stability of peptide-based inhibitors that target critical protein-protein interactions at the dimerization interface of Leishmania infantum trypanothione reductase (Li-TryR) using a backbone modification strategy. To achieve this goal we carried out the synthesis, proteolytic stability studies and biological evaluation of a small library of α/β 3 -peptide foldamers of different length (from 9-mers to 13-mers) and different α→β substitution patterns related to prototype linear α-peptides. We show that several 13-residue α/β 3 -peptide foldamers retain inhibitory potency against the enzyme (in both activity and dimerization assays) while they are far less susceptible to proteolytic degradation than an analogous α-peptide. The strong dependence of the binding affinities for Li-TryR on the length of the α,β-peptides is supported by theoretical calculations on conformational ensembles of the resulting complexes. The conjugation of the most proteolytically stable α/β-peptide with oligoarginines results in a molecule with potent activity against L. infantum promastigotes and amastigotes. Copyright © 2017 Elsevier Masson SAS. All rights reserved.

  10. AKTIVITAS PROTEOLITIK BAKTERI ASAM LAKTAT DALAM FERMENTASI SUSU KEDELAI [Proteolytic Activities of Lactic Acid Bacteria in Fermentation of Soymilk

    OpenAIRE

    Yusmarini1,2)*; R. Indrati1); T. Utami1); Y. Marsono1)

    2010-01-01

    Some lactic acid bacteria (LAB) strains had been isolated from spontaneously fermented soymilk which have proteolytic system. The purpose of this research was to study ability of isolates in fermentation of soymilk. The changes in bacterial growth, pH, titrable acidity, and proteolytic activities during fermentation were examined. Isolates of Lactobacillus plantarum 1 R.1.3.2; L. plantarum 1 R.11.1.2 and L. acidophilus FNCC 0051 (as a control) were capable growing in soymilk. The results indi...

  11. An evaluation of the proteolytic and lipolytic potential of Penicillium spp. isolated from traditional Greek sausages in submerged fermentation.

    Science.gov (United States)

    Papagianni, Maria

    2014-01-01

    A number of novel Penicillium strains belonging to Penicillium nalgiovense, Penicillium solitum, Penicillium commune, Penicillium olsonii, and Penicillium oxalicum species, isolated from the surface of traditional Greek sausages, were evaluated for their proteolytic and lipolytic potential in a solid substrate first and next in submerged fermentations, using complex media. Extracellular proteolytic activity was assessed at acid, neutral, and alkaline pH, while the lipolytic activity was assessed using olive oil, the short-chain triacylglycerol tributyrin, and the long-chain triolein, as substrates. The study revealed that although closely related, the tested strains produce enzymes of distinct specificities. P. nalgiovense PNA9 produced the highest alkaline proteolytic activity (13.2 unit (U)/ml) and the highest lipolytic activity with tributyrin (92 U/ml). Comparisons with known sources show that proteases and/or lipases can be secreted effectively by some Penicillia (P. nalgiovense PNA4, PNA7, and PNA9 and P. solitum PSO1), and further investigations on their properties and characteristics would be promising.

  12. Multi-agent sequential hypothesis testing

    KAUST Repository

    Kim, Kwang-Ki K.

    2014-12-15

    This paper considers multi-agent sequential hypothesis testing and presents a framework for strategic learning in sequential games with explicit consideration of both temporal and spatial coordination. The associated Bayes risk functions explicitly incorporate costs of taking private/public measurements, costs of time-difference and disagreement in actions of agents, and costs of false declaration/choices in the sequential hypothesis testing. The corresponding sequential decision processes have well-defined value functions with respect to (a) the belief states for the case of conditional independent private noisy measurements that are also assumed to be independent identically distributed over time, and (b) the information states for the case of correlated private noisy measurements. A sequential investment game of strategic coordination and delay is also discussed as an application of the proposed strategic learning rules.

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

    Science.gov (United States)

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

    2005-01-01

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

  14. Endocytic down-regulation of ErbB2 is stimulated by cleavage of its C-terminus

    DEFF Research Database (Denmark)

    Lerdrup, Mads; Bruun, Silas; Grandal, Michael Vibo

    2007-01-01

    inhibition of HSP90 with geldanamycin this cleavage is accompanied by proteasome-dependent endocytosis of ErbB2. However, it is unknown whether C-terminal cleavage is linked to endocytosis. To study ErbB2 cleavage and endocytic trafficking, we fused yellow fluorescent protein (YFP) and cyan fluorescent...

  15. Effects of Cysteamine on Sheep Embryo Cleavage Rates

    Directory of Open Access Journals (Sweden)

    Sinem Ö. ENGİNLER

    2015-01-01

    Full Text Available Oxidative stress during in vitro culture leads to defects in development of gametes and embryos. Several antioxidants such as cysteamine, L-ascorbic acid, beta mercaptoethanol, cysteine, glutathione, proteins, vitamins have been used to supplement culture media to counter the oxidative stress. This study was conducted to detect the effect of adding cysteamine to the maturation medium to subsequent cleavage rates of sheep embryos. Totally 604 ovaries were obtained by ten replica and 2060 oocytes were collected. The cumulus oocyte complexes were recovered by the slicing method. A total of 1818 selected oocytes were divided into two groups and used for maturation (88.25%. The first group was created as supplemented with cysteamine (Group A and second group (Group B, control without cysteamine in TCM-199. The two groups were incubated for 24 h at 38.8 °C in an atmosphere of 5% CO2 in humidified air for in vitro maturation (IVM. After IVM, oocytes were fertilized with 50 x 107 / mL fresh ram semen in BSOF medium for 18 h. After fertilization, maturation groups were divided into two subgroups with different culture media: Group AI-SOF (Synthetic Oviduct Fluid medium, Group AII-CR1aa (Charles Rosencrans medium, Group BI-SOF and Group BII-CR1aa were achieved. Cleavage rates were evaluated at day 2. post insemination. The rates of cleavage were detected as 59.54% (184/309, 55.44% (173/312, 65.34% (215/329, 59.34% (200/337 respectively, with showing no statistically significant difference between the groups at the level of P>0.05. In conclusion, supplementing cysteamine to maturation media in TCM-199 did not affect the cleavage rates of sheep embryos in SOF and CR1aa culture media.

  16. Characterization of sodium dodecyl sulfate-resistant proteolytic activity in the hyperthermophilic archaebacterium Pyrococcus furiosus

    Energy Technology Data Exchange (ETDEWEB)

    Blumentals, I.I.; Robinson, A.S.; Kelly, R.M. (Johns Hopkins Univ., Baltimore, MD (USA))

    1990-07-01

    Cell extracts from Pyrococcus furiosus were found to contain five proteases, two of which (S66 and S102) are resistant to sodium dodecyl sulfate (SDS) denaturation. Cell extracts incubated at 98{degree}C in the presence of 1% SDS for 24 h exhibited substantial cellular proteolysis such that only four proteins could be visualized by amido black-Coomassie brilliant blue staining of SDS-polyacrylamide gels. The SDS-treated extract retained 19% of the initial proteolytic activity as represented by two proteases, S66 (66 kilodaltons (kDa)) and S102 (102 kDa). Immunoblot analysis with guinea pig sera containing antibodies against protease S66 indicated that S66 is related neither to S102 nor to the other proteases. The results of this analysis also suggest that S66 might be the hydrolysis product of a 200-kDa precursor which does not have proteolytic activity. The 24-h SDS-treated extract showed unusually thermostable proteolytic activity; the measured half-life at 98{degree}C was found to be 33 h. Proteases S66 and S102 were also resistant to denaturation by 8 M urea, 80 mM dithiothreitol, and 5% {beta}-mercaptoethanol. Purified protease S66 was inhibited by phenylmethylsulfonyl fluoride and diisopropyl fluorophosphate but not by EDTA, ethylene glycol-bis({beta}-aminoethyl ether)-N,N,N{prime},N{prime}-tetraacetic acid, or iodoacetic acid. These results indicate that S66 is a serine protease. Amino acid ester hydrolysis studies showed that protease S66 was hydrolytically active towards N-benzoyl-L-arginine ethyl ester.

  17. Sequential charged particle reaction

    International Nuclear Information System (INIS)

    Hori, Jun-ichi; Ochiai, Kentaro; Sato, Satoshi; Yamauchi, Michinori; Nishitani, Takeo

    2004-01-01

    The effective cross sections for producing the sequential reaction products in F82H, pure vanadium and LiF with respect to the 14.9-MeV neutron were obtained and compared with the estimation ones. Since the sequential reactions depend on the secondary charged particles behavior, the effective cross sections are corresponding to the target nuclei and the material composition. The effective cross sections were also estimated by using the EAF-libraries and compared with the experimental ones. There were large discrepancies between estimated and experimental values. Additionally, we showed the contribution of the sequential reaction on the induced activity and dose rate in the boundary region with water. From the present study, it has been clarified that the sequential reactions are of great importance to evaluate the dose rates around the surface of cooling pipe and the activated corrosion products. (author)

  18. Differential production of proteolytic enzymes by normal human fibroblasts and their counterparts transformed by treatment with 60Co gamma rays

    International Nuclear Information System (INIS)

    Nishitani, Koji; Namba, Masayoshi; Ohkubo, Shigeki; Kimoto, Tetsuo

    1985-01-01

    Production of proteolytic enzymes by human fibroblasts in the process of transformation was investigated. The cells used were normal human fibroblasts (KSM-6) and their in vitro counterparts transformed by treatment with 60 Co gamma rays (KMST-6). Cells seeded by treatment with EDTA were cultured in a serum free medium. Proteolytic enzymes in the culture medium of cells were assayed using a synthetic substrate, N-α-(p-tosyl)-L-arginine ( 3 H) methyl ester hydrochloride. The transformed cells (KMST-6) produced a larger amount of enzymes than normal cells (KMS-6). The enzyme production in both cell lines was high in the exponential growth stage and then decreased as the cells reached confluency. The proteolytic enzymes produced by these cells were trypsin- and thrombin-like enzymes. Cell growth of KMST-6 or KMS-6 was not inhibited by the addition of protease inhibitors to the culture medium. (author)

  19. Variable context Markov chains for HIV protease cleavage site prediction.

    Science.gov (United States)

    Oğul, Hasan

    2009-06-01

    Deciphering the knowledge of HIV protease specificity and developing computational tools for detecting its cleavage sites in protein polypeptide chain are very desirable for designing efficient and specific chemical inhibitors to prevent acquired immunodeficiency syndrome. In this study, we developed a generative model based on a generalization of variable order Markov chains (VOMC) for peptide sequences and adapted the model for prediction of their cleavability by certain proteases. The new method, called variable context Markov chains (VCMC), attempts to identify the context equivalence based on the evolutionary similarities between individual amino acids. It was applied for HIV-1 protease cleavage site prediction problem and shown to outperform existing methods in terms of prediction accuracy on a common dataset. In general, the method is a promising tool for prediction of cleavage sites of all proteases and encouraged to be used for any kind of peptide classification problem as well.

  20. Proteolytic degradation of regulator of G protein signaling 2 facilitates temporal regulation of Gq/11 signaling and vascular contraction.

    Science.gov (United States)

    Kanai, Stanley M; Edwards, Alethia J; Rurik, Joel G; Osei-Owusu, Patrick; Blumer, Kendall J

    2017-11-24

    Regulator of G protein signaling 2 (RGS2) controls signaling by receptors coupled to the G q/11 class heterotrimeric G proteins. RGS2 deficiency causes several phenotypes in mice and occurs in several diseases, including hypertension in which a proteolytically unstable RGS2 mutant has been reported. However, the mechanisms and functions of RGS2 proteolysis remain poorly understood. Here we addressed these questions by identifying degradation signals in RGS2, and studying dynamic regulation of G q/11 -evoked Ca 2+ signaling and vascular contraction. We identified a novel bipartite degradation signal in the N-terminal domain of RGS2. Mutations disrupting this signal blunted proteolytic degradation downstream of E3 ubiquitin ligase binding to RGS2. Analysis of RGS2 mutants proteolyzed at various rates and the effects of proteasome inhibition indicated that proteolytic degradation controls agonist efficacy by setting RGS2 protein expression levels, and affecting the rate at which cells regain agonist responsiveness as synthesis of RGS2 stops. Analyzing contraction of mesenteric resistance arteries supported the biological relevance of this mechanism. Because RGS2 mRNA expression often is strikingly and transiently up-regulated and then down-regulated upon cell stimulation, our findings indicate that proteolytic degradation tightly couples RGS2 transcription, protein levels, and function. Together these mechanisms provide tight temporal control of G q/11 -coupled receptor signaling in the cardiovascular, immune, and nervous systems. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

  1. Eyewitness confidence in simultaneous and sequential lineups: a criterion shift account for sequential mistaken identification overconfidence.

    Science.gov (United States)

    Dobolyi, David G; Dodson, Chad S

    2013-12-01

    Confidence judgments for eyewitness identifications play an integral role in determining guilt during legal proceedings. Past research has shown that confidence in positive identifications is strongly associated with accuracy. Using a standard lineup recognition paradigm, we investigated accuracy using signal detection and ROC analyses, along with the tendency to choose a face with both simultaneous and sequential lineups. We replicated past findings of reduced rates of choosing with sequential as compared to simultaneous lineups, but notably found an accuracy advantage in favor of simultaneous lineups. Moreover, our analysis of the confidence-accuracy relationship revealed two key findings. First, we observed a sequential mistaken identification overconfidence effect: despite an overall reduction in false alarms, confidence for false alarms that did occur was higher with sequential lineups than with simultaneous lineups, with no differences in confidence for correct identifications. This sequential mistaken identification overconfidence effect is an expected byproduct of the use of a more conservative identification criterion with sequential than with simultaneous lineups. Second, we found a steady drop in confidence for mistaken identifications (i.e., foil identifications and false alarms) from the first to the last face in sequential lineups, whereas confidence in and accuracy of correct identifications remained relatively stable. Overall, we observed that sequential lineups are both less accurate and produce higher confidence false identifications than do simultaneous lineups. Given the increasing prominence of sequential lineups in our legal system, our data argue for increased scrutiny and possibly a wholesale reevaluation of this lineup format. PsycINFO Database Record (c) 2013 APA, all rights reserved.

  2. Changes in Contact Area in Meniscus Horizontal Cleavage Tears Subjected to Repair and Resection.

    Science.gov (United States)

    Beamer, Brandon S; Walley, Kempland C; Okajima, Stephen; Manoukian, Ohan S; Perez-Viloria, Miguel; DeAngelis, Joseph P; Ramappa, Arun J; Nazarian, Ara

    2017-03-01

    To assess the changes in tibiofemoral contact pressure and contact area in human knees with a horizontal cleavage tear before and after treatment. Ten human cadaveric knees were tested. Pressure sensors were placed under the medial meniscus and the knees were loaded at twice the body weight for 20 cycles at 0°, 10°, and 20° of flexion. Contact area and pressure were recorded for the intact meniscus, the meniscus with a horizontal cleavage tear, after meniscal repair, after partial meniscectomy (single leaflet), and after subtotal meniscectomy (double leaflet). The presence of a horizontal cleavage tear significantly increased average peak contact pressure and reduced effective average tibiofemoral contact area at all flexion angles tested compared with the intact state (P contact pressure after creation of the horizontal cleavage tear. Repairing the horizontal cleavage tear restored peak contact pressures and areas to within 15% of baseline, statistically similar to the intact state at all angles tested (P contact pressure and reduced average contact area at all degrees of flexion compared with the intact state (P contact area and a significant elevation in contact pressure. These changes may accelerate joint degeneration. A suture-based repair of these horizontal cleavage tears returns the contact area and contact pressure to nearly normal, whereas both partial and subtotal meniscectomy lead to significant reductions in contact area and significant elevations in contact pressure within the knee. Repairing horizontal cleavage tears may lead to improved clinical outcomes by preserving meniscal tissue and the meniscal function. Understanding contact area and peak contact pressure resulting from differing strategies for treating horizontal cleavage tears will allow the surgeon to evaluate the best strategy for treating his or her patients who present with this meniscal pathology. Copyright © 2016 Arthroscopy Association of North America. Published by Elsevier

  3. Oxidative cleavage of erucic acid for the synthesis of brassylic acid

    Energy Technology Data Exchange (ETDEWEB)

    Mohammed J. Nasrullah; Pooja Thapliyal; Erica N. Pfarr; Nicholas S. Dusek; Kristofer L. Schiele; James A. Bahr

    2010-10-29

    The main focus of this work is to synthesize Brassylic Acid (BA) using oxidative cleavage of Erucic Acid (EA). Crambe (Crambe abyssinica) is an industrial oilseed grown in North Dakota. Crambe has potential as an industrial fatty acid feedstock as a source of Erucic acid (EA). It has approximately 50-60 % of EA, a C{sub 22} monounsaturated fatty acid. Oxidative cleavage of unsaturated fatty acids derived from oilseeds produces long chain (9, 11, and 13 carbon atoms) dibasic and monobasic acids. These acids are known commercial feedstocks for the preparation of nylons, polyesters, waxes, surfactants, and perfumes. Other sources of EA are Rapeseed seed oil which 50-60 % of EA. Rapeseed is grown outside USA. The oxidative cleavage of EA was done using a high throughput parallel pressure reactor system. Kinetics of the reaction shows that BA yields reach a saturation at 12 hours. H{sub 2}WO{sub 4} was found to be the best catalyst for the oxidative cleavage of EA. High yields of BA were obtained at 80 C with bubbling of O{sub 2} or 10 bar of O{sub 2} for 12 hours.

  4. Caspase-Dependent Apoptosis Induced by Telomere Cleavage and TRF2 Loss

    Directory of Open Access Journals (Sweden)

    Asha S. Multani

    2000-07-01

    Full Text Available Chromosomal abnormalities involving telomeric associations (TAs often precede replicative senescence and abnormal chromosome configurations. We report here that telomere cleavage following exposure to proapoptotic agents is an early event in apoptosis. Exposure of human and murine cancer cells to a variety of pro-apoptotic stimuli (staurosporine, thapsigargin, anti-Fas antibody, cancer chemotherapeutic agents resulted in telomere cleavage and aggregation, finally their extrusion from the nuclei. Telomere loss was associated with arrest of cells in G2/M phase and preceded DNA fragmentation. Telomere erosion and subsequent large-scale chromatin cleavage were inhibited by overexpression of the anti -apoptotic protein, bcl-2, two peptide caspase inhibitors (BACMK and zVADfmk, indicating that both events are regulated by caspase activation. The results demonstrate that telomere cleavage is an early chromatin alteration detected in various cancer cell lines leading to drug-induced apoptosis, suggest that this event contributes to mitotic catastrophe and induction of cell death. Results also suggest that the decrease of telomeric-repeat binding factor 2 (TRF2 may be the earliest event in the ara-C-induced telomere shortening, induction of endoreduplication and chromosomal fragmentation leading to cell death.

  5. The potato carotenoid cleavage dioxygenase 4 catalyzes a single cleavage of β-ionone ring-containing carotenes and non-epoxidated xanthophylls.

    Science.gov (United States)

    Bruno, Mark; Beyer, Peter; Al-Babili, Salim

    2015-04-15

    Down-regulation of the potato carotenoid cleavage dioxygenase 4 (StCCD4) transcript level led to tubers with altered morphology and sprouting activity, which also accumulated higher levels of violaxanthin and lutein leading to elevated carotenoid amounts. This phenotype indicates a role of this enzyme in tuber development, which may be exerted by a cleavage product. In this work, we investigated the enzymatic activity of StCCD4, by expressing the corresponding cDNA in carotenoid accumulating Escherichia coli strains and by performing in vitro assays with heterologously expressed enzyme. StCCD4 catalyzed the cleavage of all-trans-β-carotene at the C9'-C10' double bond, leading to β-ionone and all-trans-β-apo-10'-carotenal, both in vivo and in vitro. The enzyme also cleaved β,β-cryptoxanthin, zeaxanthin and lutein either at the C9'-C10' or the C9-C10 double bond in vitro. In contrast, we did not observe any conversion of violaxanthin and only traces of activity with 9-cis-β-carotene, which led to 9-cis-β-apo-10'-carotenal. Our data indicate that all-trans-β-carotene is the likely substrate of StCCD4 in planta, and that this carotene may be precursor of an unknown compound involved in tuber development. Copyright © 2015 Elsevier Inc. All rights reserved.

  6. 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.

  7. Pressure modulates the self-cleavage step of the hairpin ribozyme

    Science.gov (United States)

    Schuabb, Caroline; Kumar, Narendra; Pataraia, Salome; Marx, Dominik; Winter, Roland

    2017-03-01

    The ability of certain RNAs, denoted as ribozymes, to not only store genetic information but also catalyse chemical reactions gave support to the RNA world hypothesis as a putative step in the development of early life on Earth. This, however, might have evolved under extreme environmental conditions, including the deep sea with pressures in the kbar regime. Here we study pressure-induced effects on the self-cleavage of hairpin ribozyme by following structural changes in real-time. Our results suggest that compression of the ribozyme leads to an accelerated transesterification reaction, being the self-cleavage step, although the overall process is retarded in the high-pressure regime. The results reveal that favourable interactions between the reaction site and neighbouring nucleobases are strengthened under pressure, resulting therefore in an accelerated self-cleavage step upon compression. These results suggest that properly engineered ribozymes may also act as piezophilic biocatalysts in addition to their hitherto known properties.

  8. Remarks on sequential designs in risk assessment

    International Nuclear Information System (INIS)

    Seidenfeld, T.

    1982-01-01

    The special merits of sequential designs are reviewed in light of particular challenges that attend risk assessment for human population. The kinds of ''statistical inference'' are distinguished and the problem of design which is pursued is the clash between Neyman-Pearson and Bayesian programs of sequential design. The value of sequential designs is discussed and the Neyman-Pearson vs. Bayesian sequential designs are probed in particular. Finally, warnings with sequential designs are considered, especially in relation to utilitarianism

  9. On the temperature independence of statistical model parameters for cleavage fracture in ferritic steels

    Science.gov (United States)

    Qian, Guian; Lei, Wei-Sheng; Niffenegger, M.; González-Albuixech, V. F.

    2018-04-01

    The work relates to the effect of temperature on the model parameters in local approaches (LAs) to cleavage fracture. According to a recently developed LA model, the physical consensus of plastic deformation being a prerequisite to cleavage fracture enforces any LA model of cleavage fracture to observe initial yielding of a volume element as its threshold stress state to incur cleavage fracture in addition to the conventional practice of confining the fracture process zone within the plastic deformation zone. The physical consistency of the new LA model to the basic LA methodology and the differences between the new LA model and other existing models are interpreted. Then this new LA model is adopted to investigate the temperature dependence of LA model parameters using circumferentially notched round tensile specimens. With the published strength data as input, finite element (FE) calculation is conducted for elastic-perfectly plastic deformation and the realistic elastic-plastic hardening, respectively, to provide stress distributions for model calibration. The calibration results in temperature independent model parameters. This leads to the establishment of a 'master curve' characteristic to synchronise the correlation between the nominal strength and the corresponding cleavage fracture probability at different temperatures. This 'master curve' behaviour is verified by strength data from three different steels, providing a new path to calculate cleavage fracture probability with significantly reduced FE efforts.

  10. Fibrin Clots Are Equilibrium Polymers That Can Be Remodeled Without Proteolytic Digestion

    OpenAIRE

    Chernysh, Irina N.; Nagaswami, Chandrasekaran; Purohit, Prashant K.; Weisel, John W.

    2012-01-01

    Fibrin polymerization is a necessary part of hemostasis but clots can obstruct blood vessels and cause heart attacks and strokes. The polymerization reactions are specific and controlled, involving strong knob-into-hole interactions to convert soluble fibrinogen into insoluble fibrin. It has long been assumed that clots and thrombi are stable structures until proteolytic digestion. On the contrary, using the technique of fluorescence recovery after photobleaching, we demonstrate here that the...

  11. Fractographic observations of cleavage initiation in the ductile-brittle transition region of a reactor-pressure-vessel steel

    International Nuclear Information System (INIS)

    Rosenfield, A.R.; Shetty, D.K.; Skidmore, A.J.

    1983-01-01

    This note reports the results of a fractographic study conducted on a group of 1T compact fracture toughness specimens of a heavy-section A508 steel denoted TSE6 tested in the ductile-brittle transition region (22 and 82 0 C). The fatigue-precracked specimens were loaded at a rapid rate (760 or 550 mm per second) to promote cleavage-crack growth and lower-bound toughness behavior. All specimens experienced unstable cleavage fracture prior to reaching a maximum in the load displacement curve. Some ductile crack growth occurred in half of the specimens. The objective of fractographic examinations was to understand the observed statistical variations in cleavage initiation by (a) locating the origins of unstable cleavage fracture in the vicinity of the fatigue-precrack or ductilerupture crack fronts, (b) identifying microstructural features associated with the triggering of cleavage, and (c) documenting characteristic fracture surface dimensions such as the extent of stable-crack growth prior to unstable cleavage (Δα) and the distance of the cleavage origin from the ductilerupture front, /chi/ (or fatigue-crack front when Δα = 0)

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

    DEFF Research Database (Denmark)

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

    1996-01-01

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

  13. Sequential lineup laps and eyewitness accuracy.

    Science.gov (United States)

    Steblay, Nancy K; Dietrich, Hannah L; Ryan, Shannon L; Raczynski, Jeanette L; James, Kali A

    2011-08-01

    Police practice of double-blind sequential lineups prompts a question about the efficacy of repeated viewings (laps) of the sequential lineup. Two laboratory experiments confirmed the presence of a sequential lap effect: an increase in witness lineup picks from first to second lap, when the culprit was a stranger. The second lap produced more errors than correct identifications. In Experiment 2, lineup diagnosticity was significantly higher for sequential lineup procedures that employed a single versus double laps. Witnesses who elected to view a second lap made significantly more errors than witnesses who chose to stop after one lap or those who were required to view two laps. Witnesses with prior exposure to the culprit did not exhibit a sequential lap effect.

  14. On the formation and nature of quasi-cleavage fracture surfaces in hydrogen embrittled steels

    Energy Technology Data Exchange (ETDEWEB)

    Martin, May L.; Fenske, Jamey A.; Liu, Grace S.; Sofronis, Petros [University of Illinois, Dept. of Materials Science and Engineering, 1304 W. Green St., Urbana, IL 61801 (United States); Robertson, Ian M., E-mail: ianr@illinois.edu [University of Illinois, Dept. of Materials Science and Engineering, 1304 W. Green St., Urbana, IL 61801 (United States)

    2011-02-15

    Quasi-cleavage, a common feature of hydrogen-induced fracture surfaces, is generally taken as being cleavage-like but not along a known cleavage plane. Despite the frequency with which this surface is observed, the relationship to the underlying microstructure remains unknown. Through a combination of topographical reconstruction of secondary electron microscope fractographs and a transmission electron microscopy study of the microstructure from site-specific locations, it will be shown that the features on quasi-cleavage surfaces are ridges that can be correlated with sub-surface intense and highly localized deformation bands. It will be demonstrated that the fracture surface arises from the growth and coalescence of voids that initiate at and extend along slip band intersections. This mechanism and process is fully consistent with hydrogen enhancing and localizing plastic processes.

  15. The adhesion G protein-coupled receptor G2 (ADGRG2/GPR64) constitutively activates SRE and NFκB and is involved in cell adhesion and migration

    DEFF Research Database (Denmark)

    Cornelia Peeters, Miriam; Fokkelman, Michiel; Boogaard, Bob

    2015-01-01

    Adhesion G protein-coupled receptors (ADGRs) are believed to be activated by auto-proteolytic cleavage of their very large extracellular N-terminal domains normally acting as a negative regulator of the intrinsically constitutively active seven transmembrane domain. ADGRG2 (or GPR64) which...

  16. Small heat shock protein HspB8: its distribution in Alzheimer's disease brains and its inhibition of amyloid-beta protein aggregation and cerebrovascular amyloid-beta toxicity.

    NARCIS (Netherlands)

    Wilhelmus, M.M.M.; Boelens, W.C.; Otte-Holler, I.; Kamps, B.; Kusters, B.; Maat-Schieman, M.L.; Waal, R.M.W. de; Verbeek, M.M.

    2006-01-01

    Alzheimer's disease (AD) is characterized by pathological lesions, such as senile plaques (SPs) and cerebral amyloid angiopathy (CAA), both predominantly consisting of a proteolytic cleavage product of the amyloid-beta precursor protein (APP), the amyloid-beta peptide (Abeta). CAA is also the major

  17. Robustness of the Sequential Lineup Advantage

    Science.gov (United States)

    Gronlund, Scott D.; Carlson, Curt A.; Dailey, Sarah B.; Goodsell, Charles A.

    2009-01-01

    A growing movement in the United States and around the world involves promoting the advantages of conducting an eyewitness lineup in a sequential manner. We conducted a large study (N = 2,529) that included 24 comparisons of sequential versus simultaneous lineups. A liberal statistical criterion revealed only 2 significant sequential lineup…

  18. Structure of the Integral Membrane Protein CAAX Protease Ste24p

    Energy Technology Data Exchange (ETDEWEB)

    Pryor Jr., Edward E. [Membrane Protein Structural Biology Consortium (United States); Univ. of Virginia, Charlottesville, VA (United States); Horanyi, Peter S. [Membrane Protein Structural Biology Consortium (United States); Univ. of Virginia, Charlottesville, VA (United States); Clark, Kathleen M. [Membrane Protein Structural Biology Consortium (United States); Univ. of Rochester School of Medicine and Dentistry, Rochester, NY (United States); Fedoriw, Nadia [Membrane Protein Structural Biology Consortium (United States); Univ. of Rochester School of Medicine and Dentistry, Rochester, NY (United States); Connelly, Sara M. [Membrane Protein Structural Biology Consortium (United States); Univ. of Rochester School of Medicine and Dentistry, Rochester, NY (United States); Koszelak-Rosenblum, Mary [Membrane Protein Structural Biology Consortium (United States); Hauptman-Woodward Inst., Buffalo, NY (United States); Zhu, Guangyu [Membrane Protein Structural Biology Consortium (United States); Hauptman-Woodward Inst., Buffalo, NY (United States); Malkowski, Michael G. [Membrane Protein Structural Biology Consortium (United States); Hauptman-Woodward Inst., Buffalo, NY (United States); State Univ. of New York, Buffalo, NY (United States); Wiener, Michael C. [Membrane Protein Structural Biology Consortium (United States); Univ. of Virginia, Charlottesville, VA (United States); Dumont, Mark E. [Membrane Protein Structural Biology Consortium (United States); Univ. of Rochester School of Medicine and Dentistry, Rochester, NY (United States)

    2012-10-26

    Posttranslational lipidation provides critical modulation of the functions of some proteins. Isoprenoids (i.e., farnesyl or geranylgeranyl groups) are attached to cysteine residues in proteins containing C-terminal CAAX sequence motifs (where A is an aliphatic residue and X is any residue). Isoprenylation is followed by cleavage of the AAX amino acid residues and, in some cases, by additional proteolytic cuts. We determined the crystal structure of the CAAX protease Ste24p, a zinc metalloprotease catalyzing two proteolytic steps in the maturation of yeast mating pheromone a -factor. The Ste24p core structure is a ring of seven transmembrane helices enclosing a voluminous cavity containing the active site and substrate-binding groove. The cavity is accessible to the external milieu by means of gaps between splayed transmembrane helices. We hypothesize that cleavage proceeds by means of a processive mechanism of substrate insertion, translocation, and ejection.

  19. The potato carotenoid cleavage dioxygenase 4 catalyzes a single cleavage of β-ionone ring-containing carotenes and non-epoxidated xanthophylls

    KAUST Repository

    Bruno, Mark; Beyer, Peter D.; Al-Babili, Salim

    2015-01-01

    amounts. This phenotype indicates a role of this enzyme in tuber development, which may be exerted by a cleavage product. In this work, we investigated the enzymatic activity of StCCD4, by expressing the corresponding cDNA in carotenoid accumulating

  20. Analysis of Proteolytic and Fibrinolytic Activity of the Blood Plasma in Patients with Bronchial Asthma Combined with Obesity

    Directory of Open Access Journals (Sweden)

    Fediv A.I. Fediv A.I.

    2016-09-01

    Full Text Available Aim of the study — to conduct the analysis of the performance of proteolytic and fibrinolytic activity of the blood plasma in patients with bronchial asthma (BA combined with obesity. Materials and methods. The study included 40 patients divided into groups. The basic group consisted of 20 patients with BA associated with obesity (ІI group, two comparison groups — of 10 patients with BA and normal body weight (I group and 10 patients with obesity and without pathology of the bronchopulmonary system (ІІІ group. Control group included 10 apparently healthy persons. In patients with BA, there were investigated the indexes of proteolytic and fibrinolytic activity of the blood plasma. Results. Analysis of the obtained data in patients with BA showed an increase of activity of plasma coagulation factors: total fibrinolytic activity increased in comparison to the control group, and in patients with BA it was 1.46 ± 0.13 Е440/ml/h, while in apparently healthy persons — 1.23 ± 0.16 Е440/ml/h (р < 0.05. In addition, an increase was marked and in the group of patients with the isolated obesity (by 38.5 % as compared to the group of apparently healthy persons, that can be explained by the growth of activity of inflammatory process due to the immune changes related to biological activity of fatty tissue as an additional source of proinflammatory cytokines. Conclusions. In combination of bronchial asthma and obesity, there is an activation of fibrinolytic and proteolytic systems of the blood that leads to microcirculatory and hemostatic disorders, enhancement of inflammatory process. In obesity as the state of systemic inflammatory response, the activation of fibrinolytic and proteolytic blood systems is more significant, than in bronchial asthma.

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

    International Nuclear Information System (INIS)

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

    1994-05-01

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

  2. Sequential and Multistep Substrate Interrogation Provides the Scaffold for Specificity in Human Flap Endonuclease 1

    KAUST Repository

    Sobhy, M.; Joudeh, L.; Huang, X.; Takahashi, Masateru; Hamdan, S.

    2013-01-01

    Human flap endonuclease 1 (FEN1), one of the structure-specific 5' nucleases, is integral in replication, repair, and recombination of cellular DNA. The 5' nucleases share significant unifying features yet cleave diverse substrates at similar positions relative to 5' end junctions. Using single-molecule Förster resonance energy transfer, we find a multistep mechanism that verifies all substrate features before inducing the intermediary-DNA bending step that is believed to unify 5' nuclease mechanisms. This is achieved by coordinating threading of the 5' flap of a nick junction into the conserved capped-helical gateway, overseeing the active site, and bending by binding at the base of the junction. We propose that this sequential and multistep substrate recognition process allows different 5' nucleases to recognize different substrates and restrict the induction of DNA bending to the last common step. Such mechanisms would also ensure the protection ofDNA junctions from nonspecific bending and cleavage. 2013 The Authors.

  3. Sequential and Multistep Substrate Interrogation Provides the Scaffold for Specificity in Human Flap Endonuclease 1

    KAUST Repository

    Sobhy, M.

    2013-06-06

    Human flap endonuclease 1 (FEN1), one of the structure-specific 5\\' nucleases, is integral in replication, repair, and recombination of cellular DNA. The 5\\' nucleases share significant unifying features yet cleave diverse substrates at similar positions relative to 5\\' end junctions. Using single-molecule Förster resonance energy transfer, we find a multistep mechanism that verifies all substrate features before inducing the intermediary-DNA bending step that is believed to unify 5\\' nuclease mechanisms. This is achieved by coordinating threading of the 5\\' flap of a nick junction into the conserved capped-helical gateway, overseeing the active site, and bending by binding at the base of the junction. We propose that this sequential and multistep substrate recognition process allows different 5\\' nucleases to recognize different substrates and restrict the induction of DNA bending to the last common step. Such mechanisms would also ensure the protection ofDNA junctions from nonspecific bending and cleavage. 2013 The Authors.

  4. Mass spectrometry analysis of proteome-wide proteolytic post-translational degradation of proteins

    OpenAIRE

    Shen, Yufeng; Hixson, Kim K.; Tolić, Nikola; Camp, David G.; Purvine, Samuel O.; Moore, Ronald J.; Smith, Richard D.

    2008-01-01

    Protein proteolytic degradation is an essential component to proper cell function and its life cycle. Here, we study the protein degradation in yeast Saccharomyces cerevisiae cells on a proteome-wide scale by detection of the intermediate peptides produced from the intracellular degradation of proteins using sequencing-based tandem mass spectrometry. By tracing the detected ~1,100 peptides and their ~200 protein substrate origins we obtain evidence for new insights into the proteome-wide prot...

  5. Structure of the cleavage-activated prefusion form of the parainfluenza virus 5 fusion protein.

    Science.gov (United States)

    Welch, Brett D; Liu, Yuanyuan; Kors, Christopher A; Leser, George P; Jardetzky, Theodore S; Lamb, Robert A

    2012-10-09

    The paramyxovirus parainfluenza virus 5 (PIV5) enters cells by fusion of the viral envelope with the plasma membrane through the concerted action of the fusion (F) protein and the receptor binding protein hemagglutinin-neuraminidase. The F protein folds initially to form a trimeric metastable prefusion form that is triggered to undergo large-scale irreversible conformational changes to form the trimeric postfusion conformation. It is thought that F refolding couples the energy released with membrane fusion. The F protein is synthesized as a precursor (F0) that must be cleaved by a host protease to form a biologically active molecule, F1,F2. Cleavage of F protein is a prerequisite for fusion and virus infectivity. Cleavage creates a new N terminus on F1 that contains a hydrophobic region, known as the FP, which intercalates target membranes during F protein refolding. The crystal structure of the soluble ectodomain of the uncleaved form of PIV5 F is known; here we report the crystal structure of the cleavage-activated prefusion form of PIV5 F. The structure shows minimal movement of the residues adjacent to the protease cleavage site. Most of the hydrophobic FP residues are buried in the uncleaved F protein, and only F103 at the newly created N terminus becomes more solvent-accessible after cleavage. The conformational freedom of the charged arginine residues that compose the protease recognition site increases on cleavage of F protein.

  6. Synthesis, purification, and characterization of an Arg sub 152 yields Glu site-directed mutant of recombinant human blood clotting factor VII

    Energy Technology Data Exchange (ETDEWEB)

    Wildgoose, P.; Kisiel, W. (Univ. of New Mexico, Albuquerque (USA)); Berkner, K.L. (ZymoGenetics, Inc., Seattle, WA (USA))

    1990-04-03

    Coagulation factor VII circulates in blood as a single-chain zymogen of a serine protease and is converted to its activated two-chain form, factor VIIa, by cleavage of an internal peptide bond located at Arg{sub 152}-Ile{sub 153}. Previous studies using serine protease active-site inhibitors suggest that zymogen factor VII may possess sufficient proteolytic activity to initiate the extrinsic pathway of blood coagulation. In order to assess the putative intrinsic proteolytic activity of single-chain factor VII, the authors have constructed a site-specific mutant of recombinant human factor VII in which arginine-152 has been replaced with a glutamic acid residue. Mutant factor VII was purified in a single step from culture supernatants of baby hamster kidney cells transfected with a plasmid containing the sequence for Arg{sub 152} {yields} Glu factor VII using a calcium-dependent, murine anti-factor VII monoclonal antibody column. The clotting activity of mutant factor VII was completely inhibited following incubation with dansyl-Glu-Gly-Arg chloromethyl ketone, suggesting that the apparent clotting activity of mutant factor VII was due to a contaminating serine protease. Immunoblots of mutant factor VII with human factor IXa revealed no cleavage, whereas incubation of mutant factor VII with human factor Xa resulted in cleavage of mutant factor VII and the formation of a lower molecular weight degradation product migrating at M{sup r}{approx}40 000. The results are consistent with the proposal that zymogen factor VII possesses no intrinsic proteolytic activity toward factor X or factor IX.

  7. Synthesis, purification, and characterization of an Arg152 → Glu site-directed mutant of recombinant human blood clotting factor VII

    International Nuclear Information System (INIS)

    Wildgoose, P.; Kisiel, W.; Berkner, K.L.

    1990-01-01

    Coagulation factor VII circulates in blood as a single-chain zymogen of a serine protease and is converted to its activated two-chain form, factor VIIa, by cleavage of an internal peptide bond located at Arg 152 -Ile 153 . Previous studies using serine protease active-site inhibitors suggest that zymogen factor VII may possess sufficient proteolytic activity to initiate the extrinsic pathway of blood coagulation. In order to assess the putative intrinsic proteolytic activity of single-chain factor VII, the authors have constructed a site-specific mutant of recombinant human factor VII in which arginine-152 has been replaced with a glutamic acid residue. Mutant factor VII was purified in a single step from culture supernatants of baby hamster kidney cells transfected with a plasmid containing the sequence for Arg 152 → Glu factor VII using a calcium-dependent, murine anti-factor VII monoclonal antibody column. The clotting activity of mutant factor VII was completely inhibited following incubation with dansyl-Glu-Gly-Arg chloromethyl ketone, suggesting that the apparent clotting activity of mutant factor VII was due to a contaminating serine protease. Immunoblots of mutant factor VII with human factor IXa revealed no cleavage, whereas incubation of mutant factor VII with human factor Xa resulted in cleavage of mutant factor VII and the formation of a lower molecular weight degradation product migrating at M r ∼40 000. The results are consistent with the proposal that zymogen factor VII possesses no intrinsic proteolytic activity toward factor X or factor IX

  8. Multi-agent sequential hypothesis testing

    KAUST Repository

    Kim, Kwang-Ki K.; Shamma, Jeff S.

    2014-01-01

    incorporate costs of taking private/public measurements, costs of time-difference and disagreement in actions of agents, and costs of false declaration/choices in the sequential hypothesis testing. The corresponding sequential decision processes have well

  9. Critical cleavage fracture stress characterization of A508 nuclear pressure vessel steels

    International Nuclear Information System (INIS)

    Wu, Sujun; Jin, Huijin; Sun, Yanbin; Cao, Luowei

    2014-01-01

    The critical cleavage fracture stress of SA508 Gr.4N and SA508 Gr.3 low alloy reactor pressure vessel (RPV) steels was studied through the combination of experiments and finite element method (FEM) analysis. The results showed that the value of the local cleavage fracture stress, σ F , of SA508 Gr.4N steel was significantly higher than that of SA508 Gr.3 steel. Detailed microstructural analysis was carried out using FEGSEM which revealed much smaller grains, finer and more homogenous carbide particles formed in SA508 Gr.4N steel. Compared with the SA508 Gr.3 steel currently used in the nuclear industry, the SA508 Gr.4N steel possesses higher strength and notch toughness as well as improved cleavage fracture behavior, and is considered a better candidate RPV steel for the next generation nuclear reactors. - Highlights: • Critical cleavage fracture stress was calculated through experiments and FEM. • Effects of both grain and carbide particle sizes on σ F were discussed. • The SA508 Gr.4N steel is a better candidate for the next generation nuclear reactors

  10. A set of simple cell processes is sufficient to model spiral cleavage.

    Science.gov (United States)

    Brun-Usan, Miguel; Marín-Riera, Miquel; Grande, Cristina; Truchado-Garcia, Marta; Salazar-Ciudad, Isaac

    2017-01-01

    During cleavage, different cellular processes cause the zygote to become partitioned into a set of cells with a specific spatial arrangement. These processes include the orientation of cell division according to: an animal-vegetal gradient; the main axis (Hertwig's rule) of the cell; and the contact areas between cells or the perpendicularity between consecutive cell divisions (Sachs' rule). Cell adhesion and cortical rotation have also been proposed to be involved in spiral cleavage. We use a computational model of cell and tissue biomechanics to account for the different existing hypotheses about how the specific spatial arrangement of cells in spiral cleavage arises during development. Cell polarization by an animal-vegetal gradient, a bias to perpendicularity between consecutive cell divisions (Sachs' rule), cortical rotation and cell adhesion, when combined, reproduce the spiral cleavage, whereas other combinations of processes cannot. Specifically, cortical rotation is necessary at the 8-cell stage to direct all micromeres in the same direction. By varying the relative strength of these processes, we reproduce the spatial arrangement of cells in the blastulae of seven different invertebrate species. © 2017. Published by The Company of Biologists Ltd.

  11. Sequential stochastic optimization

    CERN Document Server

    Cairoli, Renzo

    1996-01-01

    Sequential Stochastic Optimization provides mathematicians and applied researchers with a well-developed framework in which stochastic optimization problems can be formulated and solved. Offering much material that is either new or has never before appeared in book form, it lucidly presents a unified theory of optimal stopping and optimal sequential control of stochastic processes. This book has been carefully organized so that little prior knowledge of the subject is assumed; its only prerequisites are a standard graduate course in probability theory and some familiarity with discrete-paramet

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

    Science.gov (United States)

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

    2013-07-16

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

  13. Cleavage mechanoluminescence in elemental and III-V semiconductors

    International Nuclear Information System (INIS)

    Chandra, B.P.; Patel, R.P.; Gour, Anubha S.; Chandra, V.K.; Gupta, R.K.

    2003-01-01

    The present paper reports the theory of mechanoluminescence (ML) produced during cleavage of elemental and III-V semiconductors. It seems that the formation of crack-induced localized states is responsible for the ML excitation produced during the cleavage of elemental and III-V semiconductors. According to this mechanism, as the atoms are drawn away from each other in an advancing crack tip, the decreasing wave function overlap across the crack may result in localized states which is associated with increasing electron energy. If the energy of these localized states approach that of the conduction band, transition to the conduction band via tunnelling would be possible, creating minority carriers, and consequently the electron-hole recombination may give rise to mechanoluminescence. When an elemental or III-V semiconductor is cleaved, initially the ML intensity increases with time, attains a peak value I m at the time t m corresponding to completion of the cleavage of the semiconductor, and then it decreases following power law decay. Expressions are derived for the ML intensity I m corresponding to the peak of the ML intensity versus time curve and for the total ML intensity I T . It is shown that both I m and I T should increase directly with the area of the newly created surfaces of the crystals. From the measurements of the ML intensity, the velocity of crack propagation in material can be determined by using the relation v=H/t m

  14. Implementation of a combinatorial cleavage and deprotection scheme

    DEFF Research Database (Denmark)

    Nielsen, John; Rasmussen, Palle H.

    1996-01-01

    Phthalhydrazide libraries are synthesized in solution from substituted hydrazines and phthalimides in several different library formats including single compounds, indexed sub-libraries and a full library. When carried out during solid-phase synthesis, this combinatorial cleavage and deprotection...

  15. Urinary plasmin activates collecting duct ENaC current in preeclampsia

    DEFF Research Database (Denmark)

    Buhl, KB; Friis, Ulla Glenert; Svenningsen, Per

    2012-01-01

    In nephrotic syndrome, plasminogen is aberrantly filtered from plasma to the urinary space and activated along the tubular system. In vitro, plasmin increases ENaC current by proteolytic cleavage of the γ-subunit. It was hypothesized that preeclampsia is associated with plasmin-dependent ability...

  16. Flow-mediated lipoprotein retention; Pregnancy-associated plasma protein-A inhibition in atherosclerosis and its release during myocardial ischemia

    DEFF Research Database (Denmark)

    Steffensen, Lasse Bach

    , which states that retention of lipoproteins is the key initiating event of atherosclerosis development. Pregnancy-associated plasma protein-A (PAPP-A) regulates the insulin-like growth factor (IGF) system by proteolytic cleavage of a subset of IGF-binding proteins. Studies in mice have established PAPP...

  17. Exploring the sequential lineup advantage using WITNESS.

    Science.gov (United States)

    Goodsell, Charles A; Gronlund, Scott D; Carlson, Curt A

    2010-12-01

    Advocates claim that the sequential lineup is an improvement over simultaneous lineup procedures, but no formal (quantitatively specified) explanation exists for why it is better. The computational model WITNESS (Clark, Appl Cogn Psychol 17:629-654, 2003) was used to develop theoretical explanations for the sequential lineup advantage. In its current form, WITNESS produced a sequential advantage only by pairing conservative sequential choosing with liberal simultaneous choosing. However, this combination failed to approximate four extant experiments that exhibited large sequential advantages. Two of these experiments became the focus of our efforts because the data were uncontaminated by likely suspect position effects. Decision-based and memory-based modifications to WITNESS approximated the data and produced a sequential advantage. The next step is to evaluate the proposed explanations and modify public policy recommendations accordingly.

  18. Activities of indigenous proteolytic enzymes in caprine milk of different somatic cell counts.

    Science.gov (United States)

    Albenzio, M; Santillo, A; Kelly, A L; Caroprese, M; Marino, R; Sevi, A

    2015-11-01

    Individual caprine milk with different somatic cell counts (SCC) were studied with the aim of investigating the percentage distribution of leukocyte cell types and the activities of indigenous proteolytic enzymes; proteolysis of casein was also studied in relation to cell type following recovery from milk. The experiment was conducted on 5 intensively managed dairy flocks of Garganica goats; on the basis of SCC, the experimental groups were denoted low (L-SCC; 1,501,000 cells/mL) SCC. Leukocyte distribution differed between groups; polymorphonuclear neutrophilic leukocytes were higher in M-SCC and H-SCC milk samples, the percentage macrophages was the highest in H-SCC, and levels of nonviable cells significantly decreased with increasing SCC. Activities of all the main proteolytic enzymes were affected by SCC; plasmin activity was the highest in H-SCC milk and the lowest in L-SCC, and elastase and cathepsin D activities were the highest in M-SCC. Somatic cell count influenced casein hydrolysis patterns, with less intact α- and β-casein in H-SCC milk. Higher levels of low electrophoretic mobility peptides were detected in sodium caseinate incubated with leukocytes isolated from L-SCC milk, independent of cell type, whereas among cells recovered from M-SCC milk, macrophages yielded the highest levels of low electrophoretic mobility peptides from sodium caseinate. The level of high electrophoretic mobility peptides was higher in sodium caseinate incubated with polymorphonuclear neutrophilic leukocytes and macrophages isolated from M-SCC, whereas the same fraction of peptides was always the highest, independent of leukocyte type, for cells recovered from H-SCC milk. In caprine milk, a level of 700,000 cells/mL represented the threshold for changes in leukocyte distribution, which is presumably related to the immune status of the mammary gland. Differences in the profile of indigenous lysosomal proteolytic enzymes in caprine milk may influence the integrity of casein

  19. Sequential and simultaneous choices: testing the diet selection and sequential choice models.

    Science.gov (United States)

    Freidin, Esteban; Aw, Justine; Kacelnik, Alex

    2009-03-01

    We investigate simultaneous and sequential choices in starlings, using Charnov's Diet Choice Model (DCM) and Shapiro, Siller and Kacelnik's Sequential Choice Model (SCM) to integrate function and mechanism. During a training phase, starlings encountered one food-related option per trial (A, B or R) in random sequence and with equal probability. A and B delivered food rewards after programmed delays (shorter for A), while R ('rejection') moved directly to the next trial without reward. In this phase we measured latencies to respond. In a later, choice, phase, birds encountered the pairs A-B, A-R and B-R, the first implementing a simultaneous choice and the second and third sequential choices. The DCM predicts when R should be chosen to maximize intake rate, and SCM uses latencies of the training phase to predict choices between any pair of options in the choice phase. The predictions of both models coincided, and both successfully predicted the birds' preferences. The DCM does not deal with partial preferences, while the SCM does, and experimental results were strongly correlated to this model's predictions. We believe that the SCM may expose a very general mechanism of animal choice, and that its wider domain of success reflects the greater ecological significance of sequential over simultaneous choices.

  20. Proteolytic Enzymes in Detergents: Evidence of Their Presence through Activity Measurements Based on Electrophoresis

    Science.gov (United States)

    Saperas, Nuria; Fonfria-Subiros, Elsa

    2011-01-01

    This laboratory exercise uses a problem-based approach to expose students to some basic concepts relating to proteins and enzymes. One of the main applications of enzymes at the industrial level is their use in the detergent market. The students examine a detergent sample to ascertain whether proteolytic enzymes are a component and, if so, which…

  1. 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

  2. Mutation in Spike Protein Cleavage Site and Pathogenesis of Feline Coronavirus

    Science.gov (United States)

    Licitra, Beth N.; Millet, Jean K.; Regan, Andrew D.; Hamilton, Brian S.; Rinaldi, Vera D.; Duhamel, Gerald E.

    2013-01-01

    Feline coronaviruses (FCoV) exist as 2 biotypes: feline enteric coronavirus (FECV) and feline infectious peritonitis virus (FIPV). FECV causes subclinical infections; FIPV causes feline infectious peritonitis (FIP), a systemic and fatal disease. It is thought that mutations in FECV enable infection of macrophages, causing FIP. However, the molecular basis for this biotype switch is unknown. We examined a furin cleavage site in the region between receptor-binding (S1) and fusion (S2) domains of the spike of serotype 1 FCoV. FECV sequences were compared with FIPV sequences. All FECVs had a conserved furin cleavage motif. For FIPV, there was a correlation with the disease and >1 substitution in the S1/S2 motif. Fluorogenic peptide assays confirmed that the substitutions modulate furin cleavage. We document a functionally relevant S1/S2 mutation that arises when FIP develops in a cat. These insights into FIP pathogenesis may be useful in development of diagnostic, prevention, and treatment measures against coronaviruses. PMID:23763835

  3. Trichomonas vaginalis Metalloproteinase Induces mTOR Cleavage of SiHa Cells

    Science.gov (United States)

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

    2014-01-01

    Trichomonas vaginalis secretes a number of proteases which are suspected to be the cause of pathogenesis; however, little is understood how they manipulate host cells. The mammalian target of rapamycin (mTOR) regulates cell growth, cell proliferation, cell motility, cell survival, protein synthesis, and transcription. We detected various types of metalloproteinases including GP63 protein from T. vaginalis trophozoites, and T. vaginalis GP63 metalloproteinase was confirmed by sequencing and western blot. When SiHa cells were stimulated with live T. vaginalis, T. vaginalis excretory-secretory products (ESP) or T. vaginalis lysate, live T. vaginalis and T. vaginalis ESP induced the mTOR cleavage in both time- and parasite load-dependent manner, but T. vaginalis lysate did not. Pretreatment of T. vaginalis with a metalloproteinase inhibitor, 1,10-phenanthroline, completely disappeared the mTOR cleavage in SiHa cells. Collectively, T. vaginalis metallopeptidase induces host cell mTOR cleavage, which may be related to survival of the parasite. PMID:25548410

  4. Sequential memory: Binding dynamics

    Science.gov (United States)

    Afraimovich, Valentin; Gong, Xue; Rabinovich, Mikhail

    2015-10-01

    Temporal order memories are critical for everyday animal and human functioning. Experiments and our own experience show that the binding or association of various features of an event together and the maintaining of multimodality events in sequential order are the key components of any sequential memories—episodic, semantic, working, etc. We study a robustness of binding sequential dynamics based on our previously introduced model in the form of generalized Lotka-Volterra equations. In the phase space of the model, there exists a multi-dimensional binding heteroclinic network consisting of saddle equilibrium points and heteroclinic trajectories joining them. We prove here the robustness of the binding sequential dynamics, i.e., the feasibility phenomenon for coupled heteroclinic networks: for each collection of successive heteroclinic trajectories inside the unified networks, there is an open set of initial points such that the trajectory going through each of them follows the prescribed collection staying in a small neighborhood of it. We show also that the symbolic complexity function of the system restricted to this neighborhood is a polynomial of degree L - 1, where L is the number of modalities.

  5. Condensed tannins: A novel rearrangement of procyanidins and prodelphinidins in thiolytic cleavage

    Science.gov (United States)

    G. Wayne McGraw; Jan P. Steynberg; Richard W. Hemingway

    1993-01-01

    Conditions commonly used for the thiolytic cleavage of interflavanoid bonds of condensed tannins also result in cleavage of the C4 to C10 bond of flavan units. Subsequenet lectrophilic attack of the C4 carbocation on the C2' or C6' of the B-ring, and loss of phloroglucinol (the A-ring), result in the formation of a mixture of 1,3-dithiobenzyl-2,4,s,6-...

  6. Yeast ribonuclease III uses a network of multiple hydrogen bonds for RNA binding and cleavage.

    Science.gov (United States)

    Lavoie, Mathieu; Abou Elela, Sherif

    2008-08-19

    Members of the bacterial RNase III family recognize a variety of short structured RNAs with few common features. It is not clear how this group of enzymes supports high cleavage fidelity while maintaining a broad base of substrates. Here we show that the yeast orthologue of RNase III (Rnt1p) uses a network of 2'-OH-dependent interactions to recognize substrates with different structures. We designed a series of bipartite substrates permitting the distinction between binding and cleavage defects. Each substrate was engineered to carry a single or multiple 2'- O-methyl or 2'-fluoro ribonucleotide substitutions to prevent the formation of hydrogen bonds with a specific nucleotide or group of nucleotides. Interestingly, introduction of 2'- O-methyl ribonucleotides near the cleavage site increased the rate of catalysis, indicating that 2'-OH are not required for cleavage. Substitution of nucleotides in known Rnt1p binding site with 2'- O-methyl ribonucleotides inhibited cleavage while single 2'-fluoro ribonucleotide substitutions did not. This indicates that while no single 2'-OH is essential for Rnt1p cleavage, small changes in the substrate structure are not tolerated. Strikingly, several nucleotide substitutions greatly increased the substrate dissociation constant with little or no effect on the Michaelis-Menten constant or rate of catalysis. Together, the results indicate that Rnt1p uses a network of nucleotide interactions to identify its substrate and support two distinct modes of binding. One mode is primarily mediated by the dsRNA binding domain and leads to the formation of stable RNA/protein complex, while the other requires the presence of the nuclease and N-terminal domains and leads to RNA cleavage.

  7. Screening for Proteolytic Activities in Snake Venom by Means of a Multiplexing ESI-MS Assay Scheme

    NARCIS (Netherlands)

    Liesener, A.; Perchuc, Anna-Maria; Schöni, Reto; Wilmer, Marianne; Karst, U.

    2005-01-01

    A multiplexed mass spectrometry based assay scheme for the simultaneous determination of five different substrate/product pairs was developed as a tool for screening of proteolytic activities in snake venom fractions from Bothrops moojeni. The assay scheme was employed in the functional

  8. Sequential Probability Ration Tests : Conservative and Robust

    NARCIS (Netherlands)

    Kleijnen, J.P.C.; Shi, Wen

    2017-01-01

    In practice, most computers generate simulation outputs sequentially, so it is attractive to analyze these outputs through sequential statistical methods such as sequential probability ratio tests (SPRTs). We investigate several SPRTs for choosing between two hypothesized values for the mean output

  9. 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.

  10. Proteolytic processing of connective tissue growth factor in normal ocular tissues and during corneal wound healing.

    Science.gov (United States)

    Robinson, Paulette M; Smith, Tyler S; Patel, Dilan; Dave, Meera; Lewin, Alfred S; Pi, Liya; Scott, Edward W; Tuli, Sonal S; Schultz, Gregory S

    2012-12-13

    Connective tissue growth factor (CTGF) is a fibrogenic cytokine that is up-regulated by TGF-β and mediates most key fibrotic actions of TGF-β, including stimulation of synthesis of extracellular matrix and differentiation of fibroblasts into myofibroblasts. This study addresses the role of proteolytic processing of CTGF in human corneal fibroblasts (HCF) stimulated with TGF-β, normal ocular tissues and wounded corneas. Proteolytic processing of CTGF in HCF cultures, normal animal eyes, and excimer laser wounded rat corneas were examined by Western blot. The identity of a 21-kDa band was determined by tandem mass spectrometry, and possible alternative splice variants of CTGF were assessed by 5' Rapid Amplification of cDNA Ends (RACE). HCF stimulated by TGF-β contained full length 38-kDa CTGF and fragments of 25, 21, 18, and 13 kDa, while conditioned medium contained full length 38- and a 21-kDa fragment of CTGF that contained the middle "hinge" region of CTGF. Fragmentation of recombinant CTGF incubated in HCF extracts was blocked by the aspartate protease inhibitor, pepstatin. Normal mouse, rat, and rabbit whole eyes and rabbit ocular tissues contained abundant amounts of C-terminal 25- and 21-kDa fragments and trace amounts of 38-kDa CTGF, although no alternative transcripts were detected. All forms of CTGF (38, 25, and 21 kDa) were detected during healing of excimer ablated rat corneas, peaking on day 11. Proteolytic processing of 38-kDa CTGF occurs during corneal wound healing, which may have important implications in regulation of corneal scar formation.

  11. Interaction of Leptospira interrogans with Human Proteolytic Systems Enhances Dissemination through Endothelial Cells and Protease Levels

    Science.gov (United States)

    Vieira, Monica L.; Alvarez-Flores, Miryam P.; Kirchgatter, Karin; Romero, Eliete C.; Alves, Ivy J.; de Morais, Zenaide M.; Vasconcellos, Silvio A.; Chudzinski-Tavassi, Ana M.

    2013-01-01

    We have recently reported the ability of Leptospira to capture plasminogen (PLG) and generate plasmin (PLA) bound on the microbial surface in the presence of exogenous activators. In this work, we examined the effects of leptospiral PLG binding for active penetration through the endothelial cell barrier and activation. The results indicate that leptospires with PLG association or PLA activation have enhanced migration activity through human umbilical vein endothelial cell (HUVEC) monolayers compared with untreated bacteria. Leptospira cells coated with PLG were capable of stimulating the expression of PLG activators by HUVECs. Moreover, leptospires endowed with PLG or PLA promoted transcriptional upregulation matrix metalloprotease 9 (MMP-9). Serum samples from patients with confirmed leptospirosis showed higher levels of PLG activators and total MMP-9 than serum samples from normal (healthy) subjects. The highest level of PLG activators and total MMP-9 was detected with microscopic agglutination test (MAT)-negative serum samples, suggesting that this proteolytic activity stimulation occurs at the early stage of the disease. Furthermore, a gelatin zymography profile obtained for MMPs with serum samples from patients with leptospirosis appears to be specific to leptospiral infection because serum samples from patients with unrelated infectious diseases produced no similar degradation bands. Altogether, the data suggest that the Leptospira-associated PLG or PLA might represent a mechanism that contributes to bacterial penetration of endothelial cells through an activation cascade of events that enhances the proteolytic capability of the organism. To our knowledge, this is the first proteolytic activity associated with leptospiral pathogenesis described to date. PMID:23478319

  12. Characterisation of proteolytic activity of excretory-secretory products from adult Strongylus vulgaris.

    Science.gov (United States)

    Caffrey, C R; Ryan, M F

    1994-04-01

    An excretory-secretory (ES) preparation derived from adult Strongylus vulgaris in vitro was assessed for proteolytic activity using azocasein and synthetic, fluorogenic, peptide substrates. Fractionation was by molecular sieve fast protein liquid chromatography (molecular sieve FPLC) and resolution by gelatin-substrate sodium dodecyl sulphate-polyacrylamide gel electrophoresis (gelatin-substrate SDS-PAGE). The cysteine proteinase activator, dithiothreitol (DTT), enhanced azocaseinolysis and hydrolysis of carbobenzoxy-phenylalanyl-arginine-7-amido-4-methylcoumarin (Z-Phe-Arg-NMec) by the ES preparation and was a requirement for the detection of carbobenzoxy-arginyl-arginine-7-amido-4-methylcoumarin (Z-Arg-Arg-NMec) hydrolysis. Assays of FPLC-eluted fractions, with DTT, detected a broad peak of azocaseinolytic activity (22-24 kDa) and two peaks (24 and 18 kDa) of hydrolysis using the synthetic substrates. Hydrolysis by these peaks of Z-Phe-Arg-NMec was 50-fold greater than that of Z-Arg-Arg-NMec suggesting that their specificities are more like papain or cathepsin L rather than cathepsin B. In gelatin-substrate SDS-PAGE, DTT was required to detect proteolysis by the ES preparation which was optimal at pH 6.0 and resolved into eight bands (87-29 kDa). Cysteine proteinase inhibitors were the most effective in all assays. Collectively, these data indicate that cysteine-class proteolytic activity predominates in the ES preparation of adult S. vulgaris.

  13. Antifungal and proteolytic activities of endophytic fungi isolated from Piper hispidum Sw

    Directory of Open Access Journals (Sweden)

    Ravely Casarotti Orlandelli

    2015-06-01

    Full Text Available Endophytes are being considered for use in biological control, and the enzymes they secrete might facilitate their initial colonization of internal plant tissues and direct interactions with microbial pathogens. Microbial proteases are also biotechnologically important products employed in bioremediation processes, cosmetics, and the pharmaceutical, photographic and food industries. In the present study, we evaluated antagonism and competitive interactions between 98 fungal endophytes and Alternaria alternata, Colletotrichum sp., Phyllosticta citricarpa and Moniliophthora perniciosa. We also examined the proteolytic activities of endophytes grown in liquid medium and conducted cup plate assays. The results showed that certain strains in the assemblage of P. hispidum endophytes are important sources of antifungal properties, primarily Lasiodiplodia theobromae JF766989, which reduced phytopathogen growth by approximately 54 to 65%. We detected 28 endophytes producing enzymatic halos of up to 16.40 mm in diameter. The results obtained in the present study highlight the proteolytic activity of the endophytes Phoma herbarum JF766995 and Schizophyllum commune JF766994, which presented the highest enzymatic halo diameters under at least one culture condition tested. The increased activities of certain isolates in the presence of rice or soy flour as a substrate (with halos up to 17.67 mm in diameter suggests that these endophytes have the potential to produce enzymes using agricultural wastes.

  14. Polycystin-1 C-terminal Cleavage Is Modulated by Polycystin-2 Expression*

    Science.gov (United States)

    Bertuccio, Claudia A.; Chapin, Hannah C.; Cai, Yiqiang; Mistry, Kavita; Chauvet, Veronique; Somlo, Stefan; Caplan, Michael J.

    2009-01-01

    Autosomal dominant polycystic kidney disease is caused by mutations in the genes encoding polycystin-1 (PC-1) and polycystin-2 (PC-2). PC-1 cleavage releases its cytoplasmic C-terminal tail (CTT), which enters the nucleus. To determine whether PC-1 CTT cleavage is influenced by PC-2, a quantitative cleavage assay was utilized, in which the DNA binding and activation domains of Gal4 and VP16, respectively, were appended to PC-1 downstream of its CTT domain (PKDgalvp). Cells cotransfected with the resultant PKDgalvp fusion protein and PC-2 showed an increase in luciferase activity and in CTT expression, indicating that the C-terminal tail of PC-1 is cleaved and enters the nucleus. To assess whether CTT cleavage depends upon Ca2+ signaling, cells transfected with PKDgalvp alone or together with PC-2 were incubated with several agents that alter intracellular Ca2+ concentrations. PC-2 enhancement of luciferase activity was not altered by any of these treatments. Using a series of PC-2 C-terminal truncated mutations, we identified a portion of the PC-2 protein that is required to stimulate PC-1 CTT accumulation. These data demonstrate that release of the CTT from PC-1 is influenced and stabilized by PC-2. This effect is independent of Ca2+ but is regulated by sequences contained within the PC-2 C-terminal tail, suggesting a mechanism through which PC-1 and PC-2 may modulate a novel signaling pathway. PMID:19491093

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

    International Nuclear Information System (INIS)

    Cleaver, J.E.

    1983-01-01

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

  16. Sequential lineup presentation: Patterns and policy

    OpenAIRE

    Lindsay, R C L; Mansour, Jamal K; Beaudry, J L; Leach, A-M; Bertrand, M I

    2009-01-01

    Sequential lineups were offered as an alternative to the traditional simultaneous lineup. Sequential lineups reduce incorrect lineup selections; however, the accompanying loss of correct identifications has resulted in controversy regarding adoption of the technique. We discuss the procedure and research relevant to (1) the pattern of results found using sequential versus simultaneous lineups; (2) reasons (theory) for differences in witness responses; (3) two methodological issues; and (4) im...

  17. Divalent metal ion differentially regulates the sequential nicking reactions of the GIY-YIG homing endonuclease I-BmoI.

    Directory of Open Access Journals (Sweden)

    Benjamin P Kleinstiver

    Full Text Available Homing endonucleases are site-specific DNA endonucleases that function as mobile genetic elements by introducing double-strand breaks or nicks at defined locations. Of the major families of homing endonucleases, the modular GIY-YIG endonucleases are least understood in terms of mechanism. The GIY-YIG homing endonuclease I-BmoI generates a double-strand break by sequential nicking reactions during which the single active site of the GIY-YIG nuclease domain must undergo a substantial reorganization. Here, we show that divalent metal ion plays a significant role in regulating the two independent nicking reactions by I-BmoI. Rate constant determination for each nicking reaction revealed that limiting divalent metal ion has a greater impact on the second strand than the first strand nicking reaction. We also show that substrate mutations within the I-BmoI cleavage site can modulate the first strand nicking reaction over a 314-fold range. Additionally, in-gel DNA footprinting with mutant substrates and modeling of an I-BmoI-substrate complex suggest that amino acid contacts to a critical GC-2 base pair are required to induce a bottom-strand distortion that likely directs conformational changes for reaction progress. Collectively, our data implies mechanistic roles for divalent metal ion and substrate bases, suggesting that divalent metal ion facilitates the re-positioning of the GIY-YIG nuclease domain between sequential nicking reactions.

  18. Control of extracellular cleavage of ProBDNF by high frequency neuronal activity

    OpenAIRE

    Nagappan, Guhan; Zaitsev, Eugene; Senatorov, Vladimir V.; Yang, Jianmin; Hempstead, Barbara L.; Lu, Bai

    2009-01-01

    Pro- and mature neurotrophins often elicit opposing biological effects. For example, mature brain-derived neurotrophic factor (mBDNF) is critical for long-term potentiation induced by high-frequency stimulation, whereas proBDNF facilitate long-term depression induced by low-frequency stimulation. Because mBDNF is derived from proBDNF by endoproteolytic cleavage, mechanisms regulating the cleavage of proBDNF may control the direction of BDNF regulation. Using methods that selectively detect pr...

  19. Sequential Product of Quantum Effects: An Overview

    Science.gov (United States)

    Gudder, Stan

    2010-12-01

    This article presents an overview for the theory of sequential products of quantum effects. We first summarize some of the highlights of this relatively recent field of investigation and then provide some new results. We begin by discussing sequential effect algebras which are effect algebras endowed with a sequential product satisfying certain basic conditions. We then consider sequential products of (discrete) quantum measurements. We next treat transition effect matrices (TEMs) and their associated sequential product. A TEM is a matrix whose entries are effects and whose rows form quantum measurements. We show that TEMs can be employed for the study of quantum Markov chains. Finally, we prove some new results concerning TEMs and vector densities.

  20. Optimal Sequential Rules for Computer-Based Instruction.

    Science.gov (United States)

    Vos, Hans J.

    1998-01-01

    Formulates sequential rules for adapting the appropriate amount of instruction to learning needs in the context of computer-based instruction. Topics include Bayesian decision theory, threshold and linear-utility structure, psychometric model, optimal sequential number of test questions, and an empirical example of sequential instructional…

  1. Primary structure, gene organization and polypeptide expression of poliovirus RNA

    Energy Technology Data Exchange (ETDEWEB)

    Kitamura, N. (State Univ. of New York, Stony Brook); Semler, B.L.; Rothberg, P.G.

    1981-06-18

    The primary structure of the poliovirus genome has been determined. The RNA molecule is 7433 nucleotides long, polyadenylated at the 3' terminus, and covalently linked to a small protein (VPg) at the 5' terminus. An open reading frame of 2207 consecutive triplets spans over 89% of the nucleotide sequence and codes for the viral polyprotein NCVPOO. Twelve viral polypeptides have been mapped by amino acid sequence analysis and were found to be proteolytic cleavage products of the polyprotein, cleavages occurring predominantly at Gln-Gly pairs.

  2. Stimulation of NADH-dependent microsomal DNA strand cleavage by rifamycin SV.

    Science.gov (United States)

    Kukiełka, E; Cederbaum, A I

    1995-04-15

    Rifamycin SV is an antibiotic anti-bacterial agent used in the treatment of tuberculosis. This drug can autoxidize, especially in the presence of metals, and generate reactive oxygen species. A previous study indicated that rifamycin SV can increase NADH-dependent microsomal production of reactive oxygen species. The current study evaluated the ability of rifamycin SV to interact with iron and increase microsomal production of hydroxyl radical, as detected by conversion of supercoiled plasmid DNA into the relaxed open circular state. The plasmid used was pBluescript II KS(-), and the forms of DNA were separated by agarose-gel electrophoresis. Incubation of rat liver microsomes with plasmid plus NADH plus ferric-ATP caused DNA strand cleavage. The addition of rifamycin SV produced a time- and concentration-dependent increase in DNA-strand cleavage. No stimulation by rifamycin SV occurred in the absence of microsomes, NADH or ferric-ATP. Stimulation occurred with other ferric complexes besides ferric-ATP, e.g. ferric-histidine, ferric-citrate, ferric-EDTA, and ferric-(NH4)2SO4. Rifamycin SV did not significantly increase the high rates of DNA strand cleavage found with NADPH as the microsomal reductant. The stimulation of NADH-dependent microsomal DNA strand cleavage was completely blocked by catalase, superoxide dismutase, GSH and a variety of hydroxyl-radical-scavenging agents, but not by anti-oxidants that prevent microsomal lipid peroxidation. Redox cycling agents, such as menadione and paraquat, in contrast with rifamycin SV, stimulated the NADPH-dependent reaction; menadione and rifamycin SV were superior to paraquat in stimulating the NADH-dependent reaction. These results indicate that rifamycin SV can, in the presence of an iron catalyst, increase microsomal production of reactive oxygen species which can cause DNA-strand cleavage. In contrast with other redox cycling agents, the stimulation by rifamycin SV is more pronounced with NADH than with NADPH as the

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

    Science.gov (United States)

    Adam Smith, R; Sewell, Sarah L; Giorgio, Todd D

    2008-01-01

    The development and in vitro performance of a modular nanoscale system capable of specific structural modification by enzymatic activity is described in this work. Due to its small physical size and adaptable characteristics, this system has the potential for utilization in targeted delivery systems and biosensing. Nanoparticle probes were synthesized containing two distinct fluorescent species including a quantum dot base particle and fluorescently labeled cleavable peptide substrate. Activity of these probes was monitored by gel electrophoresis with quantitative cleavage measurements made by fluorometric analysis. The model proximity-activated nanoparticles studied here exhibit significant susceptibility to cleavage by matrix metalloprotease-7 (MMP-7) at physiologically relevant concentrations, with nearly complete cleavage of available substrate molecules after 24 hours. This response is specific to MMP-7 enzyme activity, as cleavage is completely inhibited with the addition of EDTA. Utilization of enzyme-specific modification is a sensitive approach with broad applications for targeted therapeutics and biosensing. The versatility of this nanoparticle system is highlighted in its modular design, as it has the capability to integrate characteristics for detection, biosensing, targeting, and payload delivery into a single, multifunctional nanoparticle structure. PMID:18488420

  4. Quantum Inequalities and Sequential Measurements

    International Nuclear Information System (INIS)

    Candelpergher, B.; Grandouz, T.; Rubinx, J.L.

    2011-01-01

    In this article, the peculiar context of sequential measurements is chosen in order to analyze the quantum specificity in the two most famous examples of Heisenberg and Bell inequalities: Results are found at some interesting variance with customary textbook materials, where the context of initial state re-initialization is described. A key-point of the analysis is the possibility of defining Joint Probability Distributions for sequential random variables associated to quantum operators. Within the sequential context, it is shown that Joint Probability Distributions can be defined in situations where not all of the quantum operators (corresponding to random variables) do commute two by two. (authors)

  5. Proteolytic activities of kiwifruit actinidin (Actinidia deliciosa cv. Hayward) on different fibrous and globular proteins: a comparative study of actinidin with papain.

    Science.gov (United States)

    Chalabi, Maryam; Khademi, Fatemeh; Yarani, Reza; Mostafaie, Ali

    2014-04-01

    Actinidin, a member of the papain-like family of cysteine proteases, is abundant in kiwifruit. To date, a few studies have been provided to investigate the proteolytic activity and substrate specificity of actinidin on native proteins. Herein, the proteolytic activity of actinidin was compared to papain on several different fibrous and globular proteins under neutral, acidic and basic conditions. The digested samples were subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and densitometry to assess the proteolytic effect. Furthermore, the levels of free amino nitrogen (FAN) of the treated samples were determined using the ninhydrin colorimetric method. The findings showed that actinidin has no or limited proteolytic effect on globular proteins such as immunoglobulins including sheep IgG, rabbit IgG, chicken IgY and fish IgM, bovine serum albumin (BSA), lipid transfer protein (LTP), and whey proteins (α-lactalbumin and β-lactoglobulin) compared to papain. In contrast to globular proteins, actinidin could hydrolyze collagen and fibrinogen perfectly at neutral and mild basic pHs. Moreover, this enzyme could digest pure α-casein and major subunits of micellar casein especially in acidic pHs. Taken together, the data indicated that actinidin has narrow substrate specificity with the highest enzymatic activity for the collagen and fibrinogen substrates. The results describe the actinidin as a mild plant protease useful for many special applications such as cell isolation from different tissues and some food industries as a mixture formula with other relevant proteases.

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

    Directory of Open Access Journals (Sweden)

    Jiangning Song

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

  7. Mechanism of Excretion of a Bacterial Proteinase: Demonstration of Two Proteolytic Enzymes Produced by a Sarcina Strain (Coccus P)

    Energy Technology Data Exchange (ETDEWEB)

    SARNER, NITZA Z; BISSELL, MINA J; GIROLAMO, MARIO Di; GORINI, LUIGI

    1970-06-29

    A Sarcina strain (Coccus P) produces two proteolytic enzymes. One is found only extracellularly, is far more prevalent, and is actively excreted during exponential growth. It is the enzyme responsible for the known strong proteolytic activity of the cultures of this strain. A second protease is, however, produced which remains associated with the intact cells but is released by the protoplasts. The two enzymes appear unrelated in their derivation. Calcium ions play an essential role in preventing autodigestion of the excreted enzyme. Bacterial proteins are found outside the cell boundary as a consequence either of passive processes such as leakage or lysis or of active excretion. Under conditions in which leakage and lysis do not occur, as during exponential growth, the cell boundary is a barrier causing a complete separation of the bulk of the intracellular proteins from the one or very few extracellular proteins, with no trace of either type being detectable on the wrong side of the boundary. Since in bacteria there is no evidence of protein being produced other than internally, the separation into intraand extracellular proteins should occur after peptide chain formation. The question arises as to whether the structure of the cell boundary or that of the excreted proteins themselves determines this separation. Coccus P, a Sarcina closely related to Micrococcus lysodeikticus (3), produces an extracellular proteinase during the exponential phase of growth so that the process appears to be active excretion. The organism grows exponentially in a defined synthetic medium (12) to relatively high cell density (10{sup 9} cells/ml); therefore the mechanism of excretion can be studied over an extended period of time without the difficulties of changing growth rates. Coagulation of reconstituted skim milk provides a simple and sensitive assay for enzyme activity (I 1). The extracellular proteinase has also been purified and partially characterized (6-8). It has been shown

  8. Fe-Catalyzed Oxidative Cleavage of Unsaturated Fatty Acids

    NARCIS (Netherlands)

    Spannring, P.

    2013-01-01

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

  9. FKBP12 regulates the localization and processing of amyloid ...

    Indian Academy of Sciences (India)

    2014-01-27

    Jan 27, 2014 ... One of the pathological hallmarks of Alzheimer's disease is the presence of insoluble extracellular amyloid plaques. These plaques ... The proteolytic cleavage of amyloid precursor protein (APP) ..... lower sAPPα/sAPPs ratio, which may lead to an increase in ..... spine density in healthy adult mouse brain.

  10. The proteolytic activity of pregnancy-associated plasma protein-A is potentially regulated by stanniocalcin-1 and -2 during human ovarian follicle development

    DEFF Research Database (Denmark)

    Jepsen, Malene R.; Kløverpris, Søren; Bøtkjær, Jane A.

    2016-01-01

    STUDY QUESTION: Is the proteolytic activity of pregnancy-associated plasma protein-A (PAPP-A) regulated by the stanniocalcins (STC1 and STC2) during human follicle maturation? SUMMARY ANSWER: The STCs and PAPP-A show similar expression by immunohistochemistry in developing follicles, and regulation...... of PAPP-A proteolytic activity is suggested by the identification of inhibited protein complexes between PAPP-A and STC1 or STC2 in human follicular fluid (FF). WHAT IS KNOWN ALREADY: The insulin-like growth factor (IGF)-regulating proteinase PAPP-A is secreted by the granulosa cells of estrogen...

  11. Sensitive microplate assay for the detection of proteolytic enzymes using radiolabeled gelatin

    International Nuclear Information System (INIS)

    Robertson, B.D.; Kwan-Lim, G.E.; Maizels, R.M.

    1988-01-01

    A sensitive, microplate assay is described for the detection of a wide range of proteolytic enzymes, using radio-iodine-labeled gelatin as substrate. The technique uses the Bolton-Hunter reagent to label the substrate, which is then coated onto the wells of polyvinyl chloride microtiter plates. By measuring the radioactivity released the assay is able to detect elastase, trypsin, and collagenase in concentrations of 1 ng/ml or less, while the microtiter format permits multiple sample handling and minimizes sample volumes required for analysis

  12. Signal peptide discrimination and cleavage site identification using SVM and NN.

    Science.gov (United States)

    Kazemian, H B; Yusuf, S A; White, K

    2014-02-01

    About 15% of all proteins in a genome contain a signal peptide (SP) sequence, at the N-terminus, that targets the protein to intracellular secretory pathways. Once the protein is targeted correctly in the cell, the SP is cleaved, releasing the mature protein. Accurate prediction of the presence of these short amino-acid SP chains is crucial for modelling the topology of membrane proteins, since SP sequences can be confused with transmembrane domains due to similar composition of hydrophobic amino acids. This paper presents a cascaded Support Vector Machine (SVM)-Neural Network (NN) classification methodology for SP discrimination and cleavage site identification. The proposed method utilises a dual phase classification approach using SVM as a primary classifier to discriminate SP sequences from Non-SP. The methodology further employs NNs to predict the most suitable cleavage site candidates. In phase one, a SVM classification utilises hydrophobic propensities as a primary feature vector extraction using symmetric sliding window amino-acid sequence analysis for discrimination of SP and Non-SP. In phase two, a NN classification uses asymmetric sliding window sequence analysis for prediction of cleavage site identification. The proposed SVM-NN method was tested using Uni-Prot non-redundant datasets of eukaryotic and prokaryotic proteins with SP and Non-SP N-termini. Computer simulation results demonstrate an overall accuracy of 0.90 for SP and Non-SP discrimination based on Matthews Correlation Coefficient (MCC) tests using SVM. For SP cleavage site prediction, the overall accuracy is 91.5% based on cross-validation tests using the novel SVM-NN model. © 2013 Published by Elsevier Ltd.

  13. Sequence specificity of DNA cleavage by Micrococcus luteus γ endonuclease

    International Nuclear Information System (INIS)

    Hentosh, P.; Henner, W.D.; Reynolds, R.J.

    1985-01-01

    DNA fragments of defined sequence have been used to determine the sites of cleavage by γ-endonuclease activity in extracts prepared from Micrococcus luteus. End-labeled DNA restriction fragments of pBR322 DNA that had been irradiated under nitrogen in the presence of potassium iodide or t-butanol were treated with M. luteus γ endonuclease and analyzed on irradiated DNA preferentially at the positions of cytosines and thymines. DNA cleavage occurred immediately to the 3' side of pyrimidines in irradiated DNA and resulted in fragments that terminate in a 5'-phosphoryl group. These studies indicate that both altered cytosines and thymines may be important DNA lesions requiring repair after exposure to γ radiation

  14. DNA Cleavage Activity of Diazonium Salts: Chemical Nucleases

    OpenAIRE

    KIZIL, Murat

    2014-01-01

    4-Fenoldiazonium tetrafluoroborate and 4-benzoicaciddiazonium tetrafluoroborate was prepared and was shown to be an effective DNA cleavage agent in the presence of the 1-electron donor copper(II) chloride. Its mechanism involves the generation of the aryl radical cleaving DNA by hydrogen atom abstraction from deoxyribose sugar.

  15. Antibody-Mediated Neutralization of uPA Proteolytic Function Reduces Disease Progression in Mouse Arthritis Models

    DEFF Research Database (Denmark)

    Almholt, Kasper; Hebsgaard, Josephine B; Nansen, Anneline

    2018-01-01

    the potential in mice of an Ab that blocks the proteolytic capacity of uPA in the CIA model and the delayed-type hypersensitivity arthritis model. A second aim was to determine the cellular origins of uPA and the uPA receptor (uPAR) in joint tissue from patients with rheumatoid arthritis. A mAb that neutralizes...

  16. Diversity of Neuropeptide Cell-Cell Signaling Molecules Generated by Proteolytic Processing Revealed by Neuropeptidomics Mass Spectrometry

    Science.gov (United States)

    Hook, Vivian; Lietz, Christopher B.; Podvin, Sonia; Cajka, Tomas; Fiehn, Oliver

    2018-04-01

    Neuropeptides are short peptides in the range of 3-40 residues that are secreted for cell-cell communication in neuroendocrine systems. In the nervous system, neuropeptides comprise the largest group of neurotransmitters. In the endocrine system, neuropeptides function as peptide hormones to coordinate intercellular signaling among target physiological systems. The diversity of neuropeptide functions is defined by their distinct primary sequences, peptide lengths, proteolytic processing of pro-neuropeptide precursors, and covalent modifications. Global, untargeted neuropeptidomics mass spectrometry is advantageous for defining the structural features of the thousands to tens of thousands of neuropeptides present in biological systems. Defining neuropeptide structures is the basis for defining the proteolytic processing pathways that convert pro-neuropeptides into active peptides. Neuropeptidomics has revealed that processing of pro-neuropeptides occurs at paired basic residues sites, and at non-basic residue sites. Processing results in neuropeptides with known functions and generates novel peptides representing intervening peptide domains flanked by dibasic residue processing sites, identified by neuropeptidomics. While very short peptide products of 2-4 residues are predicted from pro-neuropeptide dibasic processing sites, such peptides have not been readily identified; therefore, it will be logical to utilize metabolomics to identify very short peptides with neuropeptidomics in future studies. Proteolytic processing is accompanied by covalent post-translational modifications (PTMs) of neuropeptides comprising C-terminal amidation, N-terminal pyroglutamate, disulfide bonds, phosphorylation, sulfation, acetylation, glycosylation, and others. Neuropeptidomics can define PTM features of neuropeptides. In summary, neuropeptidomics for untargeted, global analyses of neuropeptides is essential for elucidation of proteases that generate diverse neuropeptides for cell

  17. Diversity of Neuropeptide Cell-Cell Signaling Molecules Generated by Proteolytic Processing Revealed by Neuropeptidomics Mass Spectrometry

    Science.gov (United States)

    Hook, Vivian; Lietz, Christopher B.; Podvin, Sonia; Cajka, Tomas; Fiehn, Oliver

    2018-05-01

    Neuropeptides are short peptides in the range of 3-40 residues that are secreted for cell-cell communication in neuroendocrine systems. In the nervous system, neuropeptides comprise the largest group of neurotransmitters. In the endocrine system, neuropeptides function as peptide hormones to coordinate intercellular signaling among target physiological systems. The diversity of neuropeptide functions is defined by their distinct primary sequences, peptide lengths, proteolytic processing of pro-neuropeptide precursors, and covalent modifications. Global, untargeted neuropeptidomics mass spectrometry is advantageous for defining the structural features of the thousands to tens of thousands of neuropeptides present in biological systems. Defining neuropeptide structures is the basis for defining the proteolytic processing pathways that convert pro-neuropeptides into active peptides. Neuropeptidomics has revealed that processing of pro-neuropeptides occurs at paired basic residues sites, and at non-basic residue sites. Processing results in neuropeptides with known functions and generates novel peptides representing intervening peptide domains flanked by dibasic residue processing sites, identified by neuropeptidomics. While very short peptide products of 2-4 residues are predicted from pro-neuropeptide dibasic processing sites, such peptides have not been readily identified; therefore, it will be logical to utilize metabolomics to identify very short peptides with neuropeptidomics in future studies. Proteolytic processing is accompanied by covalent post-translational modifications (PTMs) of neuropeptides comprising C-terminal amidation, N-terminal pyroglutamate, disulfide bonds, phosphorylation, sulfation, acetylation, glycosylation, and others. Neuropeptidomics can define PTM features of neuropeptides. In summary, neuropeptidomics for untargeted, global analyses of neuropeptides is essential for elucidation of proteases that generate diverse neuropeptides for cell

  18. Programmable RNA recognition and cleavage by CRISPR/Cas9.

    Science.gov (United States)

    O'Connell, Mitchell R; Oakes, Benjamin L; Sternberg, Samuel H; East-Seletsky, Alexandra; Kaplan, Matias; Doudna, Jennifer A

    2014-12-11

    The CRISPR-associated protein Cas9 is an RNA-guided DNA endonuclease that uses RNA-DNA complementarity to identify target sites for sequence-specific double-stranded DNA (dsDNA) cleavage. In its native context, Cas9 acts on DNA substrates exclusively because both binding and catalysis require recognition of a short DNA sequence, known as the protospacer adjacent motif (PAM), next to and on the strand opposite the twenty-nucleotide target site in dsDNA. Cas9 has proven to be a versatile tool for genome engineering and gene regulation in a large range of prokaryotic and eukaryotic cell types, and in whole organisms, but it has been thought to be incapable of targeting RNA. Here we show that Cas9 binds with high affinity to single-stranded RNA (ssRNA) targets matching the Cas9-associated guide RNA sequence when the PAM is presented in trans as a separate DNA oligonucleotide. Furthermore, PAM-presenting oligonucleotides (PAMmers) stimulate site-specific endonucleolytic cleavage of ssRNA targets, similar to PAM-mediated stimulation of Cas9-catalysed DNA cleavage. Using specially designed PAMmers, Cas9 can be specifically directed to bind or cut RNA targets while avoiding corresponding DNA sequences, and we demonstrate that this strategy enables the isolation of a specific endogenous messenger RNA from cells. These results reveal a fundamental connection between PAM binding and substrate selection by Cas9, and highlight the utility of Cas9 for programmable transcript recognition without the need for tags.

  19. Conservation of a proteinase cleavage site between an insect retrovirus (gypsy) Env protein and a baculovirus envelope fusion protein

    International Nuclear Information System (INIS)

    Pearson, Margot N.; Rohrmann, George F.

    2004-01-01

    The predicted Env protein of insect retroviruses (errantiviruses) is related to the envelope fusion protein of a major division of the Baculoviridae. The highest degree of homology is found in a region that contains a furin cleavage site in the baculovirus proteins and an adjacent sequence that has the properties of a fusion peptide. In this investigation, the homologous region in the Env protein of the gypsy retrovirus of Drosophila melanogaster (DmegypV) was investigated. Alteration of the predicted DmegypV Env proteinase cleavage site from RIAR to AIAR significantly reduced cleavage of Env in both Spodoptera frugiperda (Sf-9) and D. melanogaster (S2) cell lines. When the predicted DmegypV Env cleavage site RIAR was substituted for the cleavage sequence RRKR in the Lymantria dispar nucleopolyhedrovirus fusion protein (LD130) sequence, cleavage of the hybrid LD130 molecules still occurred, although at a reduced level. The conserved 21-amino acid sequence just downstream of the cleavage site, which is thought to be the fusion peptide in LD130, was also characterized. When this sequence from DmegypV Env was substituted for the homologous sequence in LD130, cleavage still occurred, but no fusion was observed in either cell type. In addition, although a DmegypV-Env-green fluorescent protein construct localized to cell membranes, no cell fusion was observed

  20. Sequential Generalized Transforms on Function Space

    Directory of Open Access Journals (Sweden)

    Jae Gil Choi

    2013-01-01

    Full Text Available We define two sequential transforms on a function space Ca,b[0,T] induced by generalized Brownian motion process. We then establish the existence of the sequential transforms for functionals in a Banach algebra of functionals on Ca,b[0,T]. We also establish that any one of these transforms acts like an inverse transform of the other transform. Finally, we give some remarks about certain relations between our sequential transforms and other well-known transforms on Ca,b[0,T].

  1. Drosha regulates gene expression independently of RNA cleavage function

    DEFF Research Database (Denmark)

    Gromak, Natalia; Dienstbier, Martin; Macias, Sara

    2013-01-01

    Drosha is the main RNase III-like enzyme involved in the process of microRNA (miRNA) biogenesis in the nucleus. Using whole-genome ChIP-on-chip analysis, we demonstrate that, in addition to miRNA sequences, Drosha specifically binds promoter-proximal regions of many human genes in a transcription......-dependent manner. This binding is not associated with miRNA production or RNA cleavage. Drosha knockdown in HeLa cells downregulated nascent gene transcription, resulting in a reduction of polyadenylated mRNA produced from these gene regions. Furthermore, we show that this function of Drosha is dependent on its N......-terminal protein-interaction domain, which associates with the RNA-binding protein CBP80 and RNA Polymerase II. Consequently, we uncover a previously unsuspected RNA cleavage-independent function of Drosha in the regulation of human gene expression....

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

    DEFF Research Database (Denmark)

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

    1997-01-01

    by a prior incubation of the cells with uPA inactivated by diisopropyl fluorophosphate, demonstrating a requirement for specific receptor binding of the active uPA to obtain the high-efficiency cleavage of cell-bound uPAR. Furthermore, amino-terminal sequence analysis revealed that uPAR(2+3), purified from U...

  3. The role of lysosomal proteolytic enzymes in invasion and dissemination of malignant melanoma

    International Nuclear Information System (INIS)

    Bassalyk, L.S.; Tsanev, P.E.; Parshikova, S.M.; Demidov, L.V.

    1992-01-01

    Preoperative chemo- and radiation therapy was followed by a decrease in lysosomal cathepsins activity in metastatic lymph nodes which, however, did not reach the level established for intact lymph nodes. The pathogenetic role of proteolytic endopeptidases in invasion and sissemination of malignant melanoma is discussed as well as the value of their level measurement for assessing metastatic potential of tumor and prognosis of disease of disease on the basis of tumor site, degree of invasion regional lymph node status

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

    Directory of Open Access Journals (Sweden)

    Sandeep Chakane

    2017-08-01

    Full Text Available Hemoglobin (Hb is well protected inside the red blood cells (RBCs. Upon hemolysis and when free in circulation, Hb can be involved in a range of radical generating reactions and may thereby attack several different biomolecules. In this study, we have examined the potential damaging effects of cell-free Hb on plasmid DNA (pDNA. Hb induced cleavage of supercoiled pDNA (sc pDNA which was proportional to the concentration of Hb applied. Almost 70% of sc pDNA was converted to open circular or linear DNA using 10 µM of Hb in 12 h. Hb can be present in several different forms. The oxy (HbO2 and met forms are most reactive, while the carboxy-protein shows only low hydrolytic activity. Hemoglobin A (HbA could easily induce complete pDNA cleavage while fetal hemoglobin (HbF was three-fold less reactive. By inserting, a redox active cysteine residue on the surface of the alpha chain of HbF by site-directed mutagenesis, the DNA cleavage reaction was enhanced by 82%. Reactive oxygen species were not directly involved in the reaction since addition of superoxide dismutase and catalase did not prevent pDNA cleavage. The reactivity of Hb with pDNA can rather be associated with the formation of protein based radicals. Keywords: Adult hemoglobin, Fetal hemoglobin, Supercoiled plasmid DNA, DNA cleavage, Cysteine, Protein radicals

  5. Abyssal fiction: common shares, colonial cleavages

    Directory of Open Access Journals (Sweden)

    Alexandre Montaury

    2016-12-01

    Full Text Available The paper aims to develop a reflection on the interaction between the legacies of colonialism and traditional symbolic and cultural practices in African Portuguese-speaking spaces. From a preliminary analysis of fictional texts of wide circulation in Brazil, aims to examine the cleavages, or “abyssal lines” that constitute experiences printed in the daily life of the former Portuguese colony of Cape Verde, Mozambique and Angola.---DOI: http://dx.doi.org/10.21881/abriluff.2016n17a378

  6. Forced Sequence Sequential Decoding

    DEFF Research Database (Denmark)

    Jensen, Ole Riis; Paaske, Erik

    1998-01-01

    We describe a new concatenated decoding scheme based on iterations between an inner sequentially decoded convolutional code of rate R=1/4 and memory M=23, and block interleaved outer Reed-Solomon (RS) codes with nonuniform profile. With this scheme decoding with good performance is possible as low...... as Eb/N0=0.6 dB, which is about 1.25 dB below the signal-to-noise ratio (SNR) that marks the cutoff rate for the full system. Accounting for about 0.45 dB due to the outer codes, sequential decoding takes place at about 1.7 dB below the SNR cutoff rate for the convolutional code. This is possible since...... the iteration process provides the sequential decoders with side information that allows a smaller average load and minimizes the probability of computational overflow. Analytical results for the probability that the first RS word is decoded after C computations are presented. These results are supported...

  7. Biosynthesis of intestinal microvillar proteins. Evidence for an intracellular sorting taking place in, or shortly after, exit from the Golgi complex

    DEFF Research Database (Denmark)

    Danielsen, E M; Cowell, G M

    1985-01-01

    accessibility to proteolytic cleavage. The mature polypeptide of Mr 166 000 from the latter fraction was cleaved by trypsin, proteinase K and papain, consistent with an extracellular location of the enzyme at its site of function. In contrast, both the mature form and the transient form of Mr 140 000 from...

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

    Directory of Open Access Journals (Sweden)

    Chenyu Zhang

    2009-05-01

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

  9. Effect of Cultured Celery Juice, Temperature, and Product Composition on the Inhibition of Proteolytic Clostridium botulinum Toxin Production.

    Science.gov (United States)

    Golden, Max C; Wanless, Brandon J; David, Jairus R D; Kottapalli, Bala; Lineback, D Scott; Talley, Ryan J; Glass, Kathleen A

    2017-08-01

    Clostridium botulinum may be of concern in prepared refrigerated meals, for which strict cold chain management cannot be guaranteed. This study evaluated the effect of temperature, product composition, and cultured celery juice powder (CCJP) as a source of nitrite on the inhibition of botulinum toxin formation in two experimental (meat- and vegetable-based) prepared meals. Data obtained from the challenge study were compared with a published mathematical model to determine whether the model is fail-safe with regard to the tested meals. Treatments were inoculated with proteolytic C. botulinum, vacuum packaged, cooked at 90°C for 10 min, and assayed for botulinum toxin at appropriate intervals in samples stored at 10, 15, or 20°C for up to 8 weeks. None of the treatments stored at 10°C for 8 weeks supported toxin production by proteolytic C. botulinum. The addition of CCJP delayed toxin production by 1 and 3 weeks in cauliflower potatoes and in Dijon pork, respectively, stored at 15°C. Toxin production was delayed by 1 week at 20°C when CCJP was added to the cauliflower potatoes. This study found that the predictive model was fail-safe but was overly conservative for the experimental meals described. Finally, this study confirms that product composition, the addition of nitrite via CCJP, storage time, and temperature play important roles in the inhibition of toxin formation by proteolytic C. botulinum.

  10. Sequential probability ratio controllers for safeguards radiation monitors

    International Nuclear Information System (INIS)

    Fehlau, P.E.; Coop, K.L.; Nixon, K.V.

    1984-01-01

    Sequential hypothesis tests applied to nuclear safeguards accounting methods make the methods more sensitive to detecting diversion. The sequential tests also improve transient signal detection in safeguards radiation monitors. This paper describes three microprocessor control units with sequential probability-ratio tests for detecting transient increases in radiation intensity. The control units are designed for three specific applications: low-intensity monitoring with Poisson probability ratios, higher intensity gamma-ray monitoring where fixed counting intervals are shortened by sequential testing, and monitoring moving traffic where the sequential technique responds to variable-duration signals. The fixed-interval controller shortens a customary 50-s monitoring time to an average of 18 s, making the monitoring delay less bothersome. The controller for monitoring moving vehicles benefits from the sequential technique by maintaining more than half its sensitivity when the normal passage speed doubles

  11. Cleavage strain in the Variscan fold belt, County Cork, Ireland, estimated from stretched arsenopyrite rosettes

    Science.gov (United States)

    Ford, M.; Ferguson, C.C.

    1985-01-01

    In south-west Ireland, hydrothermally formed arsenopyrite crystals in a Devonian mudstone have responded to Variscan deformation by brittle extension fracture and fragment separation. The interfragment gaps and terminal extension zones of each crystal are infilled with fibrous quartz. Stretches within the cleavage plane have been calculated by the various methods available, most of which can be modified to incorporate terminal extension zones. The Strain Reversal Method is the most accurate currently available but still gives a minimum estimate of the overall strain. The more direct Hossain method, which gives only slightly lower estimates with this data, is more practical for field use. A strain ellipse can be estimated from each crystal rosette composed of three laths (assuming the original interlimb angles were all 60??) and, because actual rather than relative stretches are estimated, this provides a lower bound to the area increase in the plane of cleavage. Based on the average of our calculated strain ellipses this area increase is at least 114% and implies an average shortening across the cleavage of at least 53%. However, several lines of evidence suggest that the cleavage deformation was more intense and more oblate than that calculated, and we argue that a 300% area increase in the cleavage plane and 75% shortening across the cleavage are more realistic estimates of the true strain. Furthermore, the along-strike elongation indicated is at least 80%, which may be regionally significant. Estimates of orogenic contraction derived from balanced section construction should therefore take into account the possibility of a substantial strike elongation, and tectonic models that can accommodate such elongations need to be developed. ?? 1985.

  12. Biased lineups: sequential presentation reduces the problem.

    Science.gov (United States)

    Lindsay, R C; Lea, J A; Nosworthy, G J; Fulford, J A; Hector, J; LeVan, V; Seabrook, C

    1991-12-01

    Biased lineups have been shown to increase significantly false, but not correct, identification rates (Lindsay, Wallbridge, & Drennan, 1987; Lindsay & Wells, 1980; Malpass & Devine, 1981). Lindsay and Wells (1985) found that sequential lineup presentation reduced false identification rates, presumably by reducing reliance on relative judgment processes. Five staged-crime experiments were conducted to examine the effect of lineup biases and sequential presentation on eyewitness recognition accuracy. Sequential lineup presentation significantly reduced false identification rates from fair lineups as well as from lineups biased with regard to foil similarity, instructions, or witness attire, and from lineups biased in all of these ways. The results support recommendations that police present lineups sequentially.

  13. Lineup composition, suspect position, and the sequential lineup advantage.

    Science.gov (United States)

    Carlson, Curt A; Gronlund, Scott D; Clark, Steven E

    2008-06-01

    N. M. Steblay, J. Dysart, S. Fulero, and R. C. L. Lindsay (2001) argued that sequential lineups reduce the likelihood of mistaken eyewitness identification. Experiment 1 replicated the design of R. C. L. Lindsay and G. L. Wells (1985), the first study to show the sequential lineup advantage. However, the innocent suspect was chosen at a lower rate in the simultaneous lineup, and no sequential lineup advantage was found. This led the authors to hypothesize that protection from a sequential lineup might emerge only when an innocent suspect stands out from the other lineup members. In Experiment 2, participants viewed a simultaneous or sequential lineup with either the guilty suspect or 1 of 3 innocent suspects. Lineup fairness was varied to influence the degree to which a suspect stood out. A sequential lineup advantage was found only for the unfair lineups. Additional analyses of suspect position in the sequential lineups showed an increase in the diagnosticity of suspect identifications as the suspect was placed later in the sequential lineup. These results suggest that the sequential lineup advantage is dependent on lineup composition and suspect position. (c) 2008 APA, all rights reserved

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

    Directory of Open Access Journals (Sweden)

    Olivier Barré

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

  15. Change in radiosensitivity of sea-urchin eggs during early cleavage stages

    International Nuclear Information System (INIS)

    Nakamura, I.

    1977-01-01

    When sea-urchin eggs were irradiated with 137 Cs γ-rays, their radiosensitivity, expressed by the percentage which formed pluteus larvae, fluctuated during the early cleavage cycle. Split-dose irradiations were made both in the sensitive and resistant phases. For eggs in the sensitive phase, the effect of the first exposure of 500 rad was not diminished during the interval before the second exposure. Eggs irradiated in the resistant phase were only slightly damaged. Results implied that fluctuations in radiosensitivity of sea-urchin eggs were caused mainly by different degrees of non-repairable damage in each phase of cleavage rather than by different recovery abilities. (author)

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

    Science.gov (United States)

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

    2016-01-01

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

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

    Science.gov (United States)

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

    2016-05-06

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

  18. Nano-zymography Using Laser-Scanning Confocal Microscopy Unmasks Proteolytic Activity of Cell-Derived Microparticles.

    Science.gov (United States)

    Briens, Aurélien; Gauberti, Maxime; Parcq, Jérôme; Montaner, Joan; Vivien, Denis; Martinez de Lizarrondo, Sara

    2016-01-01

    Cell-derived microparticles (MPs) are nano-sized vesicles released by activated cells in the extracellular milieu. They act as vectors of biological activity by carrying membrane-anchored and cytoplasmic constituents of the parental cells. Although detection and characterization of cell-derived MPs may be of high diagnostic and prognostic values in a number of human diseases, reliable measurement of their size, number and biological activity still remains challenging using currently available methods. In the present study, we developed a protocol to directly image and functionally characterize MPs using high-resolution laser-scanning confocal microscopy. Once trapped on annexin-V coated micro-wells, we developed several assays using fluorescent reporters to measure their size, detect membrane antigens and evaluate proteolytic activity (nano-zymography). In particular, we demonstrated the applicability and specificity of this method to detect antigens and proteolytic activities of tissue-type plasminogen activator (tPA), urokinase and plasmin at the surface of engineered MPs from transfected cell-lines. Furthermore, we were able to identify a subset of tPA-bearing fibrinolytic MPs using plasma samples from a cohort of ischemic stroke patients who received thrombolytic therapy and in an experimental model of thrombin-induced ischemic stroke in mice. Overall, this method is promising for functional characterization of cell-derived MPs.

  19. Caspase-2 cleavage of tau reversibly impairs memory.

    Science.gov (United States)

    Zhao, Xiaohui; Kotilinek, Linda A; Smith, Benjamin; Hlynialuk, Chris; Zahs, Kathleen; Ramsden, Martin; Cleary, James; Ashe, Karen H

    2016-11-01

    In Alzheimer's disease (AD) and other tauopathies, the tau protein forms fibrils, which are believed to be neurotoxic. However, fibrillar tau has been dissociated from neuron death and network dysfunction, suggesting the involvement of nonfibrillar species. Here we describe a novel pathological process in which caspase-2 cleavage of tau at Asp314 impairs cognitive and synaptic function in animal and cellular models of tauopathies by promoting the missorting of tau to dendritic spines. The truncation product, Δtau314, resists fibrillation and is present at higher levels in brains from cognitively impaired mice and humans with AD. The expression of tau mutants that resisted caspase-2 cleavage prevented tau from infiltrating spines, dislocating glutamate receptors and impairing synaptic function in cultured neurons, and it prevented memory deficits and neurodegeneration in mice. Decreasing the levels of caspase-2 restored long-term memory in mice that had existing deficits. Our results suggest an overall treatment strategy for re-establishing synaptic function and restoring memory in patients with AD by preventing tau from accumulating in dendritic spines.

  20. AID-induced decrease in topoisomerase 1 induces DNA structural alteration and DNA cleavage for class switch recombination.

    Science.gov (United States)

    Kobayashi, Maki; Aida, Masatoshi; Nagaoka, Hitoshi; Begum, Nasim A; Kitawaki, Yoko; Nakata, Mikiyo; Stanlie, Andre; Doi, Tomomitsu; Kato, Lucia; Okazaki, Il-mi; Shinkura, Reiko; Muramatsu, Masamichi; Kinoshita, Kazuo; Honjo, Tasuku

    2009-12-29

    To initiate class switch recombination (CSR) activation-induced cytidine deaminase (AID) induces staggered nick cleavage in the S region, which lies 5' to each Ig constant region gene and is rich in palindromic sequences. Topoisomerase 1 (Top1) controls the supercoiling of DNA by nicking, rotating, and religating one strand of DNA. Curiously, Top1 reduction or AID overexpression causes the genomic instability. Here, we report that the inactivation of Top1 by its specific inhibitor camptothecin drastically blocked both the S region cleavage and CSR, indicating that Top1 is responsible for the S region cleavage in CSR. Surprisingly, AID expression suppressed Top1 mRNA translation and reduced its protein level. In addition, the decrease in the Top1 protein by RNA-mediated knockdown augmented the AID-dependent S region cleavage, as well as CSR. Furthermore, Top1 reduction altered DNA structure of the Smu region. Taken together, AID-induced Top1 reduction alters S region DNA structure probably to non-B form, on which Top1 can introduce nicks but cannot religate, resulting in S region cleavage.

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

    Science.gov (United States)

    Ningrum, Andriati; Schreiner, Matthias

    2014-11-01

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

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

    Science.gov (United States)

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

    2003-11-14

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

  3. Tradable permit allocations and sequential choice

    Energy Technology Data Exchange (ETDEWEB)

    MacKenzie, Ian A. [Centre for Economic Research, ETH Zuerich, Zurichbergstrasse 18, 8092 Zuerich (Switzerland)

    2011-01-15

    This paper investigates initial allocation choices in an international tradable pollution permit market. For two sovereign governments, we compare allocation choices that are either simultaneously or sequentially announced. We show sequential allocation announcements result in higher (lower) aggregate emissions when announcements are strategic substitutes (complements). Whether allocation announcements are strategic substitutes or complements depends on the relationship between the follower's damage function and governments' abatement costs. When the marginal damage function is relatively steep (flat), allocation announcements are strategic substitutes (complements). For quadratic abatement costs and damages, sequential announcements provide a higher level of aggregate emissions. (author)

  4. Endothelial nitric oxide synthase gene Glu298Asp polymorphism ...

    African Journals Online (AJOL)

    Administrator

    2011-09-12

    Sep 12, 2011 ... Figure 1. The Glu298Asp polymorphism of eNOS gene was shown by .... mechanisms by which eNOS Asp298 polymorphism ... Asp298 is exposed to selective proteolytic cleavage in ... grounds, inclusion and exclusion criteria for PE women ... attention to meta analysis study, it is more probable that.

  5. Glycosaminoglycans Regulate CXCR3 Ligands at Distinct Levels: Protection against Processing by Dipeptidyl Peptidase IV/CD26 and Interference with Receptor Signaling

    Directory of Open Access Journals (Sweden)

    Mieke Metzemaekers

    2017-07-01

    Full Text Available CXC chemokine ligand (CXCL9, CXCL10 and CXCL11 direct chemotaxis of mainly T cells and NK cells through activation of their common CXC chemokine receptor (CXCR3. They are inactivated upon NH2-terminal cleavage by dipeptidyl peptidase IV/CD26. In the present study, we found that different glycosaminoglycans (GAGs protect the CXCR3 ligands against proteolytic processing by CD26 without directly affecting the enzymatic activity of CD26. In addition, GAGs were shown to interfere with chemokine-induced CXCR3 signaling. The observation that heparan sulfate did not, and heparin only moderately, altered CXCL10-induced T cell chemotaxis in vitro may be explained by a combination of protection against proteolytic inactivation and altered receptor interaction as observed in calcium assays. No effect of CD26 inhibition was found on CXCL10-induced chemotaxis in vitro. However, treatment of mice with the CD26 inhibitor sitagliptin resulted in an enhanced CXCL10-induced lymphocyte influx into the joint. This study reveals a dual role for GAGs in modulating the biological activity of CXCR3 ligands. GAGs protect the chemokines from proteolytic cleavage but also directly interfere with chemokine–CXCR3 signaling. These data support the hypothesis that both GAGs and CD26 affect the in vivo chemokine function.

  6. Applying the minimax principle to sequential mastery testing

    NARCIS (Netherlands)

    Vos, Hendrik J.

    2002-01-01

    The purpose of this paper is to derive optimal rules for sequential mastery tests. In a sequential mastery test, the decision is to classify a subject as a master, a nonmaster, or to continue sampling and administering another random item. The framework of minimax sequential decision theory (minimum

  7. SOCIAL CLEAVAGES IN THE AMERICAN SOCIETY AS A FACTOR OF 2016 PRESIDENTIAL CAMPAIGN

    Directory of Open Access Journals (Sweden)

    P. S. Kanevskiy

    2017-01-01

    Full Text Available Current article is dedicated to analysis of social cleavages in the American elections and the ways they influenced on presidential election in 2016. Originally developed by S. Rokkan and S.M. Lipset, social cleavages became a classic theme for contemporary political sociology. However, despite the fact that the theory has been developing primarily by Americans, it has been rarely used to analyze electoral system in the USA. Traditionally it’s been aimed at European and developing countries where electoral fragmentation is seen more clearly. But recent changes in the American society and the political system demonstrate the emergence of social cleavages that had not been inherent before. The article shows how American electoral space transformed since the 1980s and how it became more fragmented under the influence of social, economic and ideological factors. Elections in 2016 became a watershed for social cleavages that accumulated through time and aggravated even more considering internal crises in the Democratic and more so in the Republican parties. Donald Trump’s victory is an impersonation of the American party system crisis and of the mainstream politicians’ inability to find proper explanation of the changing electorate. Author shows that American society today is polarized even more than many European countries while group identification determines vectors of political change.

  8. Electrostatic instability of some jellium model lattices of high symmetry to their plane cleavage

    International Nuclear Information System (INIS)

    Kholopov, Eugene V; Kalashnikova, Vita V

    2007-01-01

    Jellium model structures composed of regular lattices of equal point charges immersed in a neutralizing uniform background are considered. The symmetric description eliminating the effect of potentials without transverse structural modulation is extended to the events specified by alternating distances between point-charge planes. Based on modulated potentials typical of plane-wise lattice summation, the energy of interaction between two semi-infinite hemi-crystals divided by a plane is obtained for cubic and hexagonal crystals, where all the characteristic orientations of the cleavage plane are taken into account. We found that simple cubic and hexagonal lattices, as well as cubic and hexagonal diamond structures, turn out to be unstable for certain cleavage planes. The most favourable cleavage planes for the bcc, fcc and hcp structures are also emphasized

  9. Classical and sequential limit analysis revisited

    Science.gov (United States)

    Leblond, Jean-Baptiste; Kondo, Djimédo; Morin, Léo; Remmal, Almahdi

    2018-04-01

    Classical limit analysis applies to ideal plastic materials, and within a linearized geometrical framework implying small displacements and strains. Sequential limit analysis was proposed as a heuristic extension to materials exhibiting strain hardening, and within a fully general geometrical framework involving large displacements and strains. The purpose of this paper is to study and clearly state the precise conditions permitting such an extension. This is done by comparing the evolution equations of the full elastic-plastic problem, the equations of classical limit analysis, and those of sequential limit analysis. The main conclusion is that, whereas classical limit analysis applies to materials exhibiting elasticity - in the absence of hardening and within a linearized geometrical framework -, sequential limit analysis, to be applicable, strictly prohibits the presence of elasticity - although it tolerates strain hardening and large displacements and strains. For a given mechanical situation, the relevance of sequential limit analysis therefore essentially depends upon the importance of the elastic-plastic coupling in the specific case considered.

  10. Simultaneous versus sequential penetrating keratoplasty and cataract surgery.

    Science.gov (United States)

    Hayashi, Ken; Hayashi, Hideyuki

    2006-10-01

    To compare the surgical outcomes of simultaneous penetrating keratoplasty and cataract surgery with those of sequential surgery. Thirty-nine eyes of 39 patients scheduled for simultaneous keratoplasty and cataract surgery and 23 eyes of 23 patients scheduled for sequential keratoplasty and secondary phacoemulsification surgery were recruited. Refractive error, regular and irregular corneal astigmatism determined by Fourier analysis, and endothelial cell loss were studied at 1 week and 3, 6, and 12 months after combined surgery in the simultaneous surgery group or after subsequent phacoemulsification surgery in the sequential surgery group. At 3 and more months after surgery, mean refractive error was significantly greater in the simultaneous surgery group than in the sequential surgery group, although no difference was seen at 1 week. The refractive error at 12 months was within 2 D of that targeted in 15 eyes (39%) in the simultaneous surgery group and within 2 D in 16 eyes (70%) in the sequential surgery group; the incidence was significantly greater in the sequential group (P = 0.0344). The regular and irregular astigmatism was not significantly different between the groups at 3 and more months after surgery. No significant difference was also found in the percentage of endothelial cell loss between the groups. Although corneal astigmatism and endothelial cell loss were not different, refractive error from target refraction was greater after simultaneous keratoplasty and cataract surgery than after sequential surgery, indicating a better outcome after sequential surgery than after simultaneous surgery.

  11. Stimulation of topoisomerase II mediated DNA cleavage at specific sequence elements by the 2-nitroimidazole Ro 15-0216

    International Nuclear Information System (INIS)

    Sorensen, B.S.; Jensen, P.S.; Andersen, A.H.; Christiansen, K.; Alsner, J.; Thomsen, B.; Westergaard, O.

    1990-01-01

    The effect of the 2-nitroimidazole Ro 15-0216 upon the interaction between purified topoisomerase II and its DNA substrate was investigated. The cleavage reaction in the presence of this DNA-nonintercalative drug took place with the hallmarks of a regular topoisomerase II mediated cleavage reaction, including covalent linkage of the enzyme to the cleaved DNA. In the presence of Ro 15-0216, topoisomerase II mediated cleavage was extensively stimulated at major cleavage sites of which only one existed in the 4363 base pair pBR322 molecule. The sites stimulated by Ro 15-0216 shared a pronounced sequence homology, indicating that a specific nucleotide sequence is crucial for the action of this drug. The effect of Ro 15-0216 thus differs from that of the clinically important topoisomerase II targeted agents such as mAMSA, VM26, and VP16, which enhance enzyme-mediated cleavage at a multiple number of sites. In contrast to the previous described drugs, Ro 15-0216 did not exert any inhibitory effect on the enzyme's catalytic activity. This observation might be ascribed to the low stability of the cleavage complexes formed in the presence of Ro 15-0216 as compared to the stability of the ones formed in the presence of traditional topoisomerase II targeted drugs

  12. Kinetics of phycocyanobilin cleavage from C-phycocyanin by methanolysis

    DEFF Research Database (Denmark)

    Malwade, Chandrakant Ramkrishna; Roda Serrat, Maria Cinta; Christensen, Knud Villy

    2016-01-01

    Phycocyanobilin (PCB) is an important linear tetrapyrrolic molecule for food as well as pharmaceutical industry. It is obtained from blue-green algae, where it is attached covalently to phycobiliproteins (C-PC and APC) present in the light harvesting complexes. In this work, cleavage of PCB from...

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

    Science.gov (United States)

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

    2017-01-01

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

  14. cis-Apa: a practical linker for the microwave-assisted preparation of cyclic pseudopeptides via RCM cyclative cleavage.

    Science.gov (United States)

    Baron, Alice; Verdié, Pascal; Martinez, Jean; Lamaty, Frédéric

    2011-02-04

    A new linker cis-5-aminopent-3-enoic acid (cis-Apa) was prepared for the synthesis of cyclic pseudopeptides by cyclization-cleavage by using ring-closing methatesis (RCM). We developed a new synthetic pathway for the preparation of the cis-Apa linker that was tested in the cyclization-cleavage process of different RGD peptide sequences. Different macrocyclic peptidomimetics were prepared by using this integrated microwave-assisted method, showing that the readily available cis-Apa amino acid is well adapted as a linker in the cyclization-cleavage process.

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

    Directory of Open Access Journals (Sweden)

    Susannah L. Hewitt

    2017-10-01

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

  16. Trial Sequential Methods for Meta-Analysis

    Science.gov (United States)

    Kulinskaya, Elena; Wood, John

    2014-01-01

    Statistical methods for sequential meta-analysis have applications also for the design of new trials. Existing methods are based on group sequential methods developed for single trials and start with the calculation of a required information size. This works satisfactorily within the framework of fixed effects meta-analysis, but conceptual…

  17. Sequentially pulsed traveling wave accelerator

    Science.gov (United States)

    Caporaso, George J [Livermore, CA; Nelson, Scott D [Patterson, CA; Poole, Brian R [Tracy, CA

    2009-08-18

    A sequentially pulsed traveling wave compact accelerator having two or more pulse forming lines each with a switch for producing a short acceleration pulse along a short length of a beam tube, and a trigger mechanism for sequentially triggering the switches so that a traveling axial electric field is produced along the beam tube in synchronism with an axially traversing pulsed beam of charged particles to serially impart energy to the particle beam.

  18. Metabolic Engineering to Develop a Pathway for the Selective Cleavage of Carbon-Nitrogen Bonds

    Energy Technology Data Exchange (ETDEWEB)

    John J. Kilbane II

    2005-10-01

    The objective of the project is to develop a biochemical pathway for the selective cleavage of C-N bonds in molecules found in petroleum. Specifically a novel biochemical pathway will be developed for the selective cleavage of C-N bonds in carbazole. The cleavage of the first C-N bond in carbazole is accomplished by the enzyme carbazole dioxygenase, that catalyzes the conversion of carbazole to 2-aminobiphenyl-2,3-diol. The genes encoding carbazole dioxygenase were cloned from Sphingomonas sp. GTIN11 and from Pseudomonas resinovorans CA10. The selective cleavage of the second C-N bond has been challenging, and efforts to overcome that challenge have been the focus of recent research in this project. Enrichment culture experiments succeeded in isolating bacterial cultures that can metabolize 2-aminobiphenyl, but no enzyme capable of selectively cleaving the C-N bond in 2-aminobiphenyl has been identified. Aniline is very similar to the structure of 2-aminobiphenyl and aniline dioxygenase catalyzes the conversion of aniline to catechol and ammonia. For the remainder of the project the emphasis of research will be to simultaneously express the genes for carbazole dioxygenase and for aniline dioxygenase in the same bacterial host and then to select for derivative cultures capable of using carbazole as the sole source of nitrogen.

  19. An Efficient System Based On Closed Sequential Patterns for Web Recommendations

    OpenAIRE

    Utpala Niranjan; R.B.V. Subramanyam; V-Khana

    2010-01-01

    Sequential pattern mining, since its introduction has received considerable attention among the researchers with broad applications. The sequential pattern algorithms generally face problems when mining long sequential patterns or while using very low support threshold. One possible solution of such problems is by mining the closed sequential patterns, which is a condensed representation of sequential patterns. Recently, several researchers have utilized the sequential pattern discovery for d...

  20. Membrane-bound transcription factors: regulated release by RIP or RUP.

    Science.gov (United States)

    Hoppe, T; Rape, M; Jentsch, S

    2001-06-01

    Regulated nuclear transport of transcription factors from cytoplasmic pools is a major route by which eukaryotes control gene expression. Exquisite examples are transcription factors that are kept in a dormant state in the cytosol by membrane anchors; such proteins are released from membranes by proteolytic cleavage, which enables these transcription factors to enter the nucleus. Cleavage can be mediated either by regulated intramembrane proteolysis (RIP) catalysed by specific membrane-bound proteases or by regulated ubiquitin/proteasome-dependent processing (RUP). In both cases processing can be controlled by cues that originate at or in the vicinity of the membrane.

  1. Molecular characterization of the haptoglobin.hemoglobin receptor CD163. Ligand binding properties of the scavenger receptor cysteine-rich domain region

    DEFF Research Database (Denmark)

    Madsen, Mette; Møller, Holger J; Nielsen, Marianne Jensby

    2004-01-01

    binding to SRCR domain 3 exhibited effective inhibition of ligand binding. Furthermore, analysis of purified native CD163 revealed that proteolytic cleavage in SRCR domain 3 inactivates ligand binding. Calcium protects against cleavage in this domain. Analysis of the calcium sensitivity of ligand binding...... to CD163 demonstrated that optimal ligand binding requires physiological plasma calcium concentrations, and an immediate ligand release occurs at the low calcium concentrations measured in acidifying endosomes. In conclusion, SRCR domain 3 of CD163 is an exposed domain and a critical determinant...... for the calcium-sensitive coupling of haptoglobin.hemoglobin complexes....

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

    International Nuclear Information System (INIS)

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

    1983-01-01

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

  3. [Cleavage of DNA fragments induced by UV nanosecond laser excitation at 193 nm].

    Science.gov (United States)

    Vtiurina, N N; Grokhovskiĭ, S L; Filimonov, I V; Medvedkov, O I; Nechipurenko, D Iu; Vasil'ev, S A; Nechipurenko, Iu D

    2011-01-01

    The cleavage of dsDNA fragments in aqueous solution after irradiation with UV laser pulses at 193 nm has been studied. Samples were investigated using polyacrylamide gel electrophoresis. The intensity of damage of particular phosphodiester bond after hot alkali treatment was shown to depend on the base pair sequence. It was established that the probability of cleavage is twice higher for sites of DNA containing two or more successively running guanine residues. A possible mechanism of damage to the DNA molecule connected with the migration of holes along the helix is discussed.

  4. Biochemical and Functional Characterization of Parawixia bistriata Spider Venom with Potential Proteolytic and Larvicidal Activities

    Science.gov (United States)

    Gimenez, Gizeli S.; Coutinho-Neto, Antonio; Kayano, Anderson M.; Simões-Silva, Rodrigo; Trindade, Frances; de Almeida e Silva, Alexandre; Marcussi, Silvana; da Silva, Saulo L.; Fernandes, Carla F. C.; Zuliani, Juliana P.; Calderon, Leonardo A.; Soares, Andreimar M.; Stábeli, Rodrigo G.

    2014-01-01

    Toxins purified from the venom of spiders have high potential to be studied pharmacologically and biochemically. These biomolecules may have biotechnological and therapeutic applications. This study aimed to evaluate the protein content of Parawixia bistriata venom and functionally characterize its proteins that have potential for biotechnological applications. The crude venom showed no phospholipase, hemorrhagic, or anti-Leishmania activities attesting to low genotoxicity and discrete antifungal activity for C. albicans. However the following activities were observed: anticoagulation, edema, myotoxicity and proteolysis on casein, azo-collagen, and fibrinogen. The chromatographic and electrophoretic profiles of the proteins revealed a predominance of acidic, neutral, and polar proteins, highlighting the presence of proteins with high molecular masses. Five fractions were collected using cation exchange chromatography, with the P4 fraction standing out as that of the highest purity. All fractions showed proteolytic activity. The crude venom and fractions P1, P2, and P3 showed larvicidal effects on A. aegypti. Fraction P4 showed the presence of a possible metalloprotease (60 kDa) that has high proteolytic activity on azo-collagen and was inhibited by EDTA. The results presented in this study demonstrate the presence of proteins in the venom of P. bistriata with potential for biotechnological applications. PMID:24895632

  5. HupW Protease Specifically Required for Processing of the Catalytic Subunit of the Uptake Hydrogenase in the Cyanobacterium Nostoc sp. Strain PCC 7120

    Science.gov (United States)

    Lindberg, Pia; Devine, Ellenor; Stensjö, Karin

    2012-01-01

    The maturation process of [NiFe] hydrogenases includes a proteolytic cleavage of the large subunit. We constructed a mutant of Nostoc strain PCC 7120 in which hupW, encoding a putative hydrogenase-specific protease, is inactivated. Our results indicate that the protein product of hupW selectively cleaves the uptake hydrogenase in this cyanobacterium. PMID:22020512

  6. High-resolution characterization of sequence signatures due to non-random cleavage of cell-free DNA.

    Science.gov (United States)

    Chandrananda, Dineika; Thorne, Natalie P; Bahlo, Melanie

    2015-06-17

    High-throughput sequencing of cell-free DNA fragments found in human plasma has been used to non-invasively detect fetal aneuploidy, monitor organ transplants and investigate tumor DNA. However, many biological properties of this extracellular genetic material remain unknown. Research that further characterizes circulating DNA could substantially increase its diagnostic value by allowing the application of more sophisticated bioinformatics tools that lead to an improved signal to noise ratio in the sequencing data. In this study, we investigate various features of cell-free DNA in plasma using deep-sequencing data from two pregnant women (>70X, >50X) and compare them with matched cellular DNA. We utilize a descriptive approach to examine how the biological cleavage of cell-free DNA affects different sequence signatures such as fragment lengths, sequence motifs at fragment ends and the distribution of cleavage sites along the genome. We show that the size distributions of these cell-free DNA molecules are dependent on their autosomal and mitochondrial origin as well as the genomic location within chromosomes. DNA mapping to particular microsatellites and alpha repeat elements display unique size signatures. We show how cell-free fragments occur in clusters along the genome, localizing to nucleosomal arrays and are preferentially cleaved at linker regions by correlating the mapping locations of these fragments with ENCODE annotation of chromatin organization. Our work further demonstrates that cell-free autosomal DNA cleavage is sequence dependent. The region spanning up to 10 positions on either side of the DNA cleavage site show a consistent pattern of preference for specific nucleotides. This sequence motif is present in cleavage sites localized to nucleosomal cores and linker regions but is absent in nucleosome-free mitochondrial DNA. These background signals in cell-free DNA sequencing data stem from the non-random biological cleavage of these fragments. This

  7. Embryo apoptosis identification: Oocyte grade or cleavage stage?

    Science.gov (United States)

    Bakri, Noraina Mohd; Ibrahim, Siti Fatimah; Osman, Nurul Atikah; Hasan, Nurhaslina; Jaffar, Farah Hanan Fathihah; Rahman, Zulaiha Abdul; Osman, Khairul

    2015-01-01

    Apoptosis is a programed cell death that is vital for tissue homeostasis. However, embryo apoptosis had been known to be related to embryo fragmentation which should be avoided in in vitro fertilization (IVF). The purpose of this study was to evaluate the relationship of embryo apoptosis with the grade of immature oocytes and cleavage stage of in vitro produced (IVP) cattle embryos. This study consisted of 345 oocytes collected through ovary slicing. Immature oocytes were graded as A, B and C. This grading was based on cumulus cell thickness and compactness. All oocytes then underwent an in vitro maturation (IVM) procedure. An IVF was done 24 h after IVM culture. Prior to staining, stage of cleaved embryos was determined and classified as either 2, 4, 8 or >8-cell embryo stage. Apoptosis status of cleaved IVP embryos was determined by using annexin V-FITC staining technique at 48 and 72 h post insemination (hpi). Apoptosis status for each embryo was classified as either early or late. The result showed that there was no significant difference (p > 0.05) of apoptosis status among grade A, B and C embryos. All grades of oocytes showed embryo apoptosis where 1.5% late apoptosis for grade A, 4.5% and 10.4% of early and late apoptosis for grade B and grade C. Early apoptosis was not seen in grade A embryo. We also noted no significant difference (p > 0.05) of apoptosis status between 2, 4, 8 and >8-cell embryo stage. Early apoptosis was also not seen in >8-cell stage. Even though there were no differences in apoptosis expression between the three classes, the cleavage rate of grade A oocytes was significantly higher (p < 0.01) than grade B and grade C. In conclusion, the apoptosis expression in the embryo can occur regardless of the oocyte quality and the cleavage stage of the embryo produced. PMID:26858565

  8. Cell growth and proteolytic activity of Lactobacillus acidophilus, Lactobacillus helveticus, Lactobacillus delbrueckii ssp. bulgaricus, and Streptococcus thermophilus in milk as affected by supplementation with peptide fractions.

    Science.gov (United States)

    Gandhi, Akanksha; Shah, Nagendra P

    2014-12-01

    The present investigation examined the effects of supplementation of milk peptide fractions produced by enzymatic hydrolysis on the fermentation of reconstituted skim milk (RSM). Changes in pH, cell growth, proteolytic activity, and angiotensin-converting enzyme (ACE)-inhibitory activity were monitored during fermentation of RSM by pure cultures of Lactobacillus acidophilus, Lactobacillus helveticus, Lactobacillus delbrueckii ssp. bulgaricus, and Streptococcus thermophilus. The study showed that supplementation with peptide fractions of different molecular weights did not significantly affect the bacterial growth in RSM. All bacteria showed an increased proteolytic activity in RSM supplemented with large peptides (>10 kDa), and L. helveticus in general exhibited the highest proteolytic activity among the bacteria studied. The ACE-inhibitory activity was observed to be the maximum in RSM supplemented with larger peptides (>10 kDa) for all bacteria. The results suggest that proteolysis by bacteria leads to increased production of ACE-inhibitory peptides compared to the supplemented peptides produced by enzymatic hydrolysis.

  9. Dual CRISPR-Cas9 Cleavage Mediated Gene Excision and Targeted Integration in Yarrowia lipolytica.

    Science.gov (United States)

    Gao, Difeng; Smith, Spencer; Spagnuolo, Michael; Rodriguez, Gabriel; Blenner, Mark

    2018-05-29

    CRISPR-Cas9 technology has been successfully applied in Yarrowia lipolytica for targeted genomic editing including gene disruption and integration; however, disruptions by existing methods typically result from small frameshift mutations caused by indels within the coding region, which usually resulted in unnatural protein. In this study, a dual cleavage strategy directed by paired sgRNAs is developed for gene knockout. This method allows fast and robust gene excision, demonstrated on six genes of interest. The targeted regions for excision vary in length from 0.3 kb up to 3.5 kb and contain both non-coding and coding regions. The majority of the gene excisions are repaired by perfect nonhomologous end-joining without indel. Based on this dual cleavage system, two targeted markerless integration methods are developed by providing repair templates. While both strategies are effective, homology mediated end joining (HMEJ) based method are twice as efficient as homology recombination (HR) based method. In both cases, dual cleavage leads to similar or improved gene integration efficiencies compared to gene excision without integration. This dual cleavage strategy will be useful for not only generating more predictable and robust gene knockout, but also for efficient targeted markerless integration, and simultaneous knockout and integration in Y. lipolytica. © 2018 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

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

    Science.gov (United States)

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

    2017-02-01

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

  11. Cas9-catalyzed DNA Cleavage Generates Staggered Ends: Evidence from Molecular Dynamics Simulations

    Science.gov (United States)

    Zuo, Zhicheng; Liu, Jin

    2016-11-01

    The CRISPR-associated endonuclease Cas9 from Streptococcus pyogenes (spCas9) along with a single guide RNA (sgRNA) has emerged as a versatile toolbox for genome editing. Despite recent advances in the mechanism studies on spCas9-sgRNA-mediated double-stranded DNA (dsDNA) recognition and cleavage, it is still unclear how the catalytic Mg2+ ions induce the conformation changes toward the catalytic active state. It also remains controversial whether Cas9 generates blunt-ended or staggered-ended breaks with overhangs in the DNA. To investigate these issues, here we performed the first all-atom molecular dynamics simulations of the spCas9-sgRNA-dsDNA system with and without Mg2+ bound. The simulation results showed that binding of two Mg2+ ions at the RuvC domain active site could lead to structurally and energetically favorable coordination ready for the non-target DNA strand cleavage. Importantly, we demonstrated with our simulations that Cas9-catalyzed DNA cleavage produces 1-bp staggered ends rather than generally assumed blunt ends.

  12. Isolation and Characterization of Proteolytic Ruminal Bacteria from Sheep and Goats Fed the Tannin-Containing Shrub Legume Calliandra calothyrsus

    Science.gov (United States)

    McSweeney, Christopher S.; Palmer, Brian; Bunch, Rowan; Krause, Denis O.

    1999-01-01

    Tannins in forages complex with protein and reduce the availability of nitrogen to ruminants. Ruminal bacteria that ferment protein or peptides in the presence of tannins may benefit digestion of these diets. Bacteria from the rumina of sheep and goats fed Calliandra calothyrsus (3.6% N and 6% condensed tannin) were isolated on proteinaceous agar medium overlaid with either condensed (calliandra tannin) or hydrolyzable (tannic acid) tannin. Fifteen genotypes were identified, based on 16S ribosomal DNA-restriction fragment length polymorphism analysis, and all were proteolytic and fermented peptides to ammonia. Ten of the isolates grew to high optical density (OD) on carbohydrates (glucose, cellobiose, xylose, xylan, starch, and maltose), while the other isolates did not utilize or had low growth on these substrates. In pure culture, representative isolates were unable to ferment protein that was present in calliandra or had been complexed with tannin. One isolate, Lp1284, had high protease activity (80 U), a high specific growth rate (0.28), and a high rate of ammonia production (734 nmol/min/ml/OD unit) on Casamino Acids and Trypticase Peptone. Phylogenetic analysis of the 16S ribosomal DNA sequence showed that Lp1284 was related (97.6%) to Clostridium botulinum NCTC 7273. Purified plant protein and casein also supported growth of Lp1284 and were fermented to ammonia. This is the first report of a proteolytic, ammonia-hyperproducing bacterium from the rumen. In conclusion, a diverse group of proteolytic and peptidolytic bacteria were present in the rumen, but the isolates could not digest protein that was complexed with condensed tannin. PMID:10388706

  13. Proteolytic activities in yeast after UV irradiation. Pt. 1

    International Nuclear Information System (INIS)

    Schwencke, J.; Moustacchi, E.

    1982-01-01

    Specific proteolytic activities are known to be induced in Escherichia coli following irradiation. Consequently it seemed of interest to investigate whether variations in proteinase activities occur in yeast. Among the five most well known proteinases of Saccharomyces cerevisiae, we have found that proteinase B activity increases up to three times in wild-type RAD + yeast cells after a dose of 50 Jm -2 of 254 nm ultraviolet light (40% survival). Carboxypeptidase Y and aminopeptidase I (leucin aminopeptidase) activities were only moderately increased. Proteinase A activity was only slightly enhanced, while aminopeptidase II (lysin aminopeptidase) was unaffected in both RAD + strains studied. The observed post UV-increase in proteinase B activity was inhibited by cycloheximide and was dose dependent. Increases in proteinase B levels were independent of the activation method used to destroy the proteinase B-inhibitor complex present in the crude yeast extracts. A standard method for comparison of the postirradiation levels among different proteinases, strains and methods of activation is presented. (orig.)

  14. Proteolytic activities in yeast after UV irradiation. Pt. 1

    Energy Technology Data Exchange (ETDEWEB)

    Schwencke, J.; Moustacchi, E.

    1982-04-01

    Specific proteolytic activities are known to be induced in Escherichia coli following irradiation. Consequently it seemed of interest to investigate whether variations in proteinase activities occur in yeast. Among the five most well known proteinases of Saccharomyces cerevisiae, we have found that proteinase B activity increases up to three times in wild-type RAD/sup +/ yeast cells after a dose of 50 Jm/sup -2/ of 254 nm ultraviolet light (40% survival). Carboxypeptidase Y and aminopeptidase I (leucin aminopeptidase) activities were only moderately increased. Proteinase A activity was only slightly enhanced, while aminopeptidase II (lysin aminopeptidase) was unaffected in both RAD/sup +/ strains studied. The observed post UV-increase in proteinase B activity was inhibited by cycloheximide and was dose dependent. Increases in proteinase B levels were independent of the activation method used to destroy the proteinase B-inhibitor complex present in the crude yeast extracts. A standard method for comparison of the postirradiation levels among different proteinases, strains and methods of activation is presented.

  15. Discrimination between sequential and simultaneous virtual channels with electrical hearing.

    Science.gov (United States)

    Landsberger, David; Galvin, John J

    2011-09-01

    In cochlear implants (CIs), simultaneous or sequential stimulation of adjacent electrodes can produce intermediate pitch percepts between those of the component electrodes. However, it is unclear whether simultaneous and sequential virtual channels (VCs) can be discriminated. In this study, CI users were asked to discriminate simultaneous and sequential VCs; discrimination was measured for monopolar (MP) and bipolar + 1 stimulation (BP + 1), i.e., relatively broad and focused stimulation modes. For sequential VCs, the interpulse interval (IPI) varied between 0.0 and 1.8 ms. All stimuli were presented at comfortably loud, loudness-balanced levels at a 250 pulse per second per electrode (ppse) stimulation rate. On average, CI subjects were able to reliably discriminate between sequential and simultaneous VCs. While there was no significant effect of IPI or stimulation mode on VC discrimination, some subjects exhibited better VC discrimination with BP + 1 stimulation. Subjects' discrimination between sequential and simultaneous VCs was correlated with electrode discrimination, suggesting that spatial selectivity may influence perception of sequential VCs. To maintain equal loudness, sequential VC amplitudes were nearly double those of simultaneous VCs, presumably resulting in a broader spread of excitation. These results suggest that perceptual differences between simultaneous and sequential VCs might be explained by differences in the spread of excitation. © 2011 Acoustical Society of America

  16. Sequential versus simultaneous market delineation

    DEFF Research Database (Denmark)

    Haldrup, Niels; Møllgaard, Peter; Kastberg Nielsen, Claus

    2005-01-01

    and geographical markets. Using a unique data setfor prices of Norwegian and Scottish salmon, we propose a methodologyfor simultaneous market delineation and we demonstrate that comparedto a sequential approach conclusions will be reversed.JEL: C3, K21, L41, Q22Keywords: Relevant market, econometric delineation......Delineation of the relevant market forms a pivotal part of most antitrustcases. The standard approach is sequential. First the product marketis delineated, then the geographical market is defined. Demand andsupply substitution in both the product dimension and the geographicaldimension...

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

    Directory of Open Access Journals (Sweden)

    Travis Sexton

    Full Text Available Neprilysin (NEP, a member of the M13 subgroup of the zinc-dependent endopeptidase family is a membrane bound peptidase capable of cleaving a variety of physiological peptides. We have generated a series of neprilysin variants containing mutations at either one of two active site residues, Phe(563 and Ser(546. Among the mutants studied in detail we observed changes in their activity towards leucine(5-enkephalin, insulin B chain, and amyloid β(1-40. For example, NEP(F563I displayed an increase in preference towards cleaving leucine(5-enkephalin relative to insulin B chain, while mutant NEP(S546E was less discriminating than neprilysin. Mutants NEP(F563L and NEP(S546E exhibit different cleavage site preferences than neprilysin with insulin B chain and amyloid ß(1-40 as substrates. These data indicate that it is possible to alter the cleavage site specificity of neprilysin opening the way for the development of substrate specific or substrate exclusive forms of the enzyme with enhanced therapeutic potential.

  18. Cleavage of thymine N3-H bonds by low-energy electrons attached to base π* orbitals

    International Nuclear Information System (INIS)

    Theodore, Magali; Sobczyk, Monika; Simons, Jack

    2006-01-01

    In this work, we extend our earlier studies on single strand break (SSB) formation in DNA to consider the possibility of cleaving a thymine N 3 -H bond to generate a nitrogen-centered anion and a hydrogen radical which might proceed to induce further bond cleavages. In earlier studies, we considered SSBs induced by low-energy electrons that attach to DNA bases' π* orbitals or to phosphate P=O π* orbitals to cleave sugar-phosphate C-O bonds or base-sugar N 1 -C bonds. We also studied the effects of base π-stacking on the rates of such bond cleavages. To date, our results suggest that sugar-phosphate C-O bonds have the lowest barriers to cleavage, that attachment of electrons with energies below 2 eV most likely occurs at the base π* orbitals, that electrons with energy above 2 eV can also attach to phosphate P=O π* orbitals, and that base π stacking has a modest but slowing effect on the rates of SSB formation. However, we had not yet examined the possibility that base N 3 -H bonds could rupture subsequent to base π* orbital capture. In the present work, the latter possibility is considered and it is found that the barrier to cleavage of the N 3 -H bond in thymine is considerably higher than for cleaving sugar-phosphate C-O bonds, so our prediction that SSB formation is dominated by C-O bond cleavage remains intact

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

    Science.gov (United States)

    2016-01-01

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

  20. Maximizing Selective Cleavages at Aspartic Acid and Proline Residues for the Identification of Intact Proteins

    Science.gov (United States)

    Foreman, David J.; Dziekonski, Eric T.; McLuckey, Scott A.

    2018-04-01

    A new approach for the identification of intact proteins has been developed that relies on the generation of relatively few abundant products from specific cleavage sites. This strategy is intended to complement standard approaches that seek to generate many fragments relatively non-selectively. Specifically, this strategy seeks to maximize selective cleavage at aspartic acid and proline residues via collisional activation of precursor ions formed via electrospray ionization (ESI) under denaturing conditions. A statistical analysis of the SWISS-PROT database was used to predict the number of arginine residues for a given intact protein mass and predict a m/z range where the protein carries a similar charge to the number of arginine residues thereby enhancing cleavage at aspartic acid residues by limiting proton mobility. Cleavage at aspartic acid residues is predicted to be most favorable in the m/z range of 1500-2500, a range higher than that normally generated by ESI at low pH. Gas-phase proton transfer ion/ion reactions are therefore used for precursor ion concentration from relatively high charge states followed by ion isolation and subsequent generation of precursor ions within the optimal m/z range via a second proton transfer reaction step. It is shown that the majority of product ion abundance is concentrated into cleavages C-terminal to aspartic acid residues and N-terminal to proline residues for ions generated by this process. Implementation of a scoring system that weights both ion fragment type and ion fragment area demonstrated identification of standard proteins, ranging in mass from 8.5 to 29.0 kDa. [Figure not available: see fulltext.

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

    Science.gov (United States)

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

    2017-09-01

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

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

    Directory of Open Access Journals (Sweden)

    Oliver Tiedt

    2016-08-01

    Full Text Available Complete biodegradation of the abundant and persistent fluoroaromatics requires enzymatic cleavage of an arylic C–F bond, probably the most stable single bond of a biodegradable organic molecule. While in aerobic microorganisms defluorination of fluoroaromatics is initiated by oxygenases, arylic C–F bond cleavage has never been observed in the absence of oxygen. Here, an oxygen-independent enzymatic aryl fluoride bond cleavage is described during the complete degradation of 4-fluorobenzoate or 4-fluorotoluene to CO2 and HF in the denitrifying Thauera aromatica: the ATP-dependent defluorination of 4-fluorobenzoyl-coenzyme A (4-F-BzCoA to benzoyl-coenzyme A (BzCoA and HF, catalyzed by class I BzCoA reductase (BCR. Adaptation to growth with the fluoroaromatics was accomplished by the downregulation of a promiscuous benzoate-CoA ligase and the concomitant upregulation of 4-F-BzCoA-defluorinating/dearomatizing BCR on the transcriptional level. We propose an unprecedented mechanism for reductive arylic C–F bond cleavage via a Birch reduction-like mechanism resulting in a formal nucleophilic aromatic substitution. In the proposed anionic 4-fluorodienoyl-CoA transition state, fluoride elimination to BzCoA is favored over protonation to a fluorinated cyclic dienoyl-CoA.

  3. Group-sequential analysis may allow for early trial termination

    DEFF Research Database (Denmark)

    Gerke, Oke; Vilstrup, Mie H; Halekoh, Ulrich

    2017-01-01

    BACKGROUND: Group-sequential testing is widely used in pivotal therapeutic, but rarely in diagnostic research, although it may save studies, time, and costs. The purpose of this paper was to demonstrate a group-sequential analysis strategy in an intra-observer study on quantitative FDG-PET/CT mea......BACKGROUND: Group-sequential testing is widely used in pivotal therapeutic, but rarely in diagnostic research, although it may save studies, time, and costs. The purpose of this paper was to demonstrate a group-sequential analysis strategy in an intra-observer study on quantitative FDG...

  4. Sequential logic analysis and synthesis

    CERN Document Server

    Cavanagh, Joseph

    2007-01-01

    Until now, there was no single resource for actual digital system design. Using both basic and advanced concepts, Sequential Logic: Analysis and Synthesis offers a thorough exposition of the analysis and synthesis of both synchronous and asynchronous sequential machines. With 25 years of experience in designing computing equipment, the author stresses the practical design of state machines. He clearly delineates each step of the structured and rigorous design principles that can be applied to practical applications. The book begins by reviewing the analysis of combinatorial logic and Boolean a

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

    DEFF Research Database (Denmark)

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

    1993-01-01

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

  6. Cleavage of desmin by cysteine proteases: Calpains and cathepsin B

    DEFF Research Database (Denmark)

    Baron, Caroline; Jacobsen, S.; Purslow, P.P.

    2004-01-01

    The intermediate filament protein, desmin, was purified from pork longissimus dorsi and incubated with either P-calpain, m-calpain or cathepsin B. Proteolysis of desmin was followed using SDS-PAGE and Western blotting. After incubation of desmin with the proteases, cleavage sites on the desmin mo...

  7. Structural Consistency, Consistency, and Sequential Rationality.

    OpenAIRE

    Kreps, David M; Ramey, Garey

    1987-01-01

    Sequential equilibria comprise consistent beliefs and a sequentially ra tional strategy profile. Consistent beliefs are limits of Bayes ratio nal beliefs for sequences of strategies that approach the equilibrium strategy. Beliefs are structurally consistent if they are rationaliz ed by some single conjecture concerning opponents' strategies. Consis tent beliefs are not necessarily structurally consistent, notwithstan ding a claim by Kreps and Robert Wilson (1982). Moreover, the spirit of stru...

  8. Development of a solid-phase assay for measurement of proteolytic enzyme activity

    International Nuclear Information System (INIS)

    Varani, J.; Johnson, K.; Kaplan, J.

    1980-01-01

    A solid-phase, plate assay was developed for the measurement of proteolytic enzyme activity. In this assay procedure, radiolabeled substrates were dried onto the surface of microtiter wells. Following drying, the wells were washed two times with saline to remove the nonadherent substrate. When proteolytic enzymes were added to the wells, protein hydrolysis occurred, releasing radioactivity into the supernatant fluid. The amount of protein hydrolysis that occurred was reflected by the amount of radioactivity in the supernatant fluid. When 125 I-hemoglobin was used as the substrate, it was as susceptible to hydrolysis by trypsin in the solid-phase assay as it was in solution in a standard assay procedure. Protease activity from a variety of sources (including from viable cells as well as from extracellular sources) were also able to hydrolyze the hemoglobin on the plate. 125 I-Labeled serum albumen, fibrinogen, and rat pulmonary basement membrane were also susceptible to hydrolysis by trypsin in the solid phase. When [ 14 C]elastin was dried onto the plate, it behaved in a similar manner to elastin in solution. It was resistant to hydrolysis by nonspecific proteases such as trypsin and chymotrypsin but was highly susceptible to hydrolysis by elastase. The solid-phase plate assay has several features which recommended it for routine use. It is as sensitive as standard tube assays (and much more sensitive than routinely used colormetric assays). It is quick and convenient; there are no precipitation, centrifugation, or filtration steps. In addition, very small volumes of radioactive wastes are generated. Another advantage of the solid-phase plate assay is the resistance of the dried substrates to spontaneous breakdown and to microbial contamination. Finally, this assay is suitable for use with viable cells as well as for extracellular proteases

  9. Multiplexed homogeneous assays of proteolytic activity using a smartphone and quantum dots.

    Science.gov (United States)

    Petryayeva, Eleonora; Algar, W Russ

    2014-03-18

    Semiconductor quantum dot (QD) bioconjugates, with their unique and highly advantageous physicochemical and optical properties, have been extensively utilized as probes for bioanalysis and continue to generate widespread interest for these applications. An important consideration for expanding the utility of QDs and making their use routine is to make assays with QDs more accessible for laboratories that do not specialize in nanomaterials. Here, we show that digital color imaging of QD photoluminescence (PL) with a smartphone camera is a viable, easily accessible readout platform for quantitative, multiplexed, and real-time bioanalyses. Red-, green-, and blue-emitting CdSeS/ZnS QDs were conjugated with peptides that were labeled with a deep-red fluorescent dye, Alexa Fluor 647, and the dark quenchers, QSY9 and QSY35, respectively, to generate Förster resonance energy transfer (FRET) pairs sensitive to proteolytic activity. Changes in QD PL caused by the activity of picomolar to nanomolar concentrations of protease were detected as changes in the red-green-blue (RGB) channel intensities in digital color images. Importantly, measurements of replicate samples made with smartphone imaging and a sophisticated fluorescence plate reader yielded the same quantitative results, including initial proteolytic rates and specificity constants. Homogeneous two-plex and three-plex assays for the activity of trypsin, chymotrypsin, and enterokinase were demonstrated with RGB imaging. Given the ubiquity of smartphones, this work largely removes any instrumental impediments to the adoption of QDs as routine tools for bioanalysis in research laboratories and is a critical step toward the use of QDs for point-of-care diagnostics. This work also adds to the growing utility of smartphones in analytical methods by enabling multiplexed fluorimetric assays within a single sample volume and across multiple samples in parallel.

  10. The dimer interfaces of protease and extra-protease domains influence the activation of protease and the specificity of GagPol cleavage.

    Science.gov (United States)

    Pettit, Steven C; Gulnik, Sergei; Everitt, Lori; Kaplan, Andrew H

    2003-01-01

    Activation of the human immunodeficiency virus type 1 (HIV-1) protease is an essential step in viral replication. As is the case for all retroviral proteases, enzyme activation requires the formation of protease homodimers. However, little is known about the mechanisms by which retroviral proteases become active within their precursors. Using an in vitro expression system, we have examined the determinants of activation efficiency and the order of cleavage site processing for the protease of HIV-1 within the full-length GagPol precursor. Following activation, initial cleavage occurs between the viral p2 and nucleocapsid proteins. This is followed by cleavage of a novel site located in the transframe domain. Mutational analysis of the dimer interface of the protease produced differential effects on activation and specificity. A subset of mutations produced enhanced cleavage at the amino terminus of the protease, suggesting that, in the wild-type precursor, cleavages that liberate the protease are a relatively late event. Replacement of the proline residue at position 1 of the protease dimer interface resulted in altered cleavage of distal sites and suggests that this residue functions as a cis-directed specificity determinant. In summary, our studies indicate that interactions within the protease dimer interface help determine the order of precursor cleavage and contribute to the formation of extended-protease intermediates. Assembly domains within GagPol outside the protease domain also influence enzyme activation.

  11. A smallest 6 kda metalloprotease, mini-matrilysin, in living world: a revolutionary conserved zinc-dependent proteolytic domain- helix-loop-helix catalytic zinc binding domain (ZBD

    Directory of Open Access Journals (Sweden)

    Yu Wei-Hsuan

    2012-05-01

    Full Text Available Abstract Background The Aim of this study is to study the minimum zinc dependent metalloprotease catalytic folding motif, helix B Met loop-helix C, with proteolytic catalytic activities in metzincin super family. The metzincin super family share a catalytic domain consisting of a twisted five-stranded β sheet and three long α helices (A, B and C. The catalytic zinc is at the bottom of the cleft and is ligated by three His residues in the consensus sequence motif, HEXXHXXGXXH, which is located in helix B and part of the adjacent Met turn region. An interesting question is - what is the minimum portion of the enzyme that still possesses catalytic and inhibitor recognition?” Methods We have expressed a 60-residue truncated form of matrilysin which retains only the helix B-Met turn-helix C region and deletes helix A and the five-stranded β sheet which form the upper portion of the active cleft. This is only 1/4 of the full catalytic domain. The E. coli derived 6 kDa MMP-7 ZBD fragments were purified and refolded. The proteolytic activities were analyzed by Mca-Pro-Leu-Gly-Leu-Dpa-Ala-Arg-NH2 peptide assay and CM-transferrin zymography analysis. SC44463, BB94 and Phosphoramidon were computationally docked into the 3day structure of the human MMP7 ZBD and TAD and thermolysin using the docking program GOLD. Results This minimal 6 kDa matrilysin has been refolded and shown to have proteolytic activity in the Mca-Pro-Leu-Gly-Leu-Dpa-Ala-Arg-NH2 peptide assay. Triton X-100 and heparin are important factors in the refolding environment for this mini-enzyme matrilysin. This minienzyme has the proteolytic activity towards peptide substrate, but the hexamer and octamer of the mini MMP-7 complex demonstrates the CM-transferrin proteolytic activities in zymographic analysis. Peptide digestion is inhibited by SC44463, specific MMP7 inhibitors, but not phosphorimadon. Interestingly, the mini MMP-7 can be processed by autolysis and producing ~ 6

  12. Can laccases catalyze bond cleavage in lignin?

    DEFF Research Database (Denmark)

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

    2015-01-01

    illustrations of the putative laccase catalyzed reactions, including the possible reactions of the reactive radical intermediates taking place after the initial oxidation of the phenol-hydroxyl groups, we show that i) Laccase activity is able to catalyze bond cleavage in low molecular weight phenolic lignin......-substituted phenols, benzenethiols, polyphenols, and polyamines, which may be oxidized. In addition, the currently available analytical methods that can be used to detect enzyme catalyzed changes in lignin are summarized, and an improved nomenclature for unequivocal interpretation of the action of laccases on lignin...

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

    NARCIS (Netherlands)

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

    2003-01-01

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

  14. Proteolytic action of the crude extract ofDuddingtonia flagrans on cyathostomins (Nematoda: Cyathostominae in coprocultures

    Directory of Open Access Journals (Sweden)

    Fabio Ribeiro Braga

    Full Text Available The objective of this study was to examine the action of the crude extract of Duddingtonia flagrans (isolates AC001 and CG722 on infective larvae (L3 of cyathostomins in coprocultures and to confirm its proteolytic activity by means of a zymogram. The following groups were formed in coprocultures: Group 1: 10 mL of crude extract of D. flagrans (AC001; group 2: 10 mL of crude extract of AC001 with 10 mM of Ca2+; group 3: 10 mL of crude extract of D. flagrans (CG722; group 4: 10 mL of crude extract of CG722 with 10 mM of Ca2+; and group 5: control group (distilled water. The third-stage larvae (L3 were obtained after eight days. The crude extract of D. flagrans was effective in reducing the number of L3, with the following percentage reductions: group 1, 49.5%; group 2, 52.5%; group 3, 36.8%; and group 4, 57.7%; in relation to the control group (p > 0.05. The proteolytic activity of the crude extract was confirmed through the zymogram. The results from this study confirmed that the crude extract of the fungusD. flagrans could be used for controlling cyathostomin L3, and suggested that at least one protease of approximately 38 kDa was present.

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

    OpenAIRE

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

    1989-01-01

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

  16. Force-Induced Calpain Cleavage of Talin Is Critical for Growth, Adhesion Development, and Rigidity Sensing.

    Science.gov (United States)

    Saxena, Mayur; Changede, Rishita; Hone, James; Wolfenson, Haguy; Sheetz, Michael P

    2017-12-13

    Cell growth depends upon formation of cell-matrix adhesions, but mechanisms detailing the transmission of signals from adhesions to control proliferation are still lacking. Here, we find that the scaffold protein talin undergoes force-induced cleavage in early adhesions to produce the talin rod fragment that is needed for cell cycle progression. Expression of noncleavable talin blocks cell growth, adhesion maturation, proper mechanosensing, and the related property of EGF activation of motility. Further, the expression of talin rod in the presence of noncleavable full-length talin rescues cell growth and other functions. The cleavage of talin is found in early adhesions where there is also rapid turnover of talin that depends upon calpain and TRPM4 activity as well as the generation of force on talin. Thus, we suggest that an important function of talin is its control over cell cycle progression through its cleavage in early adhesions.

  17. Generalized infimum and sequential product of quantum effects

    International Nuclear Information System (INIS)

    Li Yuan; Sun Xiuhong; Chen Zhengli

    2007-01-01

    The quantum effects for a physical system can be described by the set E(H) of positive operators on a complex Hilbert space H that are bounded above by the identity operator I. For A, B(set-membership sign)E(H), the operation of sequential product A(convolution sign)B=A 1/2 BA 1/2 was proposed as a model for sequential quantum measurements. A nice investigation of properties of the sequential product has been carried over [Gudder, S. and Nagy, G., 'Sequential quantum measurements', J. Math. Phys. 42, 5212 (2001)]. In this note, we extend some results of this reference. In particular, a gap in the proof of Theorem 3.2 in this reference is overcome. In addition, some properties of generalized infimum A sqcap B are studied

  18. Effect of culturing conditions on the expression of key enzymes in the proteolytic system of Lactobacillus bulgaricus *

    Science.gov (United States)

    Hou, Jun-cai; Liu, Fei; Ren, Da-xi; Han, Wei-wei; Du, Yue-ou

    2015-01-01

    The proteolytic system of Lactobacillus bulgaricus breaks down milk proteins into peptides and amino acids, which are essential for the growth of the bacteria. The aim of this study was to determine the expressions of seven key genes in the proteolytic system under different culturing conditions (different phases, initial pH values, temperatures, and nitrogen sources) using real-time polymerase chain reaction (RT-PCR). The transcriptions of the seven genes were reduced by 30-fold on average in the stationary phase compared with the exponential growth phase. The transcriptions of the seven genes were reduced by 62.5-, 15.0-, and 59.0-fold in the strains KLDS 08006, KLDS 08007, and KLDS 08012, respectively, indicating that the expressions of the seven genes were significantly different among strains. In addition, the expressions of the seven genes were repressed in the MRS medium containing casein peptone. The effect of peptone supply on PepX transcription was the weakest compared with the other six genes, and the impact on OppD transcription was the strongest. Moreover, the expressions of the seven genes were significantly different among different strains (PLactobacillus bulgaricus at the transcription level. PMID:25845365

  19. Effect of culturing conditions on the expression of key enzymes in the proteolytic system of Lactobacillus bulgaricus.

    Science.gov (United States)

    Hou, Jun-cai; Liu, Fei; Ren, Da-xi; Han, Wei-wei; Du, Yue-ou

    2015-04-01

    The proteolytic system of Lactobacillus bulgaricus breaks down milk proteins into peptides and amino acids, which are essential for the growth of the bacteria. The aim of this study was to determine the expressions of seven key genes in the proteolytic system under different culturing conditions (different phases, initial pH values, temperatures, and nitrogen sources) using real-time polymerase chain reaction (RT-PCR). The transcriptions of the seven genes were reduced by 30-fold on average in the stationary phase compared with the exponential growth phase. The transcriptions of the seven genes were reduced by 62.5-, 15.0-, and 59.0-fold in the strains KLDS 08006, KLDS 08007, and KLDS 08012, respectively, indicating that the expressions of the seven genes were significantly different among strains. In addition, the expressions of the seven genes were repressed in the MRS medium containing casein peptone. The effect of peptone supply on PepX transcription was the weakest compared with the other six genes, and the impact on OppD transcription was the strongest. Moreover, the expressions of the seven genes were significantly different among different strains (Pproteolytic system genes in Lactobacillus bulgaricus at the transcription level.

  20. A Subset of Membrane-Altering Agents and γ-Secretase Modulators Provoke Nonsubstrate Cleavage by Rhomboid Proteases

    Directory of Open Access Journals (Sweden)

    Siniša Urban

    2014-09-01

    Full Text Available Rhomboid proteases are integral membrane enzymes that regulate cell signaling, adhesion, and organelle homeostasis pathways, making substrate specificity a key feature of their function. Interestingly, we found that perturbing the membrane pharmacologically in living cells had little effect on substrate processing but induced inappropriate cleavage of nonsubstrates by rhomboid proteases. A subclass of drugs known to modulate γ-secretase activity acted on the membrane directly and induced nonsubstrate cleavage by rhomboid proteases but left true substrate cleavage sites unaltered. These observations highlight an active role for the membrane in guiding rhomboid selectivity and caution that membrane-targeted drugs should be evaluated for cross-activity against membrane-resident enzymes that are otherwise unrelated to the intended drug target. Furthermore, some γ-secretase-modulating activity or toxicity could partly result from global membrane effects.

  1. Protease-activated quantum dot probes

    International Nuclear Information System (INIS)

    Chang, Emmanuel; Miller, Jordan S.; Sun, Jiantang; Yu, William W.; Colvin, Vicki L.; Drezek, Rebekah; West, Jennifer L.

    2005-01-01

    We have developed a novel nanoparticulate luminescent probe with inherent signal amplification upon interaction with a targeted proteolytic enzyme. This construct may be useful for imaging in cancer detection and diagnosis. In this system, quantum dots (QDs) are bound to gold nanoparticles (AuNPs) via a proteolytically degradable peptide sequence to non-radiatively suppress luminescence. A 71% reduction in luminescence was achieved with conjugation of AuNPs to QDs. Release of AuNPs by peptide cleavage restores radiative QD photoluminescence. Initial studies observed a 52% rise in luminescence over 47 h of exposure to 0.2 mg/mL collagenase. These probes can be customized for targeted degradation simply by changing the sequence of the peptide linker

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

    Energy Technology Data Exchange (ETDEWEB)

    Weber, Nicholas D., E-mail: nweber@fhcrc.org [Vaccine and Infectious Disease Division, Fred Hutchinson Cancer Research Center, 1100 Fairview Ave N, E5-110, Seattle, WA 98109 (United States); Department of Laboratory Medicine, University of Washington, Seattle, WA 98195 (United States); Aubert, Martine, E-mail: maubert@fhcrc.org [Vaccine and Infectious Disease Division, Fred Hutchinson Cancer Research Center, 1100 Fairview Ave N, E5-110, Seattle, WA 98109 (United States); Dang, Chung H., E-mail: cdang@fhcrc.org [Vaccine and Infectious Disease Division, Fred Hutchinson Cancer Research Center, 1100 Fairview Ave N, E5-110, Seattle, WA 98109 (United States); Stone, Daniel, E-mail: dstone2@fhcrc.org [Vaccine and Infectious Disease Division, Fred Hutchinson Cancer Research Center, 1100 Fairview Ave N, E5-110, Seattle, WA 98109 (United States); Jerome, Keith R., E-mail: kjerome@fhcrc.org [Vaccine and Infectious Disease Division, Fred Hutchinson Cancer Research Center, 1100 Fairview Ave N, E5-110, Seattle, WA 98109 (United States); Department of Laboratory Medicine, University of Washington, Seattle, WA 98195 (United States); Department of Microbiology, University of Washington, Seattle, WA 98195 (United States)

    2014-04-15

    Treatment for most persistent viral infections consists of palliative drug options rather than curative approaches. This is often because long-lasting viral DNA in infected cells is not affected by current antivirals, providing a source for viral persistence and reactivation. Targeting latent viral DNA itself could therefore provide a basis for novel curative strategies. DNA cleavage enzymes can be used to induce targeted mutagenesis of specific genes, including those of exogenous viruses. Although initial in vitro and even in vivo studies have been carried out using DNA cleavage enzymes targeting various viruses, many questions still remain concerning the feasibility of these strategies as they transition into preclinical research. Here, we review the most recent findings on DNA cleavage enzymes for human viral infections, consider the most relevant animal models for several human viral infections, and address issues regarding safety and enzyme delivery. Results from well-designed in vivo studies will ideally provide answers to the most urgent remaining questions, and allow continued progress toward clinical application. - Highlights: • Recent in vitro and in vivo results for DNA cleavage enzymes targeting persistent viral infections. • Analysis of the best animal models for testing enzymes for HBV, HSV, HIV and HPV. • Challenges facing in vivo delivery of therapeutic enzymes for persistent viral infections. • Safety issues to be addressed with proper animal studies.

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

    Directory of Open Access Journals (Sweden)

    Shoba Subramanian

    Full Text Available The Plasmodium falciparum cysteine proteases falcipain-2 and falcipain-3 degrade host hemoglobin to provide free amino acids for parasite protein synthesis. Hemoglobin hydrolysis has been described as an ordered process initiated by aspartic proteases, but cysteine protease inhibitors completely block the process, suggesting that cysteine proteases can also initiate hemoglobin hydrolysis. To characterize the specific roles of falcipains, we used three approaches. First, using random P(1 - P(4 amino acid substrate libraries, falcipain-2 and falcipain-3 demonstrated strong preference for cleavage sites with Leu at the P(2 position. Second, with overlapping peptides spanning alpha and beta globin and proteolysis-dependent (18O labeling, hydrolysis was seen at many cleavage sites. Third, with intact hemoglobin, numerous cleavage products were identified. Our results suggest that hemoglobin hydrolysis by malaria parasites is not a highly ordered process, but rather proceeds with rapid cleavage by falcipains at multiple sites. However, falcipain-2 and falcipain-3 show strong specificity for P(2 Leu in small peptide substrates, in agreement with the specificity in optimized small molecule inhibitors that was identified previously. These results are consistent with a principal role of falcipain-2 and falcipain-3 in the hydrolysis of hemoglobin by P. falciparum and with the possibility of developing small molecule inhibitors with optimized specificity as antimalarial agents.

  4. Sequential analysis in neonatal research-systematic review.

    Science.gov (United States)

    Lava, Sebastiano A G; Elie, Valéry; Ha, Phuong Thi Viet; Jacqz-Aigrain, Evelyne

    2018-05-01

    As more new drugs are discovered, traditional designs come at their limits. Ten years after the adoption of the European Paediatric Regulation, we performed a systematic review on the US National Library of Medicine and Excerpta Medica database of sequential trials involving newborns. Out of 326 identified scientific reports, 21 trials were included. They enrolled 2832 patients, of whom 2099 were analyzed: the median number of neonates included per trial was 48 (IQR 22-87), median gestational age was 28.7 (IQR 27.9-30.9) weeks. Eighteen trials used sequential techniques to determine sample size, while 3 used continual reassessment methods for dose-finding. In 16 studies reporting sufficient data, the sequential design allowed to non-significantly reduce the number of enrolled neonates by a median of 24 (31%) patients (IQR - 4.75 to 136.5, p = 0.0674) with respect to a traditional trial. When the number of neonates finally included in the analysis was considered, the difference became significant: 35 (57%) patients (IQR 10 to 136.5, p = 0.0033). Sequential trial designs have not been frequently used in Neonatology. They might potentially be able to reduce the number of patients in drug trials, although this is not always the case. What is known: • In evaluating rare diseases in fragile populations, traditional designs come at their limits. About 20% of pediatric trials are discontinued, mainly because of recruitment problems. What is new: • Sequential trials involving newborns were infrequently used and only a few (n = 21) are available for analysis. • The sequential design allowed to non-significantly reduce the number of enrolled neonates by a median of 24 (31%) patients (IQR - 4.75 to 136.5, p = 0.0674).

  5. The tubulins of animals, plants, fungi and protists implications for metazoan evolution

    Science.gov (United States)

    Little, Melvyn; Ludueña, Richard F.; Morejohn, Louis C.; Asnes, Clara; Hoffman, Eugene

    1984-03-01

    α-Tubulin subunits from trout (S. gairdneri) sperm tails, sea urchin (S. purpuratus) cilia, protistan alga (C. elongatum) flagella and rose (Paul's Scarlet) cytoplasm have been characterized by limited proteolytic cleavage with the enzymeStaphylococcus aureus protease and electrophoresis of the digestion products on SDS-PAGE. The resulting patterns corresponded to either of two major types representative of animal and non-animal α-tubulins, respectively. A total of 28 α-tubulins have now been characterized by this method. They are classified in this paper according to the type of cleavage pattern generated by the enzymeS. aureus protease. The implications of these results for metazoan evolution are discussed.

  6. Group-sequential analysis may allow for early trial termination

    DEFF Research Database (Denmark)

    Gerke, Oke; Vilstrup, Mie H; Halekoh, Ulrich

    2017-01-01

    BACKGROUND: Group-sequential testing is widely used in pivotal therapeutic, but rarely in diagnostic research, although it may save studies, time, and costs. The purpose of this paper was to demonstrate a group-sequential analysis strategy in an intra-observer study on quantitative FDG-PET/CT mea......BACKGROUND: Group-sequential testing is widely used in pivotal therapeutic, but rarely in diagnostic research, although it may save studies, time, and costs. The purpose of this paper was to demonstrate a group-sequential analysis strategy in an intra-observer study on quantitative FDG...... assumed to be normally distributed, and sequential one-sided hypothesis tests on the population standard deviation of the differences against a hypothesised value of 1.5 were performed, employing an alpha spending function. The fixed-sample analysis (N = 45) was compared with the group-sequential analysis...... strategies comprising one (at N = 23), two (at N = 15, 30), or three interim analyses (at N = 11, 23, 34), respectively, which were defined post hoc. RESULTS: When performing interim analyses with one third and two thirds of patients, sufficient agreement could be concluded after the first interim analysis...

  7. Glutamic Acid Selective Chemical Cleavage of Peptide Bonds.

    Science.gov (United States)

    Nalbone, Joseph M; Lahankar, Neelam; Buissereth, Lyssa; Raj, Monika

    2016-03-04

    Site-specific hydrolysis of peptide bonds at glutamic acid under neutral aqueous conditions is reported. The method relies on the activation of the backbone amide chain at glutamic acid by the formation of a pyroglutamyl (pGlu) imide moiety. This activation increases the susceptibility of a peptide bond toward hydrolysis. The method is highly specific and demonstrates broad substrate scope including cleavage of various bioactive peptides with unnatural amino acid residues, which are unsuitable substrates for enzymatic hydrolysis.

  8. Comparison of ablation centration after bilateral sequential versus simultaneous LASIK.

    Science.gov (United States)

    Lin, Jane-Ming; Tsai, Yi-Yu

    2005-01-01

    To compare ablation centration after bilateral sequential and simultaneous myopic LASIK. A retrospective randomized case series was performed of 670 eyes of 335 consecutive patients who had undergone either bilateral sequential (group 1) or simultaneous (group 2) myopic LASIK between July 2000 and July 2001 at the China Medical University Hospital, Taichung, Taiwan. The ablation centrations of the first and second eyes in the two groups were compared 3 months postoperatively. Of 670 eyes, 274 eyes (137 patients) comprised the sequential group and 396 eyes (198 patients) comprised the simultaneous group. Three months post-operatively, 220 eyes of 110 patients (80%) in the sequential group and 236 eyes of 118 patients (60%) in the simultaneous group provided topographic data for centration analysis. For the first eyes, mean decentration was 0.39 +/- 0.26 mm in the sequential group and 0.41 +/- 0.19 mm in the simultaneous group (P = .30). For the second eyes, mean decentration was 0.28 +/- 0.23 mm in the sequential group and 0.30 +/- 0.21 mm in the simultaneous group (P = .36). Decentration in the second eyes significantly improved in both groups (group 1, P = .02; group 2, P sequential group and 0.32 +/- 0.18 mm in the simultaneous group (P = .33). The difference of ablation center angles between the first and second eyes was 43.2 sequential group and 45.1 +/- 50.8 degrees in the simultaneous group (P = .42). Simultaneous bilateral LASIK is comparable to sequential surgery in ablation centration.

  9. A Survey of Multi-Objective Sequential Decision-Making

    NARCIS (Netherlands)

    Roijers, D.M.; Vamplew, P.; Whiteson, S.; Dazeley, R.

    2013-01-01

    Sequential decision-making problems with multiple objectives arise naturally in practice and pose unique challenges for research in decision-theoretic planning and learning, which has largely focused on single-objective settings. This article surveys algorithms designed for sequential

  10. The proteolytic system of pineapple stems revisited: Purification and characterization of multiple catalytically active forms.

    Science.gov (United States)

    Matagne, André; Bolle, Laetitia; El Mahyaoui, Rachida; Baeyens-Volant, Danielle; Azarkan, Mohamed

    2017-06-01

    Crude pineapple proteases extract (aka stem bromelain; EC 3.4.22.4) is an important proteolytic mixture that contains enzymes belonging to the cysteine proteases of the papain family. Numerous studies have been reported aiming at the fractionation and characterization of the many molecular species present in the extract, but more efforts are still required to obtain sufficient quantities of the various purified protease forms for detailed physicochemical, enzymatic and structural characterization. In this work, we describe an efficient strategy towards the purification of at least eight enzymatic forms. Thus, following rapid fractionation on a SP-Sepharose FF column, two sub-populations with proteolytic activity were obtained: the unbound (termed acidic) and bound (termed basic) bromelain fractions. Following reversible modification with monomethoxypolyethylene glycol (mPEG), both fractions were further separated on Q-Sepharose FF and SP-Sepharose FF, respectively. This procedure yielded highly purified molecular species, all titrating ca. 1 mol of thiol group per mole of enzyme, with distinct biochemical properties. N-terminal sequencing allowed identifying at least eight forms with proteolytic activity. The basic fraction contained previously identified species, i.e. basic bromelain forms 1 and 2, ananain forms 1 and 2, and comosain (MEROPS identifier: C01.027). Furthermore, a new proteolytic species, showing similarities with basic bomelain forms 1 and 2, was discovered and termed bromelain form 3. The two remaining species were found in the acidic bromelain fraction and were arbitrarily named acidic bromelain forms 1 and 2. Both, acidic bromelain forms 1, 2 and basic bromelain forms 1, 2 and 3 are glycosylated, while ananain forms 1 and 2, and comosain are not. The eight protease forms display different amidase activities against the various substrates tested, namely small synthetic chromogenic compounds (DL-BAPNA and Boc-Ala-Ala-Gly-pNA), fluorogenic compounds

  11. 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.

  12. Sequential lineups: shift in criterion or decision strategy?

    Science.gov (United States)

    Gronlund, Scott D

    2004-04-01

    R. C. L. Lindsay and G. L. Wells (1985) argued that a sequential lineup enhanced discriminability because it elicited use of an absolute decision strategy. E. B. Ebbesen and H. D. Flowe (2002) argued that a sequential lineup led witnesses to adopt a more conservative response criterion, thereby affecting bias, not discriminability. Height was encoded as absolute (e.g., 6 ft [1.83 m] tall) or relative (e.g., taller than). If a sequential lineup elicited an absolute decision strategy, the principle of transfer-appropriate processing predicted that performance should be best when height was encoded absolutely. Conversely, if a simultaneous lineup elicited a relative decision strategy, performance should be best when height was encoded relatively. The predicted interaction was observed, providing direct evidence for the decision strategies explanation of what happens when witnesses view a sequential lineup.

  13. Assessment of proteolytic degradation of the basement membrane: a fragment of type IV collagen as a biochemical marker for liver fibrosis

    DEFF Research Database (Denmark)

    Veidal, Sanne S.; Karsdal, Morten A.; Nawrocki, Arkadiusz

    2011-01-01

    Collagen deposition and an altered matrix metalloproteinase (MMP) expression profile are hallmarks of fibrosis. Type IV collagen is the most abundant structural basement membrane component of tissue, which increases 14-fold during fibrogenesis in the liver. Proteolytic degradation of collagens...

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

    Science.gov (United States)

    Kotadia, Shaila; Montembault, Emilie; Sullivan, William

    2012-01-01

    Chromosome segregation must be coordinated with cell cleavage to ensure correct transmission of the genome to daughter cells. Here we identify a novel mechanism by which Drosophila melanogaster neuronal stem cells coordinate sister chromatid segregation with cleavage furrow ingression. Cells adapted to a dramatic increase in chromatid arm length by transiently elongating during anaphase/telophase. The degree of cell elongation correlated with the length of the trailing chromatid arms and was concomitant with a slight increase in spindle length and an enlargement of the zone of cortical myosin distribution. Rho guanine-nucleotide exchange factor (Pebble)–depleted cells failed to elongate during segregation of long chromatids. As a result, Pebble-depleted adult flies exhibited morphological defects likely caused by cell death during development. These studies reveal a novel pathway linking trailing chromatid arms and cortical myosin that ensures the clearance of chromatids from the cleavage plane at the appropriate time during cytokinesis, thus preserving genome integrity. PMID:23185030

  15. A radiolabel-release microwell assay for proteolytic enzymes present in cell culture media

    International Nuclear Information System (INIS)

    Rucklidge, G.J.; Milne, G.

    1990-01-01

    A modified method for the measurement of proteolytic enzyme activity in cell culture-conditioned media has been developed. Using the release of 3H-labeled peptides from 3H-labeled gelatin the method is performed in microwell plates. The substrate is insolubilized and attached to the wells by glutaraldehyde treatment, thus eliminating the need for a precipitation step at the end of the assay. The assay is sensitive, reproducible, and convenient for small sample volumes. The effect of different protease inhibitors on activity can be assessed rapidly allowing an early characterization of the enzyme. It can also be adapted to microplate spectrophotometric analysis by staining residual substrate with Coomassie blue

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

    DEFF Research Database (Denmark)

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

    2004-01-01

    We assessed the metabolic degradation kinetics and cleavage patterns of some selected CPP (cell-penetrating peptides) after incubation with confluent epithelial models. Synthesis of N-terminal CF [5(6)-carboxyfluorescein]-labelled CPP, namely hCT (human calcitonin)-derived sequences, Tat(47-57) a...

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

    Directory of Open Access Journals (Sweden)

    Oussama Ahrazem

    2016-10-01

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

  18. How to Read the Tractatus Sequentially

    Directory of Open Access Journals (Sweden)

    Tim Kraft

    2016-11-01

    Full Text Available One of the unconventional features of Wittgenstein’s Tractatus Logico-Philosophicus is its use of an elaborated and detailed numbering system. Recently, Bazzocchi, Hacker und Kuusela have argued that the numbering system means that the Tractatus must be read and interpreted not as a sequentially ordered book, but as a text with a two-dimensional, tree-like structure. Apart from being able to explain how the Tractatus was composed, the tree reading allegedly solves exegetical issues both on the local (e. g. how 4.02 fits into the series of remarks surrounding it and the global level (e. g. relation between ontology and picture theory, solipsism and the eye analogy, resolute and irresolute readings. This paper defends the sequential reading against the tree reading. After presenting the challenges generated by the numbering system and the two accounts as attempts to solve them, it is argued that Wittgenstein’s own explanation of the numbering system, anaphoric references within the Tractatus and the exegetical issues mentioned above do not favour the tree reading, but a version of the sequential reading. This reading maintains that the remarks of the Tractatus form a sequential chain: The role of the numbers is to indicate how remarks on different levels are interconnected to form a concise, surveyable and unified whole.

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

    Science.gov (United States)

    Kim, Kyungsub; Sim, Se-Hoon; Jeon, Che Ok; Lee, Younghoon; Lee, Kangseok

    2011-02-01

    RNase III, a double-stranded RNA-specific endoribonuclease, degrades bdm mRNA via cleavage at specific sites. To better understand the mechanism of cleavage site selection by RNase III, we performed a genetic screen for sequences containing mutations at the bdm RNA cleavage sites that resulted in altered mRNA stability using a transcriptional bdm'-'cat fusion construct. While most of the isolated mutants showed the increased bdm'-'cat mRNA stability that resulted from the inability of RNase III to cleave the mutated sequences, one mutant sequence (wt-L) displayed in vivo RNA stability similar to that of the wild-type sequence. In vivo and in vitro analyses of the wt-L RNA substrate showed that it was cut only once on the RNA strand to the 5'-terminus by RNase III, while the binding constant of RNase III to this mutant substrate was moderately increased. A base substitution at the uncleaved RNase III cleavage site in wt-L mutant RNA found in another mutant lowered the RNA-binding affinity by 11-fold and abolished the hydrolysis of scissile bonds by RNase III. Our results show that base substitutions at sites forming the scissile bonds are sufficient to alter RNA cleavage as well as the binding activity of RNase III. © 2010 Federation of European Microbiological Societies. Published by Blackwell Publishing Ltd. All rights reserved.

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

    Science.gov (United States)

    Zelanis, André; Huesgen, Pitter F; Oliveira, Ana Karina; Tashima, Alexandre K; Serrano, Solange M T; Overall, Christopher M

    2015-01-15

    Many snake venom toxins are serine proteases but their specific in vivo targets are mostly unknown. Various act on components of the coagulation cascade, and fibrinolytic and kallikrein-kinin systems to trigger various pathological effects observed in the envenomation. Despite showing high similarity in terms of primary structure snake venom serine proteinases (SVSPs) show exquisite specificity towards macromolecular substrates. Therefore, the characterization of their peptide bond specificity is important for understanding the active site preference associated with effective proteolysis as well as for the design of peptide substrates and inhibitors. Bothrops jararaca contains various SVSPs among which Bothrops protease A is a specific fibrinogenolytic agent and PA-BJ is a platelet-activating enzyme. In this study we used proteome derived peptide libraries in the Proteomic Identification of protease Cleavage Sites (PICS) approach to explore the peptide bond specificity of Bothrops protease A and PA-BJ in order to determine their individual peptide cleavage sequences. A total of 371 cleavage sites (208 for Bothrops protease A and 163 for PA-BJ) were detected and both proteinases displayed a clear preference for arginine at the P1 position. Moreover, the analysis of the specificity profiles of Bothrops protease A and PA-BJ revealed subtle differences in the preferences along P6-P6', despite a common yet unusual preference for Pro at P2. Taken together, these results map the subsite specificity of both SVSPs and shed light in the functional differences between these proteinases. Proteolysis is key to various pathological effects observed upon envenomation by viperid snakes. The use of the Proteomic Identification of protease Cleavage Sites (PICS) approach for the easy mapping of proteinase subsite preferences at both the prime- and non-prime sides concurrently gives rise to a fresh understanding of the interaction of the snake venom serine proteinases with peptide and

  1. A minimax procedure in the context of sequential mastery testing

    NARCIS (Netherlands)

    Vos, Hendrik J.

    1999-01-01

    The purpose of this paper is to derive optimal rules for sequential mastery tests. In a sequential mastery test, the decision is to classify a subject as a master or a nonmaster, or to continue sampling and administering another random test item. The framework of minimax sequential decision theory

  2. Evidence for the presence of proteolytically active secreted aspartic proteinase 1 of Candida parapsilosis in the cell wall

    Czech Academy of Sciences Publication Activity Database

    Vinterová, Zuzana; Šanda, Miloslav; Dostál, Jiří; Hrušková-Heidingsfeldová, Olga; Pichová, Iva

    2011-01-01

    Roč. 20, č. 12 (2011), s. 2004-2012 ISSN 0961-8368 R&D Projects: GA MŠk(CZ) LC531; GA ČR GA310/09/1945 Institutional research plan: CEZ:AV0Z40550506 Keywords : Candida parapsilosis * secreted aspartic proteinases * Sapp1p * cell wall * biotin * proteolytic activity Subject RIV: CE - Biochemistry Impact factor: 2.798, year: 2011

  3. Multichannel, sequential or combined X-ray spectrometry

    International Nuclear Information System (INIS)

    Florestan, J.

    1979-01-01

    X-ray spectrometer qualities and defects are evaluated for sequential and multichannel categories. Multichannel X-ray spectrometer has time-coherency advantage and its results could be more reproducible; on the other hand some spatial incoherency limits low percentage and traces applications, specially when backgrounds are very variable. In this last case, sequential X-ray spectrometer would find again great usefulness [fr

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

    NARCIS (Netherlands)

    Li, Qian; Browne, Wesley R.; Roelfes, Gerard

    2010-01-01

    The DNA cleavage activity of iron(II) complexes of a series of monotopic pentadentate N,N-bis(2-pyridylmethyl)-N-bis(2-pyridyl)methylamine (N4Py)-derived ligands (1-5) was investigated under laser irradiation at 473, 400.8, and 355 nm in the absence of a reducing agent and compared to that under

  5. Induction of simultaneous and sequential malolactic fermentation in durian wine.

    Science.gov (United States)

    Taniasuri, Fransisca; Lee, Pin-Rou; Liu, Shao-Quan

    2016-08-02

    This study represented for the first time the impact of malolactic fermentation (MLF) induced by Oenococcus oeni and its inoculation strategies (simultaneous vs. sequential) on the fermentation performance as well as aroma compound profile of durian wine. There was no negative impact of simultaneous inoculation of O. oeni and Saccharomyces cerevisiae on the growth and fermentation kinetics of S. cerevisiae as compared to sequential fermentation. Simultaneous MLF did not lead to an excessive increase in volatile acidity as compared to sequential MLF. The kinetic changes of organic acids (i.e. malic, lactic, succinic, acetic and α-ketoglutaric acids) varied with simultaneous and sequential MLF relative to yeast alone. MLF, regardless of inoculation mode, resulted in higher production of fermentation-derived volatiles as compared to control (alcoholic fermentation only), including esters, volatile fatty acids, and terpenes, except for higher alcohols. Most indigenous volatile sulphur compounds in durian were decreased to trace levels with little differences among the control, simultaneous and sequential MLF. Among the different wines, the wine with simultaneous MLF had higher concentrations of terpenes and acetate esters while sequential MLF had increased concentrations of medium- and long-chain ethyl esters. Relative to alcoholic fermentation only, both simultaneous and sequential MLF reduced acetaldehyde substantially with sequential MLF being more effective. These findings illustrate that MLF is an effective and novel way of modulating the volatile and aroma compound profile of durian wine. Copyright © 2016 Elsevier B.V. All rights reserved.

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

    Directory of Open Access Journals (Sweden)

    Zakia Khanam

    2015-10-01

    Full Text Available In developing countries, the increasing gap between population growth and food supply has created renewed interest in finding reliable and cheap natural resources of nutraceutical value and health promoting properties. Therefore, the present study deals with the phytochemical analyses and DNA cleavage activity of Averrhoa carambola L. fruit (starfruit extracts. The phytochemical studies involve colour tests and quantification of phenolics and flavonoids of the prepared ethanolic and aqueous extracts. Identification of phenolic acids and flavonoids present in the extracts were conducted by high performance liquid chromatography (HPLC equipped with diode array detector (DAD. DNA cleavage activity of the extracts was evaluated through gel electrophoresis against plasmid Escherichia coli DNA at different concentrations (0.125–0.60 μg/μl. The results of the study exhibited that the starfruit is a rich source of polyphenols and all the extracts exhibited a dose dependent DNA cleavage activity, whereas ethanolic extract induced more cleavage as compared to the aqueous extract. In conclusion, the present study provides preliminary evidence with regard to nutraceutical value of the fruit. So, further extensive study is a prerequisite to exploit DNA cleaving properties of the fruit extracts for therapeutic application.

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

    Directory of Open Access Journals (Sweden)

    Yujie Dai

    2017-12-01

    Full Text Available The data presented in this article are related to the research article entitled “The mechanism for cleavage of three typical glucosidic bonds induced by hydroxyl free radical” (Dai et al., 2017 [1]. This article includes the structures of three kinds of disaccharides such as maltose, fructose and cellobiose, the diagrammatic sketch of the hydrogen abstraction reaction of the disaccharides by hydroxyl radical, the structure of the transition states for pyran ring opening of moiety A and cleavage of α(1→2 glycosidic bond starting from the hydrogen abstraction of C6–H in moiety A of sucrose, the transition state structure for cleavage of α(1→2 glycosidic bond starting from the hydrogen abstraction of C1′-H in moiety B of sucrose, the transition state structure, sketch for the reaction process and relative energy change of the reaction pathway for direct cleavage of α(1→4 glycosidic bond starting from hydrogen abstraction of C6′–H of moiety B of maltose.

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

    Science.gov (United States)

    Ma, Enbo; Harrington, Lucas B.; O’Connell, Mitchell R.; Zhou, Kaihong; Doudna, Jennifer A.

    2015-01-01

    Summary Double-stranded DNA (dsDNA) cleavage by Cas9 is a hallmark of type II CRISPR-Cas immune systems. Cas9–guide RNA complexes recognize 20-base-pair sequences in DNA and generate a site-specific double-strand break, a robust activity harnessed for genome editing. DNA recognition by all studied Cas9 enzymes requires a protospacer adjacent motif (PAM) next to the target site. We show that Cas9 enzymes from evolutionarily divergent bacteria can recognize and cleave single-stranded DNA (ssDNA) by an RNA-guided, PAM-independent recognition mechanism. Comparative analysis shows that in contrast to the type II-A S. pyogenes Cas9 that is widely used for genome engineering, the smaller type II-C Cas9 proteins have limited dsDNA binding and unwinding activity and promiscuous guide-RNA specificity. These results indicate that inefficiency of type II-C Cas9 enzymes for genome editing results from a limited ability to cleave dsDNA, and suggest that ssDNA cleavage was an ancestral function of the Cas9 enzyme family. PMID:26545076

  9. Sequential Banking.

    OpenAIRE

    Bizer, David S; DeMarzo, Peter M

    1992-01-01

    The authors study environments in which agents may borrow sequentially from more than one leader. Although debt is prioritized, additional lending imposes an externality on prior debt because, with moral hazard, the probability of repayment of prior loans decreases. Equilibrium interest rates are higher than they would be if borrowers could commit to borrow from at most one bank. Even though the loan terms are less favorable than they would be under commitment, the indebtedness of borrowers i...

  10. Production of Proteolytic Enzymes by a Keratin-Degrading Aspergillus niger

    Directory of Open Access Journals (Sweden)

    Fernanda Cortez Lopes

    2011-01-01

    Full Text Available A fungal isolate with capability to grow in keratinous substrate as only source of carbon and nitrogen was identified as Aspergillus niger using the sequencing of the ITS region of the rDNA. This strain produced a slightly acid keratinase and an acid protease during cultivation in feather meal. The peak of keratinolytic activity occurred in 48 h and the maximum proteolytic activity in 96 h. These enzymes were partly characterized as serine protease and aspartic protease, respectively. The effects of feather meal concentration and initial pH on enzyme production were evaluated using a central composite design combined with response surface methodology. The optimal conditions were determined as pH 5.0 for protease and 7.8 for keratinase and 20 g/L of feather meal, showing that both models were predictive. Production of keratinases by A. niger is a less-exploited field that might represent a novel and promising biotechnological application for this microorganism.

  11. Trichomonas vaginalis Cysteine Proteinases: Iron Response in Gene Expression and Proteolytic Activity

    Science.gov (United States)

    Cárdenas-Guerra, Rosa Elena; Figueroa-Angulo, Elisa Elvira; Puente-Rivera, Jonathan; Zamudio-Prieto, Olga; Ortega-López, Jaime

    2015-01-01

    We focus on the iron response of Trichomonas vaginalis to gene family products such as the cysteine proteinases (CPs) involved in virulence properties. In particular, we examined the effect of iron on the gene expression regulation and function of cathepsin L-like and asparaginyl endopeptidase-like CPs as virulence factors. We addressed some important aspects about CPs genomic organization and we offer possible explanations to the fact that only few members of this large gene family are expressed at the RNA and protein levels and the way to control their proteolytic activity. We also summarized all known iron regulations of CPs at transcriptional, posttranscriptional, and posttranslational levels along with new insights into the possible epigenetic and miRNA processes. PMID:26090464

  12. Two-dimensional NMR evidence for cleavage of lignin and xylan substituents in wheat straw through hydrothermal pretreatment and enzymatic hydrolysis

    DEFF Research Database (Denmark)

    Yelle, Daniel J.; Kaparaju, Laxmi-Narasimha Prasad; Hunt, Christopher G.

    2013-01-01

    correlation spectroscopy, via an heteronuclear single quantum coherence experiment, revealed substantial lignin β-aryl ether cleavage, deacetylation via cleavage of the natural acetates at the 2-O- and 3-O-positions of xylan, and uronic acid depletion via cleavage of the (1 → 2)-linked 4-O....... g., further deacylation revealed by the depletion in ferulate and p-coumarate structures). Supplementary chemical analyses showed that the hydrothermal pretreatment increased the cellulose and lignin concentration with partial removal of extractives and hemicelluloses. The subsequent enzymatic...

  13. Influence of different proteolytic strains of Streptococcus thermophilus in co-culture with Lactobacillus delbrueckii subsp. bulgaricus on the metabolite profile of set-yoghurt.

    Science.gov (United States)

    Settachaimongkon, Sarn; Nout, M J Robert; Antunes Fernandes, Elsa C; Hettinga, Kasper A; Vervoort, Jacques M; van Hooijdonk, Toon C M; Zwietering, Marcel H; Smid, Eddy J; van Valenberg, Hein J F

    2014-05-02

    Proto-cooperation between Streptococcus thermophilus and Lactobacillus delbrueckii subsp. bulgaricus is one of the key factors that determine the fermentation process and final quality of yoghurt. In this study, the interaction between different proteolytic strains of S. thermophilus and L. delbrueckii subsp. bulgaricus was investigated in terms of microbial growth, acidification and changes in the biochemical composition of milk during set-yoghurt fermentation. A complementary metabolomics approach was applied for global characterization of volatile and non-volatile polar metabolite profiles of yoghurt associated with proteolytic activity of the individual strains in the starter cultures. The results demonstrated that only non-proteolytic S. thermophilus (Prt-) strain performed proto-cooperation with L. delbrueckii subsp. bulgaricus. The proto-cooperation resulted in significant higher populations of the two species, faster milk acidification, significant abundance of aroma volatiles and non-volatile metabolites desirable for a good organoleptic quality of yoghurt. Headspace SPME-GC/MS and (1)H NMR resulted in the identification of 35 volatiles and 43 non-volatile polar metabolites, respectively. Furthermore, multivariate statistical analysis allows discriminating set-yoghurts fermented by different types of starter cultures according to their metabolite profiles. Our finding underlines that selection of suitable strain combinations in yoghurt starters is important for achieving the best technological performance regarding the quality of product. Copyright © 2014 Elsevier B.V. All rights reserved.

  14. Equivalence between quantum simultaneous games and quantum sequential games

    OpenAIRE

    Kobayashi, Naoki

    2007-01-01

    A framework for discussing relationships between different types of games is proposed. Within the framework, quantum simultaneous games, finite quantum simultaneous games, quantum sequential games, and finite quantum sequential games are defined. In addition, a notion of equivalence between two games is defined. Finally, the following three theorems are shown: (1) For any quantum simultaneous game G, there exists a quantum sequential game equivalent to G. (2) For any finite quantum simultaneo...

  15. Accounting for Heterogeneous Returns in Sequential Schooling Decisions

    NARCIS (Netherlands)

    Zamarro, G.

    2006-01-01

    This paper presents a method for estimating returns to schooling that takes into account that returns may be heterogeneous among agents and that educational decisions are made sequentially.A sequential decision model is interesting because it explicitly considers that the level of education of each

  16. Simultaneous Versus Sequential Ptosis and Strabismus Surgery in Children.

    Science.gov (United States)

    Revere, Karen E; Binenbaum, Gil; Li, Jonathan; Mills, Monte D; Katowitz, William R; Katowitz, James A

    The authors sought to compare the clinical outcomes of simultaneous versus sequential ptosis and strabismus surgery in children. Retrospective, single-center cohort study of children requiring both ptosis and strabismus surgery on the same eye. Simultaneous surgeries were performed during a single anesthetic event; sequential surgeries were performed at least 7 weeks apart. Outcomes were ptosis surgery success (margin reflex distance 1 ≥ 2 mm, good eyelid contour, and good eyelid crease); strabismus surgery success (ocular alignment within 10 prism diopters of orthophoria and/or improved head position); surgical complications; and reoperations. Fifty-six children were studied, 38 had simultaneous surgery and 18 sequential. Strabismus surgery was performed first in 38/38 simultaneous and 6/18 sequential cases. Mean age at first surgery was 64 months, with mean follow up 27 months. A total of 75% of children had congenital ptosis; 64% had comitant strabismus. A majority of ptosis surgeries were frontalis sling (59%) or Fasanella-Servat (30%) procedures. There were no significant differences between simultaneous and sequential groups with regards to surgical success rates, complications, or reoperations (all p > 0.28). In the first comparative study of simultaneous versus sequential ptosis and strabismus surgery, no advantage for sequential surgery was seen. Despite a theoretical risk of postoperative eyelid malposition or complications when surgeries were performed in a combined manner, the rate of such outcomes was not increased with simultaneous surgeries. Performing ptosis and strabismus surgery together appears to be clinically effective and safe, and reduces anesthesia exposure during childhood.

  17. Forced Sequence Sequential Decoding

    DEFF Research Database (Denmark)

    Jensen, Ole Riis

    In this thesis we describe a new concatenated decoding scheme based on iterations between an inner sequentially decoded convolutional code of rate R=1/4 and memory M=23, and block interleaved outer Reed-Solomon codes with non-uniform profile. With this scheme decoding with good performance...... is possible as low as Eb/No=0.6 dB, which is about 1.7 dB below the signal-to-noise ratio that marks the cut-off rate for the convolutional code. This is possible since the iteration process provides the sequential decoders with side information that allows a smaller average load and minimizes the probability...... of computational overflow. Analytical results for the probability that the first Reed-Solomon word is decoded after C computations are presented. This is supported by simulation results that are also extended to other parameters....

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

    Science.gov (United States)

    Ma, Haojie; Zhou, Xiaoqiang; Zhan, Zhenzhen; Wei, Daidong; Shi, Chong; Liu, Xingxing; Huang, Guosheng

    2017-09-13

    Copper catalyzed chemoselective cleavage of the C(CO)-C(alkyl) bond leading to C-N bond formation with chelation assistance of N-containing directing groups is described. Inexpensive Cu(ii)-acetate serves as a convenient catalyst for this transformation. This method highlights the emerging strategy to transform unactivated alkyl ketones into amides in organic synthesis and provides a new strategy for C-C bond cleavage.

  19. ChloroP, a neural network-based method for predicting chloroplast transitpeptides and their cleavage sites

    DEFF Research Database (Denmark)

    Emanuelsson, O.; Nielsen, Henrik; von Heijne, Gunnar

    1999-01-01

    the cleavage sites given in SWISS-PROT. An analysis of 715 Arabidopsis thaliana sequences from SWISS-PROT suggests that the ChloroP method should be useful for the identification of putative transit peptides in genome-wide sequence data. The ChloroP predictor is available as a web-server at http......We present a neural network based method (ChloroP) for identifying chloroplast transit peptides and their cleavage sites. Using cross-validation, 88% of the sequences in our homology reduced training set were correctly classified as transit peptides or nontransit peptides. This performance level...

  20. Reading Remediation Based on Sequential and Simultaneous Processing.

    Science.gov (United States)

    Gunnison, Judy; And Others

    1982-01-01

    The theory postulating a dichotomy between sequential and simultaneous processing is reviewed and its implications for remediating reading problems are reviewed. Research is cited on sequential-simultaneous processing for early and advanced reading. A list of remedial strategies based on the processing dichotomy addresses decoding and lexical…

  1. Glycomics using mass spectrometry

    OpenAIRE

    Wuhrer, Manfred

    2013-01-01

    Mass spectrometry plays an increasingly important role in structural glycomics. This review provides an overview on currently used mass spectrometric approaches such as the characterization of glycans, the analysis of glycopeptides obtained by proteolytic cleavage of proteins and the analysis of glycosphingolipids. The given examples are demonstrating the application of mass spectrometry to study glycosylation changes associated with congenital disorders of glycosylation, lysosomal storage di...

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

    Science.gov (United States)

    Roda-Serrat, Maria Cinta; Christensen, Knud Villy; El-Houri, Rime Bahij; Fretté, Xavier; Christensen, Lars Porskjær

    2018-02-01

    Phycocyanins from cyanobacteria are possible sources for new natural blue colourants. Their chromophore, phycocyanobilin (PCB), was cleaved from the apoprotein by solvolysis in alcohols and alcoholic aqueous solutions. In all cases two PCB isomers were obtained, while different solvent adducts were formed upon the use of different reagents. The reaction is believed to take place via two competing pathways, a concerted E2 elimination and a S N 2 nucleophilic substitution. Three cleavage methods were compared in terms of yield and purity: conventional reflux, sealed vessel heated in an oil bath, and microwave assisted reaction. The sealed vessel method is a new approach for fast cleavage of PCB from phycocyanin and gave at 120°C the same yield within 30min compared to 16h by the conventional reflux method (P<0.05). In addition the sealed vessel method resulted in improved purity compared to the other methods. Microwave irradiation increased product degradation. Copyright © 2017 Elsevier Ltd. All rights reserved.

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

    International Nuclear Information System (INIS)

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

    2006-01-01

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

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

    Energy Technology Data Exchange (ETDEWEB)

    Hamilton, Brian S.; Chung, Changik; Cyphers, Soreen Y.; Rinaldi, Vera D.; Marcano, Valerie C.; Whittaker, Gary R., E-mail: grw7@cornell.edu

    2014-07-25

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

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

    International Nuclear Information System (INIS)

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

    2014-01-01

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

  6. Missed cleavage opportunities by FEN1 lead to Okazaki fragment maturation via the long-flap pathway

    KAUST Repository

    Zaher, Manal S.; Rashid, Fahad; Song, Bo; Joudeh, Luay I; Sobhy, Mohamed Abdelmaboud; Tehseen, Muhammad; Hingorani, Manju M; Hamdan, Samir

    2018-01-01

    RNA-DNA hybrid primers synthesized by low fidelity DNA polymerase α to initiate eukaryotic lagging strand synthesis must be removed efficiently during Okazaki fragment (OF) maturation to complete DNA replication. In this process, each OF primer is displaced and the resulting 5'-single-stranded flap is cleaved by structure-specific 5'-nucleases, mainly Flap Endonuclease 1 (FEN1), to generate a ligatable nick. At least two models have been proposed to describe primer removal, namely short- and long-flap pathways that involve FEN1 or FEN1 along with Replication Protein A (RPA) and Dna2 helicase/nuclease, respectively. We addressed the question of pathway choice by studying the kinetic mechanism of FEN1 action on short- and long-flap DNA substrates. Using single molecule FRET and rapid quench-flow bulk cleavage assays, we showed that unlike short-flap substrates, which are bound, bent and cleaved within the first encounter between FEN1 and DNA, long-flap substrates can escape cleavage even after DNA binding and bending. Notably, FEN1 can access both substrates in the presence of RPA, but bending and cleavage of long-flap DNA is specifically inhibited. We propose that FEN1 attempts to process both short and long flaps, but occasional missed cleavage of the latter allows RPA binding and triggers the long-flap OF maturation pathway.

  7. Missed cleavage opportunities by FEN1 lead to Okazaki fragment maturation via the long-flap pathway

    KAUST Repository

    Zaher, Manal S.

    2018-01-27

    RNA-DNA hybrid primers synthesized by low fidelity DNA polymerase α to initiate eukaryotic lagging strand synthesis must be removed efficiently during Okazaki fragment (OF) maturation to complete DNA replication. In this process, each OF primer is displaced and the resulting 5\\'-single-stranded flap is cleaved by structure-specific 5\\'-nucleases, mainly Flap Endonuclease 1 (FEN1), to generate a ligatable nick. At least two models have been proposed to describe primer removal, namely short- and long-flap pathways that involve FEN1 or FEN1 along with Replication Protein A (RPA) and Dna2 helicase/nuclease, respectively. We addressed the question of pathway choice by studying the kinetic mechanism of FEN1 action on short- and long-flap DNA substrates. Using single molecule FRET and rapid quench-flow bulk cleavage assays, we showed that unlike short-flap substrates, which are bound, bent and cleaved within the first encounter between FEN1 and DNA, long-flap substrates can escape cleavage even after DNA binding and bending. Notably, FEN1 can access both substrates in the presence of RPA, but bending and cleavage of long-flap DNA is specifically inhibited. We propose that FEN1 attempts to process both short and long flaps, but occasional missed cleavage of the latter allows RPA binding and triggers the long-flap OF maturation pathway.

  8. SKI2 mediates degradation of RISC 5'-cleavage fragments and prevents secondary siRNA production from miRNA targets in Arabidopsis.

    Science.gov (United States)

    Branscheid, Anja; Marchais, Antonin; Schott, Gregory; Lange, Heike; Gagliardi, Dominique; Andersen, Stig Uggerhøj; Voinnet, Olivier; Brodersen, Peter

    2015-12-15

    Small regulatory RNAs are fundamental in eukaryotic and prokaryotic gene regulation. In plants, an important element of post-transcriptional control is effected by 20-24 nt microRNAs (miRNAs) and short interfering RNAs (siRNAs) bound to the ARGONAUTE1 (AGO1) protein in an RNA induced silencing complex (RISC). AGO1 may cleave target mRNAs with small RNA complementarity, but the fate of the resulting cleavage fragments remains incompletely understood. Here, we show that SKI2, SKI3 and SKI8, subunits of a cytoplasmic cofactor of the RNA exosome, are required for degradation of RISC 5', but not 3'-cleavage fragments in Arabidopsis. In the absence of SKI2 activity, many miRNA targets produce siRNAs via the RNA-dependent RNA polymerase 6 (RDR6) pathway. These siRNAs are low-abundant, and map close to the cleavage site. In most cases, siRNAs were produced 5' to the cleavage site, but several examples of 3'-spreading were also identified. These observations suggest that siRNAs do not simply derive from RDR6 action on stable 5'-cleavage fragments and hence that SKI2 has a direct role in limiting secondary siRNA production in addition to its function in mediating degradation of 5'-cleavage fragments. © The Author(s) 2015. Published by Oxford University Press on behalf of Nucleic Acids Research.

  9. C-quence: a tool for analyzing qualitative sequential data.

    Science.gov (United States)

    Duncan, Starkey; Collier, Nicholson T

    2002-02-01

    C-quence is a software application that matches sequential patterns of qualitative data specified by the user and calculates the rate of occurrence of these patterns in a data set. Although it was designed to facilitate analyses of face-to-face interaction, it is applicable to any data set involving categorical data and sequential information. C-quence queries are constructed using a graphical user interface. The program does not limit the complexity of the sequential patterns specified by the user.

  10. Pathogenic Assay of Probiotic Bacteria Producing Proteolytic Enzymes as Bioremediation Bacteria Against Vannamei Shrimp Larvae (Litopenaeus vannamei)

    OpenAIRE

    Wilis Ari Setyati; Muhammad Zainuddin; Person Pesona Renta

    2017-01-01

    Application of bacteria in bioremediation of shrimp culture ponds is one of the methods used to clean internal pollutants. This study aimed to evaluate the pathogenicity of extracellular proteolytic enzyme produced by the probiotic bacteria as bioremediation bacteria on vannamei shrimp larvae culture. There were five probiotic bacteria, which were successfully isolated from the sediments served as substrate in mangrove area. The isolated bacteria were coded in number as 13, 19, 30, 33, and 36...

  11. 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

  12. Relationship between synthesis and cleavage of poliovirus-specific proteins.

    OpenAIRE

    Thomas, A A; Voorma, H O; Boeye, A

    1983-01-01

    Poliovirus proteinase was studied in vitro in lysates from poliovirus-infected HeLa cells. Preincubation of these lysates caused (i) a reduction in poliovirus proteinase activity and (ii) a partial dependence on exogenous mRNA for optimal translation. Proteins translated from endogenous poliovirus RNA in preincubated extracts from virus-infected HeLa cells are poorly cleaved. This cleavage deficiency is alleviated by adding fresh poliovirus RNA to the translation system, thus, allowing re-ini...

  13. Functional protease profiling with reporter peptides in serum specimens of colorectal cancer patients: demonstration of its routine diagnostic applicability.

    Science.gov (United States)

    Findeisen, Peter; Costina, Victor; Yepes, Diego; Hofheinz, Ralf; Neumaier, Michael

    2012-06-08

    The progression of many solid tumors is characterized by the release of tumor-associated proteases and the detection of tumor specific proteolytic activity in serum specimens is a promising diagnostic tool in oncology. Here we describe a mass spectrometry-based functional proteomic profiling approach that tracks the ex-vivo degradation of a synthetic endoprotease substrate in serum specimens of colorectal tumor patients. A reporter peptide (RP) with the amino acid sequence WKPYDAAD was synthesized that has a known cleavage site for the cysteine-endopeptidase cancer procoagulant (EC 3.4.22.26). The RP was added to serum specimens from colorectal cancer patients (n = 30), inflammatory controls (n = 30) and healthy controls (n = 30) and incubated under strictly standardized conditions. The proteolytic fragment of the RP was quantified with liquid chromatography / mass spectrometry (LC/MS). RP-spiking showed good intra- and inter-day reproducibility with coefficients of variation (CVs) that did not exceed a value of 10%. The calibration curve for the anchor peptide was linear in the concentration range of 0.4 - 50 μmol/L. The median concentration of the RP-fragment in serum specimens from tumor patients (TU: 17.6 μmol/L, SD 9.0) was significantly higher when compared to non-malignant inflammatory controls (IC: 11.1 μmol/L, SD 6.1) and healthy controls (HC: 10.3 μmol/L, SD 3.1). Highest area under receiver operating characteristic (AUROC) values were seen for discrimination of TU versus HC (0.89) followed by TU versus IC (0.77). IC and HC could barely be separated indicated by an AUROC value of 0.57. The proteolytic activity towards the RP was conserved in serum specimens that were kept at room temperature for up to 24 hours prior to the analysis. The proteolytic cleavage of reporter peptides is a surrogate marker for tumor associated proteolytic activity in serum specimens of cancer patients. A simple, robust and highly reproducible LC/MS method has

  14. Functional protease profiling with reporter peptides in serum specimens of colorectal cancer patients: demonstration of its routine diagnostic applicability

    Directory of Open Access Journals (Sweden)

    Findeisen Peter

    2012-06-01

    Full Text Available Abstract Background The progression of many solid tumors is characterized by the release of tumor-associated proteases and the detection of tumor specific proteolytic activity in serum specimens is a promising diagnostic tool in oncology. Here we describe a mass spectrometry-based functional proteomic profiling approach that tracks the ex-vivo degradation of a synthetic endoprotease substrate in serum specimens of colorectal tumor patients. Methods A reporter peptide (RP with the amino acid sequence WKPYDAAD was synthesized that has a known cleavage site for the cysteine-endopeptidase cancer procoagulant (EC 3.4.22.26. The RP was added to serum specimens from colorectal cancer patients (n = 30, inflammatory controls (n = 30 and healthy controls (n = 30 and incubated under strictly standardized conditions. The proteolytic fragment of the RP was quantified with liquid chromatography / mass spectrometry (LC/MS. Results RP-spiking showed good intra- and inter-day reproducibility with coefficients of variation (CVs that did not exceed a value of 10%. The calibration curve for the anchor peptide was linear in the concentration range of 0.4 – 50 μmol/L. The median concentration of the RP-fragment in serum specimens from tumor patients (TU: 17.6 μmol/L, SD 9.0 was significantly higher when compared to non-malignant inflammatory controls (IC: 11.1 μmol/L, SD 6.1 and healthy controls (HC: 10.3 μmol/L, SD 3.1. Highest area under receiver operating characteristic (AUROC values were seen for discrimination of TU versus HC (0.89 followed by TU versus IC (0.77. IC and HC could barely be separated indicated by an AUROC value of 0.57. The proteolytic activity towards the RP was conserved in serum specimens that were kept at room temperature for up to 24 hours prior to the analysis. Conclusion The proteolytic cleavage of reporter peptides is a surrogate marker for tumor associated proteolytic activity in serum specimens of cancer patients. A

  15. Endogenous proteolytic enzymes--a study of their impact on cod (Gadus morhua) muscle proteins and textural properties in a fermented product.

    Science.gov (United States)

    Yang, Fang; Rustad, Turid; Xu, Yanshun; Jiang, Qixing; Xia, Wenshui

    2015-04-01

    The aim of this study was to investigate endogenous proteolytic activities in a cod product and their impact on muscle proteins and textural properties during fermentation and storage. The result of specific proteolytic activities showed that cathepsins, especially cathepsin B, had the highest activities during fermentation and storage. SDS-PAGE indicated more degradation of myofibrillar proteins by cathepsin L than other proteases and that the hydrolysis by cathepsins was pronounced in the last stage of fermentation. Texture analysis showed that cathepsins had a negative impact on gel strength and this impact increased in the last stage of fermentation. However the product still had a firm texture. During storage (4 °C) for one week, no significant changes were seen in the gel strength. In conclusion, cathepsins had more impact on muscle proteins and textural properties than other proteases during fermentation but had little impact on gel strength during storage at 4 °C. Copyright © 2014 Elsevier Ltd. All rights reserved.

  16. Cleavage of influenza RNA by using a human PUF-based artificial RNA-binding protein–staphylococcal nuclease hybrid

    International Nuclear Information System (INIS)

    Mori, Tomoaki; Nakamura, Kento; Masaoka, Keisuke; Fujita, Yusuke; Morisada, Ryosuke; Mori, Koichi; Tobimatsu, Takamasa; Sera, Takashi

    2016-01-01

    Various viruses infect animals and humans and cause a variety of diseases, including cancer. However, effective methodologies to prevent virus infection have not yet been established. Therefore, development of technologies to inactivate viruses is highly desired. We have already demonstrated that cleavage of a DNA virus genome was effective to prevent its replication. Here, we expanded this methodology to RNA viruses. In the present study, we used staphylococcal nuclease (SNase) instead of the PIN domain (PilT N-terminus) of human SMG6 as an RNA-cleavage domain and fused the SNase to a human Pumilio/fem-3 binding factor (PUF)-based artificial RNA-binding protein to construct an artificial RNA restriction enzyme with enhanced RNA-cleavage rates for influenzavirus. The resulting SNase-fusion nuclease cleaved influenza RNA at rates 120-fold greater than the corresponding PIN-fusion nuclease. The cleaving ability of the PIN-fusion nuclease was not improved even though the linker moiety between the PUF and RNA-cleavage domain was changed. Gel shift assays revealed that the RNA-binding properties of the PUF derivative used was not as good as wild type PUF. Improvement of the binding properties or the design method will allow the SNase-fusion nuclease to cleave an RNA target in mammalian animal cells and/or organisms. - Highlights: • A novel RNA restriction enzyme using SNase was developed tor cleave viral RNA. • Our enzyme cleaved influenza RNA with rates >120-fold higher rates a PIN-fusion one. • Our artificial enzyme with the L5 linker showed the highest RNA cleavage rate. • Our artificial enzyme site-selectively cleaved influenza RNA in vitro.

  17. Top-down attention affects sequential regularity representation in the human visual system.

    Science.gov (United States)

    Kimura, Motohiro; Widmann, Andreas; Schröger, Erich

    2010-08-01

    Recent neuroscience studies using visual mismatch negativity (visual MMN), an event-related brain potential (ERP) index of memory-mismatch processes in the visual sensory system, have shown that although sequential regularities embedded in successive visual stimuli can be automatically represented in the visual sensory system, an existence of sequential regularity itself does not guarantee that the sequential regularity will be automatically represented. In the present study, we investigated the effects of top-down attention on sequential regularity representation in the visual sensory system. Our results showed that a sequential regularity (SSSSD) embedded in a modified oddball sequence where infrequent deviant (D) and frequent standard stimuli (S) differing in luminance were regularly presented (SSSSDSSSSDSSSSD...) was represented in the visual sensory system only when participants attended the sequential regularity in luminance, but not when participants ignored the stimuli or simply attended the dimension of luminance per se. This suggests that top-down attention affects sequential regularity representation in the visual sensory system and that top-down attention is a prerequisite for particular sequential regularities to be represented. Copyright 2010 Elsevier B.V. All rights reserved.

  18. 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.

  19. Cleavage/alteration of interleukin-8 by matrix metalloproteinase-9 in the female lower genital tract.

    Science.gov (United States)

    Zariffard, M Reza; Anastos, Kathryn; French, Audrey L; Munyazesa, Elisaphane; Cohen, Mardge; Landay, Alan L; Spear, Gregory T

    2015-01-01

    Interleukin-8 (IL-8, CXCL8) plays important roles in immune responses at mucosal sites including in the lower genital tract. Since several types of bacteria produce proteases that cleave IL-8 and many types of bacteria can be present in lower genital tract microbiota, we assessed genital fluids for IL-8 cleavage/alteration. Genital fluids collected by lavage from 200 women (23 HIV-seronegative and 177 HIV-seropositive) were tested for IL-8 cleavage/alteration by ELISA. IL-8 cleaving/altering activity was observed in fluids from both HIV-positive (28%) and HIV-negative women (35%). There was no clear relationship between the activity and the types of bacteria present in the lower genital tract as determined by high-throughput sequencing of the 16S rRNA gene. Protease inhibitors specific for matrix metalloproteinases (MMPs) reduced the activity and a multiplex assay that detects both inactive and active MMPs showed the presence of multiple MMPs, including MMP-1, -3, -7, -8, -9, -10 and -12 in genital secretions from many of the women. The IL-8-cleaving/altering activity significantly correlated with active MMP-9 as well as with cleavage of a substrate that is acted on by several active MMPs. These studies show that multiple MMPs are present in the genital tract of women and strongly suggest that MMP-9 in genital secretions can cleave IL-8 at this mucosal site. These studies suggest that MMP-mediated cleavage of IL-8 can modulate inflammatory responses in the lower genital tract.

  20. Mining compressing sequential problems

    NARCIS (Netherlands)

    Hoang, T.L.; Mörchen, F.; Fradkin, D.; Calders, T.G.K.

    2012-01-01

    Compression based pattern mining has been successfully applied to many data mining tasks. We propose an approach based on the minimum description length principle to extract sequential patterns that compress a database of sequences well. We show that mining compressing patterns is NP-Hard and

  1. Fast sequential Monte Carlo methods for counting and optimization

    CERN Document Server

    Rubinstein, Reuven Y; Vaisman, Radislav

    2013-01-01

    A comprehensive account of the theory and application of Monte Carlo methods Based on years of research in efficient Monte Carlo methods for estimation of rare-event probabilities, counting problems, and combinatorial optimization, Fast Sequential Monte Carlo Methods for Counting and Optimization is a complete illustration of fast sequential Monte Carlo techniques. The book provides an accessible overview of current work in the field of Monte Carlo methods, specifically sequential Monte Carlo techniques, for solving abstract counting and optimization problems. Written by authorities in the

  2. Computing sequential equilibria for two-player games

    DEFF Research Database (Denmark)

    Miltersen, Peter Bro

    2006-01-01

    Koller, Megiddo and von Stengel showed how to efficiently compute minimax strategies for two-player extensive-form zero-sum games with imperfect information but perfect recall using linear programming and avoiding conversion to normal form. Their algorithm has been used by AI researchers...... for constructing prescriptive strategies for concrete, often fairly large games. Koller and Pfeffer pointed out that the strategies obtained by the algorithm are not necessarily sequentially rational and that this deficiency is often problematic for the practical applications. We show how to remove this deficiency...... by modifying the linear programs constructed by Koller, Megiddo and von Stengel so that pairs of strategies forming a sequential equilibrium are computed. In particular, we show that a sequential equilibrium for a two-player zero-sum game with imperfect information but perfect recall can be found in polynomial...

  3. Computing Sequential Equilibria for Two-Player Games

    DEFF Research Database (Denmark)

    Miltersen, Peter Bro; Sørensen, Troels Bjerre

    2006-01-01

    Koller, Megiddo and von Stengel showed how to efficiently compute minimax strategies for two-player extensive-form zero-sum games with imperfect information but perfect recall using linear programming and avoiding conversion to normal form. Koller and Pfeffer pointed out that the strategies...... obtained by the algorithm are not necessarily sequentially rational and that this deficiency is often problematic for the practical applications. We show how to remove this deficiency by modifying the linear programs constructed by Koller, Megiddo and von Stengel so that pairs of strategies forming...... a sequential equilibrium are computed. In particular, we show that a sequential equilibrium for a two-player zero-sum game with imperfect information but perfect recall can be found in polynomial time. In addition, the equilibrium we find is normal-form perfect. Our technique generalizes to general-sum games...

  4. Two tandem RNase III cleavage sites determine betT mRNA stability in response to osmotic stress in Escherichia coli.

    Directory of Open Access Journals (Sweden)

    Minji Sim

    Full Text Available While identifying genes regulated by ribonuclease III (RNase III in Escherichia coli, we observed that steady-state levels of betT mRNA, which encodes a transporter mediating the influx of choline, are dependent on cellular concentrations of RNase III. In the present study, we also observed that steady-state levels of betT mRNA are dependent on RNase III activity upon exposure to osmotic stress, indicating the presence of cis-acting elements controlled by RNase III in betT mRNA. Primer extension analyses of betT mRNA revealed two tandem RNase III cleavage sites in its stem-loop region, which were biochemically confirmed via in vitro cleavage assays. Analyses of cleavage sites suggested the stochastic selection of cleavage sites by RNase III, and mutational analyses indicated that RNase III cleavage at either site individually is insufficient for efficient betT mRNA degradation. In addition, both the half-life and abundance of betT mRNA were significantly increased in association with decreased RNase III activity under hyper-osmotic stress conditions. Our findings demonstrate that betT mRNA stability is controlled by RNase III at the post-transcriptional level under conditions of osmotic stress.

  5. A camel-derived MERS-CoV with a variant spike protein cleavage site and distinct fusion activation properties

    Science.gov (United States)

    Millet, Jean Kaoru; Goldstein, Monty E; Labitt, Rachael N; Hsu, Hung-Lun; Daniel, Susan; Whittaker, Gary R

    2016-01-01

    Middle East respiratory syndrome coronavirus (MERS-CoV) continues to circulate in both humans and camels, and the origin and evolution of the virus remain unclear. Here we characterize the spike protein of a camel-derived MERS-CoV (NRCE-HKU205) identified in 2013, early in the MERS outbreak. NRCE-HKU205 spike protein has a variant cleavage motif with regard to the S2′ fusion activation site—notably, a novel substitution of isoleucine for the otherwise invariant serine at the critical P1′ cleavage site position. The substitutions resulted in a loss of furin-mediated cleavage, as shown by fluorogenic peptide cleavage and western blot assays. Cell–cell fusion and pseudotyped virus infectivity assays demonstrated that the S2′ substitutions decreased spike-mediated fusion and viral entry. However, cathepsin and trypsin-like protease activation were retained, albeit with much reduced efficiency compared with the prototypical EMC/2012 human strain. We show that NRCE-HKU205 has more limited fusion activation properties possibly resulting in more restricted viral tropism and may represent an intermediate in the complex pattern of MERS-CoV ecology and evolution. PMID:27999426

  6. [Cell-derived microparticles unveil their fibrinolytic and proteolytic function].

    Science.gov (United States)

    Doeuvre, Loïc; Angles-Cano, Eduardo

    2009-01-01

    Cell-derived microparticles (MP) are membrane microvesicles, 0.1-1 microm in size, shed by cells following activation or during apoptosis in a variety of pathological conditions. MPs released by blood cells or by vascular endothelial cells display molecular signatures that allow their identification and functional characterization. In addition, they provide tissue factor (TF) and a procoagulant phospholipid surface. Therefore, at present, the most strongly established applied research on MPs is their procoagulant activity as a determinant of thrombotic risk in various clinical conditions. Previous studies have indicated that MPs derived from malignant cells express matrix metalloproteinases, urokinase and its receptor (uPA/uPAR) that, in the presence of plasminogen, may act in concert to degrade extracellular matrix proteins. Recently, it was shown that MPs from TNFa-stimulated endothelial cells served as a surface for interaction with plasminogen and its conversion into plasmin by the uPA/uPAR system expressed at their surface. This capacity of MPs to promote plasmin generation confers them a new profibrinolytic and proteolytic function that may be of relevance in fibrinolysis, cell migration, angiogenesis, dissemination of malignant cells, cell detachment and apoptosis.

  7. Phosphorylation of iRhom2 Controls Stimulated Proteolytic Shedding by the Metalloprotease ADAM17/TACE

    Directory of Open Access Journals (Sweden)

    Miguel Cavadas

    2017-10-01

    Full Text Available Cell surface metalloproteases coordinate signaling during development, tissue homeostasis, and disease. TACE (TNF-α-converting enzyme, is responsible for cleavage (“shedding” of membrane-tethered signaling molecules, including the cytokine TNF, and activating ligands of the EGFR. The trafficking of TACE within the secretory pathway requires its binding to iRhom2, which mediates the exit of TACE from the endoplasmic reticulum. An important, but mechanistically unclear, feature of TACE biology is its ability to be stimulated rapidly on the cell surface by numerous inflammatory and growth-promoting agents. Here, we report a role for iRhom2 in TACE stimulation on the cell surface. TACE shedding stimuli trigger MAP kinase-dependent phosphorylation of iRhom2 N-terminal cytoplasmic tail. This recruits 14-3-3 proteins, enforcing the dissociation of TACE from complexes with iRhom2, promoting the cleavage of TACE substrates. Our data reveal that iRhom2 controls multiple aspects of TACE biology, including stimulated shedding on the cell surface.

  8. Sensitivity Analysis in Sequential Decision Models.

    Science.gov (United States)

    Chen, Qiushi; Ayer, Turgay; Chhatwal, Jagpreet

    2017-02-01

    Sequential decision problems are frequently encountered in medical decision making, which are commonly solved using Markov decision processes (MDPs). Modeling guidelines recommend conducting sensitivity analyses in decision-analytic models to assess the robustness of the model results against the uncertainty in model parameters. However, standard methods of conducting sensitivity analyses cannot be directly applied to sequential decision problems because this would require evaluating all possible decision sequences, typically in the order of trillions, which is not practically feasible. As a result, most MDP-based modeling studies do not examine confidence in their recommended policies. In this study, we provide an approach to estimate uncertainty and confidence in the results of sequential decision models. First, we provide a probabilistic univariate method to identify the most sensitive parameters in MDPs. Second, we present a probabilistic multivariate approach to estimate the overall confidence in the recommended optimal policy considering joint uncertainty in the model parameters. We provide a graphical representation, which we call a policy acceptability curve, to summarize the confidence in the optimal policy by incorporating stakeholders' willingness to accept the base case policy. For a cost-effectiveness analysis, we provide an approach to construct a cost-effectiveness acceptability frontier, which shows the most cost-effective policy as well as the confidence in that for a given willingness to pay threshold. We demonstrate our approach using a simple MDP case study. We developed a method to conduct sensitivity analysis in sequential decision models, which could increase the credibility of these models among stakeholders.

  9. The sequential structure of brain activation predicts skill.

    Science.gov (United States)

    Anderson, John R; Bothell, Daniel; Fincham, Jon M; Moon, Jungaa

    2016-01-29

    In an fMRI study, participants were trained to play a complex video game. They were scanned early and then again after substantial practice. While better players showed greater activation in one region (right dorsal striatum) their relative skill was better diagnosed by considering the sequential structure of whole brain activation. Using a cognitive model that played this game, we extracted a characterization of the mental states that are involved in playing a game and the statistical structure of the transitions among these states. There was a strong correspondence between this measure of sequential structure and the skill of different players. Using multi-voxel pattern analysis, it was possible to recognize, with relatively high accuracy, the cognitive states participants were in during particular scans. We used the sequential structure of these activation-recognized states to predict the skill of individual players. These findings indicate that important features about information-processing strategies can be identified from a model-based analysis of the sequential structure of brain activation. Copyright © 2015 Elsevier Ltd. All rights reserved.

  10. Regulation of blood vessels by prolactin and vasoinhibins.

    Science.gov (United States)

    Clapp, Carmen; Thebault, Stéphanie; Macotela, Yazmín; Moreno-Carranza, Bibiana; Triebel, Jakob; Martínez de la Escalera, Gonzalo

    2015-01-01

    Prolactin (PRL) stimulates the growth of new blood vessels (angiogenesis) either directly through actions on endothelial cells or indirectly by upregulating proangiogenic factors like vascular endothelial growth factor (VEGF). Moreover, PRL acquires antiangiogenic properties after undergoing proteolytic cleavage to vasoinhibins, a family of PRL fragments (including 16 kDa PRL) with potent antiangiogenic, vasoconstrictive, and antivasopermeability effects. In view of the opposing actions of PRL and vasoinhibins, the regulation of the proteases responsible for specific PRL cleavage represents an efficient mechanism for controlling blood vessel growth and function. This review briefly describes the vascular actions of PRL and vasoinhibins, and addresses how their interplay could help drive biological effects of PRL in the context of health and disease.

  11. A one-sided sequential test

    Energy Technology Data Exchange (ETDEWEB)

    Racz, A.; Lux, I. [Hungarian Academy of Sciences, Budapest (Hungary). Atomic Energy Research Inst.

    1996-04-16

    The applicability of the classical sequential probability ratio testing (SPRT) for early failure detection problems is limited by the fact that there is an extra time delay between the occurrence of the failure and its first recognition. Chien and Adams developed a method to minimize this time for the case when the problem can be formulated as testing the mean value of a Gaussian signal. In our paper we propose a procedure that can be applied for both mean and variance testing and that minimizes the time delay. The method is based on a special parametrization of the classical SPRT. The one-sided sequential tests (OSST) can reproduce the results of the Chien-Adams test when applied for mean values. (author).

  12. Mining Emerging Sequential Patterns for Activity Recognition in Body Sensor Networks

    DEFF Research Database (Denmark)

    Gu, Tao; Wang, Liang; Chen, Hanhua

    2010-01-01

    Body Sensor Networks oer many applications in healthcare, well-being and entertainment. One of the emerging applications is recognizing activities of daily living. In this paper, we introduce a novel knowledge pattern named Emerging Sequential Pattern (ESP)|a sequential pattern that discovers...... signicant class dierences|to recognize both simple (i.e., sequential) and complex (i.e., interleaved and concurrent) activities. Based on ESPs, we build our complex activity models directly upon the sequential model to recognize both activity types. We conduct comprehensive empirical studies to evaluate...

  13. Discrete population balance models of random agglomeration and cleavage in polymer pyrolysis

    Directory of Open Access Journals (Sweden)

    John E. J. Staggs

    2017-05-01

    Full Text Available The processes of random agglomeration and cleavage (both of which are important for the development of new models of polymer combustion, but are also applicable in a wide range of fields including atmospheric physics, radiation modelling and astrophysics are analysed using population balance methods. The evolution of a discrete distribution of particles is considered within this framework, resulting in a set of ordinary differential equations for the individual particle concentrations. Exact solutions for these equations are derived, together with moment generating functions. Application of the discrete Laplace transform (analogous to the Z-transform is found to be effective in these problems, providing both exact solutions for particle concentrations and moment generating functions. The combined agglomeration-cleavage problem is also considered. Unfortunately, it has been impossible to find an exact solution for the full problem, but a stable steady state has been identified and computed.

  14. Discrimination between sequential and simultaneous virtual channels with electrical hearing

    OpenAIRE

    Landsberger, David; Galvin, John J.

    2011-01-01

    In cochlear implants (CIs), simultaneous or sequential stimulation of adjacent electrodes can produce intermediate pitch percepts between those of the component electrodes. However, it is unclear whether simultaneous and sequential virtual channels (VCs) can be discriminated. In this study, CI users were asked to discriminate simultaneous and sequential VCs; discrimination was measured for monopolar (MP) and bipolar + 1 stimulation (BP + 1), i.e., relatively broad and focused stimulation mode...

  15. Evidence of Alternative Cystatin C Signal Sequence Cleavage Which Is Influenced by the A25T Polymorphism.

    Directory of Open Access Journals (Sweden)

    Annie Nguyen

    Full Text Available Cystatin C (Cys C is a small, potent, cysteine protease inhibitor. An Ala25Thr (A25T polymorphism in Cys C has been associated with both macular degeneration and late-onset Alzheimer's disease. Previously, studies have suggested that this polymorphism may compromise the secretion of Cys C. Interestingly, we found that untagged A25T, A25T tagged C-terminally with FLAG, or A25T FLAG followed by green fluorescent protein (GFP, were all secreted as efficiently from immortalized human cells as their wild-type (WT counterparts (e.g., 112%, 100%, and 88% of WT levels from HEK-293T cells, respectively. Supporting these observations, WT and A25T Cys C variants also showed similar intracellular steady state levels. Furthermore, A25T Cys C did not activate the unfolded protein response and followed the same canonical endoplasmic reticulum (ER-Golgi trafficking pathway as WT Cys C. WT Cys C has been shown to undergo signal sequence cleavage between residues Gly26 and Ser27. While the A25T polymorphism did not affect Cys C secretion, we hypothesized that it may alter where the Cys C signal sequence is preferentially cleaved. Under normal conditions, WT and A25T Cys C have the same signal sequence cleavage site after Gly26 (referred to as 'site 2' cleavage. However, in particular circumstances when the residues around site 2 are modified (such as by the presence of an N-terminal FLAG tag immediately after Gly26, or by a Gly26Lys (G26K mutation, A25T has a significantly higher likelihood than WT Cys C of alternative signal sequence cleavage after Ala20 ('site 1' or even earlier in the Cys C sequence. Overall, our results indicate that the A25T polymorphism does not cause a significant reduction in Cys C secretion, but instead predisposes the protein to be cleaved at an alternative signal sequence cleavage site if site 2 is hindered. Additional N-terminal amino acids resulting from alternative signal sequence cleavage may, in turn, affect the protease

  16. Hybrid Computerized Adaptive Testing: From Group Sequential Design to Fully Sequential Design

    Science.gov (United States)

    Wang, Shiyu; Lin, Haiyan; Chang, Hua-Hua; Douglas, Jeff

    2016-01-01

    Computerized adaptive testing (CAT) and multistage testing (MST) have become two of the most popular modes in large-scale computer-based sequential testing. Though most designs of CAT and MST exhibit strength and weakness in recent large-scale implementations, there is no simple answer to the question of which design is better because different…

  17. Sequential dependencies in magnitude scaling of loudness

    DEFF Research Database (Denmark)

    Joshi, Suyash Narendra; Jesteadt, Walt

    2013-01-01

    Ten normally hearing listeners used a programmable sone-potentiometer knob to adjust the level of a 1000-Hz sinusoid to match the loudness of numbers presented to them in a magnitude production task. Three different power-law exponents (0.15, 0.30, and 0.60) and a log-law with equal steps in d......B were used to program the sone-potentiometer. The knob settings systematically influenced the form of the loudness function. Time series analysis was used to assess the sequential dependencies in the data, which increased with increasing exponent and were greatest for the log-law. It would be possible......, therefore, to choose knob properties that minimized these dependencies. When the sequential dependencies were removed from the data, the slope of the loudness functions did not change, but the variability decreased. Sequential dependencies were only present when the level of the tone on the previous trial...

  18. Visual short-term memory for sequential arrays.

    Science.gov (United States)

    Kumar, Arjun; Jiang, Yuhong

    2005-04-01

    The capacity of visual short-term memory (VSTM) for a single visual display has been investigated in past research, but VSTM for multiple sequential arrays has been explored only recently. In this study, we investigate the capacity of VSTM across two sequential arrays separated by a variable stimulus onset asynchrony (SOA). VSTM for spatial locations (Experiment 1), colors (Experiments 2-4), orientations (Experiments 3 and 4), and conjunction of color and orientation (Experiment 4) were tested, with the SOA across the two sequential arrays varying from 100 to 1,500 msec. We find that VSTM for the trailing array is much better than VSTM for the leading array, but when averaged across the two arrays VSTM has a constant capacity independent of the SOA. We suggest that multiple displays compete for retention in VSTM and that separating information into two temporally discrete groups does not enhance the overall capacity of VSTM.

  19. SKI2 mediates degradation of RISC 5′-cleavage fragments and prevents secondary siRNA production from miRNA targets in Arabidopsis

    Science.gov (United States)

    Branscheid, Anja; Marchais, Antonin; Schott, Gregory; Lange, Heike; Gagliardi, Dominique; Andersen, Stig Uggerhøj; Voinnet, Olivier; Brodersen, Peter

    2015-01-01

    Small regulatory RNAs are fundamental in eukaryotic and prokaryotic gene regulation. In plants, an important element of post-transcriptional control is effected by 20–24 nt microRNAs (miRNAs) and short interfering RNAs (siRNAs) bound to the ARGONAUTE1 (AGO1) protein in an RNA induced silencing complex (RISC). AGO1 may cleave target mRNAs with small RNA complementarity, but the fate of the resulting cleavage fragments remains incompletely understood. Here, we show that SKI2, SKI3 and SKI8, subunits of a cytoplasmic cofactor of the RNA exosome, are required for degradation of RISC 5′, but not 3′-cleavage fragments in Arabidopsis. In the absence of SKI2 activity, many miRNA targets produce siRNAs via the RNA-dependent RNA polymerase 6 (RDR6) pathway. These siRNAs are low-abundant, and map close to the cleavage site. In most cases, siRNAs were produced 5′ to the cleavage site, but several examples of 3′-spreading were also identified. These observations suggest that siRNAs do not simply derive from RDR6 action on stable 5′-cleavage fragments and hence that SKI2 has a direct role in limiting secondary siRNA production in addition to its function in mediating degradation of 5′-cleavage fragments. PMID:26464441

  20. The target-to-foils shift in simultaneous and sequential lineups.

    Science.gov (United States)

    Clark, Steven E; Davey, Sherrie L

    2005-04-01

    A theoretical cornerstone in eyewitness identification research is the proposition that witnesses, in making decisions from standard simultaneous lineups, make relative judgments. The present research considers two sources of support for this proposal. An experiment by G. L. Wells (1993) showed that if the target is removed from a lineup, witnesses shift their responses to pick foils, rather than rejecting the lineups, a result we will term a target-to-foils shift. Additional empirical support is provided by results from sequential lineups which typically show higher accuracy than simultaneous lineups, presumably because of a decrease in the use of relative judgments in making identification decisions. The combination of these two lines of research suggests that the target-to-foils shift should be reduced in sequential lineups relative to simultaneous lineups. Results of two experiments showed an overall advantage for sequential lineups, but also showed a target-to-foils shift equal in size for simultaneous and sequential lineups. Additional analyses indicated that the target-to-foils shift in sequential lineups was moderated in part by an order effect and was produced with (Experiment 2) or without (Experiment 1) a shift in decision criterion. This complex pattern of results suggests that more work is needed to understand the processes which underlie decisions in simultaneous and sequential lineups.